The Structure and Pharmacological Functions of Coumarins and Their
Derivatives
L. Wu1, X. Wang
1, W. Xu
2, F. Farzaneh
3 and R. Xu*
,1
1Engineering Research Center of Molecular Medicine, Ministry of Education & Institute of Molecular Medicine,
Huaqiao University, Fujian, 362021, China
2Faculty of Science, University of New South Wales, Sydney, NSW 2052, Australia
3Department of Molecular Medicine, King’s College, London, UK
Abstract: Coumarins are of many different structures. They constitute an important class of pharmacological agents pos-sessing a range of different physiological activities including anti-cancer, anti-oxidant, anti-inflammation, anti-HIV, anti-coagulant, 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 prolifera-tion 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 S-transferases (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 anti-coagulant commonly for preventing venous thromboembolism following orthopedic surgery, recurrent myocardial infarc-tion 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 mo-lecular 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 re-lationship between structure and activity.
Coumarins (also known as 1,2-benzopyrone or, less com-monly, 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]. In-spection 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 permuta-tions offered by substitution and conjugation, and this read-ily 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 phar-macological 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,
*Address correspondence to this author at the Engineering Research Center
of Molecular Medicine, Ministry of Education, Huaqiao University Main
(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 re-cent years. However, the details of relationship between the structure and activity of coumarins remain obscure.
ANABOLIC AND CATABOLIC PATHWAYS OF COUMARINS
Coumarins naturally occur in many plants, primarily in angiosperm, including Umbelliferae, Rutaceae, Legumino-sae, 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 pyra-nocoumarins 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
The Structure and Pharmacological Functions of Coumarins Current Medicinal Chemistry, 2009 Vol. 16, No. 32 4237
O O
3
45
6
7
8
1 2
O OO
3
O OO
OO
O O
4
OO
5
Fig. (1). Chemical Structure of Coumarins: 1) simple coumarins, 2) furanocoumarins (can be further grouped into linear and angular types),
Fig. (2). The biosynthetic pathways of coumarins in plants [16, 22].
4238 Current Medicinal Chemistry, 2009 Vol. 16, No. 32 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 indi-cate that they would be catalyzed by different enzymes. Cin-namate 4-hydroxylase, a cytochrome P450 (CYP) monooxy-genase from the CYP73A family, catalyzes the conversion of cinnamic acid to 4-coumaric acid, with the formation of um-belliferone [23]. Classically, umbelliferone rather than cou-marin has been considered the parent compound of furano-coumarins. The biosynthetic route of scopoletin has been reported in Arabidopsis thaliana recently [19, 24]. It is re-ported that scopoletin is biosynthesized from ferulic acid rather than via the umbelliferone-esculetin- scopoletin path-way [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 F6’H1. 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 investi-gated the biosynthetic pathways of scopoletin by using stable isotope labeling to study post-harvest physiological deterio-ration in Cassava roots [22]. Bayoumi et al also demon-strated that the major pathway was through o-hydroxylation but not via a proposed spirolactone-dienone intermediate by feeding C
18O2-carboxylate- labelled cinnamic and ferulic
acids [22]. The biosynthesis of linear and angular furano-coumarins is still poorly understood at the molecular level, with only psoralen synthase (CYP71AJ1) identified from Ammi majus. Three new members of the CYP71AJ subfam-ily (CYP71AJ2-4) were cloned by Larbat et al (2009) who
found that psoralen synthase (CYP71AJ3) and angelicin syn-thase (CYP71AJ4) showed 70% identity in sequence com-parison [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-hydroxy-coumarins (3-, 4-, 5-, 6-, 7- and 8-HCs) and by opening of the lactone ring to yield various individual products includ-ing o-hydroxyphenylacetaldehyde (o-HPA) (a major metabo-lite of coumarin in rat and mouse liver microsomes), o-hydroxyphenylethanol (o-HPE), o-hydroxyphenylacetic acid (o-HPAA) and o-hydroxyphenyllactic acid (o-HPLA). Addi-tional metabolites of coumarin include 6,7-dihydroxy-coumarin(6,7-diHC), o-coumaric acid (o-CA), o-hydroxy-phenyl propionic acid (o-HPPA) and dihydrocoumarin (DHC) [26, 27].
Two important pathways for coumarin metabolism are 7-hydroxylation and metabolism of the lactone ring which in-volves 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, 4-epoxide 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 metabo-lized 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 7-hydroxylation 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 me-tabolism in biological systems. Enzymes from the CYP1,
O O
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DHC
O O
OH
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OH
CH2CHOHCOOH
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coumarin-3'4-epoxide
o-HPAo-HPAACO2
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CO2
OH
CH2CH2OH
o-HPE
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4-HDHC-GSHconjugate
SG
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Fig. (3). Representative pathways of coumarin metabolism [26, 27].
