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Article history: In the quest for potent antiamoebic agents, a series of hydrazone hybrids (H1eH30) have been designed
Received 1 April 2016 and sequentially synthesized. The dimethylaminoethoxy and hydrazone entities incorporated into one
Received in revised form molecule proved to be more persuasive and selective approach towards designing of antiamoebic agent.
20 July 2016
The synthesized compounds exhibited promising results against E. histolytica. The compound N0 -(2-
Accepted 11 August 2016
Available online 13 August 2016
chlorobenzylidene)-4-(2-(dimethylamino) ethoxy)benzohydrazide was most impending among the se-
ries. Cytotoxicity profile showed better cell viability on lung cancer cell line (A549 cells) by MTT assay.
© 2016 Published by Elsevier Masson SAS.
Keywords:
Hydrazones
Antiamoebic activity
Cytotoxicity
MTT assay
X-ray crystallography
http://dx.doi.org/10.1016/j.ejmech.2016.08.022
0223-5234/© 2016 Published by Elsevier Masson SAS.
446 A. Inam et al. / European Journal of Medicinal Chemistry 124 (2016) 445e455
ester group) and 1247 cm1 (AreOeC,CeOeC asym.str) respec- [24]. The distances between p clouds of the centroids in the bonds
tively. In p-hydroxymethylbenzoate (1) the peaks for ester and of benzyl groups are: dc1-c2 ¼ 3.577 Å [c1: C(5B)eC(6B) and c2:
hydroxyl groups were present. Further the formation of N0 -Benzy- C(5K)eC(6K)] and repeated for other centroids]. Table 2 contains
lidene-4-(2-(dimethylamino)ethoxy) benzohydrazide (3) can be selected bond lengths and angles for compound H23.
confirmed by the appearance of the peak for NH2 group at
3317 cm1 (NeH str) and for carbonyl group at 1605 cm1 (C]O
2.3. Biology
str). In Methyl-4-(2-(dimethylamino)ethoxy)benzoate (2) C]O
stretching band for ester group was found at 1712 cm1 which was
2.3.1. Antiamoebic activity
absent in (3) suggesting conversion of ester group to hydrazide
Preliminary experiments were carried out to determine the
group. The hydrazone derivatives (H1eH30) show all the charac-
in vitro antiamoebic activity of all the compounds (H1eH30) by
teristic bands such as the band at 3392e3061 cm1 was assigned to
microdilution method using HM1: IMSS strain of E. histolytica. The
NeH stretching and 1712e1643 cm1 to C]O stretching which
results are summarized in Table 4. The data are present in terms of
suggested the condensation of different aromatic aldehyde with
percent growth inhibition relative to untreated controls, and
hydrazide. The mass spectra of the compounds showed the ex-
plotted as probit values as a function of drug concentration. The
pected fragmentation pattern m/z corresponding to [Mþ1] which
antiamoebic effect was compared with the most widely used
confirmed their respective molecular weights.
antiamoebic medication metronidazole (Mtz) which had a 50%
inhibitory concentration (IC50) of 1.89 mM in our experiments. The
2.2. Single crystal structure structure activity relationships (SAR) can be explained on the basis
of the position, number and nature of substituent present in the
2.2.1. Single crystal structure of compound H23 phenyl ring. However, it is important to mention that the strength
ORTEP diagram for the compound 4-(2-(dimethylamino) of the group can also be considered as an interesting factor for the
ethoxy)-N-(pyridine-2-ylmethylene)benzohydrazide (H23) is activity of the compounds. As the alkyl group attached to the
showed in Fig. 5. The compound H23 crystallizes in the ortho- phenyl ring increases the electron density which makes azome-
rhombic system, with Pbca space group. In the asymmetric unit, thine group electron rich correspondingly antiamoebic activity
there are one molecule of H23 and one water molecule. Selected decreases. The same was observed in few compounds whilst from
bond distances and angles are given in Table 2. phenyl group (H1, IC50: 1.43 ± 0.012 mM) to 4-isopropylphenyl
In H23 compound, atoms, C(11), C(12), O(2), N(2) and N(3), of group (H10, IC50: 3.45 ± 0.015 mM) and 4-dimethylamino group
hydrazide group are coplanar with benzyl group, C(5), C(6), C(7), (H21, IC50: 1.89 ± 0.008 mM) where IC50 values increases and
C(8), C(9) and C(10) (mean deviation from the least-squares plane thereby slightly decreasing the antiamoebic activity. All the
being 0.0762(16) Å). Torsion angle for this part of the molecule with halogen substituted compounds showed an interesting inhibitory
pyridine group, C(13), C(14), C(15), C(16), C(17) and N(4), was found behaviour. The compounds H2, H3, H4 having chloro group at
to be 24.06(8) , which indicate a light torsion due to intermolecular ortho, meta and para positions respectively, showed activity higher
forces. than the compound H1 containing unsubstituted phenyl ring. The
Intermolecular hydrogen bonds between water molecule and ortho-Cl (H2, IC50: 0.23 ± 0.015 mM) exhibited the least IC50 value
different groups of the hydrazide derivative determine the crystal than meta-Cl (H3, IC50: 1.01 ± 0.014 mM) and para-Cl (H4 IC50:
packing of the compound (Table 3). p-p lateral stacking in- 0.79 ± 0.011 mM). The nitro group containing compounds showed
teractions between the bonds of the benzyl rings establish the slightly higher IC50 values (H5, IC50: 2.80 ± 0.021 mM & H6, IC50:
linked between frameworks and determine the structure (Fig. 6) 2.40 ± 0.029 mM), though para-nitro showed lower IC50 value than
Fig. 5. ORTEP plot for the compound 4-(2-(dimethylamino)ethoxy)-N-(pyridine-2-ylmethylene)benzohydrazide (H23). All the non-hygrogen atoms are presented by their 30%
probability ellipsoids. Hydrogen atoms are omitted for clarity.
448 A. Inam et al. / European Journal of Medicinal Chemistry 124 (2016) 445e455
Fig. 6. Intermolecular p-p interactions in H23. Dashed lines link the centres of the p clouds involving in each interaction.
meta-nitro but both can be considered as moderately active. Aldrich Chemical Company (USA). Precoated aluminum sheets
Compound H7 with eOH at ortho position was endowed with ac- (silica gel 60 F254, Merck Germany) were used for thin-layer
tivity which can be observed from its percentage inhibition value chromatography (TLC) and spots were visualized under UV light.
