Journal Book Terpene

PHYTOCHEMISTRY
www.elsevier.com/locate/phytochem
Phytochemistry Vol. 67, No. 5, 2006 Reports on Structure Elucidation
Contents
TERPENOIDS Salvidorol, a nor-abietane diterpene with a rare carbon skeleton and two abietane diterpene derivatives from Salvia dorrii Ahmed A. Ahmed *, Abou El-Hamd H. Mohamed, Joe Karchesy, Yoshinori Asakawa
1 16 7
pp 424428
OH
17
16 15 13 14
OHC H
10 5 4 8
14 13 15 12 9 11
HO 11
20 1
12
17
CO
10 9 8 7 6
Salvidorol (1), a irregular abietane-type diterpene and two epimeric diterpenes were isolated from the aerial parts of Salvia dorrii. The structures were established by high-eld NMR techniques (1H1H COSY, DEPT, HMQC, HMBC, NOESY, HRMS) and X-ray analysis.
2 3
OH
H O
6
2 3 4
H
18 19
OH
H
18 19
2 R = OMe 3 R = OMe
Iridoid glucosides from Kickxia abhaica D.A. Sutton from Scrophulariaceae Adnan J. Al-Rehaily *, Maged S. Abdel-Kader, Mohammad S. Ahmad, Jaber S. Mossa
R1O OH
From Kickxia abhaica two iridoid glucosides (12), were isolated. Their structures were established by spectral analysis, including 2D NMR data.
pp 429432
O H OR2
1- R1 = OCOCH 3 ; R2 = Glc
' 2- R1 = H ; R2 = Glc-6-OHbenzoyl
Five labdane diterpenoids from the seeds of Aframomum zambesiacum Marguerite Kenmogne, Elise Prost, Dominique Harakat, Marie-Jose Jacquier, Michel Frederich, Lucas B. Sondengam, ` Monique Zeches, Pierre Wao-Teguo *
Five labdane diterpenoids were isolated from the seeds of Aframomum zambesiacum along with the known labdanes, aframodial, aulacocarpin A and B, galanal A, and galanolactone and a linear sesquiterpene, nerolidol. Their structures were elucidated by spectroscopic analysis. Antiplasmodial activity against Plasmodium falciparum for some of the isolated compounds was evaluated.
pp 433438
O O
OH O HO R 7 : R = H, 8 : R = OH
420
Contents / Phytochemistry 67 (2006) 419423
Hydroxylation of the sesterterpene leucosceptrine by the fungus Rhizopus stolonifer Muhammad Iqbal Choudhary *, Rosa Ranjit, Atta-ur-Rahman, Krishna Prasad Devkota, Syed Ghulam Musharraf, Tirtha Maiya Shrestha
The microbial transformation of leucosceptrine (1) by Rhizopus stolonifer, aorded two leucosesterpenes, 1a-hydroxyleucosceptrine (2), and 8a-hydroxyleucosceptrine (3).
HO O OH CH3 H O
pp 439443
OH CH3 H OH
HO H3C OH H3C H O CH3 H3C HO H3C H H3C O O
HO H O CH3
H3C
H O O
Clerodane and labdane diterpenoids from Nuxia sphaerocephala Lengo Mambu *, Philippe Grellier, Loic Florent, Roger Joyeau, David Ramanitrahasimbola, Philippe Rasoanaivo, Francois Frappier
COOH H R
pp 444451
Four clerodane and three labdane diterpenoids (17) were isolated from the leaves of Nuxia sphaerocephala. Their structures have been elucidated on the basis of NMR and MS data. The antiplasmodial activity of the compounds has been evaluated.
R3
R2 R1
R R1 R2 R3 1. H,H OH H H 2 OH H H 3 O H 4. H,H OH H E-caffeoyloxy
Rings B,D-seco limonoids from the leaves of Swietenia mahogani Samir A.M. Abdelgaleil, Matsumi Doe, Yoshiki Morimoto, Munehiro Nakatani *
Three types of rings B,D-seco limonoids were isolated and structures of nine compounds were elucidated by spectroscopic methods.
pp 452458
O
O MeO2C H R
2
O OH OR 1 OTig
O O R
O
3
PHENOLICS Flavones and isoavones from the west African Fabaceae Erythrina vogelii Alain F. Kamdem Wao, Philip H. Coombes, Dulcie A. Mulholland *, Augustin E. Nkengfack, Zacharias T. Fomum
HO O
pp 459463
The stem bark of Erythrina vogelii collected in Nigeria has yielded two isoavones vogelins H (1) and I (2), a avone, vogelin J (3), and eight known avonoids.
O HO vogelin H (1) O OH
421
Phenolic compounds from the owers of Garcinia dulcis S. Deachathai, W. Mahabusarakam *, S. Phongpaichit, W.C. Taylor, Y.-J. Zhang, C.-R. Yang
O OH
pp 464469
OMe
Dulcisxanthones CF and dulcinone together with 22 known compounds were isolated from the owers of Garcinia dulcis. The radical scavenging and antibacterial activities were investigated.
MeO
O OMe
OMe
Xanthone derivatives from Cratoxylum cochinchinense roots W. Mahabusarakam *, W. Nuangnaowarat, W.C. Taylor
Xanthones and caged-prenylated xanthones, named cochinchinones AD, a synthetic known caged-prenylated xathone and seven known xanthones were isolated from the roots of Cratoxylum cochinchinense. Some of the compounds exhibited eective antioxidative properties.
pp 470474
O O O
H 3CO O OH
ALKALOIDS Alkaloids from Oriciopsis glaberrima Engl. (Rutaceae) Jean Duplex Wansi *, Jean Wandji, Alain Francois Kamdem Wao, Happi Emmanuel Ngeufa, Jean Claude Ndom, Serge Fotso, Rajendra Prasad Maskey, Dieudonne Njamen, Tanee Zacharias Fomum, Harmut Laatsch
OH O
pp 475480
OH
N R O H H N H OH
Alkaloid derivatives, oriciacridone A (1) and B (2), were isolated from the stems bark of Oriciopsis glaberrima Engl., and their structures determined spectroscopically. The extract exhibited in vitro signicant antimicrobial activity against a range of micro-organisms.
O OH
1 2
R = H R = OH
GENERAL CHEMISTRY Terpenoids and phenol derivatives from Malva silvestris Francesca Cutillo, Brigida DAbrosca, Marina DellaGreca *, Antonio Fiorentino, Armando Zarrelli
A sesquiterpene and a tetrahydroxylated acyclic diterpene were isolated from Malva silvestris. The structures of the compounds were determined by spectroscopic NMR and MS analyses. Their eects on germination and growth of Lactuca sativa L. have been studied in the concentration range 10)410)7 M.
pp 481485
OH OMe O
422
Hydroquinone diglycoside acyl esters from the stems of Glycosmis pentaphylla Junsong Wang, Yingtong Di, Xianwen Yang, Shunlin Li, Yuehu Wang, Xiaojiang Hao *
HO O O O OH
pp 486491
OMe OH O O O O OH OH OH OH OH OMe HO O O
MeO OH O O MeO O OH
OH
OH OH
From the stems of Glycosmis pentaphylla, three hydroquinone diglycoside acyl esters and one known one were isolated.
HO
OMe
OH O MeO O OH OH O O OH OH OH O O O
OMe OH
Unusual chromenes from Peperomia blanda Leosvaldo S.M. Velozo, Marcelo J.P. Ferreira, Maria Isabel S. Santos, Davyson L. Moreira, Vicente P. Emerenciano *, Maria Auxiliadora C. Kaplan
R1
9 4 1' 2 1''
pp 492496
Two chromenes were isolated and identied from the methanol extract of the aerial parts of Peperomia blanda in addition to stigmasterol, sitosterol and campesterol. Their structures were established as 2S-(4-methyl-3-pentenyl)-6-formyl-8-hydroxy-2,7-dimethyl-2Hchromene and 2S-(4-methyl-3-pentenyl)-5-hydroxy-6-formyl-2,7-dimethyl-2H-chromene through spectroscopic methods.
O
10
4'
R2 1- R1 =H; R2 =OH; 2- R1 =OH; R2 =H;
Cytotoxic and aromatic constituents from Salvia miltiorrhiza Ming-Jaw Don, Chien-Chang Shen, Wan-Jr Syu, Yi-Huei Ding, Chang-Ming Sun *
OH O O O O O
pp 497503
OH
Five naturally occurring products along with 13 known constituents were isolated from the root of Salvia miltiorrhiza. Selected compounds were evaluated for their biological activity.
O O O O CH3(CH2)14 HO O
O O HO
O O
Oligomeric secoiridoid glucosides from Jasminum abyssinicum Francesca Romana Gallo *, Giovanna Palazzino, Elena Federici, Raaella Iurilli, Franco Delle Monache, Kusamba Chifundera, Corrado Gale
7 1
pp 504510
OOC H
9" 10"
COOMe
4 3
From the root bark of Jasminum abyssinicum, three oligomeric secoiridoid glucosides, craigosides AC, were isolated and their structures established.
R2O
7"
H2C
8"
CH2
OH
10
O
1
R1 O
H2C
2" 1"
3" 4" 5"
O R3
O HO
1'
2'
3'
OH
4'
CH3
6"
OH
5' 6'
OH
423
Acetylated avonol diglucosides from Meconopsis quintuplinervia Xiao-Ya Shang, Ying-Hong Wang, Chong Li, Cheng-Zhong Zhang, Yong-Chun Yang, Jian-Gong Shi *
HO O O R5 OH O O O O OR2 HO HO OH R4
pp 511515
OH OR1 1 2 3 4 R1 = R4 = H, R2 = Ac, R3 = OH, R5 = CH2OH R1 = R4 = H, R2 = Ac, R3 = OH, R5 = CH2OAc R1 = Me, R2 = Ac, R3 = OH, R4 = H, R5 = CH2OH R1 = R3 = H, R2 = Ac, R4 = OH, R5 = H
Four acetylated avonal diglucosides 14, together with ve known avonol glycosides, have been isolated from Meconopsis quintuplinervia.
R3 HO
Lignan, phenolic and iridoid glycosides from Stereospermum cylindricum Tripetch Kanchanapoom *, Pawadee Noiarsa, Hideaki Otsuka, Somsak Ruchirawat
Lignan, phenolic and iridoid glycosides were isolated from the leaves and branches of Stereospermum cylindricum
O-Glc MeO OH
pp 516520
OH OH
HO
OMe
(+)-cycloolivil 4'-O--D-glucopyranoside
OTHER CONTENTS Corrigendum Announcement The Phytochemical Society of EuropePierre-Fabre 2006 Award for Phytochemistry Author Index Guide for Authors
* Corresponding author
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IN D E X E D /A B S T R A C T E D I N : Current Awareness in Biological Sciences (CABS), Curr Cont ASCA. Chem. Abstr. BIOSIS Data, PASCALCNRS Data, CAB Inter, Cam Sci Abstr, Curr Cont/Agri Bio Env Sci, Curr Cont/Life Sci, Curr Cont Sci Cit Ind, Curr Cont SCISEARCH Data, Bio Agri Ind ISSN 0031-9422
PHYTOCHEMISTRY Phytochemistry 67 (2006) 424428 www.elsevier.com/locate/phytochem
Salvidorol, a nor-abietane diterpene with a rare carbon skeleton and two abietane diterpene derivatives from Salvia dorrii
Ahmed A. Ahmed
b
a,*
a
, Abou El-Hamd H. Mohamed b, Joe Karchesy c, Yoshinori Asakawa
Department of Chemistry, Faculty of Science, El-Minia University, El-Minia 91516, Egypt Department of Chemistry, Aswan-Faculty of Science, South Valley University, Aswan, Egypt c Department of Wood Science and Engineering, Oregon State University, Corvallis, OR 97331, USA Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan Received 21 October 2005; received in revised form 4 December 2005 Available online 3 February 2006
Abstract Salvidorol (1), a irregular nor-abietane-type diterpene, was isolated from the aerial parts of Salvia dorrii, in addition to two epimeric abietane diterpenes (2 and 3). This is the rst report of a nor-diterpene with an irregular skeleton. The structures were established by high-eld NMR techniques (1H1H COSY, DEPT, HMQC, HMBC, NOESY and HRMS) and in case of 2 was conrmed by X-ray analysis. 2005 Elsevier Ltd. All rights reserved.
Keywords: Salvia dorrii; Lamiaceae; Nor-abietane diterpene; Salvidorol; 7a- and 7b-Methoxyrosmanol
1. Introduction The genus Salvia, a member of the family Lamiaceae, consists of about 500 species distributed throughout the world. Some species of this genus have held a place of importance from ancient times, due to their medicinal properties (Penso, 1980). They are rich in avonoids (Barberan, 1986), monoterpenes (Emboden et al., 1967) and diterpenes with abietane and clerodane skeletons (Luis, 1991; Rodriguez-Hahn et al., 1992). Many diterpenes were reported from Salvia have shown antioxidant (Nakanati, 1994) and antibacterial activities (Sosa et al., 1994). Recently, several nitrogen containing compounds were isolated from S. miltiorrhiza and were examined for cytotoxic and antimicrobial properties (Ming-Jaw et al., 2005). The avonoid constituent of S. dorrii has been studied before (Wollenweber et al., 1992). In this paper we describe from S. dorrii (Kellog) Abrams the isolation and structural elu*
cidation of salvidorol (1), a novel carbon skeletal nor-abietane diterpene and two diterpenes type abietane.
2. Results and discussion The methylene chloride extract of the air-dried aerial parts of S. dorrii was chromatographed on silica gel and Sephadex LH-20 columns to give a novel nor-diterpene (1), for which the name salvidorol was given, and two epimeric abietane diterpenes (2 and 3) (new). Compound 1, 20 yellowish oil, aD 1:53 (c 0.98, CHCl3), its IR spectrum showed absorption bands at 3409 cm1 (OH) and 1719 cm1 (C@O). The low resolution EIMS showed a molecular ion peak [M]+ at m/z 318 (100%), followed by a fragment at m/z 300 [M H2O]+. The high resolution mass spectrum exhibited a molecular ion peak [M]+ at m/z 318.1824 (calcd. 318.1817), in accord with the molecular formula of C19H26O4. The structure of salvidorol (1) was determined from careful investigation of the 1D and 2D NMR measurements. The 1H NMR spectrum revealed
Corresponding author. Tel.: +208 634 5267; fax: +208 634 2601. E-mail address: abdellaahmed@yahoo.com (A.A. Ahmed).
0031-9422/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2005.12.009
A.A. Ahmed et al. / Phytochemistry 67 (2006) 424428
425
the presence of two singlet signals at dH 1.12 (3H, H-18) and 1.11 (3H, H-19), an isopropyl group at dH 1.25 (3H, H-16), 1.26 (3H, H-17) and 3.27 (1H, H-15), a broad singlet at dH 5.80 (H-6) and a formyl proton at d 10.0 (s, H-7). The most characteristic and important signal being a one-proton signal at dH 3.45 (1H, ddd, J = 12.0, 12.0, 3.0 Hz), which correlated in 1H1H COSY with three signals at dH 1.15 (H-1a), 2.30 (H-1b) and 1.68 (H-5a). Therefore, this proton was assigned for H-10 and suggested the absence of H-20, which supported the presence of a nor-diterpene skeleton. The 13C NMR spectrum showed 19 carbon signals were classied by DEPT experiments as follows: four methyl carbon signals at dC 22.1 (C-16), 22.2 (C-17), 20.8 (C-18) and 30.1 (C-19), three methylene carbon signals at dC 36.8 (C-1), 22.8 (C-2) and 42.6 (C-3), four methine carbon signals at dC 52.6 (C-5), 91.9 (C-6), 28.5 (C10) and 27.0 (C-15). The formyl carbon signal appeared at d 191.1, while the protonated aromatic carbon signal appeared at dC 125.3 (C-14). The downeld shift of C-6 at dC 91.9 in the 13C NMR spectrum suggested the existence of a hemiacetal moiety in the structure. Moreover, all proton and carbon signals were determined by 1H1H COSY, HMQC and HMBC (Table 1). The HMBC spectrum (Fig. 1) was used to place the aldehydic group at C-8 on the basis of the correlation between the aromatic proton at dH 7.37 (H-14) with the aldehydic carbon signal at dC 191.9 (C-7). Other important correlations were observed, namely, H-5 with C-10, H-6 with C-10 and C11, H-10 with C-8 and C-11, H-18 and H-19 with C-3
Fig. 1. Selective HMBC correlations of compound 1.
Table 1 NMR data of 1 (600 MHz, CDCl3, d-values) Position 1a 1b 2a 2b 3a 3b 4 5a 6 7 8 9 10 11 12 13 14 15 16 17 18 19 dC 36.8 dH 1.15 m 2.30 dd (12.0, 3.0) 1.65 m 1.78 dt (13.8, 4.0) HBMC (HC)
and C-5 and H-15 with C-12, C-13 and C-14. The coupling constant between H-5 and H-6 was consistent with the bconguration of hydroxyl group at C-6 (Gonzalez et al., 1989). Dreiding models demonstrated the angle between H-5 and H-6 was about 90, which was in agreement with the broad singlet observed for H-6. This stereochemistry was supported by a NOESY spectrum that exhibited eects between H-6 (d 5.80) with H-1a (d 1.15) and H-19 (d 1.11), H-10 (d 3.45) with H-1b (d 2.30), H-2b (d 1.78) and H-18 (d 1.12). Also, it showed a clear eect between the formayl proton with H-10 (at d 3.45) and H-1b (at d 2.30). Although, few regular abietane diterpenes lacking the 20methyl group were reported from the genus Salvia (Lee et al., 1987), this is the rst irregular abietane diterpene which lacking the 20-methyl group.
16 7
OH
17
16 15 13 14
OHC H
10 5 4 8
14 13 15 12 9 11
HO 11
20 1
12
17
22.8
1 2
CO
10 9 8 7 6
OH
42.6
1.46 m 1.30 dd (13.8, 4.0)
H O
6
2 3 4
32.8 s 52.6 91.9 191.1 127.0 126.8 28.5 137.8 148.0 132.0 125.3 27.0 22.1 22.2 20.8 30.1
H
18
1.68 dd (12.0, 1.2) 5.80 br s 10.0 s
C-4, C-10 C-10, C-11
19
OH
H
18 19
2 R = OMe 3 R = OMe
3.45 ddd (12.0, 12.0, 3)
C-8, C-11
7.37 s 3.27 1.25 1.26 1.12 1.11 septet (7.2) d (7.2) d (7.2) s s
C-7, C-8, C-12 C-12, C-13, C-14 C-13 C-13 C-3, C-5 C-3, C-5
Compound 2 was isolated as colorless crystal. Its 1H NMR spectrum showed an isopropyl moiety as one-proton septet at d 3.07 (J = 7 Hz) and two geminal methyls doublets at d 1.21 and 1.22 (J = 7 Hz). A singlet signal at d 6.79 was assigned to an aromatic proton. Moreover, it revealed two doublets at d 4.26 and 4.71 (J = 3.0 Hz) assigned for H-7 and H-6, respectively, while H-5 appeared as singlet signal at d 2.24. Also, its 1H NMR spectrum showed a sharp three-proton singlet at d 3.66 in accord with 2 being a methoxylated derivative of rosmanol (Ahmed et al., 1995). The 13C NMR and the multiplicities
426
of the individual signals were determined using DEPT as follows: ve methyls (one oxygenated, d 58.14), three methylenes, ve methines (one aromatic, d 120.81 and two oxygenated, d 74.99 and 77.61) and eight quaternary carbons (one carbonyl and ve aromatics). The relative stereochemistry of 2 could be deduced from NOESY experiment, where H-5, H-6, OMe and H-19 correlated with each other, indicating the a-orientation of these protons. Also, it showed correlations between the aromatic proton with H7 and H-15. Additionally, the stereochemistry of 2 was conrmed by X-ray analysis (Fig. 2). Therefore, compound 2 was established to be 7a-methoxyrosmanol. Although, the NMR spectral data of 2 were identical with the previously reported data for 7a-methoxyrosmanol (Takenaka et al., 1997), compound 2 showed opposite optical rotation sign a22 6 (c = 0.35, CHCl3), while the previously D reported optical rotation was a22 24:5 (c = 0.42, D CHCl3), (Takenaka et al., 1997). Therefore, compound 2 could be enantiomer of the previously reported compound. Compound 3 was isolated as yellow oil, its CIMS exhibited a molecular ion peak [M + H]+ at m/z 361 and exact mass at m/z 361.20119 (calcd. 361.20150), established the elemental composition as C21H30O5. Its IR spectrum showed absorption bands indicative of a c-lactone group (1754 cm1) and aromatic hydroxyl groups (3360 cm1). The 1H NMR and 13C NMR spectral data of 3 were very similar to those of 2, except the optical rotation sign which 22 was opposite, aD 52 (c = 1.2, CHCl3), suggesting that 3 was an epimer of 2. Comparison of the 1H and 13C NMR spectra of 3 with those of 2 showed some dierences. The signals of H-6 (d 4.92) and H-7 (d 4.40) of 3 were detected at downeld shift (Dd + 0.21 and Dd + 0.14, respectively) in comparison with those of 2. Moreover, the carbon signal at position 5 (dC 55.39) was shifted downeld. The positions of the methoxyl group, isopropyl group and lactone
moiety were determined by HMBC spectrum. In this spectrum, H-C connectivity between the aromatic proton and C-7 (dC 78.2), C-9 (dC 123.5), C-11 (dC 142.5) and C-15 (dC 27.2); between the methoxyl and C-7 (dC 78.2), supported the location of the methoxyl group at C-7 and the isopropyl at C-12. Moreover, it displayed correlations between H-5 and C-7 (dC 78.2), C-9 (dC 123.5), C-10 (dC 47.9), C-18 (dC 21.9), C-19 (dC 31.8) and C-20 (dC 178.9); between H-6 and C-8 (dC 126.5) and C-20 (dC 178.9); between H-16, H-17 and C-15 (dC 27.2); between H-18, H-19 and C-3 (dC 37.9) and C-4 (dC 31.6). A NOESY experiment of 3 showed a cross-peak between H-5 and H-6 with H-7, indicated the a-orientation of H-7. Therefore, compound 3 was assigned to be 7b-methoxyrosmanol, a new epimer of 2.
3. Experimental 3.1. General NMR spectra were measured with a Bruker AMX-400 spectrometer and Varian Unity 600 MHz NMR spectrometry, with TMS as an internal standard. The IR spectra [oily lm, CHCl3] were taken on PerkinElmer FT-IR spectrometer. Optical rotations were measured with a Perkin Elmer 241 Polarimeter operating at sodium D line. MS were recorded on a JEOL SX102A mass spectrometer (70 eV). 3.2. Plant material Salvia dorrii (Kellog) Abrams (Lamiaceae) was collected in the owering stage near Mitchell, Oregon (voucher # 195789 Oregon State University Herbarium).
Fig. 2. ORTEP diagram of the crystal structure of 2.
427
3.3. Extraction and isolation The dichloromethane extract (20 mg) of the aerial parts (950 g) of S. dorrii was fractionated by ash column chromatography (5 55 cm) over silica gel (1 kg) eluting with nhexane with an increasing amount of CH2Cl2. The fraction (100%, n-hexane 1 L) contained hydrocarbons and waxes. The second fraction (n-hexaneCH2Cl2 3:1, 2 L) gave a crude material which was puried by a Sephadex LH-20 (3 35 cm, n-hexaneCH2Cl2MeOH 7:4:0.5, 300 mL) to give compounds 2 (14 mg), 3 (3 mg). The third fraction (CH2Cl2, 100%) was further puried by a Sephadex LH20 (3 35 cm, n-hexaneCH2Cl2MeOH 7:4:1, 500 mL) to aord compound 1 (2.5 mg). 3.3.1. Salvidorol (1) Yellowish oil; a20 1:53 (c 0.98, CHCl3); IR D (mKBr cm1 ): 3409, 3019, 2927, 1719, 1680, 1606, 1571, max 1436; EIMS [M]+m/z 318 (100), [M H2O] m/z 300 (30), 285 (40), 257 (60), 231 (30), 205 (40), 181(15); HREIMS m/z 318.1824 (calc. for C19H26O4, 318.1817). 1H and 13C NMR (see Table 1). 3.3.2. 7a-Methoxyrosmanol (2) Colorless crystal; a22 6 (c = 0.35, CHCl3); IR D (mKBr cm1 ): 3590, 2960, 1730, 1200; EIMS [M]+m/z 360 max (30), 316 (10), 285 (215); 1H NMR (400 MHz, CDCl3): d = 3.16 (1H, br. d, J = 14 Hz, H-1b), 2.00 (1H, m, H1a), 1.55 (1H, m, H-2b), 1.68 (1H, m, H-2a), 1.19 (1H, m, H-3b), 1.46 (1H, br. d, J = 14 Hz, H-3a), 2.24 (1H, s, H-5), 4.71 (1H, d, J = 3.0 Hz, H-6a), 4.26 (1H, d, J = 3.0 Hz, H-7b), 6.79 (1H, s, H-14), 3.07 (1H, septet, J = 7 Hz, H-15), 1.21 (3H, d, J = 7 Hz, H-16), 1.22 (3H, d, J = 7 Hz, H-17), 0.93 (3H, s, H-18), 1.01 (3H, s, H-19), 3.66 (3H, s, OMe); 13C NMR (100 MHz, CDCl3): d = 27.0 (t, C-1), 19.0 (t, C-2), 38.0 (t, C-3), 31.3 (s, C-4), 51.1 (d, C-5), 75.0 (d, C-6), 77.6 (d, C-7), 125.9 (s, C-8), 124.8 (s, C-9), 47.2 (s, C-10), 143.5 (s, C-11), 142.0 (s, C12), 135.6 (s, C-13), 120.8 (d, C-14), 27.1 (d, C-15), 22.2 (q, C-16), 22.5 (q, C-17), 22.0 (q, C-18), 31.5 (q, C-19), 179.9 (s, C-20), 58.2 (q, OMe). 3.3.3. 7b-Methoxyrosmanol (3) 22 Yellow material; aD 52 (c = 1.2, CHCl3); IR KBr 1 (mmax cm ): 3360, 2957, 1754, 1682, 1556, 1454; CIMS [M + H]+m/z 361 (100), 329 (60), 315 (8), 301 (12), 183 (8); HRCIMS m/z 361.20119 (calc. for C21H28O5, 361.20150). 1H NMR (400 MHz, CDCl3): d = 3.18 (1H, br. d, J = 14 Hz, H-1b), 1.91 (1H, m, H-1a), 1.51 (1H, m, H-2b), 1.61 (1H, m, H-2a), 1.18 (1H, m, H-3b), 1.42 (1H, br d, J = 14 Hz, H-3a), 1.90 (1H, s, H-5a), 4.92 (1H, d, J = 3.0 Hz, H-6a), 4.40 (1H, d, J = 3.0 Hz, H-7a), 6.77 (1H, s, H-14), 3.00 (1H, septet, J = 7 Hz, H-15), 1.01 (3H, d, J = 7 Hz, H-16), 1.12 (3H, d, J = 7 Hz, H-17), 0.92 (3H, s, H-18), 0.97 (3H, s, H-19), 3.57 (3H, s, OMe); 13 C NMR (100 MHz, CDCl3): d 27.1 (t, C-1), 18.9 (t, C2), 37.9 (t, C-3), 31.6 (s, C-4), 55.4 (d, C-5), 74.7 (d, C-6),
78.2 (d, C-7), 123.5 (s, C-8), 126.5 (s, C-9), 47.9 (s, C-10), 142.5 (s, C-11), 142.1 (s, C-12), 135.5 (s, C-13), 118.9 (d, C-14), 27.2 (d, C-15), 22.1 (q, C-16), 22.7 (q, C-17), 21.9 (q, C-18), 31.8 (q, C-19), 178.9 (s, C-20), 56.0 (q, OMe). 3.3.4. X-ray crystallography of compound 2 Crystal data: C21H28O5, formula wt. 362.466, ortho rhombic, space group P212121, a = 8.7490 (3) A, , c = 17.1930 (9) A, V = 1887.34 b = 12.5470 (5) A (14) A3, Z = 4, Dc = 1.276 Mg m3. All diagrams and calculations were performed using maXus (Brucker Nonius, Delft & Mac Science, Japan), using graphite monochro mated Mo Ka radiation (k = 0.71073 A). The structures were rened by full-matrix least-squares on F2 using Brucker SHELEXL-97 (Sheldrick, 1997). The nal R and Rw were 0.0504 and 0.1236, respectively. Crystallographic data for the structural analysis have been deposited with the Cambridge crystallographic data center. These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts /retrieving html (or from the CCDC, 250572 union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336 033; e-mail: deposit@ccdc.camac.uk).
Acknowledgements This work was supported and nanced by Matsumae Foundation (Japanese Grant for Mr. Abou El-Hamd). We thank all members of the analytical center of Tukoshima-Bunri University, Japan, for recording MS and NMR spectra. A.A.A. thanks the Alexander von Humboldt Stiftung for nancial support for the HPLC instrument.
References
Ahmed, A.A., Hussein, N.S., Adams, A.A., Mabry, T.J., 1995. Abietane diterpenes from Lepechinia urbaniana. Pharmazie 50, 279280. Barberan, F.A.T., 1986. The avonoid compounds from the Labiatae. Fitoterapia 57, 6795. Emboden Jr, W.A., Lewis, H., 1967. Terpenes as taxonomic characters in Salvia section Audibertia. Brittonia 19, 152160. Gonzalez, A.G., Castro, Z.E.A., Luis, J.G., Ravelo, A.G., 1989. New secoditerpenes from Salvia texana. Transformations of 6,7-secoabietanes in basic medium and their possible formation via oxygen singlet participation. J. Chem. Res. (S), 132133. Lee, A.R., Wu, W.L., Chang, W.L., Lin, H.C., King, M.L., 1987. Isolation and bioactivity of new tanshinones. J. Nat. Prod. 50, 157160. Luis, J.G., 1991. In: Harborne, J.B., Tomas-Baberan, F.A. (Eds.), Proceedings of Phytochemical Society of Europe: Ecological Chemistry and Biochemistry of Plant Terpenoids, vol. 31. Clarendon Press, Oxford, pp. 6382. Ming-Jaw, D., Chien-Chang, S., Yun-lian, L., Wan-Jr, S., Yi-Huei, D., Chang-Ming, S., 2005. Nitrogen-containing compounds from Salvia miltiorrhiza. J. Nat. Prod. 68, 10661070. Nakanati, N., 1994. In: Ho, C.T., Osawa, T., Huang, M.T., Rosen, R.T. (Eds.), Food Phytochemicals for Cancer Prevention II: Teas, Spices and Herbs, ACS Symposium Series, vol. 547. American Chemical Society, Washington, DC, p. 144. Penso, G. 1980. Inventory of Medicinal Plants Used in the Dierent Countries. World Health Organization, DPM 80-3, Geneva, p. 596.
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A.A. Ahmed et al. / Phytochemistry 67 (2006) 424428 Takenaka, M., Watanabe, T., Sugahara, K., Harada, Y., Yoshida, S., Sugawara, F., 1997. New antimicrobial substances against Streptomyces scabies from Rosemary (Rosmarinus ocialis L.). Biosci. Biotech. Biochem. 61, 14401444. Wollenweber, E., Doerr, M., Rustainyan, A., Roitman, J.N., Graven, E.H., 1992. Exudate avonoids of some Salvia and a Trichostema species. Zeitschrift fuer Naturforschung. C: J. Biosci. 47, 782784.
Rodriguez-Hahn, L., Esquivel, B., Cardenas, J., Ramamoorthy, T.P., 1992. In: Harley, R.M., Reynolds, T. (Eds.), Advances in Labiate Science. The Royal Botanic Gardens, Kew, UK, p. 335. Sheldrick, G.M., 1997. SHELXL97. Program for the renement of crystal structures. University of Gottingen, Germany. Sosa, M.E, Tonn, C.E., Giordano, O.S., 1994. Insect antifeedant activity of clerodane diterpenoids. J. Nat. Prod. 57, 12621265.
Iridoid glucosides from Kickxia abhaica D.A. Sutton from Scrophulariaceae

Adnan J. Al-Rehaily *, Maged S. Abdel-Kader, Mohammad S. Ahmad, Jaber S. Mossa
Department of Pharmacognosy, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia Received 17 September 2005; accepted 21 September 2005 Available online 8 November 2005
Abstract Two iridoid glucosides namely; 6-acetylantirrinoside (1), 6 0 -O-p-hydroxybenzoylantirrinoside (2) were isolated from the aerial parts of Kickxia abhaica. Beside that, three known iridoid glucosides, antirrinoside (3), antirride (4) and mussaenosidic acid (5), one avone glycoside (6) and a hexitol, D-mannitol (7) were isolated. The structures of the iridoid glucosides 12 were established by 1D and 2D NMR spectral data, including COSY, HMQC and HMBC experiments, as well as HRMS. 2005 Elsevier Ltd. All rights reserved.
Keywords: Kickxia abhaica; Scrophulariaceae; Iridoid glucosides; 6-acetylantirrinoside; 6 0 -O-p-hydroxybenzoylantirrinoside
1. Introduction The genus Kickxia is comprised of about 47 species worldwide (Mabberley, 1997). In Saudi Arabia, the genus is represented by 10 species (Kickxia elatine, Kickxia aegyptiaca, Kickxia acerbiana, Kickxia collenetteana, Kickxia corallicola, Kickxia pseudoscoparia, Kickxia scalarum, Kickxia petiolata, Kickxia hastate and Kickxia abhaica), which are distributed in dierent parts of the country (Chaudhary, 2001). Most of these species are distributed in the South and West regions including K. abhaica. Only seven Kickxia species world wide were chemically investigated and resulted in the isolation of mainly avonoids and iridoid glycosides (Khan et al., 2001; Yuldashev et al., 1996; Handjieva et al., 1995; Amer, 1993; Kassem, 1992; Khan et al., 1991; Singh and Prakash, 1987; Nicoletti et al., 1987; Toth et al., 1978a,b,c; Pinar, 1973). Up to the present time nothing has been reported about the chemistry of K. abhaica. Therefore, the present paper reports on the isolation and characterization of the two new iridoid glucosides, 6-acetylantirrinoside (1), 6 0 -O-p-hydroxybenzoylan*
tirrinoside (2) from the aerial parts of K. abhaica. In addition, the plant also yielded three known iridoid glucosides, antirrinoside (3) (Scarpati et al., 1968; Chaudhuri et al., 1980), antirride (4) (Handjieva et al., 1993) and mussaenosidic acid (5) (Damtoft et al., 1984), one avone glycoside, hispidulin 7-neohesperidoside (6) (Lee et al., 1994; Park et al., 1995) and a hexitol, D-mannitol (7) (Khan and Aqil, 1993).
2. Results and discussion Compound 1 was obtained as a gummy substance and its molecular formula C17H24O11 was determined by HRFABMS. The 17 carbons were resolved in the 13C NMR spectrum (Table 1). When compared to the spectrum of antirrhinoside (3), a very good correspondence could be seen for 15 of the signals, while the remaining two signals could be assigned to an acetyl moiety. Compound 1 was, therefore, a monoacetate of 3, in agreement with the MS data. The point of attachment was evident from the 1H NMR spectrum where the H-6 signal was seen at d 4.86, 0.9 ppm downeld from that of 3. The position of the acetate group at C-6 was further conrm by 2D NMR 1H13C
Corresponding author. Tel.: +966 1 467 7258; fax: +966 1 467 7245. E-mail address: ajalreha@ksu.edu.sa (A.J. Al-Rehaily).
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A.J. Al-Rehaily et al. / Phytochemistry 67 (2006) 429432
Table 1 1 H and 13C NMR spectral data for compounds 1-2 in CD3OD (d values, J in parenthesis in Hz)a Proton 1
1
2
13
H d (6.0) d (6.5) d (6.5) d (2.0) d (2.0) d (6.0) s d (8.0) m m m m dd (11.75, 6.5) dd (11.75, 2.5)
H d (8.0) d (6.0) d (6.0) d (1.0) br.s d (8.0) s d (8.0) m m m m dd (12.0, 7.0) dd (12.0, 2.5)
13
1 3 4 5 6 7 8 9 10 10 20 30 40 50 60a 60b 100 200 300 /700 400 /600 500

a
5.44 6.32 4.82 4.86 3.39 2.36 1.39 4.57 3.14 3.30 3.15 3.30 3.53 3.83
94.6 143.4 107.5 74.7 79.4 64.2 64.5 53.3 17.4 99.8 74.7 77.7 71.8 78.6 63.0 63.0 172.0 20.3
4.99 6.24 4.77 3.69 3.15 2.22 1.24 4.62 3.16 3.49 3.32 3.33 4.40 4.52
95.6 142.8 107.7 74.8 78.8 66.0 63.0 53.3 17.6 99.9 74.8 75.7 71.8 77.7 64.1 64.1 167.8 122.2 132.9 116.3 163.7
troscopic data with those reported in the literatures. This is the rst time that the iridoid glucosides 12 appeared in the literature and the rst report of 6 from the family scrophulariaceae. In addition, compounds 4 and 5 are reported for the second time from the genus Kickxia (Handjieva et al., 1995).
R1O HO
HO O H O R 2O HO HO O OGlc O O
4
OH H COOH
R1
1: 2: 3: CH3CO
O
R2
O
H
HO OH H OGlc
2.03 s
7.78 d (9.0) 6.73 d (9.0)
H H H
OH H OH HO HO C C C C C C OH H H H OH OH H
Assignments made by combination of COSY, DEPT, HMQC, HMBC data and comparison with the literature.
HMBC experiments. The HMBC spectrum showed 3J correlations between d 4.82 (H-4), dC-6 79.4, dC-9 53.3, and between d 4.86 (H-6) and dC-100 172.0, conrming the placement of the acetate group at C-6. These ndings unambiguously established the structure of 1 as 6acetylantirrhinoside. Compound 2, analyzed for C22H26O12 by HRFABMS, was isolated as amorphous powder and its UV spectrum exhibited absorption bands at kmax 257 and 320 nm due to the presence of a conjugated system. The 1H and 13C NMR spectra of 2 (Table 1) were diagnostic for antirrhinsoide esteried with an aromatic acid (Fauvel et al., 1995). In the 1H NMR of 2 the down eld shift of H-6 0 protons to d 4.40 and 4.52, ca. 0.8 ppm from the usual position strongly support that C-6 0 is the site of esterication. Further conrmation was made by HMBC experiment. That showed 3J correlations between d 7.78(dC300 /700 132.9), dC-100 167.8 and dC-500 163.7, and between d 4.40, 4.52 (H-6 0 a, H-6 0 b) and dC-100 167.8, conrming the attachment of aromatic acid moiety at C-6 0 .The NMR spectra of 2 were found to be similar with those reported (Dawidar et al., 1989) for 6 0 -O-cinnamoylantirrhinoside but lacking the signals for the a and b positions of cinnamoyl moiety. Based on the foregoing data, the structure of 2 was established as 6 0 -O-p-hydroxybenzoylantirrinoside. During the course of isolation of the above compounds, K. abhaica yielded three known iridoid glucosides 35, one avone glycoside (6) and one alditol (7). These compounds were identied by comparison of their physical and spec-
Rha- Glc- O
O H H
H3CO H OH O
3. Experimental 3.1. General Mp uncorr.; UV spectra were recorded on a Hewlett Packard HP-845 UVVis spectrophotometer; FTIR spectra were obtained on a Nicolet Impact 410 spectrophotometer; Specic rotation measurements were recorded on a PerkinElmer 242 MC polarimeter; NMR spectra were acquired in CD3OD or DMSO on a Bruker Avance DRX-500 instrument at 500 (1H) and 125 (13C) MHz using the residual solvent signal as internal standard. Standard Bruker pulse programs were used for APT, DEPT, 2D NMR COSY, HMQC and HMBC spectra. HRFABMS were obtained on a Bruker Bioapex-FTMS with electrospray ionization; EIMS were measured using an E.I. Finnigan model 4600 quadrupole system or a Shimadzu QP500 GC/mass spectrometer; TLC: silica gel 60 F254 (Merck) plates; solvents: dierent concentration of MeOHCHCl3 and H2OMeOH; CC: silica gel 60/230400 mesh (EM
A.J. Al-Rehaily et al. / Phytochemistry 67 (2006) 429432
431
Science); RP C-18 silica gel. Centrifugal preparative TLC (CPTLC; using Chromatotron, Harrison Research Inc. model 7924): 14 mm silica gel P254 disc. The isolated compounds were visualized under short- and long-wave UV light, followed by spraying with p-anisaldehyde reagent. 3.2. Plant material K. abhaica D.A. Sutton was collected in April, 2003 from Baljurashi, Saudi Arabia and identied by Dr. M. Atiqur Rahman, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. A voucher specimen (# 14716) was deposited at the herbarium of the College of Pharmacy, KSU. 3.3. Extraction and isolation The air-dried aerial parts (1.0 kg) of K. abhaica were exhaustedly extracted with petroleum ether (12 g), followed by EtOH at room temperature to yield after evaporation 120 g. The ethanol extract was dissolved in hot methanol to aord white precipitate (7 g) identied as D-mannitol (7). The soluble methanol fraction was concd., diluted with water and successively extracted with CHCl3 (3 300 ml), EtOAc (3 200 ml) and butanol (2 200 ml). The ethylacetate (2 g) and butanol (20 g) extracts were combined together and subjected to ash chromatography on silica gel (600 g) using chloroform and then increasing concentrations of MeOH (2050%) in CHCl3 to give 5 fractions; 1 (4.7 g), 2 (3.3 g), 3 (2.8 g), 4 (1.1 g), 5 (4.1 g). Fraction 1 (4.7 g) was rechromatographed on silica gel (60 g) using 10% CHCl3MeOH to aord sub-fractions AE. Sub-fraction A (820 mg) was separated by RP-column (30 g) using 40% H2OMeOH as a solvent to give two fractions a and b. Fraction a (150 mg) was puried by CPTLC (1 mm silica gel disc) using 8% MeOHEtOAc to yelid 1 (10 mg). Fraction b (600 mg) was separated by CPTLC (4 mm silica gel disc) using 10% MeOHEtOAc to give three fractions IIII. Fraction I (39 mg) was puried by RP-column using 40% H2OMeOH as a solvent to aord 2 (14 mg). Fraction C (800 mg) was subjected to CPTLC (4 mm silica gel disc) using 20% MeOHCHCl3 to give three sub-fractions iiii. Sub-fraction ii (400 mg) was puried by CPTLC (2 mm silica gel disc) using 20% MeOHCHCl3NH3 to give 4 (15 mg). Portion of fraction D (250 mg) was separated by CPTLC (2 mm silica gel disc) using 20% MeOHCHCl3acetic acid, further purication by LH-20 (40 g) using 30% MeOHCHCl3 as a solvent followed by repeated CC over silica gel using CHCl3 as solvent to give 3 (28 mg). Fraction 4 (1.1 g) was subjected to CPTLC (4 mm silica gel disc) using 25% MeOHCHCl3 to give two fractions A and B. Fraction B (0.5 g) was separated by CPTLC (2 mm silica gel disc) using 20% MeOHCHCl3NH3 to yielded two sub-fractions a and b. The sub-fraction a (200 mg) was puried by LH-20 (30 g) using 50% MeOHH2O to aord 6 (80 mg). The sub-fraction b (65 mg) was puried
by RP-column (30 g) using 40% H2OMeOH as a solvent to give 5 (17 mg). 3.4. 6-Acetylantirrinoside (1) Gum, [a]D 100 (c; 0.04 in MeOH); UV kmax (MeOH) nm (log e): 202 (3.61), 275 (2.24); IR (lm) mmax cm1: 3411, 2923, 1734, 1375, 1240, 1101, 1076, 1047, 1016 and 960; 1H and 13C NMR: see Table 1; EIMS m/z (rel. int. %) 241 [M 163]+ (0.25), 225 (0.58), 207 (1.8), 165 (3.3), 145 (3.5), 129 (12.4), 114 (6.5), 97 (19.9), 87 (21.9), 85 (12.1), 73 (15.8), 71 (11.7), 69 (10.1), 57 (17.3), 45 (44.7) and 43 (100); HRFABMS: 405.1393 ([M + H]+); (calc. for [C17H24O11 + H] 405.1397). 3.5. 6 0 -O-p-Hydroxybenzoylantirrinoside (2) Amorphous powder, mp. 132134 C; [a]D 51.3 (c; 0.07 in MeOH); UV kmax (MeOH) nm (log e): 202 (4.82), 257 (4.58), 320 (3.62); IR (lm) mmax cm1: 3420, 3411, 2920, 1701, 1608, 1313, 1279, 1236, 1167, 1101, 1074, 1045, 1012, 771 and 617; 1H and 13C NMR: see Table 1; EIMS m/z (rel. int. %) 483 [M + 1]+ (0.31), 198 (1.2), 177 (4.9), 173 (8.8), 163 (4.7), 138 (14.8), 121 (26), 93 (9.0), 73 (22.3), 69 (16), 60 (20.9), 57 (29.3), 55 (23.9), 45 (43.5), 44 (100) and 41 (37.8); HRFABMS: 483.1500 ([M + H]+); (calc. for [C22H26O12 + H] 483.1503). Acknowledgements The authors sincerely thank Dr. Amr Mansour, Mass Spectroscopy Unit, National Research Center, Cairo, Egypt, for HRFABMS. Also we thank Mr. Mohammed Mukhair for technical assistances. References
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A.J. Al-Rehaily et al. / Phytochemistry 67 (2006) 429432 Park, J.C., Lee, J.H., Choi, J.S., 1995. A avone diglycoside from Cirsium japonicum var. Ussuriense. Phytochemistry 39 (1), 261262. Pinar, M., 1973. 5,6,7-Trimethoxyavone and 5,6,7,4 0 -tetramethoxyavone from Kickxia lanigera. Phytochemistry 12 (12), 30144015. Scarpati, M.L., Guiso, M., Esposito, P., 1968. Isolation and characterization of iridoids. Gazzetta Chimica Italiana 98, 177. Singh, M., Prakash, L., 1987. A new avone glycoside and other chemical constituents from Kickxia ramosissima Wall. Pharmazie 42 (7), 490 491. Toth, L., Csordas, I., Papay, V., 1978a. Chemical analysis of Kickxia elatine (L.) Dum. Herba Hungarica 17 (1), 3537. Toth, L., Csordas, I., Papay, V., Bujtas, G., 1978b. Flavonoids of Kickxia elatine (L.) Dum. Pharmazie 33 (6), 374375. Toth, L., Kokovay, K., Bujtas, G., Papay, V., 1978c. Constituents of Kickxia spuria (L.) Dum. Pharmazie 33 (1), 84. Yuldashev, M.P., Batirov, E.Kh., Malikov, V.M., 1996. Flavonoids from aerial parts of Kickxia elatine. Khimiya Prirodnykh Soedinenii 1, 3841.
Khan, I.Z., Aqil, M., 1993. Isolation and identication of pectolinarin and mannitol from Kickxia ramosissima (Wall). Chemical and Environmental Research 2 (3&4), 287289. Khan, I.Z., Aqil, M., Kolo, B.G., 2001. A new avone glycoside from Kickxia ramosissima (Wall). Ultra Physical Sciences 13 (1), 112115. Khan, I.Z., Aqil, M., Khan, M.S.Y., 1991. A new avone glycoside, acetylated pectolinarigenin 7-rutinoside from Kickxia ramosissima (Wall). Discovery and Innovation 3 (2), 5960. Lee, H.B., Kwak, J.H., Zee, O.P., Yoo, S.J., 1994. Flavonoids from Cirsium rhinoceros. Archives of Pharmacal Research 17 (4), 273277. Mabberley, A.J., 1997. The Plant-book. Cambridge University Press, Cambridge. Nicoletti, M., Serani, M., Tomassini, L., Bianco, A., Passacantilli, P., 1987. Iridoids in the ora of Italy. Part II. Kickxioside, a new iridoid dlucoside from Kickxia spuria. Planta Medica 53 (3), 295297.
Five labdane diterpenoids from the seeds of Aframomum zambesiacum

