The Structure and Pharmacological Functions of Coumarins and Their Derivatives

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Current Medicinal Chemistry, 2009, 16, 4236-4260
The Structure and Pharmacological Functions of Coumarins and Their Derivatives

L. Wu1, X. Wang1, W. Xu2, F. Farzaneh3 and R. Xu*,1
Engineering Research Center of Molecular Medicine, Ministry of Education & Institute of Molecular Medicine, Huaqiao University, Fujian, 362021, China
2 3 1
Faculty of Science, University of New South Wales, Sydney, NSW 2052, Australia Department of Molecular Medicine, Kings College, London, UK
Abstract: Coumarins are of many different structures. They constitute an important class of pharmacological agents possessing a range of different physiological activities including anti-cancer, anti-oxidant, anti-inflammation, anti-HIV, anticoagulant, anti-bacterial, analgesic and comparative immune-modulation. Recently, coumarins have attracted intense research interest. Of great interest is the possibility that this class of molecules could be a source of drugs for the therapy of several diseases. These include recent insights into inhibiting cell proliferation by interfering with mitotic spindle microtubule function, decrease Matrix Metalloproteinase (MMP) activity, block the cell cycle in the S or G2/M phases to interfere with processes of cell division, suppress O2- generation in leukocytes, inhibit different protein kinases, modulate the signalings, induce carcinogen-detoxifying enzymes glutathione Stransferases (GSTs) and/or NAD(P)H quinine oxidoreductase (NQO1), suppress the phosphorylation of Akt/PKB as a mechanism inhibiting inflammation, progress in structure modification to increase in anti-fungal action, to broaden against bacteria spectrum, to enhance inhibiting activities of nitric oxide synthase (NOS) and cyclooxygenase (COX), to strengthen anti-oxidant activity and to exhibit a much higher cytotoxicity against human umbilical vein endothelial cell (HUVEC). With fewer non-hemorrhagic side effects than the indanedione derivatives, they can be applied as an oral anticoagulant commonly for preventing venous thromboembolism following orthopedic surgery, recurrent myocardial infarction and the treatment of systemic embolism in atrial fibrillation, together with the significant advances in the basis of drug action. It is therefore useful to build up some correlations with the data available in order to better explore the molecular and cellular mechanism of coumarin action in the treatment of diseases. This review will focus on recent advances in molecular and cellular mechanisms of coumarin action involved with the relationship between structure and activity.
Keywords: Coumarins, structure-activity relationship, anti-cancer, anti-oxidant, anti-coagulant, anti-inflammation, antimicroorganism, metabolism. Coumarins (also known as 1,2-benzopyrone or, less commonly, as o-hydroxycinnamic acid-8-lactone), constitute an important and large class of oxygen heterocycles, often found as plant secondary metabolites in the plant kingdom. Many of the coumarins are oxygenated at position C-7, which become 7-hydroxycoumarin, commonly known as umbelliferone, often regarded as the biogenetic precursor of more complex coumarins. The investigation of coumarin compounds has revealed a wide spectrum of medicinal plant extracts that are in use as early as 1000 A.D., contain a high content of coumarins. To date, at least 1300 such compounds have been identified (reviewed by Kostova, 2005) [1]. Inspection of the chemical structures of these compounds shows that some of the coumarins reveal that substitutions can occur at many sites. There are many possible permutations offered by substitution and conjugation, and this readily explains why so many coumarins are naturally occurring substances [2]. In addition there are a large number of other more complex compounds with the biological and the pharmacological properties of coumarin that have structures that are based on the coumarin nucleus [3-15]. The coumarins can be roughly categorized as follows: (1) simple coumarins, (2) furanocoumarins (can be further grouped into linear and angular types), (3) pyranocoumarins, (4) dicoumarins, and (5) others like phenylcoumarins. Because of their diverse pharmacological properties, coumarins have attracted increasing research interest in recent years. However, the details of relationship between the structure and activity of coumarins remain obscure. ANABOLIC AND CATABOLIC COUMARINS PATHWAYS OF
*Address correspondence to this author at the Engineering Research Center of Molecular Medicine, Ministry of Education, Huaqiao University Main Campus, Quanzhou, 362021, China; Tel: 0086-595-22691632; Fax: 0086-595-22690952; E-mail: ruianxu@hqu.edu.cn 0929-8673/09 $55.00+.00
Coumarins naturally occur in many plants, primarily in angiosperm, including Umbelliferae, Rutaceae, Leguminosae, Compositae, and Thymelaeceae. For instance, linear furanocoumarins are found primarily in the Umbelliferae, Moraceae, Rutaceae and Leguminosae families. Informations about the biosythesis of coumarins have been increasing steadily in recent years [16-21]. Coumarin is one element of phenylpropanoids. Like other pyenylpropanoids, the anabolic metabolism of simple coumarins, furanocoumarins and pyranocoumarins derive from the phenylproanoid pathway, whereas phenylalanine is formed by the shikimate pathway [16]. The most common phenylcoumarins originate from isoflavone metabolism. Coumarins can also be formed as products of the metabolism of phenylalanine via a cinnamic acid, p-coumaric acid as shown in Fig. (2) [16, 22]. It is thought that in the biosynthesis of coumarins, hydroxylation
2009 Bentham Science Publishers Ltd.
The Structure and Pharmacological Functions of Coumarins
Current Medicinal Chemistry, 2009 Vol. 16, No. 32
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8 7 6 5 1
O 3 4
O O O O O O 4 O 5
Fig. (1). Chemical Structure of Coumarins: 1) simple coumarins, 2 ) furanocoumarins (can be further grouped into linear and angular types), 3) pyranocoumarins, 4) dicoumarins, 5 ) phenylcoumarin.
OH O NH2 OH coumarin OH O O O OO
cinnamate
coumaric acid
OH O OO SCoA O O OH O O
OH OH HO OH
HO
COO-
HO
COO-
OH cinnamate
HO OH coumaroyl CoA OH coumaroyl OH caffeoylquinate SCoA
O-
OH
HO
OH
HO
OH
HO
OH
HO
OH O O OH umbelliferone O O esculetin HO R 2'-hydrox caffeic acid R E-caffeate SCoA feruloyl-CoA HO O HO OH HO O O
O O O OH
OH R O HO O SCoA 6'-hydroxyferuloyl-CoA O MeO O spirodienone
R Z-caffeate
osthenol
OH O
O Psoralen
O O Angelicin
O scopoletin
Fig. (2). The biosynthetic pathways of coumarins in plants [16, 22].
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Wu et al.
takes place at the 2-position of the ring of cinnamates, with cis-trans geometrical isomaerization of the side chain and lactonization [21]. As shown in Fig. (2), the hydroxylations in ortho- and para- positions appear different pathway, which may indicate that they would be catalyzed by different enzymes. Cinnamate 4-hydroxylase, a cytochrome P450 (CYP) monooxygenase from the CYP73A family, catalyzes the conversion of cinnamic acid to 4-coumaric acid, with the formation of umbelliferone [23]. Classically, umbelliferone rather than coumarin has been considered the parent compound of furanocoumarins. The biosynthetic route of scopoletin has been reported in Arabidopsis thaliana recently [19, 24]. It is reported that scopoletin is biosynthesized from ferulic acid rather than via the umbelliferone-esculetin- scopoletin pathway [25]. Ortho-hydroxylation is a key step in coumarin biosynthesis as a branch point from lignin bisosynthesis; Kai (2008) reported that Fe ()-and 2-oxoglutarate-dependent dioxygenase (2OGD) catalyzed ortho-hydroxylation before the lactone ring formation of scopoletin by T-DNA insertion mutants of F6H1. The substrate of 2OGD is feruloyl CoA thioester, not ferulic acid [19]. This is inconsistent with the results obtained from Bayoumi et al (2008), who investigated the biosynthetic pathways of scopoletin by using stable isotope labeling to study post-harvest physiological deterioration in Cassava roots [22]. Bayoumi et al also demonstrated that the major pathway was through o-hydroxylation but not via a proposed spirolactone-dienone intermediate by feeding C18O2-carboxylate- labelled cinnamic and ferulic acids [22]. The biosynthesis of linear and angular furanocoumarins is still poorly understood at the molecular level, with only psoralen synthase (CYP71AJ1) identified from Ammi majus. Three new members of the CYP71AJ subfamily (CYP71AJ2-4) were cloned by Larbat et al (2009) who
O O
found that psoralen synthase (CYP71AJ3) and angelicin synthase (CYP71AJ4) showed 70% identity in sequence comparison [18]. There are many pathways for involved in coumarin to decomposition (Fig. 3). Coumarin may be metabolized by hydroxylation at all six possible positions (i.e. carbon atoms 3, 4, 5, 6, 7 and 8) to yield 3-, 4-, 5-,6-, 7- and 8-hydroxycoumarins (3-, 4-, 5-, 6-, 7- and 8-HCs) and by opening of the lactone ring to yield various individual products including o-hydroxyphenylacetaldehyde (o-HPA) (a major metabolite of coumarin in rat and mouse liver microsomes), ohydroxyphenylethanol (o-HPE), o-hydroxyphenylacetic acid (o-HPAA) and o-hydroxyphenyllactic acid (o-HPLA). Additional metabolites of coumarin include 6,7-dihydroxycoumarin(6,7-diHC), o-coumaric acid (o-CA), o-hydroxyphenyl propionic acid (o-HPPA) and dihydrocoumarin (DHC) [26, 27]. Two important pathways for coumarin metabolism are 7hydroxylation and metabolism of the lactone ring which involves ring opening and cleavage of the carbon 2 atom to yield carbon dioxide. The first step in coumarin metabolism by the latter pathway is the formation of a coumarin 3, 4epoxide intermediate. However, under aqueous conditions, coumarin 3, 4-epoxide degrades rapidly, with the loss of carbon dioxide to form o-HPA, which can be further metabolized to o-HPE and o-HPAA [27]. But o-HPA formation did not accurately reflect the rate of coumairn 3.4-epoxidation. Thus, to quantitatively measure it, the glutathione (GSH) conjugate method was included, the ratio of coumarin 7hydroxylation to 3,4-epoxidation by CYP2A13 was 1.0 to 1.3 compared with a ratio of 1.0 to 0.7 [32]. CYP enzymes are ubiquitous catalysts of oxidative metabolism in biological systems. Enzymes from the CYP1,
CH2CHOHCOOH
O SG OH 4-HDHC-GSH conjugate ?
? OH
OH
o-HPLA ?
? CO2
GSH
CH2CHO OH o-HPA CH2CH2OH o-HPAA
CH2COOH OH
O coumarin-3'4-epoxide
CO2
O OH o-HPE
DHC
Fig. (3). Representative pathways of coumarin metabolism [26, 27].
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CYP2 and CYP3 families are generally regarded as representing the major catalysts for xenobiotic and drug metabolism in man. The 7-hydroxylation of coumarin by CYP2A6 is one of the most specific probe activities displayed by any of the CYP enzymes, and no other human CYP isoform has the capacity to catalyze 7-hydroxylation to any a significant degree [29, 30]. Cytochrome P450-mediated coumarin 7hydroxylation in human, specifically the role of CYP2A6, has been extensively studied [26, 27, 31]. And recently, more information has become available in the human P450s involved in coumarin 7-hydroxylation and 3, 4-epoxidation. Computer modeling and docking studies show that CYP2A6 structure has a compact, hydrophobic active site with one hydrogen bond donor, Asn297, that orients coumarin for regioselective oxidation. The potential for either the oxygen of the ether side chain or the carbonyl to hydrogen bond with Asn297 may provide less discrimination for substrate orientation. For example, the oxidation of 7-ethoxy and 7methoxy-coumarin by CYP2A6 is less regiospecific and occurs on opposite ends of the substrate, resulting in Odealkylation or 3-hydroxylation [32]. Mutation of residue Asn297 had been shown to influence substrate binding and metabolism. An N297S mutant was determined to have 4fold decreased catalytic efficiency for coumarin 7hydroxylation [33], and nearly 30-fold decreased binding affinity for coumarin [28]. CYP2A13 is also an efficient catalyst for coumarin metabolism [34]. Unlike CYP2A6, CYP2A13 catalyzes both the 7-hydroxylation and 3, 4epoxidation of coumarin with similar efficiency [35]. The active sites of CYP2A6 and CYP2A13 share several similar characteristics, which include a cluster of phenylalanine residues that line the roof of the active site and the presence of a single polar residue, Asn297 [28]. In a kinetic study, Zhuo et al reported that cDNA-expressed CYP1A1, CYP1A2, CYP2B6, CYP2E1 and CYP3A4 enzymes could catalyze the metabolism of coumarin to the 3,4-epoxidation pathway metabolite o-HPA, whereas cDNA-expressed CYP2A6 only formed 7-hydroxycoumarin (7-HC) [31]. The metabolism of coumarin to o-HPA by cDNA-expressed CYP1A1, CYP1A2 and CYP2E1 was also reported by Born et al who observed that coumarin could be metabolised to 3hydroxycoumarin by CYP3A4 and to a lesser extent by other P450s [36]. Modeling by homology with the CYP2C5 crystallographic template, CYP1, CYP2 and CYP3 famlies in human P450 were proved in agreement with the known metabolism of coumarin, and with information from sitedirected mutagenesis studies [37]. Little information about metabolism of furanocoumarins is available. Xenobioticmetabolizing P450 families in insects were CYP6, like CYP2 and CYP3 in mammals. In Papilio glaucus, CYP6B1 exhibits very high activity toward the methoxylated linear furanocoumarins, lower activity toward unsubstitued and other linear furanocoumarins, and much lower activity toward the angular furanocoumarins. Modeling programs studies showed that an aromatic network that involves residues Phe-116, His-117, Phe-484, and Phe-371 was critical for substrate binding affinity to the CYP6B1 active site [38]. STRUCTURE AND BIOLOGICAL FUNCTIONS Coumarins, a plant secondary metabolite, some of the biological activities of them are believed to relate to their
ability to act as phytoalexins. Phytoalexins accumulate in leaves and fruits to inhibit the growth and spread of bacteria or fungi, and act as repellents orantimetabolites against herbivorous insects like locust. These are naturally synthesized by plant to function as protective chemicals against traumatic injury, microorganism invasion, and insect damage. Since substitutions can occur at any of the six available sites of their basic coumarin nucleus, their structural diversity leads to multiple biological properties. The diverse biological functions of coumarins include anti-leukemia [3-4], antiinflammation [5-6], anti-platelet aggregation [7-8], anti-cancer [9-10], anti-convulsant [11], comparative immunomodulatory [12], and analgesic [13-15] properties. ANTI-CANCER AND APOPTOSIS The antineoplastic action of coumarin derivatives, all of which act at different stages of cancer formation, has been summarized in Table 1, including the different cytostatic properties and cytotoxic activity observed. The anti-tumor activity of coumarin and 7-HC against human tumor cell lines was first noted by Weber et al [39] and its use in cancer chemotherapy was first developed the application of Warfarin sodium (6) on V2 cancer cell, granulocytes, lymphocytes and macrophages in different animal models [40]. Clinical trials demonstrating activity in many different cancers, including prostate cancer, malignant melanoma and metastatic renal cell carcinoma have also been reported. Encouraginglly, Irish melanoma group first demonstrated that a daily ingestion of 50 mg of coumarin or warfarin prevented the early recurrence of high risk malignant melanoma without toxic effects [41]. Many different hypotheses have been postulated as to the mode of action for different coumarin derivatives including 4-hydroxycoumarin (4-HC) and 7-HC (Table 2). These include the observations that 4-HC decreases tyrosine phosphorylation of several proteins in melanoma cells line B16F10 [42], whereas 7-HC inhibits myosin light chain kinase [43] and disrupts the formation of the mitotic spindle microtubules in Allium cepa cells, leading to the random distribution of the chromosomes at metaphase. This is a form of cytotoxicity common to mitotic spindle poisons that inhibit mitosis through modifying microtubule dynamics [44], suggesting that different coumarin compounds may inhibit cell proliferation by interfering with mitotic spindle microtubule function [45]. To examine the effect of coumarin and its derivatives, including 2-hydroxycoumarin, 4-hydroxycoumairn, 7-HC, daphnetin (7,8-dihydroxycoumarin), and esculetin (7), on the activities of protein kinases, including epidermal growth factor (EGF) receptor tyrosine kinase, protein kinase A (PKA) and protein kinase C (PKC), Yang et al suggested that hydroxylation not at C2, C4, C6, and C7 but at C8 might be important for acting as a protein kinase inhibitor. The inhibitory kinetics by daphnetin was determined, and this compound inhibited tyrosine-specific protein kinase, EGF receptor (IC50=7.67M) and PKA (IC50=9.33M) and PKC (IC50=25.01M) in vitro [46]. Most of the antineoplastic drugs presently used in cancer therapy block the cell cycle in the S or G2/M phases. Never-
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Table 1.
Coumarins that have Been Used as Anti-Cancer Medicines so Far