The Structure and Pharmacological Functions of Coumarins Current Medicinal Chemistry, 2009 Vol. 16, No. 32 4239
CYP2 and CYP3 families are generally regarded as repre-senting the major catalysts for xenobiotic and drug metabo-lism 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 7-hydroxylation 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 in-volved 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 orien-tation. For example, the oxidation of 7-ethoxy and 7-methoxy-coumarin by CYP2A6 is less regiospecific and occurs on opposite ends of the substrate, resulting in O-dealkylation or 3-hydroxylation [32]. Mutation of residue Asn297 had been shown to influence substrate binding and metabolism. An N297S mutant was determined to have 4-fold decreased catalytic efficiency for coumarin 7-hydroxylation [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, 4-epoxidation 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 pres-ence 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 3-hydroxycoumarin by CYP3A4 and to a lesser extent by other P450s [36]. Modeling by homology with the CYP2C5 crys-tallographic template, CYP1, CYP2 and CYP3 famlies in human P450 were proved in agreement with the known me-tabolism of coumarin, and with information from site-directed mutagenesis studies [37]. Little information about metabolism of furanocoumarins is available. Xenobiotic-metabolizing 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 to-ward the angular furanocoumarins. Modeling programs stud-ies 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 her-bivorous 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], anti- inflammation [5-6], anti-platelet aggregation [7-8], anti-can-cer [9-10], anti-convulsant [11], comparative immunomodu-latory [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 War-farin sodium (6) on V2 cancer cell, granulocytes, lympho-cytes and macrophages in different animal models [40]. Clinical trials demonstrating activity in many different can-cers, including prostate cancer, malignant melanoma and metastatic renal cell carcinoma have also been reported. En-couraginglly, 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 with-out 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 in-clude the observations that 4-HC decreases tyrosine phos-phorylation of several proteins in melanoma cells line B16-F10 [42], whereas 7-HC inhibits myosin light chain kinase [43] and disrupts the formation of the mitotic spindle micro-tubules in Allium cepa cells, leading to the random distribu-tion of the chromosomes at metaphase. This is a form of cy-totoxicity common to mitotic spindle poisons that inhibit mitosis through modifying microtubule dynamics [44], sug-gesting 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, in-cluding 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=7.67 M) and PKA (IC50=9.33 M) and PKC (IC50=25.01 M) 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-
4240 Current Medicinal Chemistry, 2009 Vol. 16, No. 32 Wu et al.
Table 1. Coumarins that have Been Used as Anti-Cancer Medicines so Far
Compounds Cell lines/cancers References
B16-F10 [Velasco-Velazquez et al., 42] 4-HC
HepG2 cells [Yang et al., 46]
SK-MEL-31cells [Finn et al., 60] 6-nitro-7-hydroxycoumarin derivatives
A-498 [Finn et al., 62]
6-substituted 2-oxo-2H-1-benzopyran-3-
carboxylic acid derivatives
HT 1080 fibrosarcoma cells [Win et al., 9]
Gastric carcinoma cells [Weber et al., 39]
Caco-2 [Weber et al.,39]
Allium cepa cells [Podbielkowska et al., 44]
HepG2 cells [Yang et al., 46], [Weber et al., 39]
NSCLC [Lopez-Gonzalez et al., 47]
SK-MEL-31cells [Finn et al., 60]
7-HC
Lymphoblastic cells [Weber et al., 39]
7-diethylaminocoumarins HUVEC [Seokjoon et al., 64]
HL-60 cells [Kurakami et al., 67]
Oral and liver cancers [Tanaka et al., 68]
Esophageal cancer [Kawabata et al., 69]
Colon cancers [Kohno et al., 71],
[Kawabata et al., 72]
B16BL6 melanoma cell [Tanaka et al., 76]
Auraptene
M4Beu [Barthomeuf et al., 77]
Coumarin Malignant melanoma [Thornes et al., 41]
Compound 14 MCF-7 [Jacquot et al., 66]
HepG2 cells [Yang et al., 46] Daphnetin
A-498 [Finn et al., 48]
Human K562 erythroleukemia [Kim et al., 78], [Kim et al., 79] Decursin
U937 cells [Kim et al., 78]
U937 cells [Riveiro et al., 54]
HL-60 cells [Riveiro et al., 54]
DHMC
A549 (NSCLC) [Goel et al., 55], [Goel et al., 56]
HepG2 cells [Yang et al., 46]
U937 cells [Lee et al., 49], [Park et al., 50]
HL-60 cells [Chu et al., 3], [Wang et al., 58]
Esculetin
MCF-7 cells [Kolodziej et al., 57]
Imperatorin, isopimpinellin Mammary carcinogenesis [Prince et al., 81]
Scopoletin HL-60 cells [Kim et al., 51], [Kim et al., 52]
Silver coumarins HepG2 cells [Wang et al., 58],
[Bhumika et al., 59]
V2 cancer cells [Thornes et al., 40] Warfarin sodium
Malignant melanoma [Thornes et al., 41]
The Structure and Pharmacological Functions of Coumarins Current Medicinal Chemistry, 2009 Vol. 16, No. 32 4241
Table 2. Biological Pathways of Representative Coumarins
Compounds Biological Responses and Pathways Reference
4-HC Decreasing tyrosine phosphorylation [Velasco-Velazquez et al., 42]
6-NO2-7-HC Activation of p38, SAPK, and MAPK; blocking at sub-G1 phase [Finn et al., 62]
7-HC Inhibiting myosin light chain kinase; disrupting the formation of the mitotic spindle microtubules; blocking in G1 phase
[Wang et al., 43], [Lopez-Gonzalez et al., 47]
Auraptene Elevation in the phase enzymes GST and QR; suppressing MMP-7, -2, -9 expression [Tanaka et al.,68], [Kawabata et al., 72]
DAMC Inhibition of the catalytic activity of GST [Raj et al., 98]
Daphnetin Inhibiting activities of protein kinases, including EGF receptor tyrosine kinase, PKA, PKC; up-regulation of p38MAPK; blocking in S phase at low concentration but in G1 and early S phase at
higher concentration
[Yang et al., 46], [Finn et al., 48]
DBC Inhibiting microtubule functions and triggering apoptosis [Kim et al., 65]
Decursin Down-regulation of PKC and II [Kim et al., 78]
DHMC Activation of JNKs; inhibition of ERK1/2, PI3K/Akt; induction of p21WAF1/CIP1 [Riveiro et al., 54]
Dicoumarol Mediation of tubulin binding and stabilization of spindle microtubule dynamics; inhibiting the release of MMPs; inhibiting the pathway involving tissue factor and factor VIIa.