Marguerite Kenmogne b, Elise Prost a, Dominique Harakat d, Marie-Jose Jacquier a, c e a ` Michel Frederich , Lucas B. Sondengam , Monique Zeches , Pierre Wao-Teguo a,*
FRE 2715 CNRS, Laboratoire de Pharmacognosie, IFR 53 Biomolecules, Universite de Reims Champagne-Ardenne, Batiment 18, CPCBAI, Moulin de la Housse, BP 1039, 51687 Reims Cedex 02, France b Department of Chemistry, Faculty of Science, University of Dschang, Box 67, Dschang, Cameroon c ` ` University of Liege, Natural and Synthetic Drugs Research Center, Laboratory of Pharmacognosy, Avenue de lHopital 1, B36, B-4000 Liege, Belgium d Laboratoire Reactions Selectives et Applications, UMR CNRS 6519, Faculte des Sciences, Universite de Reims Champagne-Ardenne, B.P. 1039, 51687 Reims Cedex 2, France e Department of Organic Chemistry, Faculty of Science, University of Yaounde I, Box 812, Yaounde, Cameroon Received 6 September 2005; received in revised form 10 October 2005 Available online 29 November 2005
a
Abstract Five labdane diterpenoids, (35), zambesiacolactone A (7) and zambesiacolactone B (8), were isolated from the seeds of Aframomum zambesiacum (Baker) K. Schum., along with ve known labdanes and a linear sesquiterpene, nerolidol. Their structures were elucidated by spectroscopic analysis. Their antiplasmodial activity was evaluated in vitro against Plasmodium falciparum. Compound 3 was the most active with an IC50 value of 4.97 lM. 2005 Elsevier Ltd. All rights reserved.
Keywords: Aframomum zambesiacum; Zingiberaceae; Labdane diterpenoids; Antiplasmodial activity; Plasmodium falciparum
1. Introduction The genus Aframomum of the Zingiberaceae family includes 40 species and is most common in tropical and subtropical regions (Thomas et al., 1989). Twenty species are found in Cameroon, where they are widely used in traditional medicine, for spiritual purposes and as spices (Thomas et al., 1989). The compounds isolated from plants of this genus include avonoids (De Bernardi et al., 1976; Ayafor and Connolly, 1981), diaryl heptanoids (Kamnaing et al., 2003), sesquiterpenes (Ayafor and Connolly, 1981) and labdane diterpenoids, specially in Aframomum alboviolaceum (Abreu and Noronha, 1997), Aframomum aulacocarpos (Ayafor et al., 1994a), Aframomum daniellii
* Corresponding author. Tel.: +33 (0) 3 26 91 82 08; fax: +33 (0) 3 26 91 35 96. E-mail addresses: pierre.wao-teguo@univ-reims.fr, tteguo@yahoo.fr (P. Wao-Teguo).
(Kimbu et al., 1979, 1987), Aframomum escapum (Ayimele et al., 2004), and Aframomum sceptrum (Tomla et al., 2002). A great deal of interest has been focused on the labdanes from Aframomum species, some of which exhibit antifungal, cytotoxic, and other biological activity (Ayafor et al., 1994a,b). In general, many labdanes from terrestrial plants and marine sources show antibacterial, antifungal, anti-inammatory, antileishmanial, cardiotonic, cytotoxic, enzyme inhibitory (Singh et al., 1999), and trypanocidal (Scio et al., 2003) activities. Several Aframomum species (i.e., Aframomum angustifolium, A. danielli, Aframomum sanguineum, andAframomum sulcatum) were traditionally used to treat fevers in Africa (Iwu, 1993), and recently, the antiplasmodial activity of some labdanes from A. sceptrum and Aframomum latifolium has been investigated (Duker-Eshun et al., 2002). This paper describes the rst phytochemical investigation of the seeds of Aframomum zambesiacum (Baker) K. Schum. This species was selected in the framework of a
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screening program to discover novel active compounds from Cameroonian medicinal plants. The structural elucidation of the isolated compounds was followed by evaluation of their in vitro antiplasmodial activity against Plasmodium falciparum.
2. Results and discussion The dry, powdered seeds of A. zambeciacum were successively extracted with petroleum ether, chloroform, and methanol. Chromatographic purication of the two less polar extracts aorded 10 labdanes (110), ve of which are new, and nerolidol (11) which was previously isolated from Aframomum pruinosum (Ayafor and Connolly, 1981) and A. escapum (Ayimele et al., 2004). The structures were assigned by analysis of spectroscopic data and by comparison with literature values. The ve known labdanes were identied as aulacocarpin A (1) and aulacocarpin B (2), previously isolated from Aframomum aulacocarpus (Ayafor et al., 1994a) and A. escapum (Ayimele et al., 2004), and galanolactone (6), aframodial (9) and galanal A (10), isolated from Alpinia galanga (Morita and Itokawa, 1988).
16 COOCH3 12 13 14 O 17 3
R1 16 CH2OH
14 15
O OH
CH2OH
8 6
R2
O 15
R1 1 OH 2 OH 3 H
R2 H OH H
5
O
16
O
COOCH3
14
O R3
15
14
17 8 HO O R1 R2
6 7 8
R1 H OH OH
R2 H H OH
R3 H OH OH
Compound 3 was obtained as a white powder. Its molecular formula, C21H32O4, was established by positive HRESI-MS (m/z [M + Na]+ 371.2196). The strong IR absorptions at tmax 1724 and 1645 cm1 suggested the presence of an a,b-unsaturated ester. This is in agreement with the three 13C NMR resonances for sp2 carbons corresponding to an ester carbonyl (d 166.8) and a trisubstituted olen (d 127.5 (CH) and d 150.7 (C)). Thus, the compound is tetracyclic. The 1H NMR spectrum revealed three tertiary methyls (d 0.87, 0.9, 0.93) and a methyl ester (d 3.75). Other proton signals included a deshielded vinyl proton [d 6.81 (t, J = 6.5 Hz, H-12)], a monosubstituted epoxide [d 3.60
(brm, H-14), 2.81 (dd, J = 5.5, 2.8 Hz, H-15a) and 2.99 (dd, J = 5.5, 4.3 Hz, H-15b)], a disubstituted epoxide [d 2.30 (d, J = 3.9 Hz, H-17a) and 2.51 (d, J = 3.9 Hz, H17b)]. These data suggested that compound 3 was a labdane diterpenoid related to aulacocarpin A (1) and B (2) (Ayafor et al., 1994a). All 1H and 13C NMR signals for 3 were assigned by analysis of COSY, HSQC, and HMBC spectra. The 1H and 13C NMR data (Table 1) of 3 were almost identical to those of aulacocarpin A (1). In the 13 C NMR spectrum, the main dierence was the replacement of the C-3 methine (d 78.8) of 1 by a methylene (d 42.1) in 3. This dierence was also evident in the 1H NMR spectrum, where H-3 (d 3.24, dd) of 1 was replaced by two methylene protons at d 1.17 and 1.41 (both m). The relative conguration at C-8 was deduced from a NOESY correlation between H-17 and H-9 and by chemical shift comparison with aulacocarpin A (1) and B (2) (Ayafor et al., 1994; Morita and Itokawa, 1988). As in the case of 1, the E conguration of the 12,13 double bond was deduced from NMR data (Ayafor et al., 1994a). Thus, compound 3 is 3-deoxyaulacocarpin A, or methyl8b,17:14n,15-diepoxy-12E-labden-16-oate. Compound 4, C21H32O4 (m/z [M + Na]+ 371.2211 HRESI-MS in positive mode), a colorless oil, had bands in its IR spectrum at tmax 3417 cm1, 1714, and 1644 cm1 in agreement with the presence of a hydroxyl and an a,b-unsaturated ester. The 1H and 13C NMR spectral data of 4 (Table 1) were very similar to those of 1. The only signicant dierences were the absence of the signals of the H2-17 epoxide protons of 1 and the presence of an 8,17-exomethylene group [d 4.86 (d, J = 0.9 Hz) and 4.49 (d, J = 0.9 Hz)] in 4. The b-orientation of the hydroxyl group at C-3 was deduced from the coupling constants of H-3 [d 3.27 (dd, J = 11.7 and 4.3 Hz)] and from the NOESY spectrum, while the E conguration of the 12,13 double bond follows from the deshielded nature of H-12. Therefore, 4 is methyl-14n,15-epoxy-3b-hydroxy8(17),12E-labdadien-16-oate. The corresponding 3-deoxyderivative has been reported from the seeds of A. danielli (Kimbu et al., 1987). Compound 5, C20H34O4 (m/z [M + Na]+ 361.2349 HRESI-MS in positive mode), was obtained as white powder, m.p. 154155 C, and showed an hydroxyl absorption (tmax 3402 cm1) in its IR spectrum. The 1H and 13C NMR data (Table 1) of 5 indicated that it was also a labdane diterpenoid. The 1H NMR spectrum showed three methyl singlets at d 0.88, 0.92, and 0.92, the protons of three oxymethylene groups [dH 3.50, (dd, J = 11.3, 7.6 Hz, H-15a) and 3.60 (dd, J = 11.3, 4.6 Hz, H-15b), dH 4.04 (d, J = 12.9 Hz, H-16a) and 4.15 (d, J = 12.9 Hz, H-16b), and 2.27 (d,J = 4.1 Hz, H-17a) and dH 2.69 (d, J = 4.1 Hz, H-17b)] and a proton of an oxymethine (dH 4.6 (dd, J = 7.8, 4.3 Hz, H-14)). Comparison of the 13C NMR data of 5 with those of 3 showed that the monosubstituted epoxide had been replaced by an oxymethylene (dC 66.1, C-15) and an oxymethine (dC 71.9, C-14). Further analyses of the NMR spectra led to the assignments of all protons
Table 1 1 H and 13C NMR data of 3, 4, 5, 7 and 8 in CDCl3 3 dH 1ax 1eq 2ax 2eq 3ax/3 3eq 4 5 6ax 6eq 7ax 7eq 8 9 10 11a 11b 12 13 14 15a/15 15b 16a/16 16b 17a 17b 18 19 20 O-CH3 0.96 1.78 1.43 1.58 1.17 1.41 1.02 1.58 na 1.36 1.93 1.57 2.02 2.34 6.81 3.60 2.81 2.99 2.30 2.51 0.90 0.87 0.93 3.75 dd (13.0, 3.4) brd (13.0) m m td (13.6, 4) m dd (12, 2.8) m dq (13.8, 2.5) td (13.8, 5.3) m m dd (18.4, 6.1) t (6.5) brs dd (5.5, 2.8) dd (5.5, 4.3) dc 39.3 39.3 18.7 42.1 33.7 55.2 20.2 36.0 57.7 53.0 40.0 21.0 150.7 127.4 48.6 47.9 166.8 d (3.9) d (3.9) s s s s 49.2 33.7 21.9 14.8 52.1 4 dH 1.27 1.79 1.60 1.72 3.27 1.12 1.41 1.75 2.00 2.41 1.77 2.50 2.61 6.87 3.65 3.00 2.78 4.49 4.86 0.79 1.00 0.74 3.73 td (13.1, 3.5) dd (13.1, 3.5) qd (13.3, 3.4) dq (13.3, 3.7) dd (11.7, 4.3) dc 37.2 37.2 28.0 78.8 39.4 54.7 23.9 37.9 147.8 56.6 39.5 23.8 149.6 127.5 49 47.8 166.8 d (0.9) d (0.9) s s s s 108.2 28.5 15.6 14.6 51.9 5a dH 1.03 td (12.9, 3.1) 1.83 dd (12.7, 3.6) 1.48 dq (13.6, 3.4) 1.65 m 1.24 td (13.5, 3.7) 1.43 dtd (13.1, 3.2, 1.5) 1.10 dd (12.3, 2.3) 1.61 m 1.72 m 1.31 dq (1.39, 2.6) 1.97 td (13.7, 5) 1.60 d (10.6) 1.78 brt (8.3) 1.98 m 5.44 ddd (8.1, 4.7, 1) 4.6 dd (7.8, 4.3) 3.5 dd (11.3, 7.6) 3.6 dd (11.3, 4.6) 4.04 d (12.9) 4.15 d (12.9) 2.27 d (4.1) 2.69 d (4.1) 0.92 s 0.88 s 0.92 s dC 40.3 19.7 43.1 34.4 56.2 21.2 37.1 59.3 54.6 40.9 20.7 133.3 138.8 71.9 66.1 63.9 50.3 34.0 22.2 15.2 7 dH 1.15 1.79 1.62 1.69 3.27 1.02 1.72 1.72 1.40 1.94 1.66 2.08 2.27 6.85 5.03 4.27 4.47 2.34 2.73 1.04 0.85 0.95 td (12.9, 3.9) dt (12.9, 3.4) qd (13.3, 3.6) m dd (11.5, 4.4) dC 37.6 27.3 78.8 39.3 54.2 19.9 35.9 58.1 52.3 39.6 22.6 149.2 128.4 66.1 74.5 170.2 d (3.6) d (3.6) s s s 49.4 28.5 15.7 14.9 8b dH 1.12 1.73 1.60 1.65 3.12 dd (12.9, 3.7) dt (13.1, 3.6) m m dd (11.2, 4.6) dC 39.9 26.5 78.6 40.0 55.8 67.9 43.8 57.2 52.5 39.8 22.4 148.7 128.8 65.4 75.0 171.3 d (3.5) d (3.5) s s s 47.4 28.0 16.8 16.7 M. Kenmogne et al. / Phytochemistry 67 (2006) 433438
dd (12.5, 2.6) qd (13.1, 4.2) m td (13, 4.9) dq (13, 2.3) m ddd (16, 11.6, 7.6) ddd (16.6, 6.3, 2.8) t (7.00) t (3.4) dd (5.5, 4.4) dd (5.5, 2.8)
dd (12.5, 3.5) m m ddd (13.9, 3.7, 2.6) td (13.9, 5.8) m ddd (16.5, 9.1, 7.8) brdd (16.5, 6.8) td (7.1, 1.6) t (5.6) dd (10.4, 2) dd (10.4, 6.1)
0.94 d (1.9) 4.46 td (3, 1.8) 1.15 2.18 1.66 2.10 2.31 6.77 4.92 4.18 4.41 2.27 2.75 1.06 1.22 1.17 dd (14.8, 2.5) dd (14.8, 3.6) m m d (6.6) td (6.9, 1.8) d (6.1) dd (10.2, 4.6) dd (10.2, 6.2)
na: not assigned. a Measured in CD3OD. b Measured in CDCl3 with some drops of CD3OD.
435
436
and carbons, the E congurations of the double bond, and the b conguration of the 8,17-epoxide. Thus, structure 5 is 8b,17-epoxy-12E-labdene-14n,15,16-triol. Compound 7, C20H30O5 (m/z [M + Na]+ 373.1974 HRESI-MS in positive mode) a white powder, showed IR absorption bands at tmax 3415 and 1743 cm1. Its 1H and 13C NMR spectra data were similar to those of 6 (Morita and Itokawa, 1988) except for the absence of two methylene signals (C-3 and C-14) and the appearance of two oxymethine carbon resonances at d 66.1 (C-14) and 78.8 (C-3) in the 13C NMR spectrum of 7. H-3 appeared at d 3.27 (dd, J = 11.5,4.4 Hz) indicating that the hydroxyl attached to C-3 is b. Thus compound 7, zambesiacolactone A, is 8b,17-epoxy-3b,14n-dihydroxy-12(E)-labden-16,15olide. Analysis of the 1H and 13C NMR spectral data (Table 1) of compound 8, C20H30O6 (m/z [M + Na]+ 389.1956 HRESI-MS in positive mode; tmax 3417 and 1739 cm1), indicated that it was closely related to compound 7. The main dierence was the presence of signals for an oxymethine [dH 4.46 (td, J = 3.0,1.8 Hz, H-6); dC 67.9 (C-6)] in 8 replacing the C-6 resonances in 7. The b axial orientation of the C-6 hydroxyl of 8 was deduced from the small coupling constant (J = 1.9 Hz) between H-5 and H-6. Furthermore, the upeld resonance of H-5 (d 0.94) is reminiscent of some 6b-hydroxylated Scapania labdane derivatives (Huneck et al., 1986). Thus, compound 8, zambesiacolactone B, was assigned the structure 8b,17-epoxy-3b,6b,14ntrihydroxy-12(E)-labden-16,15-olide. The relative conguration at C-14 remains undetermined in all the new compounds, as in aulacocarpin A (1) and aulacocarpin B (2) (Ayafor et al., 1994a). The in vitro antiplasmodial activity of the labdanes 19 was determined against an FCB1 chloroquine-resistant strain of P. falciparum relative to artemisinine, chloroquine, and quinine (Table 2). The amount of compound 10 was insucient to allow evaluation of its biological activity. Compounds 1, 3, 7, and 8 showed moderate activity with IC50 values between 4 and 20 lM. Amongst the active compounds, compound 3, the least polar compound, was the most active with an IC50 of 4.97 lM (1.73 lg/ml).
Table 2 In vitro antiplasmodial activity of compounds 19, quinine, chloroquine, and artemisinine on FCB1 line of Plasmodium falciparum compound Aulacocarpin A (1) Aulacocarpin B (2) 3 4 5 Galanolactone (6) Zambesiacolactone A (7) Zambesiacolactone B (8) Aframodial (9) Quinine Chloroquine Artemisinine
a
3. Experimental 3.1. General experimental procedures Melting points were determined on a Reichert apparatus and are uncorrected. 1H and 13C NMR spectra were recorded on a Bruker Avance DRX 500 (1H at 500 MHz and 13C at 125 MHz). 2D experiments were performed using standard Bruker microprograms. ESI-MS and HRESI-MS were obtained on a Micromass Q-TOF micro spectrometer. IR spectra were obtained with a Nicolet AVATAR 320 FT-IR spectrophotometer. Optical rotations were determined in MeOH or CHCl3 with a PerkinElmer 241 polarimeter. Centrifugal TLC was carried out on a chromatotron, Model 7924T (Harrison Research). The plate was coated with silica gel 60 F254 Merck. TLC was performed on pre-coated silica gel 60 F254 Merck and detection was achieved by spraying with a vanillin sulphuric reagent containing (3 g Vanillin, 100 ml EtOH, and 3 ml H2SO4). CC was carried out on Kieselgel 60 (63200 mesh) Merck. 3.2. Plant material Seeds of A. zambesiacum (Baker) K. Schum. were collected in Nyassosso, a small locality of the district of Tombel in south-west of Cameroon in November 2003. A voucher specimen (accession number 37737HNY) has been deposited at the National Herbarium Yaounde Cameroon. The identication was conrmed by Dr. Tchiengu and P. Mezili, botanists of the Cameroon National Herbarium. 3.3. Extraction and isolation Dried and nely powdered seeds (97 g) were extracted successively with petroleum ether (2.5 l), chloroform (2.5 l), and methanol (2.5 l) at room temperature by percolation in an open column after a maceration step for 36 h. The extracts were concentrated to yield respective residues 3.55 g (petroleum ether I), 6.78 g (chloroform II) and 3.54 g (methanol III). The petroleum ether extract I (3.55 g) was subjected to column chromatography (CC) on silica gel with a mixture of hexane/EtOAc of increasing polarity to give 15 fractions (Fr. 115). Fr. 3 (50 mg) eluted with hexane/EtOAc (98/2) was puried by preparative TLC in hexane/EtOAc (8/2) to give nerolidol (11) (10 mg). Fr. 6 (60 mg) eluted with hexane/EtOAc (92.5/7.5) was puried by preparative TLC using the same conditions as above to give compound 3 (30 mg). Aframodial (9) (50 mg) was obtained from Fr. 8 (70 mg) eluted with hexane/EtOAc (9/1) after preparative TLC in hexane/EtOAc (8/2). The chloroform extract II (6.78 g) was fractionated on silica gel CC eluting with a mixture of hexane/EtOAc/ MeOH of increasing polarity to give 22 fractions (Fr. 1 22). Fr. 8 eluted with hexane/EtOAc (8/2) was further subjected to a Sephadex LH20 column, eluting with MeOH/
IC50 (lM) 13.68 6.89 21.10 4.55 4.97 2.27 39.94 12.58 >133.13 92.79 12.83 17.20 3.05 15.51 4.20 >94.33 0.55 0.10 0.30 0.03 0.01 0.00
na 3 2 2 2 2 2 3 3 3 3 5 3
n = number of experiments.
437
CH2Cl2 (1/1) and puried by preparative TLC in hexane/ EtOAc (7/3) to give galanolactone (6) (14 mg). Galanal A (10) (15 mg) and compound 4 (3.8 mg) were obtained from Fr. 9 (20 mg) eluted with hexane/EtOAc (8/2), and Fr. 11 (70 mg) eluted with hexane/EtOAc (75/25), respectively, after purication with CC over Sephadex LH20 eluting with MeOH/CH2Cl2 (1/1) and preparative TLC in hexane/EtOAc (6/4). Fr. 13 (73 mg) eluted with hexane/EtOAc (75/25) was subjected to silica CC eluting with hexane/ EtOAc (6/4) to give aulacocarpin A (1) (42 mg). Aulacocarpin B (2) (70 mg) was obtained from Fr. 17 (100 mg) eluted with hexane/EtOAc (6/4) by recrystallization in a mixture hexane/EtOAc (1/1). Fr. 19 (80 mg) eluted with hexane/EtOAc (3/7) was fractioned on Sephadex LH20 CC eluting with CH2Cl2/MeOH (1/1) and compound 7 (5.8 mg) was puried by centrifugal TLC using CHCl3/ MeOH (99/1) and preparative TLC in CH2Cl2/acetone (8/2). Fr. 20 (224 mg) eluted with EtOAc/MeOH (95/5) was fractioned on Sephadex LH20 as above to give Fr. a (37 mg) and Fr. b (34 mg) which were respectively puried by centrifugal TLC using a mixture CHCl3/MeOH (98/2) to yield compound 5 (20 mg) by recrystallisation in MeOH and compound 8 (10 mg) after a preparative TLC in CH2Cl2/acetone (6/4). 3.4. Aulacocarpin A (1) ESI-MS (positive ion mode) m/z 365 [M + Na] ; spectroscopic data as in Ayafor et al. (1994a). 3.5. Aulacocarpin B (2) ESI-MS (positive ion mode) m/z 381 [M + Na] ; spectroscopic data as in Ayafor et al. (1994a). 3.6. 3-Deoxyaulacocarpin A (3) White powder: m.p. 8889 C; aD +40.3 (CHCl3, c 1.14); IR (KBr) tmax 2952, 2923, 1724, 1645, 1433, 1389, 1262, 1245, 1214 cm1; 1H and 13C NMR (CDCl3): see Table 1; HRESI-MS: [M + Na]+ Calc. 371.2198; found 371.2196; ESI-MS (positive ion mode) m/z 371 [M + Na]+. 3.7. Methyl-14n,15-epoxy-3b-hydroxy-8(17),12Elabdadien-16-oate (4) Colorless oil: a20 + 28.2 (CHCl3, c 1.13); IR (lm) tmax D 3496, 2940, 2848, 1714, 1644, 1439, 1386, 1260 cm1 1H and 13C NMR (CDCl3): see Table 1; HRESI-MS: [M + Na]+ Calc. 371.2198; found 371.2211; ESI-MS (positive ion mode) m/z 371 [M + Na]+. 3.8. 8b,17-Epoxy-12E-labdene-14n,15,16-triol (5) White powder: m.p. 154155 C; a20 + 13.2 (MeOH, c D 1.19); IR (KBr) tmax 3402, 2929, 1647, 1434, 1387, 1218 cm1; 1H and 13C NMR (CD3OD): see Table 1;
20
HRESI-MS: [M + Na]+ Calc. 361.2355; found 361.2349; ESI-MS (positive ion mode) m/z 361 [M + Na]+. 3.9. Galanolactone (6) ESI-MS (positive ion mode) m/z 341 [M + Na]+; spectroscopic data as in Morita and Itokawa (1988). 3.10. Zambesiacolactone A (7) White powder: m.p. 164166 C; aD +73.6 (CHCl3, c 0.8); IR (KBr) tmax 3415, 2938, 2868, 1743, 1673, 1460, 1214 cm1; 1H and 13C NMR (CDCl3): see Table 1; HRESIMS: [M + Na]+ Calc. 373.1991; found 373.1974 ESI-MS (positive ion mode) m/z 373 [M + Na]+. 3.11. Zambesiacolactone B (8) White powder: m.p. 141142 C; aD +73.6 (MeOH, c 0.8); IR (KBr) tmax 3417, 2929, 2868, 1739, 1673, 1463, 1421, 1364, 1215 cm1; 1H and 13C NMR (CDCl3/CD3OD): see Table 1; HRESI-MS: [M + Na]+ Calc. 389.1940; found 389.1956 ESI-MS (positive ion mode) m/z 389 [M + Na]+. 3.12. Aframodial (9)
20 20
ESI-MS (positive ion mode) m/z 341 [M + Na]+; spectroscopic data as in Morita and Itokawa (1988). 3.13. Galanal (10)
ESI-MS (positive ion mode) m/z 341 [M + Na]+; spectroscopic data as in Morita and Itokawa (1988). 3.14. Antiplasmodial assays Continuous cultures of asexual erythrocytic stages of an FCB1 chloroquine-resistant strain of P. falciparum were maintained following the procedure of Trager and Jensen (1976) and as described previously (Frederich et al., 2002). Artemisinin (Sigma, Bornem, Belgium), chloroquine diphosphate (Sigma C6628), and quinine base (Aldrich 14590-4) were used as antimalarial references. Each test sample was applied in a series of eight fourfold dilutions (nal concentrations ranging from 20 to 0.0012 lg/ml) and was tested in duplicate and triplicate. Parasite growth was estimated by determination of lactate dehydrogenase activity as described by Delhaes et al. (1999) and Makler et al. (1993) and slightly modied. Briey, in a new microtiter plate, a 20 ll subsample of the contents of each well was mixed with 100 ll of a substrate solution containing 1 mg lithium L-lactate (Sigma), 0.2 mg 3-acetyl pyridine adenine dinucleotide (APAD, Sigma), 0.2 ll Triton X-100 (Sigma), 10 lg saponine (Merck) in TRIS buer (pH 8, Sigma). After incubation for 20 min, 20 ll of a mix of nitroblue tetrazolium (NBT, 2 mg/ml in TRIS pH 8 buer, SIGMA) and phenazine ethosulfate (PES, 0.1 mg/ml in
438
M. Kenmogne et al. / Phytochemistry 67 (2006) 433438 of labdanes from Aframomum latifolium and Aframomum sceptrum. Planta Med. 68, 642644. Frederich, M., Jacquier, M.J., Thepnier, P., De Mol, P., Tits, M., Philippe, ` G., Delaude, C., Angenot, L., Zeches-Hanrot, M., 2002. Antiplasmodial activity of alkaloids from various Strychnos species. J. Nat. Prod. 65, 13811386. Huneck, S., Connolly, J.D., Harrison, L.J., Joseph, R., Phillips, W.R., Rycroft, D.S., Ferguson, G., Parvez, M., 1986. New labdane diterpenoids from the liverwort Scapania undulata. J. Chem. Res., Synopses, 162163. Iwu, M., 1993. Handbook of African Medicinal Plants. CRC Press, Boca Raton, FL. Kamnaing, P., Tsopmo, A., Tanifum, E.A., Tchuendem, M.H.K., Tane, P., Ayafor, J.F., Sterner, O., Rattendi, D., Iwu, M.M., Schuster, B., Bacchi, C., 2003. Trypanocidal diarylheptanoids from Aframomum letestuianum. J. Nat. Prod. 66, 364367. Kimbu, S.F., Njimi, T.K., Sondengam, B.L., Akinniyi, J.A., Connolly, J.D., 1979. The structure of a labdane dialdehyde from Aframomum daniellii (Zingiberaceae). J. Chem. Soc., Perkin Trans. 1, 13031304. Kimbu, S.F., Ngadjui, B., Sondengam, B.L., 1987. A new labdane diterpenoid from the seeds of Aframomum daniellii. J. Nat. Prod. 50, 230231. Makler, M., Ries, J., Williams, J., Bancroft, J., Piper, R., Gibbins, B., Hinrichs, D., 1993. Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity. Am. J. Trop. Med. Hyg. 48, 739741. Morita, H., Itokawa, H., 1988. Cytotoxic and antifungal diterpenes from the seeds of Alpinia galanga. Planta Med. 54, 117120. Scio, E., Ribeiro, A., Alves, T.M.A., Romanha, A.J., De Souza Filho, J.D., Cordell, G.A., Zani, C.L., 2003. Diterpenes from Alomia myriadenia (Asteraceae) with cytotoxic and trypanocidal activity. Phytochemistry 64, 11251131. Singh, M., Pal, M., Sharma, R.P., 1999. Biological activity of labdane diterpenes. Planta Med. 65, 28. Tomla, C., Kamnaing, P., Ayimele, G.A., Tanifum, E.A., Tsopmo, A., Tane, P., Ayafor, J.F., Connolly, J.D., 2002. Three labdane diterpenoids from Aframomum sceptrum (Zingiberaceae). Phytochemistry 60, 197200. Thomas, D.W., Thomas, J., Bromley, W.N., Mbenkum, F.T., 1989. Korup ethnobotany survey, nal report to: The World Wide Fund for Nature, Penda House: Weyside Park, Godalming: Surrey, UK. Trager, W., Jensen, J.B., 1976. Human malaria parasites in continuous culture. Science 193, 673675.
TRIS pH 8 buer, Sigma) was added to each well. After another 30 min of incubation, the formation of the reduced form of APAD was measured at 595 nm.
Acknowledgements We are grateful to ISF (International Foundation for Science) for partial nancial support of this work, and to AUF (Agence Universitaire de la Francophonie) for a travelling fellowship (Marguerite Kenmogne).
References
Abreu, P.M., Noronha, R.G., 1997. Volatile constituents of the rhizomes of Aframomum alboviolaceum (Ridley) K. Schum from Guinea-Bissau. Flavour and Fragrance J. 12, 7983. Ayafor, J.F., Connolly, J.D., 1981. 2R,3R-(+)-3-Acetoxy-4 0 ,5-dihydroxy7-methoxyavanone and 2R,3R-(+)-3-acetoxy-4 0 ,5,7-trihydroxyavanone: two new 3-acetylated dihydroavonols from Aframomum pruinosum Gagnepain (Zingiberaceae). J. Chem. Soc., Perkin Trans. 1, 25632565. Ayafor, J.F., Tchuendem, M.H.K., Nyasse, B., 1994a. Novel bioactive diterpenoids from Aframomum aulacocarpos. J. Nat. Prod. 57, 917 923. Ayafor, J.F., Tchuendem, M.H.K., Nyasse, B., Tillequin, F., Anke, H., 1994b. Aframodial and other bioactive diterpenoids from Aframomum species. Pure & Appl. Chem. 66, 23272330. Ayimele, G.A., Tane, P., Connolly, J.D., 2004. Aulacocarpin A and B, nerolidol and b-sitosterol glucoside from Aframomum escapum. Biochem. Syst. Ecol. 32, 12051207. De Bernardi, M., Vidari, G., Vita-Finzi, P., 1976. Dehydrozyngerone from Aframomum giganteum. Phytochemistry 15, 17851786. Delhaes, L., Lazaro, J.-E., Gay, F., Thellier, M., Danis, M., 1999. The microculture tetrazolium assay: another colorimetric method of testing Plasmodium falciparum chemosensitivity. Ann. Trop. Med. Parasitol. 93, 3140. Duker-Eshun, G., Jaroszewski, J.W., Asomaning, W.A., Oppong-Boachie, F., Olsen, C.E., Christensen, S.B., 2002. Antiplasmodial activity
Hydroxylation of the sesterterpene leucosceptrine by the fungus Rhizopus stolonifer

Muhammad Iqbal Choudhary a,*, Rosa Ranjit a, Atta-ur-Rahman a, Krishna Prasad Devkota a, Syed Ghulam Musharraf a, Tirtha Maiya Shrestha
a
H.E.J. Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, Karachi 75270, Pakistan b Department of Plant Resources, Ministry of Forest and Soil Conservation, Thapathali, Kathmandu, Nepal Received 26 July 2005; received in revised form 19 November 2005 Available online 19 January 2006
Abstract The microbial transformation of leucosceptrine (1), the rst member of class leucosesterterpenes, by Rhizopus stolonifer aorded two metabolites, 1a-hydroxyleucosceptrine (2), and 8a-hydroxyleucosceptrine (3). 2005 Elsevier Ltd. All rights reserved.
Keywords: Leucosceptrine; Sesterterpene; Fungal transformation; Rhizopus stolonifer; 1a-Hydroxyleucosceptrine; 8a-Hydroxyleucosceptrine
1. Introduction Selective functionalization by chemical methods at unactivated carbon atoms has been a major challenge in organic synthesis, and thus microbiological methods have frequently been used for this purpose (Fraga et al., 1996). Biotransformations involve the use of enzymes or microorganisms to perform chemical reactions in which the starting substances and products are of comparable chemical complexity. Leucosceptrine (1), C25H36O7, the rst member of a new class of sesterterpene named as leucosesterterpenes, was isolated from a medicinal plant Leucosceptrum canum Smith, belonging to the family Lamiaceae, by our research group (Choudhary et al., 2004a,b). L. canum, locally known as Bhusure (Hooker, 1983) is traditionally used as an insecticidal agent in remote areas of Nepal. Compound 1 has exhibited prolylendopeptidase (PEP) inhibitory activity (IC50 = 80 lM) in a mechanism-based assay (Choudhary et al., 2004a,b). The novel structure of leucosceptrine (1) stimulated us to carry out microbiological transformation on this compound by employing Rhizopus stolonifer.
Corresponding author. Tel.: +92 21 4824924 5/4819010; fax: +92 21 4819018 9. E-mail address: hej@cyber.net.pk (M.I. Choudhary). 0031-9422/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2005.11.021
*
2. Results and discussion Screening scale experiments have shown that the R. stolonifer was capable of converting compound 1 into polar metabolites 23. Large scale fermentation was thus carried out to produce sucient quantities of metabolites 23 for structure elucidation (Scheme 1). Two sets of controls were used to ensure the authenticity of metabolites. Metabolites were isolated from the culture medium by chloroform extraction. The residues obtained were fractionated by column chromatography. The PEP inhibitory activity of the metabolites could not be screened due to insucient quantities after structure determination. 1a-Hydroxyleucosceptrine (2) was isolated as a colorless solid from the chloroform extract of culture broth. The compound 2 showed the strong IR absorptions at 3320 (OH), 1735 (C@O), and 1664 (CH@CH) cm1. The FAB MS of compound 2 showed the (M+ H) peak at m/z 463 (C25H36O8), 16 amu higher than the substrate 1. The HREI-MS spectrum showed an ion at m/z 446.3312 (calcd 446.3246) supporting the formula C25H34O7 and representing a loss of H2O from the M+. The 1H NMR spectrum (CDCl3) of compound 2 showed close resemblance with the substrate 1. Five methyl signals,
440
M.I. Choudhary et al. / Phytochemistry 67 (2006) 439443

22
CH3 H
6 7 8 9 11 10
O
1 2 21
OH
5
HO
12
H3C
HO H H3C
24 14
13
H O
CH3
23
15
16
H3C
25
17 18
H O
19
20
H O
1 Rhizopus stolonifer 14 days
CH3 HO
1 2 21
22
OH
6 5
H O
7 8 1 9 11 10 21 2
CH3 H
6 7 8 9 11 10
OH
5
OH
HO
12
HO
12
H 3C
HO H H3C
24 14
13
H O
H3C CH3
23
HO H H3C
24 14
13
H O
CH3
23
15
15
16 25
16 17
H3C
H O
18
H3C
25
17 18
H O
19
20
19
H O
20
H O
2
Scheme 1. Metabolism of compound 1 by Rhizopus stolonifer.
including three secondary methyl groups were resonated at d 1.15 (d, J22,6 = 6.4 Hz), 1.06 (d, J23,10 = 6.8 Hz), and 1.23 (d, J24,14 = 7.1 Hz), and assigned to the C-22, C-23, and C24 methyl protons, while the tertiary methyl singlets at dH 1.75 and 2.04 were assigned to the C-21 and C-25 methyl protons, respectively. The 13C NMR spectrum showed the resonances for ve methyl carbons, resonated at d 17.1, 12.5, 21.2, 13.8, and 12.1, which were assigned to C-21, C-22, C-23, C-24, and C-25, respectively. Two trisubstituted double bond carbons were appeared at d 142.0, 123.6, 168.3, and 117.2 and assigned to C-2, C-3, C-18, and C-19, respectively. Four methylene carbons were resonated at d 35.3, 32.4, 28.1,
and 26.3 due to C-8, C-9, C-15, and C-16, respectively. Six methine carbons were appeared at d 104.2 (C-1), 46.7 (C-6), 47.7 (C-7), 35.3 (C-10), 57.4 (C-11), and 41.7 (C14). Similarly six quaternary carbons, including two carbonyl carbons, were resonated at d 75.2, 101.2, 86.5, 218.3, and 174.1, and were assigned to C-4, C-5, C-12, C13, and C-20, respectively. The main dierence between compound 2 and the substrate 1 was the absence of oxymethylene protons and presence of a downeld proton signal at d 5.82 in compound 2 which indicated that the C-2 oxymethylene was oxidized into an OH-containing methine. The presence of a downeld signal at d 104.2 further supported the presence of a
441
22
CH3 HO H
1 2 3
OH O
5 6
H
8 7 9
HO
4 11 12 10
H3C 21 HO H
14
13
H O
23
CH3
H3C
H
15
24
16 25
H3C
18
17
O
19 20
H O
Fig. 1. Key HMBC correlations in compound 2.
hydroxyl group at C-1 in compound 2. The mass fragments at m/z 265, 247, 219, 108, and 110 further supported the presence of a hydroxyl group at C-1 in hemiacetal ring. In the HMBC spectrum (Fig. 1), H-1 was found to be coupled with C-2 (d 142.0), C-3 (d 123.6) and C-5 (d 101.2). The Horeaus method (Horeau and Kagan, 1964) was employed to deduce the stereochemistry of the newly introduced hydroxyl group at C-1 in compound 2. The sign of rotation of residual acid was negative (R), which indicated
the stereochemistry of newly formed hydroxyl group as S (a). These observations supported the structure of compound 2 as 1a-hydroxyleucosceptrine. The compound 3 was isolated as a colorless solid from the chloroform extract of broth of R. stolonifer. Compound 3 showed the strong IR absorptions at 3300 (OH), 1725 (C@O), and 1665 (C@C) cm1. The FAB MS of compound 3 showed the (M+ H) peak at m/z 463, 16 amu higher than the substrate 1. The HREI-MS showed (M+ H2O) peak at m/z 446.3012 (C25H34O7, calcd 446.2946). The 1H NMR spectrum (CDCl3) of 3 showed close resemblance with the substrate 1. The only dierence being the appearance of a downeld methine proton at d 4.29, geminal to an OH group. The broad-band (BB) decoupled 13C NMR spectrum (CDCl3) of compound 3 indicated the presence of 25 carbons including 5 methyls, 4 methylene, 10 methine and 6 quaternary carbons. The 13C NMR spectrum of 3 was distinctly similar to substrate 1, with a downeld methine carbon at d 72.1, indicating the introduction of a OH group. The mass fragment ions at m/z 265, 247 and 127 also supported the presence of an additional hydroxyl group in metabolite 3. The methine proton resonated at dH 4.29 showed COSY 45 correlations with H-7 (d 3.01) and H29 (d 1.19, 1.52), indicated the position of hydroxyl group at C-8 in ring C. The HMBC interactions between C-11 methine proton (2.19) and C-8 (dC 72.1), and between H7 (d 3.01) and C-8, further supported the assigned position of hydroxyl group at C-8 (Fig. 2). The C-9 methylene protons (d 1.19, 1.52) also showed HMBC interactions with the C-8. The relative stereochemistry in compound 3 were inferred from the cross peaks in NOESY spectrum (Fig. 2). The NOE correlations between H-6/H-8, H-8/H-11, and
22
22
CH3 H H
1 2 3
OH O
5 6
H
7
OH
8
O
H
1
H 3C H OH H H
6 5 4 7 8
OH
HO
10 11 12
HO
12 13
9 11 10
H3C21 HO H
14
H 3C
21
13
H O
23
CH3
24
H O H
15
23
CH3
H3C
H
15
H 3C
14
24
16
16
17
H3C
18
25
H O
H 3C
18 19 17
25
H O
20
19
20
(a)
H O
H
(b)
Fig. 2. (a) Key HMBC, and (b) NOESY correlations in compound 3.
442
H3-23/H-11 indicated that H-6, H-8, and H-11 were in bconguration (see Fig. 2). 3. Experimental 3.1. General methods The melting points were recorded on a micro melting point apparatus and are uncorrected. Optical rotations were measured on a digital polarimeter in methanol on a Jasco digital polarimeter (model DIP-3600). Ultraviolet spectra were recorded in methanol on a Hitachi UV 3200 spectrophotometer. Infrared spectra were recorded on a Jasco A302 IR spectrophotometer. The mass spectra were recorded on a double focusing mass spectrometer. Accurate mass measurements were performed with FAB source using glycerol as matrix. The HREI-MS were recorded on a Jeol HX 110 mass spectrometer. The 1H NMR spectra were recorded on 300 MHz, while 13C NMR spectra were recorded on Bruker AMX-500 operating at 125 MHz using CDCl3 as solvent. Methyl, methylene and methine carbons were distinguished by DEPT experiments. Homonuclear 1H connectivities were determined by using the COSY experiment. One-bond 1H13C connectivities were determined with HMQC gradient pulse factor selection. Two- and three-bond 1 H13C connectivities were determined by HMBC experiment. Chemical shifts were reported in d (ppm) and coupling constants (J) were measured in Hz. Precoated TLC plates (silica gel) were used to check the purity of compounds, and ceric sulphate spraying reagent was used for the staining of compounds on TLC. All reagents used were of analytical grades. 3.2. Organism Cultures of R. stolonifer were obtained from the American Type Culture Collection (ATCC). All cultures were maintained on Sabouraud dextrose agar (SDA) slants and stored in a refrigerator at 4 C prior to use. 3.3. Incubation of leucosceptrine (1) R. stolonifer was grown in shake cultures at 25 C in ve conical asks (250), each containing 100 mL of a sterile medium comprising (per dm3) glucose (10 g), peptone (5 g), KH2PO4 (5 g), Yeast extract (5 g), Glycerol (10 mL), and NaCl (5 g). The media solution was adjusted to pH 7.0 before sterilization by autoclaving at 121C for 15 min. Incubations were initiated by suspending the surface growth from slants in sterile medium and using the suspensions to inoculate stage I cultures. Cultures were incubated with shaking on a shaker. After two days of incubation in the above-described medium, stage I culture was used as inoculum for stage II culture. Leucosceptrine (1) (76 mg), dissolved in 5 mL of DMSO, was uniformly distributed among ve asks
(250), each containing 100 mL of a sterile medium comprising (per dm3) glucose (10 g), peptone (5 g), KH2PO4 (5 g), Yeast extract (5 g), Glycerol (10 mL), and NaCl (5 g). Culture controls consisted of fermentation blanks in which microorganisms were grown under identical conditions but without the substrate. Substrate controls consisted of sterile medium containing the same amount of substrate and were incubated under the same conditions. After 14 days, the fermentation products were ltered, extracted with chloroform, and concentrated. The organic layer were screened for biotransformation by TLC methods. 3.4. Isolation of metabolites The mycelium was ltered and the culture ltrate was extracted with CHCl3. The extract was dried over Na2SO4 and the solvent evaporated to obtain a residue (122 mg). This material was chromatographed on a silica gel column with a pet. ether-CHCl3 gradient, to aord 1a-hydroxyleucosceptrine (2) (5 mg), and 8a-hydroxyleucosceptrine (3) (2 mg). 3.4.1. 1a-Hydroxyleucosceptrine (2) Colorless crystals, m.p. 154157 C, Rf = 0.41 (5% MeOH/CHCl3), a25 143 (CHCl3, c 0.03), UV (MeOH) D nm (log e) kmax 380 (2.78), 351 (2.76); kmin nm 391 (1.06), 361 (2.16), 182 (2.56); IR (CHCl3) mmax 3456 (OH), 2935 and 2868 (CH), 1705 (C@O) cm1; 1H NMR (CDCl3, 300 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Table 1; FAB MS (M+ 1) m/z 463, HREI-MS m/z 446.3312 (calcd for C25H36O7, m/z 446.3246), EI-MS m/z (rel. int. %): 446 (30), 265 (16), 247 (100), 219 (18), 181 (18), 153 (17), 139 (19), 125 (24), 110 (46), 97 (66), 83 (62). 3.4.2. 8a-Hydroxyleucosceptrine (3) Colorless crystals, m.p. 145147 C, Rf = 0.35 (5% 25 MeOH/CHCl3), aD 240 (CHCl3, c 0.06); UV (MeOH) nm (log e): kmax 365 (2.78), 301 (2.76), kmin 382 (1.79), 346 (2.02), 261 (2.16), IR (CHCl3); mmax 3456 (OH), 2935 and 2868 (CH), 1705 (C@O) cm1; 1H NMR (CDCl3, 300 MHz) and 13C NMR (CDCl3, 100 MHz) data, see Table 1; FAB MS (M+ 1) m/z 463, HREI-MS m/z 446.3012 (calcd for C25H36O7, m/z 446.2946), EI-MS m/z (rel. int. %): 446 (30), 265 (11), 247 (19), 181 (18), 155 (10), 153 (18), 127 (22), 125 (48), 110 (42), 97 (68), 92 (17), 83 (100). 3.5. Horeaus procedure Compound 2 (5 mg, ca. 0.01 mmol) was added to a solution of 2-phenyl butyric anhydride (0.1 mL) in 0.5 mL C5H5N. The resulting mixture was stirred overnight at room temperature. Distilled water (3.0 mL) was added and the reaction mixture allowed to stand for 30 min, 0.1 M NaOH was then added dropwise until the pH became 9 and the solution was then extracted with CHCl3. The aqueous layer was acidied to pH 3 using 1.0 M HCl and the acidic layer was extracted with C6H6 (10 mL).
M.I. Choudhary et al. / Phytochemistry 67 (2006) 439443 Table 1 1 H and 13C NMR data of 1a-hydroxyleucosceptrine (2) and 8a-hydroxyleucosceptrine (3) in CDCl3 Position 2 dH (m, J in Hz) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
a 1 b 13 a
443
3 dC
b
Multiplicity CH C CH C C CH CH CH2 CH2 CH CH C C@O CH CH2 CH2 CH C CH C CH3 CH3 CH3 CH3 CH3
dH (m, J in Hz)a 3.99, 4.21 (d, 17.1) 4.96 (brs) 2.10 (m) 3.01 (m) 4.29 (m) 1.19, 1.52 (m) 1.85 (m) 2.19 (m) 3.37 (m) 1.21, 2.01 (m) 1.15, 1.85 (m) 4.81 (brd, 6.7) 5.81 (m) 1.65 (brs) 0.99 (d, 6.7) 0.71 (d, 6.9) 1.06 (d, 6.7) 2.05 (s)
dCb 64.7 137.2 121.3 73.4 96.8 41.6 42.6 72.1 38.5 36.6 52.2 82.2 221.2 38.6 28.9 26.8 84.2 167.7 117.3 181.6 14.7 10.8 17.7 13.7 13.4
Multiplicity CH2 C CH C C CH CH CH CH2 CH CH C C@O CH CH2 CH2 CH C CH C CH3 CH3 CH3 CH3 CH3
5.82 (s) 5.61 (brs) 1.95 (m) 1.55 (m) 1.45, 1.91 (m) 1.23, 1.44 (m) 1.67 (m) 1.55 (m) 3.01 (m) 1.25, 1.86 (m) 1.15, 1.75 (m) 4.81 (brd, 6.2) 5.94 (m) 1.80 (brd, 1.3) 1.15 (d, 6.4) 1.06 (d, 6.8) 1.23 (d, 7.1) 2.05 (s) H NMR data, 300 MHz. C NMR data, 125 MHz.
104.2 142.0 123.6 75.2 101.2 46.7 47.7 35.3 32.4 35.3 57.4 86.5 218.3 41.7 28.1 26.3 84.5 168.3 117.2 174.1 17.1 12.5 21.2 13.8 12.1
The benzene extract was evaporated to adjust the volume to 1.0 mL. The optical rotation of the resulting 2-phenyl butyric acid was found to be negative (R), indicating the S conguration at C-20 in compound 2. Acknowledgments One of us, Dr. Rosa Ranjit, acknowledges the support of the Third World Organization for Women in Science (TWOWS), Trieste, Italy, through a Ph.D. scholarship to study at the H.E.J. Research Institute of Chemistry, Karachi-75270, Pakistan. We are also grateful to the Third World Academy of Sciences (TWAS), Trieste, Italy, and LG Corporation Limited, Pakistan, for nancial support to Dr. Krishna P. Devkota.
References
Choudhary, M.I., Ranjit, R., Atta-ur-Rahman, Shrestha, T.M., Yasin, A., Parvez, M., 2004a. Leucosceptrine a novel sesterterpene with propylendopeptidase inhibitory activity from Leucosceptrum canum. J. Org. Chem. 69, 29062909. Choudhary, M.I., Ranjit, R., Atta-ur-Rahman, Hussain, S., Devkota, K.P., Shrestha, T.M., Parvez, M., 2004b. Novel sesterterpens from Leucosceptrum canum of Nepalese origin. Org. Lett. 6, 41394142. Fraga, B.M., Guillermo, R., Hanson, J.R., Trunch, A., 1996. Biotransformation of cerdrol and related compounds by Mucor plumbeus. Phytochemistry 42, 15831586. Hooker, J.D., 1983. Flora of British India, vol. IV. Bishen Singh Mahendra Pal Singh Publishers, Dehradun, pp. 699700. Horeau, A., Kagan, H.B., 1964. Determination des congurations par dedoublement partiel III alcohols steroides. Tetrahedron 20, 24312441.
Clerodane and labdane diterpenoids from Nuxia sphaerocephala

Lengo Mambu a,*, Philippe Grellier b, Loic Florent a, Roger Joyeau a, David Ramanitrahasimbola c, Philippe Rasoanaivo c, Francois Frappier a,z
a b
USM 0502-UMR5154 CNRS Chimie et Biochimie des substances naturelles, Departement Regulations, Developpement, Diversite Moleculaire, Museum National dHistoire Naturelle, 63 rue Buon, 75231 Paris Cedex 05, France USM 0504 Biologie fonctionnelle des protozoaires, Departement Regulations, Developpement, Diversite Moleculaire, Museum National dHistoire Naturelle, 61 rue Buon, 75231 Paris Cedex 05, France c Laboratoire de Pharmacognosie Appliquee aux Maladies Infectieuses, Institut Malgache de Recherches Appliquees, B.P. 3833, 101-Antananarivo, Madagascar Received 30 March 2005; received in revised form 17 November 2005 Available online 19 January 2006
Abstract Seven diterpenoids including four clerodane and three labdane derivatives, (13S)-ent-7b-hydroxy-3-cleroden-15-oic acid (1), ent-7bhydroxy-2-oxo-3-cleroden-15-oic acid (2), ent-2,7-dioxo-3-clero-den-15-oic acid (3), ent-18-(E)-caeoyloxy-7b-hydroxy-3-cleroden-15oic acid (4) (13S)-ent-18-(E)-coumaroyloxy-8(17)-labden-15-oic acid (5), ent-18-(E)-caeoyloxy-8(17)-labden-15-oic acid (6), ent-15(E)-caeoyloxy-8(17)-labden-18-oic acid (7), have been isolated from an ethyl acetate extract of the leaves of Nuxia sphaerocephala, together with 17 known compounds. 3-Oxolup-20(29)-en-30-al (3-oxolupenal) (8) and 3b-hydroxylup-20(29)-en-30-al (3b-hydroxy-lupenal) (9) showed the best inhibitory activity against Plasmodium falciparum with the IC50 values between 1.55 and 4.67 lg/ml in vitro, respectively. The structure and the relative stereochemistry of the compounds were established on the basis of their spectroscopic properties. The absolute conguration at C-13 of 1 and 5 was determined by the PGME amide procedure. 2005 Elsevier Ltd. All rights reserved.
Keywords: Nuxia sphaerocephala; Loganiaceae; Clerodanes; Labdanes; PGME amide method; Antiplasmodial activity
1. Introduction Nuxia sphaerocephala Baker (Loganiaceae) is a shrub growing in the Eastern rainforests of Madagascar. Leaves are used in traditional medicine to treat splenomegaly associated with malaria, and also infantile hederosyphilis (Boiteau, 1999). Previous phytochemical studies on the genus Nuxia led to the isolation of the eight-carbon iridoid glucoside unedoside and its derivatives (Jensen et al., 1998; Frederiksen et al., 1999). To the best of our knowledge, no study has been reported on N. sphaerocephala. As part of our search for novel antimalarial agents from plants, we
Corresponding author. Tel.: +33 1 40 79 56 07; fax: +33 1 40 79 31 35. E-mail address: mambu@mnhn.fr (L. Mambu). In memoriam.
investigated the leaves of N. sphaerocephala, the ethyl acetate extract of which showed antiplasmodial activity with an IC50 value of 4.2 lg/ml (Rasoanaivo et al., 2004). Seven new compounds, including four clerodane diterpenoids (1, 2, 3, 4) and three labdane diterpenoids (5, 6, 7) along with 17 known compounds were isolated, of which ten were identied as triterpenes namely 3-oxolupenal (3-oxolup20(29)-en-30-al) (8), 3b-hydroxylupenal (3b-hydroxylup20(29)-en-30-al) (9), lup-20(29)-ene-3b,30-diol (Wijeratne et al., 1981), 3-oxolupenol (30-hydroxylup-20(29)-en-3one) (10) (Bohlmann and Jakupovic, 1979), lupeol, uvaol (Dehmlow et al., 1998), ursolic acid, 3b-acetylursolic acid (Houghton and Lian, 1986), 3b-acetyloleanolic acid (11) (Chavez and Delgado, 1994) and oleanolic acid (12); three were found to be diterpenes, ent-15-hydroxy-8(17)-labden19-oic acid (13) (Zdero et al., 1991a), ent-18-hydroxy-8(17)labden-15-oic acid (14) (Zdero et al., 1991b) and ent-18-
L. Mambu et al. / Phytochemistry 67 (2006) 444451
445
hydroxy-3-cleroden-15-oic acid (15) (Tsichritzis and Jakupovic, 1990), three were identied as avones, 5hydroxy-7-methoxyavone (Mongkolsuk and Dean, 1964), 5-hydroxy-4 0 ,7-dimethoxyavone (Biftu and Stevenson, 1978) and 5,7-dihydroxy-4 0 -methoxyavone (Gaydou and Bianchini, 1978) and one as a sterol, 3-O-b-D glycopyranosylsitosterol (Ahmad et al., 1992). All clerodane and labdane diterpenoids isolated from this plant were recognized as members of ent-series through their optical rotation values. In this paper, we describe the isolation and structural elucidation of 17 as well as the inhibitory activity of all isolated compounds against Plasmodium falciparum. 2. Results and discussion A crude EtOH extract of leaves of N. sphaerocephala was partitioned successively with cyclohexane and ethyl acetate. The ethyl acetate soluble extract showed signicant inhibitory activity against P. falciparum with an IC50 of 4.2 lg/ml and was subjected to a series of bioassay-guided column chromatographic purication steps over silica gel to aord seven new compounds 17 along with 17 known compounds.
fore bicyclic. The 1H NMR spectrum (Table 2) displayed characteristic signals for an olenic proton at d 5.11 (1H, m), an oxymethine proton at d 3.98 (1H, q, J = 3.3 Hz), two tertiary methyl singlets at d 0.96 and 1.26 and three doublet methyl signals at d 0.96 (d, J = 6.9 Hz), 0.98 (3H, d, J = 7.2 Hz) and 1.59 (d, J = 1.4 Hz) and suggested a clerodane skeleton. The 13C NMR spectral data indicated that compound 1 contained 20 carbons, including ve methyls, ve methines, six methylenes and four quaternary carbons (Table 1). The COSY spectrum revealed three spin systems associated with ring A, ring B and the side chain as in 1. HMBC correlations conrmed the clerodane nucleus and the position of the functionality. Thus H-3 showed correlations with C-2, C-5 and C-18 while H-7 correlated with C-5, C-6, C-8, C-9 and C-17. The structure of the side chain was established inter alia by the HMBC correlations from H-13 to C-11, C-12, C-14, C-15 and C-16. Other useful correlations of C-5 included those from Me-18 and Me-19. The conguration of the hydroxyl group at C-7 was deduced to be a (axial) on the basis of the small coupling constants of H-7. NOEs from Me-19 to Me-20 and from H-7 to Me-17 completed the relative stereochemistry of 1 apart from the A/B ring junction which was assumed to be trans on the basis of the lack of a NOE between
COOH
11 1 H 10 4 18 19 4 13 20 17 7 R R
2 2
15 COOH
H
7
OH
18 8'
4'
OH OH
O O
7'
1'
3'
1 . R = H, H; R 1 = OH; R 2 = H 2 . R = O; R1 = OH; R2 = H 3 . R = O; R 1 , R2 = O
4' O H 8' COOH H R1 H O O 5 R1 =OH; R2 = H 6R

1=
13 20 1 H 19 O 7 5 18 OH H 17 15
O O 7'
1'
OH
R2
OH;
R2 =
OH
Diterpenoids 1-7 from N.sphaerocephala
Compound 1 was obtained as colourless oil. Its molecular formula C20H34O3 was established from its HRCIMS at m/z 340.2845 [M + NH4]+ and conrmed by 13C NMR data which also revealed the presence of a trisubstituted double bond and a carboxyl group. The molecule is there-
Me-19 and H-10. The conguration at C-13 could not be resolved by spectroscopic means. The absolute conguration at C-13 was determined by applying the phenylglycine methyl ester method (PGME) developed for carboxylic acids having a chiral center at
446 Table 1 13 C NMR data for compounds 17 Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 10 20 30 40 50 60 70 80 CO

a b
1a 17.8 26.7 119.9 144.9 37.5 42.8 73.9 39.2 38.0 46.5 36.6 29.5 30.8 41.3 178.6 19.9 12.4 18.0 21.8 20.2
2a 34.7 200.6 125.0 173.1 39.4 41.4 73.3 38.8 38.1 45.9 35.9 29.2 30.7 41.1 177.0 19.9 12.3 19.0 20.2 19.7
3a 35.0 198.9 126.0 169.5 44.3 51.2 211.3 50.1 44.3 45.7 35.3 29.2 30.5 40.8 176.0 19.9 7.7 18.8 19.4 19.1
4b 18.8 27.5 126.4 145.1 38.6 43.6 74.0 40.6 39.2 47.8 37.8 30.7 32.2 42.7 177.1 20.3 13.0 66.0 23.7 20.6 127.5 115.2 146.8 149.6 116.5 122.9 146.8 115.4 169.1
5a 38.5 18.6 36.0 37.0 49.5 24.3 38.0 148.1 56.8 39.5 20.9 35.6 30.7 41.8 178.6 19.5 106.6 72.8 17.6 14.9 127.0 130.0 115.9 157.9 115.9 130.0 144.6 115.5 167.8
6b 39.8 19.7 37.2 38.3 50.8 25.6 39.2 149.6 58.4 40.6 22.0 36.9 32.1 43.3 177.6 20.0 107.2 73.7 18.0 15.4 127.7 115.2 147.0 149.5 116.5 123.0 146.9 115.0 169.3
7b 39.5 19.5 38.4 48.0 51.2 28.0 39.1 149.0 58.7 40.0 22.2 36.9 31.7 37.0 63.9 20.3 107.4 182.2 17.1 15.2 127.8 115.2 146.8 149.5 116.5 122.9 146.8 115.3 169.4
Spectra were recorded in CDCl3. Spectra were recorded in CD3OD.
the b-position (Yabuuchi and Kusumi, 2000). Two portions of 1 (each 4 mg) were condensed in DMF with (S)and (R)-PGME in the presence of PyBOP, HOBT, and N-methylmorpholine which aorded the (S)- and (R)PGME amide derivatives (1s and 1r). Assignments of the proton signals of PGME amides were based on the correlations of the 1H1H COSY and NOESY spectra including HSQC and HMBC. The chemical shift dierence values DdRS = dR dS of the individual protons of 1s and 1r are shown in Fig. 1. The systematic arrangement of positive and negative DdRS values was observed and unambiguously established the absolute conguration of C-13 to be S as indicated by the structure 1 which belongs to the enantio series. Accordingly, compound 1 was assigned as (13S)ent-7b-hydroxy-3-cleroden-15-oic acid.
H N OCH3 O H Ph Ph H (R ) ( S)
+0.021 +0.015 +0.021 -0.001 H -0.089 +0.015 CH3
-0.059 CH3 CH3 +0.018 +0.001
CH3 CH3
OH
Fig. 1. Dd(R S) values (in ppm) for PGME amide derivatives of 1r and 1s in CDCl3.
The FABMS spectrum of 2 exhibited a pseudomolecular ion peak at m/z 337 [M + H]+ consistent with a molecular formula C20H32O4 and ve double bond equivalents. Its 13 C spectrum, UV and the IR spectra showed the presence of an a,b-unsaturated ketone (d 200.6 (C-2), 125.0 (C-3) and 173.1 (C-4); mmax 1657 cm1; kmax 238 nm). The 1H and 13C NMR data for 2 (Tables 1 and 2) were similar to those of 1 apart from the presence of a ketone at C-2, conrmed by HMBC correlations from H-10 and 2H-1 to C-2. The relative stereochemistry of 2 was established in the same way as that of 1 with additional support coming from NOEs between H-6, H-8 and H-10 indicating that all these protons were axial (b). Thus, compound 2 was established as ent-7b-hydroxy-2-oxo-3-cleroden-15-oic acid. The molecular formula of compound 3 was determined as C20H30O4 from combined analysis of HRCIMS and 13C data. Its 1H and 13C NMR data were similar to those of 1 except for the lack of an oxymethine proton (H-7) and the presence of two ketone carbonyl carbons at d 198.8 and 211.3. Long-range correlations of H-6, H-8 and H3-17 to the carbonyl at d 211.3 allowed us to place the carbonyl group at C-7 and the inuence of this group was observed by an upeld shift of C-17 (d 7.7 instead of 12.4) in the 13C spectrum. The second carbonyl group was located at C-2 as shown by the connectivities of H2-1 and H-10 with this carbon in HMBC spectrum. The relative stereochemistry of 3 was assigned by analysis of NOESY correlations as for 1 and 2. Me-20 showed strong correlations with 2H-11, Me-17 and Me-19 suggesting that they were all on the same face of the molecule. Compound 3 was therefore identied as ent-2,7-dioxo-3-cleroden-15-oic acid. For compound 4, the HRFABMS showed a pseudomolecular ion peak at m/z 523.2687 [M + Na]+ (calcd. 507.2672 for C29H40O7Na) in agreement with the molecular formula of C29H40O7. Its 1H NMR spectrum was markedly similar to that of 1 except for the presence of a caeoyl moiety, a primary oxygenated carbon (dH 4.67 (br s, 2H18), dC 66.0 (C-18)) and the lack of a vinyl methyl group. The spectroscopic data for the caeoyl residue are in Tables 1 and 2 and in Section 3. An HMBC correlation from 2H-18 to the ester carbonyl at dC 169.1 established the connection of the caeoyl group to the clerodane skeleton. The NOESY spectrum revealed the same relative stereochemistry as 1. Hence, compound 4 was elucidated as ent-18-E-caeoyloxy-7b-hydroxy-3-cleroden-15-oic acid. Compound 5 (mmax 3352, 1663, 1587 and 1416 cm1, kmax 312, 296 and 226 nm), a colorless oil, contained ester, carboxylic acid and conjugated aromatic functionality. Its molecular formula C29H40O5 was established from the [M + NH4]+ ion at m/z 486.3227 (calcd. 486.3219 for C29H44O5N) in its HRCIMS spectrum. Its 1H NMR spectrum (Table 3) revealed the presence of two tertiary methyls dH 0.84 and 0.7, a secondary methyl signal dH 0.96 (d, J = 6.6 Hz), an exomethylene group dH 4.47 and 4.80 (both s), an oxygenated methylene group dH 3.73, 3.96 (both d, J = 10.9 Hz) and a trans p-hydroxycinnamoyl moiety (cou-
L. Mambu et al. / Phytochemistry 67 (2006) 444451 Table 2 1 H NMR data of clerodane diterpenoids Position 1 2 3 6 7 8 10 11 12 13 14 16 17 18 19 20 20 50 60 70 80
a b
447
1a 1.53, m 1.96, 2.01, 5.11, 1.37, 2.07, 3.98, 1.51, 1.37, 1.28, 1.00, 1.16, 1.84, 2.14, 2.33, 0.96, 0.98, 1.59, 1.26, 0.97, m m m dd (3.3, 14.1) dd (2.6, 14.1) q (3.3) m m m m ddd (5.5, 12.3) m dd (7.9, 15.1) dd (6.2, 15.1) d (6.9) d (7.2) d (1.4) s s
2a 2.34, dd (3.9, 17.5) 2.44, dd (13.7, 17.5)
3a 2.34, m
4b 1.59, m 2.13, m
5.67, 1.50, 2.17, 4.06, 1.53, 1.89, 1.25, 1.00, 1.09, 1.83, 2.15, 2.29, 0.95, 1.00, 1.89, 1.37, 1.05,
d (1.3) dd (3.5, 14.0) dd (2.8, 14.0) ddd (2.8, 3.2, 3.5) dd (3.2, 7.2) dd (3.9, 13.7) m m m m dd (8.7, 15) dd (6.4, 15) d (6.7) d (7.2) d (1.3) s s
5.77, d (1.2) 2.34, m 2.51, d (11.9) 2.56, 2.46, 1.26, 1.09, 1.26, 1.88, 2.19, 2.28, 0.98, 0.92, 1.85, 1.08, 0.77, q (6.3, 12.8) dd (4.1, 11.2) m 1.41, dd (9.8, 12.6) m m m dd (7.3, 15.1) dd (6.4, 15.1) d (6.7) d (6.3) d (1.2) s s
5.58, 1.53, 2.10, 3.97, 1.56, 1.47, 1.31, 1.21, 1.80, 2.15, 2.23, 0.97, 1.00, 4.67, 1.40, 1.02, 7.03, 6.77, 6.93, 7.52, 6.25,
br s dd (3.1, 13.5) dd (2.8, 13.5) ddd (2.8, 3.1, 3.4) m dd (2.1, 11.4) m m m dd (14.8, 7.2) dd (14.8, 7.0) d (6.7) d (6.1) br s s s d (2.0) d (8.2) dd (2.0, 8.2) d (15.9) d (15.9)
maroyl, see Tables 1 and 3). The COSY spectrum revealed spin systems associated with rings A and B and the side chain of a labdane skeleton which was conrmed by several correlations in the HMBC spectrum, in particular from H7 to C-8, C-9 and C-17, H-5 to C-4, C-9, C-10 and C-20, H1 to C-5, C-10 and C-20, H-3 to C-4 and C-19. Support for the side chain came from correlations of H-14 to C-15 and C-16 and from H-12 to C-9 and C-11. The oxymethylene protons clearly belonged to C-18 in view of HMBC correlations to C-3, C-4, C-5 and C-19. A further correlation of these to the coumarate ester carbonyl group at d 167.8 conrmed the position of attachment of the ester. NOE interactions between Me-19 and Me-20, 2H-18 and H-5, and H-5 and H-9 indicated a typical A/B trans labdane system. The absolute conguration at C-13 was also determined by applying the phenylglycine methyl ester method (PGME). The chemical shift dierence values DdRS = dR dS of (R)-(5) and (S)-(5) PGME amides are shown in Fig. 2. From these data the absolute conguration of the chiral carbon at C-13 was identied to be S and compound 5 belongs to the enantio series. Thus, the structure of 5 was established as (13S)-ent-18-E-coumaroyloxy-8(17)-labden-15-oic acid (see Fig. 2). The HRFABMS of compound 6 gave a pseudomolecular ion peak at m/z 507.2725 [M + Na]+ (calcd. 507.2723 for C29H40O6Na) corresponding to a molecular formula C29H40O6. Its UV spectrum was characteristic of a caeoyl
moiety with absorption maxima at 327, 295, 245, 220 and 203 nm. The 1H NMR spectrum of 6 resembled that of 5 apart dierences in the aromatic region (Table 3). Examination of the proton and carbon data readily revealed that the coumaroyl moiety of 5 had been replaced by a caeoyl moiety. The structure of 6 was determined as ent-18-E-caffeoyloxy-8(17)-labden-15-oic acid. Compound 7 was obtained as colourless oil with a molecular formula of C29H40O6 (m/z 507.2739) based on HRFABMS and 13C data. The spectroscopic data, including the UV (kmax 328, 293, 245, 220, 203 nm), indicated the presence of a caeoyl moiety. The lack of 2H-14 resonances, present in 16 between dH 2.08 and 2.33, suggested that C-15 was no longer a carboxyl group but had been reduced to an alcohol. The spectroscopic properties indicated that 7 was a derivative of the C-4 epimer of ent-15hydroxy-8(17)-labden-19-oic acid (Zdero et al., 1991a). The downeld shift 2H-15 and an HMBC correlation from 2H-15 to the caeoyl carbonyl group revealed the position of attachment of the ester. NOEs between H-9 and H-5 and Me-19 and Me-20 established the trans nature of the ring junction and the presence of a C-18 carboxyl group. Thus compound 7 was assigned as 15-E-caeoyloxy-8(17)-labden-18-oic acid. All the isolated compounds were tested for their ability to inhibit in vitro the growth of the chloroquine-resistant strain FcB1 of P. falciparum. The antiplasmodial activity
448 Table 3 1 H NMR data of labdane diterpenoids Position 1 2 3 5 6 7 9 11 12 13 14 15 16 17 18 19 20 20 30 50 60 70 80

a b

29
5a 0.95, m 1.71, m 1.55, m 1.35, m 1.40, 1.35, 1.40, 1.96, 2.35, 1.59, 1.35, 1.44, 1.14, 1.31, 1.90, 2.12, 2.27, 0.96, 4.47, 4.80, 3.73, 3.96, 0.84, 0.70, 7.42, 6.82, 6.82, 7.42, 7.59, 6.30, m m m dd (4.9, 13.2) m d (9.5) m m m m m dd (7.8, 15.1) dd (6.2, 15.1) d (6.6) br s br s d (10.9) d (10.9) s s d (8.7) d (8.6) d (8.6) d (8.7) d (15.9) d (15.9)
6b 1.09, 1.83, 1.59, 1.70, 1.47, 1.51, 1.40, 1.69, 1.99, 2.38, 1.65, 1.42, 1.52, 1.16, 1.37, 1.89, 2.08, 2.24, 0.96, 4.53, 4.83, 3.75, 3.99, 0.89, 0.77, 7.06, 6.78, 6.97, 7.55, 6.29, m m m m m m m m m m m m m m m m dd (7.7; 14.6) dd (6.6, 14.6) d s s d d s s d (6.6)
7b 1.16, dd (4.7, 7.8) 1.79, m 1.51, m 1.51, 1.71, 1.96, 1.32, 1.44, 2.02, 2.36, 1.65, 1.44, 1.51, 1.24, 1.32, 1.51, 1.51, 1.71, 4.17, 0.96, 4.55, 4.84, m m dd (2.2, 12.5) m m dd (4.7, 13.1) m m m m m m m m m m d (6.3) s s
1
30
20
R2
26
H
13
19
22 28
25
H
15
R1 R
H
27
H
24 23
8 R, R1 = O , R2 = CHO 9 R = OH, R1 = H , R2 = CHO 10 R, R1 = O, R2 = CH 2OH