(H7, IC50: 1.76 ± 0.017 mM), while the compounds H8 para eOH and IR spectra were recorded on Bruker FT-IR spectrophotometer under
H28 with dihydoxy substitution were moderately active with neat condition on a Zn Se crystal. 1H NMR was recorded on Bruker
higher inhibitory concentration values (H8, IC50: 2.21 ± 0.030 mM & Spectrospin DPX 300 MHz using CDCl3 as a solvent and tetrame-
H28, IC50: 6.40 ± 0.022 mM)). The results obtained for the alkyl and thylsilane (TMS) as an internal standard. Splitting patterns are
alkoxy groups were quite similar, wherein the activity reduces from designated as follows; s, singlet; d, doublet; m, multiplet. Chemical
methyl (H15, IC50: 1.86 ± 0.010 mM) to ethyl (H9, IC50: shift values are given in ppm. Electrospray (ES) mass spectra were
5.65 ± 0.002 mM) and from methoxy (H22, IC50: 1.53 ± 0.019 mM) to carried out on Microtof-Q II 10262. Melting points were recorded
ethoxy (H11, IC50: 3.77 ± 0.011 mM) and isopropoxy (H12, IC50: on a Veego melting point apparatus (model REC-2203882) and
7.653 ± 0.003 mM) substituted compounds. The presence of the were uncorrected. Elemental analyses were performed in an Ele-
heterocyclic rings like pyridine (H23) and thiophene (H24) reduced mentar Vario analyzer and the results lay within ±0.3% of the
the activity by showing higher IC50 values. From the above dis- theoretical values.
cussion, it can be concluded that compounds with eCl substitution
H2, H3, H4 seems to be the most potent of all the compounds
screened. 4.2. Synthesis
Fig. 7. a. Measurement of cell viability following treatment with H1, H2, H3 and H4 compounds by MTT assay. b. Measurement of cell viability following treatment with H7, H15,
H17 and H18 compounds by MTT assay. c. Measurement of cell viability following treatment with H19, H21, H22, H27, H29 and H30 compounds by MTT assay.
cooled and the solid product was filtered and recrystallized in plane bending). Elemental analysis calcd (%) for C11H17N3O2: C
ethanol yielded the final product (H1eH30). 59.17, H 7.67, N 18.82; found: C 59.10, H 7.54, N 18.93.
N0 -Benzylidene-4-(2-(dimethylamino)ethoxy)benzohy- N0 -Benzylidene-4-(2-(dimethylamino)ethoxy)benzohy-
drazide (3): Cream coloured solid; 1H NMR (CDCl3, 300 MHz, drazide (H1): White solid, Yield: 85%, m.pt. 172 C; 1HNMR (CDCl3,
dppm): 7.72e7.69 (m, 2H, AreH), 7.27(bs, 1H, NH), 6.96e6.93 (m, 300 MHz, dppm): 9.43 (bs, 1H, eNH), 8.07 (d, 2H, J ¼ 6.9 Hz, AreH),
2H, AreH), 4.10 (t, 2H, J ¼ 5.7 Hz, OCH2), 2.74 (t, 2H, J ¼ 5.4 Hz,CH2), 8.31 (s, 1H, eCHN),7.88e7.72 (m, 2H, AreH), 7.48e7.37 (m, 3H,
2.34 (ss, 6H, N(CH3)2). IR nmax (cm1): 3317(NH2, NeH str), AreH), 6.98 (d, 2H, J ¼ 8.7 Hz, AreH), 4.12 (t, 2H, J ¼ 5.7 Hz, OCH2),
2973(ArCeH, CeH str), 1605(C]O str), 1250(AreOeC,CeOeC 2.77 (t, 2H, J ¼ 5.7 Hz, CH2), 2.36 (ss, 6H, N(CH3)2), 1.91 (s, 5H,
asym. str), 1172(NeN), 846(p-disubstituted benzene,CeH out of AreH). IR nmax (cm1): 3363 (NH2, NeH str), 3022 (ArCeH, CeH str),
450 A. Inam et al. / European Journal of Medicinal Chemistry 124 (2016) 445e455
(ss, 6H, N(CH3)2). IR nmax (cm1): 3367 (NeH str), 1653(C]O str),
1565(N]CH), 1247 (AreOeC, CeOeC asym. str), 1168(NeN);
Elemental analysis calcd (%) for C18H20N4O4: C 60.66, H 5.66, N
15.72; found: C 60.34, H 5.54, N 15.43; ES-MS: m/z 357.32 (Mþ1).
N0 -(2-hydroxybenzylidene)-4-(2-(dimethylamino)ethoxy)
benzohydrazide (H7): Yellow solid, Yield: 70%, m.pt. 150 C; 1H
NMR (CDCl3, 300 MHz, dppm): 11.06 (bs, 1H), 9.12 (s, 1H), 8.46 (s,
1H), 7.82 (d, 2H,J ¼ 8.7), 7.33e7.33 (m, 2H), 7.23e7.20 (m, 1H),
7.00e6.97 (m, 3H), 4.13 (t, 2H, J ¼ 5.4 Hz), 2.77 (t, 2H,J ¼ 5.7 Hz),
2.35 (ss, 6H). IR nmax (cm1): 3355 (NeH str), 1646(C]O str),
1561(N]CH), 1252 (AreOeC, CeOeC asym. str), 1150(NeN);
Elemental analysis calcd (%) for C18H21N3O3: C 66.04, H 6.47, N
12.84; found: C 66.14, H 6.47, N 12.80 ES-MS: m/z 328.00 (Mþ1).
N0 -(4-hydroxybenzylidene)-4-(2-(dimethylamino)ethoxy)
benzohydrazide (H8): Yellow solid, Yield: 62%, m.pt.111 C; 1H
Fig. 8. IC50 Value of compounds (H1eH30) for MTT assay.