[Madari et al.,45], [Li et al., 84]
Esculetin Induction of p21; decreasing the activity of Cdks; inhibition of pRB phosphorylation; mitochon-drial dysfunction; activation of caspase-3
[Lee et al., 49], [Park et al., 50], [Chu et al., 3]
Imperatorin Inhibition of CYP1A1/1B1 and induction hepatic GSTs to block the formation of DNA adducts [Prince et al., 81]
Scopoletin Activation of NF- B and caspase-3 [Kim et al., 51],
[Kim et al., 52]
theless, Lopez-Gonzalez reported that coumarin and umbel-liferone (8) showed significant inhibition of proliferation of non-small cell lung carcinoma (NSCLC), and that umbellif-erone blocked the cell cycle in the G1 phase and inducing apoptosis [47]. Daphnetin was demonstrated to have the anti-prolierative 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 concentra-tions 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 ap-proached 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 ac-tion 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 apopto-sis in human promyeloleukemic cells and found that sco-poletin 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 inter-mediates (ROIs) might be an important factor in scopoletin-induced apoptosis of HL-60 cells. The activation of caspase-3 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 adeno-carcinoma cell lines via the same pathway [47]. 7, 8-dihydroxy-4- methylcoumarin (DHMC) was reported to in-duce selective and concentration-dependent apoptosis in hu-man 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 proto-oncogene c-myc as well as induction of the cell cycle inhibi-tor 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=160 g/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-4-methylcoumarin (DAMC) and its thiocoumarin derivative
4242 Current Medicinal Chemistry, 2009 Vol. 16, No. 32 Wu et al.
7,8-diacetoxy-4-methylthiocoumarin (DAMTC) was found to exhibit similar toxic activity (A549 IC50=160 g/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 sub-stitution in esculetin formation [57]. This activity is further confirmed by a recent report regarding esculetin-induced inhibition of cell cycle progression in human leukemia HL-60 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 cis-platin [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 irre-versible cytotoxic agents and to inhibit the S phase regula-tory protein, cycliin A, so as to inhibit DNA synthesis, but 7-HC 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, stress-activated protein kinase (SAPK) and MAP kinase in the hu-man 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 pharma-cotherapy 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 4-position greatly promoted the bioactivity and compound (10) could strongly inhibits the proliferation of various different
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The Structure and Pharmacological Functions of Coumarins Current Medicinal Chemistry, 2009 Vol. 16, No. 32 4243
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 bio-chemical assays to show that 6-hydroxycoumarin -3-carboxylatosilver was capable of inducing apoptotic cell death in Hep-G2 cells by increasing the activity of pro-apoptosis caspases 3, and through the cleavage of PARP, which is one of the substrates for caspases 3, thereby de-creasing the percentage of cells entering G0/G1 [59]. Jacquot et al reported that 2, 4-diaryl-4H, 5H-pyrano [3,2-c]benzopyran-5-ones (14) exhibited a strong antiproliferative activities in MCF-7 breast carcinoma cells [66].