Fig. 3. Structures of lupane derivatives 810.
Table 4 In vitro antiplasmodial activity of compounds 112 Compound IC50 SD (lg/ml) FcB1 14.6 1.4 4.3 0.9 8.0 0.2 7.3 0.8 11.4 1.1 21.0 1.85 16.0 0.87 1.55 0.06 3.15 0.07 9.05 1.06 7.65 0.49 9.83 3.1 0.05 0.002 IC50 SD (lg/ml) FcM29
(10.8) (10.8) 1.12, s 0.74, s 7.04, d (2.0) 6.77, 6.94, 7.52, 6.24, d (8.3) dd (2.0, 8.3) d (15.9) d (15.9)
(2.0)
d (8.2) dd (2.0, 8.2) d (15.9) d (15.9)
1 2 3 4 5 6 7 8 9 10 11 12 Chloroquine
4.67 0.09 4.06 0.53 15.56 2.11
H +0.053 +0.023 +0.014 +0.025 -0.023 H +0.121 N
O OCH3
Results are expressed as IC50 and IC90 values (lg/ml) standard deviations. All experiments were realised in triplicate.
-0.054 +0.035 +0.021
O H Ph Ph (R) H (S)
H O OH
Fig. 2. Dd(R S) values (in ppm) for PGME amide derivatives of 5r and 5s in CDCl3.
of new compounds 17 and the most active known compounds (812) are summarized in Table 4. The other compounds displayed IC50 value over 10 lg/ml. Compounds 8 and 9 displayed signicant antimalarial activity with IC50 value between 1.55 and 4.67 lg/ml depending upon the P. falciparum strain tested (Fig. 3). In comparison to chloroquine which was used as a positive control, these compounds have to be considered as moderately active (Table 4).
For 8 and 9, the C-30 aldehyde and the C-3 ketone probably have a major inuence on the activity. The activity of 10, with a C-30 hydroxyl group, was lower and with hydroxyls at both C-3 and C-30 the activity diminished considerably. Ursolic acid exhibited antiplasmodial activity with IC50 value of 16 lg/ml which is consistent with that published (Azas et al., 2002). In vitro antiplasmodial activity of 12 has been previously reported with IC50 value similar to those found in the present work (Sairaanpour et al., 2003). The lupane triterpenoids betulinic acid and lupeol, as well as labdane diterpenoids have been reported to exhibit moderate antiplasmodial activity in vitro (Bringmann et al., 1997; Ziegler et al., 2002; Asili et al., 2004). It has been demonstrated that terpenoids can be incorporated into the lipid bilayer of erythrocyte membranes irreversibly and induce shape transformation of this membrane (Ziegler et al., 2002; Asili et al., 2004). In this case,
449
the inhibition of parasite growth observed in vitro could be attributed to indirect eects due to stomatocytic or echinocytic modications of the host cell membrane. Non-parasitized erythrocytes were then incubated with increasing concentrations of compounds 8, 9 and 10 under the same conditions as previously described (Ziegler et al., 2002). No lysis of cells and no change of erythrocyte membrane shape in echnocytic forms were observed at the concentrations up to 50 lg/ml by phase-contrast microscopy. There was also no evidence of stomatocytic forms. However, such modication might not be clearly detectable by photonic microscopy, further investigation using transmission electron microscopy will be required to conrm our observations.
3.3. Bioassay The in vitro antiplasmodial tests, based on the inhibition of [3H]-hypoxanthine uptake by P. falciparum cultured in human blood, were conducted as previously described (Frappier et al., 1996). 3.4. Extraction and isolation Air-dried powdered leaves of N. sphaerocephala (600 g) was exhaustively extracted with ethanol (3 500 ml) at room temperature to give 31 g of crude extract. The ethanolic extract exhibited an IC50 value of 7.1 lg/ml against the growth of P. falciparum. A portion of the crude extract (14 g) was partitioned between ethyl acetate (3 500 ml) and water (300 ml). The organic fraction (10.8 g) showed an IC50 value of 4.2 lg/ml whereas the aqueous fraction was inactive (1.27 g; IC50 > 25 lg/ml). A portion (5 g) of the ethyl acetate extract was chromatographed on a silica gel column using a mixture of cyclohexaneEtOAc of increasing polarity as eluant to give 22 fractions. The purication of the most potent fractions F1 and F2 on silica gel column chromatography (cyclohexaneEtOAc 90:10) yielded 27 mg of compound 8. Chromatography of F6 with cyclohexaneEtOAc 85:15 followed by crystallization (acetone) aorded 42 mg of compound 9. Repeated chromatography of fraction F11 on MPLC column (AIT, France) with CH2Cl2MeOH 98:2 furnished 8 mg of compound 1. Chromatography of fraction F12 yielded additional compound 1. From fraction F15, compound 5 (8 mg) was obtained after repeated purication on silica gel with cyclohexaneEtOAc 75:25 followed by CH2Cl2 MeOH 97:3. F16 was puried by silica gel column chromatography (CH2Cl2MeOH 98:2) to aord 13 mg of compound 7. Compound 6 (9 mg) was isolated from fraction F17 by successive chromatography on silica gel with cyclohexaneEtOAc 60:40 and on a Sephadex column using MeOH as eluting solvent. F18 was puried by silica gel column chromatography (cyclohexaneEtOAc 60:40 and CH2Cl2MeOH 95:5) to aord 11 mg of compound 4. Fraction F22 aorded compound 3 (2 mg) and compound 2 (5 mg) by further purication by column chromatography on silica gel eluting with CH2Cl2MeOH 98:2. 3.4.1. (13S)-ent-7b-Hydroxy-3-cleroden-15-oic acid (1) 20 Colourless oil; aD 32:3 (c 0.4, CHCl3); IR (CHCl3) mmax 2925, 1712, 1470, 1066 cm1; UV (MeOH) kmax (loge) 237 (3.06), 203 (3.48) nm; 1H NMR and 13C NMR data, see Tables 1 and 2; HRCIMS, m/z: 340.2845 ([M + NH4]+) (calcd. for C20H38O3N, 340.2852). 3.4.2. ent-7b-Hydroxy-2-oxo-3-cleroden-15-oic acid (2) 20 Colourless oil; aD 30 (c 0.195, CHCl3); IR (CHCl3) mmax 2925, 1657, 1470, 1029 cm1; UV (MeOH) kmax (loge) 281 (2.90), 238 (3.56), 203 (3.52) nm; 1H NMR and 13C NMR data, see Tables 1 and 2; HRFABMS, m/z: 337.2373 ([M + H]+) (calcd. for C20H33O4, 337.2379).
3. Experimental 3.1. General experimental procedures Optical rotations were measured with a PerkinElmer model 341 polarimeter at 20 C. IR spectra were taken on a Nicolet Impact 400D spectrophotometer. The UV spectra were recorded on a Kontron spectrometer. 1H and 13C NMR spectra were recorded at 400.13 and 100.61 MHz, respectively, on a Bruker AVANCE-400 spectrometer at 298 K, equipped with 1H-broad-band reverse gradient probehead. Temperature was controlled by a Bruker BCU-05 refrigeration unit and a BVT 3000 control unit. The 1H and 13C NMR chemical shifts were expressed in ppm relative to TMS, with coupling constants (J) given in Hz. High-resolution mass spectra and FAB-MS were recorded on a JEOL MS700 apparatus. Mass spectra data were recorded using an electrospray time of ight mass spectrometer (ESI-TOF-MS) operating in the positive mode (QSTAR Pulsar I of Applied Biosystems). TLC was carried out on precoated Si gel 60 F254 plates (Merck). Spots were detected under UV (254 and 366 nm) before spraying with phosphomolybdic acid solution in EtOH or LiebermannBurchard reagent or vanillin-sulfuric solution followed by heating the plate at 110 C. Column chromatography was performed on 200400 mesh silica gel 60 (Merck). Preparative medium-pressure liquid chromatography (MPLC) was performed with a pump K-120 (Knauer) and Flashsmart cartridges (Si gel 2040 lm, AIT, France). 3.2. Plant material The plant material was collected in March 2000 in Ankazobe, middle West located at 100 km North from Antananarivo (Madagascar) and was identied by Armand Rakotozafy by comparison with authentic specimens held in the Department of Botany, Parc Botanique et Zoologique de Tsimbazaza, Antananarivo. A voucher specimen (ANKA 15/AR/2000) was deposited at the Institut Malgache de Recherches Appliquees.
450
3.4.3. ent-2,7-Dioxo-3-cleroden-15-oic acid (3) 20 Colourless oil; aD 10 (c 0.215, CHCl3); IR (CHCl3) mmax 2931, 2372; 1663, 1458, 1079 cm1; UV (MeOH) kmax (loge) 276 (2.70), 229 (3.29), 203 (3.47) nm; 1H NMR and 13 C NMR data, see Tables 1 and 2; HRCIMS, m/z: 335.2224 ([M + H]+) (calcd. for C20H31O4, 335.2222). 3.4.4. ent-18-E-Caeoyloxy-7b-hydroxy-3-cleroden-15-oic acid (4) 20 Colourless oil; aD 29 (c 0.165, MeOH); IR (CHCl3) mmax 2926, 1660, 1610, 1418, 1279, 1165, 1032, 797 cm1; UV (MeOH) kmax (loge) 329 (4.02), 295 (3.91), 245 (3.81), 218 (3.97), 204 (4.02) nm; 1H NMR and 13C NMR data, see Tables 1 and 2; HRFABMS, m/z: 523.2687 ([M + Na]+) (calcd. for C29H40O7Na, 523.2672). 3.4.5. (13S)-ent-18-E-Coumaroyloxy-8(17)-labden-15-oic acid (5) 20 Colourless oil: aD 2:4 (c 0.365, MeOH); IR (CHCl3) mmax 2928, 1663, 1587, 1416, 1158, 1024 cm1; UV (MeOH) kmax (loge) 312 (3.70), 296 (3.69), 226 (3.58), 203 (3.83) nm; 1 H NMR and 13C NMR data, see Tables 1 and 3; HRCIMS, m/z: 486.3227 ([M + NH4]+) (calcd. for C29H44O5N, 486.3219). 3.4.6. ent-18-E-Caeoyloxy-8(17)-labden-15-oic acid (6) 20 Colourless oil; aD 8:9 (c 0.373, MeOH); IR (CHCl3) mmax 2371, 1640, 1270, 1165, 1038 cm1; UV (MeOH) kmax (loge) 327 (3.93), 295 (3.84), 245 (3.76), 220 (3.96), 203 (4.15) nm; 1H NMR and 13C NMR data, see Tables 1 and 3; HRFABMS, m/z: 507.2725 ([M + Na]+) (calcd. for C29H40O6Na, 507.2723). 3.4.7. ent-15-E-Caeoyloxy-8(17)-labden-18-oic acid (7) 20 Colourless oil; aD 21 (c 0.21, MeOH); IR (CHCl3) mmax 2371, 1659, 1273, 1170, 1026 cm1; UV (MeOH) kmax (loge) 328 (3.78), 294 (3.84), 245 (3.64), 220 (3.83), 203 (4.00) nm; 1H NMR and 13C NMR data, see Tables 1 and 3; HRFABMS, m/z: 507.2739 ([M + Na]+) (calcd. for C29H40O6Na, 507.2723). 3.4.8. ent-15-Hydroxy-8(17)-labden-19-oic acid (13) 20 Colourless oil; aD 23 (c 0.165, CHCl3), no lit. value 1 available. H NMR and 13C NMR spectral data consistent with the literature values (Zdero et al., 1991a). 3.4.9. ent-18-Hydroxy-8(17)-labden-15-oic acid (14) Colourless oil; a20 34 (c 0.40, CHCl3); lit. a20 37 (c D D 2.3, CHCl3) as methyl ester (Hugel et al., 1966). 1H NMR and 13C NMR spectral data consistent with the literature values (Zdero et al., 1991b). 3.4.10. ent-18-Hydroxy-3-cleroden-15-oic acid (15) 20 Colourless oil; aD 28:4 (c 0.25, CHCl3), no lit. value 1 available. H NMR and 13C NMR spectral data consistent with the literature values (Tsichritzis and Jakupovic, 1990).
3.4.11. Preparation of the (R)- and (S)-PGME amide derivatives of 1 and 5 (R)- and (S)-phenylglycine methyl ester were obtained by esterication of phenylglycine (Nagai and Kusumi, 1995). Two portions of compound 1 (each 4 mg; 0.0124 mmol) were separately stirred with (R)-PGME (3.1 mg; 0.0155 mmol) and (S)-PGME (3.1 mg, 0.0155 mmol) in dry DMF (0.5 ml). To these solutions were added successively 1H-benzotriazol-1-yloxytripyrrolidinophosphonium hexauorophosphate (6.5 mg, 0.0155 mmol), 1-hydroxybenzotriazole (2.4 mg, 0.0155 mmol) and N-methylmorpholine (5 ll) at 0 C. After stirring at room temperature for 5 h, ethyl acetate was added to the reaction mixture which was successively washed with HCl 0.1 N, saturated NaHCO3 solution and brine. The organic layers were dried over Na2SO4 and concentrated under vacuum. Purication on silica gel column chromatography (cyclohexaneEtOAc 75:25) aorded 3 mg of compound 1r and 1s in 52% yield. 1H NMR data of the (R)-PGME amide derivative (1r): (400 MHz, CDCl3) d 7.3417.312 (5H, m, Ar-H), 6.358 (1H, d, J = 7.2 Hz, NH), 5.570 (1H, d, J = 7.2 Hz, H-a), 5.113 (1H, d, J = 1.4 Hz, H-3), 3.971 (1H, q, J = 3.3 Hz, H-7), 3.708 (3H, s, CO2CH3), 2.249 (1H, dd, J = 5.7, 13.9 Hz, H-14a), 2.043 (1H, dd, J = 2.7, 14.2 Hz, H-6a), 1.952 (1H, m, H-2a), 1.930 (1H, dd, J = 5.3, 13.9 Hz, H-14b), 1.872 (1H, m, H-2b), 1.806 (1H, m, H-13), 1.590 (3H, d, J = 1.3 Hz, CH3-18), 1.476 (3H, m, 2H-1, H-8), 1.312 (2H, m, H-6b, H-10), 1.255 (3H, s, CH3-19), 1.233 (2H, m, 2H-11), 1.072 (1H, m, H-12a), 0.953 (3H, d, J = 7.3 Hz, CH3-17), 0.949 (4H, br s, H12b, CH3-20), 0.859 (3H, d, J = 6.5 Hz, CH3-16); 1H NMR data of the (S)-PGME amide derivative (1s): (400 MHz, CDCl3) d 7.3447.320 (5H, m, Ar-H), 6.327 (1H, d, J = 7.1 Hz, NH), 5.570 (1H, d, J = 7.1 Hz, H-a), 5.099 (1H, d, J = 1.4 Hz, H-3), 3.957 (1H, q, J = 3.3 Hz, H-7), 3.713 (3H, s, CO2CH3), 2.213 (1H, dd, J = 5.7, 14.1 Hz, H-14a), 2.037 (1H, dd, J = 2.7, 14.1 Hz, H-6a), 1.952 (3H, m, H-2a, H-14b), 1.864 (1H, m, H-2b), 1.824 (1H, m, H-13), 1.572 (3H, d, J = 1.2 Hz, CH3-18), 1.485 (2H, m, 2H-1), 1.476 (1H, m, H-8), 1.313 (2H, m, H-6b, H-10), 1.248 (3H, s, CH3-19), 1.212 (2H, m, 2H-11), 1.051 (1H, m, H-12a), 0.934 (7H, m, H-12b, CH3-17, CH3-20), 0.917 (3H, d, J = 6.6 Hz, CH3-16). 3.4.12. Preparation of the (R)- and (S)-PGME amide derivatives of 5 Compound 5 (each 6 mg) was condensed with (R)- and (S)-PGME under the same conditions above to yield after purication on silica gel column chromatography (cyclohexaneEtOAc 80:20) 3 mg of 5r and 5s. 1H NMR data of the (R)-PGME amide derivative (5r): (400 MHz, CDCl3) d 7.3557.326 (5H, m, Ar-H), 7.273 (2H, d, J = 8.4 Hz, H2 0 , H-6 0 ), 6.939 (1H, d, J = 12.4 Hz, H-7 0 ), 6.774 (2H, d, J = 8.6 Hz, H-3 0 , H-5 0 ), 6.517 (1H, d, J = 6.8 Hz, NH), 5.768 (1H, d, J = 12.4 Hz, H-8 0 ), 5.567 (1H, d, J = 7.0 Hz, H-a), 4.713 (1H, br s, H-17a), 4.363 (1H, br s,
451
H-17b), 3.795 (1H, d, J = 11.4 Hz, H-18a), 3.716 (3H, s, CO2CH3), 3.553 (1H, d, J = 11.4 Hz, H-18b), 2.199 (1H, m, H-7a), 2.152 (2H, m, 2H-14), 1.855 (1H, m, H-13), 1.630 (1H, m, H-7b), 1.579 (1H, m, H-1a), 1.480 (1H, m, H-3a), 1.420 (2H, m, 2H-2), 1.350 (1H, m, H-6a), 1.257 (2H, m, 2H-11), 1.176 (1H, m, H-6b), 1.154 (3H, m, H-9, 2H-12), 1.132 (1H, m, H-3b), 0.896 (1H, m, H-1b), 0.876 (1H, m, H-5), 0.860 (3H, d, J = 6.5 Hz, CH3-16), 0.686 (3H, s, CH3-19), 0.563 (3H, s, CH3-20); 1H NMR data of the (S)-PGME amide derivative (5s): (400 MHz, CDCl3) d 7.3607.321 (5H, m, Ar-H), 7.113 (2H, d, J = 8.3 Hz, H-2 0 , H-6 0 ), 6.922 (1H, d, J = 12.5 Hz, H-7 0 ), 6.534 (2H, d, J = 8.6 Hz, H-3 0 , H-5 0 ), 6.647 (1H, d, J = 6.7 Hz, NH), 5.754 (1H, d, J = 12.5 Hz, H-8 0 ), 5.580 (1H, d, J = 6.8 Hz, H-a), 4.691 (1H, br s, H-17a), 4.328 (1H, br s, H-17b), 3.754 (1H, d, J = 11.4 Hz, H-18a), 3.722 (3H, s, CO2CH3), 3.539 (1H, d, J = 11.4 Hz, H-18b), 2.314 (1H, m, H-14a), 2.277 (1H, m, H-14b), 2.125 (2H, m, 2H7), 1.873 (1H, m, H-13), 1.554 (2H, m, 2H-1), 1.482 (3H, m, 2H-2, H-3a), 1.267 (2H, m, 2H-6), 1.233 (2H, m, 2H11), 1.089 (2H, m, 2H-12), 1.033 (1H, m, H-9), 0.913 (3H, d, J = 6.5 Hz, CH3-16), 0.809 (1H, m, H-5), 0.669 (3H, s, CH3-19), 0.548 (3H, s, CH3-20). Acknowledgment This work was supported by a grant from the pro` gramme VIH-Pal, Ministere Francais de la Recherche.
References
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Triphyophyllum peltatum and Ancistrocladus heyneanus, antimalarial activity and crystal structure of the benzyl ester. Planta Med. 63, 255 257. Chavez, M.I., Delgado, G., 1994. Isolation and relay synthesis of 11ahydroperoxy-diacetylhederagenin, a novel triterpenoid derivative from Serjania triquetra (Sapindaceae). Biogenetic implications. Tetrahedron 50, 38693878. Dehmlow, E.V., Ree, T.V., Jakupovic, J., Take, E., Kunsting, H., 1998. Notes on activity tests and constituents of two supposed medicinal plants from South Africa, Englerophytum magalismontanum and Diospyros lycioides des. Subsp. Sericea. J. Chem. Res. (S) 5, 252253. Frappier, F., Jossang, A., Soudon, J., Calvo, F., Rasoanaivo, P., Ratsimamanga-Urverg, S., Saez, J., Schrevel, J., Grellier, P., 1996. Bisbenzylisoquinolines as modulators of chloroquine resistance in Plasmodium falciparum and multidrug resistance in tumor cells. Antimicrob. Agents Chemother. 40, 14761481. Frederiksen, L.B., Damtoft, S., Jensen, S.R., 1999. Biosynthesis of iridoids lacking C-10 and the chemotaxonomic implications of their distribution. Phytochemistry 52, 14091420. Gaydou, E.M., Bianchini, J.-P., 1978. Etudes de composes avoniques. Bull. Soc. Chim. Fr. II, 4347. Houghton, P.J., Lian, L.M., 1986. Triterpenoids from Desfontainia spinosa. Phytochemistry 25, 19391944. Hugel, G., Oehlschlager, A.C., Ourisson, G., 1966. The structure and stereochemistry of diterpenes from Trachylobium verrucosum. Tetrahedron 22 (Suppl. 8), 203216. Jensen, S.R., Ravnkilde, L., Schripsema, J., 1998. Unedoside derivatives in Nuxia and their biosynthesis. Phytochemistry 47, 10071011. Mongkolsuk, S., Dean, F.M., 1964. Pinostrobin and alpinetin from Kaempferia pandurata. J. Chem. Soc., 46544655. Nagai, Y., Kusumi, T., 1995. New chiral anisotropic reagents for determining the absolute conguration of carboxylic acids. Tetrahedron Lett. 36, 18531856. Rasoanaivo, P., Ramanitrahasimbola, D., Rafatro, H., Rakotondramanana, D., Robijaona, B., Rakotozafy, A., Ratsimamanga-Urverg, S., Labaed, M., Grellier, P., Allorge, L., Mambu, L., Frappier, F., 2004. Screening extracts of Madagascan plants in search of antiplasmodial compounds. Phytother. Res. 18, 742747. Sairaanpour, M., Bahreininejad, B., Witt, M., Ziegler, H.L., Jaroszewski, J.W., Staerk, D., 2003. Terpenoids of Salvia hydrangea: two new, rearranged 20-norabietanes and the eect of oleanolic acid on erythrocyte membranes. Planta Med. 69, 846850. Tsichritzis, F., Jakupovic, J., 1990. Diterpenes and other constituents from Relhania species. Phytochemistry 29, 31733187. Wijeratne, D.B.T., Kumar, V., Sultanbawa, M.U.S., 1981. 3-Oxolup20(29)-en-30-al, a new lupane from Gymnosporia emarginata (Celastraceae). J. Chem. Soc., Perkin Trans. I, 27242726. Yabuuchi, T., Kusumi, T., 2000. Phenylglycine methyl ester, a useful tool for absolute conguration determination of various chiral carboxylic acids. J. Org. Chem. 65, 397404. Zdero, C., Bohlmann, F., King, R.M., 1991a. Diterpenes and norditerpenes from the Aristeguetia group. Phytochemistry 30, 29913000. Zdero, C., Bohlmann, F., King, R.M., 1991b. Guaianolides and labdanes from Brickellia species. Phytochemistry 30, 15911595. Ziegler, H.L., Staerk, D., Christensen, J., Hviid, L., Hagerstrand, H., Jaroszewski, J.W., 2002. In vitro Plasmodium falciparum drug sensitivity assay: inhibition of parasite growth by incorporation of stomatocytogenic amphiphiles into the erythrocyte membrane. Antimicrob. Agents Chemother. 46, 14411446.
Rings B,D-seco limonoids from the leaves of Swietenia mahogani

Samir A.M. Abdelgaleil a, Matsumi Doe b, Yoshiki Morimoto b, Munehiro Nakatani
c
c,*
Department of Pesticide Chemistry, Faculty of Agriculture, Alexandria University, Alexandria, Egypt Analytical Division, Graduate School of Science, Osaka City University, 3-3-7 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan Department of Chemistry and Bioscience, Faculty of Science, Kagoshima University, 1-21-35 Korimoto, Kagoshima 890-0065, Japan
b
Received 17 March 2005; received in revised form 6 October 2005 Available online 26 January 2006
Abstract Seven phragmalin limonoids of swietephragmins A-G, and two other dierent types of 2-hydroxy-3-O-tigloylswietenolide and deacetylsecomahoganin, were isolated along with three known limonoids from the leaves of Swietenia mahogani (Meliaceae). Their structures were determined by spectroscopic methods. 2005 Elsevier Ltd. All rights reserved.
Keywords: Swietenia mahogani; Meliaceae; Phragmalins; Swietephragmins A-G; Mexicanolide; 2-Hydroxy-3-O-tigloylswietenolide; Rings B,D-seco limonoid; Deacetylsecomahoganin
1. Introduction Swietenia mohogani JACQ. is a valuable meliaceous timber tree closely related to the African genus Khaya and one of the most popular traditional medicines in Africa. The decoction of the bark of these mahoganies is extensively used as febrifuge, which could be associated with its use as an antimalarial drug (Nagalakshmi et al., 2001). This genus is one of the main sources of rings B,D-seco limonoids of mexicanolides and phragmalins. Ever since a mexicanolide was isolated (Adeoye and Bekoe, 1965), many limonoids having bicyclo[3,3,1] and tricyclo[3.3.1.1] ring systems have been reported (Taylor, 1984). In our continuing search for limonoid antifeedants from the family Meliaceae, we have reported the isolation of ring D opened phragmalin-type limonoids from the stem bark of Swietenia mahogani, collected at Alexandria, Egypt (Saad et al., 2003). A subsequent study of the leaves isolated seven new phragmalins possessing an orthoester group at 8,9,30-position, named swietephragmins A (1)
F (6) and G (9), together with two new dierent type rings B,D-seco limonoids, 2-hydroxy-3-O-tigloylswietenolide (7) and deacetylsecomahoganin (8), along with three known limonoids of methyl 6-hydroxyangolensate (10) (Adesogan and Taylor, 1968), swietemahonin G (11) (Kadota et al., 1990b) and 7-deacetoxy-7-oxogedunin (12) (Kadota et al., 1990a). We describe herein the isolation, and structural elucidation of these new limonoids. The antifeedant activity of the isolated compounds was also described briey.
2. Results and discussion After partition with hexane and methylene chloride of the ether extract of the leaves dissolved in H2OMeOH (1:1), the methylene chloride layer was subjected to chromatographic separation using SiO2 with MeOHCH2Cl2 as an eluant system, with the resulting limonoid fraction divided into four fractions by SiO2 rechromatography with hexaneAcOEt (1:1) for elution. The rst limonoid fraction was puried by a combination of TLC and reversed phase HPLC to give nine new compounds, 19, and three known compounds, 1012.
Corresponding author. Tel.: +81 99 285 8114; fax: +81 99 285 8117. E-mail address: nakat@mail.sci.kagoshima-u.ac.jp (M. Nakatani).
S.A.M. Abdelgaleil et al. / Phytochemistry 67 (2006) 452458

O
453
21 18 12 13 14 8 15
23 22
O Tig =
20 17
19
11 9
O
16
MeO2C R2 H
6
5 29 1 3
O OH
30 2
O R O
3
28
OR1
OTig 1: R1 = Ac, R 2= H, R3 = CH(CH3)2
2: R1 = Ac, R 2 = H, R3 = CH(CH 3)CH2CH3 1 3: R = H, R 2 = H, R3 = CH(CH 3)CH 2 3 CH 1 4: R = H, R 2 = H, R 3= CH(CH 3) 2 5: R1 = H, R 2 = OH, R 3 = CH(CH3 )CH2CH3 6: R1 = H, R 2 = H, R3 = CH 2 3 CH 9: R1 = H, R 2 = H, R3 = CH 3 O
MeO2C H
O OH
9
6 5 3 1
HO
8 30
R OTig
7: R = OH 13: R = H
O
30 1 9
O O
H
3 5 7
15
CO2Me CH2OR
8: R = H 14: R = Ac 15: R= -D-glu
Swietephragmin A (1) was found to possess a molecular formula of C38H46O13 (16 unsaturations) as determined from the HRFAB-MS (m/z: 711.3009[M + 1]+, D 0.8 mmu) and 13C NMR spectrum. The IR spectrum revealed absorption bands for hydroxyl (36003200 cm1) saturated (1740 cm1) and unsaturated ester carbonyl (1724 and 1715 sh cm1) groups. The UV spectrum also indicated the presence of an a,b-unsaturated ester group at 215 nm. From the 1H and 13C NMR spectra, it was clear that eight of the elements of unsaturation were present as double bonds: four carboncarbon double bonds (one furan ring) and four CO (as esters). Therefore, the molecule is octacyclic. The presence of a b-furyl group was recognized together with each one of acetyl, tigloyl, methoxy and hydroxyl groups. All protons directly bonded with carbon atoms were assigned by analysis of the HMQC spectrum. From the subsequent 2D NMR spectroscopic studies of the 1H1H
COSY, HMBC and NOESY spectra, it was strongly suggested that 1 was a phragmalin limonoid (Tables 1 and 2). Thus, a characteristic low-eld singlet at d 5.71 due to H-17 was observed and the H2-6 protons at d 2.34 (dd, J = 16.4 and 11.7 Hz) and 2.40 (br d, J = 16.4 Hz), attached to a methylene carbon adjacent to an ester carbonyl, were coupled with the H-5 broad doublet proton at d 2.48 (br d, J = 11.7 Hz). These observations strongly suggested that 1 was a rings B,D-seco limonoid. In addition to this knowledge, the absence of two tertiary methyl sig~ nals due to 4b-Me (Me-29) and 8b-Me (Me-30) groups in the basic limonoid skeleton, and the presence of two proton resonances at d 1.73 and 1.94 (each d, J = 11.5 Hz) assigned to the 29-methylene group, strongly supported that 1 had a tricyclo [3.3.12,10.11,4] decane ring system. The presence of an orthoester group in 1 was also presumed from the characteristic orthocarbon resonance of d 122.9. Almost all of the phragmalins isolated so far have been reported to be 1,8,9- or 8,9,14-orthoacetates except for some exceptions (Olmo et al., 1997; Nakatani et al., 2001; Saad et al., 2004), and their orthocarbon signals have been observed around d 119 (Taylor, 1984). The 1H and 13C NMR spectra due to the tricyclodecane ring of 1 were similar to those of swietenialide A (Saad et al., 2003) isolated from the stem bark of the same plant. All of the carbons due to the ring system, including Me-19 and 28 were, respectively, assigned by long-range CH correlations (Fig. 1) of a broad doublet (J = 11.7 Hz, H-5) and two methine singlets at d 5.34 (H-3) and 5.44 (H-30) with the corresponding carbon signals. A W-type long range coupling between the H-5 signal and one (Pro-S) of 29-methylene signals observed at d 1.73 and the NOE correlation of the other H-29 signal at d 1.94 (Pro-R) with the 10a-Me (Me-19), conrmed the ring structure and their relative stereochemistry. The presence of 3-tigloyl and 1OH groups was elucidated by HMBC correlations of the H-3 and OH signals with the tigloyl carbonyl carbon and C-1 signals, respectively. On the other hand, compound 1 showed the presence of one trisubstituted double bond at dC151.8 (s, C-14) and 123.4 (d, C-15) conjugated with a lactone carbonyl. An olenic proton at d 6.32 (s, H-15) correlating to the carbon at 163.6 (C-16) in the HMBC spectrum, showed additional correlations with two quaternary carbons of C-8 and C-13. In the HMBC correlations (Fig. 1), the H2-11, H2-12 and H-17 signals correlated with C-8, C-9 and C-11C-17 resonances, and characterized the second fragment of C and D rings including 13-Me (Me-18) and a furan ring. Finally, the orthoester moiety, identied as an isobutylate group, was located at the positions 8,9,30 by the HMBC correlation of the H-30 resonance with the orthocarbon signal at d 122.9 and a consideration of the molecular model. This was supported further by the NOEs of the acetyl signal of 2-OAc with the H-3 and H-30 signals. Stereochemistry of 1 was elucidated by the consideration of NOE correlations (Fig. 2) using a molecular model. Strong cross-peaks of H-5 with H-12b and H-17, and
454
Table 1 1 H NMR spectroscopic data for swietephragmins AF (16) and G (9) No. 3 5 6 11a 11b 12a 12b 15a 17 18 19 21 22 23 28 29pro-R 29pro-S 30 OMe 2-OAc 1-OH 2-OH 6-OH Tigloyl 30 40 2 0 -Me 1 5.34 2.48 2.34 2.40 1.87 2.17 1.16 1.57 6.32 5.71 1.36 1.30 7.48 6.46 7.42 0.68 1.94 1.73 5.44 3.68 2.19 3.44 s br d (11.7) dd (16.4, 11.7) br d (16.4) ddd (15.8, 15.0, 4.0) dt (15.0, 3.5) dt (14.1, 3.5) ddd (15.8, 14.1, 3.2) s s s s br s br d (1.1) br t (1.5) s d (11.5) br d (11.5) s s s s 2 5.30 2.48 2.34 2.40 1.87 2.11 1.16 1.56 6.39 5.71 1.35 1.29 7.48 6.46 7.42 0.73 1.94 1.72 5.43 3.68 2.19 3.45 s br d (11.5) dd (16.5, 11.7) br d (16.3) dt (4.2, 15.4) dt (15.0, 3.0) dt (14.1, 3.7) dt (3.7, 14.1) s s s s br s br d (1.1) br t (1.3) s d (11.6) br d (11.6) s s s s 3 4.83 2.55 2.38 2.43 1.86 2.08 1.22 1.51 5.97 5.68 1.33 1.28 7.47 6.44 7.42 0.82 1.82 1.78 4.49 3.72 s br d (11.5) dd (16.6, 11.5) dd (16.6, 2.0) m dt (14.9, 3.2) dt (14.3, 3.6) br dt (2.7, 14.7) s s s s br s br d (1.4) t (1.5) s d (11.4) br d (11.4) s s 4 4.83 2.55 2.38 2.44 1.85 2.08 1.13 1.51 5.97 5.68 1.33 1.29 7.47 6.44 7.41 0.82 1.83 1.78 4.50 3.72 s br d (11.6) dd (16.6, 11.6) dd (16.6, 1.8) dt (3.9, 14.9) dt (14.9, 3.4) dt (14.5, 3.7) br dt (3.6, 14.8) s s s s br s br d (1.1) t (1.5) s d (11.1) br d (11.1) s s 5 4.73 s 2.63 br s 4.57 br s 1.89 2.21 1.21 1.58 5.94 5.52 1.29 1.54 7.47 6.40 7.43 0.95 2.27 1.73 4.47 3.85 2.17 3.53 3.57 2.80 dt (3.9, 15.5) dt (14.9, 3.2) dt (13.8, 3.3) br dt (2.9, 14.5) s s s s br s br s br s s d (10.7) d (10.7) s s s s s s 6 4.83 2.55 2.38 2.43 1.89 2.09 1.13 1.50 5.97 5.69 1.34 1.28 7.47 6.44 7.41 0.82 1.83 1.78 4.51 3.72 s br d (11.5) dd (16.5, 11.5) br d (16.5) dt (3.7, 14.7) dt (14.9, 1.9) ddd (14.3, 3.7, 1.9) br dt (2.0, 14.5) s s s s br s br s br s s d (11.3) br d (11.3) s s 9 4.83 2.54 2.38 2.43 1.91 2.10 1.13 1.50 5.97 5.69 1.35 1.28 7.47 6.44 7.41 0.82 1.83 1.78 4.51 3.72 s br d (11.4) dd (16.6, 11.4) br d (16.6) dt (3.6, 14.7) dt (14.9, 2.0) dt (14.3, 3.0) br t (14.5) s s s s br s br s br s s d (11.5) br d (11.5) s s
3.49 s 3.55 s
3.49 s 3.56 s
3.50 s 3.56 s
3.50 s 3.56 s
6.62 qq (6.9, 1.4) 1.71 br d (6.9) 1.87 br s
6.62 qq (6.9, 1.4) 1.71 br d (7.0) 1.87 br s 1.94 m 1.24 1.68 0.93 1.02 m m t (7.5) d (6.9)
6.91 qq (7.1, 1.2) 1.75 br d (7.1) 1.85 br s 1.92 m 1.22 1.71 0.93 1.02 dq (9.6, 7.5) m t (7.5) d (6.9)
6.91 qq (7.0, 1.3) 1.75 dq (7.0, 0.9) 1.85 br s 2.17 quint (7.0) 1.04 d (7.0)
6.75 br q (7.0) 1.74 br d (6.9) 1.85 br s 1.93 m 1.23 1.71 0.93 1.02 m m t (7.5) d (6.9)
6.91 br q (7.0) 1.75 br d (7.0) 1.85 br s 1.96 dq (14.5, 7.6) 1.93 dq (14.5, 7.6) 1.03 t (7.6)
6.91 br q (6.8) 1.75 br d (6.8) 1.85 br s 1.67 s
Orthoesters 20 0 2.19 quint (6.9) 300 -a 300 -b 400 200 -Me 1.04 d (6.9)
1.04 d (6.9)
1.04 d (7.0)
All spectra were measured in CDCl3 at 600 MHz. Chemical shifts are expressed in ppm. J values in parentheses are in Hz.
S.A.M. Abdelgaleil et al. / Phytochemistry 67 (2006) 452458 Table 2 13 C NMR spectroscopic data for swietephragmis AF (16) and G (9) No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 28 29 30 OMe OAc Tiglate 10 20 30 40 2 0 -Me 1 84.7 84.0 84.8 44.7 39.6 33.7 173.9 83.9 85.7 48.1 26.9 29.3 37.7 151.8 123.4 163.6 79.8 19.2 15.4 119.9 142.0 110.2 143.3 13.8 39.0 73.9 52.1 170.3 21.8 167.9 130.8 136.1 13.5 12.9 2 84.6 84.0 84.7 44.7 39.6 33.7 173.9 83.7 85.7 48.0 26.0 29.3 37.7 151.8 123.4 163.6 79.8 19.2 15.4 119.9 142.0 110.2 143.2 13.8 39.0 73.7 52.1 170.2 21.8 167.9 130.8 136.1 13.5 12.8 123.0 35.4 23.6 13.5 12.8 3 84.6 75.7 86.6 43.7 40.1 33.7 173.9 83.6 86.6 47.2 25.7 29.0 37.7 153.1 122.5 163.2 79.7 19.8 15.5 119.8 142.0 110.1 143.2 14.4 38.5 77.9 52.1 4 84.6 75.7 86.6 43.7 40.1 33.7 173.9 83.8 86.7 47.2 25.7 29.0 37.7 153.0 122.5 163.2 79.7 19.8 15.4 119.8 142.0 110.1 143.2 14.4 38.5 78.0 52.1 5 84.6 75.6 87.5 43.4 45.5 71.6 174.5 83.6 87.0 48.4 25.6 29.4 37.8 153.1 122.1 162.7 80.0 19.8 17.4 119.6 141.4 109.8 143.2 15.6 39.8 77.7 52.3 6 84.7 75.7 86.7 43.7 40.1 33.7 173.9 83.8 86.9 47.3 25.9 29.0 37.7 153.0 122.6 163.1 79.7 19.8 15.4 119.8 142.1 110.1 143.2 14.4 38.6 78.1 52.1 9 84.6 75.7 86.7 43.8 40.2 33.8 173.6 84.0 87.2 47.3 26.2 29.2 37.9 152.7 122.5 162.8 79.7 20.1 15.6 119.4 141.9 110.0 143.1 14.6 38.7 78.1 52.2
7 6 22 23
455
H O H 21 H CO2Me H H H 12
11 9 17 20
CH2
19
Me
28
Me
H
5 10
H O H
13 16 14 18
Me
4 29 1 3
Tig OO
30 2
O
8 1'
15
HO
H OAc
O H
O H R
Fig. 2. Signicant NOE correlations in 1.
168.1 130.0 139.9 14.2 12.3 122.9 35.5 23.6 11.4 13.1
168.2 130.0 139.9 14.2 12.3 122.8 28.9 16.6 16.6
167.7 130.2 139.0 14.4 12.5 122.5 35.5 23.7 11.6 13.3
168.2 130.1 139.9 14.2 12.3 121.3 23.2 7.6
167.9 129.9 139.8 14.4 12.4 119.7 16.6
Orthoester 122.9 100 200 28.9 16.6 300 400 200 -Me 16.6
13
C
H H
H H
MeO2C H
Me
H
O
Me H H H H Me H
OTig O OH O O O
H CH(CH3)2
OCOCH3
Fig. 1. Selected HMBC correlations in 1.
H-12b with H-17 in the NOESY spectrum indicated a b orientation for these three protons. In addition to a cross-peak between H-30 and H-15, a NOE correlation of H-11b with H3-19 (10a-Me) indicated that 1 was present in a folded conformation containing a quasi chair C ring as shown in Fig. 2. The NOE observation of 2-OAc with H-3 and H-30 also supported this structure assignment. Thus, swietephragmin A was identied as the 8,9,30-ortho-isobutylate (1) of methyl 2-acetoxy-3b-tigloyloxy-1,8,9,30-tetrahydroxy-[3.3. 12,10. 11,4]-tricyclomeliac-16,17-lactonic-14 (15)-en-7-oate. Swietephragmin B (2) was obtained as a white amorphous powder. The molecular formula (C39H48O13) was determined by the HRFAB-MS (m/z: 725.3154 [M + 1]+ , D 1.9 mmu) and NMR spectra, which suggested the presence of one additional CH2 unit in 2 compared to 1. The IR and NMR (Tables 1 and 2) spectroscopic data of 2 were extremely similar to those of 1, with the only dierence being observed in the change of the orthoester group to a 2-methylbutanoate moiety in 2. The 8,9,30-orthoester bonding in 2 was also conrmed by both the similar HMBC correlation between the H-30 signal and the orthoester carbon resonance observed at d 123.0, as well as the NOEs of the 2-OAc signal with the 1-OH and H-3 resonances. Swietephragmin C (3) exhibited the molecular formula of C37H46O12 by HRFAB-MS (m/z: 683.3059 [M + 1]+, D 0.8 mmu) and NMR spectroscopic data. The NMR spectra were similar to those of 2 including the presence of tigloyl and 2-methylbutanoyl orthoester groups, except for the change of an acetoxy group to a hydroxyl group. In the HMBC spectrum, two OH groups were correlated to C-1, C-2 and C-29 and to C-1, C-2 and C-3, respectively, to elucidate clearly the structure having the 8,9,30-orthoester group. Although the NOE correlations in 3 resembled those in 1 and 2 to suggest not so large conformation change in 3, the H-30 signal was observed
456
S.A.M. Abdelgaleil et al. / Phytochemistry 67 (2006) 452458 Table 3 1 H and 13C NMR spectroscopic data for compounds 7 and 8 No. 7 dH 1 2 3 4 5 6 7 8 9 10 11a 11b 12a 12b 13 14 15a 15b 16 17 18 19 20 21 22 23 28 29 30a 30b OMe 2-OH 6-OH Tig 10 20 30 40 2 0 -Me dC 217.2 77.9 87.5 39.7 45.0 73.1 175.3 126.4 53.0 52.5 18.8 29.6 38.2 132.8 33.2 169.0 80.9 17.7 17.7 120.7 141.1 109.7 143.2 23.3 22.2 44.7 53.2 8 dH 6.67 d (10.6) 5.93 d (10.6) dC 152.6 126.5 203.6 44.9 51.5 61.1 176.7 43.1 43.1 50.8 21.5 32.5 37.8 68.2 51.4 166.9 78.4 19.3 18.2 120.0 141.2 109.9 143.3 24.2 23.2 15.1 53.3
at a high-eld of d 4.49 compared to d 5.44 and 5.43 in 1 and 2. This high-eld shift was attributed to an anisotropic eect of the carbonoxygen double bond of the 3-tigloyl group xed by a seven membered hydrogen bonding with the 2-OH group. The molecular formula of swietephragmin D (4) was determined as C36H44O12 by HRFAB-MS (m/z: 669.2914 [M + 1]+, D +0.3 mmu) and NMR data. The 1H and 13C NMR spectroscopic data (Tables 1 and 2) were extremely similar to those of 3 except for the replacement of 2-methylbutanoyl orthoester in 3 by 2-methylpropanoate in 4. Swietephragmin E (5) was shown to have the molecular formula C37H46O13 by HRFAB-MS (m/z: 699.3021 [M + 1]+, D +0.6 mmu). Although the NMR spectra of 5 were also similar to those of 3 and 4, it showed the presence of an additional hydroxyl group. The structure of 5 as a 6-hydroxyl derivative of 3 was readily elucidated from that of a hydroxymethine signal at d 4.57 (H-6) weakly coupled to an OH resonance at d 2.80 and showed a HMBC correlation with an ester carbonyl carbon at d 174.5 (C-7). The R conguration at C-6 was inferred from that of some mexicanolides, swietenins and swietemahonins (Connolly et al., 1965; Kadota et al., 1990a,b; Saad et al., 2004) isolated from the same plant S. mahogani. This was supported by the NOE correlations observed between the H-6 signal and the H-5, H-12b and 10-Me (Me-19) resonances, the 6-OH signal and the H-28Pro-S and 4a-Me (Me-28) resonances, and the 7-carboxymethyl signal and the H-17 and tigloyl 3 0 -H and 3 0 -Me resonances. The latter implied that the 7-CO2Me group was oriented to the same b-side as H-17 and 3-tigloyl of the molecule. The H-28Pro-S signal showed a W-type long range coupling with the H-5 signal and a strong NOE with the H-3a resonance, which also accounted well for the stereochemistry of 5. The structure of swietephragmin F (6), C35H42O12; HRFAB-MS (m/z: 655.2736[M + 1]+, D 1.9 mmu) was readily deduced from the spectroscopic data. The 1H and 13 C NMR spectra were very similar to those of compounds 3 and 4 except for the change of the orthoester groups to an orthopropyonate moiety in 6. The molecular formula of 7 (2-hydroxy-3-O-tigloylswietenolide) was determined to be C32H40O10 by HRFABMS (m/z: 585.2673 [M + 1]+, D 2.6 mmu). The UV and IR spectra showed similar absorption bands to those of the phragmalins, (1)(6), and the NMR spectra (Table 3) suggested a mexicanolide structure for 7. Thus the presence of four tertiary methyls, due to the basic limonoid skeleton, and one methyl ester moiety was observed along with each keto and lactonic carbonyl and tigloyl groups and two hydroxyl groups. The 1H NMR spectrum resembled 3-Otigloylswietenolide (13) isolated from the same species (Kadota et al., 1990) except for the presence of an additional hydroxyl group in 7. The presence of C-8/C-14 double bond was elucidated by HMBC correlations of the H-15b and H-30 signals with the C-8 and C-14 resonances. An additional OH group at d 4.14 was located in C-2 by
4.78 s 3.30 br s 4.54 br s
1.94 br t (4.7) 3.94 m, 4.11 m
2.10 m 1.80 1.90 1.19 1.74 m m ddd (15.0, 10.3, 3.1) dd (15.0, 3.1)
3.32 d (11.1) 1.57 1.42 1.45 1.77 m m m m
3.26 br d (21.3) 3.60 d (21.3) 5.41 s 0.99 s 1.53 s 6.40 7.41 7.43 0.84 1.05 1.76 3.04 3.86 4.14 2.81 br s br s br t (1.4) s s br d (14.8) d (14.8) s br s s
3.67 s
5.42 s 1.22 s 1.19 s 7.38 6.33 7.38 1.26 1.07 1.25 s br s s s s s
3.80 s 2.16 br s
6.91 br q (7.0) 1.82 br d (7.0) 1.90 br s
167.0 129.2 138.7 14.5 12.4
All spectra were measured in CDCl3 at 600 MHz. Chemical shifts are expressed in ppm. J value in parentheses are in Hz.
HMBC correlations of the OH signal with the C-1, C-2 and C-3 resonances at d 217.2, 77.9 (s) and 87.5 (d). Significant NOE correlations of the H-5 signal with the 4b-Me (28) and the two tigloyl methyl signals and the 10a-Me (19) signal with the H-6 and H-9 resonances claried the relative stereochemistry of these protons in the dicyclo[3.3.1]nonane ring system. Finally, the conguration at C-6 was assumed to be the same R as that of known mexicanolides (Taylor, 1969; Kadota et al., 1990a,b) from the same specimen. The molecular formula of compound 8, 6-O-deacetoxysecomahoganin, was determined to be C27H34O8 by HRFAB-MS (m/z: 487.2327 [M + 1]+, D 0.5 mmu). Compound 8 showed UV and IR absorptions due to a conjugated enone system at kmax 230 nm and mmax 1680 cm1
457
dierent from the above compounds, and the NMR spectrum (Tables 3) also revealed several dierences. The 1H and 13C NMR spectroscopic data showed the presence of six methyls (ve tertiary and one methoxy), three methylenes, nine methines (ve olenic), and nine quaternary carbons (one olenic and one keto and two ester carbonyls). Thus 9 is tetracyclic. The 1H NMR spectrum showed a characteristic H-15 epoxide proton as a singlet at 3.67 and ve methyls due to the basic limonoid skeleton at d 1.07, 1.19, 1.22, 1.25 and 1.26. The presence of the lactonic D ring was also conrmed by the characteristic H-17 singlet at d 5.42. A conjugated enone system at dH 6.67 and 5.93 (each d, J = 10.6 Hz); dC152.6 (d), 126.5 (d) and 203.6, was assigned to ring A by analysis of the HMBC correlations. The H-1 signal at d 6.67 showed correlations with the C-3, C-5, C-10 and C-19 signals. On the other hand, the HMBC and NOESY spectra claried that ring B was opened at C6C7. Thus, the HMBC correlations of the H-9 signal with the C-1, C-5, C-7, C-19 and C-30 resonances, and the NOE correlations of the H-9 signal with the H-5 and H-19 resonances, the H-1 signal with the H-11b and Me-30 resonances, and the H-5 signal with the H-11a resonance, suggested that rings A and C in the molecule were twisted about 90 through the C9C10 bonding in a preferential conformer. These data claried 9 to have the same aglycone moiety as secomahoganin (12) (Kadota et al., 1990b) and khayanoside (13) (Nakatani et al., 2002). The structure of the last compound, swietephragmin G (9), C34H40O12; HRFAB-MS (m/z: 641.2604 [M + 1]+, D 0.6 mmu) was readily elucidated from analysis of the spectroscopic data. The 1H and 13C NMR spectra were very similar to those of compounds 4, 5 and 7 except for the orthoester group being orthoacetate in 9. The antifeedant activity of the isolated compounds (1 12) was tested by a conventional leaf disk method (Wada and Munakata, 1968) against the third-instar larvae of Spodoptera littoralis (Boisd.). All of the compounds except for 7-deacetoxy-7-oxogedunin (12) were active at 1000 ppm, corresponding to a concentration of ca. 20 lg/ leaf-cm2, in which swiemahonin G (11) was most active and swietephragmins 16 and 9, showed moderate activities. Details will be reported together with another biological activities of cytotoxity and antiviral activity against HIV-1 replication in the near future.
3.2. Plant material The leaves of S. mahogani were collected in April 2001 at Alexandria in Egypt. The plant material was identied by Dr. Khaleil Darweish of Alexandria University and a voucher specimen is deposited in the Faculty of Agriculture, Alexandria University. 3.3. Extraction and isolation of compounds 16 Air-dried leaves of S. mahogani (1.9 kg) were extracted with Et2O (15 l) at room temperature for four weeks to give a crude extract (102.2 g). The Et2O extract was suspended in 1 l of H2OMeOH (1:2), fractionated successfully with hexane (3 500 ml) and CH2Cl2(3 500 ml) to give hexane (67.7 g) and CH2Cl2 (32.3 g) extract. The CH2Cl2 extract (10 g) was subjected to SiO2 (500 g) cc with a 010% MeOH/CH2Cl2 gradient eluent to give 50 fractions. The limonoid fractions of 1841 eluted with 2.5% MeOH/ CH2Cl2 were further applied to SiO2 (150 g) with hexane/ AcOEt (1:1) to give 50 fractions, which were combined as needed to give three limonoid and two non-limonoid fractions. The rst fraction (1.4 g) was roughly separated into 13 fractions through HPLC with 25% H2O/MeOH as solvent, followed by TLC separation with 3% MeOH/CH2Cl2 and HPLC purication with 2030% H2O/MeOH to give 1 (31.5 mg), 2 (40 mg), 3 (16 mg), 4 (12.5 mg), 5 (14.5 mg), 6 (9.5 mg), 7 (5 mg), 8 (5.5 mg), 9 (6 mg), 10 (15 mg), 11 (12 mg) and 12 (0.5 mg). 3.3.1. Swietephragmin A (1) White amorphous powder; C38H46O13; HRFAB-MS m/z: 711.3009 [M + 1]+ (calc. 711.3017); UV kmax nm (e): 215 (16,000); IR mmax cm1: 36003200, 1740, 1724, 1715 sh, 1635; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2. 3.3.2. Swietephragmin B (2) White amorphous powder; C39H48O13;HRFAB-MS m/z: 725.3154 [M + 1]+ (calc. 725.3173); UV kmax nm (e): 215 (14,000); IR mmax cm1: 36003300, 17401710; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2. 3.3.3. Swietephragmin C (3) White amorphous powder; C37H46O12;HRFAB-MS m/z: 683.3059 [M + 1]+ (calc. 683.3067); UV kmax nm (e): 215 (14,000); IR mmax cm1: 35503200, 17351710; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2. 3.3.4. Swietephragmin D (4) White amorphous powder; C36H44O12;HRFAB-MS m/z: 669.2914 [M + 1]+ (calc. 669.2911); UV kmax nm (e): 215 (14,000); IR mmax cm1: 35003200, 17351710; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2.
3. Experimental 3.1. General H and 13C NMR spectra were measured at 600 and 150 MHz in CDCl3 on JEOL FX-600 spectrophotometer. IR (KBr) and UV (MeOH) spectra were recorded on JASCO FT/IR 5300 and Shimadzu UV-210A spectrophotometers. HPLC were performed on Waters lBondapak C18 column by using 3565% H2O/MeOH as solvent.
1
458
3.3.5. Swietephragmin E (5) White amorphous powder; C37H46O13;HRFAB-MS m/z: 669.3021 [M + 1]+ (calc. 669.3015); UV kmax nm (e): 215 (16,000); IR mmax cm1: 36003200, 17401710; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2. 3.3.6. Swietephragmin F (6) White amorphous powder; C35H42O12; HRFAB-MS m/z: 655.2736 [M + 1]+ (calc. 655.2755); UV kmax nm (e): 215 (16,000); IR mmax cm1: 36003300, 17401710; for 1 H and 13C NMR spectroscopic data, see Tables 1 and 2. 3.3.7. 3-O-Tigloylswietenolide (7) White amorphous powder; C32H40O10;HRFAB-MS m/z: 585.2673 [M + 1]+ (calc. 585.2699); UV kmax nm (e): 215 (12,000); IR mmax cm1: 35003200, 17451710; for 1 H and 13C NMR spectroscopic data, see Tables 3. 3.3.8. 6-O-Deacetylsecomahoganin (8) White amorphous powder; C27H34O8;HRFAB-MS m/z: 487.2327 [M + 1]+ (calc. 487.2332); UV kmax nm (e): 230 (12,000); IR mmax cm1: 35003300, 1740, 1720, 1680; for 1H and 13C NMR spectroscopic data, see Tables 3. 3.3.9. Swietephragmin G (9) White amorphous powder; C34H40O12;HRFAB-MS m/z: 641.2604 [M + 1]+ (calc. 641.2598); UV kmax nm (e): 215 (16,000); IR mmax cm1: 36003300, 17401710; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2. 3.4. Antifeedant test The antifeeding potential of the isolated compounds was tested three times by a conventional leaf disk method against thrid-instar larvae of S. littoralis. Five leaf disks (diameter 1.2 cm) of Chinese cabbage (Brassica campestris var. chinensis) were immersed in an acetone solution of the sample for 2 s. The treated disks were arranged alternatively with another ve control disks (immersed only in acetone) close to the wall of a Petri dish. Ten larvae were placed in the centre of each Petri dish. The eaten areas of treated and untreated leaf disks were evaluated at appropriate intervals for 310 h. The experiment was terminated
after the larvae had eaten approximately 50% of one of the disks.
Acknowledgements We are grateful to Mr. K. Takezaki, Kagoshima Prefectural Agricultural station, for the supply of the insects.
References
Adeoye, S.A., Bekoe, D.A., 1965. The molecular structure of Cedrela odorata substance. Biol. J. Chem. Soc. Commun., 301302. Adesogan, E.K., Taylor, D.A.H., 1968. Extractives from Khaya senegalensis (Desr.) A. Juss.. J. Chem. Soc. (C), 19741981. Connolly, J.D., Henderson, R., McCrindle, R., Overton, K.H., Bacca, N.S., 1965. Tetranortriterpenoids Part I. The constitution of swietenine. J. Chem. Soc., 69356939. Kadota, S., Marpaung, L., Kikuchi, T., Ekimoto, H., 1990a. Constituents of the seeds of Swietenia mahogani JACQ.I. Isolation, structures, and 1 H- and 13C-nuclear magnetic resonance signal assignments of new tetranortriterpenoids related to swietenine and swietenolides. Chem. Pharm. Bull. 38, 639651. Kadota, S., Marpaung, L., Kikuchi, T., Ekimoto, H., 1990b. Constituents of the seeds of Swietenia mahogani JACQ. II. Structures of swietemahoganin A, B, C, D, E, F, and G and swietemahonolide. Chem. Pharm. Bull. 38, 894901. Nagalakshmi, M.A.H., Thangadurai, D., Muralidara, D., Pullaiah, R.T., 2001. Phytochemical and antimicrobial study of Chukrasia tabularis leaves. Fitoterapia 72, 6264. Nakatani, M., Abdelgaleil, S.A.M., Kurawaki, J., Okamura, H., Iwagawa, T., Doe, M., 2001. Antifeedant rings B and D opened limonoids from Khaya senegalensis. J. Nat. Prod. 64, 12611265. Nakatani, M., Abdelgaleil, S.A.M., Kassem, Sh.M.I., Takezaki, K., Okamura, H., Iwagawa, T., Doe, M., 2002. Three new modied limonoids from Khaya senegalensis. J. Nat. Prod. 65, 12191221. Olmo, L.R.V., da Silva, M.F.G.F., Fo, E.R., Vieira, P.C., Fernandes, J.B., Pinheiro, A.L., Vilela, E.F., 1997. Limonoids from the leaves of Khaya senegalensis. Phytochemistry 44, 11571165. Saad, M.M.G., Iwagawa, T., Doe, M., Nakatani, M., 2003. Swietenialides, novel ring D opened phragmalin limonoid orthoesters from Swietenia mahogani JACQ. Tetrahedron 59, 80278033. Saad, M.M.G., Iwagawa, T., Okamura, H., Doe, M., Nakatani, M., 2004. Three new mexicanolides from the stem bark of Swietenia mahogani JACQ. Heterocycles 63, 389399. Taylor, D.A.H., 1969. Extractives from Swietenia mahogani (L) Jacq. J. Chem. Soc. Chem. Commun., 5859. Taylor, D.A.H., 1984. In: Herz, W., Grisebach, H., Kirby, G.W. (Eds.), Progress in the Chemistry of Organic Natural Products. Springer, New York, pp. 1102. Wada, K., Munakata, K., 1968. Naturally occurring insect control chemicals. J. Agr. Food Chem. 16, 471474.
Flavones and isoavones from the west African Fabaceae Erythrina vogelii
Alain F. Kamdem Wao a,b,c, Philip H. Coombes a, Dulcie A. Mulholland Augustin E. Nkengfack c, Zacharias T. Fomum c
a
a,d,*
Natural Products Research Group, School of Chemistry, University of KwaZulu-Natal, Howard College Campus, 4041, Durban, South Africa b Department of Chemistry, Faculty of Science, University of Douala, P.O. Box 24157 Douala, Cameroon c Department of Organic Chemistry, Faculty of Science, University of Yaounde I, P.O. Box 812 Yaounde, Cameroon d School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, UK Received 18 August 2005; received in revised form 20 September 2005 Available online 17 November 2005
Abstract The CH2Cl2/MeOH extract of the stem bark of Erythrina vogelii (Fabaceae) from Nigeria has yielded two novel isoavones, 7,4 0 -dihydroxy-8-(c,c-dimethylallyl)-200 n-(400 -hydroxyisopropyl)dihydrofurano[100 ,300 :5,6]isoavone (vogelin H) (1) and 7,4 0 -dihydroxy-8-[(2000 n, 3000 -dihydroxy-3000 -methyl)butyl]-200 ,200 -dimethyl-300 ,400 -dehydropyrano[100 ,400 :5,6]isoavone (vogelin I) (2), a novel avone, 7,4 0 -dihydroxy-200 ,200 -dimethyl-300 ,400 -dehydropyrano[100 ,400 :5,6]avone (vogelin J) (3), and eight known avonoids. 2005 Elsevier Ltd. All rights reserved.
Keywords: Erythrina vogelii; Fabaceae; Stem bark; Isolation; Isoavones; Flavones; 7,4 0 -Dihydroxy-8-(c,c-dimethylallyl)-200 n-(400 -hydroxyisopropyl)dihydrofurano[100 ,300 :5,6]isoavone (Vogelin H); 7,4 0 -Dihydroxy-8-[(2000 n,3000 -dihydroxy-3000 -methyl)butyl]-200 ,200 -dimethyl-300 ,400 -dehydropyrano[100 ,400 :5,6]isoavone (Vogelin I); 7,4 0 -Dihydroxy-200 ,200 -dimethyl-300 ,400 -dehydropyrano[100 ,400 :5,6]avone (Vogelin J); 6,8-Diprenylgenistein; 8-Prenylluteone; Warangalone; Scandenone; Auriculatin; 2,3-Dihydroauriculatin; Carpachromene
1. Introduction The genus Erythrina L. (Fabaceae), comprising approximately 130 species of coral trees distributed throughout the tropical and subtropical regions of the world, has been widely studied. More than 340 extractives have been isolated to date, with some 30 of these secondary metabolites variously reported to display antimicrobial (Ingham and Markham, 1980; Kamat et al., 1981; Mitscher et al., 1988a,b; Chacha et al., 2005), antibacterial (Fomum et al., 1986), antifungal (Tahara et al., 1984; Bojase et al., 2001), anti-inammatory (Wandji et al., 1994; Chacha et al., 2005), antiemetic and antitussive (Abbasoglu et al., 1991), and cytotoxic (Hou et al., 2001) properties, and also
Corresponding author. Tel.: +27 31 260 1395; fax: +27 31 260 3091. E-mail addresses: mulholld@ukzn.zc.za, d.mulholland@surrey.ac.uk (D.A. Mulholland). 0031-9422/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2005.09.022
*
to act as phytoalexins (Dagne et al., 1993) and phospholipase A2 inhibitors (Hegde et al., 1997). Previous studies (Atindehou et al., 2002; Queiroz et al., 2002) on the root bark of Erythrina vogelii Hook. from Ivory Coast have yielded vogelins AG, seven new ring B prenylated isoavonoids, and ve known isoavonoids.
2. Results and discussion In continuation (Fomum and Ayafor, 1983; Kamdem Wao, 2000; Njamen et al., 2004) of our studies on species of this genus, we now report on the isolation of two novel isoavones and a novel avone, together with eight known avonoids, from the CH2Cl2/MeOH (1:1) extract of the stem bark of E. vogelii collected in Nigeria. Compound 1 was assigned a molecular formula of C25H26O6 on the basis of HREIMS data, and showed a
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A.F. Kamdem Wao et al. / Phytochemistry 67 (2006) 459463