NMR (CDCl3, 300 MHz, dppm): 11.51 (s, 1H, eNH), 9.92 (s, 1H, OH),
8.33 (s, 1H, CHN), 7.88 (d, 2H, J ¼ 6.3 Hz, AreH), 7.55 (d,
1699 (C]O str), 1250 (AreOeC, CeOeC asym. str), 1171 (NeN); 2H,J ¼ 8.7 Hz, AreH), 7.06 (d, 2H, J ¼ 6.3 Hz, AreH), 6.84 (d, 2H,
Elemental analysis calcd (%) for C18H21N3O2: C 69.43, H 6.80, N J ¼ 6.0 Hz, AreH), 4.13 (t, 2H, J ¼ 5.4 Hz, OCH2), 2.81 (t, 2H,
13.49; found: C 69.14, H 6.54, N 13.33; ES-MS: m/z 312.52 (Mþ1). J ¼ 5.7 Hz, CH2), 2.22 (ss, 6H, N(CH3)2); IRnmax (cm1): 3590 (eOH),
N0 -(2-chlorobenzylidene)-4-(2-(dimethylamino)ethoxy)ben- 3301 (NeH str), 1644 (C]O str), 1557 (N]CH), 1283 (AreOeC,
zohydrazide (H2): Colourless solid, Yield: 85%, m.pt. 100 C; 1H CeOeC asym. str), 1166 (NeN); . Elemental analysis calcd (%) for
NMR (CDCl3, 300 MHz, dppm): 9.36 (bs, 1H, eNH), 8.19 (s, 1H, CHN), C18H21N3O3: C 66.04, H 6.47, N 12.84; found: C 66.34, H 6.45, N
7.89(bs, 2H, AreH), 7.38e7.29 (m, 4H, AreH), 7.01 (d, 2H, J ¼ 8.7, 12.83; ES-MS: m/z 328.55 (Mþ1).
AreH), 4.13 (t, 2H, J ¼ 6 Hz, OCH2), 2.76 (t, 2H, J ¼ 5.7 Hz, CH2), 2.35 N0 -(4-ethylbenzylidene)-4-(2-(dimethylamino)ethoxy)ben-
(ss, 6H, N(CH3)2). IRnmax (cm1): 3184 (NeH str), 1649 (C]O str), zohydrazide (H9): solid, Yield: 60%, m.pt.155 C; 1H NMR (CDCl3,
1554 (N]CH), 1256 (AreOeC, CeOeC asym. str), 1177(NeN); 300 MHz, dppm): 9.11 (bs, 1H), 8.24 (s, 1H), 7.85 (bs, 2H), 7.65 (bs,
Elemental analysis calcd (%) for C18H20N3O2Cl: C 62.52, H 5.83, N 2H),7.26e7.21 (m, 2H), 6.99 (d, 2H, J ¼ 7.2 Hz), 4.14 (t, 2H,
12.15; found: C 62.34, H 5.54, N 12.33; ES-MS: m/z 346.16 (Mþ1). J ¼ 5.7 Hz), 2.77 (t, 2H, J ¼ 5.4 Hz), 2.71 (m, 2H), 2.35 (ss, 6H), 1.27 (t,
N0 -(3-chlorobenzylidene)-4-(2-(dimethylamino)ethoxy)ben- 3H, J ¼ 7.5 Hz). IRnmax (cm1): 3357 (NeH str), 1648 (C]O str),
zohydrazide (H3): Colourless solid, Yield: 82%, m.pt. 99 C; 1H NMR 1560 (N]CH), 1270 (AreOeC, CeOeC asym. str), 1173 (NeN);
(CDCl3, 300 MHz, dppm): 9.21 (bs, 1H, eNH), 8.26 (s, 1H, CHN), 7.87 Elemental analysis calcd (%) for C20H25N3O2: C 70.77, H 7.42, N
(bs, 2H, AreH), 7.73 (bs, 1H, AreH),7.58 (bs, 1H, AreH), 7.37e7.29 12.38; found: C 70.64, H 7.54, N 12.43; ES-MS: m/z 340.10 (Mþ1).
(m, 2H, AreH), 7.00 (bs, 1H, CHN), 4.13 (t, 2H, J ¼ 5.7 Hz, OCH2), 2.76 N0 -(4-isopropylbenzylidene)-4-(2-(dimethylamino)ethoxy)
(t, 2H, J ¼ 5.4 Hz, CH2), 2.35(ss, 6H, N(CH3)2). IR nmax (cm1): benzohydrazide (H10): solid, Yield: 60%, m.pt.150 C; 1H NMR
3061(NeH str), 3061(ArCeH, CeH str), 1643(C]O str), (CDCl3, 300 MHz, dppm): 9.41 (bs, 1H), 8.24 (s, 1H), 7.96e7.84 (m,
1248(AreOeC, CeOeC asym. str), 1552(eN]CH), 1182(NeN); 2H), 7.68e7.61 (m, 2H), 7.25e7.24 (m, 2H), 6.99 (d, 2H, J ¼ 8.7 Hz),
Elemental analysis calcd (%) for C18H20N3O2Cl: C 62.52, H 5.83, N 4.14 (t, 2H, J ¼ 5.4 Hz), 2.97e2.91 (m, 1H), 2.78 (t, 2H, J ¼ 5.7 Hz),
12.15; found: C 62.34, H 5.54, N 12.33; ES-MS: m/z 346.15 (Mþ1). 2.35 (s, 6H), 1.27 (d, 6H, J ¼ 6.9 Hz); IRnmax (cm1): 3384 (NeH str),
N0 -(4-chlorobenzylidene)-4-(2-(dimethylamino)ethoxy)ben- 1647 (C]O str), 1557 (N]CH), 1250 (AreOeC, CeOeC asym. str),
zohydrazide (H4): Colourless solid, Yield: 84%, m.pt. 188 C; 1H 1176 (NeN); Elemental analysis calcd (%) for C21H27N3O2: C 71.36, H
NMR (CDCl3, 300 MHz, dppm): 9.37(bs, 1H, eNH), 8.28 (s, 1H, CHN), 7.70, N 11.89; found: C 71.35, H 7.70, N 11.88; ES-MS: m/z 354.25
7.86(bs, 2H, AreH), 7.65 (bs, 2H, AreH),7.37 (d, 2H, J ¼ 8.1, AreH), (Mþ1).