Cell lines/cancers B16-F10 HepG2 cells References [Velasco-Velazquez et al., 42] [Yang et al., 46] [Finn et al., 60] [Finn et al., 62] [Win et al., 9]
Compounds 4-HC
6-nitro-7-hydroxycoumarin derivatives
SK-MEL-31cells A-498
6-substituted 2-oxo-2H-1-benzopyran-3carboxylic acid derivatives 7-HC
HT 1080 fibrosarcoma cells
Gastric carcinoma cells Caco-2 Allium cepa cells HepG2 cells NSCLC SK-MEL-31cells Lymphoblastic cells
[Weber et al., 39] [Weber et al.,39] [Podbielkowska et al., 44] [Yang et al., 46], [Weber et al., 39] [Lopez-Gonzalez et al., 47] [Finn et al., 60] [Weber et al., 39] [Seokjoon et al., 64] [Kurakami et al., 67] [Tanaka et al., 68] [Kawabata et al., 69] [Kohno et al., 71], [Kawabata et al., 72]
7-diethylaminocoumarins Auraptene
HUVEC HL-60 cells Oral and liver cancers Esophageal cancer Colon cancers
B16BL6 melanoma cell M4Beu Coumarin Compound 14 Daphnetin Malignant melanoma MCF-7 HepG2 cells A-498 Decursin Human K562 erythroleukemia U937 cells DHMC U937 cells HL-60 cells A549 (NSCLC) Esculetin HepG2 cells U937 cells HL-60 cells MCF-7 cells Imperatorin, isopimpinellin Scopoletin Silver coumarins Mammary carcinogenesis HL-60 cells HepG2 cells
[Tanaka et al., 76] [Barthomeuf et al., 77] [Thornes et al., 41] [Jacquot et al., 66] [Yang et al., 46] [Finn et al., 48] [Kim et al., 78], [Kim et al., 79] [Kim et al., 78] [Riveiro et al., 54] [Riveiro et al., 54] [Goel et al., 55], [Goel et al., 56] [Yang et al., 46] [Lee et al., 49], [Park et al., 50] [Chu et al., 3], [Wang et al., 58] [Kolodziej et al., 57] [Prince et al., 81] [Kim et al., 51], [Kim et al., 52] [Wang et al., 58], [Bhumika et al., 59]
Warfarin sodium
V2 cancer cells Malignant melanoma
[Thornes et al., 40] [Thornes et al., 41]
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Table 2.
Biological Pathways of Representative Coumarins