4 ''' 3''' 2 ''' 1 8a 5'''
kmax at 271 nm in the UV spectrum. A 1H singlet resonance at dH 7.75 and corresponding olenic oxymethine signal at dC 152.5 are characteristic of H-2 and C-2, respectively, in an isoavone skeleton (Mabry et al., 1970; Agrawal, 1989). From a pair of coupled doublets at dH 7.28 and 6.83 (each 2H, J = 8.5 Hz, H-2 0 /H-6 0 , H-3 0 /H-5 0 ); dC 130.2 (each CH, C-2 0 /6 0 ), 115.4 (each CH, C-3 0 /5 0 ) was deduced the presence of a para disubstituted ring B, while that of a hydroxyl group at C-4 0 was established from a peak at m/z 118 in the EIMS. Further inspection of the NMR and mass spectra showed compound 1 to possess a c,c-dimethylallyl (prenyl) substituent (m/z 379 [M 43]+ and 367 [M 55]+; dH 3.29 (2H, d, J = 7.3 Hz, 2H-1000 ), 5.23 (1H, m, H-2000 ), 1.64 (3H, s, 3H-4000 ), 1.73 (3H, s, 3H-5000 ); dC 21.8 (CH2, C-1000 ), 121.4 (CH, C-2000 ), 132.2 (C, C-3000 ), 17.7 (CH3, C-4000 ), 25.5 (CH3, C-5000 )). The presence of a hydroxyisopropyldihydrofuran ring, suggested by characteristic peaks in the EIMS at m/z 363 [M 59]+ and 59 (Tahara et al., 1984, 1989), was supported by the observation of resonances attributable to two non-equivalent geminal methyl groups (dH 1.20/1.30 (each 3H, s, 3H-500 /600 ); dC 25.5/24.0 (each CH3, C-500 /600 )), an oxymethine proton (dH 4.74 (1H, d, J = 8.3 Hz, H-200 ); (dC 91.0 (CH, C-200 )), two diastereotopic protons (dH 3.20 (2H, br d, J = 8.1 Hz, 2H-300 ; dC 27.1 (CH2, C-300 )) and a fully substituted oxygenated carbon (dC 72.2 (C, C-400 )) (Tahara et al., 1989). The absence of signals in the region dH 5.7 6.1, normally attributed to H-6 and H-8, placed both the prenyl group and dihydrofuran ring on ring A, while the lack of both the downeld singlet at dH 12.013.2 and the absorption band at 3280 cm1 in the IR spectrum, characteristic of a chelated OH group at C-5, suggested the latter substituent to be fused at C-5/C-6, and thus, given the normal biosynthetic requirement of a hydroxy group at C-7, that the prenyl group be located at C-8. This placement was supported by correlations in the HMBC spectrum (Fig. 1) between both the H-2 and 2H-1000 resonances and a downeld fully substituted carbon signal at dC 151.8, which can only be C-8a, and by further correlations between both 2H-1000 and 2H-300 to a second downeld fully substituted carbon signal at dC 164.5, which must then be C-7. Correlations between the 2H300 resonance and fully substituted carbon signals at dC 101.8 and 159.7, assigned to C-6 and C-5, respectively, and between H-200 and both of these, conrmed these assignments. Compound 1 is thus the novel 7,4 0 -dihydroxy-8-(c,c-dimethylallyl)-200 n-(400 -hydroxyisopropyl)dihydrofurano[100 ,300 :5,6]isoavone, which we name vogelin H, and is regioisomeric with senegalensin, from E. senegalensis DC. (Wandji et al., 1990), euchrenone b10, from Euchresta horseldii (Lesch.) Bennet (Mizuno et al., 1990), and lupinisoavone G, from Lupinis albus L. (Tahara et al., 1989) and Derris scandens Benth. (Sekine et al., 1999); the structures of the former two compounds, transposed when originally reported, were revised on recent isolation from E. suberosa var glabresence Haines (Tanaka et al., 2001).
OH
1'''
HO
6 3''
O
4
2 1' 6' 5' 2' 3' 4'
HO
4 ''
OH O
4a 2'' 4'' 5 ''
6 ''
O '' 1
OH
3 '' 6''
2 ''
O '' 1
5''
OH
HO
vogelin H (1)
vogelin I (2) OH
HO
O vogelin J (3) OH
H H HO H H H O H HO (1) (2) O OH O O OH H O H HO H H OH O H
Fig. 1. Selected HMBC correlations in vogelins H (1) and I (2).
In similar fashion, compound 2, assigned the molecular formula C25H26O7 by HREIMS, possesses an isoavone skeleton (dH 8.01 (1H, s, H-2); dC 154.2 (CH, C-2)) and a para disubstituted C-4 0 -hydroxy ring B (dH 7.32 and 6.84 (each 2H, J = 8.6 Hz, H-2 0 /6 0 , H-3 0 /5 0 ); dC 130.9 (each CH, C-2 0 /6 0 ), 116.0 (each CH, C-3 0 /5 0 )). However, the signals of both the prenyl group and dihydrofuran ring in vogelin H (1) were absent, having been replaced by those attributable to a 200 ,200 -dimethyl-300 ,400 -dehydropyran ring (dH 5.64 (1H, d, J = 10.0 Hz, H-300 ), 6.67 (1H, d, J = 10.0 Hz, H-400 ), 1.46 (6H, s, 3H-500 /3H-600 ); dC 78.9 (C, C-200 ), 128.7 (CH, C-300 ), 116.1 (CH, C-400 ), 28.5/28.5 (each CH3, C-500 /C-600 )), and a 2n, 3-dihydroxy-3-methylbutyl group (dH 3.60 (1H, m, H-2000 ), 2.88 (1H, m, H-1a000 ), 2.85 (1H, m, H-1b 000 ), 1.28 (6H, s, 3H-4000 /3H-5000 ); dC 25.5 (CH2, C-1000 ), 78.6 (CH, C-2000 ), 73.6 (C, C-3 000 ), 24.4/26.0 (each CH3, C-4000 /C-5000 )) (Takashima and Ohsaki, 2002). As in vogelin H (1), the absence of the resonances normally attributable to H-6 and H-8 placed both of these groups on the A ring. Correlations in the HMBC spectrum (Fig. 1) between both H-2 and 2H-1000 and a downeld fully substituted carbon resonance at dC 156.4, which must be C8a, and between 2H-1000 and a second downeld fully substituted carbon resonance at dC 158.1, which must then be C7, place the 2n,3-dihydroxy-3-dimethylbutyl group at C-8.
461
Further correlations between H-400 and C-7, and between H-400 and fully substituted carbon signals at dC 105.5 and 155.5, assigned to C-6 and C-5, respectively, locate the dehydropyran ring at C-5/C-6. Compound 2 is thus the novel 7,4 0 -dihydroxy-8-[(2000 n,3000 -dihydroxy-3000 -methyl)butyl]-200 ,200 -dimethyl-300 ,400 -dehydro-pyrano[100 ,400 :-5,6]isoavone, which we name vogelin I. The UV and NMR spectra of vogelin J (3), assigned the molecular formula C20H16O5 from HREIMS data, displayed kmax peaks at 272, 313 and 330 nm, a 1H singlet resonance at dH 6.46, and corresponding upeld olenic signal at dC 103.1 (CH) characteristic of H-3 and C-3, respectively, in a avone skeleton (Mabry et al., 1970; Agrawal, 1989). In common with vogelins H (1) and I (2), vogelin J (3) possesses a para disubstituted 4 0 -hydroxy ring B (dH 7.60 and 6.85 (each 2H, J = 8.9 Hz, H-2 0 /H-6 0 , H-3 0 /H5 0 ); dC 127.7 (each CH, C-2 0 /6 0 ), 116.2 (each CH, C-3 0 / 5 0 )) and a 200 ,200 -dimethyl-300 ,400 -dehydropyran ring (dH 5.54 (1H, d, J = 10.0 Hz, H-300 ), 6.62 (1H, d, J = 10.0 Hz, H-400 ), 1.40 (6H, s, 3H-500 /3H-600 ); dC 78.0 (C, C-200 ), 127.3 (CH, C-300 ), 115.7 (CH, C-400 ), 27.9/28.0 (each CH3, C-500 / C-600 )). The latter was placed at C-5/C-6 when a bathochromic shift with NaOAc, but not AlCl3, indicated the presence of a hydroxyl group at C-7 only. Vogelin J (3) is thus 7,4 0 -dihydroxy-200 ,200 -dimethyl-300 ,400 -dehydropyr00 00 ano[1 ,4 :5,6]avone, a novel regioisomer of limonianin from Citrus limon [L.] Burn. (Chang, 1990), carpachromene from Flindersia laevicarpa C.T.White (Picker et al., 1976; Jain et al., 1978) and yinyanghuo C from Vancouveria hexandra (Hook.) C. Morren & Decne (Linuma et al., 1993) and Epimedium sagittatum (Siebold & Zucc.) Maxim. (Chen et al., 1996). The known compounds were identied as 6-prenylapigenin (Abegaz et al., 1998), 6,8-diprenylgenistein (Shirataki et al., 1982), 8-prenylluteone (Nkengfack et al., 1989), warangalone (scandenone) (Nkengfack et al., 1989), auriculatin (Shabbir et al., 1968; Subba Raju et al., 1981), 2,3dihydroauriculatin (Shabbir et al., 1968; Taylor et al., 1986), limonianin (Chang, 1990) and carpachromene (Saraswathy et al., 1998) by comparison of their physical properties and spectral data with the literature values.
recorded on a Nicolet Impact 400D Fourier-Transform Infrared (FT-IR) spectrometer, using NaCl windows with CHCl3 as solvent against an air background. LREIMS and HREIMS were taken on PerkinElmer 6890-Agilent 5975 GCMS and Micromass VG 70 SEQ instruments, respectively. Optical rotations were measured at room temperature in CHCl3 on a PerkinElmer 341 Polarimeter, using a 100 mm quartz microcell ow tube. 3.2. Plant material Erythrina vogelii Hook. was collected at Ogbomoso, Nigeria, in May 2003 and identied at the University of Ibadan Herbarium and the Cameroon National Herbarium, Yaounde, where a voucher specimen (20693/SRF Cam.) is retained for verication purposes. 3.3. Extraction and isolation of compounds The air-dried, ground stem bark material of E. vogelii (3 kg) was extracted for 72 h at room temperature with CH2Cl2:MeOH (1:1) and concentrated under reduced pressure to give 201.3 g of extract. Repeated combinations of vacuum liquid and gravity column chromatography on Merck 7729 and 9385 silica gels, and PTLC on aluminium backed analytical TLC (Merck 5554) plates, using various mixtures of hexane:EtOAc:MeOH, aorded vogelins H (1) (4.0 mg), I (2) (4.9 mg) and J (3) (15.2 mg), together with 6prenylapigenin (6.8 mg), 6,8-diprenylgenistein (9.7 mg), 8prenylluteone (10.2 mg), warangalone (scandenone) (9.0 mg), auriculatin (10.0 mg), 2,3-dihydroauriculatin (10.1 mg), limonianin (6.7 mg) and carpachromene (6.8 mg). 3.3.1. 7,4 0 -Dihydroxy-8-(c,c-dimethylallyl)-200 n-(400 hydroxyisopropyl)dihydrofurano-[100 ,300 :5,6]isoavone, vogelin H (1) 20 Pale yellow powder; m.p. 195197 C; aD 38 (c, 1 0.0015 in CHCl3); mmax(NaCl) cm 3545, 1642, 1610, 1512, 1425, 1382, 1270, 1215, 1172, 1075, 836; HREIMS (70 eV) m/z 422.1720 (calc. for C25H26O6 422.1729); EIMS (70 eV) m/z (rel. int.) 422 (98), 407 (20), 379 (70), 367(100), 363 (25), 352 (33), 349 (28), 335 (18), 320 (14), 307 (30), 295 (40), 118 (35), 59 (10); kmax (MeOH) nm (log e): 203 (4.48), 216 (4.37), 271 (4.48), (MeOH + NaOAc) 275; 1H NMR spectral data (400 MHz, CD3OD) dH 7.75 (1H, s, H-2), 7.28 (2H, d, J = 8.5 Hz, H-2 0 /H-6 0 ), 6.83 (2H, d, J = 8.5 Hz, H-3 0 /H-5 0 ), 5.23 (1H, m, H-2000 ), 4.74 (1H, d, J = 8.3 Hz, H-200 ), 3.29 (2H, d, J = 7.3 Hz, 2H-1000 ), 3.20 (2H, br d, J = 8.1 Hz, 2H-300 ), 1.73 (3H, s, 3H-5000 ), 1.64 (3H, s, 3H-4000 ), 1.30/1.20 (each 3H, s, 3H-500 /600 ); 13C NMR spectral data (100 MHz, CD3OD) (Table 1). 3.3.2. 7,4 0 -Dihydroxy-8-[(2000 n,3000 -dihydroxy-3000 -methyl)butyl]-200 ,200 -dimethyl-300 ,400 -de-hydropyrano[100 ,400 :5,6]isoavone, vogelin I (2) Pale yellow powder; m.p. 248249 C; a20 42 (c, D 0.0050 in CHCl3); mmax(NaCl) cm1 3525, 1640, 1610,
3. Experimental 3.1. General Melting points were determined on a Koer micro-hot stage melting point apparatus and are uncorrected. NMR spectra were recorded at room temperature on a 400 MHz Varian UNITY-INOVA spectrometer. Chemical shifts are expressed in d (ppm) units relative to tetramethylsilane (TMS) as internal standard and coupling constants are given in Hz. 1H NMR, 13C, HMBC, HSQC and NOESY spectra were recorded in CDCl3 and CD3OD. UV spectra were obtained on a Varian DMS 300 UVvisible spectrometer with MeOH as solvent. IR spectra were
462
Table 1 13 C NMR spectral data for vogelins H (1), I (2), and J (3) (100 MHz) Carbon 2 3 4 4a 5 6 7 8 8a 10 20 30 40 50 60 200 300 400 500 600 1000 2000 3000 4000 5000
A
Acknowledgements We thank Mr. Dilip Jagjivan for the running of NMR spectra, Mr. Bret Parel and Mr. Tommy van der Merwe for GCMS and HRMS, respectively, Mr. Ernest Makhaza for his technical assistance, and the University of KwaZulu-Natal and the National Research Foundation (NRF) for nancial aid. Dr. Alain Kamdem gratefully acknowledges a post-doctoral Fellowship from the NRF.
1 152.5 123.0 180.7 104.0 159.7 101.8 164.5 107.0 151.8 (CH) (C) (C) (C) (C) (C) (C) (C) (C)
2 154.2 124.0 182.4 105.8 155.5 105.5 158.1 106.5 156.4 (CH) (C) (C) (C) (C) (C) (C) (C) (C)
3 164.5 103.1 182.5 102.8 155.7 105.4 159.3 95.2 156.9 (C) (CH) (C) (C) (C) (C) (C) (CH) (C)
121.4 (C) 130.2(CH) 115.4 (CH) 156.0 (C) 115.4 (CH) 130.2(CH) 91.0 27.1 72.2 25.5 24.0 21.8 121.4 132.2 17.7 25.5 (CH) (CH2) (C) (CH3)A (CH3)A (CH2) (CH) (C) (CH3) (CH3)
122.7(C) 130.9(CH) 116.0 (CH) 158.0 (C) 116.0 (CH) 130.9(CH) 78.9 128.7 116.1 28.5 28.5 25.5 78.6 73.6 26.0 24.4 (C) (CH) (CH) (CH3) (CH3) (CH2) (CH) (C) (CH3)A (CH3)A
122.0(C) 127.7 (CH) 116.2 (CH) 160.8 (C) 116.2 (CH) 127.7 (CH) 78.0 127.3 115.7 28.0 27.9 (C) (CH) (CH) (CH3)A (CH3)A
References
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Values interchangeable within column.
1085 and 840; HREIMS (70 eV) m/z 438.1665 (calc. for C25H26O7 438.1678); EIMS (70 eV) m/z (rel. int.) 438 (46), 423 (64), 398 (3), 379 (29), 349 (100), 335 (20), 321 (60), 295 (13), 236 (5), 203 (8), 166 (9), 152 (7), 118 (4), 91 (18), 57 (27), 28 (61); kmax (MeOH) nm (log e): 215 (4.41), 268 (4.40), 294 (3.93); 1H NMR spectral data (400 MHz, CDCl3) dH 8.01 (1H, s, H-2), 7.32 (2H, d, J = 8.6 Hz, H-2 0 /H-6 0 ), 6.84 (2H, d, J = 8.5 Hz, H-3 0 /H5 0 ), 6.67 (1H, d, J = 10.0 Hz, H-400 ), 5.64 (1H, d, J = 10.0 Hz, H-300 ), 3.60 (1H, m, H-2000 ), 2.88 (1H, m, H1a000 ), 2.85 (1H, m, H-1b000 ), 1.46 (6H, s, 3H-500 /3H-600 ), 1.28 (6H, s, 3H-4000 /3H-5000 ), 13C NMR spectral data (100 MHz, CDCl3) (Table 1). 3.3.3. 4 0 ,7-Dihydroxy-200 ,200 -dimethyl-300 ,400 dehydropyrano[100 ,400 :5,6]avone, vogelin J (3) Pale yellow needles; m.p. 238239 C; mmax(NaCl) cm1 3450, 1652, 1589, 1540, 1400, 1272, 1235; HREIMS (70 eV) m/z 336.0932 (calc. for C20H16O5 336.0998); EIMS (70 eV) m/z (rel. int.) 336 (25), 321 (100), 203 (19), 135 (27), 118 (43); kmax (MeOH) nm (log e): 236 (4.42), 272 (4.36), 313 (4.23), 330 (4.25), 356 (3.78), (MeOH + NaOAc) 277; 1 H NMR spectral data (400 MHz, CDCl3)dH 7.60 (2H, d, J = 8.9 Hz, H-2 0 /6 0 ), 6.85 (2H, d, J = 8.9 Hz, H-3 0 /H-5 0 ), 6.62 (1H, d, J = 10.0 Hz, H-400 ), 6.46 (1H, s, H-3), 6.34 (1H, s, H-8), 5.54 (1H, d, J = 10.0 Hz, H-300 ), 1.40 (6H, s, 3H-500 /3H-600 ); 13C NMR spectral data (100 MHz, CDCl3) (Table 1).
A.F. Kamdem Wao et al. / Phytochemistry 67 (2006) 459463 Mitscher, L.A., Gollapudi, S.R., Gerlach, D.C., Drake, S.D., Verliz, E.A., Ward, A.J., 1988a. Erycristin, a new antimicrobial pterocarpan from Erythrina crista-galli. Phytochemistry 27, 381385. Mitscher, L.A., Okwute, S.A., Gollapudi, S.R., Drake, S.D, Avona, E., 1988b. Antimicrobial pterocarpans of Nigerian Erythrina mildbraedii. Phytochemistry 27, 34493452. Mizuno, M., Tanaka, T., Tamura, K., Matsuura, N., Iinuma, M., Phengklai, C., 1990. Flavonoids in the roots of Euchresta horseldii in Thailand. Phytochemistry 29, 26632665. Njamen, D., Mbafor, J.T., Fomum, Z.T., Kamanyi, A., Mbanya, J.C., Giner, R.M., Recio, M.C., Manez, S., Rios, J.L., 2004. Antiinammatory activities of two avanones, sigmoidin A and sigmoidin B from Erythrina sigmoidea. Planta Medica 70, 15. Nkengfack, A.E., Sanson, D.R., Fomum, Z.T., Tempesta, M.S., 1989. 8Prenylluteone, a prenylated isoavone from Erythrina eriotricha. Phytochemistry 28, 25222526. Picker, K., Ritchie, E., Taylor, W.C., 1976. The chemical constituents of Australian Flindersia species. XXI. An examination of the bark and the leaves of F. laevicarpa. Australian Journal of Chemistry 29, 20232036. Queiroz, E.F., Atindehou, K.K., Terreaux, C., Antus, S., Hostettmann, K., 2002. Prenylated isoavonoids from the root bark of Erythrina vogelii. Journal of Natural Products 65, 403406. Saraswathy, A., Balakrishna, K., Bhima Rao, R., Alliranti, T., Patra, A., Pichai, R., 1998. Carpachomene from Atalantia monophylla. Fitoterapia 69, 463464. Sekine, T., Inagaki, M., Ikegami, M., Fujii, Y., Ruangrungsi, N., 1999. Six diprenylisoavones, derrisisoavones AF, from Derris scandens. Phytochemistry 52, 8794. Shabbir, M., Zaman, A., Crombie, L., Truck, B., Whiting, D.A., 1968. Structure of auriculatin, an extractive of Milletia auriculata. Journal of the Chemical Society, 18991901.
463
Shirataki, Y., Manaka, A., Yokoe, I., Komatsu, M., 1982. Two prenylated avanones from Euchresta japonica. Phytochemistry 21, 29592963. Subba Raju, K.V., Srimannarayana, G., Ternai, B., Stanley, R., Markham, K.R., 1981. 13C NMR studies of some complex natural oxygen heterocycles. Structure of millettin, a novel isoavone isolated from Millettia auriculata. Tetrahedron 37, 957962. Tahara, S., Ingham, J.L., Nakahara, S., Mizutani, J., Harborne, J.B., 1984. Fungitoxic dihydrofuranoisoavones and related compounds in white lupin, Lupinus albus. Phytochemistry 23, 18891900. Tahara, S., Orihara, S., Ingham, J.L., Mizutani, J., 1989. Seventeen isoavonoids from Lupinus albus roots. Phytochemistry 28, 901911. Takashima, J., Ohsaki, A., 2002. Brosimacutins AI, nine new avonoids from Brosimum acutifolium. Journal of Natural Products 64, 1336 1340. Tanaka, H., Doi, M., Etoh, H., Watanabe, N., Shimizu, H., Hirata, M., Ahmad, M., Qurashi, I., Khan, M.R., 2001. Revised structures for senegalensin and euchrenone b10. Journal of Natural Products 65, 18431847. Taylor, R.B., Corley, D.G., Tempesta, M.S., Fomum, Z.T., Ayafor, J.F., Wandji, J., Lfeadike, P.N., 1986. 2,3-Dihydroauriculatin, a new prenylated isoavanone from Erythrina senegalensis. Application of the selective INEPT technique. Journal of Natural Products 49, 670 673. Wandji, J., Fomum, Z.T., Tillequin, F., Baudouin, B., Koch, M., 1994. Epoxy-isoavones from Erythrina senegalensis. Phytochemistry 35, 15731577. Wandji, J., Nkengfack, A.E., Fomum, Z.T., Ubillas, R., Killday, K.B., Tempesta, M.S., 1990. A new prenylated isoavone and long chain esters from two Erythrina species. Journal of Natural Products 53, 14251429.
Phenolic compounds from the owers of Garcinia dulcis

S. Deachathai a, W. Mahabusarakam a,*, S. Phongpaichit b, W.C. Taylor c, Y.-J. Zhang d, C.-R. Yang d
a b
Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand c School of Chemistry, University of Sydney, NSW 2006, Australia d Laboratory of Phytochemistry, Kunming Institute of Botany, Chinese Academy of Sciences, China Received 7 July 2005; received in revised form 14 September 2005 Available online 1 December 2005
Abstract Dulcisxanthones CF (14) and dulcinone (5) together with 22 known compounds were isolated from the owers of Garcinia dulcis. Their structures were determined by spectroscopic methods. The abilities of some of these compounds to act as radical scavengers and antibacterial agents were investigated. 2005 Elsevier Ltd. All rights reserved.
Keywords: Garcinia dulcis; Guttiferae; Xanthones; Chromones; Radical scavenging; Antibacterial
1. Introduction The sub-woody plant Garcinia dulcis Kurz. (Guttiferae), local Thai name Ma-Phut, grows mainly in southeast Asia. Its leaves and seeds have been used in traditional medicine against lymphatitis, parotitis, struma and other disease conditions (Kasahara and Henmi, 1986). In our previous work (Deachathai et al., 2005), we have investigated the chemical constituents from its fruit and their biological activities. In our continuing work, we have examined the chemical constituents from the owers. This investigation had led to the isolation and structural determination of ve new and 22 known compounds.
2. Results and discussion The owers of G. dulcis were sequentially extracted with acetone. Purication of the extract, produced four new
Corresponding author. Tel.: +66 7428 8432; fax: +66 7421 2918. E-mail addresses: mwilawan@ratree.psu.ac.th, wilawan.m@psu.ac.th (W. Mahabusarakam). 0031-9422/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2005.10.016
*
xanthones named dulcisxanthones CF (14), one new chromone named dulcinone (5), along with 22 known compounds: volkensiavone (6), morelloavone (7) (Ansari et al., 1976), 1-hydroxy-3,4,5-trimethoxyxanthone (8) (Stout et al., 1973), rhamnazin (9) (Subhadhirasakul et al., 2003), quercetin 3-O-b-galactopyranoside (10) (Kartnig et al., 1985), podocarpusavone A (11) (Harrison et al., 1994), xanthochymusside (12), fukugeside (13) (Konoshima et al., 1970), cowaxanthone (14) (Deachathai et al., 2005), GB-2a (15) (Ansari et al., 1976), xanthochymol (16) (Blount and Williams, 1976), BR-xanthone A (17), 1,3,6-trihydroxy-7-methoxy-2,5-bis(3-methyl-2-butenyl)xanthone (18) (Deachathai et al., 2005), guttiferone E (19) (Gustafson et al., 1992), rheediaxanthone A (20) (Delle Monache et al., 1981), a-mangostin (21), 1,7-dihydroxy-3methoxy-2-(3-methyl-2-butenyl)xanthone (22) (Deachathai et al., 2005), 3-isomangostin (23) (Mahabusarakam et al., 1987), kaempferol 3-O-glucopyranosyl-7-O-rhamnopyranoside (24), garcinone B (25), morusignin J (26) (Deachathai et al., 2005) and b-mangostin (27) (Mahabusarakam et al., 1987). Dulcisxanthone C (1) is 1-hydroxy-2,3,4,6-tetramethoxyxanthone, and a yellow solid, m.p. 125128 C. Its
S. Deachathai et al. / Phytochemistry 67 (2006) 464469

2'''
465
O
7 8a 9a
OH
1
4''' 8a 6
O
9a
OH
1 4'
OCH 3 OCH 3
O HO
H3CO
10a
O 4a OCH 3 1
10a O 4a 1''
O 2'
4''
5''
O HO
6 8a 9a
OH
1 1'
9'
10'
2''
4'' 8a 6
O
9a
OH
1 1'
5'
O
8'
4' 10a O 4a
HO
10a
O 4a
OH
HO
OCH 3
1''
4'' 5''
3 O HO
6 4 2 8
H 3C
CH3
OH 5
molecular formula of C17H16O7 was established on the basis of its mass spectrum ([M]+ m/z 332). The UV spectrum showed absorption maxima at 376, 309, 273, 241 nm. The IR spectrum exhibited OH stretching at 3400 cm1 and C@O stretching at 1646 cm1. The 1H NMR spectrum showed a singlet signal of a chelated hydroxy proton (1-OH) at d 12.60. The signals in the aromatic region, d 7.84 (dd), 7.32 (d) and 7.26 (d) that appeared as an ABX type were proposed for the signals of H-7, H-8 and H-5, respectively. These assignment were supported by the correlations of H-7 to C-5, C-6, C-9; H8 to C-6, C-8a, C-10a and H-5 to C-6, C-7, C-10a from an HMBC experiment. Four singlet signals at d 4.04, 4.16, 3.97 and 4.05 were assigned for the methoxy protons of 2-OCH3, 3-OCH3, 4-OCH3 and 6-OCH3, respectively, and conrmed by the 3J coupling of the methoxy protons to C-2, C-3, C-4 and C-6, respectively, in HMBC. The assigned structure was further conrmed by analysis of HMBC correlations (Table 1). Dulcisxanthone D (2) is l,6-dihydroxy-5-(3-methyl-2butenyl)-2 0 ,2 0 -dimethylchromeno(5 0 ,6 0 :2,3)-2000 ,2000 -dimethylchromeno(5000 ,6000 :8,7)xanthone. It is an orange solid, m.p. 218220 C, with the molecular formula C28H28O6. The UV spectrum showed absorption maxima at 335, 301, 289, 221 nm. Absorption bands of OH stretching and C@O stretching were shown in the IR spectrum at 3424 cm1 and at 1615 cm1. The 1H NMR spectrum showed the signals of a chelated hydroxy proton at d 13.70 (1-OH), a non-chelated hydroxy proton at d 6.34 (6-OH) and an isolated aromatic proton at d 6.30 (H-4). The characteristic signals of a prenyl group were present at d 3.57 (H-100 ), 5.28 (H-200 ), 1.69 (H-400 ) and 1.88 (H-500 )
Table 1 HMBC correlation data of compounds 1 and 5 H-position 3 5 7 8 2-OMe 3-OMe 4-OMe 6-OMe 2-Me 7-Me 1 C-6, C-7, C-10a C-5, C-6, C-9 C-6, C-8a, C-10a C-2 C-3 C-4 C-6 5 C-2, C-4, C-4a, 2-Me C-4, C-4a, C-6, C-7, C-8a
C-2, C-3 C-6, C-7, C-8
and it was located at C-5 according to the HMBC correlation of H-100 to C-6, and C-10a. The signals of four methyl groups at d 1.49 (6H, s, 2000 -Me) and 1.47 (6H, s, 2 0 -Me) and vicinal olenic protons at d 5.77 (d, H-3000 ), 7.98 (d, H-4000 ), 5.57 (d, H-3 0 ) and 6.73 (d, H-4 0 ) associated with a chromene rings were present. The 3J correlations of H-4000 to C-7 and H-4 0 to C-1 suggested that the chromene rings were connected to the parent structure at C-7, C-8 and C-2, C-3. The complete HMBC data (Table 2) conrmed this structure. Dulcisxanthone E (3) is 1,3,6,7-tetrahydroxy-2-(3,7dimethyl-2,6-octadienyl)-5-(3-methyl-2-butenyl)xanthone. It is a yellow solid with the molecular formula C28H32O6. The UV spectrum showed absorption maxima at 369, 322, 258, 242 nm. The IR spectrum showed absorption bands of OH stretching at 3450 cm1 and C@O stretching at 1690 cm1. The 1H NMR spectrum exhibited resonances of a chelated hydroxy proton 1-OH at d 13.19 and aromatic protons H-4 at d 6.42 and H-8 at d 7.33. The characteristic
466 Table 2 HMBC correlation data of compounds 24 H-position 4 5 8 10 20 30 40 50 60 80 90 10 0 100 200 300 400 500 3000 4000 1-OH 3-OMe 2 0 -Me 200 -Me 2000 -Me 2
3
0
4 C-2, C-3, C-4a, C-9a C-6, C-7, C-8a, C-10a C-1, C-2, C-3, C-2 0 , C-3 0 C-2, C-4 0 , C-5 0 C-2 0 , C-3 0 , C-5 0 C-2 0 , C-3 0 , C-4 0
C-2, C-3, C-4a, C-9, C-4
C-2, C-3, C-4a, C-9a C-6, C-7, C-8a, C-9, C-10a C-1, C-2, C-3, C-2 0 , C-3 0 C-1 0 , C-4 0
C-2, C-2 0 , 2 0 -Me C-1, C-2 0
C-6, C-8a, C-10a, C-200 , C-300 C-5, C-400 , C-500 C-200 , C-300 , C-500 C-200 , C-300 , C-400 C-8, C-2000 C-7, C-2000 C-1, C-2, C-9a C-2 0 , C-3 0
C-3 0 , C-5 0 , C-6 0 C-3 0 , C-4 0 , C-6 0 C-5 0 , C-8 0 , C-10 0 C-6 0 , C-7 0 , C-10 0 C-2 0 , C-3 0 , C-4 0 , C-5 0 C-6 0 , C-7 0 , C-8 0 C-5, C-6, C-10a, C-200 , C-300 C-100 , C-400 , C-500 C-200 , C-300 , C-500 C-200 , C-300 , C-400
C-8, C-400 C-200
C-9a, C-1, C-2 C-3 C-300
C-2000 , C-3000
signals of a prenyl unit were displayed at d 6.30 (d, H-100 ), 5.28 (br t, H-200 ), 1.69 (s, H-400 ) and 1.88 (s, H-500 ). The prenyl unit was linked at C-5 according to the HMBC correlation of H-100 to C-5, C-6 and C-10a. It also exhibited the typical signal of a geranyl group: three singlets of three methyl groups at d 1.57 (H-8 0 ), 1.80 (H-9 0 ) and 1.52 (H10 0 ), a doublet of methylene protons at d 3.37 (H-1 0 ), two multiplets of methylene protons at d 1.99 (H-4 0 ) and 2.03 (H-5 0 ) and two broad triplets of two olenic methine protons at d 5.28 (H-2 0 ) and 5.02 (H-6 0 ). The correlation of H-1 0 to C-1, C-2 and C-3 in the HMBC indicated that the geranyl side chain was at C-2. The complete HMBC (Table 2) supported this structure. Dulcisxanthone F (4) is 1,6-dihydroxy-2-(3-methyl2-butenyl)-3-methoxy-200 ,200 -dimethylchromeno(500 ,600 :8,7)xanthone. It is a yellow solid with the molecular formula C24H24O6. The UV spectrum showed absorption maxima at 331, 322, 265, 244 nm. The IR spectrum exhibited absorption bands of OH stretching at 3479 cm1 and C@O stretching at 1675 cm1. As was found for compound 2, the 1H NMR spectrum indicated the presence of a chelated hydroxy group 1-OH at d 13.35 (s), aromatic protons H-4 at d 6.36 (s), H-5 at d 6.83 (s), methoxy protons 3OCH3 at d 3.91 (s) and proton resonances corresponded to a prenyl group at d 1.80 (H-5 0 , s), 1.68 (H-4 0 , s), 3.36 (H-1 0 , d) and 5.23 (H-2 0 , br t). The characteristic signals of two methyl groups (H-500 , H-600 ) and vicinal olenic protons (H-300 and H-400 ) associated with a chromene ring were shown at d 1.50, 5.83 and 8.04, respectively. The deshielded eect on the resonance of H-400 indicated that the chromene ring was attached to the xanthone nucleus close to its carbonyl group. The HMBC correlation of H-1 0 to C-1, C-2
and C-3 indicated that the prenyl unit was at C-2. The methoxy group was assigned at C-3 according to the HMBC correlations of H-4 to C-3 and methoxy protons to C-3. Irradiation of the methoxy protons at d 3.91 in a NOE experiment, enhanced the signal of H-4 and this also conrmed the placement of the methoxy group. The complete HMBC (Table 2) supported the assigned structure. Dulcinone (5) is 6,8-dihydroxy-2,7-dimethyl-4H-chromen-4-one. It is a yellow solid with molecular formula C11H10O4. The IR spectrum showed absorption bands of OH stretching at 3343 cm1 and C@O stretching at 1657 cm1. The 13C NMR spectrum also showed the resonance of a carbonyl carbon at d 181.5. The 1H NMR spectrum showed a singlet signal of an olenic proton H-3 at d 5.99, a singlet signal of an aromatic proton H-5 at d 6.33, a singlet signal of methyl protons 2-Me at d 2.39 and a singlet signal of methyl protons 7-Me at d 2.13. These assignment were supported by the HMBC correlations: H-3 to C-2, C4, C-4a; H-5 to C-4a, C-6, C-7, C-8a; 2-Me to C-2, C-3 and 7-Me to C-6, C-7, C-8. The assigned structure was further conrmed by HMBC correlations (Table 1). Compounds 15 are new natural products. Compounds 6, 7, 11, 14, 15, 17, 18, 21, 22, 24, 25, and 26 were previously isolated from G. dulcis (Ansari et al., 1976; Harrison et al., 1994; Deachathai et al., 2005). This is the rst report of the known compounds 8, 9, 10, 12, 13, 16, 19, 20, 23, 27 in G. dulcis. Evaluation of the radical scavenging activities of some of the compounds at the concentration of 10 lM (Table 3) revealed that none of the newly isolated compounds showed any antioxidant activity. However 10, 13, 16 and 19 had potent antioxidant properties produced 57%, 56%, 60% and 59% scavenging properties, respec-
S. Deachathai et al. / Phytochemistry 67 (2006) 464469 Table 3 Radical scavenging activity of compounds from the ower of G. dulcis (10 lM) Compounds 1 2 3 4 5 6 8 9 10 11 12 13 15 16 19 20 23 27 BHT % Scavenging of DPPH 2 16 15 2 3 5 2 8 57 5 36 56 33 61 59 8 3 2 43 IC50 (lM) 10.50 11.40 8.50 10.10 19.00
467
specimen (Coll. No. 02, Herbarium No. 0012652) has been deposited at Prince of Songkla University Herbarium, Biology Department, Faculty of Science, Prince of Songkla University, Thailand. 3.3. Extraction and isolation The owers of G. dulcis (1.2 kg) were extracted at room temperature sequentially with acetone (5 and 7 days). Removal of the solvents from the extracts yielded the acetone extracts A (148.2 g) and B (38.4 g). An aliquot of the acetone extract A (92.0 g) was further separated by QCC and eluted with CH2Cl2, CH2Cl2Me2CO, Me2CO, Me2COMeOH gradient solvent system. The eluted fractions were combined into 17 fractions (A1A17) on the basis of their TLC behaviour. Fraction A6 (7.7 g) was subjected to CC on sephadex LH-20 and eluted with a gradient of H2OMeOH to give compounds 1 (3.0 mg), 8 (9.0 mg), 9 (62.0 mg), 10 (27.0 mg) and 11 (10.0 mg). Fractions A5 (2.2 g), A7 (4.8 g) and A13 (1.0 g) were further puried producing 6 (29.1 mg), 7 (1.5 g), 12 (148.0 mg) and 13 (51.0 mg). The acetone extract A (55.8 g) was also fractionated by dissolving in CH2Cl2 to give a soluble (15.2 g) and insoluble (40.2 g) fraction. This CH2Cl2 soluble fraction was subjected to silica gel CC and eluted sequentially with CH2Cl2, and a CH2Cl2Me2CO gradient to aord 2 (10.0 mg), 14 (21.5 mg), 15 (38.2 mg) and 16 (2.3 g). The CH2Cl2 insoluble fraction after CC produced 17 (15.2 mg) and 18 (5.4 mg). Extraction of the acetone extract B (38.4 g) with CH2Cl2, EtOAc and then Me2CO gave CH2Cl2 soluble (8.7 g), EtOAc soluble (8.5 g) and Me2CO soluble (3.5 g) fractions. After CC of CH2Cl2 soluble fraction and eluting with CH2Cl2Me2CO in a polarity gradient manner, compounds 19 (10.2 mg), 20 (5.5 mg), 3 (14.3 mg), 21 (31.7 mg), 22 (20.2 mg), 23 (3.2 mg) and 24 (12.3 mg) were obtained. The EtOAc soluble fraction was subjected to silica gel CC eluted with CH2Cl2Me2CO in a polarity gradient manner to aord 4 (23.8 mg) and 5 (5.2 mg), whereas the Me2CO soluble fraction subjected to silica gel CC and eluted with CH2Cl2 MeOH in a polarity gradient manner gave 25 (2.6 mg), 26 (2.3 mg) and 27 (15.2 mg). 3.3.1. Dulcisxanthone C (1) Yellow solid, m.p. 125128 C. HRESIMS m/z 332.0892 [M]+ (calcd. for C17H16O7, 332.0896). UV (CH3OH) kmax (nm) (log e): 376 (3.37), 309 (3.78), 273 (3.87), 241 (3.91). IR (neat), m (cm1): 3400, 1646. 1H NMR (CDCl3) (d ppm): 12.60 (1H, s, 1-OH), 7.84 (1H, dd, J = 7.8, 1.5 Hz, H-7), 7.32 (1H, d, J = 7.8 Hz, H-8), 7.26 (1H, d, J = 1.5 Hz, H-5), 4.16 (3H, s, 3-OMe), 4.05 (3H, s, 6OMe), 4.04 (3H, s, 2-OMe), 3.97 (3H, s, 4-OMe). EIMS m/z (% rel. int): ([M]+ 332, 100), 317 (98), 302 (21), 287 (15), 259 (17), 203 (10), 175 (13). 13C NMR (CDCl3) (d ppm): 181.7 (C-9), 154.1 (C-3), 150.6 (C-1), 148.7 (C-6), 146.4 (C-10a), 145.7 (C-4a), 135.4 (C-4), 132.7 (C-2), 123.7 (C-8), 120.9 (C-8a), 116.5 (C-7), 115.9 (C-5), 104.5
tively. These values corresponded to IC50 values of 10.50, 11.40, 8.50 and 10.10 lM, respectively. Compound 9 and 16 were tested for the antibacterial activity. Compound 16 was found to inhibit the growth of both the penicillinsensitive strain, ATCC 25923, and the methicillin-resistant strain, MRSA SK1, of Staphylococcus aureus with MIC values of 8 lg/mL whereas 9 showed only weak activity with MIC values greater than 128 lg/mL. The results of radical scavenging and antibacterial activities of compounds 7, 14, 17, 18, 21, 22, 24, 25, 26 have been reported in the previous work (Deachathai et al., 2005). 3. Experimental 3.1. General methods Melting points were measured on a digital Electrothermal 9100 Melting Point Apparatus and are uncorrected. Infrared spectra were recorded on an FTS 165 FT-IR spectrometer. Ultraviolet absorption spectra were recorded using a UV-160A spectrometer (SHIMADZU). 1H and 13 C NMR spectra were performed on a Varian UNITY INOVA 500 spectrometer and on a 300 MHz FTNMR, Bru ker spectrometer. The high resolution mass spectra were recorded on an MS25RFA spectrometer. Pre-coated TLC sheets of silica gel 60 PF254 were used. Quick column chromatography (QCC) was performed with silica gel 60H. Column chromatography (CC) was performed with silica gel 100 and sephadex LH-20. 3.2. Plant material The owers of G. dulcis were collected from Songkhla province in the southern part of Thailand. The voucher
468
(C-9a), 61.9 (2-OMe), 61.7 (3-OMe), 61.2 (4-OMe), 56.5 (6OMe). 3.3.2. Dulcisxanthone D (2) Yellow solid, m.p. 218220 C. HRFABMS m/z 460.1929 [M]+ (calcd. for C28H28O6 460.1886). UV (CH3OH) kmax (nm) (log e): 335 (4.10), 301 (4.24), 289 (4.30), 221 (4.21), 205 (4.25). IR (neat) m (cm1): 3424, 1615, 1440 cm1. 1H NMR (CDCl3) (d ppm): 13.70 (1H, s, 1-OH), 7.98 (1H, d, J = 10.2 Hz, H-4000 ), 6.73 (1H, d, J = 10.2 Hz, H-4 0 ), 6.34 (1H, s, 6-OH), 6.30 (1H, s, H-4), 5.77 (1H, d, J = 10.2 Hz, H-3000 ), 5.57 (1H, d, J = 10.2 Hz, H-3 0 ), 5.28 (1H, br t, J = 5.7 Hz, H-200 ), 3.57 (2H, d, J = 7.5 Hz, H-100 ), 1.88 (3H, s, H-500 ), 1.69 (3H, s, H-400 ), 1.49 (6H, s, 2000 -Me), 1.47 (6H, s, 2 0 -Me). FABMS m/z (% rel. int.): ([M]+ 460, 100), 444 (50), 61 (42). 13C NMR (CDCl3) (d ppm): 182.8 (C-9), 159.8 (C-1), 157.7 (C-3), 156.5 (C-10a), 150.9 (C-4a), 148.6 (C-6), 136.5 (C-7), 132.6 (C-300 ), 131.3 (C-3000 ), 127.0 (C-3 0 ), 120.9 (C-4000 ), 120.9 (C-200 ), 117.1 (C-8), 115.7 (C-4 0 ), 115.3 (C-8a), 108.3 (C-5), 104.2 (C-9a), 103.8 (C-2), 94.2 (C-4), 77.9 (C-2 0 ), 76.8 (C-2000 ), 28.3 (2 0 -Me), 27.4 (2000 -Me), 25.8 (C400 ), 22.5 (C-100 ), 18.0 (C-500 ). 3.3.3. Dulcisxanthone E (3) Yellow solid, m.p. 185187 C. HRESIMS m/z 464.2199 [M]+ (calcd. for C28H32O6, 464.2199). UV (CH3OH) kmax (nm) (log e): 369 (4.06), 322 (4.28), 258 (4.46), 242 (4.50). IR (neat), m (cm1): 3450, 2923, 1690, 1456. 1H NMR (CDCl3 + CD3OD one drop) (d ppm): 13.19 (1H, s, 1OH), 7.33 (1H, s, H-8), 6.42 (1H, s, H-4), 6.30 (2H, d, J = 6.0 Hz, H-100 ), 5.28 (2H, br t, H-2 0 , H-200 ), 5.02 (1H, br t, H-6 0 ), 3.37 (2H, d, J = 6.0 Hz, H-1 0 ), 2.03 (2H, m, H-5 0 ), 1.99 (2H, m, H-4 0 ), 1.88 (3H, s, H-500 ), 1.80 (3H, s, H-9 0 ), 1.69 (3H, s, H-400 ), 1.57 (3H, s, H-8 0 ), 1.52 (3H, s, H-10 0 ). EIMS m/z (% rel. int.): ([M]+ 464, 80), 421 (50), 409 (100), 339 (40), 297 (55), 285 (54), 69 (26). 13C NMR (CDCl3 + CD3OD one drop) (d ppm): 180.6 (C-9), 162.5 (C-3), 159.8 (C-1), 156.1 (C-4a), 150.7 (C-6), 150.2 (C10a), 141.9 (C-7), 135.9 (C-300 ), 132.2 (C-3 0 ), 131.5 (C-7 0 ), 124.2 (C-6 0 ), 122.4 (C-2 0 ), 121.3 (C-200 ), 115.5 (C-8a), 112.7 (C-5), 110.4 (C-2), 105.1 (C-8), 102.5 (C-9a), 93.4 (C-4), 39.8 (C-4 0 ), 25.8 (C-400 ), 25.5 (C-8 0 ), 22.4 (C-100 ), 21.7 (C-5 0 ), 21.4 (C-1 0 ), 17.8 (C-9 0 ), 17.6 (C-10 0 ), 16.2 (C500 ). 3.3.4. Dulcisxanthone F (4) Yellow solid, m.p. 213215 C. HRESIMS m/z 408.1573 [M]+ (calcd. for C24H24O6, 408.1573). UV (CH3OH) kmax (nm) (log e): 331 (4.44), 322 (4.43), 265 (4.53), 244 (4.54). IR (neat) m (cm1): 3479, 1675, 1587, 1447. 1H NMR (CDCl3 + DMSO-d6 one drop) (d ppm): 13.35 (1H, s, 1OH), 8.04 (1H, d, J = 9.7 Hz, H-400 ), 6.83 (1H, s, H-5), 6.36 (1H, s, H-4), 5.83 (1H, d, J = 9.7 Hz, H-300 ), 5.23 (1H, br t, J = 7.0 Hz, H-2 0 ), 3.91 (3H, s, 3-OMe), 3.36 (2H, d, J = 6.7 Hz, H-1 0 ), 1.80 (3H, s, H-5 0 ), 1.68 (3H, s, H-4 0 ), 1.50 (6H, s, 200 -Me). EIMS m/z (% rel. int.): ([M]+
408, 60), 393 (55), 365 (70), 353 (100), 169 (15). 13C NMR (CDCl3 + DMSO-d6 one drop) (d ppm): 182.4 (C9), 163.6 (C-3), 159.6 (C-1), 155.5 (C-4a), 153.0 (C-10a), 150.7 (C-6), 136.7 (C-7), 132.2 (C-300 ), 131.7 (C-3 0 ), 122.2 (C-2 0 ), 121.0 (C-400 ), 119.7 (C-8), 111.4 (C-2), 108.7 (C-8a), 102.2 (C-9a, C-5), 88.9 (C-4), 71.0 (C-200 ), 55.8 (3OCH3), 29.6 (200 -Me), 27.3 (200 -Me), 25.8 (C-4 0 ), 21.3 (C1 0 ), 17.7 (C-5 0 ). 3.3.5. Dulcinone (5) Yellow solid, m.p. 227229 C. HRESIMS m/z 206.0570 [M]+ (calcd. for C11H10O4, 206.0579). UV (CH3OH) kmax (nm) (log e): 327 (3.51), 300 (3.62), 258 (4.25), 226 (3.96). IR (neat) m (cm1): 3343, 1657, 1598, 1417. 1H NMR (CDCl3 + DMSO-d6 one drop) (d ppm): 6.33 (1H, s, H5), 5.99 (1H, s, H-3), 2.39 (3H, s, H-2), 2.13 (3H, s, H-7). EIMS m/z (% rel. int.): ([M]+ 206, 100), 205 (95), 177 (11), 165 (8). 13C NMR (CDCl3 + DMSO-d6 one drop) (d ppm): 181.5 (C-4), 165.6 (C-2), 160.9 (C-6), 158.2 (C8a), 154.6 (C-8), 106.8 (C-3), 102.9 (C-4a), 101.2 (C-7), 97.5 (C-5), 19.3 (2-CH3), 6.4 (7-CH3). 3.4. Radical scavenging activity This was carried out according to the procedure of Deachathai et al. (2005). 3.5. Antibacterial activity This was carried out according to the procedure of Mahabusarakam et al. (2004). Acknowledgements This research was supported by a scholarship to S.D. from the Postgraduate Education and Research Program in Chemistry (PERCH), funded by The Royal Thai Government and the Graduate School, Prince of Songkla University.
References
Ansari, W.H., Rahman, W., Barraclough, D., Maynard, M.R., Scheinmann, F., 1976. Biavonoids and a avanonechromone from the leaves of Garcinia dulcis (Roxb.). Kurz. J. Chem. Soc. Perkin Trans I., 14581463. Blount, J.F., Williams, T.H., 1976. Revised structure of xanthochymol. Tetrahedron Lett. 34, 29212924. Deachathai, S., Mahabusarakam, W., Towatana, N., Phongpaichit, S., Taylor, W.C., 2005. Phenolic compounds from the fruit of Garcinia dulcis. Phytochemistry 66, 23682375. Delle Monache, F., Botta, B., Nicoletti, M., de Barros Coelho, J.S., de Andrade Lyra, F.D., 1981. Three new xanthones and macluraxanthone from Rheedia benthamiana PI. Triana (Guttiferae). J. Chem. Soc. Perkin Trans. I., 484488. Gustafson, K.R., Blunt, J.W., Munro, M.H.G., Fuller, R.W., McKee, T.C., Cardellina II, J.H., McMahon, J.B., Cragg, G.M., Boyd, M.R., 1992. The guttiferones, HIV-inhibitory benzophenones from Sympho-
S. Deachathai et al. / Phytochemistry 67 (2006) 464469 nia globulifera, Garcinia livingstonei, Garcinia ovalifolia and Clusia rosea. Tetrahedron 48, 1009310102. Harrison, L.J., Leong, L.-S., Leong, Y.-W., Sia, G.-L., Sim, K.-Y., Tan, H.T.-W., 1994. Xanthone and avonoid constituents of Garcinia dulcis (Guttiferae). Nat. Prod. Lett. 5, 111116. Kartnig, Th., Gruber, A., Stachel, J., 1985. Zur kenntnis des avonoidmusters von Asparagus ocinalis. Planta Med. 3, 288. Kasahara, S., Henmi, S., 1986. Medicine Herb Index in Indonesia. Eisai Indonesia, Jakarta, p. 92. Konoshima, M., Ikeshiro, Y., Miyahara, S., Yen, K.-Y., 1970. The constitution of biavonoids from Garcinia plants. Tetrahedron Lett. 48, 42034206.
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Mahabusarakam, W., Wiriyachitra, P., Taylor, W.C., 1987. Chemical constituents of Garcinia mangostana. J. Nat. Prod. 50, 474478. Mahabusarakam, W., Deachathai, S., Phongpaichit, C., Jansakul, C., Taylor, W.C., 2004. A benzil and isoavone derivatives from Derris scandens Benth. Phytochemistry 65, 11851191. Stout, G.H., Christensen, E.N., Balkenhol, W.J., Stevens, K.L., 1973. Xanthones of the Gentianaceae-II Frasera albicaulis Dougl. EX Griesb. Tetrahedron 25, 19611973. Subhadhirasakul, S., Jankeaw, B., Malinee, A., 2003. Chemical constituents and antioxidative activity of the extracts from Dyera costulata leaves. Songklanakarin J. Sci. Technol. 25, 351353.
Xanthone derivatives from Cratoxylum cochinchinense roots

W. Mahabusarakam
a
a,*
, W. Nuangnaowarat a, W.C. Taylor
Department of Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand b School of Chemistry, University of Sydney, New South Wales 2006, Australia Received 5 April 2005; received in revised form 6 September 2005 Available online 28 November 2005
Abstract Two xanthones and two caged-prenylated xanthones, named cochinchinones AD, respectively, and a synthetically known cagedprenylated xanthone, together with seven known compounds were isolated from the roots of Cratoxylum cochinchinense (Lour.) Blume. Their structures were assigned on the basis of analyses of spectroscopic data. Some of the compounds exhibited eective antioxidative properties. 2005 Elsevier Ltd. All rights reserved.
Keywords: Cratoxylum cochinchinense; Clusiaceae; Xanthones; Caged-prenylated xanthones; Radical scavenger
1. Introduction Cratoxylum cochinchinense (Lour.) Blume is called tuegliang locally in Thailand. In traditional medicine, it has been used to treat fevers, coughs, diarrhoea, itches, ulcers and abdominal complaints (Vo, 1997). The bark of this plant was previously shown to contain triterpenoids, tocotrienols and xanthones (Bennett et al., 1993; Sia et al., 1995; Nguyen and Harrison, 1998). The use of this plant as a traditional medicine, and the results from a preliminary screening of the biological activity of crude extracts from its roots, led us to examine them further for substances that act as radical scavengers. Two new xanthones (12), two new caged-prenylated xanthones (34), a synthetically known caged-prenylated xanthone (5) and seven known compounds (612) were isolated. Their structures were elucidated from analyses of 1D and 2D NMR spectroscopic data, including 1H, 13C NMR, NOE, COSY, HMQC and HMBC. Radical scavenging activity of the compounds was investigated.
2. Results and discussion Separation of a dichloromethane extract of the roots of C. cochinchinense produced cochinchinones AD (14), the caged-prenylated xanthone (5) (Thoison et al., 2000), bmangostin (6) (Mahabusarakam et al., 1987), l,3,7-trihydroxy-2,4-bis (3-methyl-2-butenyl)xanthone (7) (Iinuma et al., 1996), mangostin (8) (Mahabusarakam et al., 1987), macluraxanthone (9) (Iinuma et al., 1994), garcinone B (10) (Sen et al., 1982), celebixanthone (11) (Stout et al., 1962), and garcinone D (12) (Bennett et al., 1993). Cochinchinone A, 1,3,7-trihydroxy-2-(3-methyl-2-butenyl)-4-(3,7-dimethyl-2,6-octadienyl)xanthone (1), was a yellow solid, m.p. 119120 C. Its molecular ion of m/z 448.2299 was in agreement with the molecular formula C28H32O5. The IR spectrum showed the presence of OH (3413 cm1) and C@O (1641 cm1) groups. The 1H NMR spectrum (Table 1) exhibited signals of a hydrogen-bonded hydroxy proton at d12.95 (s, 1-OH) and three aromatic protons which coupled as an ABX system at d7.59 (d, J = 3.0 Hz, H-8), 7.36 (d, J = 9.0 Hz, H-5) and 7.24 (dd, J = 9.0, 3.0 Hz, H-6). The lower eld resonance at d7.59 was assigned for H-8 according to the anisotropic eect from C@O. Since H-8 showed only m-coupling in the 1H NMR spectrum, a hydroxyl group was placed at C-7.
Corresponding author. Tel./fax: +66 7421 2918. E-mail address: wilawan.m@psu.ac.th (W. Mahabusarakam).
W. Mahabusarakam et al. / Phytochemistry 67 (2006) 470474