6.99 (d, 2H, J ¼ 8.4, AreH), 4.12 (t, 2HJ ¼ 5.1 Hz, OCH2), 2.75 (t, 2H, N0 -(4-ethoxybenzylidene)-4-(2-(dimethylamino)ethoxy)ben-
J ¼ 5.7 Hz, CH2), 2.35(ss, 6H, N(CH3)2). IR nmax (cm1): 3392 (NeH zohydrazide (H11): solid, Yield: 65%, m.pt.162 C; 1H NMR (CDCl3,
str), 1695 (C]O str), 1565 (N]CH), 1257 (AreOeC, CeOeC asym. 300 MHz, dppm): 9.24 (bs, 1H), 8.21 (s, 1H), 7.84 (bs, 2H), 7.67 (bs,
str), 1178 (NeN); Elemental analysis calcd (%) for C18H20N3O2Cl: C 2H), 6.98 (d, 2H, J ¼ 8.7 Hz), 6.90 (d, 2H, J ¼ 8.7 Hz), 4.13 (t, 2H,
62.52, H 5.83, N 12.15; found: C 62.34, H 5.54, N 12.33; ES-MS: m/z J ¼ 5.4 Hz), 4.07e4.02 (m, 2H), 2.77 (t, 2H, J ¼ 5.7 Hz), 2.34 (s, 6H),
346.28 (Mþ1). 1.44 (t, 3H, J ¼ 6.9 Hz); IRnmax (cm1): 3301 (NeH str), 1644 (C]O
N0 -(3-Nitrobenzylidene)-4-(2-(dimethylamino)ethoxy)ben- str), 1557 (N]CH), 1250 (AreOeC, CeOeC asym. str), 1167 (NeN);
zohydrazide (H5): Colourless solid, Yield: 82%, m.pt.130 C; 1H Elemental analysis calcd (%) for C20H25N3O3: C 67.58, H 7.09, N
NMR (CDCl3, 300 MHz, dppm): 9.43(bs, 1H, eNH), 8.46 (s, 1H, CHN), 11.82; found: C 67.58, H 7.09, N 11.85; ES-MS: m/z 356.10 (Mþ1).
8.24 (d, 2H,J ¼ 8.7, AreH), 8.13(bs, 1H, AreH) 7.89 (d, 2H,J ¼ 7.5, N0 -(4-propoxybenzylidene)-4-(2-(dimethylamino)ethoxy)
AreH), 7.58 (t, 1H, J ¼ 8.1, AreH), 7.01 (d, 2H, J ¼ 8.7, AreH), 4.13 (t, benzohydrazide (H12): solid, Yield: 72%, m.pt.190 C; 1H NMR
2H, J ¼ 5.7 Hz, OCH2), 2.78 (t, 2H, J ¼ 5.7 Hz,eCH2), 2.35 (ss, 6H, (CDCl3, 300 MHz, dppm): 9.08 (bs, 1H), 8.19 (s, 1H), 7.96e7.83 (m,
N(CH3)2). IR nmax (cm1): 3204(NeH str), 1684(C]O str), 1562(N] 2H), 7.65e7.60 (m, 2H), 6.99 (d, 2H, J ¼ 8.7 Hz), 6.90 (d, 2H,
CH), 1259(AreOeC, CeOeC asym. str), 1177(NeN); Elemental J ¼ 9.0 Hz), 4.65 (m, 1H), 4.14 (t, 2H, J ¼ 5.4 Hz), 2.77 (t, 2H,
analysis calcd (%) for C18H20N4O4: C 60.66, H 5.66, N 15.72; found: C J ¼ 5.7 Hz), 2.34 (s, 6H), 1.36 (d, 6H, J ¼ 6.0 Hz); IRnmax (cm1): 3361
60.34, H 5.54, N 15.43; ES-MS: m/z 357.11 (Mþ1). (NeH str), 1644 (C]O str), 1563 (N]CH), 1240 (AreOeC, CeOeC
N0 -(4-Nitrobenzylidene)-4-(2-(dimethylamino)ethoxy)ben- asym. str), 1142 (NeN); Elemental analysis calcd (%) for
zohydrazide (H6): Yellow solid, Yield: 82%, m.pt.99 C; 1H NMR C21H27N3O3: C 68.27, H 7.37, N 11.37; found: C 68.27, H 7.39, N 11.34;
(CDCl3, 300 MHz, dppm): 9.84(bs, 1H, eNH), 8.46 (s, 1H, CHN), 8.25 ES-MS: m/z 370.22 (Mþ1).
(d, 2H,J ¼ 8.7, AreH), 7.87 (d, 4H,J ¼ 6.9, AreH) 6.98 (d, 2H,J ¼ 8.7, N0 -(4-hydroxy-3-methoxybenzylidene)-4-(2-(dimethyla-
AreH), 4.19 (t, 2H, J ¼ 5.4 Hz, OCH2), 2.87 (t, 2H,J ¼ 5.7 Hz, CH2), 2.42 mino)ethoxy)benzo-hydrazide (H13): solid, Yield: 76%,
m.pt.147 C; 1H NMR (CDCl3, 300 MHz, dppm): 11.02 (s, 1H), 8.24 (s,
A. Inam et al. / European Journal of Medicinal Chemistry 124 (2016) 445e455 451
2H), 7.91e7.49 (m, 3H), 7.02e6.87 (m, 4H), 4.13 (t, 2H, J ¼ 5.4 Hz), J ¼ 5.4 Hz), 3.89e3.84 (m, 3H), 2.77 (t, 2H, J ¼ 5.7 Hz), 2.37 (s, 6H),
3.94 (s, 3H) 2.78e2.74 (m, 2H), 2.33 (s, 6H); IRnmax (cm1): 3358 1.40 (d, 6H, J ¼ 6.3 Hz); IRnmax (cm1): 3386 (NeH str), 1642 (C]O
(NeH str), 1640 (C]O str), 1504 (N]CH), 1241 (AreOeC, CeOeC str), 1536(N]CH), 1238 (AreOeC, CeOeC asym. str), 1150 (NeN);
asym. str), 1173 (NeN); Elemental analysis calcd (%) for C19H23N3O4: Elemental analysis calcd (%) for C22H29N3O4: C 66.14, H 7.32, N
C 63.85, H 6.49, N 11.76; found: C 63.89, H 6.52, N 11.75; ES-MS: m/z 10.52; found: C 66.12, H 7.30, N 10.51; ES-MS: m/z 400.16 (Mþ1).