Auraptene (15) and umbelliprenin (16) are both coumar-ins 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-13-acetate (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 devel-opment of oral neoplasms induced by 4-nitroquinoline 1-oxide (4-NQO). This mechanism might be related to eleva-tion in the phase enzymes glutathione S-transferases (GST) and quinine reductase (QR) of the liver and tongue [68]. Die-tary auraptene was also reported to be effective in inhibiting the development of esophageal tumors by N-nitrosomethyl-benzylamine (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 epithe-lium [69]. Besides, auraptene was also reported be a poten-tial 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 ex- pression in lipopolysaccharide-stimulated inflammatory cells [74] and decrease lipid peroxidation and experimental car- cinogenesis 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 dephos- phorylation of constitutively activated extracellular signal--
regulated kinase (ERK)1/2, without affecting its mRNA ex- pression level [72]. Tanaka et al reported that auraptene could significantly reduce the growth and number of metas- tatic 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 caspase- inpendent 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 tu-mor-suppressing PKC activator that induces the down-regulation of PKC and II and promotes their translocation into the nuclear membrane of K562 cells [78], and competi-tively inhibits the binding of PKC and phorbol 12,13-dibutyrate (PDBu), which induces the megakaryocytic dif-ferentiation of K562 through PKC activation [79]. The struc-ture activity relationship of decursin has led to suggestion that the coumarin structure is required for anti-leukemic ac-tivity 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 proc-esses of cell division in order to disrupt tumor cell prolifera-tion, 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 iden-tified as MAP kinase, ERK kinase inhibitors (MEK1 inhibi-tors).This has led to the development of two chemical ana-logue (19) and (20) which also show anti-cancer activity through inhibition of MEK1[80]. These two coumarin de-rivatives display high antiangiogenic activity both in vitro and in vivo [53]. Several 6-substituted 2-oxo-2H-1-benzopyran-3-carboxylic acid derivatives had been synthe-sized, 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 OO
O
O
17
O OON
O
F
18 19 X=Cl 20 X=Br
O O
O
O XO
O
4244 Current Medicinal Chemistry, 2009 Vol. 16, No. 32 Wu et al.
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 effi-ciency, 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 effi-cacy, 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. Un-til recently, the pyranocoumarin compound (±)-3-angeloyl -4-acetoxy- cis-khel-lactone was isolated from Radix peuce-dani and caused apoptotic cell death for drug sensitive KB-3-1 and MDR KB-V1 cancer cell lines by the mechanism of being a P-gp inhibitor [82].
Some coumarins possess enhancing effects on lympho-cyte 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 de-crease 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. Sco-poletin-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 (en-hanced lymphocyte activation) and type 3 (enhanced secre-tion of IFN ). Isopimpinellin, bergapten, and xanthotoxin (72) may also serve as candidates for the immuno-stimulating activity of IFN secretion. Among the three coumarins tested, two types of immunomodulation were ex-hibited 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 immuno-modulation of bergapten and xanthotoxin (one methoxy group and elevation of IFN secretion), whereas the position
of the methoxy group affected the immunomodulating po-tency such as bergapten (5-methoxypsoralen) and xantho-toxin (8-methoxypsoralen) [12].
ANTI-COAGULANT AND ANTI-PLATELET
Some coumarins, primarily 4-HCs and their derivatives, are conferred to function as anti-coagulants. Coumarin anti-coagulants 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 mo-lecular and cellular mechanism of action of coumarin anti-coagulants in the treatment of malignancies. Tumor cell in-vasion is dependent on angiogenesis and requires both cell migration and the digestion of basement membrane via pro-teases. Dicoumarol (3, 3’-methylenebis [4-hydroxy-cou-marin] (24) is a natural anti-coagulant drug. The antiprolif-erative 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 ex-tracellular matrix proteins, it is logical to assume that dicou-marol might enhance the antiadhesive activity of taxanes. Coumarin anti-coagulants inhibit the pathway involving tis-sue 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 demon-strated 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, dicouma-rol and other coumarin anti-coagulants might have other new tumor specificities other than those currently used as anti-mitotic 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 cou- marin 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 thromboem- bolism following orthopedic surgery. The coumarin deriva- tives 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-
O O
OCH3
H3CO
21
O
O
22
O O
OO O
OCH3
OCH3
23
O OO
OHOH
O
24
The Structure and Pharmacological Functions of Coumarins Current Medicinal Chemistry, 2009 Vol. 16, No. 32 4245
terial as biologically active. As more applications are devel- oped, 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 re-sponsible 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 pre-formed ring system simulating cyclic acetals. (3) Coumarin derivatives, devoid of an oxygen function in the 4-position substitute, like 3- and 4-phenylcoumarin and 4-methylcoumarins. Loading the molecule with additional methoxyl groups either potentiated or inhibited activity de-pending on the positions of the substitute [88]. Thus there is considerable difference in activity between the isomeric di-coumarol derivatives, 3, 3’-methylenebis (4-hydroxy-5, 7- dimethoxycoumarin (25) and 3, 3’-methylenebis (4-hydroxy-7,8-dimethoxycoumarin) (26) . The higher activity of the compound with substitutes in position 8 showed the impor-tance of location. Similar findings exist for the 4-hydroxycoumarins, 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 de-rivatives and that of their corresponding methyl ethers. Thus, invariably, the methylation of a free hydroxyl group in-creased 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 antico-agulant activity, whereas introduction of a hydroxyl, methoxyl or benzamido group in position 3 of coumarin de-creases the anti-coagulant activity [88] (Table 3).
8-methyl-4-(1-piperazinyl)-7-(3-pyridinylmethoxy)-2H-1-benzopyran-2-one (RC414) (28) was tested for its effect on human platelets. The results revealed that RC414 in a dose-dependent 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, 0.98±0.36 uM and 1.00 ± 0.15 uM, respectively [89]. RC414 increased cAMP levels, through the specific inhibition of the cAMP high affinity phosphodiesterase (IC50=1.73±0.35 uM). RC414 reduced [Ca
2+]i transients and PKC activation induced by thrombin.