5' 5'
471
O HO
7 8a 9 9a
OH
1 1 3
'
4'
O HO
7 8a 9a 9
OH
1 1 3
'
4'
4b
4a 1''
OH
HO
1''
4b
4a
OH
9''
4''
9''
4''
10''
8''
10'' 18
8''
19
O
12
15 5 4b
O
10
O
9
4a 3
R1
11
3
8
8a
9a
O
3
3 R1= H, R2= OH, R = OCH3 4 R1= R2= OH, R3= OCH3 5 R1= R2= OH, R3= H
The characteristic signals of protons in a prenyl unit were displayed at d3.47 (H-1 0 , d), 5.29 (H-2 0 , br t), 1.84 (H-4 0 , s) and 1.76 (H-5 0 , s). In addition, the presence of a geranyl side chain was indicated from the resonances at d3.57 (H100 , d), 5.27 (H-200 , br t), 2.04 (H-400 , m), 2.09 (H-500 , m),
Table 1 The 1H NMR and HMBC spectroscopic data of cochinchinones AB (12) H-position Cochinchinone A (1)
1
5.05 (H-600 , br t), 1.57 (H-800 , s), 1.88 (H-900 , s) and 1.64 (H-1000 , s). The correlation of H-1 0 to C-1, C-2 and C-3 in the HMBC spectrum (Table 1) established that the location of the prenyl unit was at C-2, whereas the correlation of H-100 to C-3, C-4 and C-4a indicated that the geranyl
Cochinchinone B (2) HMBC C-7, C-8a, C-4b C-7, C-4b C-6, C-7, C-9, C-4b C-1, C-2, C-3, C-2 0 , C-3 0 C-4 0 , C-5 0 C-2 0 , C-3 0 , C-5 0 C-2 0 , C-3 0 , C-4 0 C-3, C-4, C-4a, C-2 0 , C-3 0 C-4, C-400 C-200 , C-500 , C-600 C-300 , C-400 , C-600 C-500 , C-l000 C-600 , C-700 , C-1000 C-200 , C-300 , C-400 C-600 , C-700 , C-800 C-l, C-2, C-9a
1
H NMR
H NMR
HMBC C-2, C-3, C-4a, C-9, C-9a C-6, C-7, C-9, C-4b C-1, C-2, C-3, C-2 0 , C-3 0 C-1 0 , C-4 0 , C-5 0 C-2 0 , C-3 0 , C-5 0 C-2 0 , C-3 0 , C-4 0 C-5, C-6, C-4b, C-200 , C-300 C-100 , C-400 C-200 , C-300 , C-600 C-400 , C-600 , C-700 C-800 , C-1000 C-600 , C-700 , C-1000 C-200 , C-300 , C-400 C-600 , C-700 , C-800 C-1, C-2, C-9a
4 5 6 8 10 20 40 50 100 200 400 500 600 800 900 1000 1-OH
7.36 (d, 1H, J = 9.0 Hz) 7.24 (dd, 1H, J = 9.0, 3.0 Hz) 7.59 (d, 1H, J = 3.0 Hz) 3.47 (d, 2H, J = 7.0 Hz) 5.29 (br t, 1H, J = 7.0 Hz) 1.84 (s, 3H) 1.76 (s, 3H) 3.57 (d, 2H, J = 7.0 Hz) 5.27 (br t, lH, J = 7.0 Hz) 2.04 (m, 2H) 2.09 (m, 2H) 5.05 (br t, 1H, J = 7.0 Hz) 1.57 (s, 3H) 1.88 (s, 3H) 1.64 (s, 3H) 12.95 (s, 1H)
6.40 (s, 1H) 7.48 (s, 1H) 3.35 (d, 2H, J = 7.0 Hz) 5.26 (br t, 1H, J = 7.0 Hz) 1.78 (s, 3H) 1.66 (s, 3H) 3.56 (d, 2H, J = 7.0 Hz) 5.26 (br t, 1H, J = 7.0 Hz) 1.92 (m, 2H) 2.01 (m, 2H) 5.00 (br t, 1H, J = 7.0 Hz) 1.50 (s, 3H) 1.84 (s, 3H) 1.56 (s, 3H) 13.33 (s, 1H)
472
side chain was at C-4. Thus, the structure of cochinchinone A was deduced as 1. Cochinchinone B, 1,3,6,7-tetrahydroxy-2-(3-methyl-2butenyl)-5-(3,7-dimethyl-2,6-octadienyl)xanthone (2), was a yellow solid, m.p. 221222 C. The molecular formula was determined as C28H32O6 by HR-MS. The IR spectrum again showed the presence of OH (3350 cm1) and C@O (1640 cm1) groups. The 1H NMR spectrum (Table 1) indicated the presence of a hydrogen-bonded hydroxy group at d13.33 (s, 1-OH) and two isolated aromatic protons at d6.40 (s, H-4) and 7.48 (s, H-8). The resonances at d3.35 (d, H-1 0 ), 5.26 (br t, H-2 0 ), 1.78 (s, H-4 0 ) and 1.66 (s, H5 0 ) revealed the presence of a prenyl unit. The characteristic signals of a geranyl group were observed at d3.56 (d, H-100 ), 5.26 (br t, H-200 ), 1.92 (m, H-400 ), 2.01 (m, H-500 ), 5.00 (br t, H-600 ), 1.50 (s, H-800 ), 1.84 (s, H-900 ) and 1.56 (s, H-1000 ). The prenyl group was assigned at C-2 according to the HMBC correlation (Table 1) of H-1 0 to C-1, C-2 and C-3 whereas the geranyl group was placed at C-5 due to the correlation of H-100 to C-5, C-6 and C-4b. The structure 2 was therefore indicated for cochinchinone B. Cochinchinone C (3) was a yellow solid, m.p. 147 148 C and a29 50 (c 0.089, CHCl3). A pseudomolecuD lar ion [M CO]+ at m/z 382.1745 was consistent for the molecular formula of C24H26O6 with the loss of 28 amu (CO). The IR spectrum also showed absorption bands of OH stretching at 3467 cm1, conjugated C@O stretching at 1642 cm1 and unconjugated C@O stretching at 1749 cm1. The 13C NMR spectroscopic data (Table 3) exhibited carbon resonances at d180.73 (C-9) and 201.16 (C-6), conrming the presence of conjugated- and unconjugated carbonyl groups. The 1H NMR spectrum (Table 2) revealed resonances of a hydrogen-bonded hydroxy proton at d12.00 (s, 1-OH) and three aromatic protons which coupled as an ABM system at d6.55 (dd, J = 8.4, 0.9 Hz, H-2), 7.41 (t, J = 8.4 Hz, H-3), and 6.52 (dd, J = 8.4, 0.9 Hz, H-4). The spectrum further displayed the presence of an olenic
proton (d7.51, s, H-8), a methoxy group (d3.65, s, 7-OCH3), a pair of non-equivalent methylene protons (d1.59, dd, Hb10 and 2.39, d, Ha-10), a methine proton (d2.54, d, H-11) and a prenyl unit [d4.39 (br t, H-16), 2.64 (d, H-15), 1.37 (s, H-18) and 1.01 (s, H-19)]. The prenyl unit was placed at C-5 according to the correlation of H-15 to C-4b and C-5. The HMBC correlations (Table 2) of Ha-10 to C-4b, C-8, C-11; Hb-10 to C-7, C-8, C-11 and H-11 to C-4b, C5, C-10 suggested that CH2-10 and CH-11 were in between C-7 and C-4b. The signals of the methine proton H-11 and the gem-dimethyl groups (H-13, d1.69, s and H-14, d1.33, s) revealed a 2,2-dimethyltetrahydrofuran ring. It was suggested as being fused at C-5, C-4b and C-11 from the correlations of H-13 to C-11 and of H-11 to C-4b and C-5. Cochinchinone D (4) was shown to be a methoxy derivative of the synthetically known compound (5) (Thoison et al., 2000). It was a yellow solid, m.p. 218219 C. and 29 aD 58 (c 6.90 102 in CHCl3). The molecular ion [M CO]+ at m/z 398.1713 was in agreement to the molecular formula C24H26O7 with the loss of 28 amu (CO). The IR spectrum showed absorption bands of OH stretching at 3467 cm1 and of C@O stretching at 1646 (conjugated C@O) and 1738 (unconjugated C@O) cm1. In the 13C NMR spectrum (Table 3), the resonances of the carbonyl carbons were at d178.00 (C-9) and 201.00 (C-6). The 1H NMR spectrum (Table 2), showed a singlet for 1-OH at d12.39 and a broad singlet for 3-OH at d8.05, whereas the aromatic part was tetrasubstituted as evidence from signals at d6.05 (d, H-4) and 6.03 (d, H-2) with meta coupling (J = 2.1 Hz). The data from the 1H NMR and HMBC spectra (Table 2) and 13C NMR spectra (Table 3) indicated that the non-aromatic part contained a methoxy group (d3.62, 7-OCH3), an olenic proton H-8 (d7.44, s), a prenyl unit (d4.43, H-16; 2.63, H-15; 1.40, H-18 and 1.13, H-19), a pair of methylene protons Ha-10 (d2.35, d), Hb-10 (d1.59, dd), a methine proton H-11 (d2.50, d) and gem-dimethyl protons H-13 (d1.66) and H-14 (d1.31).
Table 2 The 1H NMR and HMBC spectroscopic data of cochinchinones CD (34) H-position Cochinchinone C (3)
1
Cochinchinone D (4) HMBC C-1, C-4 C-1, C-4a C-2, C-4a, C-9, C-9a C-4b, C-7, C-8a, C-9 C-4b, C-8, C-11 C-7, C-8, C-11 C-4b, C-5, C-10 C-11, C-12, C-14 C-11, C-12, C-13 C-4b, C-5, C-16, C-17 C-15, C-18, C-19 C-16, C-17, C-19 C-16, C-17, C-18 C-1, C-2, C-9a C-7
1
H NMR
H NMR
HMBC C-1, C-4, C-9a C-2, C-3, C-4a, C-9, C-9a C-4b, C-6, C-8a, C-9 C-4b, C-7, C-8, C-12 C-6,C-7,C-8,C-11 C-4b, C-7, C-12, C-13 C-11, C-12, C-14 C-11, C-12, C-13 C-4b, C-5, C-6, C-16, C-17 C-15, C-18, C-19 C-16,C-17,C-19 C-16,C-17,C-18 C-1, C-2, C-9a C-7
2 3 4 8 10a 10b 11 13 14 15 16 18 19 1-OH 3-OH 7-OCH3
6.55 (dd, 1H, J = 8.4, 0.9 Hz) 7.41 (1H, J = 8.4 Hz) 6.52 (dd, 1H, J = 8.4, 0.9 Hz) 7.51 (s, 1H) 2.39 (d, 1H, J = 12.9 Hz) 1.59 (dd, 1H, J = 12.9, 9.9 Hz) 2.54 (d, 1H, J = 9.9 Hz) 1.69 (s, 3H) 1.33 (s, 3H) 2.64 (d 2H, J = 8.1 Hz) 4.39 (br t, 1H, J = 8.1 Hz) 1.37 (s, 3H) 1.01 (s, 3H) 12.00 (s, 1H) 3.65 (s, 3H)
6.03 (d, 1H, J = 2.1 Hz) 6.05 (d, 1H, J = 2.1 HZ) 7.44 (s, 1H) 2.35 (d 1H, J = 132 Hz) 1.59 (dd, lH, J = 13.2, 9.6 Hz) 2.50 (d, 1H, J = 9.6 Hz) 1.66 (s, 3H) 1.31 (s, 3H) 2.63 (d 2H, J = 7.5 Hz) 4.43 (br t, 1H, J = 7.5 Hz) 1.40 (s, 3H) 1.13 (s, 3H) 12.39 (s, 1H) 8.05 (br s, 1H) 3.62 (s, 3H)
W. Mahabusarakam et al. / Phytochemistry 67 (2006) 470474 Table 3 The 13C NMR spectroscopic data of cochinchinones AD (14) C-position 1 2 3 4 4a 4b 5 6 7 8 8a 9 9a 10 20 30 40 50 100 200 300 400 500 600 700 800 900 1000 1 158.27 108.90 161.13 105.00 152.97 150.34 118.87 124.12 152.43 108.89 120.45 180.90 102.96 21.58 121.58 134.92 17.94 25.86 21.59 121.58 137.94 39.72 26.43 123.85 131.50 17.69 16.27 25.66 2 160.00 110.02 162.28 93.34 155.73 149.76 115.32 150.23 141.60 105.95 112.85 180.11 102.33 21.28 122.63 131.40 17.79 25.72 22.32 121.43 135.41 39.66 26.56 124.12 131.19 17.57 16.23 25.52 C-position 1 2 3 4 4a 4b 5 6 7 8 8a 9 9a 10 11 12 13 14 15 16 17 18 19 7-OCH3 3 162.90 109.57 138.97 107.41 159.44 88.76 84.18 201.16 84.86 135.25 132.14 180.73 106.15 29.73 49.43 83.96 30.37 29.04 29.21 118.48 135.73 25.51 16.69 54.09 4 160.50 95.49 167.88 96.95 164.50 88.50 83.78 201.00 84.50 133.25 132.25 178.00 100.50 29.87 49.27 83.76 30.26 28.94 28.98 117.86 135.50 25.46 16.88 53.84
473
more eective than butylated hydroxytoluene (BHT) with an IC50 of 28.9 lM. The greater eectiveness of compounds 2, 9 and 11 than the others was possibly due to the presence ortho-dihydroxy groups which upon donating hydrogen radicals will give higher stability to their radical forms (Shahidi and Wandasundara, 1992). Caged-prenylated xanthones have been exclusively found in the genus Garcinia (Cao et al., 1998; Thoison et al., 2000; Rukachaisirikul et al., 2000). The present work is the rst report of the caged-prenylated xanthones isolated from the genus Cratoxylum.
3. Experimental 3.1. General method Melting points were recorded with a digital electrothermal melting point apparatus (Electrothermal 9100) and are uncorrected. Optical rotations were measured on an AUTOPOL II automatic polarimeter. Ultraviolet spectra were measured with a UV-160A spectrophotometer (SHMADZU). Infrared spectra (IR) were obtained with a FTS165 FT-IR spectrometer. 1H and 13C-Nuclear Magnetic Resonance spectra were recorded with a FT-NMR Bruker Avance 300 MHz or Varian UNITY INOVA 500 MHz spectrometer, whereas high resolution mass spectra were obtained using a MAT 95 XL. Column and quick CC were performed on silica gel 100 and silica gel 60H (Merck), respectively. Precoated TLC sheets of silica gel 60 F254 were used. Known compounds were identied by comparison of their spectroscopic data with those in the literature. 3.2. Plant material The roots of C. cochinchinense (Clusiaceae) were collected from Amphur Bannasan, Suratthani Province in the southern part of Thailand in February 2003. The voucher specimen (No. W. Nuangnaowarat 1 Suratthani: Bannasan 31/3/04) was identied by Dr. Kitichate Sridith and has been deposited in the herbarium of the Department of Biology, Faculty of Science, Prince of Songkla University, Thailand. 3.3. Extraction and isolation Chopped, dried roots of C. cochinhinense (11 kg) were sequentially extracted at room temperature with CH2Cl2 (42 L) and MeOH (39 L) (3 days each). Removal of solvents in vacuo produced a yellow-brown, viscous, CH2Cl2 extract (294 g) and a MeOH extract (292 g), respectively. An aliquot of the CH2Cl2 extract (66 g) was subjected to quick CC over silica gel 60H using hexane, hexane CH2Cl2, CH2Cl2, CH2Cl2Me2CO and Me2CO as eluents to give fractions A1A14. Fractions A4 (8.51 g) and A6 (7.54 g) were further puried by crystallization in hexane
In their work on bractatin and derivatives, Thoison et al. (2000) have shown that caged-prenylated xanthones can exist in pure enantiomeric form (with relatively large optical rotation) or as mixtures of enantiomers, with low optical rotation values. It is concluded therefore that cochinchinone C and D are dierent mixtures of the two possible enantiomers of the structures 3 and 4, respectively, in which only the relative stereochemistry is shown. At the concentration of 50 lM (Table 4), the twelve isolated compounds were able to scavenge the DPPH radical in the range of 1.781.0%. Compounds 2, 9 and 11 exhibited the most potent radical scavenging activity with IC50 values of 9.4, 19.0 and 12.3 lM, respectively. These are
Table 4 Radical scavenging activity of compounds 112 (at 50 lM) Compounds 1 2 3 4 5 6 7 8 9 10 11 12 BHT % Scavenging of DPPH 20.7 79.3 1.7 5.2 5.2 1.7 20.7 5.2 75.9 6.9 79.3 6.9 51.7
474
CH2Cl2 to give the yellow solid 6 (738 mg). Fraction A7 (18.96 g) was subjected to silica gel column and eluted with hexaneCH2Cl2, CH2Cl2, CH2Cl2Me2CO to give fractions A7A (5.33 g), A7B (10.77 g) and A7C (1.94 g). Repeated chromatography of fraction A7A (2.00 g) on silica gel column using hexaneMe2CO (9:1) as mobile phase yielded the yellow solids 7 (55 mg), 1 (20 mg), 3 (15 mg) and 8 (60 mg). Fraction A7B (1.20 g) was submitted to CC over silica gel, eluted with hexaneMe2CO (17:3) to give the yellow solids 9 (6 mg), 5 (25 mg) and 4 (35 mg). Fraction A9 (4.06 g) was separated by CC over silica gel and eluted with hexaneMe2CO (4:1) to give the yellow solids 10 (15 mg), 2 (67 mg) and 11 (22 mg). Fraction A11 (300 mg) was applied to a silica gel column using CH2Cl2MeOH (49:1) as eluent to yield a yellow solid 12 (40 mg). 3.3.1. Cochinchinone A (1) Yellow solid, m.p. 119120 C. HREIMS m/z 448.2299 for C28H32O5 (calcd. 448.2250). UV (EtOH) kmax nm (log e): 232 (4.44), 268 (4.42), 316 (4.04), 384 (3.70). IR (KBr) m (cm1): 3413, 1641. EIMS m/z (% rel int): 449 ([M + H]+, 53), 448 ([M]+, 15), 447 (10), 393 (18), 325 (12), 323 (30), 309 (13), 281 (10), 277 (18), 270 (23), 269 (100), 257 (13), 253 (10), 185 (75), 93 (60). For 1H NMR and 13C NMR spectroscopic data, see Tables 1 and 3. 3.3.2. Cochinchinone B (2) Yellow solid, m.p. 221222 C. HRMS m/z 464.2189 for C28H32O6 (calcd. 464.2199). UV (EtOH) kmax nm (log e): 243 (4.35), 259 (4.32), 323 (4.10), 372 (3.98). IR (KBr) m (cm1): 3350, 1640. EIMS m/z (% rel int): 465 ([M + H+, 18), 464 ([M]+, 63), 422 (10), 421(39), 409 (76), 379 (17), 353 (11), 342 (24), 339 (31), 325 (24), 297 (46), 285 (45), 257 (25), 207 (13), 178 (58), 161 (32), 121 (37), 108 (50), 91 (76), 79 (77), 69 (96), 57 (100). For 1H NMR and 13C NMR spectroscopic data, see Tables 1 and 3. 3.3.3. Cochinchinone C (3) Yellow solid, m.p. 147148 C. HREMS [M CO]+ m/ z 382.1745 for C23H26O5 [M CO]+ (calcd. 382.1780). 29 Optical rotation: aD 50 (c 8.9 102 in CHCl3). UV (EtOH) kmax nm (log e): 206 (4.46), 222 (4.30), 307 (3.98), 346 (3.70). IR (KBr) m (cm1): 3467, 1749, 1642. EIMS m/z (% rel int): 382 ([M CO]+, 14), 381 (52), 312 (100), 285 (35), 243 (19), 68.9 (9). For 1H NMR and 13C NMR spectroscopic data, see Tables 2 and 3. 3.3.4. Cochinchinone D (4) Yellow solid, m.p. 218219 C. HREIMS m/z 398.1713 for (C23H26)6 [M CO]+ (calcd. 318.1729). Optical rota29 tion: aD 58 (c 6.90 102 in CHCl3). UV (EtOH) kmax nm: 212 (4.47), 275 (4.09), 332 (4.06), 357 (4.10). IR (KBr) m (cm1): 3392, 1738, 1646. EIMS m/z (% rel int): 398 ([M CO]+, 10), 397 (36), 329(16), 328 (100), 300 (29), 259 (16), 68.9 (13). For 1H NMR and 13C NMR spectroscopic data, see Tables 2 and 3.
3.4. Radical scavenging activity The experiments were modied from the method of Yamasaki et al., 1994. The sample solution (3.0 mM in absolute ethanol, 50 lL) was mixed with DPPH solution (0.05 mM, 3 mL) and allowed to stand at room temperature for 30 min. The absorbance was then measured at 517 nm. The results were expressed as percentage radical scavenging, % radical scavenging = [(Acontrol Asample)/ Acontrol] 100. The DPPH solution without sample was used as control. The IC50 values were obtained by linear regression analysis of the dose response curves, which were plots of % radical scavenging versus concentration. Measurements were performed in triplicate.
Acknowledgement This research was supported by a scholarship to W.N. from the Postgraduate Education and Research Program in Chemistry (PERCH), funded by The Royal Thai Government and the Graduate School, Prince of Songkla University.
References
Bennett, G.J., Harrison, L.J., Sia, G.-L., Sim, K.-Y., 1993. Triterpenoids, tocotrienols and xanthones from the bark of Cratoxylum cochinchinense. Phytochemistry 32, 12451251. Cao, S.-G, Sng, V.H.L., Wu, X.-H, Sim, K.-Y., Tan, B.H.K., Pereira, J.T., Goh, S.H., 1998. Novel cytotoxic polyprenylated xanthonoids from Garcinia gaudichaudii (Guttiferae). Tetrahedron 54, 1091510924. Iinuma, M., Tosa, H., Tanaka, T., Yonemori, S., 1994. Two xanthones from root bark of Calophyllwn inophyllum. Phytochemistry 35, 527532. Iinuma, M., Tosa, H., Tanaka, T., Riswan, S., 1996. Three new xanthones from the bark of Garcinia dioica. Chem. Pharm. Bull. 44, 232234. Mahabusarakam, W., Wiriyachitra, P., Taylor, W.C., 1987. Chemical constituents of Garcinia mangostana. J. Nat. Prod. 50, 474478. Nguyen, L.H.D., Harrison, L.J., 1998. Triterpenoid and xanthone constituents of Cratoxylum cochinchinense. Phytochemistry 50, 471476. Rukachaisirikul, V., Kaewnok, W., Koysomboon, S., Phongpaichit, S., Taylor, W.C., 2000. Caged-tetraprenylated xanthones from Garcinia scortechinii. Tetrahedron 56, 85398543. Sia, G.-L, Bennett, G.J., Harrison, L.J., Sim, K.-Y., 1995. Minor xanthones from the bark of Cratoxylum cochinchinense. Phytochemistry 38, 15211528. Sen, A.K., Sarkar, K.K., Mazumder, P.C., Banerji, N., Uusvuori, R., Hase, T.A., 1982. The structures of garcinones A, B and C: three new xanthones from Garcinia mangostana. Phytochemistry 21, 17471750. Shahidi, F., Wandasundara, P.K., 1992. Phenolic antioxidants. Crit. Rev. Food Sci. Nutr. 32, 67103. Stout, G.H., Stout, V.F., Welsh, M.J., 1962. The structure of celebixanthone. Tetrahedron Lett. 13, 541544. Thoison, O., Fahy, J., Dumontet, V., Chiaroni, A., Riche, C., Tri, M.V., Sevenet, T., 2000. Cytotoxic prenylxanthones from Garcinia bracteata. J. Nat. Prod. 63, 441446. Vo, V.V., 1997A Dictionary of Medicinal Plants in Vietnam, vol. 435. Y Hoc Publisher, HoChiMinh City. Yamasaki, K., Hashimoyo, A., Kokusenya, Y., Miyamoto, T., Sato, T., 1994. Electrochemical method for estimating the antioxidative eects of methanol extracts of crude drugs. Chem. Pharm. Bull. 42, 1663 1665.
Alkaloids from Oriciopsis glaberrima Engl. (Rutaceae)
Jean Duplex Wansi a,*, Jean Wandji b, Alain Francois Kamdem Wao a, a a Happi Emmanuel Ngeufa , Jean Claude Ndom , Serge Fotso c, Rajendra Prasad Maskey c, Dieudonne Njamen b, Tanee Zacharias Fomum b, Harmut Laatsch c
c a Department of Chemistry, Faculty of Science, University of Douala, P.O. Box 24157, Douala, Cameroon Department of Organic Chemistry, Faculty of Science, University of Yaounde I, P.O Box 812, Yaounde, Cameroon Department of Organic and Biomolecular Chemistry, Georg August University, Tammannstrasse 2, D-37077 Gottingen, Germany b
Received 9 March 2005; received in revised form 16 September 2005 Available online 21 November 2005
Abstract Two alkaloid derivative, oriciacridone A and B, were isolated from the stem bark of Oriciopsis glaberrima (Rutaceae). The structures were elucidated by a detailed spectroscopic analysis. The extract exhibited in vitro signicant antimicrobial activity against a range of micro-organisms. 2005 Elsevier Ltd. All rights reserved.
Keywords: Oriciopsis glaberrima; Rutaceae; Oriciacridone A and B; Antimicrobial activity
1. Introduction Oriciopsis glaberrima Engl. (Rutaceae) is a monotypic genus endemic to the humid rain forests of Cameroon (Letouzey, 1963). It is used as medicinal plant against infections, hypotension, mycoses, dermatitis and many other diseases (Bouquet, 1969). Previous phytochemical studies of O. glaberrima resulted in the isolation of one tretranortriterpenoid namely oriciopsin, and the furoquinoline alkaloid (Ayafor et al., 1982). This paper describes the isolation and structural elucidation of two new alkaloids; only the antimicrobial activities of the isolates and the extract were examined. 2. Results and discussion Ground, air-dried, stem bark of O. glaberrima was extracted with CH2Cl2/MeOH (1:1) at room temperature. The extract was concentrated under reduced pressure and
q *
Part 1 in the series Oriciopsis studies (Pygmees plant). Corresponding author. Tel.: +237 7817731. E-mail address: jdwansi@yahoo.fr (J.D. Wansi).
its anti-microbial activities against a range of micro-organisms were evaluated in vitro, using the agar diusion test. Following bioassay-directed chromatographic fractionation, two new alkaloid, oriciacridone A (1) and oriciacridone B (2), were isolated, together with the known lichexanthone (3). 25 Oriciacridone A (1), m.p. 294 C, aD 45:7 , was obtained as yellow crystals and reacted positively with FeCl3, thereby indicating the presence of a phenolic hydroxyl group. Alkaloid 1 was shown to have the molecular formula of C36H32N2O9 by HR-EIMS ([M]+; m/z = 636.2108; calc. 636.21024). The IR (m = 3360, 2972, 1649, 1620, 1563 cm1) and UV (kmax = 401, 352, 323, 285, 272, 269 nm) spectra suggested the presence of a 9-acridone moiety (Takemura et al., 1998) and a xanthone skeleton (Peres and Nagem, 1997; Peres et al., 2000). The characteristic signals of two chelated hydroxyl protons at d 14.40 and d 13.15 (s, each 1H) in the 1H NMR spectrum, coupled with its molecular ion, suggested a dimeric structure. Similarly, the resonances at d 11.32 (s br, 1H) and d 10.70 (s br, 1H) indicated the presence of two further D2O exchangeable protons. The 1H NMR spectrum also showed signals of ve aromatic protons at d 8.14 (dd, J = 7.8, 1.8 Hz,
476
J.D. Wansi et al. / Phytochemistry 67 (2006) 475480
Structures of compounds 1, 2 and 3

O
8 9 10 11 7a 11a 12 12a 7 6a
OH
6 5
N H
15
4a 12b 1 4
O
3 13
OH
11' 10' 9' 14' 8' 7' 6a' 6' 10a' 11a' 5a'
O
12' 12a' 4a' 5'
H OH
14
1' 2' 3' 4'
O
13'
OH
Oriciacridone A(1)
O
8 9 10 11 7a 11a 12 12a 7 6a
OH
6 5
N OH OH
11' 10' 9' 14' 8' 7' 6a' 6' 10a' 11a' 5a' 5' 12' 12a' 4a' 1'
4a 12b 1 15 4
O
3 13
H O H N H
2 14
OH
2' 3' 4'
O
13'
OH
Oriciacridone B (2)
CH3 O OH
(C-13), 23.7 (C-14), 42.5 (C-1), 70.9 (C-2), and 76.9 (C-3) (Magiatis et al., 1999). 2D NMR techniques (HSQC and HMBC) indicated a hydroxydimethylchroman ring. In the HMBC spectrum (Fig. 1), the proton at d 5.29 showed cross-peaks with carbon signals at C-12a (d 141.5), C-12b (d 96.7), and C-4a (d 158.8). This nding clearly indicated that the hydroxydimethylchroman ring was fused in an angular fashion. All this information is in agreement with the acridone skeleton (Bahar et al., 1982; Magiatis et al., 2001). The presence of an acridone skeleton was also conrmed by the EI MS mass spectrum, which showed a peak at m/z 311 (C18H16NO4). Furthermore, the 1H NMR spectrum showed signals corresponding ortho-coupled aromatic protons at d 7.13 and 6.50 (d, J = 8.8 Hz, 1H each), an aromatic proton singlet at d 6.28 (s, 1H) and a dimethylchroman ring [d 1.14 (s, 6H), 1.48 (m, 2H), 2.54 (m, 2H)] indicating the presence of a dihydro-6-desoxyjacareulin skeleton (Locksley et al., 1971). The latter was conrmed by 13C NMR, DEPT, and 2D NMR techniques (HMBC, HSQC and COSY). The HMBC spectrum showed cross-peaks of the chelated hydroxyl signal at d 13.15 with the carbon signals at C-11a 0 (d 100.9) and C-10a (d 110.9), and between the aromatic proton singlet at d 6.28 (H-6 0 ) and C-11a 0 (d 100.9), C-10a 0 (d 110.9). Further correlations were observed between the aromatic proton at d 6.50 (H-2 0 ) and carbon signals at C-4 0 (d 150.6) and C-12a 0 (d 105.2), and between the proton at d 7.13 (H-3 0 ) and carbons C-1 0 (d 136.3), and C-4a 0 (d 133.3). Thus, 1 has a dihydro-6-desoxyjacareulin group linked to an acronycine skeleton. To conrm the linkage of the two skeletons, 2D NMR (HMBC and NOESY) experiments were used. In the HMBC spectrum, cross-peaks between the proton signal at d 9.56 (H-15) and carbons C-2 0 (d 105.2) and C-2 (d 70.9), and between the proton at d 5.29 (H-1) and carbon signals at C-1 0 (d 136.3), C-12a (d 141.5), and C-4a
H
8 9 7a 11a 11
O
7 6a 12a 12
OH
6 5
H3CO
OCH 3
10
Lichexanthone (3)
OH
11'
N H
15
4a 12b 1 4
O
3 13
O
12'
N H
2 14
OH
11a' 5a' 12a' 4a' 5' 1' 2' 3' 4'
1H), 7.80 (s br, 1H), 7.78 (s br, 1H), 7.30 (m, 1H), and 5.98 (s, 1H), the signals of a substituted pyran ring at d 1.62 (s, 3H), 1.42 (s, 3H), 5.29 (dd, J = 3.7, 3.8 Hz, 1H), 4.30 (d, J = 3.8 Hz, 1H), and a doublet at d 9.56 (d, J = 5.2 Hz, 1H). The aromatic loweld signal at d 8.14 was deshielded by a carbonyl group (Takemura et al., 1998). The presence of a pyran ring was further conrmed by the 13C NMR spectrum, which showed characteristic signals at d 22.2
10' 9' 14' 8' 7' 6a' 6' 10a'
O
13'
O H
OH
Fig. 1. Signicant long-range correlations observed in 1H13C HMBC for compound 1 in DMSO-d6.
477
(d 158.8), suggested that the two substructures were linked by nitrogen, furthermore, in the NOESY spectrum, there were cross-peaks between the proton H-15 (d 9.56) and the aromatic proton H-2 0 (d 6.50), and between the same proton H-15 (d 9.56) and protons at d 5.29 (H-1) and d 4.30 (H-2), indicating clearly the linkage position of the two fragments. The coupling constant of J = 3.7 Hz between protons H-1 and H-2 showed that they are in a cis relationship (Magiatis et al., 1999). From the above spectroscopic studies, compound 1 was characterized as ()-cis-2-
hydroxy-1-(3,4-dihydro-6-desoxyjacareulin)amino-1,2dihydroacronycine named oriciacridone A. 25 Alkaloid 2, m.p. 309 C, aD 85:3 , isolated as a yellow solid, had the molecular formula C36H32N2O10 (HREIMS, m/z found 652.2061; calc. 652.20515) and thus one oxygen more than oriciacridone A (1). The 13C NMR spectrum revealed 36 carbon signals, which were sorted by DEPT and APT experiments into four methyl, two methylene, eight sp2 methine, two sp3 methine, and 20 quaternary carbons; among the latter, two were
Table 1 1 H (300 MHz) and Attribution
13
C (75.5 MHz) assignments for oriciacridone A (1) and oriciacridone B (2) in DMSO-d6 1
13
2 C
1
H [m, J (Hz)]
13
H [m, J (Hz)] (m) (d, 3.9)
1 2 3 4 4a 5 6 6a 7 7a 8 9 10 11 11a 12 12a 12b 13 14 10 20 30 40 4a 0 50 5a 0 60 6a 0 70 80 90 10 0 10a 0 11 0 11a 0 12 0 12a 0 13 0 14 0 2-OH 6-OH 4 0 -OH 11 0 -OH 11-OH 12-NH 15-NH
42.5 70.9 76.9 158.8 95.7 162.7 104.6 180.2 118.8 124.7 121.9 133.3 117.5 140.4 141.5 96.7 22.2 23.7 136.3 105.2 121.9 150.6 133.3 154.5 92.6 163.2 68.9 42.3 17.0 110.9 159.3 100.9 182.6 101.9 29.1 29.0
5.29 (dd, 3.7, 3.8) 4.30 (d, 3.7) 5.98 (s) 8.14 (br d, 7.8) 7.30 (ddd, 6.8, 5.6, 2.3) 7.78 (br d, 5.7) 7.82 (td, 8.7, 1.5) 1.42 (s) 1.62 (s) 6.50 (d, 8.8) 7.13 (d, 8.8) 6.28 (s) 1.48 (m) 2.54 (m) 1.14 (s) 1.14 (s) 4.14 (br s) 14.40 (s) 11.32 (br s) 13.15 (s) 10.70 (br s) 9.56 (d, 5.2)
42.8 71.5 76.2 159.0 94.9 163.9 105.2 181.6 119.5 115.5 123.3 119.9 148.8 141.5 142.8 96.7 22.9 24.9 136.3 105.7 122.0 151.2 134.3 155.4 93.2 163.2 68.9 43.2 15.2 110.9 159.2 100.6 183.8 101.9 25.4 27.4
5.72 4.13 5.99 7.57 7.22 7.17 1.39 1.62 6.58 7.18 6.30 1.45 2.57
(s)
(dd, 9.0, 3.0) (d, 9.0) (d, 9.0)
(s) (s) (d, 11.4) (d, 11.4)
(s)
(m) (m)
1.18 (s) 1.18 (s) 10.72 (br 14.60 (s) 10.84 (br 13.12 (s) 10.59 (br 10.42 (br 9.28 (m)
s) s) s) s)
478
carbonyls. The IR spectrum showed vibration bands at 3428, 3250, and 1664 cm1, due to hydroxyl groups, a methine, and a chelated carbonyl group, respectively. These data, together with those obtained from UV (kmax 257, 275, 278, 295, 312, 372, 404 nm), 1H NMR (two singlets, 1H each at d 14.60 and 13.12 due to chelated OH) and 13 C NMR data (Table 1), suggested that alkaloid 2 has the same skeleton as oriciacridone A (1). Furthermore, in the 1 H NMR spectrum, an ABM spin system corresponding to a 1,2,3-trisubstituted benzene ring [d 7.57 (dd, J = 9.0, 3.0 Hz, 1H), 7.22 (d, J = 9.0 Hz, 1H), 7.17 (d, J = 9.0 Hz, 1H)], three hydroxyl signals [d 10.84, 10.59, 10.72 (s br, each 1H)], and signals corresponding to hydroxyldimethylchroman and dimethylchroman units were also observed. These data suggested that the additional oxygen atom in 2 is attached as a hydroxy group in the acronycine moiety. Since the loweld signal at d 7.57 of the ABM-type aromatic protons was deshielded by a carbonyl group and the presence of H-8, H-9 and H-10 of acridone skeleton was revealed, this indicated that the free phenolic hydroxyl group was attached to C-11 (Basa, 1975). This location was conrmed by the NOESY spectrum in which a cross-peak was observed between the hydroxyl proton H-11 (d 10.59) and the proton at H-12 (d 10.42) and by HMBC experiments. Therefore, the structure of alkaloid 2 was concluded to be ()-cis-2,11-dihydroxy-1-(3,4-dihydro-6-desoxyjacareulin)amino-1,2-dihydroacronycine, named oriciacridone B. Compounds 13 were tested for their antimicrobial potential against four bacteria (Bacillus subtilis, Streptomyces viridochromogenes, Staphylococcus aureus, and Escherichia coli), two fungi (Mucor miehei and Candida albicans), and three microalgae (Chlorella vulgaris, Chlorella sorokiniana, and Scenedesmus subspicatus) in the agar diusion test, as shown in Table 2. The extract exhibited in vitro signicant antimicrobial activity against C. albicans, M. miehei and S. aureus. Oriciacridone B (2) also exhibited in vitro signicant
antimicrobial activity against M. miehei compared to the Nystatin as reference.
3. Experimental section 3.1. General Melting points are uncorrected; optical rotations: PerkinElmer model 241 polarimeter. 1H (300 and 600 MHz) and 13C NMR spectra (75.5 and 125.7 MHz) were measured on a Bruker AMX 300 and on a Varian Inova 600 (599.740 MHz) spectrometer, respectively. ESI mass spectra were recorded on a Quattro Triple Quadrupole Mass Spectrometer, Finnigen TSQ 7000 with nano-ESI-APIion source. ESI HRMS was measured on a Micromass LCT mass spectrometer coupled with a HP 1100 HPLC with a Diode Array Detector. Reserpin (MW = 608) and LeucinEnkephalin (MW = 555) were used as standards in positive and negative modes. EI-MS was recorded on Varian MAT 95 Finnigan (70 eV), high resolution with peruorokerosine as standard. IR spectra were recorded on a PerkinElmer 1600 Series FT-IR spectrometer as KBr pellets. UVVIS spectra were recorded on a Perkin Elmer Lambda 15 UV/VIS spectrometer. Flash chromatography was carried out on silica gel (230400 mesh, Merck) and silica gel (70230 mesh, Merck) was used for column chromatography. Thin layer chromatography (TLC) was performed on Polygram SIL G/UV254 (Macherey-Nagel & Co.). 3.2. Plant material Stem bark of O. glaberrima was collected in January 2002, at Bertoua, Cameroon. The plant was identied by Nana Victor of National Herbarium. A voucher specimen (1888/HNC) documenting the collection is deposited in the National Herbarium, Yaounde, Cameroon.
Table 2 Diameter of inhibition zones of compounds 13 from Oriciopsis glaberrima in the agar diusion test with 20 lg/disk (B in mm, paper disk B 9 mm) Sample Micro-organism tested BSa CH2Cl2MeOH (1/1) Oriciacridone A (1) Oriciacridone B (2) Lichexanthone (3) Nystatin
a b c d e f g h i
SVb 13 11 11 10 14
SAc 12 9 11 11 12
ECd 11 10 9 9 14
MMe 17 11 15 12 15
CAf 18 14 12 11 18
CVg 15 12 10 12
CSh 11 9 9 10
SSi 13 9 10 9
10 10 12 9 16
Bacillus subtilis. Streptomyces viridochromogenes. Staphylococcus aureus. Escherichia coli. Mucor miehei. Candida albicans. Chlorella vulgaris. Chlorella sorokiniana. Scenedesmus subspicatus.
479
4. Extraction and isolation Air-dried; powdered stem bark of O. glaberrima (10 kg) was extracted with a mixture of CH2Cl2/MeOH (1/1) at room temperature during 48 h. The extract was concentrated under reduced pressure to yield a brown viscous extract (99 g). This extract was evaluated for its antimicrobial activity and then subjected to ash column chromatography on silica gel (500 g) (70230 mesh, Merck) with a hexaneEtOAcMeOH mixture of increasing polarity. A total of 90 sub-fractions (ca. 250 ml each) were collected and combined on the basis of TLC analysis leading to four main fractions AD. Sub-fractions 120, eluting with a mixture of hexaneEtOAc (17:3) gave main fraction A (15 g). Fraction B (20 g) was constituted of sub-fractions 2140 eluted with a mixture of hexaneEtOAc (1:1), main fraction C (10 g) was constituted of sub-fractions 4166 eluted with hexaneEtOAc (1:4), and fraction D (4 g) was constituted of sub-fractions 6790 eluted with EtOAc MeOH (19:1). Main fraction A was chromatographed on a silica gel column (250 g) with a hexaneEtOAc gradient. A total of 20 fractions of ca. 100 ml each were collected and combined on the basis of TLC. Fractions 110 eluted with a mixture of hexaneEtOAc (9:1) yielded lichexanthone (3) (10 mg). Main fraction C was column chromatographed over silica gel with hexaneEtOAc (2:1). A total of 30 fractions of ca. 100 ml each were collected and combined on the basis of TLC. Fractions 110, eluted with a mixture of hexaneEtOAc (1:9), yielded oriciacridone A (1) (30 mg). Fractions 2030 eluted with EtOAc yielded oriciacridone B (2) (5 mg). 4.1. Oriciacridone A (1) Yellow crystals; m.p. 249 C; aD 45:7 (DMSO, c 0.070); UV (DMSO) kmax nm (log e) 269 (4.84), 272 (4.85), 285 (4.62), 323 (4.28), 352 (4.15), 401 (4.14); IR (KBr) mmax cm1: 3360, 2972, 2362, 1920, 1731, 1649, 1619,1558, 1489, 1394, 1289, 1156, 1025; 1H NMR (300 MHz, DMSO-d6) and 13C NMR (75.5 MHz, DMSO-d6) see Table 1; HR-EIMS m/z 636.2108 (calc. for C36H32N2O9, 636.21024); EI MS (70 eV) m/z (%): 636 [M]+ (15), 619 (40), 580 (18), 410 (45), 363 (5), 346 (60), 311 (100), 298 (80), 245 (20), 191 (30), 149 (35), 119 (28), 101 (50). 4.2. Oriciacridone B (2) Yellow crystals, m.p. 309 C; aD 85:3 (DMSO, c 0.075); UV (DMSO) kmax nm (log e) 239 (4.48), 257 (4.60), 275 (4.40), 278 (4.40), 295 (4.12), 312 (4.13), 372 (3.80), 404 (3.88); IR (KBr) mmax cm1: 3425, 2923, 2853, 2376, 2246, 1650, 1620,1558, 1502, 1471, 1380, 1360, 1279, 1171, 1025, 819; 1H NMR (300 MHz, DMSO-d6) and 13C NMR (75.5 MHz, DMSO-d6) see Table 1; HR EIMS m/z 652.2061 (calc. for C36H32N2O10, 652.20515); EI MS
25 25
(70 eV) m/z (%): 652 [M]+ (20), 635 (10), 620 (30), 537 (40), 394 (25), 345 (15), 317 (100), 294 (80), 150 (45), 97 (18). 4.3. Lichexanthone (3) M.p. 187188 C; IR (KBr), 1H NMR and EI MS data were identical with those reported by Garcia et al. (1976).
5. Antimicrobial assay Agar diusion tests were performed in the usual manner (Maskey et al., 2002) with B. subtilis and E. coli (on peptone agar), S. aureus (Bacto nutrient broth), S. viridochromogenes (M2 agar), the fungi M. miehei and C. albicans (Sabouraud agar), and three microalgae (C. vulgaris, C. sorokiniana and S. subspicatus). Compounds 1, 2, 3 and Nystatin were dissolved in MeOH/chloroform (87:18) at a concentration of 500 lg/ ml. Paper disks (B 9 mm) were impregnated with 40 ll each using a HPLC syringe, dried for 1 h under sterile conditions and placed on the pre-made agar test plates. Bacteria and fungi plates were kept in an incubator at 37 C to 12 h, micro algae plates for three days at room temperature in a day light incubator. The diameter of inhibition zones was measured.
Acknowledgments One of the authors (J.D.W.) thanks the DAAD (Deutscher Akademischer Austanschdienst) for a visiting grant. The authors are also grateful to Mr. R. Machinek for the NMR measurements, to Dr. H. Frauendorf for the mass spectra, and to Mrs. F. Lissy for the antimicrobial screening.
References
Ayafor, F.J., Sondengam, L.B., Kimbu, F.S., Tsamo, E., Connolly, D.J., 1982. Phytochemistry 21, 26022603. Bahar, M.H., Shringarpure, J.D., Kulkarni, G.H., Sabata, B.K., 1982. Structure and synthesis of atalaphylline and related alkaloids. Phytochemistry 21, 27292731. Basa, S.C., 1975. Atalaphyllinine, a new acridone base from Atalantia monophylla. Phytochemistry 14, 835836. Bouquet, A., 1969. Feticheurs et Medecines Traditionnelles du Congo Brazzaville, ORSTON, Paris, 220. Garcia, M., Ruben, F., Brown Jr., K.S., 1976. Alkaloids of three Aspidosperma species. Phytochemistry 15, 10931095. Letouzey, R., 1963. Flore du Cameroun 1. Museum National dHistoire Naturelle, Paris. Locksley, H.D., Quillinan, A.J., Scheinmann, F., 1971. Extractives from Guttiferae, Part XXIII. Unambiguous synthesis of 6-deoxyjacareulin and related 3,3- and 1,1-dimethylallyl and annulated xanthones. J. Chem. Soc. (C), 38043814. Magiatis, P., Mitaku, S., Skaltsounis, A.L., Tillequin, F., Koch, M., Pierre, A., Atassi, G., 1999. Synthesis and cytotoxic activity of 1alkoxy- and 1-amino-2-hydroxy-1,2-dihydro-acronycine derivatives. Chem. Pharm. Bull. 47, 611614.
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J.D. Wansi et al. / Phytochemistry 67 (2006) 475480 Peres, V., Nagem, T.J., 1997. Trioxygenated naturally occurring xanthones. Phytochemistry 44, 191214. Peres, V., Nagem, T.J., Faustino de Oliveria, F.F., 2000. Tetraoxygenated naturally occurring xanthones. Phytochemistry 55, 683 710. Takemura, Y., Wada, M., Ju-Ichi, M., Ito, C., Furukawa, H., 1998. A new bimeric acridone alkaloids from Citrus pardisi MACF. Chem. Pharm. Bull. 46, 693696.
Magiatis, P., Mitaku, S., Skaltsounis, A.L., Tillequin, F., 2001. 1-Oxo-2hydroxy-1,2-dihydroacronycine: a useful synthon in the acronycine series for the introduction of amino subsistent at 6-position and for the conversion into isopropyl furanoacridones. Chem. Pharm. Bull. 49, 13041307. Maskey, R.P., Asolkar, R.N., Kapaun, E., Wagner-Dobler, I., Laatsch, H., 2002. Phytotoxic arylethylamides from limnic bacteria using a screening with microalgae. J. Antibiot. 55, 643649.
Terpenoids and phenol derivatives from Malva silvestris

Francesca Cutillo a, Brigida DAbrosca b, Marina DellaGreca Antonio Fiorentino b, Armando Zarrelli a
a
a,*
` Dipartimento di Chimica Organica e Biochimica, Universita Federico II, via Cynthia 4, I-80126 Napoli, Italy b ` Dipartimento di Scienze della Vita Seconda Universita di Napoli, via Vivaldi 43, I-81100 Caserta, Italy Received 5 September 2005; received in revised form 21 November 2005 Available online 5 January 2006
Abstract A sesquiterpene and a tetrahydroxylated acyclic diterpene as well as two known monoterpenes, 6 C13 nor-terpenes and 11 aromatic compounds were isolated from the water extract of Malva silvestris. The structures of the compounds were determined by spectroscopic NMR and MS analysis. Eects of these compounds on germination and growth of dicotyledon Lactuca sativa L. (lettuce) were studied in the 104107 M concentration range. 2005 Elsevier Ltd. All rights reserved.
Keywords: Malva silvestris; Lactuca sativa; Phytotoxic activity; Spectroscopic analysis; Terpenes; Phenols
1. Introduction Malva silvestris is a species widely distributed in Italy and used in traditional phytotherapy (Guarrera, 2005) and cosmetic treatments (Pauque, 2000). Fluidextract of M. silvestris owers and leaves are used as a valuable remedy for cough and inammatory diseases of mucous membranes (Farina et al., 1995). The chemical composition of a water extract of M. silvestris has been investigated, and resulted in the isolation and structure elucidation of a novel sesquiterpene and a new tetrahydroxylated linear diterpene as well as two monoterpenes, six C13 nor-terpenes and eleven aromatic compounds. 2. Results and discussion Fresh plants of M. silvestris were extracted with water using a Naviglio extractor (Naviglio, 2003). This extractor is based on the suction eect, generated by the compression of the solvent used for extraction on solids at a pressure of about 89 bars for a particular time period, followed by
*
Corresponding author. Tel.: +39 081 674162; fax: +39 081 674393. E-mail address: dellagre@unina.it (M. DellaGreca).
immediate decompression at atmospheric pressure. The rapid release of the liquid used for extraction from the inside of a solid matrix, because of pressure gradient, transports the extractable compounds within the solid matrix towards the outside. After extraction of the aqueous portion shaken with EtOAc, the organic fraction was chromatographed on a silica gel column, the fractions were puried by preparative thin layer chromatography and HPLC yielding 21 compounds. A test of extraction conducted using conventional procedure (Cutillo et al., 2005) resulted in similar amount of extract. The compounds were identied as 4-hydroxybenzoic acid (1), 4-methoxybenzoic acid (2), 4-hydroxy-3-methoxybenzoic acid (3), 2-hydroxybenzoic acid (4), and 4hydroxy-2-methoxybenzoic acid (5), compounds 6 and 9 as 4-hydroxybenzyl alcohol and tyrosol, compounds 7 and 8 as 4-hydroxydihydrocinnamic acid and 4-hydroxy3-methoxydihydrocinnamic acid, and 10 and 11 as 4hydroxycinnamic acid and ferulic acid by comparison of their spectral data with those of authentic samples. Monoterpenes 12 and 13 were linalool and linalool-1-oic acid (Nicoletti et al., 1989). The EIMS of compound 14 showed a molecular ion peak at m/z 262, and prominent peaks at m/z 245 [M OH]+, 234 [M CO]+, and 219 [M C3H7]+. The molecular formula
482 Table 1 NMR spectral data of compound 14 in CDCl3 Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 OMe

a 1
F. Cutillo et al. / Phytochemistry 67 (2006) 481485
dHa
NOESY
dC 125.1 142.9 144.0 127.0 115.8 139.5 74.7 27.3 23.6 27.8 31.2 18.5 17.8 67.9 (q) (q) (q) (q) (t) (q) (q) (s) (s) (t) (t) (p) (p) (s)
c
HMBCb
6.69 s
12, 13, 15
1, 3, 7
2.12 1.97 3.45 2.43 1.14 1.13 4.00 3.45 2.32 3.81
m, 1.30 m m, 1.49 m m q (6.8) d (6.8) d (6.8) dd (8.3, 2.4), m s s
5 5
6, 1 2, 6, 7, 7, 1,
7, 9 6 7, 12, 13 11 11 7, 9, 10
5, 12, 13, OMe 15
16.1 (p) 60.7 (p)
3, 4, 5 3
H chemical shift values (d ppm from SiMe4) followed by multiplicity and then the coupling constants (J in Hz). b HMBC correlations from H to C. c Letters, p, s, t and q, in parentheses indicate, respectively, the primary, secondary, tertiary and quaternary carbons, assigned by DEPT.
was determined to be C16H22O3 by HREIMS. Its IR spectrum showed absorption bands of hydroxyl group (3300 cm1) and phenyl group (1600 cm1). The structure of compound 14 was established using 1H NMR and 13C NMR including COSY, NOESY, HMQC, and HMBC experiments (Table 1). The 1H1H COSY experiment showed a correlation series beginning with signal of a methine at d 2.43 assigned to H-11 and coupled to two methyls at d 1.14 and 1.13. The signals at d 4.00 and 3.45 assigned to methylene H-14 were correlated with H-10 at d 3.45, which in turn was coupled to the two H-9 protons at d 1.97 and 1.49. These were correlated with H-8 at d 2.12 and 1.30. Present also, were three singlets attributed to two methyls and a methine. The 13C NMR spectrum of 14 showed 16 carbon signals due to four methyls, three methylenes, and three methines. An HMQC experiment allowed to assign the protons to the corresponding carbons. In the HMBC spectrum H-5 was correlated with C-1, C-3, and C-7, while the methoxyl group at d 3.81 and the methyl at d 2.32 were correlated with the C-3. The multiplet at d 3.45 (H-10) was correlated with C-2 and C-6. While H-14 was correlated with C-1, C7, C-9, and C-10, thus completely dating the structure of 14. The analysis of NOESY spectrum evidenced NOEs of
OH
10 9 8 1 14 3 6 11 12 4 2
the methyls at d 1.14 and 1.13 with H-5 methine, and the methyl at d 2.32 with the methoxyl at d 3.81 (Fig. 1). Compounds 15 and 16 were identied as (6R,7E,9S)9-hydroxy-4,7-megastigmadien-3-one and blumenol A (Cutillo et al., 2005) and compound 17 as (+)-dehydrovomifoliol (Mori, 1974) on the basis of their spectral data. Compounds 1820 had spectral data identical with those reported for (3R,7E)-3-hydroxy-5,7-megastigmadien-9-one, (3S,5R,6S,7E,9R)-5,6-epoxy-3,9-dihydroxy-7-megastigmene and (3S,5R,6R,7E,9R)-3,5,6,9-tetrahydroxy-7-megastigmene isolated from Chenopodium album and Cestrum parqui (DellaGreca et al., 2004; DAbrosca et al., 2004a). Compound 21 showed a molecular ion peak at m/z 340, and peaks at m/z 325 [M CH3]+, 322 [M H2O]+, and 297 [M C3H7]+. The molecular formula was determined to be C20H36O4 by HREIMS. The four oxygen functions were ascribed to two secondary hydroxyl groups and the remaining two were attributed to tertiary hydroxyl groups (Table 2). The structure of 21 was characterized by 1H NMR and 13C NMR including COSY, NOESY, HMQC, and HMBC experiments. Five singlet methyls, ten aliphatic protons of ve methylenes, two methines bearing oxygen, ve olenic protons, two as broad triplets, and three as double doublets were present in the 1H NMR spectrum of 21. The 13C NMR spectrum showed 19 carbon signals identied as ve methyls, six methylenes, and ve methines. All the carbons were correlated to the corresponding protons on the basis of an HMQC experiment. The tertiary hydroxyl groups were positioned at C-3 and C-15 on the basis of an HMBC experiment that showed correlations between the C-3 carbon with the H-1, H-2, H-4 protons, and C-15 with the H-14, H-16, and H-17 protons. Furthermore, NOESY correlations of H-8 with H-6, H-10, and CH3-19, and H-14 with CH3-16 and CH3-17 conrmed the structure of diterpene 21 (Fig. 1). The absolute congurations at the C-8 and C-14 secondary carbinol carbons have been established by Moshers method (Dale and Mosher, 1973) by converting compound 21 into the diasteromeric MTPA diesters. The chemical shift dierences of protons, at b position of C-8 and C-14 chiral carbons, were assigned by a 1H1H COSY experiment (Ohtani et al., 1991). The chemical shifts comparison of the signals due to H-9 and H-6/H-19 protons in both the (R) and the (S) MPTA derivatives and the calculation of the corresponding dierences, expressed as DdRS, were in agreement with the S conguration for C-8. For the C-14
OMe
16
OH
15
OH
10 12
OH
4 7
OH
1
O
15
18
19
20
14
21
Fig. 1. Structure of terpenes isolated from M. silvestris.
F. Cutillo et al. / Phytochemistry 67 (2006) 481485 Table 2 NMR spectral data of compound 21 in CD3OD Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
a 1
483
dHa 5.19 dd, 5.03 dd (17.0, 10.5, 1.5) 5.94 dd (17.0, 10.5) 1.52 dd (8.0, 7.6) 2.08 ddd (8.0, 7.6, 7.0) 5.34 brt (7.0) 3.93 t (6.8) 2.24 m 5.16 brt (7.0) 2.24 m, 2.05 m 1.70 m, 1.34 m 3.23 dd (10.4, 2.0) 1.16 1.15 1.63 1.60 1.25 s s s s s
NOESY
dC 112.6 (s)
c
HMBCb 2, 3 3 2, 3, 5, 6, 20 4, 6, 7 4, 5, 8, 19 6, 7, 9, 10, 19 7, 8, 10, 11 12, 18 10, 11, 13, 18 12 12 14, 15, 17 14, 15, 16 10, 12 6, 7, 8 2, 3, 4
4, 8 6, 10, 19 8, 12
12, 16, 17 14 14 8
146.7 (t) 74.3 (q) 43.5 (s) 23.8 (s) 127.9 (t) 138.4* (q) 79.2 (t) 35.2 (s) 122.4 (t) 138.2* (q) 38.5 (s) 31.2 (s) 79.5 (t) 74.3 (q) 25.5 (p) 26.1 (p) 16.9 (p) 11.9 (p) 28.1 (p)
in CDCl3 or CD3OD at 25 C. MS spectra were obtained with a HP 6890 spectrometer equipped with a MS 5973 N detector. IR spectra were recorded on a Jasco FT/IR430 instrument. UVVis spectra were recorded in CHCl3 or MeOH on a PerkinElmer Lambda 7 spectrophotometer. HPLC was performed on an Agilent 1100 by using an UV detector. Silica gel 60 (230400 mesh, Merck) was used for CC, and preparative TLC was performed on silica gel (UV-254 precoated) plates with 0.5 and 1.0 mm thickness (Merck). Preparative HPLC was performed using RP-18 (LiChrospher 10 lm, 250 10 mm i.d., Merck) column. 3.2. Plant material Aerial parts of M. silvestris were collected near Caserta (Italy) in the spring of 2003 and identied by Professor Antonino Pollio of the Dipartimento di Biologia Vegetale of University of Naples. Voucher specimens (HERBNAQA650) are deposited at the Dipartimento di Biologia Vegetale of University Federico II of Naples. 3.3. Extraction and isolation Fresh leaves (10.0 kg) of the plant were extracted with H2O at room temperature using the Naviglio extractor. The water was reduced in volume and partitioned between EtOAc and H2O. The organic extract (19 g) was subjected to silica gel column chromatography, by using CHCl3 and successively increasing the EtOAc concentration by 25%, 50% and 80% in CHCl3. Fractions of 200 ml were collected and fractions with similar TLC proles were combined. The rst fraction eluted with 100% CHCl3 was puried by ash silica gel column chromatography with hexane ethyl ether (1:1) to give fractions containing compounds 1420. Fraction containing crude 14 was puried by reverse phase C-18 HPLC with MeOHMeCNH2O [(1:6:3), 25 5 mg]: aD 2.0 (CHCl3; c 0.5), MS: m/z 262 [M]+; HREIMS m/z 262.1498 (Calcd. for C16H22O3, 262.1569); NMR data: see Table 1. Compounds 15 (10 mg), 17 (14 mg), and 18 (19 mg) were puried by preparative TLC with CHCl3 Me2CO (7:3). The fraction containing crude 20 (5 mg) was puried by preparative TLC with CH2Cl2MeOHH2O (11:10:9). Compounds 16 (20 mg) and 19 (20 mg) were puried by preparative TLC with CHCl3MeOH (19:1). The second fraction eluted with 100% CHCl3 was extracted with 2 N NaOH. After neutralization this fraction was extracted with EtOAc to give 600 mg of residual material. Column chromatography on silica gel gave a fraction containing 4, 8 and 13. Compound 4 (20 mg) was puried by preparative TLC with CH2Cl2MeOHH2O (33:30:35) lower layer. Compounds 8 (40 mg) and 13 (10 mg) were puried by C-18 HPLC with H2OMeCNMeOH (7:2:1). The fth fraction eluted with 50% EtOAc was puried by ash column chromatography on Si gel using CH2Cl2 and successively increasing the Me2CO concentration from 0% to 50% in CH2Cl2. Fractions eluted with 100% CH2Cl2
H chemical shift values (d ppm from SiMe4) followed by multiplicity and then the coupling constants (J in Hz). b HMBC correlations from H to C. c Letters, p, s, t and q, in parentheses indicate, respectively, the primary, secondary, tertiary and quaternary carbons, assigned by DEPT. * Assignments may be interchanged.
carbon, the positive DdRS for the H-16/H-17 and negative value for H-13 were found, indicating R conguration for C-14. Therefore, the structure of 21 was deduced to be (6E,8S,10E,14R)-3,7,11,15-tetramethylhexadeca-1,6,10trien-3,8,14,15-tetraol. The phytotoxicity of compounds 1, 3, 710, and 1520 on the seeds of Lactuca sativa was previously reported (DellaGreca et al., 2004; DAbrosca et al., 2004a,b). Compounds 2, 5, and 1114 were tested for their activities on the seeds of L. sativa. Aqueous solutions, ranging between 104 and 107 M, were tested on germination, root length and shoot length of treated lettuce seeds (Fig. 2). Compound 2 and sesquiterpene 14 reduced the germination by 20% at 104 M respect to the control. Compounds 5, 11, and 13 were inactive at all tested concentrations. Linalool showed about 80% inhibition on germination at the higher concentration tested, while no eects were observed on root and shoot length. Among compounds tested, only 2 reduced root length signicantly. Activities of 1530% on the shoot length at a concentration of 104 M have been observed for compounds 2, 5, and 1113 as compared to the control.
3. Experimental 3.1. General experiment procedures H and 13C NMR spectra were run on a Varian INOVA 500 NMR spectrometer at 500 and 125 MHz, respectively,
1
484
Fig. 2. (A) Eect of compounds 2, 5, and 1114 on germination of L. sativa L. Value presented as percentage dierences from control and are not signicantly dierent with P > 0.05 for Students t test: (a) P < 0.01; (b) 0.01 < P < 0.05. (B) Eect of compounds 2, 5, and 1114 on root length of L. sativa L. Value presented as percentage dierences from control and are not signicantly dierent with P > 0.05 for Students t test: (a) P < 0.01; (b) 0.01 < P < 0.05. (C) Eect of compounds 2, 5, and 1114 on shoot length of L. sativa L. Value presented as percentage dierences from control and are not signicantly dierent with P > 0.05 for Students t test: (a) P < 0.01; (b) 0.01 < P < 0.05.
were rechromatographed on silica gel under the same conditions. The subfraction eluted with 10% Me2CO was puried by C-18 HPLC with MeOHH2O (4:3) to give compounds 1 (5 mg), 2 (3 mg), 7 (20 mg), 10 (5 mg), and 11 (5 mg). The subfraction eluted with 15% Me2CO was puried by C-18 HPLC with MeOHH2O (4:3) to give compounds 3 (2 mg), and 5 (2 mg). The subfraction eluted
with 50% Me2CO was puried by C-18 HPLC with MeOHMeCNH2O (4:1:5) to give compound 6 (5 mg). The fraction eluted with 50% Me2CO was rechromatographed on silica gel using CH2Cl2 and successively increasing the Me2CO concentration by 20%, 40%, and 100% in CH2Cl2. The fraction eluted with 20% Me2CO was puried by C-18 HPLC with MeOHMeCNH2O
485
(4:1:5) to give 12 (20 mg). The fraction eluted with 40% Me2CO was puried by preparative TLC with EtOAc Me2CO (19:1) to give 9 (12 mg). The fraction eluted with 100% Me2CO was puried by C-18 HPLC with MeOH 25 MeCNH2O (3:2:5) to give 21 (2 mg): aD +21.0 (MeOH; + c 0.05), MS: m/z 340 [M] ; HREIMS m/z 340.2598 (Calcd. for C20H36O4, 240.2614); NMR data: see Table 2. 3.4. Bioassays Seeds of L. sativa L. (cv Napoli V.F.) collected during 2003, were obtained from Ingegnoli S.p.a. All undersized or damaged seeds were discarded and the assay seeds were selected for uniformity. Bioassays used Petri dishes (50 mm diameter) with one sheet of Whatman No. 1 lter paper as support. In four replicate experiments, germination and growth were conducted in aqueous solutions at controlled pH, using MES (2-[N-morpholino]ethanesulfonic acid, 10 mM, pH 6). Test solutions (104 M) were prepared in MES and the rest (105107 M) were obtained by dilution. Parallel controls were performed. After adding 25 seeds and 5 ml test solutions, Petri dishes were sealed with Paralm to ensure closed-system models. Seeds were placed in a growth chamber KBW Binder 240 at 25 C in the dark. Germination percentage was determined daily for ve days (no more germination occurred after this time). After growth, plants were frozen at 20 C to avoid subsequent growth until the measurement process. Data are reported as percentage dierences from control in the graphics and tables. Thus, zero represents the control; positive values represent stimulation of the control; positive values represent stimulation of the parameter studied and negative values represent inhibition. 3.5. Statistical treatment The statistical signicance of dierences between groups was determined by a Students t test, calculating mean values for every parameter (germination average, shoot and root elongation) and their population variance within a Petri dish. The level of signicance was set at P < 0.05.
Acknowledgement NMR experiments have been performed at Centro Interdipartimentale di Metodologie Chimico-Fisiche of University Federico II of Naples on a 500 MHz spectrometer of Consortium INCA Lab.
References
Cutillo, F., DellaGreca, M., Previtera, L., Zarrelli, A., 2005. C13 norisoprenoids from Brassica fruticulosa. Nat. Prod. Res. 19, 99103. DAbrosca, B., DellaGreca, M., Fiorentino, A., Monaco, P., Temussi, F., 2004a. Structure elucidation and phytotoxicity of C13 nor-isoprenoids from Cestrum parqui. Phytochemistry 65, 497505. DAbrosca, B., DellaGreca, M., Fiorentino, A., Monaco, P., Zarrelli, A., 2004b. Low molecular weight phenols from bioactive aqueous fraction of Cestrum parqui. J. Agric. Food Chem. 52, 41014108. Dale, J.A., Mosher, H.S., 1973. Nuclear magnetic resonance enantiomer reagents. Congurational correlations via nuclear magnetic resonance chemical shifts of diastereomeric mandelate, O-methylmandelate, and a-methoxy-a-triuoromethylphenylacetate (MTPA) esters. J. Am. Chem. Soc. 95, 512519. DellaGreca, M., Di Marino, C., Zarrelli, A., DAbrosca, B., 2004. Isolation and phytotoxicity of apocarotenoids from Chenopodium album. J. Nat. Prod. 67, 14921495. Farina, A., Doldo, A., Cotichini, V., Rajevic, M., Quaglia, M.G., Mulinacci, N., Vincieri, F.F., 1995. HPTLC and reectance mode densitometry of anthocyanins in Malva silvestris L.: a comparison with gradient-elution reversed-phase HPLC. J. Pharmaceut. Biomed. Anal. 14, 203211. Guarrera, P.M., 2005. Traditional phytotherapy in central Italy (Marche, Abruzzo, and Latium). Fitoterapia 76, 125. Mori, K., 1974. Carotenoids and degraded carotenoids. IV. Syntheses of optically active grasshopper ketone and dehydrovomifoliol as a synthetic support for the revised absolute conguration of (+)-abscisic acid. Tetrahedron 30, 10651072. Naviglio, D., 2003. Naviglios Principle and presentation of an innovative solidliquid extraction technology: extractor Naviglio. Anal. Lett. 36, 16471659. Nicoletti, M., Tommassini, L., Serani, M., 1989. Two linalool-1-oic acids from Kickxia spuria. Fitoterapia 60, 252254. Ohtani, I., Kusumi, T., Kashman, Y., Kakisawa, H., 1991. High-eld NMR application of Moshers method. The absolute congurations of marine terpenoids. J. Am. Chem. Soc. 113, 40924096. Pauque, J.J., 2000. Method for extracting a active principle based on Malva sylvestris, the active principle obtained, and cosmetic treatment using it. Patent Application: FR 2000-11973 20000920.
Hydroquinone diglycoside acyl esters from the stems of Glycosmis pentaphylla