358.11 (Mþ1). N0 -(4-(dimethylamino)benzylidene)-4-(2-(dimethylamino)
N0 -(2-bromo-4-hydroxy-3-methoxybenzylidene)-4-(2- ethoxy)benzohydrazide (H21): solid, Yield: 56%, m.pt.216 C; 1H
(dimethylamino)ethoxy) benzohydrazide (H14): solid, Yield: 68%, NMR (CDCl3, 300 MHz, dppm): 8.94(bs, 1H), 8.10 (s, 1H), 7.91 (bs,
m.pt.197 C; 1H NMR (CDCl3, 300 MHz, dppm): 11.68 (s, 1H), 10.12 2H), 7.63 (bs, 2H), 6.98 (d, 2H, J ¼ 8.7 Hz), 6.70 (d, 2H, J ¼ 8.7 Hz),
(bs, 1H), 8.29 (s, 1H), 7.89 (d, 2H, J ¼ 5.7 Hz), 7.40e7.30 (m, 2H), 4.31 (t, 2H, J ¼ 5.4 Hz), 3.02 (ss, 6H), 2.79 (t, 2H, J ¼ 5.7 Hz), 2.37 (s,
7.10e7.07 (m, 2H), 4.14 (t, 2H, J ¼ 5.4 Hz), 3.89 (s, 3H), 2.64 (t, 2H, 6H); IRnmax (cm1): 3352 (NeH str), 1644 (C]O str), 1517 (N]CH),
J ¼ 5.7 Hz), 2.22 (s, 6H); IRnmax (cm1): 3356 (NeH str), 1640 (C]O 1253 (AreOeC, CeOeC asym. str), 1177 (NeN); Elemental analysis
str), 1502 (N]CH), 1250 (AreOeC, CeOeC asym. str), 1169 (NeN); calcd (%) for C20H26N4O2: C 67.77, H 7.39, N 15.81; found: C 67.75, H
Elemental analysis calcd (%) for C19H22BrN3O4: C 52.30, H 5.08, N 7.40, N 15.81; ES-MS: m/z 355.16 (Mþ1).
9.63; found: C 52.33, H 5.18, N 9.66; ES-MS: m/z 437.36 (Mþ1). N0 -(4-methoxybenzylidene)-4-(2-(dimethylamino)ethoxy)
N0 -(4-methylbenzylidene)-4-(2-(dimethylamino)ethoxy) benzohydrazide (H22): solid, Yield: 69%, m.pt.120 C; 1H NMR
benzohydrazide (H15): solid, Yield: 55%, m.pt.164 C; 1H NMR (CDCl3, 300 MHz, dppm): 9.24 (bs, 1H), 8.22 (s, 1H), 7.84(bs, 2H),
(CDCl3, 300 MHz, dppm): 9.11 (bs, 1H), 8.22 (s, 1H), 7.83 (bs, 2H), 7.68 (bs, 2H), 6.97e6.89 (m, 4H), 4.13 (t, 2H, J ¼ 5.4 Hz), 3.83(ss, 3H),
7.65 (bs, 2H), 7.22 (d, 2H, J ¼ 8.1 Hz), 7.00 (d, 2H, J ¼ 8.1 Hz), 4.14 (t, 2.77 (t, 2H, J ¼ 5.7 Hz), 2.34 (s, 6H); IRnmax (cm1): 3390 (NeH str),
2H, J ¼ 5.4 Hz), 2.78 (t, 2H, J ¼ 5.7 Hz), 2.38 (ss, 9H); IRnmax (cm1): 1644 (C]O str), 1559 (N]CH), 1241 (AreOeC, CeOeC asym. str),
3353 (NeH str), 1647 (C]O str), 1562(N]CH), 1276 (AreOeC, 1164 (NeN); Elemental analysis calcd (%) for C19H23N3O4: C 66.84, H
CeOeC asym. str), 1176 (NeN); Elemental analysis calcd (%) for 6.79, N 12.31; found: C 66.84, H 6.79, N 12.31; ES-MS: m/z 342.66
C19H23N3O2: C 70.13, H 7.12, N 12.91; found: C 70.15, H 7.16, N 12.89; (Mþ1).
ES-MS: m/z 326.41 (Mþ1). 4-(2-(dimethylamino)ethoxy)-N-(pyridine-2-ylmethylene)
N0 -(3,4-dimethoxybenzylidene)-4-(2-(dimethylamino) benzohydrazide (H23): solid, Yield: 52%, m.pt.102 C; 1H NMR
ethoxy)benzohydrazide (H16): solid, Yield: 64%, m.pt.125 C; 1H (CDCl3, 300 MHz, dppm): 9.38(bs, 1H), 8.61 (s, 1H), 8.23e8.05 (m,
NMR (CDCl3, 300 MHz, dppm): 9.43 (bs, 1H), 8.22 (s, 1H), 7.85 (bs, 2H), 8.00e7.90 (m, 2H), 7.76 (t, 1H, J ¼ 7.8 Hz), 7.31e7.28 (m, 1H),
2H), 7.48 (bs, 1H), 7.10 (d, 1H, J ¼ 8.4 Hz), 6.97 (d, 2H, J ¼ 8.4 Hz), 7.013 (d, 2H, J ¼ 8.7 Hz), 4.152 (t, 2H, J ¼ 5.4 Hz), 2.781 (t, 2H,
6.85 (d, 1H, J ¼ 8.1 Hz), 4.12 (t, 2H, J ¼ 5.4 Hz), 3.91 (ss, 6H), 2.77 (t, J ¼ 5.7 Hz), 2.35 (s, 6H); IRnmax (cm1): 3379 (NeH str), 1649 (C]O
2H, J ¼ 5.4 Hz), 2.34 (s, 6H); IRnmax (cm1): 3354 (NeH str), 1641 str), 1560 (N]CH), 1272 (AreOeC, CeOeC asym. str), 1179 (NeN);
(C]O str), 1540 (N]CH), 1240 (AreOeC, CeOeC asym. str), 1165 Elemental analysis calcd (%) for C17H20N4O2: C 65.37, H 6.45, N
(NeN); Elemental analysis calcd (%) for C20H25N3O4: C 64.67, H 6.78, 17.94; found: C 65.36, H 6.46, N 17.96; ES-MS: m/z 313.23 (Mþ1).