In addition RC414 was able to increase nitric oxide forma-tion 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 de-rivative (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
OC2H5 OCH(CH3)2 or OCH2C6H5. In turn, the 8-methyl substitution increased the anti-platelet activity of the 7-alkoxy substituted compounds [90]. Two coumarins deriva-tives (30) and (31) have been reported to allow the relaxation of vascular smooth muscle and inhibiting platelet aggrega-tion with a profle similar to that of trans-resveratrol (t-RESV) (32). Compound (31) has a vaso-relaxant activity that
O OH3CO
OCH3 OH
O OCH3
OCH3OH
O
H2C
25
O OH3CO
OH
O OCH3
OCH3OH
O
H2C
26
OCH3
O OHO
CH327
O OO
CH3
N28
N
NH
O O
N NH
29 OH
OH
OH
32
O OR2
R1
OH
OH
30: R1=H, R2=OH
31: R1=OH, R2=H
O OH3CO
O
H
O
33
O OH3CO
34
O
O
O
4246 Current Medicinal Chemistry, 2009 Vol. 16, No. 32 Wu et al.
is twice as high as that of t-resveratrol and a platelet anti-aggregant 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 se-necioate (34), were isolated from the leaves of Murraya om-phalocarpa 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 plate-let 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 substitu-tions in the basic molecule may influence the structure-related biological activities of coumarins [93]. They act as anti-oxidants, enzyme inhibitors and precursors of toxic sub-stances. 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 anti-oxidants 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, es-culetin, scopoletin, DHC, 4-methylesculetin, and 7-hydroxy-4-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 mo-lecular 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 sub-stituted 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 at-tributed 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 en-zyme proteins which could putatively result in the modifica-tion 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 acetyla-tion 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 experi-ments. 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 ob-tained when the TAase catalyzed activation of NADPH cy-tochrome c reductase assay and AflatoxinB1 (AFB1)-DNA binding inhibitory assay were performed with 4-phenyl-coumarins and 4-methylcoumarins [96]. These results con-firmed the hypothesis that DAMC was found to modulate the activities of enzymes including cytochrome P-450-linked mixed-function-oxidase (MFO), NADPH cytochrome c re-ductase 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 com-pares the specificities of the acetylated coumarins, biscou-marins, 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].
O OAcO
35
OAc O OAcO
36
OAc
The effects of coumarin anti-oxidants on tumor-modulation 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 struc-ture activity relationship [100]. It is known that GST induc-tion can occur through different mechanisms, such as via the xenobiotic response element (XRE) or the anti-oxidant re-sponse element (ARE). Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a member of the cap’n’ 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 en-zymes 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 activi-ties 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
The Structure and Pharmacological Functions of Coumarins Current Medicinal Chemistry, 2009 Vol. 16, No. 32 4247
in Jurkat cells [104]. The mode of procaspase-9 activation in order to inhibit apoptosis was probably through the reversi-ble 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 radi-cal 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 5-lipoxygenase bioassays and turned out to be a moderate and selective a 5-lipoxygenase inhibitor (IC50=36.2 μM) [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 ara-chidonate metabolism) and were found to inhibit selectively the proinflammatory 5-lipoxygenase enzyme with 5, 7-dihydroxy-4-methylcoumarin demonstrating a higher po-tency against cyclooxygenase [2]. It is well known that lipoxygenase (LOX) possess regiospecificity during interac-tion with substrates and on this basis have been primarily designed as arachidonate 5-, 12-, and 15-LOX. 5-LOX repre-sents a dioxygenase that possesses two distinct enzymatic activities leading to the formation of LTA4, which is con-verted to 5-hydroxyeicosatetraenoic acid (5-HETE) or five lipoxygenase activating protein (FLAP).
Modeling and sequence comparisons of the human 12- and 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 sub-strate 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 pre-dicted 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 simu-lation and found that the inhibitors should share some analo-gous features: a polar head and tail interacting with the hy-drophilic portion of 5-LOX through hydrogen bonds or elec-trostatic interactions, and a hydrophobic body stretching into the large hydrophobic channel of 5-LOX to form strong hy-drophobic interactions with its surrounding lipophilic resi-dues [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 prolifera-tion and survival [111]. Esculetin can selectively inhibit LOXs with different IC50. In platelet, esculetin inhibit 12-LOX with IC50=0.65 μM [112]. Whereas esculetin inhibits the formation of 5-HETE with IC50= 1.46mM in polymor-phonuclear leukocytes, although more strongly than HHT (IC50= 57.3mM) [113]. The roles of 15-LOX in cancer de-velopment are unclear. However, esculetin can also inhibit the formation of 15-HETE from 15-LOX.