Junsong Wang, Yingtong Di, Xianwen Yang, Shunlin Li, Yuehu Wang, Xiaojiang Hao
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Heilong tan, Kunming 650204, PR China Received 1 August 2005; received in revised form 30 October 2005 Available online 19 January 2006
Abstract Four hydroquinone diglycoside acyl esters, glypentosides AC (13) and seguinoside F (4), were isolated from the stems of Glycosmis pentaphylla. Glypentosides AB (12) were identied as compounds and designated as methoxyquinol 4-O-[(5-O-trans-p-coumaroyl)-bD-apiofuranosyl-(1 ! 2)-b-D-glucopyranoside] (1) and 4-demethylantiarol 4-O-[(3-methoxy-4-hydroxy-benzoyl)-b-D-apiofuranosyl(1 ! 2)-b-D-glucopyranoside] (2). Glypentoside C (3) is a hydroquinone diglycoside acyl ester with a neolignan moiety in the acyl unit. Their structures were elucidated by the combination of one- and two-dimensional NMR analysis, mass spectrometry and chemical evidences. 2005 Elsevier Ltd. All rights reserved.
Keywords: Glycosmis pentaphylla; Rutaceae; Glypentosides AC; Hydroquinone glycosides; Acyl esters; Apiosyl-(1 ! 2)-glucosides
1. Introduction The genus Glycosmis of the family Rutaceae is represented in China by nearly 11 species (Huang, 1997). Glycosmis pentaphylla (Retz.) DC. is a shrub or small (1.55 m) tree widely distributed from India, Malaysia and southern China to the Philippine Islands where it occurs in tropical forests at low altitudes. It has been used as a folk medicine in the treatment of fever, liver complaints and certain other diseases (Sastri, 1956). Phytochemical researches of this species were mainly focused on hydrophobic alkaloids, including those of the quinolone (Bhattacharyya and Chowdhury, 1985), quinazoline (Muthukrishnan et al., 1999; Sarkar and Chakraborty, 1979), acridone (Quader et al., 1999) and carbazole (Jash et al., 1992; Chowdhury et al., 1987) types, of leaves, root and stem bark. No study on glycosidic constituents of G. pentaphylla has been
reported. In this paper, we present results of an study on the polar constituents of the stem wood of the plant.
2. Results and discussion The EtOAc-soluble fraction of the MeOH extract was subjected to a succession of chromatographic procedures and nally by preparative ODS-HPLC to give three new hydroquinone diglycosides, namely glypentoside A (1), glypentoside B (2) and glypentoside C (3), besides the known compound seguinoside F (4). The known compound was identied by comparing its spectral data with those previously reported (Zhong et al., 1998). Compound 1 was isolated as amorphous powder. Its molecular formula C27H32O14 was deduced from the negative HRFABMS spectrum and 13C NMR spectral data. The IR spectrum showed absorption bands due to hydroxyl (3420 cm1) and carbonyl groups (1690 cm1). The 1 H NMR signals due to aromatic and olenic protons at d 7.54 (1H, d, J = 15.7 Hz), 7.37 (2H, d, J = 8.4 Hz), 6.79
Corresponding author. Tel.: +86 871 522 3263; fax: +86 871 515 0227. E-mail address: haoxj@mail.kib.ac.cn (X. Hao).
J. Wang et al. / Phytochemistry 67 (2006) 486491
487
OMe
3 2
1
HO
4'''
3''' 2'''
1''' 6'''
6'
5'''
OH O O OH 1' O
1''
OH
Table 1 The 1H NMR (400 MHz) and 13C NMR (100 MHz) data of 1 and 2 in CD3OD (J in Hz within parentheses) No. 1 dH (J in Hz) 1 2 3 4 5 6 10 20 30 40 50 60 100 200 300 400 500 1000 2000 3000 4000 5000 6000 a b c OMe
a
2 dC 142.9 s 149.2 s 103.4 d 152.6 s 109.6 d 116.1 d 102.0 d 78.8 d 78.5 d 71.7 d 78.1 d 62.6 t 110.5 d 78.7 d 79.2 s 75.4 t 67.6 t 127.1 s 131.2 d 116.9 d 161.3 s 116.9 d 131.2 d 168.8 s 114.8 d 146.9 d 56.4 q dH (J in Hz) 5.99 s dC 155.5 s 94.5 d 154.8 s 128.5 s 154.8 s 94.5 d 102.7 d 78.8 d 78.5 d 71.7 d 78.1 d 62.6 t 110.5 d 78.7 d 79.6 s 75.7 t 67.8 t 122.4 113.6 152.9 148.6 115.9 125.2 168.1 s d s s d d s
5''
OH O
O OH OH
6.70 d (2.4) 6.48 dd (2.4, 8.2) 6.62 d (8.2) 4.80 d (7.3) 3.583.65 3.583.65 3.333.36 3.333.36 3.583.65, 3.85a 5.48 d (1.8) 4.02 d (1.8) Ha 3.88 d (9.5) Hb 4.28 d (9.5) Ha 4.26 d (11.2) Hb 4.37 d (11.2) 7.37 d (8.4) 6.79 d (8.4) 6.79 d (8.4) 7.37 d (8.4) 6.20 d (15.7) 7.54 d (15.7) 3.86 s
1
HO
4'''' 5'''' 6''''
OMe
3'''' 2'''' 1'''' 7'''' 9''''
OH
8'''' 5''' 6''' 8''' 1''' 2''' 7''' 9'''
OMe
1
O
4''' 3'''
6'
OH O O 1' OH O
1''
4 5
6
OH
MeO
5''
OH O
5.99 s 4.94 d (7.6) 3.623.72 3.623.72 3.333.37 3.333.37 3.623.72, 3.82a 5.50 d (2.2) 4.00 d (2.2) Ha 3.90 d (9.8) Hb 4.38 d (9.8) Ha 4.36 d (10.8) Hb 4.43 d (10.8) 7.44 d (1.8)
O OH OH
OMe OH O O OH O O O OH OH OH O OH
HO
6.78 d (8.2) 7.47 dd (8.2,1.8)
3.83 s (MeO-3000 ) 3.70 s (MeO-3,5)
56.3 q (MeO-3000 ) 56.7 q (MeO-3,5)
Signal pattern unclear due to overlapping.
Fig. 1. Selected HMBC correlations of 1.
(2H, d, J = 8.4 Hz), 6.20 (1H, d, J = 15.7 Hz), as well as one ester carbonyl carbon at d 168.8, suggested the presence of one trans-p-coumaroyl moiety. The 1H and 13C NMR spectra of 1 (Table 1) also revealed the presence of glucopyranose and apiofuranose moieties. The b-anomeric conguration for the glucopyranose was determined from a large coupling constant value (7.3 Hz) of the anomeric proton (Agrawal, 1992; Ishii and Yanagisawa, 1998). The banomeric conguration for the apiofuranose was indicated from the anomeric signals at dC 110.5 (Kitagawa et al., 1993) with dH 5.48 (1H, d, J = 1.8 Hz) (Mbararoua et al., 1994; Otsuka et al., 1994). On acid hydrolysis, 1 aorded D-glucose and D-apiose as component sugars, which was identied by TLC and GLC analysis. The apiosyl-(1 ! 2)-glucosyl linkage of the glycosidic moiety was assigned from the cross-peaks observed between apiose H-1 and glucose H-2 in the NOESY spectrum. Also in the HMBC spectrum (Fig. 1) of 1, a correlation was evident between apiose H-1 (d 5.48) and glucose C-2 (d 78.8). The position was conrmed by the chemical shift (d 78.8) of glucose C-2, as compared with a nonsubstituted C-2, which is ca. d 74.0. All chemical shifts of this sugar moiety are in good agreement with the literature data
(Zhong et al., 1998). The signicant deshielding of H-5 of apiose (4.26 and 4.37 ppm) and the HMBC cross-peak between the proton at 4.37 ppm and the carbonyl carbon at 168.8 ppm conrmed that the coumaroyl unit was attached to position 5 of apiose. The 13C NMR spectrum of 1 showed, for the aglycon portion, seven signals. These were assigned to a methoxy group, three to aromatic CH, and three to phenolic functions. The 1H NMR spectrum contained the signals for three aromatic protons at d 6.70 (1H, d, J = 2.4 Hz), 6.62 (1H, d, J = 8.2 Hz), and 6.48 (1H, dd, J = 2.4, 8.2 Hz), along with a signal for a methoxy group at d 3.86, which correlated in the HMBC spectrum with a signal at dC 149.2, corresponding to a typical methoxyquinol. The site of glycosidation was revealed to be C-4 by HMBC experiment, which showed a long-range correlation between C-4 (d 152.6) and the anomeric proton (d 4.80) of glucose, which was further supported by the NOE cross-peaks (Fig. 2) observed from the anomeric proton (d 4.80) of glucose to two aromatic protons at dH 6.48 (1H, dd, J = 2.4, 8.2 Hz) and 6.70 (1H, d, J = 2.4 Hz). Based on the above results, the structure of glycopentoside A (1) was established as methoxyquinol 4-O-(5-O-trans-p-coumaroyl)-bD-apiofuranosyl-(1 ! 2)-b-D-glucopyranoside.
488
MeO
3 2 1 6
OMe HO
4''' 5''' 6''' 3''' 2''' 1'''
OH O 4 O OH 1' 5 MeO OH 5'' O O

6' 1''
OH
O OH
2
OH
OMe OMe HO OH O O O OH
4 OMe OH HO OH O O O OH OH OH O O O OH
OH O
OH
OH
OH
Fig. 2. Key NOEs correlations of 1.
Glycopentoside B (2) was isolated as amorphous powder, whose elemental composition was determined to be C27H34O16. Its IR spectrum showed hydroxyl groups (3424 cm1), a conjugated ester group (1706 cm1), and aromatic ring (1605; 1513 cm1). Comparison of its 1H NMR and 13C NMR data (Table 1) with those of compound 1 suggested the same sugar portion. Furthermore, the signals attributable to a ABX-coupling system were observed at d 7.44 (1H, d, J = 1.8 Hz), 6.78 (1H, d, J = 8.2 Hz), and 7.47 (1H, dd, J = 1.8, 8.2 Hz), in combination with the signal for a carbonyl carbon at d 168.1 in the 13 C NMR spectrum, suggested a 1,3,4-substituted benzoyl moiety in 2. HMBC correlations (Fig. 3) were observed between the proton signals at d 7.44 (H-2000 ) and 7.47 (H6000 ) and the carbonyl carbon, as well as methoxy signals
at d 3.83 and the carbon signal at 152.9, which conrmed the presence of a vanillic acid unit in the compound. The HMBC spectrum of 2 conrmed that the ester linkage is on the hydroxyl group on C-5 of apiose, since signicant cross-peaks were observed between dC 168.1 and dH 4.36 and 4.43. In the 1H NMR spectrum of 2, the only features for the aglycon moiety were a signal at d 5.99 (2H, s) and a signal for a methoxy group at d 3.70 (6H, s). The shielded aromatic protons allowed us to propose the presence of isolated aromatic protons in symmetrical relationship with the rest of the molecule, positioned in an electron donor environment. The 13C NMR spectrum of 2 exhibited, for the aglycon moiety, ve signals. These were indicative for two methoxy group, two aromatic CH, and four phenolic functions. The location of the methoxy groups at C-3,5 and of the disaccharide chain at C-4 were deduced from the HMBC correlations between the proton signal at d 3.70 (OMe) and the carbon resonance at d 154.8 (C-3,5), and between the anomeric signal of the glucose unit at d 4.94 and the carbon resonance at d 128.5 (C-4). Thus, the structure 4demethylantiarol 4-O-(3-methoxy-4-hydroxy-benzoyl)-bD-apiofuranosyl-(1 ! 2)-b-D-glucopyranoside was assigned to 2. Glycopentoside C (3) was isolated as an amorphous powder. The molecular formula was established as C38H44O18 by HRFABMS. In comparison of the 1H and 13 C NMR spectra (Table 2) of 3 with those of 1, the signals due to sugars and the aglycon were superimposable. On acid hydrolysis, 3 aorded D-glucose and D-apiose as component sugars. These suggested that 3 was also methoxyquinol apiosyl-(1 ! 2)-glucoside acyl ester. The 1H NMR
Table 2 The 1H NMR (400 MHz) and 13C NMR (100 MHz) data of 3 in CD3OD (J in Hz within parentheses) No 1 2 3 4 5 6 10 20 30 40 50 60 100 200 300 400 500 1000 2000
a
dH (J in Hz)
dC 142.9 s 149.2 s 103.2 d 152.7 s 109.5 d 116.1 d 102.0 d 78.8 d 78.3 d 71.7 d 78.1 d 62.6 t 110.5 d 78.7 d 79.2 s 75.4 t 67.6 t 129.6 s 113.6 d
No 3 4000 5000 6000 7000 8000 9000 10000 20000 30000 40000 50000 60000 70000 80000 90000 2-OMe 3000 -OMe 30000 -OMe
000
dH (J in Hz)
dC 145.8 s 152.1 s 131.7 s 119.3 d 147.1 d 115.5 d 168.7 s 134.1 s 110.6 d 149.2 s 147.8 s 116.2 d 119.9 d 89.9 d 54.7 d 64.6 t 56.4 q 56.8 q 56.4 q
6.68 d (2.2) 6.46 d (8.2, 2.2) 6.62 d (8.2) 4.81 d (7.8) 3.543.65 3.543.65 3.34 m 3.34 m 3.86a, 3.543.65 5.53 d (2.0) 4.01 d (2.0) Ha 3.78 d (10.2) Hb 4.24 d (10.2) Ha 4.20 d (11.0) Hb 4.28 d (11.0) 7.04 s
7.08 s 7.55 d (15.8) 6.25 d (15.8)
6.94 d (2.0)
MeO OMe HO O O O OH OH OH O O MeO O OH
6.77 d (8.2) 6.82 dd (2.0, 8.2) 5.57 d (6.4) 3.54 m 3.83a 3.81 s 3.88 s 3.75 s
OH OH
Signal pattern unclear due to overlapping.
489
spectrum revealed the remaining signals of ve aromatic protons enclosed in two aromatic systems: an AX system corresponding to a 1,2,3,5-tetrasubstituted ring and an AMX system corresponding to a 1,2,4-trisubstituted ring. The aromatic region of the 1H NMR spectrum of 3 showed one AB set of signals at d 6.25 and 7.55 (1H, d, J = 15.8 Hz). The coupling constants of the vinylic system indicated that they have a trans conguration. HMBC correlations (Fig. 4) between the proton resonances of the vinylic system with the carbon resonance of the ester group, suggested that compound 3 have one a,b-unsaturated ester moieties in its structure. The presence of these moieties was also conrmed by the presence of a band at 1699 cm1 in the IR spectrum of 3. In the 1H NMR spectrum of 3, an ABC set of signals can also be found. They are due to proton resonances of a methine group [dH 3.54 (m); dC 54.7], one benzylic methine group [dH 5.57 (d, J = 6.4 Hz); dC 89.9] and also a methylene group (dH 3.83; dC 64.6). The 13C NMR spectrum of 3 displayed characteristic signals for three methoxy groups. Besides these substituent signals, 18 skeletal carbon resonances appeared in the 13C NMR spectrum of the acyl unit of 3, which, in combination with its 1H NMR data, suggested that the acyl moiety of 3 is a dihydrobenzo[b]furan neolignan (Chakravarty et al., 1996; Li et al., 1997). HMBC correlations (Fig. 4) led to the planar structure of the acyl moiety. The NOE interactions (Fig. 5) between OMe/H-3, OMe/ H-2000 and OMe/ H-20000 allowed the locations of the methoxy at C-2, C-3000 and C-30000 . Since the coupling constant (J = 6.4 Hz) of H-70000 was similar to J = 6.2 Hz of H-7 in trans-dehydrodiconiferyl alcohol (Wang et al., 1992), the relative conguration of C-70000 and C-80000 in 3 was deter-
HO
OMe
OH O MeO O OH OH O OH O O OH OH O O
OMe OH
mined as trans form which was also conrmed by a lack of correlation between H-70000 and H-80000 and a pronounced coupling between the two protons at C-90000 and C-70000 from the NOESY spectrum (Fig. 5). The absolute conguration of the dihydrofuran ring was determined using CD spectroscopic evidence. The CD spectrum of 3 showed a negative Cotton eect at 287 nm, providing evidence that the conguration in 3 must be 70000 S, 80000 R (Lynn et al., 1987; Wang and Jia, 1997). The linkage of acyl moiety with sugar moiety was solved by analysis of the HMBC spectrum. In the HMBC spectrum, the carbonyl group (dC 168.7) not only showed correlation with protons assigned to double bond but also had correlation with methylene protons at d 4.20 (1H, d, J = 11.0) and 4.28 (1H, d, J = 11.0 Hz), suggesting the acyl moiety is connected to the hydroxyl group on C-5 of apiose. This conrmed the structure of glycopentoside C is shown as that of 3. The four hydroquinone diglycoside acyl esters identied in this investigation of glycosidic metabolites from the genus Glycosmis is the rst report of a compound of this type in the Rutaceae. However, since no studies on the polar constituents of the stems of other species have been carried out, whether this has taxonomic value remains unsure. These phenolic glycoside esters may store in the plant as precursors of post-inhibitin and has a defensive ecological role. The sugar moiety appears to stabilize the molecules, preventing dehydrogenation to give hydroquinones which have been reported to show antimicrobial (Jin and Sato, 2003; Ma et al., 1999; Perry and Brennan, 1997) and cytotoxic activities (Perry and Brennan, 1997) and to be allelopathic agents (Weidenhamer and Romeo, 2004). However, glycoside form of these hydroquinone generally exhibited low activities (Jin and Sato, 2003; Ma et al., 1999; Perry and Brennan, 1997; Weidenhamer and Romeo, 2004). Active phenolic toxins are released from the corresponding glycosides by enzymic hydrolysis caused by microbial invasion or herbivore attack on foliage against the invasion of pathogens under environmental conditions (Harborne, 1988).
3. Experimental 3.1. General procedures
HO
OMe
OH O MeO O OH OH O OH O O OH OH O O
OMe OH
Fig. 5. Key NOEs correlations of 3.
H, 13C, and 2D NMR spectra were recorded on a Bruker AM 400 NMR and a DRX-500 spectrometer with TMS as internal standard. MS data were obtained on a VG AutoSpec 3000 spectrometers. UV spectra were obtained on a Shimadzu double-beam 210A spectrophotometer. The IR (KBr) spectra were obtained on a Bio-Rad FTS-135 spectrometer. HPLC separations were performed on a HP 1100 apparatus equipped with Diode array UV detector and XTERRA C18 (Waters, 10 lm, 15 200 mm, ow rate: 15 mL/min) column. GCMS
490
was run on a FISONS MD-800 instrument. The CD spectra were measured with a JASCO-715 spectropolarimeter. Optical rotations were measured with a JASCO DIP-370 digital polarimeter in MeOH solution. 3.2. Plant material The stems of G. pentaphylla were collected in Xishuangbanna, Yunnan, China., in March 2003. The plant material was identied by Prof. De-Ding Tao, and a voucher specimen (BN20030412) was deposited in the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences. 3.3. Extraction and isolation The air-dried material (10 kg) was nely pulverized and extracted by percolation with MeOH for one month at room temperature. The combined extracts were ltered and concentrated under vacuum to obtain a crude extract (350 g). The extract was partitioned between water and CHCl3 and then further extracted with EtOAc. The EtOAc-soluble fraction (25 g) was fractionated by column chromatography over D101 porous resin using gradient aqueous ethanol to give six fractions (fractions IVI). Fraction II (5 g) was subjected to column chromatography over D101 porous resin and eluted with 530% Me2CO in H2O, yielding fractions II-1II-5. Fraction II-2 (956 mg) was subjected to medium pressure chromatography (MPLC) over C18 Si gel and eluted with MeOHH2O (1:95:5) under gradient conditions yielding fractions II2-1II-2-6. Fraction II-2-4 (66 mg) was applied to Sephadex LH-20 with MeOH. The major component eluted as a yellow band (32 mg) and was further puried by RP-18 preparative HPLC with MeOHH2O (2:8), yielded compound 2 (7 mg) at 26.4 min. Fraction II-3 (3 g) was applied to RP-18 MPLC and eluted with Me2COH2O (1:93:7) under gradient conditions yielding fractions II-3-1II-3-6. Fraction II-3-1 (1.3 g) was again applied to RP-18 MPLC and eluted with MeOHH2O (1:95:5) yielding fractions II-3-1-1II-3-1-6. Fraction II-3-1-1 (600 mg) and Fraction II-3-1-5 (90 mg) were chromatographed on Sephadex LH20 with MeOH and further puried by successive RP-18 preparative HPLC with 40% MeOH to obtain 1 (5 mg, tR 26.1 min), and with 30% MeOH to aord 3 (10 mg, tR 41.4 min), respectively. 3.3.1. Methoxyquinol 4-O-[(5-O-trans-p-coumaroyl)-b-Dapiofuranosyl-(1 ! 2)-b-D-glucopyranoside] (Glycopentoside A), 1 25 Amorphous powder; aD : 7.5 (c 0.33, MeOH); UV (MeOH) kmax nm (loge): 313 (4.12), 289 (4.15), 206 (4.55); IR mmax (KBr) cm1: 3420, 2937, 2076, 1690, 1605, 1514, 1454, 1361, 1271, 1200, 1168, 1110, 1074, 1030, 945, 833; 1 H and 13C NMR spectral data: see Table 1; HRFABMS (negative-ion mode) m/z: 579.1726 [M H] (C27H31O14 requires 579.1714).
3.3.2. 4-Demethylantiarol 4-O-[(3-methoxy-4-hydroxybenzoyl)-b-D-apiofuranosyl-(1 ! 2)-b-D-glucopyranoside] (Glycopentoside B), 2 25 Amorphous powder; aD :60.7 (c 0.48, MeOH); UV (MeOH) kmax nm (loge): 267 (4.03), 206 (4.62); IR mmax (KBr) cm1: 3424, 2940, 2850, 2075, 1706, 1605, 1513, 1465, 1429, 1336, 1284, 1219, 1119, 1073, 1029, 817; 1H and 13C NMR spectral data: see Table 1; HRFABMS (negative-ion mode) m/z: 613.1773 [M H] (C27H33O16 requires 613.1769). 3.3.3. Glycopentoside C, 3 25 Amorphous powder; aD :49.7 (c 0.84, MeOH); UV (MeOH) kmax nm (loge): 329 (4.34), 290 (4.14), 224 (4.44), 205 (4.70); IR mmax (KBr) cm1: 3424, 2937, 2885, 2059, 1699, 1630, 1606, 1514, 1454, 1432, 1336, 1274, 1144, 1072, 1030, 943, 837, 803; 1H and 13C NMR spectral data: see Table 2; HRFABMS (negative-ion mode) m/z: 787.2462 [M H] (C38H43O18 requires 787.2449). CD (MeOH): [h]287 = 12400. 3.4. Acid hydrolysis, TLC and GC analysis of 13 Each solution of 13 (each 2 mg), in 1 M HCl (dioxane H2O, 1:1, 2 mL) was heated at 95 C for 2 h in a water bath. After removing the solution under a stream of nitrogen, the residue was suspended with H2O and extracted with EtOAc three times. The aqueous layer was then neutralized with NaHCO3 and concentrated to dryness under reduced pressure. The residue was compared with standard sugars by cothin layer chromatography (CHCl3MeOHH2OHOAc, 16:9:2:2; detection with spray agent: 4% a-naphthol EtOH5% H2SO4). Hexoses gave purple spots and pentoses blue spots. The Rf values of each sugar are as follows: glucose, 0.42 and apiose, 0.52. The residue was dried and dissolved in pyridine (0.5 mL). Then trimethylchlorosilane (0.5 mL) was added and the reaction mixture was kept at ambient temperature for 20 min. After concentrated to dryness under reduced pressure, the residue was dissolved in diethyl ether and then directly subjected to GCMS [column: 30 m 0.32 mm (30QC2/AC5)] analysis under the following conditions: electron-impact (EI) mode (70 eV), temperature programming from 180 to 240 C at 5 C/min; carrier N2 gas. In the acid hydrolysate of 13, D-glucose and D-apiose were conrmed by comparison of the retention times of their TMSi derivatives with those of D-glucose and D-apiose derivatives prepared in a similar way, which showed retention times of 6.86 and 3.08 min, respectively.
Acknowledgments The authors gratefully acknowledge nancial support from the National Natural Science Foundation of China (NSFC) for Distinguished Young Scientists to X.-J. Hao
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(39525025, 30070087). We are grateful to members of the analytical group in the Laboratory of Phytochemistry, Kunming Institute of Botany for the spectral measurements. References
Agrawal, P.K., 1992. NMR spectroscopy in the structural elucidation of oligosaccharides and glycosides. Phytochemistry 31, 33073330. Bhattacharyya, P., Chowdhury, B.K., 1985. Glycolone, a quinolone alkaloid from Glycosmis pentaphylla. Phytochemistry 24, 634635. Chakravarty, A.K., Mukhopadhyay, S., Saha, S., Pakrashi, S.C., 1996. A neolignan and sterols in fruits of Solanum sisymbrifolium. Phytochemistry 41, 935939. Chowdhury, B.K., Mustapha, A., Garba, M., Bhattacharyya, P., 1987. Carbazole and 3-methylcarbazole from Glycosmis pentaphylla. Phytochemistry 26, 21382139. Harborne, J.B., 1988. In Introduction to Ecological Biochemistry, third ed. Academic, London, pp. 312315. Huang, C.C., 1997. Flora of China, 43 (2), Science Press, pp. 117126. Ishii, T., Yanagisawa, M., 1998. Synthesis, separation and NMR spectral analysis of methyl apiofuranosides. Carbohydr. Res. 313, 189192. Jash, S.S., Biswas, G.K., Bhattacharyya, S.K., Bhattacharyya, P., Chakraborty, A., Chowdhury, B.K., 1992. Carbazole alkaloids from Glycosmis pentaphylla. Phytochemistry 31, 25032505. Jin, S.G., Sato, N., 2003. Benzoquinone, the substance essential for antibacterial activity in aqueous extracts from succulent young shoots of the pear Pyrus spp. Phytochemistry 62, 101107. Kitagawa, I., Hori, K., Sakagami, M., Hashiuchi, F., Yoshikawa, M., Ren, J., 1993. Saponin and Sapogenol. XLIX. On the constituents of the roots of Glycyrrhiza inate Batalin from Xinjiang, China. Characterization of two sweet oleanane-type triterpene oligoglycosides, apioglycyrrhizin and araboglycyrrhizin. Chem. Pharm. Bull. 41, 13501357. Li, S., Iliefski, T., Lundquist, K., Wallis, A.F.A., 1997. Reassignment of relative stereochemistry at C-7 and C-8 in arylcoumaran neolignans. Phytochemistry 46, 929934.
Lynn, D.G., Chen, R.H., Manning, K.S., Wood, H.N., 1987. The structural characterization of endogenous factors from Vinca rosea crown gall tumors that promote cell division of tobacco cells. Proc. Natl. Acad. Sci. USA 84, 615619. Ma, W.G., Fukushi, Y., Ducrey, B., Hostettmann, K., Tahara, S., 1999. Phenolic glycosides from Eriosema tuberosum. Phytochemistry 51, 10871093. ` Mbararoua, O., Ton-That, T., Tapiero, C., 1994. Syntheses de 6-O-b-D apiofuranosyl-b-D-glucopyranosides de monoterpenyle. Carbohydr. Res. 253, 7999. Muthukrishnan, J., Seifert, K., Homann, K.H., Lorenz, M.W., 1999. Inhibition of juvenile hormone biosynthesis in Gryllus bimaculatus by Glycosmis pentaphylla leaf compounds. Phytochemistry 50, 249 254. Otsuka, H., Kamada, K., Ogimi, C., Hirata, E., Takushi, A., Takeda, Y., 1994. Alangionosides A and B, ionol glycosides from leaves of Alangium premnifolium. Phytochemistry 35, 13311334. Perry, N.B., Brennan, N.J., 1997. Antimicrobial and cytotoxic phenolic glycoside esters from the New Zealand Tree Toronia toru. J. Nat. Prod. 60, 623626. Quader, M.A., Nutan, M.T.H., Rashid, M.A., 1999. Antitumor alkaloid from Glycosmis pentaphylla. Fitoterapia 70, 305307. Sarkar, M., Chakraborty, D.P., 1979. Glycophymoline, a new minor quinazoline alkaloid from Glycosmis pentaphylla. Phytochemistry 18, 694695. Sastri, B.N., 1956. The wealth of India: Raw Materials, vol. IV. CSIR, New Delhi, p. 150. Wang, C.Z., Jia, Z.J., 1997. Lignan, phenylpropanoid and iridoid glycosides from Pedicularis Torta. Phytochemistry 45, 159166. Wang, H.B., Yu, D.Q., Liang, X.T., 1992. The structure of a new neolignan glycoside from Stauntonia chinensis. J. Nat. Prod. 55, 214 216. Weidenhamer, J.D., Romeo, J.T., 2004. Allelochemicals of Polygonella myriophylla: chemistry and soil degradation. J. Chem. Ecol. 30, 1067 1082. Zhong, X.N., Otsuka, H., Ide, T., Hirata, E., Takushi, A., Takeda, Y., 1998. Hydroquinone glycosides from leaves of Myrsine seguinii. Phytochemistry 49, 21492153.
Unusual chromenes from Peperomia blanda

Leosvaldo S.M. Velozo a, Marcelo J.P. Ferreira b, Maria Isabel S. Santos c, Davyson L. Moreira a, Vicente P. Emerenciano b,*, Maria Auxiliadora C. Kaplan
b c
a Nucleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, Brazil Instituto de Qumica, Qumica Fundamental, Universidade de Sao Paulo, Caixa Postal 26077, CEP: 05513-970, Sao Paulo, SP, Brazil Departamento de Produtos Naturais e Alimentos, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, Brazil
Received 23 June 2005; received in revised form 9 September 2005 Available online 3 February 2006
Abstract From the methanol extract of the aerial parts of Peperomia blanda (Piperaceae), two chromenes were isolated and characterized mainly through application of 2D-NMR spectroscopy. The structures were 2S-(4-methyl-3-pentenyl)-6-formyl-8-hydroxy-2,7dimethyl-2H-chromene and 2S-(4-methyl-3-pentenyl)-5-hydroxy-6-formyl-2,7-dimethyl-2H-chromene named as blandachromenes I and II, respectively. 2006 Published by Elsevier Ltd.
Keywords: Peperomia blanda; Piperaceae; Chromenes; Aerial parts
1. Introduction The Peperomia genus belongs to the Piperaceae family that comprises some 600 species (Mabberley, 1993) widely distributed in southeast Brazil. Chemical studies carried out on Piperaceae species have revealed the occurrence of a variety of compounds including essential oils, pyrones, lignoids, polyphenols, unsaturated amides and alkaloids (Parmar et al., 1997, 1998; Moreira et al., 1998a,b; Baldoqui et al., 1999). These species have been extensively investigated as a source of new natural products with potential antimicrobial, antitumor and insecticidal activities (Costantin et al., 2001; Min et al., 2004; Konishi et al., 2005; Sacchetti et al., 2005). In contrast to the extensive studies of the Piper compounds (Parmar et al., 1997, 1998), few phytochemical studies of Peperomia have been reported. Previous phytochemical investigations on dierent species of Peperomia have shown the presence of avonoids (Aqil et al., 1993), benzopyran derivatives (Seeram et al., 1998;
Mbah et al., 2002; Salazar et al., 2005), secolignans (Chen et al., 1989; Monache and Compagnone, 1996; Govindachari et al., 1998), terpenes, arylpropanoids, phenolic compounds (Tanaka et al., 1998; Moreira et al., 1999; Bayma et al., 2000; Li et al., 2003) and essential oils (Bessiere et al., 1994; Silva et al., 1999; Zoghbi et al., 2005). Some biological activities were found in compounds isolated from the Peperomia genus, e.g., prenylated phenols, with antiparasitic activity, from P. galioides (Mahiou et al., 1995, 1996), as well as analgesic activity with mice that encountered extracts of aerial parts from P. pellucida (Aziba et al., 2001). As part of our studies on Brazilian Piperaceae species, we have performed a phytochemical examination of the aerial parts of P. blanda collected in the Brazilian Atlantic Forest. In this paper, we describe the isolation and structural elucidation of two chromenes from the non-polar fraction of P. blanda.
2. Results and discussion

*
Corresponding author. Tel.: +55 11 30912056; fax: +55 11 38155579. E-mail address: vdpemere@iq.usp.br (V.P. Emerenciano).
The hexane solubles of a methanol extract, obtained from the aerial parts of P. blanda, aorded a mixture of
0031-9422/$ - see front matter 2006 Published by Elsevier Ltd. doi:10.1016/j.phytochem.2005.12.012
L.S.M. Velozo et al. / Phytochemistry 67 (2006) 492496
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three sterols stigmasterol, sitosterol and campesterol and two unusual chromenes (compounds 1 and 2, Fig. 1), which were isolated and identied. Compound 1 (Blandachromene I) was obtained as a yellowish oil and its molecular formula was assigned as C18H22O3 by HREIMS showing an [M+] ion at m/z 286.1536. The mass spectral fragmentation pattern showed a base peak at m/z 203.0695 [C12H11O3]+ and a signal at m/z 271 due to the fragment [M-15]+ that resulted from loss of a methyl group. Its IR spectrum showed a broad band at 3350 cm1 and an absorption at 1738 cm1 which indicated the presence of a hydroxyl group and an aldehyde carbonyl group, respectively. The absorptions at 1651 and 1584 cm1 suggested an aromatic ring. The 1H NMR spectrum (Table 1) showed only one resonance due to an aromatic proton at d 6.95 indicating a pentasubstituted aromatic ring that was correlated with carbon signals of C4, C4a, C7, C8a and C9. An AB system (d 5.65 and 6.55, Ja,b = 10.0 Hz) suggested a cis-olen with the signal at d 5.65 being attributed to H3 which was correlated with C2, C4, C4a, C1 0 and C100 . On the other hand, the resonance at d 6.55 exhibited correlations with C2, C4a, C5 and C8a conrming the location of the aromatic proton
in C5. The presence of an aldehyde carbonyl group is indicated from the singlet at d 10.3 and its correlations which were observed with C5, C6 and C7, demonstrating that the aldehyde group should be attached to C6. The aromatic methyl group (C10) at d 2.40 was located at C7 based on observed correlations with C6, C7 and C8. Additional signals included an olenic proton at d 5.10 (1H, m), four coupled aliphatic protons at d 2.05 (2H, m, H-2 0 ) and d 1.70 (2H, t, H-1 0 ), besides three singlets corresponding to methyl groups at d 1.35, 1.50 and 1.60. The correlations with C2, C3, C1 0 observed for the rst methyl group conrmed its position to an oxygen-bearing carbon. The other data suggested a 4-methyl-3-pentenyl substitution in position C2 of the chromene skeleton. The 13C NMR spectroscopic data of 1 (Table 1) are in agreement with the proposed assignments and conrm the presence of an aldehyde carbonyl group (d 190.0), an oxygen-bearing aromatic carbon (C-8) at d 160.0 and a methyl (C-10) at d 22.0 substituted aromatic carbon (C-7) at d 142.5. Based on the HMBC analysis (Table 1), the methyl and the 4methyl-3-pentenyl units were attached to C2 (d 79.9) of a pyrane ring. Comparative phytochemistry using 5hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl-benzopyran
OH
9 4 1' 9 4 1'
O
10
O
4' 10
O 2
1''
O 2
1''
4'
OH 1
Fig. 1. Chromenes isolated from Peperomia blanda.
Table 1 NMR spectroscopic data of chromenes 1 and 2 isolated from Peperomia blanda (500 MHz for 1H NMR and 125 MHz for No. 1
1
13
C NMR, DMSO-d6) HMBC C2, C4, C4a, C1 0 , C100 C2, C4a, C5, C8a
2
13
H NMR
C NMR
HMBC C2, C4, C4a, C1 0 , C100 C2, C4a, C5, C8a C4, C4a, C7, C8a, C9
H NMR
13
C NMR
2 3 4 4a 5 6 7 8 8a 9 10 10 20 30 40 50 60 100 OH
5.65 d (J = 10.0 Hz) 6.55 d (J = 10.0 Hz) 6.95 s
10.30 s 2.40 s 1.70 t (J = 6.9 Hz) 2.05 m 5.10 m 1.60 s 1.50 s 1.35 s
79.9 127.5 116.5 106.1 111.3 116.0 142.5 160.0 158.0 190.0 22.0 40.0 22.5 124.2 131.2 25.7 17.5 26.0
5.65 d (J = 10.0 Hz) 6.60 d (J = 10.0 Hz)
6.25 s C5, C6, C7 C6, C7, C8 C2, C2 0 , C3 0 C2, C1 0 , C3 0 , C4 0 C1 0 , C2 0 , C5 0 , C6 0 C3 0 , C4 0 , C6 0 C3 0 , C4 0 , C5 0 C2, C3, C1 0 10.0 2.45 1.65 2.00 5.05 s s t (J = 6.9 Hz) m m
1.60 s 1.50 s 1.37 s 12.6
80.0 127.5 115.0 106.4 160.0 113.0 145.0 111.0 160.0 196.2 18.0 40.0 22.5 124.0 131.5 25.8 17.5 27.0
C4a, C6, C7, C8a, C10 C5, C6, C7 C6, C7, C8 C2,C2 0 , C3 0 C2, C1 0 , C3 0 , C4 0 C1 0 , C2 0 , C5 0 ,C6 0 C3 0 , C4 0 , C6 0 C3 0 , C4 0 , C5 0 C2, C3, C1 0 C4a, C5, C6
494
6 5 4 3 2 1 0 -1 -2 -3 -4 180
200
220
240 nm
260
280
300
320
Fig. 2. CD spectra of compounds 1 and 2 (in CHCl3) d Compound 1; h Compound 2.
(Ferraz et al., 2001), lhotzchromene (Moreira et al., 1998a), clusifoliol (Seeram et al., 1998), methyl 8-hydroxy-2,2dimethyl-2H-chromene-6-carboxylate (Orjala et al., 1993) and galopiperone (Mahiou et al., 1996) as models, further supports the proposed structure for 1 as 2-(4-methyl-3pentenyl)-6-formyl-8-hydroxy-2,7-dimethyl-2H-chromene. The optical rotation, a25 + 26.0 was very similar to that D observed for sargatriol (Kikuchi et al., 1983), although this compound is not appropriate for such direct comparison since its side-chain has two additional chiral centers. Nevertheless, the CD curve for blandachromene I (Fig. 2) was fully opposite to that observed for the sargatriol and daurichromenes (Iwata et al., 2004) including the positive Cotton eect at 260280 nm. Therefore, despite dierences in the substitution pattern in the aromatic ring of blandachromene I, the S conguration at C-2 was suggested. Compound 2 (Blandachromene II), also identied as a chromene, was obtained as a yellowish oil. The UV spectrum revealed a phenolic compound with an extended chromophore. The molecular ion [M]+ at m/z 286.1478 is compatible with the molecular formula of C18H22O3. Its IR spectrum showed a broad band at 3441 cm1 typical of a hydroxyl group chelated to a conjugated carbonyl aldehyde group at 1725 cm1. The major dierence in the 1 H NMR spectrum of 2 as compared to 1, was a hydroxyl group signal at d 12.6, due to a hydrogen bonded phenolic hydroxyl. This signal was correlated with carbon signals of C4a, C5 and C6, while the aldehyde resonance at d 10.0 has HMBC correlations (C5, C6 and C7) that conrmed the hydroxyl and aldehyde groups should be attached to C5
and C6 positions, respectively. The aromatic hydrogen at d 6.25, correlated with C4a, C6, C7, C8a and the methyl carbon (C10), was located at C8. The aromatic methyl group (C10) was located at C7 based on observed correlations with C6, C7 and C8. The signal of H4 was correlated with C2, C4a, C5 and C8a, and nally, the correlations between H3 and C1 0 and C100 conrmed the location of all substituents in the aromatic ring. The 13C NMR spectrum assignments of 2 are in agreement with the proposed structure and also conrm the presence of the aldehyde carbonyl group with a signal at d 196.2, the aromatic carbon (C-5) bearing an oxygen-substituent at d 160.0 and the methyl substituted aromatic carbon (C-7) at d 145.0. These spectroscopic data are in agreement with that for a racemic mixture previously described (Dike and Merchant, 1978). The placement of the hydroxyl group was conrmed by analysis of the NOE eects. The NOE eects (7-Me/H-8 and CHO) conrmed the structure for the compound 2 as 2S-(4-methyl-3-pentenyl)-5-hydroxy-6-formyl-2,725 dimethyl-2H-chromene. The optical rotation, aD + 22.0 was observed for the compound 2 and the CD spectra of the substance showed the same pattern obtained for the compound 1 (Fig. 2); thus the S conguration at C-2 was also suggested.
3. Conclusions Both chromenes isolated from P. blanda are rare examples of the occurrence of benzaldehyde derivatives in the
495
Piperaceae species, since their members usually produce compounds more oxidized, for example benzoic acid derivatives (Moreira et al., 1998a,b; Baldoqui et al., 1999; Salazar et al., 2005). These chromenes were biosynthesized probably through the polyketide route and rearm the possible hypothesis of horizontal gene transfer suggested in P. villipetiola (Salazar et al., 2005).
4.4. 2S-(4-Methyl-3-pentenyl)-6-formyl-8-hydroxy-2,7dimethyl-2H-chromene (1) Yellowish oil. aD +26 (MeOH, c 0.1); UV kMeOH nm max (log e): 211 (2.36), 266 (1.99); IR mfilm : 3350, 2962, 2924, max 2855, 1738, 1651, 1584, 1385; CD (CHCl3) kmax De287 +4.92, De254 2.87; HREIMS, m/z: 286.1536 (C18H22O3 requires 286.1506); GC-MS m/z (rel. int.): 286 [M+] (3), 271 [MCH3]+ (2), 203 [MC6H11]+ (100), 173 (4), 145 (3), 128 (3), 115 (7), 91 (11), 69 (33); For 1H and 13C NMR spectra (DMSO-d6), see Table 1; 1H NMR (CDCl3): d 1.44 (3H, s, H-1), 1.56 (3H, s, H-6), 1.62 (3H, s, H-5), 1.77 (2H, t, J = 6.9 Hz, H-1), 2.10 (2H, m, H-2), 2.52 (3H, s, H10), 5.09 (1H, m, H-3), 5.57 (1H, d, J = 10.0 Hz, H-3), 6.98 (1H, s, H-5), 6.64 (1H, d, J = 10.0 Hz, H-4), 10.42 (1H, s, CHO); 13C NMR (CDCl3): d 79.7 (s, C-2), d 127.3 (d, C3), d 116.6 (d, C-4), d 106.8 (s, C-4a), d 111.0 (d, C-5), d 116.1 (s, C-6), d 143.1 (s, C-7), d 159.6 (s, C-8), d 155.6 (s, C-8a), d 190.5 (d, C-9), d 22.0 (q, C-10), d 41.0 (t, C1 0 ), d 22.6 (t, C-2 0 ), d 123.6 (d, C-3 0 ), d 131.9 (s, C-4 0 ), d 25.5 (q, C-5 0 ), d 17.5 (q, C-6 0 ), d 26.1 (q, C-100 ). 4.5. 2S-(4-methyl-3-pentenyl)-5-hydroxy-6-formyl-2,7dimethyl-2H-chromene (2) Yellowish oil. aD +22 (MeOH, c 0.1); UV kMeOH nm (log max e): 203 (0.72), 276 (1.03), 319 (0.49); IR mfilm : 3441, 2969, 2875, max 1725, 1653, 1568, 1379; CD (CHCl3) kmax De292 +5.63, De260 3.78; HR-MS, m/z: 286.1478 (C18H22O3 requires 286.1506); GC-MS m/z (rel. int.): 286 [M+] (3), 271 [MCH3]+ (5), 203 [MC6H11]+ (100), 173 (9), 145 (5), 115 (13), 91 (17), 69 (66), 55 (84); for 1H and 13C NMR spectra (DMSO-d6), see Table 1; 1H NMR (200 MHz, CDCl3): 1.44 (3H, s, H-1), 1.59 (3H, s, H-6), 1.68 (3H, s, H-5), 1.76 (2H, t, J = 6.9 Hz, H-1), 2.08 (2H, m, H-2), 2.50 (3H, s, H10), 5.11 (1H, m, H-3), 5.51 (1H, d, J = 10.0 Hz, H-3), 6.19 (1H, s, H-8), 6.72 (1H, d, J = 10.0 Hz, H-4), 10.0 (1H, s, CHO); 13C NMR (CDCl3): d 80.5 (s, C-2), d 126.1 (d, C-3), d 115.7 (d, C-4), d 106.5 (s, C-4a), d 160.8 (s, C-5), d 113.0 (s, C-6), d 143.5 (s, C-7), d 110.8 (d, C-8), d 160.4 (s, C-8a), d 192.7 (d, C-9), d 18.2 (q, C-10), d 41.6 (t, C-1 0 ), d 22.5 (t, C-2 0 ), d 123.6 (d, C-3 0 ), d 131.8 (s, C-4 0 ), d 25.5 (q, C-5 0 ), d 17.5 (q, C-6 0 ), d 27.2 (q, C-100 ). Acknowledgements We are grateful to Eduardo M.B. da Silva and Marco A. Schiavon for obtaining, respectively, the NMR and HRMS spectra and to Lus Carlos Giordano for helping us with collecting the plant material. Financial support from CNPq, FAPERJ and FAPESP are also acknowledged. References
Aqil, M., Khan, I.Z., Ahmad, M.B., 1993. Flavonoids from Peperomia pellucida. Sci. Phys. Sci. 5, 213215.
25 25
4. Experimental 4.1. General procedures Optical rotations were determined using the Perkin Elmer 243B; polarimeter UV and IR spectra were measured on a Shimadzu UV-1601 and a PerkinElmer 599B, respectively. CD spectra were measured in methanol with a JASCO ORD/UV-6 spectropolarimeter. GC-MS analysis was performed in a GC-MS-QP5000 Shimadzu using fused capillary column (DB-1 30 m 0.20 mm), H2 as carrier gas and temperature programing from 40 to 240 C (5 C/min). High-resolution mass spectra were recorded on a Micromass spectrometer, VG-Autospec model; 1H- and 13C NMR were obtained on a Varian Gemini 200 NMR spectrometer operating at 200 MHz for 1H NMR and 50.2 MHz for13C NMR in CDCl3, using TMS as internal standard. The 1H and 13C NMR data were recorded too on a Bruker Avance DRX 500 spectrometer (500 MHz for 1H NMR and 125 MHz for 13C NMR) with TMS as internal standard, using DMSO-d6 as solvent. 4.2. Plant material Aerial tissue of P. blanda (Jacq.) Humb. Bonpl. and Kunth were collected near Parati, Rio de Janeiro State, Brazil. The identication of the plant was done by Prof. Elsie F. Guimaraes, Botanical Garden of Rio de Janeiro. A voucher sample (RB: 325983) was deposited in the Herbarium of Rio de Janeiro Botanical Garden, Rio de Janeiro, Brazil. 4.3. Extraction and isolation The dried aerial parts of the plant were exhaustively extracted with MeOH. The MeOH extract was suspended in a MeOHH2O (7:3) mixture and extracted in successive steps using hexane, CH2Cl2, EtOAc and n-BuOH. The hexane-soluble part was submitted to silica gel CC eluting with hexane/EtOAc mixtures of increasing polarity. The fraction eluted with hexane/EtOAc 510% was puried through a Sephadex LH-20 column yielding compounds 1 (18 mg) and 2 (22 mg) that were shown to be pure by TLC and GC/MS analysis. The sterols stigmasterol, sitosterol and campesterol were isolated as a mixture of 172 mg and identied from the literature (Blunt and Stothers, 1977).
496
L.S.M. Velozo et al. / Phytochemistry 67 (2006) 492496 Mbah, J.A., Tchuendem, M.H.K., Tane, P., Sterner, O., 2002. Two chromenes from Peperomia vulcanica. Phytochemistry 60, 799 801. Min, K.R., Kim, K.S., Ro, J.S., Lee, S.H., Kim, J.A., Son, J.K., Kim, Y., 2004. Piperlonguminine from Piper longum with inhibitory eects on amelanocyte-stimulating hormone-induced melanogenesis in melanoma B16 cells. Planta Med. 70, 11151118. Monache, F.D., Compagnone, R.S., 1996. A secolignan from Peperomia glabella. Phytochemistry 43, 10971098. Moreira, D.L., Guimaraes, E.F., Kaplan, M.A.C., 1998a. Non-polar constituents from leaves of Piper lhotzkianum. Phytochemistry 49, 13391342. Moreira, D.L., Guimaraes, E.F., Kaplan, M.A.C., 1998b. A chromene from Piper aduncum. Phytochemistry 48, 10751077. Moreira, D.L., De Souza, P.O., Kaplan, M.A.C., Guimaraes, E.F., 1999. Essential oil analysis of four Peperomia species (Piperaceae). Acta Hortic. 500, 6569. Orjala, J., Erdelmeier, C.A.J., Wright, A.D., Rali, T., Sticher, O., 1993. Two chromenes and a prenylated benzoic acid derivative from Piper aduncum. Phytochemistry 34, 813818. Parmar, V.S., Jain, S.C., Bisht, K.S., Jain, R., Taneja, P., Jha, A., Tyagi, O.D., Prassad, A.K., Wengel, J., Olsen, C., Boll, P.M., 1997. Phytochemistry of genus Piper. Phytochemistry 46, 597 673. Parmar, V.S., Jain, S.C., Gupta, S., Talwar, S., Rajwanshi, V.K., Kumar, R., Azim, A., Malhotra, S., Kumar, N., Jain, R., Sharma, N.K., Tyagi, O.D., Lawrie, S.J., Errington, W., Howarth, O.W., Olsen, C.E., Singh, S.K., Wengel, J., 1998. Polyphenols and alkaloids from Piper species. Phytochemistry 49, 10691078. Sacchetti, G., Maietti, S., Muzzoli, M., Scaglianti, M., Manfredini, S., Radice, M., Bruni, R., 2005. Comparative evaluation of 11 essential oils of dierent origin as functional antioxidants, antiradicals and antimicrobials in foods. Food Chem. 91, 621632. Salazar, K.J.M., Paredes, G.E.D., Lluncor, L.R., Young, M.C.M., Kato, M.J., 2005. Chromenes of polyketide origin from Peperomia villipetiola. Phytochemistry 66, 573579. Seeram, N.P., Jacobs, H., Mclean, S., Reynolds, W.F., 1998. A prenylated benzopyran derivative from Peperomia clusifolia. Phytochemistry 49, 13891391. da Silva, M.H.L., Zoghbi, M.G.B., Andrade, E.H.A., Maia, J.G.S., 1999. The essential oils of Peperomia pellucida Kunth and P. circinnata Link var. circinnata. Flav. Frag. J. 14, 312314. Tanaka, T., Asai, F., Iinuma, M., 1998. Phenolic compounds from Peperomia obtusifolia. Phytochemistry 49, 229232. Zoghbi, M.G.B., Andrade, E.H.A., Lobato, R.C.L., Tavares, A.C.C., Souza, A.P.S., Conceicao, C.C.C., Guimaraes, E.F., 2005. Peperomia circinnata Link and Peperomia rotundifolia (L.) Kunth growing on dierent host-trees in Amazon: volatiles and relationship with bryophytes. Biochem. Syst. Ecol. 33, 269274.
Aziba, P.I., Adedeji, A., Ekor, M., Adeyemi, O., 2001. Analgesic activity of Peperomia pellucida aerial parts in mice. Fitoterapia 72, 5758. Baldoqui, D.C., Kato, M.J., Cavalheiro, A.J., Bolzani, V.S., Young, M.C.M., Furlan, M., 1999. A chromene and prenylated benzoic acid from Piper aduncum. Phytochemistry 51, 899902. Bayma, J.de C., Arruda, M.S.P., Muller, A.H., Arruda, A.C., Canto, W.C.C., 2000. A dimeric ArC2 compound from Peperomia pellucida. Phytochemistry 55, 779782. Bessiere, J.M., Menut, C., Lamaty, G., Joseph, H., 1994. Variations in the volatile constituents of Peperomia rotundifolia Schlecht. & Cham. grown on dierent host-trees in Guadeloupe. Flav. Frag. J. 9, 131133. Blunt, J.W., Stothers, J.B., 1977. 13C NMR spectra of steroids A survey and commentary. Org. Magn. Res. 9, 439464. Chen, C.M., Jan, F.Y., Chen, M.T., Lee, T.J., 1989. Peperomins A, B, and C, novel secolignans from Peperomia japonica. Heterocycles 29, 411 414. Costantin, M.B., Sartorelli, P., Limberger, R.P., Henriques, A.T., Steppe, M., Ferreira, M.J.P., Ohara, M.T., Emerenciano, V.P., Kato, M.J., 2001. Essential oils from Piper cernuum and Piper regnellii: Antimicrobial activities and analysis by GC/MS and 13C NMR. Planta Med. 67, 771773. Dike, S.Y., Merchant, J.R., 1978. Reactions of orcinol derivatives with citral and dihydrocitral. Indian J. Chem. B 16, 11111112. Ferraz, A.B.F., Bordignon, S.A.L., Staats, C., Schripsema, J., Poser, G.L.V., 2001. Benzopyrans from Hypericum polyanthemum. Phytochemistry 57, 12271230. Govindachari, T.R., Kumari, K.G.N., Partho, P.D., 1998. Two secolignans from Peperomia dindigulensis. Phytochemistry 49, 21292131. Iwata, N., Wang, N., Yao, X., Kitanaka, S., 2004. Structures and histamine release inhibitory eects of prenylated orcinol derivatives from Rhododendron dauricum. J. Nat. Prod. 67, 11061109. Kikuchi, T., Mori, Y., Yokoi, T., Nakazawa, S., Kuroda, H., Masada, Y., Kitamura, K., Kuriyama, K., 1983. Structure and absolute conguration of sargatriol, a new isoprenoid chromenol from a brown alga, Sargassum tortile AGARDH, C. Chem. Pharm. Bull. 31, 106113. Konishi, T., Konoshima, T., Daikonya, A., Kitanaka, S., 2005. Neolignans from Piper futokadsura and their inhibition of nitric oxide production. Chem. Pharm. Bull. 53, 121124. Li, N., Wu, J.-L., Sakai, J., Ando, M., 2003. Dibenzylbutyrolactone and dibenzylbutanediol lignans from Peperomia duclouxii. J. Nat. Prod. 66, 14211426. Mabberley, D.J., 1993. The Plant Book. Cambridge University Press, Cambridge, UK. Mahiou, V., Roblot, F., Hocquemiller, R., Cave, A., Barrios, A.A., Fournet, A., Ducrot, P.H., 1995. Peperogalin, a new prenylated diphenol from Peperomia galioides. J. Nat. Prod. 58, 324328. Mahiou, V., Roblot, F., Hocquemiller, R., Cave, A., 1996. New prenylated quinones from Peperomia galioides. J. Nat. Prod. 59, 694697.
Cytotoxic and aromatic constituents from Salvia miltiorrhiza