N 11.31; found: C 64.65, H 6.79, N 11.32; ES-MS: m/z 372.10 (Mþ1). 4-(2-(dimethylamino)ethoxy)-N-((3-methylthiophene-2-yl)
N0 -(benzo[1,3]dioxol-5-ylmehylene)-4-(2-(dimethylamino) methylenen) benzohydrazide (H24): solid, Yield: 61%, m.pt.141 C;
ethoxy)benzohydrazide (H17): solid, Yield: 69%, m.pt.149 C; 1H 1
H NMR (CDCl3, 300 MHz, dppm): 9.31 (bs, 1H), 9.20 (s, 1H), 7.83 (bs,
NMR (CDCl3, 300 MHz, dppm): 9.21(bs, 1H), 8.20 (s, 1H), 7.84 (bs, 1H), 7.27 (bs, 1H), 6.97 (d, 2H, J ¼ 8.7 Hz), 6.84 (d, 2H, J ¼ 5.1 Hz),
2H), 7.39 (bs, 1H), 7.06 (d, 1H, J ¼ 8.1 Hz), 6.98 (d, 2H, J ¼ 8.7 Hz), 4.13 (t, 2H, J ¼ 5.4 Hz), 2.77 (t, 2H, J ¼ 5.7 Hz), 2.34 (s, 9H); IRnmax
6.81 (d 1H, J ¼ 8.1 Hz), 6.00 (ss, 2H), 4.13 (t, 2H, J ¼ 5.4 Hz), 2.77 (t, (cm1): 3324 (NeH str), 1642 (C]O str), 1558 (N]CH), 1244
2H, J ¼ 5.7 Hz), 2.34 (s, 6H); IRnmax (cm1): 3299 (NeH str), 1641 (AreOeC, CeOeC asym. str), 1177 (NeN); Elemental analysis calcd
(C]O str), 1561 (N]CH), 1250 (AreOeC, CeOeC asym. str), 1171 (%) for C17H21N3O2S: C 61.61, H 6.39, N 12.68; found: C 61.61, H 6.39,
(NeN); Elemental analysis calcd (%) for C19H21N3O4: C 64.21, H 5.96, N 12.65; ES-MS: m/z 332.49 (Mþ1).
N 11.82; found: C 64.20, H 5.97, N 11.80; ES-MS: m/z 356.10 (Mþ1). Ferrocenyl-4-(2-(dimethylamino)ethoxy)benzohydrazide
N0 -(3,5-dibromo-4-hydroxybenzylidene)-4-(2-(dimethyla- (H25): solid, Yield: 60%, m.pt.192 C; 1H NMR (CDCl3, 300 MHz,
mino)ethoxy) benzohydrazide (H18): solid, Yield: 56%, dppm): 8.84 (bs, 1H), 8.22 (bs, 1H), 7.81 (bs, 2H), 6.99 (d, 2H,
m.pt.152 C; 1H NMR (CDCl3, 300 MHz, dppm): 11.63 (s, 1H), 8.19 (s, J ¼ 8.7 Hz),4.69 (s, 2H), 4.41 (s, 2H), 4.221 (s, 4H), 4.15 (t, 2H,
1H), 7.89e7.87 (m, 2H), 7.77e7.61 (m, 2H), 7.06e7.04 (m, 2H), 4.27 J ¼ 5.4 Hz), 2.78 (t, 2H, J ¼ 5.4 Hz), 2.36 (s, 6H); IRnmax (cm1): 3397
(t, 2H, J ¼ 5.4 Hz), 2.84 (t, 2H, J ¼ 5.7 Hz), 2.43 (s, 6H); IRnmax (NeH str), 1641 (C]O str), 1558 (N]CH), 1256 (AreOeC, CeOeC
(cm1): 3356 (NeH str), 1612 (C]O str), 1571(N]CH), 1252 asym. str), 1188 (NeN); Elemental analysis calcd (%) for
(AreOeC, CeOeC asym. str), 1175 (NeN); Elemental analysis calcd C22H25FeN3O2: C 62.96, H 5.39, N 10.01; found: C 62.95, H 5.39, N
(%) for C18H19Br2N3O3: C 44.56, H 3.95, N 8.66; found: C 44.56, H 10.04; ES-MS: m/z 421.50 (Mþ1).
3.95, N 8.66; ES-MS: m/z 486.05 (Mþ1). N0 -(3,4-dichlorobenzylidene)-4-(2-(dimethylamino)ethoxy)
N0 -(2,4,6-trimethylbenzylidene)-4-(2-(dimethylamino) benzohydrazide (H26): solid, Yield: 69%, m.pt. 96 C; 1H NMR
ethoxy)benzohydrazide (H19): Solid, Yield: 60%, m.pt.196 C; 1H (CDCl3, 300 MHz, dppm): 8.34 (bs, 1H), 7.84 (s, 1H), 7.81 (bs, 3H),
NMR (CDCl3, 300 MHz, dppm): 11.57 (s, 1H), 8.73 (s, 1H), 7.90e7.89 7.54 (bs, 1H), 7.47 (d, 1H, J ¼ 8.1 Hz), 6.98 (d, 2H, J ¼ 8.4 Hz), 4.14 (t,
(m, 2H), 7.07e7.05 (m, 2H), 6.91 (s, 2H), 4.12 (t, 2H, J ¼ 5.4 Hz), 2.65 2H, J ¼ 5.4 Hz), 2.74 (t, 2H, J ¼ 5.7 Hz), 2.35 (s, 6H); IRnmax (cm1):
(t, 2H, J ¼ 5.7 Hz), 2.42 (s, 6H), 2.24 (s, 3H), 2.21 (s, 6H); IRnmax 3260 (NeH str), 1649 (C]O str), 1554 (N]CH), 1247 (AreOeC,
(cm1): 3227 (NeH str), 1643 (C]O str), 1542 (N]CH), 1255 CeOeC asym. str), 1173 (NeN); Elemental analysis calcd (%) for
(AreOeC, CeOeC asym. str), 1179 (NeN); Elemental analysis calcd C18H19Cl2N3O2: C 56.85, H 5.04, N 11.05; found: C 56.85, H 5.02, N
(%) for C21H27N3O2: C 71.36, H 7.70, N 11.89; found: C 71.35, H 7.73, N 11.02; ES-MS: m/z 381.28 (Mþ1).