On investigation of specific products of the LOX path-way mediating the autoregulatory effect of glucose and glu-cose transport in vascularsmoothmuscle cells (VSMC) and vascularendothelial cells (VEC), esculetin (100 μM) was found to inhibit the formation of 12- and 15-HETE and de-crease 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 rela-tionship, the inhibitory potency on LOX is not related to the oxidation potential of the compound [115]. Esculin, glucosi-dation at one of the hydroxy group of esculetin, decreases the inhibitory potency markedly with IC50=290 μM. 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 μM) and prostaglandin syn-thesis [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-heptade-catrienoic acid (HHT)) in polymorphonuclear leukocytes
O OMeO
37
O O
38
39
O OHONHC
40
OO
N
O O
41
O OH3CHNN
O O
42
N
RO
Ha: R1=H, R=2-CO2Ac
b: R1=H, R=2-CO2Me
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
R1
4248 Current Medicinal Chemistry, 2009 Vol. 16, No. 32 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 anti-inflammatory 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 3-carboxamidocoumarins (42) had been synthesized through a Knoevenangel condensation of nitriles with salicylaldehyde
with salicylaldehydes in the presence of piperidine in etha-nol. 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 54±6.5%, 51±5.05%, 48±6.51% and 47±7.13% inhibition respectively (at doses of 10 mg/kg p.o.; piroxicam control showed 57±6.61% inhibition). In acetic acid peritonitis tests, the most active compounds (42a) and (42c) showed 42±7.20%, 40±6.43% and 39±5.09% inhibi-tion (at doses of 10 mg/kg p.o., piroxicam revealed a protec-tion of 29±7.24%). 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 se-lected to show potent inhibitory effects on iNOS protein ex-pression 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 re-ported that scopoletin (1–50 μg/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 concentration-dependent 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 lipopolysaccha-ride (LPS) challenged macrophages, and to block the LPS-induced activation of nuclear factor- B (NF- B) through the prevention of inhibitor- B degradation [120]. This com-pound also showed anti-oxidant properties and at a dose of 40mg/kg, and could inhibit iNOS and IL-1 gene expres-sion 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 inhibi-tors, 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 com-pounds (43) were 20-30 times more potent than the corre-sponding nonsubstituted counterpart. Alkyl groups, except a methyl group, at this position tended to decrease the activi-ties. 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 ac-tivity [121], (Table 3).
One of the more promising approaches to discover in-hibitors of TNF is the inhibition of a zinc containing metal-loproteinase, TNF converting enzyme (TACE), which con-verts membrane bound pro-TNF to mature and soluble TNF . A series of couamrin-base analogues (44) were pre-pared, 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 com-pounds paralleled TACE activity. Furthermore, 6-position methyl or methoxyl substituted analogues (46) were pre-pared. The analogues (46a) and (46b) showed IC50 at 36nM and 3nM, and 220nM and 320nM for TACE enzyme inhibi-tion and TNF inhibition, respectively. Allyl (46c) and ben-zylated (46d) analogues showed similar results at a range of 30nM and 5nM in the same assay [122].
O O
ArR
ON
O
43
O O
44
RNHOH
O
OO
NHO
O
H
45O O
R HN
OH
O
R2
46
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 4249
In addition, inflammation is well known as an important factor in the development of diabetes; since coumarin sup-presses 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 de-rivatives (oxycoumarin derivatives) activate peroxisome proliferator-activated receptor-c (PPAR-c) and exert a bene-ficial 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 bac-terial 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 de-rived from various Streptomyces species. Each compound contains an individual noviosyl sugar component that im-parts 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 in-creases in both anti-bacterial activity and in vitro activity against DNA gyrase. The aminocoumarin antibiotics novo-biocin and clorobiocin consist of a 3-amino-4, 7-dihydroxycoumarin (ADHC) moiety flanked on one side by L-noviose and on the other side by a 3-dimethylallyl-4- hy-droxybenzoyl (DMAHB) moiety. Both ADHC and L-noviose are essential for anti-bacterial activity and that the
Table 3. The Relationships Between the Structure and the Function of Coumarins
A free 7-OH was important for anti-bacterial activity [Sardari et al., 133]
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.
[Kayser et al., 131]
The lipophilic character and a planar structure are requirements for high anti-bacterial activities
[Kayser et al., 131]
Anti-bacterial
coumarins
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
[Hishmat et al., 128]
Anti-fungal
coumarins
A free 6-OH was essential for anti-fungal activity. [Sardari et al., 133]
Anti-HIV coumar-ins
SARs of DCK and Calanolides have been reviewed and summarized [Yu et al., 145]
Modeling and docking: carbonyl pointed toward the Arg880, and the ester O atom formed hydrogen bonds with Thr1010 in region of XO
[Masamoto et al., 94]
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
[Nakamura et al., 119]
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.