Ming-Jaw Don a, Chien-Chang Shen
a
a,b
, Wan-Jr Syu c, Yi-Huei Ding a, Chang-Ming Sun
a,b,*
National Research Institute of Chinese Medicine, No 155-1, Section 2, Li-Nong Street, Shih-Pai, Taipei 112, Taiwan b Department of Biochemistry, National Yang-Ming University, Taipei 112, Taiwan c Institute of Microbiology and Immunology, National Yang-Ming University, Taipei 112, Taiwan Received 28 May 2005; received in revised form 1 October 2005 Available online 20 December 2005
Abstract As part of an ongoing study of traditional Chinese medicinal plants, the root tissue of Salvia miltiorrhiza was further investigated for its chemical constituents. Five naturally occurring products along with 13 known constituents were isolated from an ethyl acetate-soluble portion of its ethanol extract. Their structures were elucidated by means of spectroscopic methods. Some selected compounds were also evaluated for biological activity. 2005 Elsevier Ltd. All rights reserved.
Keywords: Salvia miltiorrhiza; Labiatae; Danshen; Cytotoxicity
1. Introduction The traditional Chinese medicine Salvia miltiorrhiza Bunge (Labiatae) has drawn attention by natural product chemists and medicinal clinicians as it has been used for treatment of menstrual disorder, menostasis, menorrhalgia, insomnia, arthritis, and coronary heart diseases, particularly angina pectoris and myocardial infarction (Jiangsu New Medical College, 1988; Chen, 1984; Chang and But, 2001). Numerous diterpenoid tanshinones have also been isolated from S. miltiorrhiza (Kakisawa et al., 1969; Chang et al., 1990) and many of them were shown to have various biological activities including antitumor (Chang and But, 2001; Ryu et al., 1997b; Yang et al., 1981; Wu et al., 1991) and antimicrobial (Honda et al., 1988; Gao et al., 1979) activities. Previously, we reported a novel compound with antitumor activity from S. miltiorrhiza (Wang et al., 2004). In the present paper, we describe the isolation and structural determination of several natural products from
the EtOAc fraction of the ethanolic extract of this plant. Some selected compounds have also been evaluated for their biological activity.
2. Results and discussion The further chemical investigation of S. miltiorrhiza was focused on the ethyl acetate-soluble portion of an ethanolic extract of the dried roots. Further fractionation by repeated column chromatography of the EtOAc extract resulted in the isolation of 18 components including ve new naturally occurring products (15) along with 13 known compounds. Compound 1 was obtained as a yellow oil with a molecular formula of C18H20O4 as determined by HREIMS. The 1 H NMR spectrum of 1 showed ve aromatic protons at d 8.33, 7.45, 7.40, 7.67, and 7.39, one aliphatic methine signal, two methylene groups (one oxygenated), and three methyl protons at d 2.65, 2.02, and 1.07 (Table 1). Furthermore, one OH group signal with intramolecular hydrogen bonding was observed at d 14.00. The 13C NMR and DEPT spectra of 1 indicated the presence of 18 carbon
Corresponding author. Tel.: +886 2 28201999; fax: +886 2 28264276. E-mail address: cmsun@nricm.edu.tw (C.-M. Sun).
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M.-J. Don et al. / Phytochemistry 67 (2006) 497503
Table 1 1 H NMR (500 MHz) spectroscopic data of 15 in CDCl3 Position d(J)a 1 1 2 3 4 5 6 7 9 11 12 13 14 15 16 17 18 19 20 22 Palmitate 2 3 16
a b
2 8.30 m 7.457.46 m 7.457.46 m
3 ax 1.54 eq 2.53 5.17 m eq 1.86 ax 1.33 t (11.5) m dd (3.5, 13.5) t (13.5)
5 2.28 s
8.33 d (7.5) 7.40 t (7.5) 7.45 d (7.5)
2.90 t (7.0) 2.03 t (7.0)
6.56 d (16.0) 7.19 d (16.0)
7.39 d (9.0) 7.67 d (9.0)
7.78 d (9.0) 7.95 d (9.0)
1.86 dd (3.5, 13.5) ax 2.58 dd (13.5, 18.0) eq 2.68 dd (3.5, 18.0)
7.43 d (9.0) 7.95 d (9.0)
6.59 d (3.0) 6.35 d (3.0) 4.61 s
6.71 s a 2.89 dd (8.0, 16.0) b 3.18 dd (6.0, 16.0) 2.62 m a 4.01 dd (6.5, 11.0) b 4.08 dd (5.5, 11.0) 2.02 s 1.07 d (6.5) 2.65 s
3.37 m a 4.47 dd (1.0, 11.5) b 4.76 dd (3.5, 11.5)
7.90 s 3.18 1.20 1.21 0.97 1.04 1.26 2.05 m d (7.0)b d (7.0)b s s s s
7.25 s 3.13 1.27 1.27 1.42 1.42 sept (7.0) d (7.0) d (7.0) s s
1.42 d (7.5) 2.62 s
2.67 t (7.5) 1.83 m 0.86 t (7.0)
J = Coupling constant in Hertz. Interchangeable.
signals (Table 2), which were assigned to three methyl, two methylene (one oxygenated), one saturated methine, one ketone (d 205.1), one ester (d 171.0), and 10 aromatic (ve protonated and ve quaternary) carbons. The connectivities of the 1H and 13C signals were determined by analysis of its HMQC spectrum, whereas 2D COSY correlations (H-1/H-2; H-6/H-7) and HMBC correlations (H-1/C-3, C-5, C-9; H-18/C-3, C-4, C-5; H-6/C-8, C-10; H-7/C-5, C-9) (Fig. 1) revealed the presence of a naphthalene ring. In addition, 2D COSY correlations (H-13/H-12, H-14, H-17) and HMBC correlations (H-13/C-11; H-14/C-15; H-16/C-15) suggested the presence of a 4-acetoxy-3-methylbutanoyl moiety. The moiety was also supported from analysis of the EIMS data which showed a molecular ion peak at m/z 300 and major fragment peaks at m/z 240, 225 and 185, suggesting [MCH3COOH]+, [MCH3COOHCH3]+ and [MCH3CO2C4H8]+, respectively. Furthermore, the IR spectrum exhibited one carbonyl absorption of a saturated aliphatic ester at 1739 cm1and the other carbonyl group with intramolecular hydrogen bonding and conjugation to an aromatic ring at 1623 cm1. The HMBC correlations of the OH group (dH 14.00) with C-8 and C-10 and H-7 with C-11 (Fig. 1) indicated that the 4-acetoxy-3-methylbutanoyl and hydroxyl groups were located on C-8 and C-9, respectively.
The structure of 1 was therefore assigned as 4-(1hydroxy-5-methylnaphthalen-2-yl)-2-methyl-4-oxobutyl acetate and compound 1 was given the trivial name salvianonol. Compound 2 was obtained as orange crystals with a molecular formula of C18H14O4 as determined by HREIMS. The 1H NMR spectrum exhibited ve aromatic protons at d 8.30, 7.95, 7.78, and 7.457.46, one aliphatic methylene group at d 4.47 and 4.76, one aliphatic methine proton at d 3.37, and two methyl signals at d 2.62 and 1.42 (Table 1). The 13C NMR and DEPT spectra of 2 showed 18 carbon signals (Table 2), which were assigned to two methyl, one oxygenated methylene, one saturated methine, ve protonated aromatic, and nine sp2 quaternary carbons. The connectivities of the 1H and 13C signals were determined by analysis of its HMQC spectrum. Analysis of the COSY, HMQC and HMBC data indicated that 2 was partially similar to 1 having a naphthalene ring. The COSY spectrum showed that the methine proton (H-13) was correlated with the methylene group (H-14) and the methyl protons (H-17). In the HMBC spectrum, the correlations (H-17/C-12, C-14; H-13/C-11, C-16; H-14/C-12, C-15; H7/C-9, C-11) were observed (Fig. 1), which suggested that a 5,6-dihydropyran-2-one moiety was fused to the bezochromen-4-one ring. In addition, the IR spectrum of 2
M.-J. Don et al. / Phytochemistry 67 (2006) 497503 Table 2 13 C NMR spectroscopic data of 18 in CDCl3 Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Palmitate 1 2 3 16
a
499
1 122.5 125.7 130.9 134.0 136.4 114.7 123.9 112.8 163.0 125.5 205.1 42.2 29.7 68.6 171.0 20.8 17.1 19.5
2 120.8 127.1 130.8 134.7 135.0 122.2 119.5 119.6 153.6 123.8 175.9 131.1 26.3 73.1 158.6 144.2 16.5 19.4
3 42.8 68.7 46.2 34.6 48.9 35.6 198.4 123.5 155.0 39.3 109.5 159.5 133.7 126.9 26.7 22.1a 22.4a 32.4 22.0 23.9 171.1 21.4
4 205.0 36.5 35.4 36.2 157.6 122.9 137.5 132.2 121.4 127.7 145.1 138.1 141.7 117.3 28.0 22.8 22.8 29.6 29.6
5 28.1 198.4 124.3 129.7 150.9 117.0 110.6 157.3 57.7
6 125.6 130.1 129.0 134.8 135.6 131.5 121.7 133.6 126.3 130.7 181.2 160.9 124.9 182.7 35.6 80.4 18.9 20.0
7 125.4 130.3 129.1 135.1a 135.0a 132.3 122.5 133.8 124.0 130.3 184.1 153.0 125.5 185.2 24.5 20.0 20.0 19.9
8 24.9 22.5 128.6 131.0 139.1 128.2 120.7 127.3 126.2 144.5 184.4 176.3 120.2 161.7 121.2 141.3 8.8 19.8
171.9 34.2 25.1 14.1
Interchangeable.
OH
O O
O O O
Fig. 1. Selected HMBC correlations of 1 and 2.
showed the presence of a lactone (1745 cm1) and a conjugated ketone (1644 cm1) absorptions. The structure of 2 was therefore assigned as 4,8-dimethyl-8,9-dihydro-10, 12-dioxa-benzo[a]anthracene-7,11-dione and compound 2 was given the trivial name salviamone. Compound 3 was obtained as an orange oil with a molecular formula of C22H30O4 as determined by HREIMS. The 13C NMR and DEPT spectra of 3 exhibited 22 carbon signals (Table 2), which were assigned to six methyl, three methylene, three aliphatic methine, two aliphatic quaternary, two carbonyl (d 171.1 and 198.4), and six aromatic (two protonated and four quaternary) carbons. The 1H NMR spectrum exhibited two aromatic protons (d 6.71 and 7.90), two doublets of two methyl signals (d 1.20 and 1.21), four singlets of four methyl groups, three aliphatic methine protons, and three methylene signals, which was ` partially similar to those of 2a-hydroxysugiol (Gonzalez
et al., 1988). The HMBC spectrum showed correlations of the carbonyl group (dC 171.1) with the methine (H-2, dH 5.17) and methyl groups (Me-22, dH 2.05), which indicated that an acetoxyl group was located on C-2. The relative stereochemistry of the acetoxyl group was determined on the basis of a NOESY experiment, which showed correlations of H-2 with Me-19 and Me-20 and suggested that the acetoxyl group was located in an equatorial orientation at C-2. In addition, the IR spectrum showed the presence of a hydroxyl group at 3286 cm1 and the carbonyl groups of saturated aliphatic ester and conjugated ketone at 1733 and 1652 cm1, respectively. Thus, compound 3 was established as 2a-acetoxysugiol. Compound 4 was obtained as a yellow oil with a molecular formula of C35H52O4 as determined by HREIMS. The EIMS of 4 gave a molecular ion peak at m/z 536 and a major fragment peak at m/z 298, suggesting a [Mpalmitoyl moiety + H]+. In the 1H NMR spectrum, the palmitoyl signals were observed at d 0.86, 1.42, 1.83, and 2.67. The presence of the palmitoyl moiety was further supported by analysis of the 13C NMR spectrum and comparison of the NMR spectroscopic data with those of methyl palmitate (Vandevoorde et al., 2003). In additional to the palmitoyl signals, the 1H NMR spectroscopic data (Table 1) showed an AB pattern for two ortho-aromatic protons at d 7.43 and 7.95, one aromatic signal as a singlet at d 7.25, a geminal dimethyl group at d 1.42 (6H), two methylene groups at d 2.03 and 2.90, and an isopropyl group at d 1.27 (6H) and 3.13 (1H), which were similar to those of arucadiol (Majetich et al., 1997). The IR spectrum of 4 indicated presence of a carbonyl stretch of an ester at higher frequency at 1762 cm1, suggesting the presence of aromatic group conjugation with an alcohol (OH) moiety. In addition, a carbonyl absorption was observed at lower frequency at 1642 cm1, due to the eects of conjugation with the aromatic ring and intramolecular hydrogen bonding. Furthermore, an OH signal with intramolecular hydrogen bonding (dH 10.55) suggested that an hydroxyl group was located on C-11, which was conrmed by its HMBC correlations with C-9, C-11 and C-12. Thus, compound 4 was established as palmitoyl arucadiol. Compound 5 was obtained as a yellow oil with a molecular formula of C9H10O3 as determined by HREIMS. The EIMS of 5 gave a molecular ion peak at m/z 166 and major fragment peaks at m/z 135, suggesting [MCH3O]+. The 1 H NMR spectrum exhibited two trans-olenic protons at d 7.19 and 6.56, two aromatic signals at d 6.59 and 6.35, one oxygenated methylene group as a singlet at d 4.61, and one methyl singlet at d 2.28 (Table 1). The 13C NMR and DEPT spectra of 5 showed nine carbon signals (Table 2), which were assigned to one methyl, one oxygenated methylene, four sp2 methine, and two oxygenated quaternary sp2 carbons. The connectivities of H and C were determined by analysis of an HMQC experiment. The HMBC data revealed the correlations of H-4 with C-2, 3, -5, and -6 and H-9 with C-7 and -8, indicating that 5 is a 2,5 disubstituted furan. The structure of 5 was therefore
500
assigned as (E)-4-[5-(hydroxymethyl)furan-2-yl]but-3-en-2one. Thirteen known compounds were also identied as dihydroisotanshinone I (6) (Kong and Liu, 1984), danshenxinkun B (7) (Luo et al., 1994), 1,2-dihydrotanshinone I (8) (Feng and Li, 1980), tanshinone I (Ryu et al., 1997a), tanshinone IIA (Ryu et al., 1997a), methylenetanshinquinone (Luo et al., 1994), 15,16-dihydrotanshinone I (Ikeshiro et al., 1991), cryptotanshinone (An et al., 2002), ferruginol (Harrison and Asakawa, 1987), sugiol (Gao and Han, 1997), norsavioxide (Li et al., 1991), and a mixture of danshenspiroketallactone and its epi-isomer (Asari et al., 1990) by analysis of their MS, 1D and 2D NMR spectroscopic data and by comparison with those in the literature; however, 13C NMR spectroscopic data of compounds 68 have not been previously reported and are reported herein. Compounds 1, 2, and 68 might be biosynthesized from abietic acid (9) (Scheme 1). Abietic acid may rstly lead to 7 and further to danshenxinkun A (10) via several steps of oxidation. Dehydration of 10 potentially aords 6 and dihydrotanshinone I (11), of which the latter might lead to 8 by hydrogenation and dehydrogenation. After BaeyerVilliger oxidation, compound 10 is envisaged to produce a seven-membered lactone intermediate 12, which is then hydrolyzed, followed by esterication and dehydration with ring closure to result in compound 2. Alterna-
tively, the lactone intermediate 12 can be followed in turn by hydrogenation, dehydration, retro-aldol reaction, hydration, hydrolysis and acetylation to aord compound 1. Compounds 14 and 7 were evaluated for their cytotoxicity against selected cancer cell lines by using MTT method and cisplatin as positive control. Among the tested compounds, 4 was the most potent compound with CD50 values of 3.2 and 4.1 lg/ml against the HeLa and OVCAR-3 cells, respectively, which had slightly lower CD50 values than cisplatin (Table 3). The cytotoxicities of compounds 14 and 7 against the above cancer cell lines have not been reported so far. In addition, compounds 14 were subjected to evaluation of antibacterial activity against Gram-positive Staphylococcus aureus and Enterococcus faecalis, and Gram-negative Escherichia coli, using paper disk methods. However, all tested compounds were considered inactive (inhibition zone <10 mm/100 lg/disk).
3. Conclusions So far about 50 abietanoids and diterpenoid tanshinones have been identied from the root of S. miltiorrhiza. The occurrence of tanshinones, however, can be used as a taxonomic characteristic for the genus Salvia (Patudin et al., 1974). Some Salvia species such as Salvia yunnanensis (Qian
O OH O
1 12 3 6 18 18 7 13 17
O
15 16 12 9 11
14 13 17 22 20 11
OH
1 9 3 5 7 14
16 15
15
16
O O
O O
3 6 7
H
19 18
1 O CH3(CH2)14 O HO
7 6
O O
1 3 9 1 3 6 18 11
O
14
16 15
HO
O
7
O 4 5
OH O O O
501
OH O [O] [O] [O] O
COOH
COOH
Dehydroabietic acid
OH
7
[O]
O O
OH
O OH [H] O
OH
OH OH [O] O O
O O H2O O
12
H 2O H2O O OH O O O OH O OH OH O
10
H 2O O
11
H2O O OH O OH HO OH O O O O
8
H2O OH O O O O O
1 2
Scheme 1. Proposed biosynthetic pathways for 1, 2, 68 of S. miltiorrhiza.
et al., 2002; Yang et al., 1996), Salvia przewalskii (Li et al., 1991), and Salvia paramiltiorrhiza f. purpureorubra (Wang, 1981), which have been used as substitutes in Chinese folk
medicine for Danshen, were also reported to contain the pharmacologically active tanshinones. We report here that ve new natural products, one furan derivative and four
502
Table 3 Cytotoxicity of 14 and 7 against human cancer cell lines Compound CD50 (lg/ml) HeLa 1 2 3 4 7 Cisplatin 17.4 >100 25.5 3.2 40.5 7.2 HepG2 37.5 >100 37.5 25.1 34.5 7.1 OVCAR-3 >100 >100 30.2 4.1 33.5 9.0
abietanoids, and 13 known diterpenoid tanshinones were isolated from an ethyl acetate-soluble portion of ethanol extract of S. miltiorrhiza. These new components could provide a support of the chemotaxonomic signicance for the species of S. miltiorrhiza.
4. Experimental 4.1. General experimental procedures Melting points were measured using a Yanaco MP-S9 micro-melting point apparatus and uncorrected. UV spectra were performed on a Hitachi U-3310 spectrophotometer. IR spectra were recorded on a Nicolet Avatar 320 FT-IR spectrometer. The 1H and 13C NMR spectra were recorded on a Varian Unity Inova 500 spectrometer in CDCl3 with tetramethylsilane (TMS) as an internal standard. COSY, HMQC, HMBC, and DEPT spectra were obtained using standard Varian pulse sequences. EIMS spectra were measured with a direct insertion probe on a Finnigan GCQ spectrometer at 30 eV. HREIMS data were taken on a Finnigan MAT 95XL mass spectrometer. Silica gel (Kieselgel 60, 70230 mesh, Macherey-Nagel) was used for column chromatography. TLC was carried out on aluminum sheets precoated with silica gel 60 F254 (layer thickness 0.2 mm, Merck Art. 5554). The chromatograms were visualized under UV light (254 or 365 nm) or by spraying with 5% phosphomolybdic acid in 5% H2SO4 containing a trace of ceric sulfate, followed by heating on a hot plate (120 C). 4.2. Plant material The dried and sliced roots of S. miltiorrhiza (5 kg) were purchased from the Cherng-Chi Chinese herbal shop in Taipei in April 2004. A voucher specimen (NRICM 04024) was deposited in the herbarium of National Research Institute of Chinese Medicine, Taipei. 4.3. Extraction and isolation The root of S. miltiorrhiza was extracted with EtOH (30 l) three times at 60 C for 24 h. The EtOH extracts were combined and concentrated in vacuo to 1 l. The concentrated extract was suspended in H2O (4 l) and partitioned
successively with EtOAc. After concentration of the EtOAc extract, the concentrate was mixed with 700 g of silica gel (230400 mesh). The air-dried mixture was subjected to silica gel column chromatography (cc) using a mixture of hexane-EtOAc of increasing polarity as eluents. Fractions were collected, with similar fractions (monitored by TLC) combined to give 5 fractions (F-1 to F-5). F-1 was further applied to silica gel cc to give danshenxinkun B (7) (12 mg) and ferruginol (153 mg). F-2 was resubjected to silica gel cc to give tanshinone IIA (1.2 g), 1,2-dihydrodanshinone I (8) (35 mg), palmitoyl arucadiol (4) (10 mg), methylenetanshinquinone (63 mg), and tanshinone I (480 mg), respectively. F-3 was separated by further silica gel cc to yield dihydroisotanshinone I (6) (12 mg), sugiol (75 mg), norsalvioxide (18 mg), and a mixture of danshenspiroketallactone and epi-danshenspiroketallactone (30 mg). F-4 was reapplied to silica gel cc to aord salvianonol (1) (28 mg), cryptotanshinone (700 mg), 2a-acetoxysugiol (3) (41 mg), and 15,16dihydrotanshinone I (175 mg). F-5 was also subjected to silica gel cc to give salviamone (2) (15 mg) and (E)-4-[5(hydroxymethyl)furan-2-yl]but-3-en-2-one (5) (43 mg). 4.4. Salvianonol, 4-(1-hydroxy-5-methylnaphthalen-2-yl)-2methyl-4-oxobutyl acetate (1) Yellow oil; aD 30 (CHCl3, c 0.1); UV (CH3OH) kmax (log e) 215.0 (5.06), 256.8 (5.00), 371.4 (4.17) nm; IR (lm) mmax 2963, 2925, 2853, 1739, 1623, 1576, 1471, 1384, 1238, 1081, 1038, 796 cm1; for 1H and 13C NMR spectra, see Tables 1 and 2, respectively; HREIMS m/z 300.1363 (calcd. for C18H20O4 300.1356); EIMS m/z (rel. int.): 300 [M]+ (80), 240 (67), 225 (100), 197 (8), 185 (33), 128 (10). 4.5. Salviamone, 4,8-dimethyl-8,9-dihydro-10,12-dioxabenzo[a]anthracene-7,11-dione (2) Orange crystals, m.p. 203204 C, aD 35 (CHCl3, c 0.2); UV (CH3OH) kmax (log e) 228.8 (4.83), 275.0 (4.36), 360.4 (3.87) nm; IR (lm) mmax 2967, 2925, 1745, 1644, 1510, 1466, 1400, 1266, 1211, 1169, 1120, 772 cm1; For 1 H and 13C NMR spectra, see Tables 1 and 2, respectively; HREIMS m/z 294.0887 (calcd. for C18H14O4 294.0887); EIMS m/z (rel. int.): 294 [M]+ (100), 249 (72), 235 (12). 4.6. 2a-Acetoxysugiol, (3S,4aS,10aS)-1,2,3,4,4a,9,10,10aoctahydro-6-hydroxy-7-isopropyl-1,1,4a-trimethyl-9oxophenanthren-3-yl acetate (3) Orange oil; aD 16 (CHCl3, c 1.0); UV (CH3OH) kmax (log e) 205.2 (4.43), 232.2 (4.36), 282.2 (4.25) nm; IR (neat) mmax 3286, 2963, 2925, 2870, 1733, 1652, 1596, 1464, 1366, 1268, 1179, 1129, 757 cm1; for 1H and 13C NMR spectra, see Tables 1 and 2, respectively; HREIMS m/z 358.2146 (calcd. for C22H30O4 358.2144); EIMS m/z (rel. int.): 358 [M]+ (68), 298 (38), 283 (100), 241 (53).
25 25 25
503
4.7. Palmitoyl arucadiol, 1,2,3,4-tetrahydro-5-hydroxy-7isopropyl-1,1-dimethyl-4-oxophenanthren-6-yl palmitate (4) Yellow oil; UV (CH3OH) kmax (log e) 216.8 (4.66), 271.6 (4.30) nm; IR (neat) mmax 2959, 2925, 2854, 1762, 1642, 1594, 1465, 1412, 1282, 1136 cm1; for 1H and 13C NMR spectra, see Tables 1 and 2, respectively, 13C NMR of palmitoyl moiety d 14.11, 22.69, 25.11, 29.27, 29.34, 29.36, 29.52, 29.70, 31.92, 34.25, 171.90; HREIMS m/z 536.3870 (calcd. for C35H52O4 536.3866); EIMS m/z (rel. int.): 536 [M]+ (3), 298 (100). 4.8. (E)-4-[5-(Hydroxymethyl)furan-2-yl]but-3-en-2-one (5) Yellow oil; for 1H and 13C NMR spectra, see Tables 1 and 2, respectively; HREIMS m/z 166.0633 (calcd. for C9H10O3 166.0624); EIMS m/z (rel. int.): 166 [M]+ (31), 151 (4), 135 (100), 67 (3).
Acknowledgements We gratefully acknowledge the nancial support through the NRICM (94-DMC-03) for this work. The authors also thank Ms. Shu-Chi Lin of the Instrumentation Center, National Tsing Hua University for HREIMS data.
References
An, L.-K., Bu, X.-Z., Wu, H.-Q., Guo, X.-D., Ma, L., Gu, L.-Q., 2002. Reaction of tanshinones with biogenic amine metabolites in vitro. Tetrahedron 58, 1031510321. Asari, F., Kusumi, T., Zheng, G.-Z., Cen, Y.-Z., Kakisawa, H., 1990. Cryptoacetalide and epicryptoacetalide, novel spirolactone diterpenoids from Salvia miltiorrhiza. Chem. Lett. 10, 18851888. Chang, H.-M., But, P., 2001. In: Chang, H.M., But, P. (Eds.), Pharmacology and Applications of Chinese Materia Medica, vol. 1. World Scientic, Singapore, pp. 237249. Chang, H.-M., Cheng, K.-P., Choang, T.F., Chow, H.-F., Chui, K.-Y., Hon, P.-M., Lau Tan, F.-W., Yang, Y., Zhong, Z.-P., Lee, C.-M., Sham, H.-L., Chan, C.-F., Cui, Y.-X., Wong, H.N.-C., 1990. Structure elucidation and total synthesis of new tanshinones isolated from Salvia miltiorrhiza Bunge (Danshen). J. Org. Chem. 55, 35373543. Chen, W.-Z., 1984. Pharmacology of Salvia miltiorrhiza (Yao Xue Xue Bao). Acta Pharm. Sinica 19, 876880. Feng, B.-S., Li, S.-R., 1980. Studies on the chemical components of Danshen (Salvia miltiorrhiza Bunge). Acta Pharm. Sinica (Yao Xue Xue Bao) 15, 489494. Gao, J., Han, G., 1997. Cytotoxic abietane diterpenoids from Caryopteris incana. Phytochemistry 44, 759761.
Gao, Y.-G., Song, Y.-M., Yang, Y.-Y., Liu, W.-F., Tang, J.-X., 1979. Pharmacology of tanshinone. Acta Pharm. Sinica (Yao Xue Xue Bao) 14, 7582. ` Gonzalez, A.G., Herrera, J.R., Luis, J., Ravelo, A.G., Ferro, E.A., 1988. Terpenes and avones of Salvia cardiophylla. Phytochemistry 27, 15401541. Harrison, L.J, Asakawa, T., 1987. 18-Oxoferruginol from the leaf of Torreya nucifera. Phytochemistry 26, 12111212. Honda, G., Koezuka, Y., Tabata, M., 1988. Isolation of an antidermatophytic substance from the root of Salvia miltiorrhiza. Chem. Pharm. Bull. 36, 408411. Ikeshiro, Y., Hashimoto, I., Iwamoto, Y., Mase, I., Tomita, Y., 1991. Diterpenoids from Salvia miltiorrhiza. Phytochemistry 30, 27912792. Jiangsu New Medical College, 1988. Dictionary of Chinese Material Medica (Zhong Yao Da Ci Dian). Shanghai Scientic and Technological Publishers, Shanghai, pp. 478482. Kakisawa, H., Hayashi, T., Yamazaki, T., 1969. Structures of isotanshinones. Tetrahedron Lett. 5, 301304. Kong, D.-Y., Liu, X.-J., 1984. Structure of dihydroisotanshinone I of danshen. Acta Pharm. Sinica (Yao Xue Xue Bao) 19, 755759. Li, B., Niu, F.-D., Lin, Z.-W., Zhang, H.-J., Wang, D.-Z., Sun, H.-D., 1991. Diterpenoids from the roots of Salvia przewalskii. Phytochemistry 30, 38153817. Luo, H.-W., Sun, X.-R., Niwa, M., 1994. Diterpenoids from Salvia paramiltiorrhiza. Heterocycles 38, 24732479. Majetich, G., Liu, S., Fang, J., Siesel, D., Zhang, Y., 1997. Use of conjugated dienones in cyclialkylations: total syntheses of arucadiol, 1,2-didehydromiltirone, ()-hinokione, ()-nimbidiol, sageone, and miltirone. J. Org. Chem. 62, 69286951. Patudin, A.V., Romanova, A.S., Sokolov, V.S., Pribylova, G.V., 1974. Occurrence of phenanthren-quinones in the genus Salvia. Planta Med. 26, 201207. Qian, Z., Liang, X., Hou, A., Ruan, Z., 2002. Medicinal resources of Salvia yunnanensis. Zhong Yao Cai 25, 628629. Ryu, S.Y., No, Z.S., Kim, S.H., Ahn, J.W., 1997a. Two novel abietane diterpenes from Salvia miltiorrhiza. Planta Med. 63, 4446. Ryu, S.Y., Lee, C.O., Choi, S.U., 1997b. In vitro cytotoxicity of tanshinones from Salvia miltiorrhiza. Planta Med. 63, 339342. Vandevoorde, S., Tsuboi, K., Ueda, N., Jonsson, K.O., Fowler, C.F., Lambert, D.M., 2003. Esters, retroesters, and a retroamide of palmitic acid: pool for the rst selective inhibitors of N-palmitoylethanolamine selective acid amidase. J. Med. Chem. 46, 43734376. Wang, X., Bastow, K.F., Sun, C.-M., Lin, Y.-L., Yu, H.-J., Don, M.-J., Wu, T.-S., Nakamura, S., Lee, K.-H., 2004. Antitumor agents. 239. Isolation, structure elucidation, total synthesis, and anti-breast cancer activity of neo-tanshinlactone from Salvia miltiorrhiza. J. Med. Chem. 47, 58165819. Wang, X.-M., 1981. Preliminary comparison of the quality between Salvia paramiltiorrhiza f. purpureorubra and Salvia miltiorrhiza Bunge. Chin. Pharm. Bull. 16, 89. Wu, W.-L., Chang, W.-L., Chen, C.-F., 1991. Cytotoxic activities of tanshinones against human carcinoma cell lines. Am. J. Chin. Med. 19, 207216. Yang, B.-J., Qian, M.-K., Qin, G.-W., Chen, Z.-Y., 1981. Studies on the active principles of Dan-Shen. V. Isolation and structures of przewaquinone A and przewaquinone B. Acta Pharm. Sinica (Yao Xue Xue Bao) 16, 837841. Yang, M.H., Blunden, G., Xu, Y.X., Nagy, G., Mathe, I., 1996. Diterpenoids from Salvia species. Pharm. Sci. 2, 6972.
Oligomeric secoiridoid glucosides from Jasminum abyssinicum
Francesca Romana Gallo a,*, Giovanna Palazzino a, Elena Federici a, Raaella Iurilli a, Franco Delle Monache b, Kusamba Chifundera c, Corrado Gale a
c
` Dipartimento del Farmaco, Istituto Superiore di Sanita, V. le Regina Elena 299, 00161 Rome, Italy ` CNR, Centro Chimica dei Recettori, Universita Cattolica S. Cuore, L.F. Vito 1, 00168 Rome, Italy Institut Superieur dEcologie pour la Conservation de la Nature, Lwiro, P.O. Box 293 Cyangugu, Rwanda
b
Received 29 March 2005; received in revised form 28 September 2005 Available online 27 December 2005
Abstract From the root bark of Jasminum abyssinicum (Oleaceae) collected in Congo was isolated tree oligomeric secoiridoid glucosides named craigosides AC. The three compounds are esters of a cyclopentanoid monoterpene with an iridane skeleton, esteried with three, two and two, respectively, units of oleoside 11-methyl ester. The structures were elucidated by spectroscopic methods and chemical correlations. 2005 Elsevier Ltd. All rights reserved.
Keywords: Jasminum abyssinicum; Oleaceae; Root bark; Oligomeric secoiridoid glucosides; Craigoside A; Craigoside B; Craigoside C
1. Introduction Genus Jasminum (Oleaceae) includes beyond 200 species, some of which are used in folk medicine or cultivated to obtain essential oil from the fragrant owers. The term Jasminum (Oleaceae) was rst mentioned in the Materia Medica of Dioscoride (I A.D.). The phytochemical studies of the aerial parts of some species, J. sambac [Soland.] (Tanahashi et al., 1988), J. mesnyi Hance (Tanahashi et al., 1989), J. urophyllum Hemsl. (Shen and Hsieh, 1997) and J. nudiorum Lindl. (Tanahashi et al., 2000), resulted in the isolation of some secoiridoid glucosides, in particular of oligomeric consisting of oleoside units linked to a cyclopentanoid monoterpene named iridane. This study deals with the structure elucidation of three oligomeric secoiridoid glucosides, two trimer and one tetramer, isolated from the root bark of Jasminum abyssinicum R. Br. (= Hochst. ex DC.) and named craigosides A, 1, B,
q ` Presented at FITOMED 2004, 1 Congresso Intersocieta sulle Piante Medicinali, Trieste, Italy, 1619 September, 2004. * Corresponding author. Tel.: +39 06 49903055; fax: +39 06 49903060. E-mail address: gallo@iss.it (F.R. Gallo).
2, and C, 3, as a tribute to L.C. Craig (Craig and Post, 1949) and his apparatus of counter-current distribution (CCD) utilised for our separations. The aerial parts of this plant are used in Traditional Medicine in the South Kivu Province, Congo against endoparasitic worms and for treatment of mumps (Chifundera, 2001).
2. Results and discussion Craigoside A, 1, is an amorphous powder, C61H86O34 (ICR-FTMS, m/z 1385.48602 [M + Na]+), a20 185 D (MeOH), kmax 233 nm (log e 4.54). Its 1H and 13C NMR spectra (Tables 1 and 2) showed inter alia a pattern of signals corresponding to oleoside methyl ester, viz., an acetalic methine and an anomeric methine, a vinylic oxymethine, an ethylidene and a carbomethoxy. In agreement with the molecular formula of 1, the signals of some carbons (6, 8, 9, 1 0 , 4 0 and 6 0 ) of this iridoid moiety appeared in triplicate and in particular the methoxy group, d 52.05, 52.03 and 52.00. This accounted for the presence of three oleoside methyl ester units and thus the presence of cyclic esters engaging two carboxylic groups of the same oleoside could
F.R. Gallo et al. / Phytochemistry 67 (2006) 504510

OGl O
505
O=C H O
7"
COOMe
9" 10"
H2C
8"
CH2
OH
HO
H2C
CH2
OH
O MeOOC H C=O
H2C
2" 1"
3" 4" 5"
O
7 11
O
H
5
H2C
O O=C CH3 H COOMe
O=C
6
COOMe MeOOC
4 3
C=O
CH3 O OGl
6"
10
9 8
O
1
O OGl
O OGl
OGl
Craigoside A (1)
OGl O
Craigoside B (2)
O=C H HO H2C CH2 O
COOMe
O MeOOC H C=O
H2C
OH
Craigoside C (3)
CH3
O OGl
be ruled out. The LR HETCOR observed in 1 between the a-b unsaturated carboxyl group, d 168.6, and the methoxy group, d 3.72, accounted for the methyl ester in position 11 of the iridoid and therefore the other carboxyl group, (C-7, d 173.1), was engaged in the linkage with the non-iridoidic moiety. The 10 minor 13C signals of craigoside A due to this last part of the molecule corresponded to a cyclopentanoid monoterpene, endowed with three oxymethylene groups, d 68.4, 65.1 and 61.2, an oxymethine, d 80.0, and a methyl group, d 19.7. By alkaline hydrolysis of 1 and subsequent methylation with diazomethane, dimethyl oleoside, 4, and tetraol iridane 5, C10H20O4 (ICR-FTMS, m/z 227.12552 [M + Na]+), 26 aD 2:4 (MeOH), were obtained. This last substance appeared dierent from the two known tetraols 6 and 7 obtained by saponication of sambacosides A, E and F of J. sambac (Tanahashi et al., 1988) and of jasurosides C and D of J. urophyllum (Shen and Hsieh, 1997), respectively. In particular, the cyclic methylene of 5, instead of at d 37.7 and 37.0, as in 6 and 7, respectively, resonated at higher chemical shift (d 43.2), as in the known triol 8 (d 43.7) obtained from nudiosides AC of J. nudiorum
(Tanahashi et al., 2000) and having an hydroxy group in position 400 instead of 500 .
7 11
MeOOC
6 10
H
5 9 8
COOMe
4 3
O
1 2'
O HO
1'
3'
OH
4'
OH
5' 6'
OH
Dimethyl oleoside (4)
The HETCOR and selective 1H-1H decoupling of the tetra-acetyl derivative of 5, 9, gave full account of its structure and relative stereochemistry. Thus the irradiation of H-400 of the acetoxymethine (d 5.12, quintet, J = 6.0, 3.0 and 3.0) made the two signals of the adjacent methylene, d 1.52, dq, J = 13.5, 10.5 and 6.0, Ha-500 , and d 1.83, m, J = 13.5, 6.8 and 3.0, Hb-500 , into two dd with the loss of the couplings of J = 6.0 and 3.0, respectively. Moreover,
506
Table 1 1 H NMR spectroscopic data of compounds 1, 2, 3 and 5 in CD3OD Position 1 3 5 6a 6b 8 10 1 5.97, 5.94, 7.54, 7.53, 4.10, bs bs s s dd (11.0; 4.8) 2 5.96, 5.94, 7.54, 7.55, 4.16, 2.76, 2.70, 2.51, 2.47, 6.12, 6.11, 1.76, s s s s dd (11.0; 4.8) dd (13.0; dd (13.0; dd (13.0; dd (13.0; bq (6.8) bq (6.8) d (6.9) 4.8) 4.8) 11.0) 11.0) 3 5.95, s 7.55, s 4.29, dd (11.0; 4.8) 4.19, dd (11.0; 4.8) 2.76, dd (13.0; 4.8) 2.52, 2.48, 6.11, 6.10, 1.77, 1.75, dd (13.0; 11.0) dd (13.0;11.0) bq (6.8) bq (6.8) d (6.8) d (6.8) 5
2.72, dd (13.0; 4.8) 2.49, dd (13.0; 11.0) 6.10, bq (6.8) 1.77, 1.75, 1.73, 3.72, 4.82,
A
MeO 10 2 0 -5 0 60 a 60 b 100 200 300 400 500 a 500 b 600 700 800 900 1000
A
d d d s d
(6.8) (6.8) (6.8) (7.6)
3.74, s 4.83, d (7.6)

A
3.73, s 4.83, d (7.6)

A
3.66, m 3.94, m 1.93, m 1.69, m 1.91, m 5.11, m 1.52, ddd (12.6; 6.7; 3.6) 1.95, m 1.09, d (6.5) 4.05 m 1.89 m 4.12 m 3.59 m
3.66, m 3.99, m 1.94, m 1.68, m 1.90, m 5.11, m 1.53, ddd (13.0; 6.7; 2.5) 1.86, m 1.10, d (6.6) 4.06, m 1.83, m 3.66 m 3.68 m
3.66 m 3.99 m 1.93, m 1.69, m 1.90, m 4.10, m 1.52, ddd (12.5; 6.7; 3.5) 1.88, m 1.07, d (6.6) 4.05, m 1.86, m 3.63 m 4.11 m
1.97, 1.51, 1.75, 4.08, 1.48, 1.70, 1.03, 3.70, 1.70, 3.63, 3.61, 3.59,
m (6.9; 6.9) m m (5.1) q (5.1; 5.1; 5.1) m (11.9; 6.9; 5.1) m (11.9; 5.1) d (6.9) m m m m m
In the range 3.33.5.
the irradiation in the range of d 4.04.2 corresponding to the three acetoxymethylenic groups made the signal of H800 (d 2.11, m) into a doublet with J = 6.3 and the signal of H-200 (d 1.63, m) into a perfect triplet, J = 9.0. The dihedral angle of H-200 both with H-100 and H-300 consistent with this last coupling was about 140 (trans relationship with both) and it corresponded to a position of C-200 out of the plane of the other four carbons of the cyclopentanoid ring, thus allowing a quasi equatorial allocation of the substituents in 200 and 300 . This ring conformation in 9 was in agreement with the coupling constants J = 3.0 Hz between H-300 and H-400 and between H-400 and Hb-500 corresponding to the dihedral angle of about 115 (trans relationship for both). In tetraol 5, the signal d 4.08 of H-400 was a quartet, due to the identical coupling constant, J = 5.1, with H-300 , Ha500 and Hb-500 . The cis relationship between HO-400 and Hb-500 was further conrmed by the downeld shift of the latter, d 1.70, respect to Ha-500 , d 1.48, owing to the anisotropic eect of the former. In order to establish the absolute conguration of tetraol 5, according the Moshers method through esterication of the secondary alcoholic function with (S)-MTPA and (R)-MTPA (Ohtani et al., 1991), tetraacyl derivatives 10 and 11 were prepared, respectively. The results of Dd
(1H NMR) (dS dR) showed, in line with the models of Fig. 1, the b conguration of the hydroxyl group in 400 . The structure 5 was thus unambiguously established for the tetraol iridane of craigoside A.
9"
" 10 H C
3"
H2C
CH2
8" 4"
5
RO
7" H2C
2" 1"
" R
3
6"
B= (S)-MTPA radical C= (R)-MTPA radical
R1 5 6 7 8 9 10 11 -- Me -- Me Me --Me --Me --Me --Me
R2
R3
R4 H H H H Ac B C
R5 OH OH OH H OAc OB OC
R6 OH OH OH OH OAc OB OC
OH H H OH H -- OH OH H OAc H OB H OC H
The three 11-methyl oleoside units on tetraol 5 in craigoside A, 1, were assigned in positions 400 , 700 and 900 on the
F.R. Gallo et al. / Phytochemistry 67 (2006) 504510 Table 2 13 C NMR spectroscopic data of compounds 1, 2,3 and 5 in CD3OD Position 1 3 4 5 6 7 8 9 10 11 MeO 10 20 30 40 50 60 100 200 300 400 500 600 700 800 900 1000 1 95.2 155.1 109.4; 109.3 31.8; 31.7 41.4; 41.3; 41.2 173.1 125.0; 124.9; 124.8 130.9; 130.7;130.6 13.8 168.6 52.05; 52.03; 52.00 100.9; 100.8; 100.7 74.7 78.4; 78.3 71.5; 71.4; 71.3 77.9 62.9; 62.8; 62.7 37.0 49.5 49.9 80.0 41.3 19.7 68.4 43.5 65.1 61.2 2 95.3; 95.2 155.5 109.8; 109.7 31.9; 31.8 41.6; 41.4 173.0; 172.9 125.1; 125.0 130.6; 130.5 13.9;13.8 168.8; 168.7 52.1; 52.0 100.9; 100.8 74.9; 74.8 78.6; 78.5 71.6 78.0 62.8; 62.7 37.3 49.8 49.7 80.3 41.8 20.0 68.7 46.8 62.7 62.0 3 95.2; 95.1 155.3 109.5 32.0 41.4; 41.3 173.4; 173.3 125.0 130.8; 130.7 13.9 168.8; 168.7 52.2; 52.1 100.9; 100.8 74.9; 74.8 78.5 71.6; 71.5 78.0 62.6 35.7 49.6 52.6 74.4 43.6 20.7 67.7 42.8 61.6 64.2 5
507
35.1 51.9 52.6 75.5 43.2 20.9 65.8 46.3 62.7 61.9
(5'') HC 2 downfield
CH (3'') C (4'')
upfield
(5'') 2HC upfield
CH (3'')
downfield
MeO
H
Ph
(R)
C (4'')
and C-700 , d 68.7, respect to the corresponding values of the tetraol, d 75.5 and 65.8, respectively, showed unambiguously the positions of the two iridoid moieties in the molecule of craigoside B. Respect to craigoside A, 1, the absence of an iridoid unit at C-900 in craigoside B, 2, resulted in the upeld shift of C-900 itself from d 65.1 to 62.7 and the downeld shift of C-800 from d 43.5 to 46.8. Craigoside C, 3, is an amorphous powder, C44H64O24 (ICR-FTMS, m/z 999.37081 [M + Na]+), a20 160 D (MeOH), kmax 236 nm (log e 4.26 ). Its 1H and 13C NMR data are reported in Tables 1 and 2, respectively. The alkaline hydrolysis of this trimer, isomer of 2, and the subsequent methylation with diazomethane likewise gave tetraol 5 and dimethyl oleoside, 4. The downeld shifts observed in 3 only for C-700 , d 67.7, and C-1000 , d 64.2, respect to the corresponding values of 5, d 65.8 and 61.9, besides the b upeld shifts for C-200 and C-800 in the former, gave account of the positions 700 and 1000 for the two oleoside 11-methyl ester units. The CD curve of craigoside A, having the same conguration at C-800 as molihuaside E from J. sambac (Zhang et al., 1995), showed an additional band at 247 nm besides the band at 228 nm. In summary, the oligomeric secoiridoid glucosides, new respect to the previously described ones occurring in the aerial parts of Jasminum genus plants, have been isolated from the root bark of J. abyssinicum from Congo. The three oligomeric, craigoside A, tetramer, and craigosides B and C, trimer, have the same cyclopentanoid monoterpene, which is a tetraol iridane, 5, esteried by oleoside 11-methyl ester units.
Ph
H
OMe
(S)
3. Experimental
CF3 CF3
3.1. General
(3'') HC (5'') H2C
4''
MeO O H O
Ph
(5'')
(R)
(3'') HC
Ph O
(S)
4''
OMe CF3
H2C H
CF3
Fig. 1. Congurational correlation models for the (R)-MTPA derivatives and the (S)-MTPA derivatives proposed by Mosher.
A Craig-Post apparatus, 200 stages, 10:10 ml, upper and lower phase, for the CCD. 1H NMR, 300 MHz, 13C NMR, 75 MHz, TMS as internal standard, chemical shifts (d) in ppm, coupling constants (J) in Hz, Varian Gemini 300. ICR-FTMS, high resolution, APEX II Bruker; ESI-MS, Thermo Finnigan, and FAB-MS, VG 7070 EQ-HF. CD, Jasco 710. 3.2. Plant material Root barks of J. abyssinicum R. Br. were collected in March 1999 near Bukavu (South Kivu Province, Congo). The plant material was identied in the Institut Superieur dEcologie pour la Conservation de la Nature, Lwiro (Cyangugu, Rwanda), where a voucher specimen (Mubeza, B 346) is deposited. 3.3. Extraction and isolation Air-dried root barks (310 g) were extracted three times with MeOH. The residue from the evaporation of the sol-
basis of the a downeld eects (and the b upeld eects) on the 13C resonances respect to the corresponding ones of the tetraol. Thus the chemical shifts of C-400 , C-700 and C-900 in 5, d 75.5, 65.8 and 62.7, respectively, moved to d 80.0, 68.4 and 65.1 in craigoside A. Craigoside B, 2, is an amorphous powder, C44H64O24 20 (ICR-FTMS, m/z 999.36655 [M + Na]+), aD 170 1 13 (MeOH), kmax 236 nm (log e 4.29). Its H and C NMR spectra (Tables 1 and 2) showed the signals typical of 11methyl oleoside, the most of them in duplicate. By alkaline hydrolysis and subsequent methylation, the aforementioned tetraol 5, and dimethyl oleoside, 4, were obtained. The downeld shifts observed in 2 only for C-400 , d 80.3,
508
vent (32.6 g) was dissolved in water (350 ml) and extracted with EtOAc (2 300 ml). The aqueous phase evaporated to dryness under vacuum gave as residue 26.5 g. Six grams of this was submitted to CCD with the biphase system H2O:EtOAc:n-PrOH discontinuously changing the ratio from 10:9:1 to 10:7:3. The separations were monitored by TLC, silica gel F254, solvent n-BuOH:H2O:HOAc = 4:5:1 (upper phase); detection by uorescence quenching and/or by spray reagent anisaldehyde: H2SO4:HOAc:EtOH = 0.5:0.5:0.1:9. Three of the nine collected fractions, J6, 514 mg, J7, 441 mg, and J6/7, 138 mg, were submitted to CCD on recycling with the solvent system H2O:EtOAc:n-PrOH = 10:7:3 and three pure compounds, craigoside C, 3, 203 mg, craigoside B, 2, 170 mg, and craigoside A, 1, 366 mg, were obtained. 3.4. Craigoside A (1) Amorphous powder, aD 185 (MeOH, c 0.5), UV (MeOH), kmax nm (log e): 233 (4.54); CD (MeOH), k nm ([H]): 211 (8.1 107), 228 (12.7 107), 247 (8.8 107). Molecular formula C61H86O34, ICR-FTMS m/z: 1385.48602 [M + Na]+, calcd 1385.48927; ESI-MS m/z: 1386.6, 16 [M + Na]+, 1224.3, 100 [M + Na 162]+, 1062.4, 7 [M + Na 162 2]+, 982.4, 67 [M + Na an iridoid H2O]+, 819.3, 32 [M + Na an iridoid H2O 162]+. 1H and 13C NMR data in Tables 1 and 2, respectively. 3.5. Craigoside B (2) Amorphous powder, a20 170 (MeOH, c 0.4), UV D (MeOH), kmax nm (log e): 236 (4.29); CD (MeOH), k nm ([H]): 233 (7.0 107). Molecular formula C44H64O24, ICR-FTMS m/z: 999.36655 [M + Na]+, calcd 999.36797; ESI-MS m/z: 1000.3, 41 [M + Na]+, 837.3, 55 [M + Na 162]+, 595.3, 100 [M + Na an iridoid H2O]+. 1 H and 13C NMR data in Tables 1 and 2, respectively. 3.6. Craigoside C (3) Amorphous powder, aD 160 (MeOH, c 0.4), UV (MeOH), kmax nm (log e): 236 (4.26); CD (MeOH), k nm ([H]): 232 (5.7 107). Molecular formula C44H64O24, ICR-FTMS m/z: 999.37081 [M + Na]+, calcd 999.36797; ESI-MS m/z: 999.5, 34 [M + Na]+, 837.3, 100 [M + Na 162]+, 595.3, 15 [M + Na an iridoid H2O]+. 1H and 13 C NMR data in Tables 1 and 2, respectively. 3.7. Acetylation of craigosides AC Each substance (50 mg) was acetylated with pyridine and Ac2O (each, 0.5 ml). After evaporation of the reagents under vacuum, the compound was puried by CC (silica gel, solvents cyclohexane:EtOAc = 2:8) to give the corresponding pure peracetate.
20 20
3.7.1. Craigoside A tredeca-acetate 24 Crystals from cyclohexane, mp 8789 C, aD 153 1 (CHCl3, c 0.4). H NMR (CDCl3) d: 7.46 (3H, s, H-3 3); 6.01, 5.99 (3H, bq, J = 6.7, H-8 3); 5.72 (3H, bs, H-1 3); 5.28 (3H, t, J = 9.3, H-3 0 3); 5.13 (3H, t, J = 9.3, H4 0 3); 5.12 (3H, dd, 9.3, 7.8, H-2 0 3); 5.11 (1H, m, H-400 ); 5.06 (3H, d, J = 7.8, H-1 0 3); 4.33 (3H, dd, J = 12.3, 3.2, Ha-6 0 3); 4.10 (3H, dd, J = 12.3, 1.5, Hb-6 0 3); 3.96 (3H, dd, J = 11.0, 4.8, H-5 3); 3.78 (3H, m, J = 9.3, 3.2, 1.5, H-5 0 3); 3.71 (9H, s, MeO 3); 2.66 (3H, dd, J = 13.0, 4.8, Ha-6 3); 2.42 (3H, dd, J = 13.0, 11.0, Hb-6 3); 2.11 (1H, m, H-800 ); 2.08, 2.04 (39H, s, CH3CO 13); 1.94 (1H, m, Hb-500 ); 1.86 (1H, m, H-300 ); 1.75 (9H, d, J = 6.9, H310 3); 1.65 (1H, m, H-200 ); 1.51 (1H, m, Ha-500 ); 1.05 (3H, d, J = 6.3, H3-600 ). 13C NMR d: 170.9, 170.4, 170.0 (C7 3); 169.3, 169.2 (CH3CO 13); 166.6 (C-11 3); 152.9 (C-3 3); 128.5, 128.2 (C-9 3); 124.7, 124.5 (C-8 3); 108.5, 108.4 (C-4 3); 97.0 (C-100 3); 93.7 (C-1 3); 77.9 (C-400 ); 72.4 (C-5 0 3); 72.1 (C-3 0 3); 70.6 (C-2 0 3); 68.0 (C-4 0 3); 67.0 (C-700 ); 63.2 (C-900 ); 62.4 (C-1000 ); 61.5 (C6 0 3); 51.4 (MeO 3); 48.5 (C-300 ); 47.7 (C-200 ); 40.2 (C500 ); 39.8 (C-6 3); 38.9 (C-800 ); 35.8 (C-100 ); 29.9 (C-5 3); 21.0, 20.6 (CH3CO 13); 19.4 (C-600 ); 13.5 (C-10 3). 3.7.2. Craigoside B deca-acetate 24 Crystals from cyclohexane, mp 7375 C, aD 133 1 (CHCl3, c 0.4). H NMR (CDCl3) d: 7.46, 7.45 (2H, s, H-3 2); 6.01 (2H, bq, J = 6.6, H-8 2); 5.73, 5.72 (2H, s, H-1 2); 5.28 (2H, t, J = 9.4, H-3 0 2); 5.15 (2H, t, J = 9.4, H-4 0 2); 5.13 (1H, m, H-400 ); 5.12 (2H, dd, J = 9.4, 8.1, H-2 0 2); 5.06, 5.04 (2H, d, J = 8.1, H1 0 2); 4.33 (2H, dd, J = 12.3, 4.5, Ha-6 0 2); 4.11 (2H, dd, J = 12.3, 2.1, Hb-6 0 2); 4.00, 3.96 (2H, dd, J = 11.0, 4.8, H-5 2); 3.80 (2H, m, J = 9.4, 4.6, 2.1, H-5 0 2); 3.71 (6H, s, MeO 2); 2.71, 2.62 (2H, dd, J = 13.0, 4.8, Ha-6 2); 2.45, 2.40 (2H, dd, J = 13.0, 11.0, Hb-6 2); 2.11 (1H, m, H-800 ); 2.09, 2.04, 2.03 (30H, s, CH3CO 10); 1.96 (1H, m, Hb-500 ); 1.84 (1H, m, H-300 );1.76 (6H, d, J = 6.9, H3-10 2); 1.65 (1H, m, H- 200 ); 1.52 (1H, m, Ha500 ); 1.05 (3H, d, J = 6.3, H3-600 ). 13C NMR d: 171.3, 170.9 (C-7 2); 170.8, 170.7, 170.3, 169.5, 169.0 (CH3CO 10); 166.9 (C-11 2); 153.2 (C-3 2); 128.8, 128.7 (C-9 2); 125.0, 124.8 (C-8 2); 108.8 (C-4 2); 97.4, 97.3 (C-1 0 2); 94.2, 94.0 (C-1 2); 77.8 (C-400 ); 72.8 (C-5 0 2); 72.4 (C-3 0 2); 71.0 (C-2 0 2); 68.5 (C-4 0 2); 67.3 (C-700 ); 63.6 (C-900 ); 62.8 (C-1000 ); 61.9 (C-6 0 2); 51.6 (MeO 2); 49.1 (C-300 ); 48.1 (C-200 ); 40.7 (C-500 ); 40.2, 40.1 (C-6 2); 39.2 (C-800 ); 36.0 (C-100 ); 30.4, 30.3 (C-5 2); 21.0, 20.8 (CH3CO 10); 19.6 (C-600 ); 13.8, 13.7 (C-10 2). 3.7.3. Craigoside C deca-acetate 24 Crystals from cyclohexane, mp 6971 C, aD 114 1 (CHCl3, c 0.4). H NMR (CDCl3) d: 7.47 (2H, s, H-3 2); 6.01 (2H, bq, J = 7.2, H-8 2); 5.71 (2H, bs, H-1 2); 5.28 (2H, t, J = 9.6, H-3 0 2); 5.14 (2H, t, J = 9.6, H-4 0 2); 5.13 (1H, m, H-400 ); 5.12 (2H, dd, J = 9.6, 8.1, H-2 0 2); 5.04 (2H, d, J = 8.1, H-1 0 2); 4.33 (2H, dd, J = 12.3, 4.2,
509
Ha-6 0 2); 4.07 (2H, dd, J = 12.3, 1.8, Hb-6 0 2); 4.02 (2H, dd, J = 11.0, 4.8, H-5 2); 3.78 (2H, m, J = 9.6, 4.2, 1.8, H-5 0 2); 3.72 (6H, s, MeO 2); 2.70, 2.69 (2H, dd, J = 13.0, 4.8, Ha-6 2); 2.44 (2H, dd, J = 13.0, 11.0, Hb6 2); 2.11 (1H, m, H-800 ); 2.08, 2.03, 2.02, 2.00 (30H, s, CH3CO 10); 1.96 (1H, m, Hb-500 ); 1.84 (1H, m, H-300 ); 1.76 (6H, d, J = 6.9, H3-10 2); 1.63 (1H, m, H-200 ); 1.54 (1H, m, Ha-500 ); 1.05 (3H, d, J = 6.6, H3-600 ). 13C NMR d: 171.0, 170.8 (C-7 2); 170.5, 170.4, 170.2, 170.0, 169.2, 169.1 (CH3CO 10); 166.5 (C-11 2); 152.9 (C-3 2); 128.4 (C-9 2); 124.7 (C-8 2); 108.5 (C-4 2); 97.0, 96.9 (C-1 0 2); 93.7, 93.6 (C-1 2); 76.9 (C-400 ); 72.4 (C-5 0 2); 72.1 (C-3 0 2); 70.7 (C-2 0 2); 68.1 (C-4 0 2); 66.1 (C-700 ); 63.2 (C-900 ); 62.5 (C-1000 ); 61.6 (C-6 0 2); 51.3 (MeO 2); 48.4 (C-300 ); 48.0 (C-200 ); 40.5 (C-500 ); 39.8, 39.7 (C-6 2); 38.9 (C-800 ); 35.2 (C-100 ); 30.0, 29.9 (C-5 2); 21.0, 20.4 (CH3CO 10); 19.1 (C-600 ); 13.4 (C-10 2). 3.8. Alkaline hydrolysis of craigosides AC. Methylation with diazomethane Each compound (300 mg) was treated with 0.5 M NaOH (5 ml). After 20 h the solution was neutralized with weakly acid cation-exchanger (H+ form) and concentrated in vacuum to dryness. The residue was dissolved in MeOH and methylated with an ethereal solution of diazomethane. After 2 days the residue obtained by evaporation of the solvents was submitted to CCD with solvent system H2O:nBuOH:EtOAc = 10:7.5:2.5 and dimethyl oleoside, 4, and tetraol iridane, 5, were separated. The former was identied by NMR and rotatory power (Tanahashi et al., 1988). 3.8.1. Tetraol iridane (5) 26 Syrop, aD 2:4 (MeOH, c 0.4). Molecular formula C10H20O4, ICR-FTMS m/z: 227.12552 [M + Na]+, calcd 227.12538; FAB-MS m/z: 205, 100 [M + 1]+, 187, 66 [M 17]+. 1H and 13C NMR data in Tables 1 and 2, respectively. 3.9. Acetylation of 5: iridane tetra-acetate (9) Tetraol iridane 5 (28 mg) was acetylated with pyridine and Ac2O (each, 1 ml). After evaporation of the reagents under vacuum, the product was puried by CC (silica gel, solvents cyclohexane:EtOAc = 3:7) to give the correspond23 ing tetra-acetate. Oily, aD 14:2 (CHCl3, c 0.3). Molecular formula C18H28O8, FAB-MS m/z: 372 (1, M), 329 (3, M-Ac), 313 (3, M-AcO), 269 (45, M-Ac-AcOH), 150 (100). 1 H NMR (CDCl3) d: 5.12 (1H, quintet, J = 6.0, 3.0, 3.0, H400 ); 4.17, 4.05 (2H, m, H2-900 ); 4.13, 3.99 (2H, m, H2-1000 ); 4.11, 4.00 (2H, m, H2-700 ); 2.11 (1H, m, J = 6.3, H-800 ); 2.08, 2.06, 2.05, 2.02 (12H, s, CH3CO 4); 1.96 (1H, m, J = 9.0, 6.3, 3.0, H-300 ); 1.94 (1H, m, J = 10.5, 9.0, 6.8, 6.6, H-100 ); 1.83 (1H, m, J = 13.5, 6.8, 3.0, Hb-500 ); 1.63 (1H, m, J = 9.0, 9.0, H-200 ); 1.52 (1H, dq, J = 13.5, 10.5, 6.0, Ha -500 ); 1.07 (3H, d, J = 6.6, H3-600 ). 13C NMR d: 169.9 (CH3CO 4); 77.1 (C-400 ); 66.1 (C-700 ); 63.4 (C-900 ); 62.4 (C-1000 ); 48.6
(C-300 ); 48.1 (C-200 ); 40.5 (C-500 ); 39.0 (C-800 ); 35.2 (C-100 ); 21.1, 20.7 (CH3CO 4); 19.1 (C-600 ). 3.10. (S)-MTPA tetra-ester of 5 (10) A suspension of 5 (40 mg) in anhydrous CH2Cl2 (15 ml) was added with (S)-MTPA (188 mg), DMAP (26 mg) and then with DCC (176 mg). After 2 days of stirring, more (S)- MTPA (45 mg) and DCC (46 mg) were added. After 2 days the mixture was diluted with water and extracted with additional CH2Cl2. The residue of the evaporation of the organic phase was submitted to CC (silica gel, solvents cyclohexane:EtOAc=8:2) and the tetra acyl derivative 10 was obtained. Molecular formula C50H48O12F12, ICR-FTMS m/z: 1091.28111 [M + Na]+, calcd 1091.28464. 1H NMR (CDCl3) d: 7.47-7.37 (20H, m, ArH5 4); 5.15 (1H, m, H400 ); 4.26 (1H, dd, J = 11.4, 4.9, Hb-1000 ); 4.22 (1H, dd, J = 12.0, 4.4, Ha-1000 ); 4.13, 4.11 (2H, m, H2-700 ); 4.08 (1H, m, Hb-900 ); 3.93 (1H, dd, J = 11.4, 6.8, Ha-900 ); 3.50, 3.47, 3.45 (12H, s, MeO 4); 2.05 (1H, m, H-800 ); 1.81 (1H, m, H-300 ); 1.69 (1H, m, Hb-500 ); 1.65 (1H, m, H-100 ); 1.46 (1H, m, H-200 ); 1.16 (1H, m, Ha-500 ); 0.83 (3H, d, J = 6.2, H3-600 ). 13 C NMR d: 166.2 (CO 4); 132.2 (C Ar1 4); 129.8, 128.7, 127.4, 127.3 (C Ar2-6 4); 122.3 (CF3 4); 84.3 (C1 4); 79.6 (C-400 ); 68.0 (C-700 ); 64.3 (C-900 ); 63.3 (C-1000 ); 55.6, 55.3 (MeO 4); 48.9 (C-300 ); 48.1 (C-200 ); 40.0 (C-500 ); 38.8 (C-800 ); 35.2 (C-100 ); 18.7 (C-600 ). 3.10.1. (R)-MTPA tetra-ester of 5 (11) Tetraol 5 was likewise treated with (R)-MTPA and tetra acyl derivative 11 was obtained. Molecular formula C50H48O12F12, ICR-FTMS m/z: 1091.27944 [M + Na]+, calcd 1091.28464. 1H NMR (CDCl3) d: 7.477.36 (20H, m, ArH5 4); 5.09 (1H, m, H-400 ); 4.36 (1H, dd, J = 11.7, 5.1, Hb-1000 ); 4.10 (1H, m, Ha-1000 ); 4.08, 4.06 (2H, m, H2-700 ); 3.98, 3.92 (2H, m, H2-900 ); 3.49, 3.47 (12H, s, MeO 4); 2.04 (1H, m, H-800 ); 1.81 (1H, m, Hb-500 ); 1.74 (1H, m, H100 ); 1.73 (1H, m, H-300 ); 1.54 (1H, m, H-200 ); 1.37 (1H, m, Ha-500 ); 0.86 (3H, d, J = 6.0, H3-600 ). 13C NMR d: 165.9 (CO 4); 132.0 (C Ar1 4); 129.7, 128.5, 127.2 (C Ar26 4);121.4 (CF3 4); 84.4 (C-1 4); 79.3 (C-400 ); 67.9 (C700 ); 63.9 (C-900 ); 63.5 (C-1000 ); 55.3 (MeO 4); 48.7 (C-300 ); 47.8 (C-200 ); 39.8 (C-500 ); 38.9 (C-800 ); 35.4 (C-100 ); 18.7 (C-600 ). Acknowledgements The authors thank Professor Marcello Nicoletti (Univer` sita La Sapienza Rome, Italy) for his NMR data support.
References
Chifundera, K., 2001. Contribution to the inventory of medicinal plants from the Bushi area, South Kivu Province, Democratic Republic of Congo. Fitoterapia 72, 351368. Craig, L.C., Post, O., 1949. Apparatus for countercurrent distribution. Anal. Chem. 21, 500504.
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F.R. Gallo et al. / Phytochemistry 67 (2006) 504510 Tanahashi, T., Nagakura, N., Kuwajima, H., Takaiski, K., Inoue, K., Inouye, H., 1989. Secoiridoid glucosides from Jasminum mesnyi. Phytochemistry 28, 14131415. Tanahashi, T., Takenaka, Y., Nagakura, N., Nishi, T., 2000. Five secoiridoid glucosides esteried with a cyclopentanoid monoterpene unit from Jasminum nudiorum. Chem. Pharm. Bull. 48, 12001204. Zhang, Y.J., Liu, Y.Q., Pu, X.Y., Yang, C.R., 1995. Iridoidal glycosides from Jasminum sambac. Phytochemistry 38, 899903.
Ohtani, I., Kosumi, T., Kashman, Y., Kakisawa, H., 1991. High-eld FTNMR application of Moshers method. The absolute conguration of marine terpenoids. J. Am. Chem. Soc. 13, 40924096. Shen, Y.C., Hsieh, P.W., 1997. Four new secoiridoid glucosides from Jasminum urophyllum. J. Nat. Prod. 60, 453457. Tanahashi, T., Nagakura, N., Inoue, K., Inouye, H., 1988. Sambacosides A, E and F, novel tetrameric iridoid glucosides from Jasminum sambac. Tetrahedron Lett. 29, 17931796.
Acetylated avonol diglucosides from Meconopsis quintuplinervia