11.86; ES-MS: m/z 354.16 (Mþ1). 2-methoxy-4-[2-{4-(dimethylamino)ethoxybenzoyl)hydrazi-
N0 -(3-methoxy-4-propoxybenzylidene)-4-(2-(dimethyla- nylidene}methyl]phenyl acetate (H27): solid, Yield: 56%, m.pt.
mino)ethoxy)benzohydrazide (H20): Solid, Yield: 55%, 83 C; 1H NMR (CDCl3, 300 MHz, dppm): 9.66 (bs, 1H), 8.10 (bs, 1H),
m.pt.126 C; 1H NMR (CDCl3, 300 MHz, dppm): 9.24 (bs, 1H), 8.20 (s, 7.87(bs, 2H), 7.47 (s, 1H), 6.99e6.93 (m, 4H), 4.13 (t, 2H, J ¼ 6.0 Hz),
1H), 7.85 (bs, 2H), 7.48 (bs, 1H), 7.09 (d, 1H, J ¼ 6.3 Hz), 6.98 (d, 2H, 3.82 (s, 3H), 2.78e2.75 (m, 2H), 2.35 (s, 6H), 2.31 (s, 3H); IRnmax
J ¼ 9.0 Hz), 6.87 (d, 1H, J ¼ 8.4 Hz), 4.63 (m, 1H), 4.13 (t, 2H, (cm1): 3329 (NeH str), 1646 (C]O str), 1554 (N]CH), 1284
452 A. Inam et al. / European Journal of Medicinal Chemistry 124 (2016) 445e455
(AreOeC, CeOeC asym. str), 1171 (NeN); Elemental analysis calcd Table 1
(%) for C21H25N3O5: C 63.14, H 6.31, N 10.52; found: C 63.14, H 6.33, Crystal Data and Structure Refinement for the compound 4-(2-(dimethylamino)
ethoxy)-N-(pyridine-2-ylmethylene)benzohydrazide (H23$H2O).
N 10.55; ES-MS: m/z 400.07 (Mþ1).
N0 -(3,4-dihydroxybenzylidene)-4-(2-(dimethylamino) Formula C17 H22 N4 O3 (H23$H2O)
ethoxy)benzohydrazide (H28): solid, Yield: 64%, m.pt. 195 C; 1H Formula weight 330.39
T, K 100 (2)
NMR (CDCl3, 300 MHz, dppm): 11.47 (s, 1H), 9.73e9.29 (bs, 2H, OH), Wavelength, Å 0.71073
8.24 (s, 1H), 7.78e7.66 (m, 2H), 7.30 (s, 1H), 7.05e7.03 (m, 2H), Crystal system Orthorhombic
6.78e6.76 (m, 1H), 6.60e6.56 (m, 1H), 4.13 (t, 2H, J ¼ 5.4 Hz), 2.63 (t, Space group Pbca
2H, J ¼ 5.7 Hz), 2.22 (s, 6H) IRnmax (cm1): 3314 (NeH str), 1645 a/Å 8.4474 (10)
b/Å 11.8401 (16)
(C]O str), 1505 (N]CH), 1248 (AreOeC, CeOeC asym. str), 1176
c/Å 34.235 (4)
(NeN); Elemental analysis calcd (%) for C18H21N3O4: C 62.96, H 6.16, V/Å3 3424.2 (7)
N 12.24; found: C 62.93, H 6.17, N 12.25; ES-MS: m/z 344.64 (Mþ1). Z 8
N0 -(2,5-dimethoxybenzylidene)-4-(2-(dimethylamino) F000 1408
ethoxy)benzohydrazide (H29): solid, Yield: 59%, m.pt. 124 C; 1H Dcalc/g cm3 1.282
m/mm1 0.090
NMR (CDCl3, 300 MHz, dppm): 9.22 (bs, 1H), 8.57 (bs, 1H), 7.95 (bs, q/( ) 1.19e26.83
2H), 7.64 (bs, 1H), 6.99e6.93 (m, 3H), 6.86e6.83 (m, 1H), 4.14- (t, Rint 0.1243
2H, J ¼ 4.8 Hz), 3.82 (ss, 6H), 2.78 (t, 2H, J ¼ 4.5 Hz), 2.36 (s, 6H); Crystal size/mm3 0.27 0.25 0.17
IRnmax (cm1): 3457 (eOH), 3336 (NeH str), 1642 (C]O str), 1563 Goodness-of-fit on F2 1.036
R1 [I > 2s(I)]a 0.0542
(N]CH), 1246 (AreOeC, CeOeC asym. str), 1170 (NeN); Elemental
wR2 (all data)b 0.1582
analysis calcd (%) for C20H25N3O4: C 64.67, H 6.78, N 11.31; found: C Largest differences peak and hole (eÅ3) 0.337 and 0.290
64.67, H 6.78, N 11.31; ES-MS: m/z 372.18 (Mþ1). a
R1 ¼ SrrForrFcrr/SrFor.
N0 -(4-(diethoxymethyl)benzylidene)-4-(2-(dimethylamino) b
wR2 ¼ {S[w (rrFor2rFcr2r)2]r/S[w (F2o)2]}1/2.
ethoxy)benzohydrazide(H30): solid, Yield: 69%, m.pt.75 C; 1H
NMR (CDCl3, 300 MHz, dppm): 9.05 (bs, 1H), 7.86 (s, 1H), 7.72e7.52
(m, 4H), 7.50 (d, 2H, J ¼ 8.1 Hz), 7.00 (d, 2H, J ¼ 8.7 Hz), 5.52 (s, 1H), Table 2
4.15 (t, 2H, J ¼ 5.7 Hz), 3.62e3.49 (m, 4H), 2.79 (t, 2H, J ¼ 5.7 Hz), Bond lengths [Å] and angles [ ] for the compound 4-(2-
(dimethylamino)ethoxy)-N-(pyridine-2-ylmethylene)benzo-
2.36 (s, 6H), 1.26 (m, 6H); IRnmax (cm1): 3389 (NeH str), 1649 (C]
hydrazide (H23·H2O).