Biological Responses and Pathways Decreasing tyrosine phosphorylation Activation of p38, SAPK, and MAPK; blocking at sub-G1 phase Inhibiting myosin light chain kinase; disrupting the formation of the mitotic spindle microtubules; blocking in G1 phase Elevation in the phase enzymes GST and QR; suppressing MMP-7, -2, -9 expression Inhibition of the catalytic activity of GST Inhibiting activities of protein kinases, including EGF receptor tyrosine kinase, PKA, PKC; upregulation of p38MAPK; blocking in S phase at low concentration but in G1 and early S phase at higher concentration Inhibiting microtubule functions and triggering apoptosis Down-regulation of PKC and II Activation of JNKs; inhibition of ERK1/2, PI3K/Akt; induction of p21
WAF1/CIP1
Compounds 4-HC 6-NO2-7-HC 7-HC Auraptene DAMC Daphnetin
Reference [Velasco-Velazquez et al., 42] [Finn et al., 62] [Wang et al., 43], [Lopez-Gonzalez et al., 47] [Tanaka et al.,68], [Kawabata et al., 72] [Raj et al., 98] [Yang et al., 46], [Finn et al., 48]
DBC Decursin DHMC Dicoumarol Esculetin Imperatorin Scopoletin
[Kim et al., 65] [Kim et al., 78] [Riveiro et al., 54] [Madari et al.,45], [Li et al., 84] [Lee et al., 49], [Park et al., 50], [Chu et al., 3] [Prince et al., 81] [Kim et al., 51], [Kim et al., 52]
Mediation of tubulin binding and stabilization of spindle microtubule dynamics; inhibiting the release of MMPs; inhibiting the pathway involving tissue factor and factor VIIa. Induction of p21; decreasing the activity of Cdks; inhibition of pRB phosphorylation; mitochondrial dysfunction; activation of caspase-3 Inhibition of CYP1A1/1B1 and induction hepatic GSTs to block the formation of DNA adducts Activation of NF-B and caspase-3
theless, Lopez-Gonzalez reported that coumarin and umbelliferone (8) showed significant inhibition of proliferation of non-small cell lung carcinoma (NSCLC), and that umbelliferone blocked the cell cycle in the G1 phase and inducing apoptosis [47]. Daphnetin was demonstrated to have the antiprolierative effects against renal cell carcinoma (RCC) line, A-498[48] by inhibiting S phase cell cycle transition at low concentrations and G1 and early S phase at higher concentrations through modulation of extracellular-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (p38MAPK) signaling. This results in a hypothesis that daphnetin could cause inhibition of EGFR signaling, coupled with a modulation of PKC and PKA signaling, which approached to a down-regulation of proliferative signals and an associated up-regulation of differentiation signals, including p38MAP kinase. Lee et al studied the anti-proliferative action of esculetin on cultured human monocytic leukemia U937 cells, and found that the ERK pathway participates in p21 induction [49]. This subsequently leads to a decrease in the kinase activity of cyclin-dependent kinases (Cdks) and to the inhibition of pRB phosphorylation in esculetin-mediated G1 cell-cycle arrest of U937 cells [49]. Esculetin-induced apoptosis has been correlated with mitochondrial dysfunction, leading to the release of cytochrome c from the mitochondria in to the cytosol, as well as to the proteolytic activation of caspases. In addition, esculetin selectively increased the phosphorylations of ERK and c-Jun N-terminal kinase (JNK), which are key regulators of apoptosis in response to esculetin in human leukemia U937 cells [50]. Esculetin has been found to inhibit the survival of human promyelocytic leukemia HL-60 cells in a concentration and time-dependent manner. It induces apoptosis in human leukemia cells by increasing the
leukemia cells by increasing the translocation of cytochrome c from mitochondria into the cytosol and via activation of a cysteine protease 32 kDa proenzyme (CPP32), which is one of caspase-3 family [3] (Table 2). Kim et al investigated scopoletin (6-methoxy-7-hydroxycoumarin) induced apoptosis in human promyeloleukemic cells and found that scopoletin activated NF- B and caspase-3, and finally initiated apoptosis in HL-60 cells [51, 52] (Table 2). In addition, it was suggested that the generation of reactive oxygen intermediates (ROIs) might be an important factor in scopoletininduced apoptosis of HL-60 cells. The activation of caspase3 by ROIs was responsible for the proteolytic cleavage of cellular substrates including actin, lamin, poly (ADP-ribose) polymerase (PARP) and inhibitors of deoxyribonuclease (such as DFF45 or ICAD) [53]. Based on the structural similarity between esculetin and 7-HC, it is likely that 7-HC induces apoptosis in the adenocarcinoma cell lines via the same pathway [47]. 7, 8dihydroxy-4- methylcoumarin (DHMC) was reported to induce selective and concentration-dependent apoptosis in human leukemic cells. The pro-apoptotic effect of DHMC was mediated by activation of the JNKs and inhibition of the ERK1/2 and PI3K/Akt pathways, with no participation of the p38 cascade [54]. In addition, down-regulation of the protooncogene c-myc as well as induction of the cell cycle inhibitor p21 WAF1/CIP1 through a p53 independent mechanism was observed in U-937 cells [54]. DHMC was also reported to induce apoptosis of A549 (NSCLC) (IC50=160g/ml) by reactive oxygen species (ROS)-independent mitochondrial pathway through partial inhibition of ERK/MAPK signaling [55]. Therefore, acetylation of DHMC to 7,8-diacetoxy-4methylcoumarin (DAMC) and its thiocoumarin derivative
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O HO O O HO O O
ONa
HO 7 8
R3 R2 R1 9 O O O OH
Et2N
O O
Et2N
O S
CN
N 11
CN
10
O N O O H N N N 12 13 14 Et2N O O O O
R'
15
16
7,8-diacetoxy-4-methylthiocoumarin (DAMTC) was found to exhibit similar toxic activity (A549 IC50=160g/ml) [56]. The presence and the position of the hydroxyls in the structures of coumarins will greatly influence the cytotoxic values. A comparative study of various hydroxyl coumarin derivatives on the growth of breast carcinoma MCF-7 cells has underlined the functional importance of dihydroxy substitution in esculetin formation [57]. This activity is further confirmed by a recent report regarding esculetin-induced inhibition of cell cycle progression in human leukemia HL60 cells [58]. Tumor specific cytotoxicity of the naturally occurring esculetin can be further enhanced by substitutions at 3- and/or 4-position. Hydroxylation in positions of 6, 7 or 8 coumarin-3-carboxylic acid (9) and their associated silver complex induced a concentration-dependent cytotoxic effect. The silver complex dramatically enhanced cytotoxicity in HepG2 cells with IC50 values 2-5.5 times greater than cisplatin [59]. Finn et al found that 7-HC and coumarin had similar toxic activity on treated SK-MEL-31cells, and that novel, synthetic, nitrated coumarins, including 6-nitro-7-hydroxy-
coumarin (6-NO2-7-HC) and 3,6,8-nitro-7-hydroxycoumarin (3,6,8-NO2-7-HC) showed significantly more toxicity as well as being dose and time dependent [60]. Furthermore, 7-HC, 6-NO2-7-HC and 3,6,8-NO2-7-HC were found to be irreversible cytotoxic agents and to inhibit the S phase regulatory protein, cycliin A, so as to inhibit DNA synthesis, but 7HC was the only nitro-derivative which acted in a selective manner [61]. 6-NO2-7-HC has been shown to be a selective anti-proliferative agent capable of activating p38, stressactivated protein kinase (SAPK) and MAP kinase in the human renal carcinoma cell line, A-498. The participation of p38 MAP kinase was involved in 6-NO2-7-HC induced apoptosis of A-498 cells by altering cell cycle progression, leading to the appearance of a sub-G1 peak [62] (Table 2). Coumarin derivatives exhibit great functions in pharmacotherapy of breast cancer (reviewed by Musa et al 2008) [63]. A series of 7-diethylaminocoumarin compounds have been tested and exhibited a cytotoxicity effect against human umbilical vein endothelial cell (HUVEC) and some other cancer cell lines. Introduction of cyano groups at the 4position greatly promoted the bioactivity and compound (10) could strongly inhibits the proliferation of various different
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cancer cell lines, whereas compound ( 11) and (12) showed a high selectivity for HUVEC [64]. But without cyano groups at C4, DBC (13) has a broad spectrum of antiproliferative activity toward various multidrug resistant (MDR) cancer cell lines (IC50=44.8-475.2nM) by inhibiting microtubule functions and triggering apoptosis. This can be explained by the suggestion that DBC is a poor substrate of P-gp drug efflux pump and retains substantial activity against P-gp overexpressing [65]. Bhumika et al used a selection of biochemical assays to show that 6-hydroxycoumarin -3carboxylatosilver was capable of inducing apoptotic cell death in Hep-G2 cells by increasing the activity of proapoptosis caspases 3, and through the cleavage of PARP, which is one of the substrates for caspases 3, thereby decreasing the percentage of cells entering G0/G1 [59]. Jacquot et al reported that 2, 4-diaryl-4H, 5H-pyrano [3,2c]benzopyran-5-ones (14) exhibited a strong antiproliferative activities in MCF-7 breast carcinoma cells [66]. Auraptene (15) and umbelliprenin (16) are both coumarins with the only difference being the increase length of the 7-prenyloxy chain which contains 15 instead of 10 carbons in auraptene. Both auraptene and umbelliprenin have been isolated from cold-pressed oil of natsumikan and proved to inhibit tumor promoter 12-O-tetradecanoylphorhol-13acetate (TPA)-induced Epstein-Barr virus by the mechanism of suppressing O2-generation in leukocytes [67]. Citrus auraptene was found to be effective in inhibiting the development of oral neoplasms induced by 4-nitroquinoline 1oxide (4-NQO). This mechanism might be related to elevation in the phase enzymes glutathione S-transferases (GST) and quinine reductase (QR) of the liver and tongue [68]. Dietary auraptene was also reported to be effective in inhibiting the development of esophageal tumors by N-nitrosomethylbenzylamine (NMBA) when given during the initiation as well as post-initiation phases, and such inhibition was related to suppression of cell proliferation in the esophageal epithelium [69]. Besides, auraptene was also reported be a potential chemopreventive agent against rat hepatocarcinogenesis induced by N, N-diethylnitrosamine [70] and colitis-related colon cancer in rodents [71]. Both Auraptene and umbelliprenin have been reported as being able to decrease Matrix Metalloproteinase (MMP) activity [72, 73] and auraptene has been shown to reduce inducible nitric oxide synthase and cyclooxygenase-2 expression in lipopolysaccharide-stimulated inflammatory cells [74] and decrease lipid peroxidation and experimental carcinogenesis in rats [75]. In 2006, Kawabata et al reported that auraptene showed great efficacy in suppressing MMP-7, -2,-9 expression in the human colorectal adenocarcinoma cell line HT-29 by the mechanism of remarkable inhibition of the production of proMMPs proteins through the dephosphorylation of constitutively activated extracellular signal--
regulated kinase (ERK)1/2, without affecting its mRNA expression level [72]. Tanaka et al reported that auraptene could significantly reduce the growth and number of metastatic lung tumors in mice bearing B16BL6 murine melanoma [76]. Metastatic pigmented malignant melanoma (M4Beu) cell-proliferation is inhibited by umbelliprenin (IC50 12.3uM) via a G1 cell-cycle arrest and through induction of caspaseinpendent apoptosis. The finding that the cytotoxic effect of umbelliprenin is markedly more pronounced in M4Beu cells than in primary fibroblasts, suggests it could be used as a potential therapeutic agent [77]. PKC has always been considered an attractive target for anti-cancer drug screening because it is the cellular receptor for tumor-promoting phorbol esters. Decursin (17) is a tumor-suppressing PKC activator that induces the downregulation of PKC and II and promotes their translocation into the nuclear membrane of K562 cells [78], and competitively inhibits the binding of PKC and phorbol 12,13 dibutyrate (PDBu), which induces the megakaryocytic differentiation of K562 through PKC activation [79]. The structure activity relationship of decursin has led to suggestion that the coumarin structure is required for anti-leukemic activity and the side chain is a determinant of PKC activation and the cytotoxic mechanism seen in leukemia cells [78] (Table 2). Simple natural and synthetic coumarins such as esculetin and the synthetic 6-nitro-substituted coumarin are known to exert anti-cancer properties. Anti-cancer chemotherapies often consist of using cytotoxic agents to interfere with processes of cell division in order to disrupt tumor cell proliferation, invasion and metastasis. However, the majority of drugs that target the latter stages of tumor progression are still in clinical trials, including vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) inhibitors (monoclonal antibodies) and their receptor tyrosine kinase inhibitors (small-molecule inhibitors) as well as MMP, urokinase (uPA), cycloogygenase-2(COX-2) or methionine aminopeptidase inhibitors [80]. Recently it has been reported that a coumarin derivative (18) (IC50=0.3 M) has been identified as MAP kinase, ERK kinase inhibitors (MEK1 inhibitors).This has led to the development of two chemical analogue (19) and (20) which also show anti-cancer activity through inhibition of MEK1[80]. These two coumarin derivatives display high antiangiogenic activity both in vitro and in vivo [53]. Several 6-substituted 2-oxo-2H-1benzopyran-3-carboxylic acid derivatives had been synthesized, evaluated and found that they exhibit great potency in reducing the invasive behaviour of HT 1080 fibrosarcoma cells [10]. Isabelle et al also found that the replacement of the ester function in the 6-position of (19) and (20) by an amide function was allowed without a loss of potency in the Boyden chamber chemoinvasion assay with HT 1080 [10].
