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Life Sciences 78 (2005) 454 – 466 www.elsevier.com/locate/lifescie Minireview Natural cannabinoids: Templates for drug discovery Ganesh A. Thakur, Richard I. Duclos Jr., Alexandros Makriyannis * Center for Drug Discovery, Northeastern University, 360 Huntington Avenue, 116 Mugar Life Sciences Building, Boston, MA-02115, United States Abstract Recent studies have elucidated the biosynthetic pathway of cannabinoids and have highlighted the preference for a C-3 n-pentyl side chain in the most prominently represented cannabinoids from Cannabis sativa and their medicinally important decarboxylation products. The corresponding C-3 n-propyl side chain containing cannabinoids are also found, although in lesser quantities. Structure – activity relationship (SAR) studies performed on D9-tetrahydrocannabinol (D9-THC), the key psychoactive ingredient of Cannabis, and its synthetic analogues have identified the C-3 side chain as the key pharmacophore for ligand affinity and selectivity for the known cannabinoid receptors and for pharmacological potency. Interestingly, the terminal n-pentyl saturated hydrocarbon side chain of endocannabinoids also plays a corresponding crucial role in conferring similar properties. This review briefly summarizes the biosynthesis of cannabinoids and endocannabinoids and focuses on their side chain SAR. D 2005 Elsevier Inc. All rights reserved. Keywords: D9-Tetrahydrocannabinol; Biosynthesis; Anandamide; Endocannabinoids; Side chain SAR Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biosynthesis of cannabinoids and endocannabinoids . . . . . . . . . . . . Side chain SAR of cannabinoids and endocannabinoids . . . . . . . . . . Side chain length and branching. . . . . . . . . . . . . . . . . . . . . Effects of side chain substituents . . . . . . . . . . . . . . . . . . . . Conformationally restricted side chain analogues . . . . . . . . . . . . Cyclic substituents at C-1V for probing a novel pharmacophoric subsite Effects of other cyclic and bulky side chains . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction Natural products have long been the source of a great majority of drugs and drug candidates. Cannabis sativa L. is one of the oldest known medicinal plants and has been extensively studied with respect to its phytochemistry. The plant biosynthesizes a total of 483 identified chemical entities * Corresponding author. Tel.: +1 617 373 4200; fax: +1 617 373 7493. E-mail address: [email protected] (A. Makriyannis). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.09.014 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454 456 458 458 459 460 462 463 464 464 464 belonging to different chemical classes (ElSohly, 2002), of which the cannabinoids are the most distinctive class of compounds, known to exist only in this plant. There are 66 known plant-derived cannabinoids, the most prevalent of which are the tetrahydrocannabinols (THCs), the cannabidiols (CBDs), and the cannabinols (CBNs). The next most abundant cannabinoids are the cannabigerols (CBGs), the cannabichromenes (CBCs), and cannabinodiols (CBNDs) (Fig. 1). Most cannabinoids contain 21 carbon atoms, but there are some variations in the length of the C-3 side chain attached to the aromatic ring. In the most common homologues, the n-pentyl G.A. Thakur et al. / Life Sciences 78 (2005) 454 – 466 OH OR1 OH R1 O 455 R2 R1 R2 ∆9-Tetrahydrocannabinols (THCs) R1 or R3 = H or COOH R2 = C1, C3, C4, C5 side chain O R2 OR3 R3 R3 Cannabinols (CBNs) R1 = H or CH3 R2 = H or COOH R3 = C1, C3, C4, C5 side chain Cannabidiols (CBDs) R1 = H or COOH R2 = C1, C3, C4, C5 side chain R3 = H or CH3 OH R1 OH R1 O R 3O OH R2 R2 R HO Cannabichromenes (CBCs) Cannabinodiols (CBNDs) R = C3, C5 side chain Cannabigerols (CBGs) R1 = H or COOH R2 = C3, C5 