[Cheng et al., 121]
Coumarins
The presence of a strong electron-withdrawing group at the 3-position facilitates the nucleophilic attack by the serine hydroxyl residue in protease
[Béchet et al., 157]
DAMC Acetylation of several lysine residues in its active site of GST, but phenyl presences in C4 position will drastically reduce its specificity
[Raj et al., 98],
[Kumar et al., 99]
Modeling and docking: 6-hydroxyl interacts with the E802 residue of XO [Masamoto et al., 94] Esculetin
The hydroxylation at C6 is important for high selectivity of 12-LOX [Hardt et al., 116]
7-hydroxy-4-
methylcoumarin
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 7-hydroxy-4-methlcoumarin increases the anti-coagulant activity
[Arora et al., 88]
Osthole Methoxy-group at position-7 and the 3-methyl-2-butenyl- group at position-8 are essen-tial to lower plasma ALT in hepatitis
[Okamoto et al., 137]
4250 Current Medicinal Chemistry, 2009 Vol. 16, No. 32 Wu et al.
substituents attached to these fragments have a significant impact on their bioactivities. X-ray crystallographic exami-nation of antibiotic-enzyme complexes showed that the ADHC and L-noviose moieties are each involved in antibi-otic 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 patho-gens, and the proclivity of staphylococci to develop endoge-nous 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-dimethylallyl-4- hydroxybenzoyl moiety reduced biological activity [127]. The highest activities were shown by compounds containing a hydrophobic alkyl group at position 3 of the 4-hydroxybenzoyl moiety. Polar groups in this side chain, es-pecially 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-3 to O-2 of L-noviose only moderately reduced activ-ity, 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-amino-4,7-dihydroxycoumarin moiety also showed low activity. Lack of a methyl group at O-4 of 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
the size of the acyl group display an increasing range of anti-bacterial 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 modi-fied 6,8-dibromo-3-(w-bromoacetyl) coumarin increased further its anti-bacterial activity. The dichloroacetamido moi-ety 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 coumar-ins 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 diffu-sion 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 con-sidered. 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 7-OH was important for anti-bacterial activity [133] (Table 3).
O O
OHHN
O
OH
O
CH3
47
OH3CO
O O
H2N
OH2''
3''
1''
O O
OHHN
O
OH
O
ClO
H3CO
O O
48NH
OH
1''
2''
3''
O O
OHHN
OO
CH3O
OHO
O
H3CO
49NH
O
NH
OO
OH
CH3
O
O
HOO
OCH3
O
HN
The Structure and Pharmacological Functions of Coumarins Current Medicinal Chemistry, 2009 Vol. 16, No. 32 4251
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 streptococ-cus, streptococcus pneumoniae and haemophilus influenzae [131]).
A combination of these structural features, two methoxy functions and at least one additional phenolic group, as re-flected 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 addi-tion 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)-4- methyl-7-substituted coumarins (54) were synthesized and their anti-fungal activity investigated. Their anti-fungal ac-tivities were measured against a phytopathologic fungus, Botrytis cinerea [134]. Compounds with a substituted ben-zoyloxy, benzoyloxyethoxy, methylbenzoylaminoethoxy, or benzoylaminophenoxy group at the 7-position had an inhibi-tory 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 pyranocou-marin analogues were evalutated in vitro for antiviral effi-cacy against measles virus (MV). The structural activity rela-tionship study showed that the O-substituted 5,7-dihydroxycoumarins 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 in-hibitory effect upon oral administration. In a Con A-induced hepatitis mouse model, intraperitoneally administered ost-hole (100 mg/kg dose) resulted in 85% inhibition of Con A-induced 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 sub-stitution 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-butenyl- group at position-8 are essential to lower plasma ALT in hepatitis. Okamoto et al has screened 28 synthetic deriva-tives of osthole and found that most of them increased pro-tective abilities. Three compounds (57), (58), (59) exhibited 68.7 %, 62.5% and 88.3% inhibition of Con A-induced ele-vation 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 C-related 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 anti-HIV 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 inhi-bition and integration in the HIV replication cycle [140].
Warfarin and other 4-HC derivatives are the first genera-tion of HIV-PR inhibitors. Inspired by the structure, 3-phenyl-, 3-benzyl-, 3-phenoxy-, 3-benzenesulfonyl-, and 3-(7-coumairnyloxy)-4-hydroxycoumarin derivatives had been screened. These results revealed the importance of substitu-ents at position 5 and 7 of the coumarin ring on the inhibi-tory potency of the HIV-1-PR [141]. Position 5 and 3 were