O O O 17 O O O N O 18 O O O F O O
O O O X
19 X=Cl 20 X=Br
Wu et al.
H3CO
O O
OCH3 OH O OCH3 OH
OCH3 21
O O OO 24 O
22
23
A blockage in the formation of DNA adducts has been shown to inhibit tumorigenesis and Prince et al proposed that the inhibition of both CYP1A1/1B1 and induced hepatic GSTs could be most effective [81]. To compare the efficiency, simple coumarin (e.g. coumarin and limettin (21) which induced mouse hepatic GSTs, but had little effect on CYP1A1/1B1) and linear furanocoumarins (e.g. imperatorin (22) and isopimpinellin (23) which induced hepatic GSTs and were potent inhibitors of CYP1A1/1B1) were taken into consideration. The results showed that both were high efficacy, but that linear furanocoumarins had a greater inhibitory effect on 7,12-dimethylbenz[a]anthracene(DMBA)-DNA adduct formation in mouse mammary glands. This raises the possibility that a combination of both P450 inhibition and GST induction might lead to more effective inhibition of polycyclic aromatic hydrocarbon mammary carcinogenesis [81]. Less is known about the effects of the pyranocoumarins on carcinogenesis, as their availablitiy has been limited. Until recently, the pyranocoumarin compound ()-3-angeloyl 4-acetoxy- cis-khel-lactone was isolated from Radix peucedani and caused apoptotic cell death for drug sensitive KB-31 and MDR KB-V1 cancer cell lines by the mechanism of being a P-gp inhibitor [82]. Some coumarins possess enhancing effects on lymphocyte mitogen responsiveness. Scopoletin had dual effects on both concanavalin A-stimulated murine T-cell proliferation and an antiproliferative activity in a lymphoma cell line [83]. The antiproliferative action exerted by scopoletin on tumor cells was through a dual action mechanism which revealed that at low concentrations cell viability was not affected, whereas at high concentrations a cytotoxic effect and a decrease in cell viability were described [83]. The latter event could be related to an induction of apoptosis, where cells with apoptotic morphology were observed in significant amounts at concentrations that show a cytotoxic effect. Scopoletin-inducing cell proliferation in normal T lymphocytes was found to be due to the interaction with PKC [83]. There are three types of immunomodulation, type 1 (enhanced lymphocyte activation and secretion of IFN), type 2 (enhanced lymphocyte activation) and type 3 (enhanced secretion of IFN ). Isopimpinellin, bergapten, and xanthotoxin (72) may also serve as candidates for the immunostimulating activity of IFN secretion. Among the three coumarins tested, two types of immunomodulation were exhibited and correlated with the number of methoxy groups including, a type 2 response of isopimpinellin (two methoxy group and lymphocyte activation), and a type 3 immunomodulation of bergapten and xanthotoxin (one methoxy group and elevation of IFN secretion), whereas the position
of the methoxy group affected the immunomodulating potency such as bergapten (5-methoxypsoralen) and xanthotoxin (8-methoxypsoralen) [12]. ANTI-COAGULANT AND ANTI-PLATELET Some coumarins, primarily 4-HCs and their derivatives, are conferred to function as anti-coagulants. Coumarin anticoagulants have been demonstrated to inhibit metastasis in several animal models, but the mechanism of this effect is obscure [1]. Little information has been acquired on the molecular and cellular mechanism of action of coumarin anticoagulants in the treatment of malignancies. Tumor cell invasion is dependent on angiogenesis and requires both cell migration and the digestion of basement membrane via proteases. Dicoumarol (3, 3-methylenebis [4-hydroxy-coumarin] (24) is a natural anti-coagulant drug. The antiproliferative mechanism of action of dicoumarol and any possible related pharmacophores may be mediated by tubulin binding and the stabilization of spindle microtubule dynamics [45]. As Taxol has been reported to inhibit migration of human ovarian and prostate carcinoma cells [84], and that coumarin anti-coagulants can inhibit the thrombin-induced release of matrix metalloproteinases that cause the breakdown of extracellular matrix proteins, it is logical to assume that dicoumarol might enhance the antiadhesive activity of taxanes. Coumarin anti-coagulants inhibit the pathway involving tissue factor and factor VIIa. Tissue factor VIIa appears to be a major contributor to the regulation of angiogenic growth properties of tumor cells, and in vivo studies have demonstrated a significant role for warfarin in the regulation of the inhibition of angiogenesis. Dicoumarol and coumarins could provide a new structural class for synthetic agents that could lead to improved antineoplastic drugs. In addition, dicoumarol and other coumarin anti-coagulants might have other new tumor specificities other than those currently used as antimitotic agents [45]. Oral anti-coagulants are the most commonly used agents in the long-term prophylaxis and treatment of arterial and venous thrombosis disorders [85]. There are two distinct chemical groups of oral anti-coagulants: the 4-hydroxy coumarin derivatives (e.g., warfarin sodium) and the indane-1, 3-dione derivatives (e.g., phenindione). Oral anti-coagulants are also used commonly for preventing venous thromboembolism following orthopedic surgery. The coumarin derivatives are the oral anti-coagulants of choice, because they are associated with fewer nonhemorrhagic side effects than the indanedione derivatives. Warfarin is highly protein-bound (primarily albumin), leaving only the nonprotein-bound ma-
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terial as biologically active. As more applications are developed, including prevention of recurrent myocardial infarction and the treatment of systemic embolism in atrial fibrillation, the use of oral anti-coagulants is rising. Any drug or chemical that is also bound to albumen may displace warfarin from its protein-binding sites and thereby increase the availability of biologically active material [86]. Warfarin is metabolized in the liver by the p450 (CYP2C9) system of enzymes. Interference with the CYP2C9 enzymes by various drugs or a mutation in the gene coding for one of the common CYP2C9 enzymes can interfere markedly with the metabolism of warfarin [87]. The structural features responsible for the anti-coagulant activity of coumarins, using 3 major methods, have been examined [88]. These are: (1) the influence of substitutions, such as hydroxyl and methoxyl groups. (2) The examination of compounds in which the oxygen atom in position 4 was involved in a preformed ring system simulating cyclic acetals. (3) Coumarin derivatives, devoid of an oxygen function in the 4-position substitute, like 3- and 4-phenylcoumarin and 4methylcoumarins. Loading the molecule with additional methoxyl groups either potentiated or inhibited activity depending on the positions of the substitute [88]. Thus there is considerable difference in activity between the isomeric dicoumarol derivatives, 3, 3-methylenebis (4-hydroxy-5, 7dimethoxycoumarin (25) and 3, 3-methylenebis (4-hydroxy7,8-dimethoxycoumarin) (26) . The higher activity of the compound with substitutes in position 8 showed the importance of location. Similar findings exist for the 4hydroxycoumarins, and furthermore, in 4-hydroxycoumarins a bromine atom at position 3 slightly increases the molar activity, while an acetyl group in the same position conferred even greater activity. Interestingly, 3- and 4-phenylcoumarins were very weak anti-coagulants. These compounds exerted a significant dif-
ference between the activity of the various hydroxylated derivatives and that of their corresponding methyl ethers. Thus, invariably, the methylation of a free hydroxyl group increased the anti-coagulant activity. This result may indicate the importance of ionization as one of the factors aiding the vitamin-K-like activity. Introduction of an 8-acetyl group into 7-hydroxy-4-methylcoumarin (27) increased the anticoagulant activity, whereas introduction of a hydroxyl, methoxyl or benzamido group in position 3 of coumarin decreases the anti-coagulant activity [88] (Table 3). 8-methyl-4-(1-piperazinyl)-7-(3-pyridinylmethoxy)-2H1-benzopyran-2-one (RC414) (28) was tested for its effect on human platelets. The results revealed that RC414 in a dosedependent manner inhibited aggregation both in platelet rich plasma and in washed platelets. It was particularly effective in platelets challenged by collagen, ADP and thrombin: IC50 values are 0.51 0.12 uM, 0.980.36 uM and 1.00 0.15 uM, respectively [89]. RC414 increased cAMP levels, through the specific inhibition of the cAMP high affinity phosphodiesterase (IC50=1.730.35 uM). RC414 reduced [Ca2+]i transients and PKC activation induced by thrombin. In addition RC414 was able to increase nitric oxide formation involving the stimulation of constitutive nitric oxide synthase enzyme [89]. The introduction of a 6- or 7- alkoxy substituent could increase the activity of the coumarin derivative (29) [90]. Furthermore, it seemed to indicate that the effectiveness of a 7-alkoxy substitution rose in accordance with its volume/lipophilicity ration in the order OCH3 OC2H5OCH(CH3)2 or OCH2C6H5. In turn, the 8-methyl substitution increased the anti-platelet activity of the 7alkoxy substituted compounds [90]. Two coumarins derivatives (30) and (31) have been reported to allow the relaxation of vascular smooth muscle and inhibiting platelet aggregation with a profle similar to that of trans-resveratrol (tRESV) (32). Compound (31) has a vaso-relaxant activity that
OCH3 H3CO O OO H2 C OCH3 OH 25 N CH3 O O O N 28 N NH 29 NH OH 32 O OH OCH3 OH 26 O OH O OCH3 H3CO O OO H2 C OH OCH3 27 CH3 O OCH3 HO O O
OH
R2 R1
O OH H H3CO
O O O O H3CO
O O
30: R1=H, R2=OH 31: R1=OH, R2=H
OH
33
34
Wu et al.
is twice as high as that of t-resveratrol and a platelet antiaggregant activity that is six times higher [91]. More studies have been carried out to search for more effective chemical biological structures from natural plants. Chia et al used a bioactivity-guided fractionation method, two coumarins: minumicroline acetonide (33) and epimurpaniculol senecioate (34), were isolated from the leaves of Murraya omphalocarpa Hayata (Rutaceae) [92]. The biological activity research reported that compounds (33) showed significant inhibition of arachidonic acid (AA)- and collagen- induced platelet aggregation, and strong inhibition induced via platelet activating factor (PAF). In addition, compound (34) also displayed significant inhibitory activity on collagen- and AA- induced platelet aggregation, but not those potentiated by PAF [92]. ANTI-OXIDANT It has been noted that a large variety of possible substitutions in the basic molecule may influence the structurerelated biological activities of coumarins [93]. They act as anti-oxidants, enzyme inhibitors and precursors of toxic substances. For monohydroxycoumarins, their anti-oxidant properties have been related to radical-scavenging activity, and inhibition of tyrosine kinases [2]. But, accumulating data of studies reveal that dihydroxycoumarins are better antioxidants than monohydroxycoumarins and the OH groups positioned near C6 and C7 in the coumarin skeleton play an important role in the inhibition of the mushroom tyrosinase [94]. Coumarins derivatives (coumarin, 4-HC, 7-HC, esculetin, scopoletin, DHC, 4-methylesculetin, and 7-hydroxy4-methylcoumarin) were analysed for their anti-oxidant properties and their ability to scavenge free radicals. The results showed that esculetin was the most potent radical scavenger, followed by 4-methylesculetin [95]. The number of hydroxyl groups on the coumarin ring structure correlates with the ROS suppressor function. The structure-based molecular modeling revealed interactions between coumarins and the molybdopterin region of xanthine oxidase (XO). The carbonyl pointed toward the Arg880, and the ester O atom formed hydrogen bonds with Thr1010. Esculetin, which bears two hydroxyl moieties on its benzene rings, had the highest affinity toward the binding site of XO, and this was mainly due to the interaction of 6-hydroxyl with the E802 residue of XO [95]. The chemical structure of scopoletin is similar to that of esculetin except with methoxy moiety substituted for the 6-hydroxyl, which results in a diminished potency of scopoletin to inhibit XO. This further enhances the hypothesis that the H atom of the 6-hydroxyl plays a more significant role than the O atom [95]. Although the anti-oxidant activity has been primarily attributed to the presence of free hydroxyl groups, a significant anti-oxidant effect has also been reported for compounds where these groups are acetylated [96]. A novel enzyme in the microsomes of the liver catalyzed the transfer of acetyl groups from acetylated polyphenols to certain receptor enzyme proteins which could putatively result in the modification of their catalytic activities [97]. Protein transacetylase (TAase) was found to catalyze the transfer of the acetyl group from DAMC (35) to GST. This results in the acetylation of several lysine residues in its active site and subse-