R3 = H, CH3 R1 = H or COOH R2 = C3, C5 side chain Fig. 1. Subclasses of cannabinoids present in Cannabis sativa (ElSohly, 2002). side chain is replaced with an n-propyl (De Zeeuw et al., 1972; Vree et al., 1972). These analogues are named using the suffix ‘‘varin’’ and are designated as THCV, CBDV, or CBNV, as examples. Cannabinoids with one (Vree et al., 1972) and four (Smith, 1997) carbons also exist but are minor components. Classical cannabinoids (CCs) are ABC tricyclic terpenoid compounds bearing a benzopyran moiety (Fig. 2) and are insoluble in water but soluble in lipids, alcohols, and other nonpolar organic solvents. These phenolic derivatives are more water soluble as their phenolate salts formed under strong alkaline conditions. ( )-D9-Tetrahydrocannabinol (D9-THC, 2) the key psychoactive constituent of marijuana (Gaoni and Mechoulam 1964) interacts with the two known cannabinoid receptors CB1 (Devane et al., 1988; Gerard et al., 1990, 1991; Matsuda et al., 1990) and CB2 (Munro et al., 1993), both of which belong to the super family of G-protein coupled receptors, and produce a broad spectrum of physiological effects (Grotenhermen, 2002) including antiemetic, appetite enhancing, analgesic, and lowering of intraocular pressure. D9THC (2) is formed by the decarboxylation of its nonpsychoactive precursor D9-THCA (1) by the action of light or heat during storage or smoking (Claussen and Korte, 1968; Yamauchi et al., 1967), or under alkaline conditions. D9-THCA (1) is biosynthesized by a well-established pathway involving the action of several specific enzymes which will be presented in the next section. The identification of cannabinoid receptors in the brain suggested the presence of an endogenous ligand. The search for such a compound led to the discovery of the first endocannabinoid N-arachidonoylethanolamine (AEA, anandamide, 3) (Fig. 3) (Devane et al., 1992), a highly lipophilic compound susceptible to both oxidation (Burstein et al., 2000; Kozak et al., 2004) and hydrolysis (Cravatt et al., 1996; Giang and Cravatt, 1997; Willoughby et al., 1997). Anandamide (3) was shown to bind to the CB1 receptor with modest affinity (K i = 61 nM), to have low affinity for the CB2 receptor (K i = 1930 nM) (Lin et al., 1998), and behaves as a partial agonist in the biochemical and pharmacological tests used to characterize cannabinoid activity. Its role as a neurotransmitter or neuromodulator is supported by its pharmacological profile as well as its mechanisms of biosynthesis and bioinactivation. A second important endocannabinoid, 2-arachidonoylglycerol (2-AG, 4), binds weakly to both CB1 (K i = 472 nM) and CB2 (K i = 1400 nM) receptors (Mechoulam et al., 1995). 2-AG (4) was isolated from intestinal (Mechoulam et al., 1995) and brain tissues (Stella et al., 1997) and is present in the brain at concentrations approximately 170-fold higher than AEA (3) (Stella et al., 1997). 11 Polar Head 9 OH COOH Action of light, heat during storage or smoking OH C 6a O -CO2 1 (-)-∆9-Tetrahydrocannabinolic acid (THCA) 6 10a 1 A B O 3 2 1' 5' Hydrophobic side chain (-)-∆9-Tetrahydrocannabinol (THC) Fig. 2. Non-enzymatic formation of D9-THC from its precursor. 456 G.A. Thakur et al. / Life Sciences 78 (2005) 454 – 466 O 8 1' 2 11 Polar Head 5 N H OH O OH O OH 14 Tail 3 Anandamide (AEA) Ki = 61 nM (CB1) = 1930 nM (CB2) 4 2-Arachidonoylglycerol (2-AG) Ki = 472 nM (CB1) = 1400 nM (CB2) Fig. 3. Endogenous cannabinoid receptor ligands. The common structural features between the plant-derived cannabinoid receptor agonist D9-THC (2) and the endocannabinoid agonists {AEA (3) and 2-AG (4)} are that both classes of compounds have a polar head group and a hydrophobic chain with a terminal n-pentyl group. Recent work has provided evidence that ligands from both classes have common binding sites (Li et al., 2005; Tian et al., 2005; Picone et al., in press). This is substantiated by structure –activity relationship (SAR) work which revealed a number of similarities between the two classes of cannabinergics. The SAR studies performed on classical cannabinoids represented by D9-THC (2) and its next generation analogues, the non-classical (Johnson and Melvin, 1986; Little et al., 1988) and hybrid cannabinoids (Chu et al., 2003; Drake et al., 1998; Harrington et al., 2000; Makriyannis and Rapaka, 1990; Thakur et al., 2002; Tius et al., 1997, 1994), have recognized four pharmacophores within the cannabinoid prototype: a phenolic hydroxyl (PH), a lipophilic side chain (SC), a northern aliphatic hydroxyl (NAH), and a southern aliphatic hydroxyl (SAH) (for reviews see: Howlett et al., 2002; Khanolkar et al., 2000; Makriyannis and Rapaka, 1990; Palmer et al., 2000 , 2002; Thakur et al., 2005a,b). This review focuses on the SAR of the ‘‘lipophilic side chain,’’ the key pharmacophore which plays a crucial role in determining ligand affinity and selectivity towards cannabinoid receptors as well as pharmacological potency. In order to provide a clear picture regarding the biochemical origin of the n-pentyl side chain and how it is incorporated into the cannabinoid template, the biosynthesis of D9-THCA (1) and other major cannabinoid components of Cannabis will be summarized below. The key steps in the biosynthesis of the endocannabinoids will also be highlighted. Biosynthesis of cannabinoids and endocannabinoids Prior to 1990, the precursors of all terpenoids, isopentenyl diphosphate (IPP, 5) and dimethylallyl diphosphate (DMAAP, 6) were believed to be biosynthesized via the mevalonate pathway (Shoyama et al., 1975). Later, it was shown that many plant terpenoids including cannabinoids are biosynthesized via the deoxyxylulose phosphate pathway (Eisenreich et al., 1998; Fellermeier et al., 2001; Rohmer, 1999). The established biosynthetic pathway of D9-THC acid (1) and other major cannabinoids has been summarized in Scheme 1. Geranyl pyrophosphate (GPP, 7), obtained from IPP (5) and DMAPP (6), was also shown to partially isomerize to neryl pyrophosphate (NPP, 8). Although there is no direct evidence about the biosynthesis of olivetolic acid (12), its chemical structure suggests a biosynthetic route involving cyclization of a polyketide compound 11 (Raharjo et al., 2004a,b,c). This polyketide is formed by condensation of one molecule of nhexanoyl-CoA (9) with three molecules of malonyl-CoA (10) catalyzed by polyketide synthase (PKS). Condensation of olivetolic acid (12) and GPP (7) gives cannabigerolic acid (CBGA, 13) and is catalyzed by the enzyme geranylpyrophosphate:olivetolate geranyltransferase (GOT) (Fellermeier and Zenk, 1998). This enzyme also accepts NPP (8) as a minor substrate to give cannabinerolic acid (CBNA, 14), which is the precursor for cannabidiolic acid (CBDA, 16) (Taura et al., 1996). Oxidocyclization of CBGA (13) to cannabichromenic acid (CBCA, 15) is catalyzed by the oxidoreductase enzyme cannabichromenic acid synthase (Morimoto et al., 1998). CBNA (14) is also converted to CBCA (15), although to a lesser extent by the action of this same enzyme. Cannabidiolic acid synthase (CBDA synthase) catalyzes the oxidocyclization of CBGA (13) to CBDA (16) (Taura et al., 1996). The above studies showed that cannabidiolic acid (CBDA, 16) is predominately biosynthesized from cannabigerolic acid (CBGA, 13) and to a lesser extent from cannabinerolic acid (CBNA, 14). The key cannabinoid precursor D9-tetrahydrocannabinolic acid (THCA, 1) is formed from CBGA (13) through stereoselective oxidocyclization involving tetrahydrocannabinolic acid synthase (THCA synthase) (Sirikantaramas et al., 2004; Taura et al., 1995). THCA synthase is a monomeric flavinylated enzyme