Xiao-Ya Shang a, Ying-Hong Wang a, Chong Li b, Cheng-Zhong Zhang b, Yong-Chun Yang a, Jian-Gong Shi a,*
a
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China b Lanzhou Medical College, Lanzhou University, Lanzhou 730000, China Received 11 March 2005; received in revised form 17 August 2005 Available online 18 January 2006
Abstract Four acetylated avonol diglucosides, quercetin 3-O-[2000 -O-acetyl-b-D-glucopyranosyl-(1 ! 6)-b-D-glucopyranoside] (1), quercetin 3O-[2000 ,6000 -O-diacetyl-b-D-glucopyranosyl-(1 ! 6)-b-D-glucopyranoside] (2), isorhamnetin 3-O-[2000 -O-acetyl-b-D-glucopyranosyl-(1 ! 6)b-D-glucopyranoside] (3), and quercetin 3-O-[2000 -O-acetyl-a-L-arabinopyranosyl-(1 ! 6)-b-D-glucopyranoside] (4), together with ve known avonol glycosides quercetin 3-O-b-D-glucopyranoside, kaempferol 3-O-b-D-glucopyranoside, quercetin 3-O-[b-D-galactopyranosyl-(1 ! 6)-glucopyranoside], isorhamnetin 3-O-[b-D-galactopyranosyl-(1 ! 6)-b-D-glucopyranoside], and kaempferol 3-O-[b-D-glucopyranosyl-(1 ! 2)-b-D-glucopyranoside] have been isolated from Meconopsis quintuplinervia. Their structures were determined using chemical and spectroscopic methods including HRFABMS, 1H1H COSY, HSQC and HMBC experiments. 2005 Elsevier Ltd. All rights reserved.
Keywords: Meconopsis quintuplinervia Regel; Papaveraceae; Acetylated avonol glycosides
1. Introduction Meconopsis quintuplinervia Regel, a plant belonging to the Papaveraceae family and widely distributed in the Qingzang plateau of the northwest of China (Luo et al., 1984), is used as a traditional Tibetan medicine for treatments of various diseases, such as inammation, pain, hepatitis and tuberculosis (Luo et al., 1984). There are, however, very few reports (Wang et al., 1991; Wang and Chen, 1995) concerning secondary metabolites of M. quintuplinervia. As part of our program to assess systematically the chemical and biological diversity of medicinal plants distributed at higher altitude, we carried out a chemical investigation of this plant. In previous papers (Shang et al., 2002, 2003a,b), we described the isolation and structural identication of three alkaloids, norsan*
Corresponding author. Tel.: +86 10 83154789; fax: +86 10 63017757. E-mail address: shijg@imm.ac.cn (J.-G. Shi).
guinarine, O-methylavinantine and meconoquintupline, and seven avonoids, quercetin, dihydroquercetin, luteolin, chrysoeriol, apigenin, huazhongilexone and hydnocarpin from the less polar fraction of the ethanolic extract of the plant. In continuation of this work, four new acetylated avonol diglucosides (14), together with ve known avonol glycosides, have been isolated from the polar fraction of the same material. By comparison of the spectroscopic data with those reported in the literature, the known compounds were characterized as quercetin 3-O-b-D-glucopyranoside (Veit et al., 1990), kaempferol 3-O-b-D-glucopyranoside (Chaurasia and Wichtl, 1987), quercetin 3-O-[b-D-galactopyranosyl(1 ! 6)-glucopyranoside] (Waage and Hedin, 1985), isorhamnetin 3-O-[b-D-galactopyranosyl-(1 ! 6)-b-Dglucopyranoside] (Degot et al., 1971) and kaempferol 3O-[b- D -glucopyranosyl-(1 ! 2)-b- D -glucopyranoside] (Song, 1990). The present paper deals with isolation and structural elucidation of compounds 14.
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X.-Y. Shang et al. / Phytochemistry 67 (2006) 511515
OH HO O O R5 OH O
1'"
OR1
R4 R3 HO
OO
1"
OH
OR2 HO HO 1 2 3 4 R1 = R4 = H, R2 = Ac, R3 = OH, R5 = CH2OH R1 = R4 = H, R2 = Ac, R3 = OH, R5 = CH2OAc R1 = Me, R2 = Ac, R3 = OH, R4 = H, R5 = CH2OH R1 = R3 = H, R2 = Ac, R4 = OH, R5 = H
2. Results and discussion The water soluble portion of the ethanolic extract of M. quintuplinervia Regel was subjected successively to column chromatography on macroporous adsorbent resin, normal phase and reversed phase silica gels and Sephadex LH-20, to aord two mixtures which were further puried by preparative reversed phase HPLC to yield compounds 14 and the known compounds. Compound 1 was isolated as a yellow amorphous powder. Its IR spectrum showed the presence of hydroxyl (3400 cm1), conjugated carbonyl (1734 and 1655 cm1) and aromatic ring (1506 and 1604 cm1) functional groups. Its UV spectrum exhibited absorption bands characteristic for avonols at 207, 257, 270, 296, and 362 nm. The posi-
tive FABMS exhibited a quasi-molecular ion peak at m/z 669 [M + H]+, with the molecular formula established as C29H32O18 by the positive HRFABMS at m/z 669.1615[M + H]+ (calcd. for C29H33O18 669.1666). The 1 H NMR spectrum of 1 showed two anomeric proton doublets at d 5.06 (1H, d, J = 7.8 Hz, H-100 ) and 4.15 (1H, d, J = 8.1 Hz, H-1000 ) and an acetyl singlet at d 1.61 (3H, s), in addition to resonances characteristic for a quercetin aglycone moiety (Table 1), as well as signals attributed to remaining protons of two glycosyl units between d 2.84 and 4.40. These data indicated that compound 1 was an acetylated quercetin diglycoside, which was conrmed by analysis of the 13C NMR spectroscopic data of 1 (Table 2). Acid hydrolysis of 1 produced glucose as the sole sugar as identied by TLC and PC comparison with authentic sugar samples. The 1H1H COSY and HSQC experiments of 1 led to unambiguous assignments of signals of the protons and protonated carbons in the NMR spectra of 1, while the resolvable axial-axial couplings between vicinal protons of the glycosyl units, excluding H-500 , H2-600 , H5000 and H2-6000 (Table 1), conrmed that both glycosyl units were b-glucopyranosyls. The chemical shift of C-3 suggested that the quercetin aglycone was glycosylated at C3, which was conrmed by a long range correlation from H-100 to C-3 (d 136.0) in the HMBC spectrum of 1. Meanwhile, HMBC correlations from H-1000 to C-600 (d 69.2) and H2-600 to C-1000 (d 102.1) unequivocally revealed an 1 ! 6 connectivity between the two b-glucopyranosyls. In addition, the carbonyl (d 169.1) of the acetoxyl group correlated to H-2000 of the outer b-glucopyranosyl unit at d
Table 1 1 H NMR spectroscopic data for compounds 14 No. 6 8 20 50 60 100 200 300 400 500 600 1000 2000 3000 4000 5000 6000 Ac Ac OMe
1
1 6.15 6.38 8.01 6.84 7.65 5.06 3.80 3.54 3.72 3.51 3.68 4.15 4.40 3.04 3.20 2.84 d (1.8) d (1.8) d (2.4) d (9.0) dd (2.4, d (7.8) dd (7.8, dd (8.4, dd (7.5, m m d (8.1) dd (8.1, dd (9.0, dd (9.3, m
4 6.15 d (2.1) 6.38 d (2.1) 8.09 d (2.1) 6.84 d (9.0) 7.69 dd (2.1, 9.0) 5.01 d (7.8) 3.83 dd (7.8, 7.8) 3.55 dd (7.8, 7.5) 3.69 dd (7.5, 7.2) 3.53 m 3.64 m 4.01 d (7.8) 4.63 dd (7.8, 9.3) 2.94 dd (9.3, 9.3) 3.52 m (a) 3.00 brd (12.3) (b) 3.70 m
9.0) 8.4) 7.5) 7.2)
8.5) 7.8) 7.5) 7.8)
8.7) 8.1) 9.4) 7.2)
9.0) 9.3) 9.6)
8.4) 9.3) 9.3)
9.3) 9.3) 9.6)
(a) 3.69 m (b) 3.57 m 1.61 s
(a) 4.20 dd (2.1, 12.3) (b) 4.03 dd (5.5,12.3) 1.59 s 1.98 s
(a) 3.64 dd (2.4, 11.7) (b) 3.48 m 1.64 s 3.92 s
1.56 s
H NMR data were measured in methanol-d4 at 300 MHz. Proton coupling constants (J) in Hz are given in parentheses. The assignments were based on DEPT,1H1H COSY, HSQC and HMBC experiments.
X.-Y. Shang et al. / Phytochemistry 67 (2006) 511515 Table 2 1 C NMR spectroscopic data for compounds 14 No. 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 100 200 300 400 500 600 1000 2000 3000 4000 5000 6000 Ac Ac OMe
13
513
1 158.2 136.0 179.2 162.9 100.0 166.2 94.9 158.3 105.6 122.6 117.9 145.9 150.2 116.5 122.9 105.7 73.2 74.8 70.5 77.3 69.2 102.1 75.3 75.7 71.3 77.2 62.2 20.3 169.1
2 156.9 134.8 178.1 161.8 98.8 165.1 93.8 157.2 104.4 121.4 116.7 144.8 149.2 115.4 121.6 104.4 72.0 73.5 69.6 76.6 68.0 100.8 74.0 74.3 70.2 73.6 63.1 19.3 170.5 19.6 171.8
3 158.1 135.5 179.4 163.0 100.0 166.2 95.0 158.4 105.8 122.8 114.5 148.5 151.1 116.2 123.6 104.5 73.1 74.7 70.4 77.3 68.8 101.8 75.3 75.8 71.3 77.3 62.1 20.6 171.8
4 157.8 136.2 179.2 162.9 100.1 166.7 95.0 158.3 105.4 122.6 117.9 146.0 150.3 116.3 122.8 106.0 73.2 74.8 70.7 77.8 69.1 102.7 73.7 72.0 69.8 67.0 20.6 172.0
57.0
C NMR data were measured in methanol-d4 at 75 MHz. The assignments were based on DEPT, 1H1H COSY, HSQC and HMBC experiments.
4.40 (1H, dd, J = 8.1 and 9.0 Hz), demonstrating that the acetoxyl group was located at C-2000 of the glucopyranosyl. Thus, 1 was quercetin 3-O-[2000 -O-acetyl-b-D-glucopyranosyl-(1 ! 6)-b-D-glucopyranoside]. Compound 2 was obtained as a yellow powder with a molecular formula C31H34O19 as determined by the positive FABMS at m/z 711.1810 [M + H]+ (calcd. for C31H35O19, 711.1772). The UV, IR and NMR spectra of 2 were similar to those of 1, except for the appearance of signals due to one more acetoxyl unit at dH 1.98 (3H, s) and dC 19.6 (q) and 171.8 (s) in the NMR spectra of 2, indicating that it was an acetylated cognate of 1. This was supported by acid hydrolysis and 2D NMR spectroscopic experiments of 2. In the HMBC spectrum of 2 correlations from H-2000 to one acetoxyl carbonyl and from H2-6000 to the other unequivocally established that the two acetyls were esteried at C-2000 and C-6000 of the outer glucosyl moiety, respectively. Therefore, 2 was quercetin 3-O-[2000 ,6000 -O-diacetyl-b-D-glucopyranosyl-(1 ! 6)-b-D-glucopyranoside]. Compound 3 was obtained as a yellow powder. Its molecular formula was determined as C30H34O18 by the positive FABMS at m/z 683.1854 [M + H]+ (calcd. for
C30H35O18 683.1823). The UV, IR and NMR spectra of 3 were similar to those of 1, except for the appearance of signals attributed to an aromatic methoxyl group at dH 3.92 (3H, s) and dC 57.0 in the NMR spectra of 3, indicating that it was a methylated derivative of 1. A comparison of the NMR spectroscopic data of 3, with those of the co-occurring isorhamnetin 3-O-[b-D-galactopyranosyl(1 ! 6)-b-D-glucopyranoside] (Degot et al., 1971), demonstrated that the aglycone of 3 was isorhamnetin. Therefore, 3 was isorhamnetin 3-O-[2000 -O-acetyl-b-D-glucopyranosyl(1 ! 6)-b-D-glucopyranoside]. Compound 4 was obtained as a yellow powder with a molecular formula C28H30O17 as established by the positive HRFABMS at m/z 639.1546 [M + H]+. The UV and IR spectra of 4 were similar to those of 1. A comparison of its NMR spectroscopic data with those of 1 (Tables 1 and 2) indicated that the only dierence between 1 and 4 was replacement of the outer glucopyranosyl of 1 by an arabinopyranosyl (Simon et al., 1993) in 4. This was supported by acid hydrolysis of 4 yielding glucose and arabinose as the sugars. The location of the acetyl linkage between the glycosyls in 4 was further conrmed by 2D NMR experiments (1H1H COSY, HSQC and HMBC). Consequently, 4 was quercetin 3-O-[2000 -O-acetyl-a-L-arabinopyranosyl-(1 ! 6)-b-D-glucopyranoside]. Previous studies indicated that plants of the genus Meconopsis contained alkaloids (Hemingway et al., 1981; Allais et al., 1983; Liu and Wang, 1986; Wang and Chen, 1995), triterpenoids (Zhang et al., 1997) and avonoids (Tanaka et al., 2001), although the emphasis of the chemical investigations thus far was focused on the alkaloids in species of this genus. However, our systematical chemical investigation of M. quintuplinervia has revealed that diverse avonoids represent the main metabolites in this species while two morphinane alkaloids, O-methylavinantine and meconoquintupline, and a benzophenanthrindine alkaloid norsanguinarine, were obtained (Shang et al., 2002, 2003a,b). The structures of the acetylated avonol diglycosides and meconoquintupline from M. quintuplinervia were distinctive by the number and/or substitution position of acetyl in the acetylated avonol diglycosides and the 8,14-dihydrogenation of the morphinane skeleton in meconoquintupline, i.e., even though avonoids from Meconopsis grandis (Tanaka et al., 2001) and the morphinane/benzophenanthrindine alkaloids from several Meconopsis species (Hemingway et al., 1981) have been reported, respectively. Both alkaloids and avonoids may, therefore, have chemotaxonomically important roles in the genus Meconopsis though avonoids from this genus have received relatively little attention. In the cytotoxic and antioxidant assays compounds 14 and the known avonoids showed neither cytotoxicity against human colon cancer (HCT-8), hepatoma (Bel7402), stomach cancer (BGC-823), and lung adenocarcinoma (A549) cell lines (IC50 > 10 lg/mL) nor signicant antioxidant activity inhibiting rat liver microsomal lipid peroxidation (IC50 > 5 lg/mL).
514
3. Experimental 3.1. General Melting points were determined on an XT-4 micro melting point apparatus and are uncorrected. IR spectra were recorded as KBr disks on a Nicolet Impact 400 FT-IR Spectrophotometer. 1D- and 2D NMR spectra were obtained at 300 and 75 MHz for 1H and 13C, respectively, on Inova 300 or 500 MHz spectrometers in methanol-d4 or DMSO-d6 with solvent peaks as references. FABMS and HRFABMS data were measured with a Micromass Autospec-Ultima ETOF spectrometer. Column chromatography was performed with silica gel (200300 mesh), RP-18 reversed phase silica gel (4360 lm) and Sephadex LH20. HPLC separation was performed on an instrument consisting of a Waters 600 controller, a Waters 600 pump, and a Waters 2487 dual k absorbance detector with an Alltima (250 22 mm) preparative column packed with C18 (10 lm). TLC was carried out with glass precoated silica gel GF254 plates. Spots were visualized under UV light or by spraying with 3% FeCl3 in EtOH or 7% H2SO4 in 95% EtOH followed by heating. 3.2. Plant material M. quintuplinervia Regel (4 kg) was collected at Daban mountain at an altitude of 34003600 m, Qinghai province, China, in August of 1999. The plant was identied by Prof. Guo-liang Zhang (Department of Biology, Lanzhou University, Lanzhou 730000, China). A voucher specimen (No. 200025) was deposited at the Herbarium of the Department of Medicinal Plants, Institute of Materia Medica, Beijing, China. 3.3. Extraction and isolation Air dried aerial parts of M. quintuplinervia (4 kg) were extracted with 11.0 L of 90% EtOH at room temperature for 3 48 h. The ethanolic extract was evaporated to almost dryness in vacuo to yield a dark brown viscid residue (470 g). The residue was suspended in H2O (1100 mL) and then partitioned successively with petroleum ether (4 800 mL), and EtOAc (4 650 mL). The aq. phase resulting from the partition was applied to a macroporous adsorbent resin (RA, Seventh Factory of Beijing Chemical Industry, China) (650 g, dried weight) column using H2O and EtOHH2O (6:4) as eluents. After solvent removal, the fraction (7.8 g) eluted by EtOH H2O (6:4) was subjected to normal phase silica gel CC eluting with a gradient of increasing MeOH in CHC13. The CHCl3MeOH (4:1) eluent gave a mixture that was separated into three subfractions by gel chromatography over Sephadex LH-20 eluted with CHCl3MeOH (1:1). The third subtraction was puried by reversed-phase HPLC using MeOHH2O (45:55) as mobile phase to give quercetin 3-O-[b-D-galactopyranosyl-(1 ! 6)-glucopyranoside]
(35 mg) and quercetin 3-O-b-D-glucopyranoside (21 mg). The CHCl3MeOH (2:1) eluent was separated into four subfractions by gel chromatography over Sephadex LH20 eluted with CHCl3MeOH (1:1). The third and fourth subfractions were further puried, respectively, by preparative reversed phase HPLC using MeOHH2O (40:60) as the mobile phase to aord 1 (18 mg), 2 (21 mg), 3 (17 mg), 4 (15 mg), kaempferol 3-O-b-D-glucopyranoside (27 mg), isorhamnetin 3-O-[b-D-galactopyranosyl-(1 ! 6)-b-Dglucopyranoside] (31 mg) and kaempferol 3-O-[b-D-glucopyranosyl-(1 ! 2)-b-D-glucopyranoside] (13 mg). 3.4. Quercetin 3-O-[2000 -O-acetyl-b-D-glucopyranosyl(1 ! 6)-b-D-glucopyranoside] (1) Amorphous yellow powder; a20 +20.6 (MeOH c 0.16); D UV kMeOH nm (log e): 207 (4.37), 257 (4.14), 270 (4.03), 296 max (3.73), 362 (4.08); IR mKBr cm1 : 3400, 2910, 1734, 1655, max 1604, 1506, 1444, 1360, 1304, 1200, 1169, 1074, 1022. For 1 H and 13C NMR spectroscopic data, see Tables 1 and 2; FABMS (m/z): 669 [M + H]+. HRFABMS (m/z): 669.1615 [M + H]+, C29H33O18 requires 669.1666. 3.5. Quercetin 3-O-[2000 ,6000 -O-diacetyl-b-D-glucopyranosyl(1 ! 6)-b-D-glucopyranoside] (2) Amorphous yellow powder; aD +30.8 (MeOH; c 0.25); UV kMeOH nm (log e): 206 (4.45), 257 (4.18), 270 (4.07), 296 max (3.77), 363 (4.13); IR mKBr cm1 : 3419, 2908, 1732, 1653, max 1604, 1506, 1444, 1361, 1244, 1078. For 1H and 13C NMR spectroscopic data, see Tables 1 and 2; FABMS (m/z): 711[M + H]+, HRFABMS (m/z): 711.1810 [M + H]+, C31H35O19 requires 711.1773. 3.6. Isorhamnetin 3-O-[2000 -O-acetyl-b-D-glucopyranosyl(1 ! 6)-b-D-glucopyranoside] (3) Amorphous yellow powder; aD +19.4 (MeOH; c 0.15); UV kMeOH nm (log e): 206 (4.39), 255(4.08), 269(3.99), max 298(3.75), 357(4.03); IR mKBr cm1 : 3415, 2908, 1732, max 1653, 1604, 1514, 1431, 1356, 1290, 1203, 1074, 1028. For 1 H and 13C NMR spectroscopic data, see Tables 1 and 2; FAB MS m/z: 683 [M + H]+; HRFABMS m/z: 683.1854 [M + H]+, C30H35O18 requires 683.1823. 3.7. Quercetin 3-O-[2000 -O-acetyl-a-L-arabinopyranosyl(1 ! 6)-b-D-glucopyranoside] (4) Amorphous yellow powder; a20 +32.9 (MeOH; c 0.04); D UV kMeOH nm (log e): 207 (4.55), 257 (4.32), 269 (4.24), 298 max (3.94), 363 (4.26); IR mKBr cm1 : 3400, 2918, 1732, 1653, max 1604, 1498, 1446, 1360, 1304, 1201, 1171, 1072, 1020. For 1 H and 13C NMR spectroscopic data, see Tables 1 and 2; FABMS m/z: 639 [M + H]+; HRFABMS m/z: 639.1546 [M + H]+, C28H31O17 requires 639.1561.
20 20
515
3.8. Acid hydrolysis of 14 A solution of each compound (5 mg) in 2 N HCl (2 mL) was individually reuxed for 16 h at 94 C. The reaction mixture was partitioned with EtOAc, with the aqueous phase neutralized with 1 N NaOH and dried using a stream of N2. The resulting residue was dissolved in EtOH (0.5 mL) and analyzed by TLC and PC together with authentic sugar samples, using as developing solvent systems CHCl3MeOH (2.5:1) for TLC and the upper layer of n-BuOHAcOHH2O (4:1:5) for PC; products were visualized by spraying aniline hydrogen phthalate followed by heating at 105 C.
Acknowledgements The authors are grateful to A. Zeper for mass spectra measurements. Financial support is from the NSFC (Grant No. 20432030).
References
Allais, D.P., Guinaudeau, H., Freyer, A.J., Shamma, M., Ganguli, N.C., Talapatra, B., Talapatra, S.K., 1983. Limogine and himalayamine: a new class of alkaloids. Tetrahedron Lett. 24, 24452448. Chaurasia, N., Wichtl, M., 1987. Flavonol glycosides from Urtia dioica. Planta Med. 53, 432434. Degot, A.V., Litvinenko, V.I., Kurinnaya, N.V., 1971. Flavonoids of Orthantha lutea. Khim. Prir. Soedin. 7, 117119.
Hemingway, S.R., Phillipson, J.D., Verpoorte, R., 1981. Meconopsis cambrica alkaloids. J. Nat. Prod. 44, 6774. Liu, S.Y., Wang, X.K., 1986. Studies on chemical constituents of Meconopsis punicea. Zhong Yao Tong Bao 11, 360362. Luo, D.S., Sun, A.L., Xia, G.C., 1984. Tibetan drug in Qingzang plateau, a preliminary investigation of resources Meconopsis. Zhong Cao Yao 15, 359360. Shang, X.Y., Zhang, C.Z., Li, C., Yang, Y.C., Shi, J.G., 2002. Studies on chemical constituents of Meconopsis quintuplinervia Regel. Zhong Yao Cai 25, 250252. Shang, X.Y., Shi, J.G., Yang, Y.C., Liu, X., Li, C., Zhang, C.Z., 2003a. Alkaloids from a Tibetan medicine Meconopsis quintuplinervia Regel. Acta Pharmaceut. Sin. 38, 276278. Shang, X.Y., Jiao, H.S., Yang, Y.C., Shi, J.G., 2003b. A morphinane alkaloid from Meconopsis quintuplinervia. Chin. Chem. Lett. 14, 597598. Simon, A., Chulia, A.J., Kaouadji, M., Allais, D.P., Delage, C., 1993. Further avonoid glycosides from Calluna vulgaris. Phytochemistry 32, 10451049. Song, C.Q., 1990. Chemical constituents of saron (Crocus sativus). II. The avonol compounds of petals. Zhong Cao Yao 21, 439440. Tanaka, M., Fujimori, T., Uchida, I., Yamaguchi, S., Takeda, K., 2001. A malonylated anthocyanin and avonols in blue Meconopsis owers. Phytochemistry 56, 373376. Veit, M., Geiger, H., Czygan, F., Markham, K.R., 1990. Malonylated avone 5-O-glucosides in the barren sprouts of Equisetum arvense. Phytochemistry 29, 25552560. Waage, S.K., Hedin, P.A., 1985. Quercetin 3-O-galactopyranosyl-(1 ! 6)glucopyranoside, a compound from narrowleaf vetch with antibacterial activity. Phytochemistry 24, 243245. Wang, M.A., Chen, Y.Z., 1995. A new alkaloid from Meconopsis quintuplinervia Regel. Nat. Prod. Res. Develop. 7, 3234. Wang, M.A., Chen, S.N., Zhang, H.D., Chen, Y.Z., 1991. Studies on the chemical constituents of Meconopsis quintuplinervia Regel, a Tibetan medicinal herb. J. Lanzhou Univ. (Nat. Sci.) 27, 8092. Zhang, G.L., Li, B.G., Zhou, Z.Z., 1997. Non-alkaloidal constituents from Meconopsis punicea Maxim. Nat. Prod. Res. Develop. 9, 46.
Lignan, phenolic and iridoid glycosides from Stereospermum cylindricum

Tripetch Kanchanapoom
a
a,*
, Pawadee Noiarsa a, Hideaki Otsuka b, Somsak Ruchirawat
Department of Pharmaceutical Botany and Pharmacognosy, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand b Department of Pharmacognosy, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan c Chulabhorn Research Institute, Vipavadee Rangsit Highway, Bangkok 10210, Thailand Received 21 February 2005; received in revised form 25 August 2005 Available online 28 November 2005
Abstract A lignan glycoside [(+)-cycloolivil 4 0 -O-b-D-glucopyranoside], a phenolic glycoside [3,4-dimethoxyphenyl 1-O-b-D-xylopyranosyl(1 ! 6)-b-D-glucopyranoside] and a iridoid glycoside (stereospermoside) were isolated from the leaves and branches of Stereospermum cylindricum, together with (+)-cycloolivil, (+)-cycloolivil 6-O-b-D-glucopyranoside, ()-olivil, ()-olivil 4-O-b-D-glucopyranoside, ()olivil 4 0 -O-b-D-glucopyranoside, vanilloloside, decaeoyl-verbascoside, isoverbascoside, 3,4,5-trimethoxyphenyl 1-O-b-D-xylopyranosyl-(1 ! 6)-b-D-glucopyranoside, ajugol, verminoside, and specioside. The structure elucidations were based on spectroscopic evidence. 2005 Elsevier Ltd. All rights reserved.
Keywords: Stereospermum cylindricum; Bignoniaceae; Lignan glucoside; Phenolic glycoside; Iridoid glucoside; Stereospermoside
1. Introduction As part of our systematic investigation on Thai Bignoniaceous plants, especially in tribe Tecomeae, we reported the constituents of Fernandoa adneophylla (Kanchanapoom et al., 2001), Markhamia stipulata (Kanchanapoom et al., 2002a) and Barnettia kerrii (Kanchanapoom et al., 2002b). To further study plants in the same tribe, we investigated the constituents of Stereospermum cylindricum Pierre ex P. Dop. (Thai name: Khae-Khao), collected from the Botanical garden, Faculty of Pharmaceutical Sciences, Khon Kaen University, Thailand. S. cylindricum is a tree up to 25 m high, distributed in South-east Asia. The bark of this plant is used in Thai traditional medicine for antifever purposes, as well as an anti-inammatory agent. No phytochemical study has been carried out on this species. Preliminary studies on plants in this genus have led to isolation of several compounds such as lignans (Ghogomu
et al., 1985), and quinones (Onegi et al., 2002; Kumar et al., 2003). The present paper deals with the isolation of 15 compounds, including a new lignan glycoside, a new phenolic diglycoside and a new iridoid glycoside, as well as 12 known compounds.
2. Results and discussion The methanolic extract was suspended in H2O and defatted with Et2O. The aqueous layer was subjected to Diaion HP-20 column chromatography, using H2O, MeOH and Me2CO as eluants, successively. The portion eluted with MeOH was repeatedly subjected to silica gel, RP-18, and preparative HPLC-ODS chromatography to aord 15 compounds. Twelve were identied as (+)-cycloolivil (1) (Abe et al., 1988), (+)-cycloolivil 6-O-b-D-glucopyranoside (2) (Sugiyama et al., 1993), ()-olivil (4) (Abe et al., 1988), ()-olivil 4-O-b-D-glucopyranoside (5) (Abe et al., 1988), ()-olivil 4 0 -O-p-glucopyranoside (6) (Abe et al., 1988), vanilloloside (7) (Ida et al., 1993), decaeoyl-verbascoside (8) (Karasawa et al., 1986), isoverbascoside (9)
Corresponding author. Tel.: +66 43 202378; fax: +66 43 202379. E-mail address: trikan@kku.ac.th (T. Kanchanapoom).
T. Kanchanapoom et al. / Phytochemistry 67 (2006) 516520
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(Kanchanapoom et al., 2002a), 3,4,5-trimethoxyphenyl 1O-b-D-xylopyranosyl-(1 ! 6)-b-D-glucopyranoside (10) (Kosuge et al., 1994), ajugol (12) (Nishimura et al., 1989), verminoside (13) (Sticher and A-Yazar, 1979), specioside (14) (Compadre et al., 1982) by comparison of physical data with literature values and from spectroscopic evidence. The molecular formula of compound 3 was determined as C26H34O12 by negative ion HR-FAB mass spectrometric analysis. The 1H and 13C NMR spectroscopic data showed the presence of a b-glucopyranosyl unit from the anomeric proton signal at dH 4.83 (d, J = 7.3 Hz) and from the carbon signals at dC 102.9, 78.1, 77.8, 74,9, 71.3 and 62.5, in addition to the signals of the aglycone moiety. The chemical shifts of the aglycone moiety were similar to those of (+)-cycloolivil (1), suggesting that compound 3 is (+)cycloolivil glucoside. This was supported by enzymatic hydrolysis of 3 with crude hesperidinase, providing compound 1. The location of the sugar unit was assigned by an NOESY experiment, in which NOESY correlation was found between dH 4.83 (d, J = 7.3 Hz, H-1 0 0 ) and dH 7.08 (d, J = 8.3 Hz, H-5 0 ), indicating that the sugar part was attached at C-4 0 . Moreover, HMBC spectrum provided the further conrmation of the structure from the three-bond correlation between H-1 0 0 (dH 4.83) and C-4 0 (dC 146.6) as illustrated in Fig. 1. Therefore, structure of compound 3 was elucidated as (+)-cycloolivil 4 0 -O-b-Dglucopyranoside. The molecular formula of compound 11 was determined as C19H28O12 by negative ion HR-FAB mass spectrometric analysis. The 1H NMR spectrum indicated the presence of a tri-substituted aromatic ring (ABX system) from the signals at dH 6.83 (d, J = 8.8 Hz), 6.64 (d, J = 2.1 Hz) and 6.59 (dd, J = 8.8, 2.7 Hz), two singlet methoxyl signals at dH 3.72 and 3.68, and two anomeric proton signals at dH 4.69 (d, J = 7.6 Hz) and 4.16 (d, J = 7.3 Hz). The 13C NMR spectrum displayed 19 carbon signals, of which three were assignable to three oxy-aryl
Fig. 1. Signicant HMBC correlations of compound 3.
carbons at dC 151.9, 149.3 and 144.0, three aryl-methines at dC 112.8, 107.3 and 102.3, and two methoxyl groups at dC 56.1 and 55.5 for the aglycone moiety. The remaining carbon signals belonged to the sugar moiety, and could be identied as b-D-xylopyranosyl-(1 ! 6)-b-D-glucopyranosyl unit by comparing chemical shifts with those of 3,4,5-trimethoxyphenyl 1-O-b-D-xylopyranosyl-(1 ! 6)-bD-glucopyranoside (10). All protonated carbons were assigned by the result from HSQC spectrum. From these spectral data, compound 11 is a glycoside of dimethoxylphenol. The locations of two methoxyl groups and the sugar moiety were assigned by NOESY experiment The signicant correlations observed between the signals were at: (i) dH 4.69 (H-1 0 ) and dH 6.64 (H-2), (ii) dH 4.69 (H-1 0 ) and dH 6.59 (H-6), (iii) dH 6.64 (H-2) and dH 3.72 (MeO3), and (iv) dH 6.83 (H-5) and dH 3.68 (MeO-4); indicating that two methoxyl groups were attached at C-3 and C-4, as well as the sugar moiety was substituted at C-1. Consequently, the structure of compound 11 was identied to be 3,4-dimethoxyphenyl 1-O-b-D-xylopyranosyl(1 ! 6)-b-D-glucopyranoside. The molecular formula of compound 15 was determined as C24H30O13 negative ion HR-FAB mass spectrometric analysis. The 13C NMR spectroscopic data showed the presence of one b-glucopyranosyl unit, one coumaroyl moiety in addition to nine carbons signal for the aglycone moiety. DEPT experiments indicated that compound 15 contained one quarternary carbinolic carbon (dC 80.7), seven methines (dC 140.8, 106.0, 93.0, 85.8, 84.3, 48.3 and 36.5) and one methylene (dC 64.3) for the aglycone part, consistent with cyclopentanopyran ring of an iridoid skeleton. The chemical shift at dC 93.0 was characteristic of an acetal group of C-l. The methine signals at dC 140.8 and 106.0 were assigned to a disubstituted olen group at C-3 and C-4, respectively. The coumaroyl moiety was assigned as trans by the coupling constant of the proton signals at dH 7.60 and 6.32 (each d, J = 15.9 Hz). The remaining signals in the 1H NMR spectrum at dH 5.50 (d, J = 4.4 Hz), 6.22 (dd, J = 6.1, 1.8 Hz), 5.19 (dd, J = 6.1, 3.4 Hz), 2.71 (m), 4.71 (dd, J = 5.6, 5.4 Hz), 4.09 (d, J = 5.6 Hz), and 2.45 (dd, J = 9.8, 4.4 Hz) were assignable to H-1, H-3, H-4, H-5, H-6, H-7 and H-9, respectively. Also, it showed the AB type of methylene protons at dH 3.98 and 3.78 (each d, J = 12.0 Hz), ascribable to H-10. The 1H and 13C NMR spectroscopic data were closely related to those of decinnamoyl-globularimin (16) (Chaudhuri and Sticher, 1981), except for a set of additional signals arising from the coumaroyl moiety. This ester moiety was assigned to be located at C-6 since the chemical shifts of C-6, C-5 and C-7 were signicantly changed by +2.7, 0.8, and 2.1 ppm, respectively. Moreover, the HMBC spectrum (Fig. 2.) provided further conrmation from the three-bond correlation from H-6 (dH 4.71) to C-9 0 0 (dC 169.1). Therefore, the structure of compound 15 was determined as 6-O-trans-p-coumaroyl-decinnamoylglobularimin, namely stereospermoside.
518
Fig. 2. Signicant HMBC correlations of compound 15.
3. Experimental 3.1. General H, 13C and 2D NMR spectra were recorded using a JEOL JNM a-400 spectrometer (400 MHz for 1H NMR and 100 MHz for 13C NMR). The NMR spectroscopic data were measured in CD3OD and DMSO-d6 with tetramethylsilane (TMS) as internal standard. The negative-ion mode FAB-MS spectra were recorded on a JEOL JMS-SX 102 spectrometer. IR spectra were measured with a Perkin Elmer Spectrum one FT-IR spectrometer. Optical rotations were determined on a Union PM-1 digital polarimeter. For column chromatography, silica gel G (Merck no. 7734), YMC-gel ODS (50 lm, YMC) and highly porous copolymer resin of styrene and divinylbenzene (Mitsubishi Chem. Ind. Co. Ltd.) were used. HPLC (Jasco PU-980 pump) were carried on columns of ODS (150 20 mm i.d., YMC) and Polyamine II (250 4.6 mm i.d.) with a Toyo Soda refractive index (RI-8000) detector. The ow rate was 6 ml/min. The solvent systems were (I) EtOAc MeOH (9:1), (II) EtOAcMeOHH2O (40:10:1), (III) EtOAcMeOHH2O (70:30:3), (IV) 1050% aqueous MeOH, (V) 10% aqueous MeCN, (VI) 12% aqueous MeCN, (VII) 15% aqueous MeCN, (VIII) 20% aqueous MeCN, (IX) 20% aqueous MeOH and (X) 80% aqueous MeCN, respectively. 3.2. Plant material The leaves and branches of S. cylindricum Pierre ex. P. Dop were collected from Botanical Garden, Faculty of Pharmaceutical Sciences, Khon Kaen University in July, 2004, and identied by Mr. Bamrung Tavinchiua of the Department of Pharmaceutical Botany and Pharmacognosy, Faculty of Pharmaceutical Sciences, Khon Kaen University, Thailand. A voucher sample (PSKKU-0050) is kept in the Herbarium of the Faculty of Pharmaceutical Sciences, Khon Kaen University, Thailand.
1
Among the compounds isolated, the present study yielded two phenylethanoid glycosides (89) and four iridoid glucosides (1215). The phenylethanoid glycosides, decaeoyl-verbascoside (8) and isoverbascoside (9), are widely distributed in Bignoniaceous plants, such as in Thailand (Kanchanapoom et al., 2001, 2002a,b). Iridoids are also widespread in the family Bignoniaceae (von Poser et al., 2000), especially those lacking a carboxylic acid functionality at C-4. These appears to be the most common for tribe Tecomeae such as ajugol (12), verminoside (13), specioside (14), and stereospermoside (15). Thus, these two classes of compounds may serve as useful chemotaxonomical methods in tribe Tecomeae of the family Bignoniaceae.
519
3.3. Extraction and isolation Dried leaves and branches (2.5 kg) of S. Cylindricum were extracted with hot MeOH three times/(under reux, 5 l, for each extraction). After removal of the solvent by evaporation, with Et2O. The aqueous layer was applied to a column of Diaion HP-20 and eluted successively with H2O, MeOH and Me2O. The fraction eluted with MeOH (49.3 g) was subjected to a silica gel cc using solvent systems I (2.0 l), II (5.0 l) and III (4.0 l). Five fractions were collected. Fraction 2 (5.1 g) was located onto a column of RP-18 using solvent system IV to provide compounds 1 (1.23 g) and 4 (519.9 mg). Fraction 3 (18.9 g) was applied to a RP-18 column using solvent system IV, aording 12 fractions. Fraction 31 was puried by preparative HPLC-ODS with solvent system V to give compounds 7 (195.6 mg), 8 (154.8 mg) and 12 (36.4 mg). Fraction 33 was puried by preparative HPLC-ODS with solvent system VII to obtain compounds 5 (353.8 mg) and 6 (92.3 mg). Fraction 35 was further puried by preparative HPLC-ODS with solvent system VIII to aord compounds 9 (68.8 mg), 13 (627.5 mg) and 15 (49.2 mg). Compound 14 (430.0 mg) was crystallized from fractions 37 and 38. Fraction 4 (15.4 mg) was similarly separated on a column of RP18 using solvent system IV to give eight fractions. Fraction 43 was puried by preparative HPLC-ODS with solvent system VI to give compound 10 (135.4 mg) and fraction 4-3-2. This fraction was further puried by preparative HPLC-ODS with solvent system IX to give compound 2 (54.2 mg) and fraction 4-3-2-2, which was nally puried by analytical HPLC-Polyamine II with solvent system X to provide compounds 3 (26.3 mg) and 11 (13.7 mg). 3.4. (+)-Cycloolivil-4 0 -O-b-D-glucopyranoside (3) Amorphous powder, aD +17.6 (MeOH, c 1.59); IR cm1 : 3434, 2920, 1617, 1514, 764; for 1H and 13C NMR (CD3OD) spectra, see Table 1; negative HR-FABMS, m/z: 537.1967 [M H] (calcd for C26H33O12, 537.1972). 3.5. Enzymatic hydrolysis of compound 3 Compound 3 (13.0 mg) was hydrolyzed with crude hesperidinase (25 mg) in 2 ml of H2O. After stirring at 37 C for 24 h, the reaction mixture was extracted with EtOAc, and then evaporated to dryness to provide (+)-cycloolivil 24 (5.2 mg), aD +85.4), whose structure was identied using physical and NMR spectral analyses. 3.6. 3,4-Dimethoxyphenyl 1-O-b-D-xylopyranosyl-(1 ! 6)b-D-glucopyranoside (11) Amorphous powder, a24 100.7 (DMSO, c 0.45); IR D cm1 : 3533, 3380, 3275, 2921, 1602, 1522, 833; for
24
Table 1 NMR Spectroscopic data of compound 3 No. 1 2 2a 3 3a 4 5 6 7 8 9 10 MeO-7 MeO-3 0 10 20 30 40 50 60 100 200 300 400 500 600
a
dC 39.9 74.9 69.3 47.5 60.8 45.0 117.3 145.4 147.6 113.0 133.1 126.5 56.7 56.4 142.0 114.9 150.8 146.6 117.9 123.6 102.9 74.9 77.8 71.3 78.1 62.5
dH 3.17 (1H, d, J = 16.8 Hz) 2.57 (1H, d, J = 16.8 Hz) 3.75 3.55 2.00 3.75 3.49 4.03 6.11 (1H)a (1H, d, J = 11.2 Hz) (1H, br d, J = 11.5 Hz) (1H)a (1H, dd, J = 11.2, 4.2 Hz) (1H, br d, J = 11.5 Hz) (1H, s)
6.59 (1H, s)
3.75 (3H, s) 3.74 (3H, s) 6.75 (1H, d, J = 1.9 Hz)
7.08 (1H, d, J = 8.3 Hz) 6.72 (1H, dd, J = 8.3, 1.9 Hz) 4.83(1H, d, J = 7.3 Hz) 3.43 (1H, dd, J = 8.3, 7.3 Hz) 3.42 (1H, dd, J = 9.0, 8.3 Hz) 3.36 (1H, dd, J = 9.0, 8.3 Hz) 3.26 (1H, m) 3.84 (1H, br d, J = 12.0 Hz) 3.65 (1H, dd, J = 12.0, 5.6 Hz)
Chemical shifts obtained approximately by HSQC.
H and 13C NMR (DMSO-d6) spectra, see Table 2; negative HR-FAB-MS, m/z: 447.1477 [M H] (calcd for C19H27O12 447.1503).
Table 2 NMR Spectroscopic data of compound 11 (DMSO-d6) No. 1 2 3 4 5 6 MeO-3 MeO-4 10 20 30 40 50 60 100 200 300 400 500
a
dC 151.9 102.3 149.3 144.0 112.8 107.3 55.5 56.1 101.2 73.2 76.5 69.8 75.9 68.5 103.9 73.4 76.6 69.6 65.7
dH 6.64 (1H, d, J = 2.7 Hz)
mKBR max
6.83 6.59 3.72 3.68 4.69 3.21 3.22 3.11 3.45 3.93 3.56 4.16 2.94 3.05 3.25 3.65 2.95
(1H, d, J = 8.8 Hz) (1H, d, J = 8.8, 2.7 Hz) (3H, s) (3H, s) (1H, d, J = 7.6 Hz) (1H)a (1H)a (1H, dd, J = 9.0, 8.1 Hz) (1H, m) (1H, br d, J = 11.0 Hz) (1H, dd, J = 11.0, 6.6 Hz) (1H, d, J = 7.3 Hz) (1H, dd, J = 8.8, 7.3 Hz) (1H, d, J = 8.8, 8.5 Hz) (1H, m) (1H, dd, J = 11.2, 5.1 Hz) (1H, dd, J = 11.2, 3.9 Hz)
mKBr max
Chemical shifts obtained approximately by HSQC.
520 Table 3 NMR Spectroscopic data of compound 15 No. 1 3 4 5 6 7 8 9 10 10 20 30 40 50 60 100 200 , 600 300 , 500 400 700 800 900 dC 93.0 140.8 106.0 36.5 85.8 84.3 80.7 48.3 64.3 99.5 74.7 77.9 71.6 78.1 62.8 127.1 131.2 116.8 161.3 146.9 115.0 169.1 dH
References
Abe, F., Yamauchi, T., Wan, A.S.C., 1988. Lignans related to olivil from genus Cerbera (Cercera.) VI). Chemical and Pharmaceutical Bulletin 36, 795799. Chaudhuri, R.K., Sticher, O., 1981. New iridoid glucosides and a lignan diglucoside from Globularia alypum L. Helvetica Chimica Acta 64, 3 15. Compadre, C.M., Jauregui, J.F., Nathan, P.J., Enrquez, R.G., 1982. Isolation of 6-O-(p-coumaroyl)-catapol from Tabebuia rosea. Planta Medica 46, 4244. Ghogomu, R.T., Bodo, B., Nyasse, B., Sondengam, B.L., 1985. Isolation and identication of ()-olivil and (+)-cycloolivil from Stereospermum kunthianum. Planta Medica 45, 464. Ida, Y., Satoh, Y., Ohtsuka, M., Nagasao, M., Shoji, J., 1993. Phenolic constituents of Phellodendron amurense bark. Phytochemistry 35, 209 215. Kanchanapoom, T., Kasai, R., Yamasaki, K., 2001. Lignan and phenylpropanoid glycosides from Fernandoa adenophylla. Phytochemistry 57, 12451248. Kanchanapoom, T., Kasai, R., Yamasaki, K., 2002a. Phenolic glycosides from Markhamia stipulata. Phytochemistry 59, 557563. Kanchanapoom, T., Kasai, R., Yamasaki, K., 2002b. Phenolic glycosides from Barnettia kerrii. Phytochemistry 59, 565570. Karasawa, H., Kobayashi, H., Takizawa, N., Miyase, T., Fukushima, S., 1986. Studies on the constituents of Cistanchis herba. VII. Isolation and structures of cistanosides H and I. Yakugaku Zasshi 106, 562566. Kosuge, K., Mitsunaga, K., Koiki, K., Ohmoto, T., 1994. Studies on the constituents of Ailanthus integrifolia. Chemical and Pharmaceutical Bulletin 42, 16691671. Kumar, U.S., Aparna, P., Rao, R.J., Rao, T.P., Rao, J.M., 2003. 1Methyl anthraquinones and their biogenetic precursors from Stereospermum personatum. Phytochemistry 63, 925929. Nishimura, H., Sasaki, H., Morota, T., Chin, M., Mitsuhashi, H., 1989. Six iridoids from Rehmannia glutinosa. Phytochemistry 28, 27052709. Onegi, B., Kraft, C., Kohler, L., Freund, M., Jenett-Siems, K., Siems, K., Beyer, G., Melzig, M.F., Bienzle, U., Eich, E., 2002. Antiplasmodial activity of naphthoquinones and one anthraquinone from Stereospermum kunthianum. Phytochemistry 60, 3944. Sticher, O., A-Yazar, F.U., 1979. Minecoside and verminoside, two new iridoid glucosides from Veronica ocinale L. (Scrophulariaceae). Helvetica Chimica Acta 62, 535539. Sugiyama, M., Nagayama, E., Kikuchi, M., 1993. Lignan and phenylpropanoid glycosides from Osmanthus asiaticus. Phytochemistry 33, 12151219. von Poser, G.L., Schripsema, J., Henriques, A.T., Jensen, S.R., 2000. The distribution of iridoids in Bigniniaceae. Biochemical Systematics and Ecology 28, 351366.
5.50 6.22 5.19 2.71 4.71 4.09
(1H, (1H, (1H, (1H, (1H, (1H,
d, J = 4.4 Hz) dd, J = 6.1, 1.8 Hz) dd, J = 6.1, 3.4 Hz) m) dd, J = 5.6, 5.4 Hz) d, J = 5.6 Hz)
2.45 (1H, dd, J = 9.8, 4.4 Hz) 3.98 (1H, d, J = 12.0 Hz) 3.78 (1H, d, J = 12.0 Hz) 4.62 (1H, d, J = 7.8 Hz) 3.16 (1H, dd, J = 8.8, 7.8 Hz) 3.22 (1H, dd, J = 9.0, 8.8 Hz) 3.26 (1H, dd, J = 9.0, 8.0 Hz) 3.27 (1H, m) 3.83(1H, br d, J = 11.7 Hz) 3.62 (1H, dd, J = 11.7, 3.9 Hz) 7.41 (2H, d, J = 8.8 Hz) 6.76 (2H, d, J = 8.8 Hz) 7.60 (1H, d, J = 15.9 Hz) 6.32 (1H, d, J = 15.9 Hz)
3.7. Stereospermoside (15) Amorphous powder, a24 90.8 (MeOH, c 1.74); IR D cm1 : 3427, 2927, 1692, 1605, 832; for 1H and 13C NMR (CD3OD) spectra, see Table 3; negative HR-FABMS, m/z: 525.1599 [M H] (calcd for C24H29O13, 525.1608).
mKBr max
Acknowledgements The authors are grateful to Khon Kaen University, and NRCT-JSPS Core University Program for nancial support of this work. We also thank Mr. Savian Jantaviset of the Faculty of Pharmaceutical Sciences, Khon Kaen University, for help in obtaining the plant material.
Corrigendum
Corrigendum to Flavones and avone synthases [Phytochemistry 66 (2005) 23992407]

Stefan Martens a, Axel Mithofer
a
b,*
Institut fur Pharmazeutische Biologie, PhilippsUniversitat Marburg, Deutschhausstr. 17A, D-35037 Marburg/Lahn, Germany b Bioorganische Chemie, Max-Planck-Institut fur Chemische Okologie, Hans-Knoll Str. 8, D-07745 Jena, Germany Available online 30 January 2006
The authors regret that Fig. 2 (p. 2400) was published incorrectly. The correct gure is given below.
6-Deoxychalcones
CHI
5-Deoxyflavanones
IFS + IFD
5-Deoxyflavonoids
Isoflavones Flavan-4-ols
O
3-Deoxyflavonoids
3x MalonylCoA
CHS
CHS CHKR IFS + IFD
Chalcones
CHI
FHT
Flavanones
Dihydroflavonols
O OH
DFR
ANS Leucoanthocyanins
Anthocyanidins
O
FGT
Anthocyanins
O
p-CoumaroylCoA
O
O OH OH
+
OH
+
O-Glc
FNS I or FNS II
FLS
LAR
ANR
Flavones
O
Flavonols
O OH
trans Flavan-3-ols
O OH
cis Flavan-3-ols
O OH
Fig. 2.
DOI of original article: 10.1016/j.phytochem.2005.07.013. Corresponding author. Tel.: +49 0 3641 571263; fax: 49 0 3641 571256. E-mail address: amithoefer@ice.mpg.de (A. Mithofer).
Announcement
The Phytochemical Society of EuropePierre-Fabre 2006 Award for Phytochemistry

The Phytochemical Society of Europe and Pierre-Fabre Laboratories are pleased to publish this call for candidates for the 2006 Award, to be presented to a young European scientist who has made an outstanding contribution to phytochemistry or plant biochemistry. Candidatures are invited from all disciplines within this general field. The Award aims to reward an exceptional contribution to the research field in which the candidate is working. The Award consists of a cheque for 2000 euros, together with a parchment certificate. Recipients will be invited to present their research in the form of an award lecture to be delivered during one of the Societys meetings in 2007. Expenses for attending the meeting are paid by the Awarding Committee. Previous PSEPF Award winners are: 2001Prof. Robert NASH (UK) 2002Dr Wolfgang EISENREICH (DE) 2003Dr Virginia LANZOTTI (IT) 2004Dr Deniz TASDEMIR (CH/TU) 2005Dr Simon GIBBONS (UK) All members of the PSE are strongly encouraged to make a nomination. This should consist of: a CV, a short summary of the significant points of the candidates research career (12 pages); a list of publications; the addresses of 2 or 3 independent referees; a letter of support. In particular, Professors and Directors of Institutes are strongly encouraged to nominate colleagues or promising former PhD students or post-doctoral researchers. Nominations for the 2006 Award should be sent before 15 April 2006preferably in electronic formatto the PSE Vice-Chairman: Dr Richard Robins, LAIEM, CNRS UMR6006, Faculte des Sciences et des Techniques, Universite de Nantes, B.P. 92208, F-44322 Nantes 03, France. E-mail: richard.robins@univ-nantes.fr Further information about the PSE and its Awards can be found on the website http://www.phytochemicalsociety. org Further information about Pierre-Fabre Laboratories can be found on the website http://www.pierre-fabre. com
doi: 10.1016/j.phytochem.2006.01.029
PHYTOCHEMISTRY
www.elsevier.com/locate/phytochem
Phytochemistry Vol. 67, No. 5, 2006 Author Index

Abdelgaleil, S.A.M., 452 Abdel-Kader, M.S., 429 Ahmad, M.S., 429 Ahmed, A.A., 424 Al-Rehaily, A.J., 429 Asakawa, Y., 424 Atta-ur-Rahman, 439 Chifundera, K., 504 Choudhary, M.I., 439 Coombes, P.H., 459 Cutillo, F., 481 DAbrosca, B., 481 Deachathai, S., 464 DellaGreca, M., 481 Devkota, K.P., 439 Di, Y., 486 Ding, Y.-H., 497 Doe, M., 452 Don, M.-J., 497 Duplex, W.J., 475 Emerenciano, V.P., 492 Federici, E., 504 Ferreira, M.J.P., 492 Fiorentino, A., 481 Florent, L., 444 Fomum, Z.T., 459, 475 Fotso, S., 475 Frappier, F., 444 Frederich, M., 433 Gale, C., 504 Gallo, F.R., 504 Grellier, P., 444 Hao, X., 486 Harakat, D., 433 Harmut, L., 475 Iurilli, R., 504 Jacquier, M.-J., 433 Joyeau, R., 444 Kamdem Wao, A.F., 459, 475 Kanchanapoom, T., 516 Kaplan, M.A.C., 492 Karchesy, J., 424 Kenmogne, M., 433 Laatsch, H., 475 Li, C., 511 Li, S., 486 Mahabusarakam, W., 464, 470 Mambu, L., 444 Martens, S., 521 Maskey, R.P., 475 Mithofer, A., 521 Mohamed, A.E.H., 424 Monache, F.D., 504 Moreira, D.L., 492 Morimoto, Y., 452 Mossa, J.S., 429 Mulholland, D.A., 459 Musharraf, S.G., 439 Nakatani, M., 452 Ndom, J.C., 475 Nguefa, H.E., 475 Njamen, D., 475 Nkengfack, A.E., 459 Noiarsa, P., 516 Nuangnaowarat, W., 470 Otsuka, H., 516 Palazzino, G., 504 Phongpaichit, S., 464 Prost, E., 433 Ramanitrahasimbola, D., 444 Ranjit, R., 439 Rasoanaivo, P., 444 Ruchirawat, S., 516 Santos, M.I.S., 492 Shang, X.-Y., 511 Shen, C.-C., 497 Shi, J.-G., 511 Shrestha, T.M., 439 Sondengam, L.B., 433 Sun, C.-M., 497 Syu, W.-J., 497 Taylor, W.C., 464, 470 Velozo, L.S.M., 492 Wao-Teguo, P., 433 Wandjii, J., 475 Wang, J., 486 Wang, Y., 486 Wang, Y.-H., 511 Wansi, J.D., 475 Yang, C.-R., 464 Yang, X., 486 Yang, Y.-C., 511 Zarrelli, A., 481 ` Zeches, M., 433 Zhang, C.-Z., 511 Zhang, Y.-J., 464
doi:10.1016/S0031-9422(06)00050-1

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Phytochemistry Vol. 67, No. 5, 2006 Reports on Structure Elucidation

Contents / Phytochemistry 67 (2006) 419423

HO H3C OH H3C H O CH3 H3C HO H3C H H3C O O

R R1 R2 R3 1. H,H OH H H 2 OH H H 3 O H 4. H,H OH H E-caffeoyloxy

Contents / Phytochemistry 67 (2006) 419423

Contents / Phytochemistry 67 (2006) 419423

R2 1- R1 =H; R2 =OH; 2- R1 =OH; R2 =H;

3" 4" 5"

Contents / Phytochemistry 67 (2006) 419423

p 521 p 522 pI pp IIIII

PHYTOCHEMISTRY Phytochemistry 67 (2006) 424428 www.elsevier.com/locate/phytochem

, Abou El-Hamd H. Mohamed b, Joe Karchesy c, Yoshinori Asakawa

A.A. Ahmed et al. / Phytochemistry 67 (2006) 424428

Fig. 1. Selective HMBC correlations of compound 1.

1.46 m 1.30 dd (13.8, 4.0)

1.68 dd (12.0, 1.2) 5.80 br s 10.0 s

C-4, C-10 C-10, C-11

3.45 ddd (12.0, 12.0, 3)

A.A. Ahmed et al. / Phytochemistry 67 (2006) 424428

Fig. 2. ORTEP diagram of the crystal structure of 2.

A.A. Ahmed et al. / Phytochemistry 67 (2006) 424428

PHYTOCHEMISTRY Phytochemistry 67 (2006) 429432 www.elsevier.com/locate/phytochem

Iridoid glucosides from Kickxia abhaica D.A. Sutton from Scrophulariaceae

A.J. Al-Rehaily et al. / Phytochemistry 67 (2006) 429432

1 3 4 5 6 7 8 9 10 10 20 30 40 50 60a 60b 100 200 300 /700 400 /600 500

7.78 d (9.0) 6.73 d (9.0)

A.J. Al-Rehaily et al. / Phytochemistry 67 (2006) 429432

PHYTOCHEMISTRY Phytochemistry 67 (2006) 433438 www.elsevier.com/locate/phytochem

Five labdane diterpenoids from the seeds of Aframomum zambesiacum

M. Kenmogne et al. / Phytochemistry 67 (2006) 433438

M. Kenmogne et al. / Phytochemistry 67 (2006) 433438

M. Kenmogne et al. / Phytochemistry 67 (2006) 433438

PHYTOCHEMISTRY Phytochemistry 67 (2006) 439443 www.elsevier.com/locate/phytochem

Hydroxylation of the sesterterpene leucosceptrine by the fungus Rhizopus stolonifer

M.I. Choudhary et al. / Phytochemistry 67 (2006) 439443

1 Rhizopus stolonifer 14 days

M.I. Choudhary et al. / Phytochemistry 67 (2006) 439443

Fig. 2. (a) Key HMBC, and (b) NOESY correlations in compound 3.

M.I. Choudhary et al. / Phytochemistry 67 (2006) 439443

PHYTOCHEMISTRY Phytochemistry 67 (2006) 444451 www.elsevier.com/locate/phytochem

Clerodane and labdane diterpenoids from Nuxia sphaerocephala

L. Mambu et al. / Phytochemistry 67 (2006) 444451

4' O H 8' COOH H R1 H O O 5 R1 =OH; R2 = H 6R

Diterpenoids 1-7 from N.sphaerocephala

446 Table 1 13 C NMR data for compounds 17 Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 10 20 30 40 50 60 70 80 CO

L. Mambu et al. / Phytochemistry 67 (2006) 444451

Spectra were recorded in CDCl3. Spectra were recorded in CD3OD.

+0.021 +0.015 +0.021 -0.001 H -0.089 +0.015 CH3

-0.059 CH3 CH3 +0.018 +0.001

2a 2.34, dd (3.9, 17.5) 2.44, dd (13.7, 17.5)

Spectra were recorded in CDCl3. Spectra were recorded in CD3OD.

448 Table 3 1 H NMR data of labdane diterpenoids Position 1 2 3 5 6 7 9 11 12 13 14 15 16 17 18 19 20 20 30 50 60 70 80

L. Mambu et al. / Phytochemistry 67 (2006) 444451

8 R, R1 = O , R2 = CHO 9 R = OH, R1 = H , R2 = CHO 10 R, R1 = O, R2 = CH 2OH

d (8.2) dd (2.0, 8.2) d (15.9) d (15.9)

Spectra were recorded in CDCl3. Spectra were recorded in CD3OD.

4.67 0.09 4.06 0.53 15.56 2.11

H +0.053 +0.023 +0.014 +0.025 -0.023 H +0.121 N

-0.054 +0.035 +0.021

L. Mambu et al. / Phytochemistry 67 (2006) 444451

L. Mambu et al. / Phytochemistry 67 (2006) 444451

L. Mambu et al. / Phytochemistry 67 (2006) 444451