quent inhibition of the catalytic activity of GST [98]. Twelve acetoxy coumarins and dihydrocoumarins bearing a phenyl ring and methyl group at C-4 were involved into the experiments. The results of acetylation of GST by the the coumarin derivatives by TAase showed that DAMC had the highest catalytic activity, and that acetoxy 4-phenylcoumarins (36) had significantly less activity [96]. Similar results were obtained when the TAase catalyzed activation of NADPH cytochrome c reductase assay and AflatoxinB1 (AFB1)-DNA binding inhibitory assay were performed with 4-phenylcoumarins and 4-methylcoumarins [96]. These results confirmed the hypothesis that DAMC was found to modulate the activities of enzymes including cytochrome P-450-linked mixed-function-oxidase ( MFO), NADPH cytochrome c reductase and cytosolic GST when catalyzed by TAase [99]. To elucidate the structure activity relationship (SAR) of the phenyl ring on the benzopyran nucleus, Kumar et al compares the specificities of the acetylated coumarins, biscoumarins, chromones, flavones/isoflavones and xanthones for TAase activity. The results demonstrated that the presence of the phenyl ring on the pyran nucleus of polyphenolic acetates drastically reduces their specificity to TAase modification [99].
OAc OAc AcO O O AcO O O
35 36
The effects of coumarin anti-oxidants on tumormodulation also have been studied using carcinogens [1]. Structural insights into hydroxycoumarin-induced apoptosis in U937 cells were studied by Riveiro et al. The results showed that the presence of two adjacent phenolic hydroxyl groups was the most important factor in terms of the structure activity relationship [100]. It is known that GST induction can occur through different mechanisms, such as via the xenobiotic response element (XRE) or the anti-oxidant response element (ARE). Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a member of the capn collar family of basic region leucine zipper (bZIP) transcription factor [101]. The ARE is a 41-bp element in the 5-flanking region of the rat GST Ya gene, that is positively regulated by Nrf2 [101]. Naturally occurring coumairns possess anti-carcinogenic activities in part by inducing carcinogen- detoxifying enzymes GST and/or NAD(P)H quinine oxidoreductase (NQO1) [102,103]. To determine whether citrus coumarins induce hepatic GST and/or NQO1 via activ ation of Nrf2 and ARE in HepG2 cell line. Prince et al found that auraptene and imperatorin induced murine liver cytosolic GST activities via the Nrf2/ARE mechanism, whereas isopimpinellin, although structurally similar, did not appear to activated HepG2-ARE-GFP and the Nrf2 knockout mouse, which might indicate that isopimpinellin induced GST and NQO1 via additional mechanism [103]. In addition, the presence of low concentrations of H2O2, one of ROIs, was able to inhibit caspase-mediated apoptosis
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in Jurkat cells [104]. The mode of procaspase-9 activation in order to inhibit apoptosis was probably through the reversible oxidation of sensitive cysteine residues by the presence of H2O2 in an iron-dependent reaction [105]. Coumarins are known to have the ability to act as an anti-oxidant and a radical scavenger. As a result, coumarins might be related to activation of caspase-9 and induction of cell apoptosis. But this signal transduction mechanism remians unclear, and now is under investigation by our research group. ANTI-INFLAMMATION Osthole (37) was tested in cyclooxygenase and 5lipoxygenase bioassays and turned out to be a moderate and selective a 5-lipoxygenase inhibitor (IC50=36.2 M) [106]. 4-methylesculetin and 4-methyldaphnetin were tested on ionophore-activated rat leukocytes (a cell system that express both cyclooxygenase and 5-lipoxygenase pathways of arachidonate metabolism) and were found to inhibit selectively the proinflammatory 5-lipoxygenase enzyme with 5, 7dihydroxy-4-methylcoumarin demonstrating a higher potency against cyclooxygenase [2]. It is well known that lipoxygenase (LOX) possess regiospecificity during interaction with substrates and on this basis have been primarily designed as arachidonate 5-, 12-, and 15-LOX. 5-LOX represents a dioxygenase that possesses two distinct enzymatic activities leading to the formation of LTA4, which is converted to 5-hydroxyeicosatetraenoic acid (5-HETE) or five lipoxygenase activating protein (FLAP). Modeling and sequence comparisons of the human 12and 15-LOX has led to suggestions as to which residues are required for determination of the positional specificity [107]. Few studies have presented the direct binding data of LOX enzyme to the inhibitors, and details of their interactions remain unclear[108]. Using the crystalline structure of rabbit 15-LOX, it was noted that the base of the boot shaped substrate binding pocket wad lined by the side chains of Phe353, Ile418, Met419, and Ile593 and the volume of the pocket could be important for positional specificity. It is predicted that 12-LOX has a slightly larger substrate binding pocket compared to 15-LOX, whereas the pocket of 5-LOX is predicted to be 20% larger [107]. Based on the crystallographic data of rabbit 15-LOX, Du et al (2006) investigated the bind-
ing of human 5-LOX with its inhibitor, including esculetin, by SPR technology correlating with molecular docking simulation and found that the inhibitors should share some analogous features: a polar head and tail interacting with the hydrophilic portion of 5-LOX through hydrogen bonds or electrostatic interactions, and a hydrophobic body stretching into the large hydrophobic channel of 5-LOX to form strong hydrophobic interactions with its surrounding lipophilic residues [108]. Accumulating evidences suggest that the 5-LOX pathway has profound influence on the development and progression of human cancers [109]. The 5-LOX pathway interacts with multiple intracellular signaling pathways that control cancer cell proliferation [110]. Experimental data reveal that 12-LOX is involved in both cancer cell proliferation and survival [111]. Esculetin can selectively inhibit LOXs with different IC50. In platelet, esculetin inhibit 12LOX with IC50=0.65 M [112]. Whereas esculetin inhibits the formation of 5-HETE with IC50= 1.46mM in polymorphonuclear leukocytes, although more strongly than HHT (IC50= 57.3mM) [113]. The roles of 15-LOX in cancer development are unclear. However, esculetin can also inhibit the formation of 15-HETE from 15-LOX. On investigation of specific products of the LOX pathway mediating the autoregulatory effect of glucose and glucose transport in vascularsmoothmuscle cells (VSMC) and vascularendothelial cells (VEC), esculetin (100 M) was found to inhibit the formation of 12- and 15-HETE and decrease the productions to 23.0% and 37.7%, respectively [114]. These indicate that esculetin is the most potent in blocking 12-LOX. Further insight into structure activity relationship, the inhibitory potency on LOX is not related to the oxidation potential of the compound [115]. Esculin, glucosidation at one of the hydroxy group of esculetin, decreases the inhibitory potency markedly with IC50=290 M. 7-HC, a metabolite of coumarin, has the ability to reduce edema in the rat paw carrageenan test. It was also shown to inhibit rat platelet lipoxygenase (IC50=502 M) and prostaglandin synthesis [116]. However, without hydroxyl in the position of C6 and C7, coumarin and 4-HC had no inhibitory effect on either enzyme at concentrations up to 1mM [112]. The effect of daphnetin and fraxetin on the formation of 5-HETE and the cyclooxygenase product (12-hydroxy-5,8 ,10-heptadecatrienoic acid (HHT)) in polymorphonuclear leukocytes
O MeO O O
HONHC
O N
39 38 37 O O H3CHNN O 41 42 O R1 O H N R a: R1=H, R=2-CO2Ac R=2-CO2Me b: R1=H, c: R1=H, R=2-COOH d: R1=6-OMe, R=2-COOH e: R1=8-OMe, R=2-COOH f: R1=6-NO2, R=2-COOH g: R1=6-Cl, R=2-COOH h: R1=8-allyl, R=2-COOH
40
Wu et al.
were studied [113]. The results showed that daphnetin and fraxetin inhibit the formation of 5-HETE more strongly than HHT; the concentrations of IC50 were, respectively, 6.90mM, 2.57mM and 139.0mM, 532.5mM. In addition, scopoletin were also show to inhibit the formation of 5-HETE and HHT, but less strongly [113]. Therefore, that hydroxylation at C6 is very important for esculetin to inhibit LOX. Some coumarin derivatives with substitutions at position 7, compounds (38), (39), (40) and (41) were subjected to the induced carrageenan paw edema test to analyse their antiinflammatory activity, and the results obtained showed that they possessed significant protection (55.1%, 58.5%, 54.0% and 54.7%, respectively)[117]. Some N-Aryl substituted 3carboxamidocoumarins (42) had been synthesized through a Knoevenangel condensation of nitriles with salicylaldehyde with salicylaldehydes in the presence of piperidine in ethanol. These coumarin derivatives were found to be potent anti-inflammatory agents. In carrageenan-induced rat paw edema assays, compounds (42b), (42g) and (42h) were found most active with 546.5%, 515.05%, 486.51% and 477.13% inhibition respectively (at doses of 10 mg/kg p.o.; piroxicam control showed 576.61% inhibition). In acetic acid peritonitis tests, the most active compounds (42a) and (42c) showed 427.20%, 406.43% and 395.09% inhibition (at doses of 10 mg/kg p.o., piroxicam revealed a protection of 297.24%). All tested compounds were essentially non-toxic at the highest dose graded [118]. Both nitric oxide (NO) and prostaglandin E2 (PGE2), and their associated enzymes, nitric oxide synthase (NOS) and Cyclooxygenase (COX), are involved in the development of inflammation. Sixty-three naturally occurring oxycoumairns were screened and four 5,7-dimethoxycoumarins were selected to show potent inhibitory effects on iNOS protein expression and NO generation. It was suggested that the methoxyl group on C5 and C7 and the short alkyl chain (1-5 carbons) on C6 might be essential for the potent activities. These compounds also showed inhibitory effects on nitric oxide generation and mRNA expression of inflammatory mediators, namely, iNOS and COX-2 [119]. It has been reported that scopoletin (150 g/ml) inhibits the release of PGE2, TNF, IL-1 and IL-6 and suppresses expression of COX-2, but not COX-1 protein, in a concentrationdependent manner in RAW 264.7 cells [52]. 3-(1-1-dimethyl-allyl)-6-hydroxy-7-methoxy-coumarin, obtained from the methanol (50%) extraction of the Ruta
graveolens L. plant, was observed to inhibit both the protein and mRNA expression of iNOS and IL-1 in lipopolysaccharide (LPS) challenged macrophages, and to block the LPSinduced activation of nuclear factor- B (NF- B) through the prevention of inhibitor-B degradation [120]. This compound also showed anti-oxidant properties and at a dose of 40mg/kg, and could inhibit iNOS and IL-1 gene expression significantly in an endotoxin-induced inflammatory model of BALB/c mice [120]. When studying the relationship between the structure and the activities of coumarin derivatives, such as TNF inhibitors, Cheng et al found that substitution at position C-6 of the coumarin ring system most dramatically influenced TNF inhibitory activity [121]. The 6-halo coumarin compounds (43) were 20-30 times more potent than the corresponding nonsubstituted counterpart. Alkyl groups, except a methyl group, at this position tended to decrease the activities. No clear trend was observed for any electronic effects of the substitutuions. Electron withdrawing groups such as CHO, CN, NO2, and COOH either improved or diminished the TNF inhibitory activities. Electron donating groups like MeO increased activity by three-fold, similar to the CN group. A C-8 methyl derivative is 25% less potent than the corresponding C-6 derivative. Similarly, a methoxy group at C-5 position completely abolished the TNF inhibitory activity [121], (Table 3). One of the more promising approaches to discover inhibitors of TNF is the inhibition of a zinc containing metalloproteinase, TNF converting enzyme (TACE), which converts membrane bound pro-TNF to mature and soluble TNF. A series of couamrin-base analogues (44) were prepared, with structural similarites to gelasatin hydroxamates (45). These showed good binding to the TACE binding in the docking model [122]. Similarly, methyl and methoxyl substituted analogues had comparable activity, and were more active than other analogues for in vitro TACE enzyme inhibition. Larger substitutions, like the tert-butyl group on the chromene core decreased the inhibitory activities. For the inhibitory activities of MMP-2 and MMP-9, these compounds paralleled TACE activity. Furthermore, 6-position methyl or methoxyl substituted analogues (46) were prepared. The analogues (46a) and (46b) showed IC50 at 36nM and 3nM, and 220nM and 320nM for TACE enzyme inhibition and TNF inhibition, respectively. Allyl (46c) and benzylated (46d) analogues showed similar results at a range of 30nM and 5nM in the same assay [122].
O
O N O R 43 O R O 44 O O O Ar R
O NHOH O
HO
N H
O 45 R2 H N O a: R1=6-OMe, R2=Me b: R1=6-Me, R2=Me c: R1=6-Me, R2=allyl d: R1=6-Me, R2=benzyl
OH
46
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Table 3.
The Relationships Between the Structure and the Function of Coumarins