that oxidizes CBGA (13) via a mechanism similar to that of berberine bridge enzyme (BBE) and requires molecular oxygen (Sirikantaramas et al., 2004), unlike CBDA synthase and CBCA synthase, which do not require other coenzymes, co-factors, or molecular oxygen for enzymatic action (Morimoto et al., 1998; Taura et al., 1995, 1996). CBNA (14) is also converted to THCA (1) by the action of this enzyme, and it was proposed that THCA (1) is formed via a common intermediate in the reactions of CBGA (13) and CBNA (14) (Taura et al., 1995). It is worth noting here that THCA synthase does not convert the corresponding neutral cannabinoids, cannabigerol and cannabinerol to D9-THC (2), indicating that the presence of the carboxyl group in these substrates is essential for the enzymatic cyclization of terpene moieties. The biosynthesis of anandamide (AEA, 3) and 2-arachidonoylglycerol (2-AG, 4) in neural cells has been well-reviewed (De Petrocellis et al., 2004; Piomelli, 2004; Ueda et al., 2005). G.A. Thakur et al. / Life Sciences 78 (2005) 454 – 466 O O COO O O P O O + H OH 457 O HO 3 O S CoA + O O Malonyl-CoA (10) n-Hexanoyl-CoA (9) Deoxyxylulose phosphate pathway O S CoA Polyketide synthase (PKS) + O O O P O P O O O O O O P O P O O O O O O S CoA O IPP (5) O Polyketide intermediate (11) DMAPP (6) OH O O O P O P O O O isomerase O O O P O P O O O COOH + HO Olivetolic acid (12) Geranyl pyrophosphate (GPP, 7) Neryl pyrophosphate (NPP, 8) Geranylpyrophosphate:olivetolate geranyltransferase OH OH COOH COOH HO HO Cannabinerolic acid (CBNA, 14) Cannabigerolic acid (CBGA, 13) Cannabichromenic acid synthase Cannabidiolic acid synthase Tetrahydrocannabinolic acid synthase OH OH COOH O Cannabichromenic acid (CBCA, 15) OH COOH O ∆ -Tetrahydrocannabinolic acid (THCA, 1) 9 COOH HO Cannabidiolic acid (CBDA, 16) Scheme 1. Biosynthesis of D9-THCA and other cannabinoids. The biosynthesis of AEA (3) involves the enzymatic transfer of an arachidonoyl group from the sn-1 position of phosphatidylcholine (PC) to the head group of a phosphatidylethanolamine (PE) by an N-acyltransferase (NAT) (Di Marzo et al., 1994). Anandamide (3) is then released by an N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) (Okamoto et al., 2004; Ueda et al., 2005), and possibly to some extent via the corresponding N-arachidonoyllysophosphatidy- lethanolamine (NA-lyso-PE) (Sun et al., 2004). The biosynthesis of 2-AG (3) from 1,2-diacylglycerols (1,2-DAG) is enzymatically catalyzed by an sn-1-selective diacylglycerol lipase (sn-1-DAGL) (Bisogno et al., 2003; Sugiura et al., 2002). The two known endocannabinoids AEA (3) and 2-AG (4) differ only in their head groups, are both derived from arachidonic acid, and each has the biologically important terminal n-pentyl tail. 458 G.A. Thakur et al. / Life Sciences 78 (2005) 454 – 466 Side chain SAR of cannabinoids and endocannabinoids Variation of the n-pentyl group of natural cannabinoids and endocannabinoids can lead to wide variations in affinity and selectivity for the cannabinoid receptors as well as their pharmacological potencies. In a very early SAR finding, the importance of the side chain was first demonstrated by Adams (Adams, 1942; Adams et al., 1949) where the 1V,1V-dimethylheptyl analogue was shown to be 100-fold more potent than the n-hexyl analogue in the D6a,10a-THC series. Subsequent SAR studies on classical cannabinoids (CCs) have recognized the C3 alkyl side chain as the most critical pharmacophoric group (Howlett et al., 2002; Khanolkar et al., 2000; Makriyannis and Rapaka, 1990; Palmer et al., 2000, 2002; Thakur et al., 2005a). Therefore, the focus of this review of the SAR studies of analogues derived from cannabinoids and endocannabinoids is on the terminal n-pentyl tail. Side chain length and branching Decreasing the length of the n-pentyl side