O O
R
NH
O
CHCl250
O O
51
HN
O
O
N
O O
52
N N
NH
NH
OH
4252 Current Medicinal Chemistry, 2009 Vol. 16, No. 32 Wu et al.
important for not allowing both the introduction of a hy-droxyl 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) deriva-tives have been synthesized and examined for inhibition of HIV-1 replication in H9 lymphocytes [143]. Of the ten syn-thesized 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 re-viewed 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. How-ever, (-)-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 reso-nance of the coumarin system, resulting in decreased or completely lost activity. (4) Methyl or other aliphatic substi-tutions 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) 3-Hydroxymethyl and 3-dibromomethyl analogs retain po-tency, 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 activ-ity [146]. In a continuing effort to identify the pharmacopho-res 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 Calo-phyllum lanigerum were found to be HIV-1 reverse tran-scriptase inhibitors [148]. They utilize a novel mechanistic pathway that inhibited RT in two different template primer systems, primed ribosomal RNA template and homopolym-eric poly rA-oligodT12–18 template/primer [148]. Another group of tetracyclic pyranocoumarins, the inophyllums iso-lated from the genus Calophyllum ionophyllum P, are capa-ble of potent inhibition of HIV-1 RT, though the most active inophyllums (64) and (65) have slightly different stereo-chemistry 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 ino-phyllums 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
O OO
53
O O
54
OH
N
O O
55
O
HO
O O
56
O
O
OH
OH
OH
O OEtO
57
O OMeO
58Me
O2N
NMeO
59
NO2
O O
R'
H3CH2CH2CO
CH2
NH2
60
The Structure and Pharmacological Functions of Coumarins Current Medicinal Chemistry, 2009 Vol. 16, No. 32 4253
COOH-terminal zinc finger motifs. Imperatorin showed in-hibitory 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-1-LTR 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 ex-pression 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 pluri-costata, 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 FOR NEW COUMARIN DRUGS
Since coumarins have been approved for used as a thera-peutic 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 dif-ferences 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 indi-cates 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 uri-nary metabolites.In rat, only 35% is found in the urine given
an equivalent dose [152]. The significance of the 7-hydroxylation pathway is that it appears inversely related to species differences in hepatotoxicity. Epoxidation of the 3,4-double 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 enzy-matically by GST [154]. To obtain insight in whether de-creased 7-HC formation would result in increased liver lev-els 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 7-hydroxylation 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 metabo-lism- and species-dependent, the rat may not be a suitable model for evaluating the pharmacological hazards of cou-marin 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 ac-tivity. Halomethyldihydrocoumarin was the first suicide sub-strate of a serine protease [157]. Based on halomethyldihy-drocoumarin, a new series of coumarin-type inhibitors, char-acterized by an alkyl, aryl ester, thioester, amide or ketone function in position 3 and electrophilic moiety in the 6-position, were synthesized [158-160]. The presence of a strong electron-withdrawing group at the 3-position is ex-
O
61
O
O
H3C
O
O
O
O
O
O
OO
O
O OO
O
62
O OO
O
63
OH
O OO
O
64
OH
O OO
O
OH
O OHO
OH
66
O
O OHO
OH
67
O
65
4254 Current Medicinal Chemistry, 2009 Vol. 16, No. 32 Wu et al.
pected to increase the electrophilicity of the lactone group, thus facilitating the nucleophilic attack by the serine hy-droxyl residue. It appeared that esters in position 3 of the 6-chloromethylcoumarin had more potent capacity to inacti-vate -chymotrypsin ( -CT) and human leukocyte elastase (HLE) than amides. Moreover, the presence of an aromatic group (aryl alkyl cycloalkyl) strongly enhanced the in-hibitory 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 po-tent -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 exo-cyclic ester function led to compounds with a lower inhibi-tory potency against -CT and HLE. To evaluate the impor-tance of a latent alkylating function of position 6, the sub-stituent in this position was modulated for -CT, the re-placement of the chloromethyl moiety then led to the com-plete loss of inhibitory potency [159].
Scopoletin, derived from P. sabulosa, produced a dose-related anti-nociception in the acetic acid-induced model of visceral pain in mice. Two structural derivatives of sco-poletin, 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 relation-ship 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 antinocicep-tive properties. The addition of an acetyl group, as in ace-tylscopoletin, decreases the anti-nociceptive properties of the compound by approximately 26-fold compared to the origi-nal coumarin [161]. Sets of coumarinyl ethers having chro-mone, 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 benzo-furanyl ethers of coumarins were found to be most active
amongst all the compounds. The Chloro and methoxy sub-stitution in coumarin ring was found to increase activity [161]. Three major coumarins, edgeworin (EdN) (69), edge-worosides 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 (p 0.05-0.01) and analgesic (p 0.001) effects, while EdeC showed only an analgesic effect [13].
Bergapten was also reported to exhibit significant anti-convulsant activity [162]. Umbelliferone extracted from the root bark of Adina cordifolia exhibited a strong anti-amoebic activity, and its derivatives, thiosemicarbazones, when sub-stituted with adamantamine, p-benzyl piperidine and N-methyl 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 com-pounds 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 impor-tant class of pharmacological agents possessing a range of different physiological activities including anti-cancer, anti-leukemia, anti-inflammation, anti-HIV, anti-coagulant, anti-bacterial, analgesic and comparative immunomodulation.
Of great interest is the possibility that this class of mole-cules could be a source of drugs for the therapy of several diseases, including cancer, mycosis fungoides. To fully de-scribe the recent progress in structure modification and the structure activity relationship is not an easy task. Neverthe-less, it is useful to build up some correlations with the data available in order to help researchers in discovering and de-
O O
68
R
MeO
a: OAc
b: OBz
O O
69
HO
O OO
O O
70
HO
O OO
O OOO
OHOH
OH
Me
O O
71
HO
O OOO
OHOH
OH
Me
O OO
R2
R1
R1 R2
Bergapten OCH3 H
Isopimpinellin OCH3 OCH3
Xanthotoxin H OCH372
The Structure and Pharmacological Functions of Coumarins Current Medicinal Chemistry, 2009 Vol. 16, No. 32 4255
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.
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