Structure Activity Relationships (SARs) A free 7-OH was important for anti-bacterial activity A methoxy at C-7 and, where present, an OH group at either the C-6 or C-8 position was invariably effective against a broad spectrum of bacteria. The lipophilic character and a planar structure are requirements for high anti-bacterial activities 3-acetylamino coumarin derivatives show antibacterial activity which through an ascending increase in the size of the acyl group display an increasing range of anti-bacterial action Reference [Sardari et al., 133] [Kayser et al., 131] [Kayser et al., 131] [Hishmat et al., 128]
Compounds Anti-bacterial coumarins
Anti-fungal coumarins Anti-HIV coumarins Coumarins
A free 6-OH was essential for anti-fungal activity.
[Sardari et al., 133]
SARs of DCK and Calanolides have been reviewed and summarized Modeling and docking: carbonyl pointed toward the Arg880, and the ester O atom formed hydrogen bonds with Thr1010 in region of XO Methoxyl group on C5 and C7 and the short alkyl chain (1-5 carbons) on C6 might be essential for the potent inhibiting activities of NOS and COX No clear trend was observed for any electronic effects of the substitutuions to influence TNF inhibitory activity. However, substitution at position C-6 of the coumarin ring system most dramatically influenced TNF inhibitory activity. The presence of a strong electron-withdrawing group at the 3-position facilitates the nucleophilic attack by the serine hydroxyl residue in protease
[Yu et al., 145] [Masamoto et al., 94] [Nakamura et al., 119] [Cheng et al., 121]
[Bchet et al., 157] [Raj et al., 98], [Kumar et al., 99] [Masamoto et al., 94] [Hardt et al., 116] [Arora et al., 88]
DAMC
Acetylation of several lysine residues in its active site of GST, but phenyl presences in C4 position will drastically reduce its specificity Modeling and docking: 6-hydroxyl interacts with the E802 residue of XO The hydroxylation at C6 is important for high selectivity of 12-LOX
Esculetin
7-hydroxy-4methylcoumarin Osthole
Introduction of a hydroxyl, methoxyl or benzamido group in position 3 of coumarin decreases the anticoagulant activity; but introduction of a 8-acetyl group into 7hydroxy-4-methlcoumarin increases the anti-coagulant activity Methoxy-group at position-7 and the 3-methyl-2-butenylgroup at position-8 are essential to lower plasma ALT in hepatitis
[Okamoto et al., 137]
In addition, inflammation is well known as an important factor in the development of diabetes; since coumarin suppresses the phosphorylation of Akt/PKB as a mechanism inhibiting inflammation, it may be hypothesized that this could be a factor in insulin resistance [123]. On the other hand, Hu and co-workers demonstrated that benzopyran derivatives (oxycoumarin derivatives) activate peroxisome proliferator-activated receptor-c (PPAR-c) and exert a beneficial effect on insulin action on glucose uptake and lipid metabolism. It was shown that, as a mechanism accelerating insulin-stimulated glucose uptake, oxycoumarin increased the phosphorylation of Akt and p38 MAP kinase in skeletal muscle [124]. It should be noted that not all oxycoumarins derivatives could suppress the level of LPS-induced iNOS, and differences in the structures of oxycoumarins were associated with this contradiction [123]. ANTI-MICROORGANISM The indiscriminate use of antibiotics has led to many bacterial strains becoming drug resistant. Development of new and effective antibiotic compounds to target resistant micro-
organisms has become critically important and new the products in development should alleviate these concerns. Glycosylation of many natural products is required for the associated antibiotic and/or anti-tumor activities. Novobiocin (47), clorobiocin (48), and coumermycin A1 (49) are all members of the coumarin family of antibiotics and are derived from various Streptomyces species. Each compound contains an individual noviosyl sugar component that imparts the functionality essential for biological activity. This family of antibiotics exerts its anti-bacterial activity via the inhibition of the type II DNA topoisomerase, DNA gyrase. Removal of the carbamoyl group from novobiocin or its transference to the 2-hydroxy group of novobiose, leads to a complete loss of activity [125]. However, replacement of the carbamoyl group with a 5-methyl-2-pyrrolylcarbamoyl group (which is present in coumermycin), leads to >10-fold increases in both anti-bacterial activity and in vitro activity against DNA gyrase. The aminocoumarin antibiotics novobiocin and clorobiocin consist of a 3-amino-4, 7dihydroxycoumarin (ADHC) moiety flanked on one side by L-noviose and on the other side by a 3-dimethylallyl-4- hydroxybenzoyl (DMAHB) moiety. Both ADHC and Lnoviose are essential for anti-bacterial activity and that the
Wu et al.
substituents attached to these fragments have a significant impact on their bioactivities. X-ray crystallographic examination of antibiotic-enzyme complexes showed that the ADHC and L-noviose moieties are each involved in antibiotic binding to the B subunit of DNA gyrase [126]. The toxicity of novobiocin in eukaryotes, as well as its poor activity towards most gram-negative bacterial pathogens, and the proclivity of staphylococci to develop endogenous resistance to aminocoumarins during therapy, led pharmaceutical companies to direct their anti-bacterial drug development resources towards other antibiotic classes. However, the emergence of antibiotic resistant organisms has resulted in a reevaluation of the old antibiotic classes. For example, analysis of the structure activity relationship of clorobiocin found that modifications of the 3-dimethylallyl4- hydroxybenzoyl moiety reduced biological activity [127]. The highest activities were shown by compounds containing a hydrophobic alkyl group at position 3 of the 4hydroxybenzoyl moiety. Polar groups in this side chain, especially amide, strongly reduced anti-bacterial activity and replacement of the alkyl side chain with a halogen atom or a methoxy group at the same position markedly reduced the activity. Transfer of the pyrrole carboxylic acid moiety from O-3to O-2 of L-noviose only moderately reduced activity, whereas the complete absence of the pyrrole carboxylic acid moiety led to a complete loss of activity. Desclorobiocin derivatives lacking the chlorine atom at C-8 of the 3-amino4,7-dihydroxycoumarin moiety also showed low activity. Lack of a methyl group at O-4of L-noviose resulted in a completely inactive compound [127]. 3-acetylamino coumarin derivatives was reported to show anti-bacterial activity which through an ascending increase in
OH H N OH
the size of the acyl group display an increasing range of antibacterial action [128]. Introduction of a bromine atom in to the side chain, i.e. 6-bromo-3-(w-bromoacetyl coumarin, maintained its anti-bacterial potency, while the further modified 6,8-dibromo-3-(w-bromoacetyl) coumarin increased further its anti-bacterial activity. The dichloroacetamido moiety has also been inserted in to the allylic position of the pyran ring of coumarin, and the resulting compound (50) have moderate activity against B. cirroflagellosus, E. coli, A. niger, and C. albicans [129]. New 3-acylamido coumarins derived from 3-amino-5-substituted isoxazoles (51) have been designed and synthesized for possible anti-microbial agents [130]. Promisingly, Radulovic et al screened a new 3,4-annelated coumarin derivative (52), which showed a greater antibiotic activity than both Ampicillin and Nystatin, and four times as much anti-oxidant activity as -tocopherol acetate [130]. In vitro structure activity relationship studies of coumarins have shown that the lipophilic character and a planar structure are requirements for high antibacterial activities [131]. Anti-microbial potency may be due to passive diffusion facilitated by both the lipophilic character and the planar molecular structure. Increased lipophilicity of compounds may be associated with easier penetration into Gram-positive bacteria, but other factors such as shape also have to be considered. It has been suggested that simply an aromatic substitution and the avoidance of bulky side-chains could aid in penetration through bacterial cell walls [132]. It has also been reported that a free 6-OH of the coumarin nucleus was essential for anti-fungal activity, while a free 7OH was important for anti-bacterial activity [133] (Table 3).
OH 1'' O O H3CO O 3'' O 2'' OH H3CO O 3'' O NH 48 H2N O NH OH H N O HO O O O CH3 O H3CO O O 49 NH O OH CH3 O O O O O OCH3 OH Cl O 2'' OH 47 O O O 1'' O CH3 H N O
OH
HN
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O O R O 50 N H CHCl2 O 51 52 O O O H N N O N
O NH N NH OH
Angelicin (53), a naturally occurring furanocoumarin, which showed anti-fungal activity, was considered to be a lead structure for a group of synthetic coumarins. Simple long chained hydrocarbons are connected to the furanocoumarin skeleton of angelicin, and the anti-microbial activities are more efficacious than other furanocoumarins, protection of 6-OH by groups that change the electronic contribution of oxygen 6 to the ring, or change the polarity of the functional groups to a favored pattern improves the anti-fungal activity [133]. Studies have shown that a free 6-OH moiety in the coumarin nucleus was a necessity to combat bacteria [131]. Systemic analysis of the structural activity relationships have revealed that coumarins with a methoxy function at C-7 and, where present, an OH group at either the C-6 or C-8 position were invariably effective against a broad spectrum bacteria. The presence of an aromatic dimethoxy arrangement was shown to be favorable against those microorganisms which required special growth factors (beta-hemolytic streptococcus, streptococcus pneumoniae and haemophilus influenzae [131]). A combination of these structural features, two methoxy functions and at least one additional phenolic group, as reflected by the highly oxygenated coumarins, has led to some promising candidates with a broad-spectrum of anti-bacterial activity to be identified [131]. The development of azoles has revolutionized the treatment of many fungal infections, but still treatment of many of them necessitates application of the highly toxic drug, amphotericin B or a combination thereof. Emergence of new resistant species of fungi, in addition to the poor safety and pharmacokinetics profile, present challenges to clinicians in the best way to handle the fungal infections. Fifty-three new 3-(2-diethylaminoethyl)-4methyl-7-substituted coumarins (54) were synthesized and their anti-fungal activity investigated. Their anti-fungal activities were measured against a phytopathologic fungus, Botrytis cinerea [134]. Compounds with a substituted benzoyloxy, benzoyloxyethoxy, methylbenzoylaminoethoxy, or benzoylaminophenoxy group at the 7-position had an inhibitory effect (MIC: 50 ppm) on the germination of spores in an in vitro screening system. It also indicated that modification of 7-hydroxy group was effective in generating anti-fungal activity and suggested that length of substitutents and the number of benzene rings greatly affected their anti-fungal activity [134]. Efforts to modify coumarins with a hydroxyl group in the benzene ring have resulted in an increase in anti-fungal activity. A series of coumarin and pyranocoumarin analogues were evalutated in vitro for antiviral efficacy against measles virus (MV). The structural activity relationship study showed that the O-substituted 5,7dihydroxycoumarins but not C-substituted or unsubstituted were generally active against MV, and that the free hydrox-
yls in benzodipyranone (55) and benzotripyranone (56) were essential for anti-MV activity [135]. Counteracting the effects of Hepatitis C Virus (HCV) through the use of coumarins has proved challenging as only a small number, primarily osthole, are capable of inhibiting HCV replication and/or proliferation, this counteracting the progression of hepatitis C into hepatocarcinoma [136]. The action of osthole causes a strong reduction of plasma alanine aminotransferase and also inhibits caspase-3 activation [137]. However, osthole exhibits low solubility in water and this property may lead to a decrease in the anti-hepatitis inhibitory effect upon oral administration. In a Con A-induced hepatitis mouse model, intraperitoneally administered osthole (100 mg/kg dose) resulted in 85% inhibition of Con Ainduced elevation of plasma alanine aminotransferase (ALT). However, oral administration of osthole at a 100 mg/kg dose caused only a 38% inhibition of Con A-induced elevation of plasma ALT [138]. Osthenol, an osthole derivative with substitution of a 7-methoxy group for 7-hydroxy of osthole, caused 32% inhibition of Con A-induced elevation of plasma ALT at the dose of 100 mg/kg (i.p.), whereas 7-HC caused only 9% inhibition [137] (Table 3). These indicated that methoxy-group at position-7 and the 3-methyl-2-butenylgroup at position-8 are essential to lower plasma ALT in hepatitis. Okamoto et al has screened 28 synthetic derivatives of osthole and found that most of them increased protective abilities. Three compounds (57), (58), (59) exhibited 68.7 %, 62.5% and 88.3% inhibition of Con A-induced elevation of plasma ALT at dose of 100 mg/kg respectively [138]. These chemicals could contribute to the development of hepatoprotective drugs for various types of liver diseases, including viral hepatitis. Some 7-propyloxy derivatives (60) showed structural similarity to osthole and were endowed with inhibitory properties against both HCV and Hepatitis Crelated virus by targetting HCV surrogate viruses (BVDV) activity [139]. The HIV/AIDS pandemic is a serious threat to health and development of mankind, and searching for effective antiHIV agents remains ongoing. Considerable progress has been made in recent years in the field of drug development against HIV. Anti-HIV coumarins have been identified to inhibit viral adsorption, reverse transcription, protease inhibition and integration in the HIV replication cycle [140]. Warfarin and other 4-HC derivatives are the first generation of HIV-PR inhibitors. Inspired by the structure, 3phenyl-, 3-benzyl-, 3-phenoxy-, 3-benzenesulfonyl-, and 3(7-coumairnyloxy)-4-hydroxycoumarin derivatives had been screened. These results revealed the importance of substituents at position 5 and 7 of the coumarin ring on the inhibitory potency of the HIV-1-PR [141]. Position 5 and 3 were