chain of D9-THC by two carbons (i.e. C-3 n-propyl) reduces potency by 75% (Razdan, 1986). Shortening the length of the side chain in D8THC by one carbon atom (i.e. C-3 n-butyl) results in reduction of affinity for CB1 (K i = 65 nM) as well as potency, while increasing the side chain length to hexyl, heptyl and octyl provides a systematic increase in affinity (K D values ranging from 41 to 8.5 nM) and potency (Martin et al., 1999). The effect of side chain branching with the presence of 1V,1V-dimethyl groups was also further explored for other side chains in tetrahydrocannabinols (Fig. 4, 17 – 23). Much of the tetrahydrocannabinol SAR involves D8-THC (17), the equipotent and thermodynamically more stable isomer of D9-THC (2). Thus, 1V,1V-dimethylethyl-D8-THC (CB1, K i = 14.0 nM), 1V,1Vdimethylbutyl-D8-THC (CB1, K i = 10.9 nM), 1V,1V-dimethylpentyl-D8-THC (18) (CB1, K i = 3.9 nM), 1V,1V-dimethylhexyl-D8THC (CB1, K i = 2.7 nM), and 1V,1V-dimethylheptyl-D8-THC (19) (CB1, K i = 0.77 nM) show significant increases in their binding affinities for the CB1 receptor (Huffman et al., 2003) compared to their non-branched congeners. Affinities increase with increasing chain length up until 1V,1V-dimethyloctyl-D8THC, where it then decreases with further increasing side chain length. The dimethylheptyl side chain was found to be optimal based on pharmacological testing. Replacing the C-3 n-pentyl side chain of THC with a 1V,1V-dimethylheptyl side chain increased the binding affinity 53-fold (Martin et al., 1993). This enhancement was also reflected in the tetrad of the behavioral tests for these compounds namely, spontaneous activity (SA) (14-fold), tail flick (TF) (35-fold), hypothermia (RT) (35-fold) and ring immobility (30-fold) (Martin et al., 1993). Thus, the 1V,1V-dimethyl branching in the side chain brings about significant improvements in affinity and potency of cannabinoids. For this reason the dimethylheptyl side chain has found extensive use during the exploration of other pharmacophores in classical, non-classical, and hybrid cannabinoids (Palmer et al., 2002; Thakur et al., 2005a). Early on, Adams et al. (1948) found that 1V,2V-dimethyl substitution in the hexyl side chain of the D6a,10a-THC analogue (synhexyl) greatly increased potency. This study was performed with a mixture of the eight steroisomers from the three chiral centers. Aaron and Ferguson (1968) first described the synthesis of these eight individual isomers (D6a,10a-THCs) and their pharmacological studies which found two of them to be very potent. Subsequently, the synthesis of stereoisomeric mixtures of 3-(1V,2V-dimethylheptyl)-D8- and D9-THC analogues was reported (Edery et al., 1972; Petrzilka et al., 1969). More recently, the synthesis of all four isomers of 1V,2V-dimethylheptylresorcinol and their corresponding D8THC analogues was carried out by Huffman et al. (1997a), Liddle and Huffman (2001) and Liddle et al. (1998), who showed that the 1VS,2VR isomer 20 has the greatest affinity for CB1 (K i = 0.46 nM) and is the most potent diastereomer in vivo. The stereochemical features of an alkyl substituent on the side chain were further explored in a series of D8-THC analogues bearing a methyl group at either the C-1V (21, 22), C-2V, C-3V or C-4V (23) positions of the side chain (Huffman et al. OH OH OH O O 17 (-)-∆8-THC Ki = 47.6 nM (CB1) = 39.3 nM (CB2) O 18 Ki = 3.9 nM (CB1) 19 (-)-∆8-THC-DMH Ki = 0.77 nM (CB1) OH OH O O 20 Ki = 0.46 nM (CB1) 21, 1' R; Ki = 7.6 nM (CB1) 22, 1' S; Ki = 20 nM (CB1) OH O 23 Ki = 141 nM (CB1) Fig. 4. D8-THC analogues with variations in side chain length and branching. G.A. Thakur et al. / Life Sciences 78 (2005) 454 – 466 1997b). Analogues possessing C-1V (21, 22) and C-2V methyl groups exhibited greater affinities for the CB1 receptor than D8THC (17), whereas two other