O str), 1558 (N]CH), 1259 (AreOeC, CeOeC asym. str), 1176
(NeN); Elemental analysis calcd (%) for C23H31N3O4: C 66.81, H 7.56, Bond lengths H23$H2O
N 10.16; found C 66.85, H 7.55, N 10.14; ES-MS: m/z 414.11 (Mþ1). O(1)eC(5) 1.357 (2)
O(1)eC(4) 1.432 (3)
N(1)eC(1) 1.457 (3)
4.3. Pharmacological evaluation
N(1)eC(2) 1.462 (3)
N(1)eC(3) 1.463 (3)
4.3.1. Antiamoebic activity O(2)eC(11) 1.224 (2)
All the compounds were screened in vitro for antiamoebic ac- N(2)eC(11) 1.364 (3)
tivity against HM1:IMSS strain of E. histolytica by micro dilution N(2)eN(3) 1.379 (2)
N(3)eC(12) 1.281 (3)
method [25]. E. histolytica trophozoites were cultured in wells of
96-well microtiter plate by using Diamond TYIS-33 growth me- Angles H23·H2O
dium [26]. The test compounds (1 mg) were dissolved in ethanol C(5)eO(1)eC(4) 117.90 (15)
(50 mL, level at which no inhibition of amoeba occurs) [27]. The C(3)eN(1)eC(1) 108.62 (17)
stock solutions of the compounds were prepared freshly before use C(3)eN(1)eC(2) 111.50 (17)
C(1)eN(1)eC(2) 109.78 (17)
at a concentration of 1 mg/mL. Each plate included metronidazole C(11)eN(2)eN(3) 117.98 (17)
as standard amoebicidal drug, negative control (culture medium C(12)eN(3)eN(2) 115.31 (18)
plus amoeba) and a blank (culture medium only). All the com- N(1)eC(3)eC(4) 115.99 (19)
pounds were used in triplicate concentrations. The parasite sus- O(1)eC(4)eC(3) 108.45 (18)
pension was adjusted to 105 cells per mL by adding fresh medium
and then distributed to a 96-wells microtiter plate (Corning).
Compounds were added to the respective wells and the plate was Table 3
sealed, gassed for 10 min with nitrogen and then incubated at 37 C Hydrogen bonds in the compound 4-(2-(dimethylamino)ethoxy)-N-(pyridine-2-
ylmethylene)benzohydrazide (H23·H2O).
for 72 h. After incubation, the amoeba growth was checked under
microscope. The culture medium was removed by inverting the DeH…A d(DeH) d(H … A) d(D … A) <(DHA)
plate and shaking gently. Plate was then immediately washed with N(2)eH(2N)…O(1W)#1 0.94 (2) 1.91 (2) 2.828 (2) 162 (2)
sodium chloride (0.9%) at 37 C, dried at room temperature and the O(1W)eH(1WB)…O(2)#2 0.92 (4) 2.00 (3) 2.849 (2) 152 (3)
cells fixed with methanol and once dried, stained with aqueous O(1W)eH(1WB)…N(3)#2 0.92 (4) 2.46 (4) 3.171 (2) 134 (3)
O(1W)eH(1WA)…N(1)#3 1.04 (4) 1.74 (4) 2.775 (2) 174 (3)
eosin (0.5%) for 15 min. The optical density of the resulting solution
in each well was determined at 490 nm in a micro plate reader. The Symmetry transformations used to generate equivalent atoms:
#1 xþ1/2,yþ1/2,z #2 x,y1,z #3 x1/2, yþ1/2, z.
percentage inhibition of parasite growth was calculated from the
optical densities of the control and test compounds. A non-linear
regression analysis was used to determine the best fitting line
from which the IC50 values were estimated. Each compound was
all the compounds were between 5 and 640 mm. Compounds were
tested at least in two independent experiments.
reconstituted initially in ethanol, but further dilutions were made
in culture medium. A549 cells were cultured in 96 well plates at a
4.3.2. Cytotoxicity
density of 8000 cells/well in Ham's F-12 medium (Hi Media) sup-
Cytotoxic effects of the series (H1eH30) were determined in
plemented with 10% FBS. Once the cells attached to 80e90%
A549 cells (lung epithelial cells). The concentration range used for
A. Inam et al. / European Journal of Medicinal Chemistry 124 (2016) 445e455 453
Table 4
In vitro antiamoebic activity of (H1eH30) against HM1:IMSS strain of E. histolytica and cytotoxicity profile of active compounds.
Compound R Antiamoebic activity IC50 ± S.D. (mM) Cytotoxicity IC50 ± S.D. (mM)
Table 4 (continued )
Compound R Antiamoebic activity IC50 ± S.D. (mM) Cytotoxicity IC50 ± S.D. (mM)
Table 4 (continued )
Compound R Antiamoebic activity IC50 ± S.D. (mM) Cytotoxicity IC50 ± S.D. (mM)
confluence, medium was aspirated off and replaced with medium Commission, New-Delhi, India for BSR (SRF) Fellowship.
containing desired concentration of each drug and 1%FBS. Each
drug concentration was taken in triplicates. Cells treated only with
medium were used as negative controls. Plates were incubated at Appendix A. Supplementary data
37 C with 5% CO2. At 48 h post drug treatment, cell viability was
measured by MTT assay. In brief, cells in each well were incubated Supplementary data related to this article can be found at http://
with 20 ml of MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H- dx.doi.org/10.1016/j.ejmech.2016.08.022.
tetrazolium bromide) reagent at a concentration of 5 mg/ml in 5%
methanol for 4 h. Then, MTT reagent was removed and the resulting
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One of the authors (AI) is thankful to University Grants [30] SHELX, G.M. Sheldrick, Acta Crystallogr. Sect. A 64 (2008) 112.