HO O O O H O O O O O
Wu et al.
N 53 54
55
OH O O O MeO OH OH O 57 NH2 CH2 MeO N H3CH2CH2CO NO2 59 60 R' O O EtO O O O2N 56 58 Me O O
important for not allowing both the introduction of a hydroxyl group and to allow direct fixation of an aromatic group. NF-B and Tat cooperate in driving HIV replication from the state of latency. Inhibition of the activity of these critical proteins will result in an effective blocking of viral replication. Eleven natural 4-phenylcoumarins isolated from Marila pluricostata were screened and found to show the inhibition of NF- B and Tat [142]. Several disubstituted 3',4'-di-O-(s)-camphanoyl-(+)-cis-khellactone (DCK) derivatives have been synthesized and examined for inhibition of HIV-1 replication in H9 lymphocytes [143]. Of the ten synthesized 5-methoxy-4-methyl DCK compounds, (61) was the most active derivative and outperforms the lead compound DCK in the same assay [144]. SAR of DCK has been comprehensive studied and reviewed by Yu et al (2003) [145]: (1) Stereochemistry at the 3 and 4 position should be R-configured. (2) The volume, size, and shape of the camphanoyl group are more important than the absolute configuration of its chiral carbon. However, (-)-camphanoyl analogs are most potent. (3) A planar coumarin system is probably an essential fearture for potent anti-HIV activity. Steric compression between C4 and C5 substituents can reduce the overall planarity and, thus resonance of the coumarin system, resulting in decreased or completely lost activity. (4) Methyl or other aliphatic substitutions on the coumarin nucleus are favorable for anti-HIV activity, whereas aromatic substituents are not. (5) Thio and lactam 4-methyl-DCKs retain activity. In the CEM-SS cell lines, the thio analog is more potent 4-methyl-DCK. (6) 3Hydroxymethyl and 3-dibromomethyl analogs retain potency, but 3-carboxyl and 3-amino analogs do not. Thus, polar but not negatively or positively charged substitutents can be tolerated. Chen et al reported that the position of C6
might be crucial for potency against HIV replication and sensitive to modification. A tert-butyl group at C6 position showed no anti-HIV-1 activity. In addition, two methyl groups at C8 position might be favorable for anti-HIV activity [146]. In a continuing effort to identify the pharmacophores in this class of potent anti-HIV agents, three 9,10-di-O (-)-camphanoyl-7,8,9,10-tetrahydro-benzo[h] chromen-2-one (7-carbon-DCK) analogs, which replaced the oxygen in the C ring of DCK with a methylene group, were synthesized and showed to be potential HIV-1 inhibitors [147]. Two diastereomeric tetracyclic coumarins Calanolide A1 (62) and Calanolide B2 (ostatolide) (63), isolated from Calophyllum lanigerum were found to be HIV-1 reverse transcriptase inhibitors [148]. They utilize a novel mechanistic pathway that inhibited RT in two different template primer systems, primed ribosomal RNA template and homopolymeric poly rA-oligodT1218 template/primer [148]. Another group of tetracyclic pyranocoumarins, the inophyllums isolated from the genus Calophyllum ionophyllum P, are capable of potent inhibition of HIV-1 RT, though the most active inophyllums (64) and (65) have slightly different stereochemistry to the equally potent Calanolides [148]. Structure activity relationship studies show that bulky substituents are required at C4 position, both calanolides and inophyllums require methyls at C10 and C11 of the chromanol ring to be trans-diaxial, and both require a hydrogen bond acceptor at C12. In case of calanolides, the C12 hydroxyl should be S configured, or carbonyl can be present. C12 hydroxyl of inophyllums can be either S or R configured, but cannot be a carbonyl [145]. The transcription factor Sp1 is a member of a multigene family that binds DNA GC boxes and related motifs through
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H3C O
O O O O O O
O O O O O O O O O
OH O O O
OH O O
61
62
63 O
64
OH HO O O O HO O O O O
OH O O OH
65
66
67
COOH-terminal zinc finger motifs. Imperatorin showed inhibitory activity of Sp1 in HIV by inhibiting both PMA (a known activator of the ERK and DNA-dependent protein kinase pathways) and Tat-induced Sp1-dependent HIV-1LTR transcription. It can also inhibit phorbol 12-myristate 13-acetate-induced transcriptional activity of the Gal4-Sp1 fusion protein, as well as strongly inhibiting cyclin D1 expression and arresting HeLa cells in the G1 phase of the cell cycle [149]. Mesuol (66) and isomesuol (67), two 4-phenyl coumarins, isolated from leaves and stems of Marila pluricostata, were demonstrated to combat HIV by inhibiting the transcriptional activity of the HIV-1-LTR promoter through a signaling pathway that involves the phosphorylation of the p65 subunit of the NF-kB transcription factor [150]. RESEARCH AND DEVELOPMENT COUMARIN DRUGS FOR NEW
an equivalent dose [152]. The significance of the 7hydroxylation pathway is that it appears inversely related to species differences in hepatotoxicity. Epoxidation of the 3,4double bond is involved in the coumarin metabolic pathway in the rats. Its role as a liver toxicant has been demonstrated using DHC, which shows no signs of toxicity in the rat liver in vivo in contrast to coumarins [153]. Coumarin 3,4-epoxide is detoxified via GSH conjugation and this reaction is mediated non-enzymatically and enzymatically by GST [154]. To obtain insight in whether decreased 7-HC formation would result in increased liver levels of the hepatotoxic o-HPA, Reitjens et al (2008) built a physiologically based biokinetic (PBBK) model for both rat and man. The results showed that even when 7hydroxylation was deficient, the chances on formation of the hepatotoxic o-HPA metabolite would be significantly lower in the liver of humans than those expected in the liver of rats [155]. Thus, the cytotoxicity of coumarin is both metabolism- and species-dependent, the rat may not be a suitable model for evaluating the pharmacological hazards of coumarin in humans [156]. The search for new drugs requires a deep understanding of the molecular basis of drug action, and is necessary for elucidating the mechanisms of action involved with respect to understanding the relationship between structure and activity. Halomethyldihydrocoumarin was the first suicide substrate of a serine protease [157]. Based on halomethyldihydrocoumarin, a new series of coumarin-type inhibitors, characterized by an alkyl, aryl ester, thioester, amide or ketone function in position 3 and electrophilic moiety in the 6position, were synthesized [158-160]. The presence of a strong electron-withdrawing group at the 3-position is ex-
Since coumarins have been approved for used as a therapeutic drug, concerns have been raised as to its side effects regarding toxicity, metabolism and pharmacokinetics. The toxicology of coumarin began to receive intensive attention after it was revealed that coumarins had marked species differences in both metabolism and hepatotoxicity [151]. There are important, quantitative differences between species in the routes of elimination of coumarin metabolites. After oral administration 83% of the dose (200mg/kg) is excreted in in the human urine within 24h, primarily as 7-HC, which indicates that there is little or no biliary excretion of coumarin metabolites in humans. 7-HC and its glucuronide and sulfate conjugates are non-toxic and represent 40-97% of total urinary metabolites.In rat, only 35% is found in the urine given
Wu et al.
OH MeO O O a: OAc b: OBz O O O Me
HO O O
O O
O O
HO
O 69
OH OH
70
68
R1
OH Me
HO O O
O O
O O
O R2
O R1 OCH3 OCH3 H R2 H OCH3 OCH3
OH OH
71
72
Bergapten Isopimpinellin Xanthotoxin
pected to increase the electrophilicity of the lactone group, thus facilitating the nucleophilic attack by the serine hydroxyl residue. It appeared that esters in position 3 of the 6chloromethylcoumarin had more potent capacity to inactivate -chymotrypsin (-CT) and human leukocyte elastase (HLE) than amides. Moreover, the presence of an aromatic group (arylalkylcycloalkyl) strongly enhanced the inhibitory potency when the aryl side-chain was directly linked to the oxycarbonyl moiety [158-159]. The meta chlorophenyl derivative appeared to be one of the most potent -CT inactivators, and among the phenyl derivatives, the most efficient compounds possess a dichlorophenyl ester in the position 3. However, the presence of a substituent on the para position relative to the oxygen atom of the exocyclic ester function led to compounds with a lower inhibitory potency against -CT and HLE. To evaluate the importance of a latent alkylating function of position 6, the substituent in this position was modulated for -CT, the replacement of the chloromethyl moiety then led to the complete loss of inhibitory potency [159]. Scopoletin, derived from P. sabulosa, produced a doserelated anti-nociception in the acetic acid-induced model of visceral pain in mice. Two structural derivatives of scopoletin, acetylscopoletin (68a) and benzoylscopoletin (68b) also exhibited antinociceptive activity. Benzoylscopoletin decreased the acetic acid-induced abdominal constriction 2-fold more than the original molecule. Structure activity relationship studies showed that the addition of a benzoyl group might increase the absorption of the compound or facilitate its binding to the active site in order to increase antinociceptive properties. The addition of an acetyl group, as in acetylscopoletin, decreases the anti-nociceptive properties of the compound by approximately 26-fold compared to the original coumarin [161]. Sets of coumarinyl ethers having chromone, benzofuranyl and 4-hydroxy coumarins were prepared and tested for analgesic and anti-inflammatory activity. The results showed that these heterocyclic derivatives exhibited both anti-inflammatory and analgesic activity. The benzofuranyl ethers of coumarins were found to be most active
amongst all the compounds. The Chloro and methoxy substitution in coumarin ring was found to increase activity [161]. Three major coumarins, edgeworin (EdN) (69), edgeworosides A and C (EdeA (70) and EdeC (71)), isolated from Edgeworthia chrysantha L., were evaluated for both anti-inflammatory and analgesic activities. The results showed that EdN and EdeA had anti-inflammatory (p0.050.01) and analgesic (p0.001) effects, while EdeC showed only an analgesic effect [13]. Bergapten was also reported to exhibit significant anticonvulsant activity [162]. Umbelliferone extracted from the root bark of Adina cordifolia exhibited a strong anti-amoebic activity, and its derivatives, thiosemicarbazones, when substituted with adamantamine, p-benzyl piperidine and Nmethyl benzyl amine showed an even greater anti-amoebic activity [163]. In addition, coumarins with weak estrogenic activity have been explored for potential medical interest. Previous work showed that the derivatives of these compounds could be used as therapeutic agents to prevent the emergence of adverse effects associated with menopause, such as osteoporosis, cardiovascular risk (atherosclerosis) and cognitive deficiency [164]. CONCLUSION Coumarins have many different structures, due to the various types of substitutions at their core, which can in turn influence their biological activity. They constitute an important class of pharmacological agents possessing a range of different physiological activities including anti-cancer, antileukemia, anti-inflammation, anti-HIV, anti-coagulant, antibacterial, analgesic and comparative immunomodulation. Of great interest is the possibility that this class of molecules could be a source of drugs for the therapy of several diseases, including cancer, mycosis fungoides. To fully describe the recent progress in structure modification and the structure activity relationship is not an easy task. Nevertheless, it is useful to build up some correlations with the data available in order to help researchers in discovering and de-
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veloping new active compounds. In summary, the simple chemical structure of the coumarins allows great potential to clinically explore combinations of coumarin analogues with other agents in an attempt to improve efficacy for improving clinical outcomes. ACKNOWLEDGEMENTS This work is supported by National High-tech Grant No.2005AA0216050 and No. 2008AA02Z135 and Quanzhou City Science and Technology Grant 2007Z41 to RAXU. ABBREVIATIONS ADHC AFB1 ALT ARE BVDV bZIP -CT CYP DAMC DAMTC DCK DHC DHMC 6,7-diHC DMAHB EGF ERK FLAP GSH GST 4-HC 7-HC HCV 5-HETE HHT HLE o- HPA o- HPAA o- HPE o- HPLA o- HPPA = 3-amino-4, 7-dihydroxycoumarin = AflatoxinB1 = alanine aminotransferase = antioxidant response element = HCV surrogate viruses = basic region leucine zipper = -chymotrypsin = Cytochrome P450 = 7,8-diacetoxy-4-methylcoumarin = 7,8-diacetoxy-4-methylthiocoumarin = 3',4'-di-O-(s)-camphanoyl-(+)-ciskhellactone = dihydrocoumarin = 7, 8-dihydroxy-4- methylcoumarin = 6,7-dihydroxycoumarin = 3-dimethylallyl-4- hydroxybenzoyl = epidermal growth factor = extracellular-regulated kinase = lipoxygenase activating protein = glutathione = glutathione S-transferase = 4-hydroxycoumarin = 7-hydroxycoumarin = Hepatitis C Virus = 5-hydroxyeicosatetraenoic acid = 12-hydroxy-5, 8, 10-heptadecatrienoic acid = human leukocyte elastase = o-hydroxyphenylacetaldehyde = o-hydroxyphenylacetic acid = o-hydroxyphenylethanol = o-hydroxyphenyllactic acid = o-hydroxyphenyl propionic acid
HUVEC JNK LOX M4Beu MDR MMP MV NMBA NSCLC 6-NO2-7-HC NOS NQO1 4-NQO Nrf2 2OGD PAF PARP PBBK PPAR-c QR RCC ROIs ROS SAPK SAR TACE TPA VEC VEGF VSMC XO XRE REFERENCES
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= human umbilical vein endothelial cell = c-Jun N-terminal kinase = lipoxygenase = Metastatic pigmented malignant melanoma = multidrug resistant = Matrix Metalloproteinase = measles virus = N-nitrosomethylbenzylamine = non-small cell lung carcinoma = 6-nitro-7- hydroxycoumarin = nitric oxide synthase = NAD(P)H quinine oxidoreductase = 4-nitroquinoline 1-oxide = Nuclear factor-erythroid 2-related factor 2 = 2-oxoglutarate-dependent dioxygenase = platelet activating factor = poly (ADP-ribose) polymerase = physiologically based biokinetic = peroxisome proliferator-activated receptor-c = quinine reductase = renal cell carcinoma = reactive oxygen intermediates = reactive oxygen species = stress-activated protein kinase = structure activity relationship = TNF converting enzyme = 12-O-tetradecanoylphorhol-13-acetate = vascularendothelial cells = vascular endothelial growth factor = vascularsmoothmuscle cells = xanthine oxidase = xenobiotic response element
3,6,8-NO2-7-HC = 3,6,8-nitro-7-hydroxycoumarin
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Wu et al. xylic acid as a core structure for specific inhibitors of human leukocyte elastase. J. Med. Chem. 1999, 42(20), 4161-71. Pochet, L.; Doucet, C.; Schynts, M.; Thierry, N.; Boggetto, N.; Pirotte, B.; Jiang, K.Y.; Masereel, B.; de Tullio, P.; Delarge, J.; Reboud-Ravaux, M. Esters and amides of 6-(chloromethyl)-2-oxo2H-1- benzopyran-3- carboxylic acid as inhibitors of alphachymotrypsin: Significance of the ''aromatic'' nature of the novel ester-type coumarin for strong inhibitory activity. J. Med. Chem. 1996, 39(13), 2579-85. Pochet, L.; Doucet, C.; Dive, G.; Wouters, J.; Masereel, B.; Reboud-Ravaux, M.; Pirotte, B. Coumarinic derivatives as mechanism-based inhibitors of alpha-chymotrypsin and human leukocyte elastase. Bioorg. Med. Chem. 2000, 8(6), 1489-501. Meotti, F.C.; Ardenghi, J.V.; Pretto, J.B.; Souza, M.M.; AvILA, M.J.; Junior, A.C.; Soldi, C.; Pizzolatti, M.G.; Santos, A.R.S. Antinociceptive properties of coumarins, steroid and dihydrostyryl-2pyrones from Polygala sabulosa (Polygalaceae) in mice. J. Pharm. Pharmacol. 2006, 58, 107-112. Tosun, F.; Kizilay, C.A.; Erol, K.; Kilic, F.S.; Kurkcuoglu, M.; Baser, K.H.C. Anticonvulsant activity of furanocoumarins and the essential oil obtained from the fruits of Heracleum crenatifolium. Food Chem. 2008, 107, 990-993. Prince, F.I.; Abdul, R.B.; Amir, A. Antiamoebic coumarins from the root bark of Adina cordifolia and their new thiosemicarbazone derivatives. Eur. J. Med. Chemi. 2009, 44(5), 2252-2259. Usui, T. Pharmaceutical prospects of phytoestrogens Endocr. J. 2006, 53, 7.
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[158]
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Received: June 08, 2009
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Current Medicinal Chemistry, 2009, 16, 4236-4260

The Structure and Pharmacological Functions of Coumarins and Their Derivatives

The Structure and Pharmacological Functions of Coumarins

Current Medicinal Chemistry, 2009 Vol. 16, No. 32

HO OH coumaroyl CoA OH coumaroyl OH caffeoylquinate SCoA

OH O O OH umbelliferone O O esculetin HO R 2'-hydrox caffeic acid R E-caffeate SCoA feruloyl-CoA HO O HO OH HO O O

OH R O HO O SCoA 6'-hydroxyferuloyl-CoA O MeO O spirodienone

Fig. (2). The biosynthetic pathways of coumarins in plants [16, 22].

4238 Current Medicinal Chemistry, 2009 Vol. 16, No. 32

CH2CHO OH o-HPA CH2CH2OH o-HPAA

Fig. (3). Representative pathways of coumarin metabolism [26, 27].

The Structure and Pharmacological Functions of Coumarins

Current Medicinal Chemistry, 2009 Vol. 16, No. 32

4240 Current Medicinal Chemistry, 2009 Vol. 16, No. 32

Coumarins that have Been Used as Anti-Cancer Medicines so Far

6-substituted 2-oxo-2H-1-benzopyran-3carboxylic acid derivatives 7-HC

HT 1080 fibrosarcoma cells

V2 cancer cells Malignant melanoma

[Thornes et al., 40] [Thornes et al., 41]

The Structure and Pharmacological Functions of Coumarins

Current Medicinal Chemistry, 2009 Vol. 16, No. 32

Biological Pathways of Representative Coumarins

Compounds 4-HC 6-NO2-7-HC 7-HC Auraptene DAMC Daphnetin

DBC Decursin DHMC Dicoumarol Esculetin Imperatorin Scopoletin

4242 Current Medicinal Chemistry, 2009 Vol. 16, No. 32

The Structure and Pharmacological Functions of Coumarins

Current Medicinal Chemistry, 2009 Vol. 16, No. 32

4244 Current Medicinal Chemistry, 2009 Vol. 16, No. 32

The Structure and Pharmacological Functions of Coumarins

Current Medicinal Chemistry, 2009 Vol. 16, No. 32

30: R1=H, R2=OH 31: R1=OH, R2=H

4246 Current Medicinal Chemistry, 2009 Vol. 16, No. 32

The Structure and Pharmacological Functions of Coumarins

Current Medicinal Chemistry, 2009 Vol. 16, No. 32

4248 Current Medicinal Chemistry, 2009 Vol. 16, No. 32

O 45 R2 H N O a: R1=6-OMe, R2=Me b: R1=6-Me, R2=Me c: R1=6-Me, R2=allyl d: R1=6-Me, R2=benzyl

The Structure and Pharmacological Functions of Coumarins

Current Medicinal Chemistry, 2009 Vol. 16, No. 32

The Relationships Between the Structure and the Function of Coumarins

Compounds Anti-bacterial coumarins

Anti-fungal coumarins Anti-HIV coumarins Coumarins

A free 6-OH was essential for anti-fungal activity.

[Sardari et al., 133]

[Okamoto et al., 137]

4250 Current Medicinal Chemistry, 2009 Vol. 16, No. 32

The Structure and Pharmacological Functions of Coumarins

Current Medicinal Chemistry, 2009 Vol. 16, No. 32

4252 Current Medicinal Chemistry, 2009 Vol. 16, No. 32

OH O O O MeO OH OH O 57 NH2 CH2 MeO N H3CH2CH2CO NO2 59 60 R' O O EtO O O O2N 56 58 Me O O

The Structure and Pharmacological Functions of Coumarins

Current Medicinal Chemistry, 2009 Vol. 16, No. 32

4254 Current Medicinal Chemistry, 2009 Vol. 16, No. 32

OH MeO O O a: OAc b: OBz O O O Me

O R1 OCH3 OCH3 H R2 H OCH3 OCH3

Bergapten Isopimpinellin Xanthotoxin

The Structure and Pharmacological Functions of Coumarins

Current Medicinal Chemistry, 2009 Vol. 16, No. 32

Current Medicinal Chemistry, 2009 Vol. 16, No. 32

Current Medicinal Chemistry, 2009 Vol. 16, No. 32

Received: June 08, 2009

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