analogues, 3V-methyl-D8-THC (CB1, K i = 54 nM for racemic, K i = 38 nM for 3VR, K i = 53 nM for 3VS) and 4V-methyl-D8-THC (23, K i = 141 nM), showed decreased affinities. Of the two C-1V methyl analogues, the 1VR isomer 21 exhibited higher CB1 affinity (K i = 7.6 nM) and potency (TF and RT tests) compared with the 1VS analogue 22 (K i = 20 nM). The in vivo pharmacological data (SA, TF and RT test) for 1V-methyl, 2V-methyl, and 4V-methyl isomers were found to be consistent with their receptor affinities. In another study it was observed that the 3VS epimer of 3V-hydroxy-D8-THC was significantly more potent than the 3VR isomer (Martin et al., 1984). Thus, it can be concluded that as the branching moves away from the aromatic A ring, the binding affinities and potencies of the ligands consistently decrease, and that both factors, branching site and stereochemistry within the chain, influence the affinity and potency of cannabinoids. In the early 1990s soon after the discovery of anandamide (3), it was realized that the terminal n-pentyl tail of endocannabinoids may play an analogous role to that of the n-pentyl side chain of THC (2). It was observed by Felder et al. (1993) that when the arachidonic acid moiety of AEA (3) was replaced with docosahexaenoic acid, the resulting structurally related amide analogue with a terminal ethyl group compared with the n-pentyl tail of AEA (3) exhibited a great reduction in affinity for CB1 and potency. Replacement of the n-pentyl side chain in the AEA (3) template (Fig. 5) with n-hexyl (K i = 24 nM), n-heptyl (K i = 55 nM) and n-octyl chains (24, K i = 18 nM) resulted in a similar trend in CB1 binding affinities as in the classical cannabinoids (Ryan et al., 1997). However, these three analogues failed to produce significant pharmacological effects in vivo. The replacement of 5-carbon chain with the dimethylalkyl chain improved affinity and increased potency in both in vitro and in vivo. Like the THCs, the dimethylheptyl analogue 25 exhibited the highest potency (Ryan et al., 1997; Seltzman et al., 1997). When the geminal dimethyl branching was moved one carbon towards the terminal methyl, the resulting ligand 26 exhibited a decrease in CB1 binding affinity compared to 25. This observation is also analogous to that observed for classical cannabinoids. The corresponding monomethyl substituted analog 27 showed a smaller decrease in CB1 binding affinity. Thus, a comparison of affinities and potencies of AEA analogues with THC analogues revealed that similar trends were found when the chain length and branching were varied. However, the magnitude of enhanced potency when the side chain was varied from straight to branched was greater for THC analogues compared to the AEA analogues (Ryan et al., 1997). Effects of side chain substituents The side chain seems to be a place of choice for halogen substitution and a considerable enhancement in affinity for CB1 was observed by substitution at the terminal carbon of the side chain with bulkier halogens (Br, I) producing the largest effects (Charalambous et al., 1991a; Martin et al., 1993; Nikas et al., 2004). Thus, the 5V-Br-D8-THC and 5V-Br-1V,1V-dimethylpentylD8-THC (AM087, 28, Fig. 6) exhibited 7.6 and 0.43 nM affinities, respectively, for the CB1 receptor, whereas 5V-Br1V,1V-dimethylheptyl-D8-THC showed a slight reduction in affinity (1.27 nM) compared to 28. 5V-I-D8-THC has high affinity (CB1, K i = 7.8 nM) as compared to ( )-1V-iodoheptylD8-THC (CB1, K i = 328 nM) which exhibits an 8-fold reduction in affinity compared to D8-THC (17) (Nikas et al. 2004). Although ( )-5V-18F-D8-THC had a slightly reduced affinity (CB1, K i = 57 nM) compared to D8-THC (17), this fluorinated compound has served as a positron imaging (PET) probe for O O N H OH 24 Ki = 18 nM (CB1) N H OH 25 Ki = 7.0 nM (CB1) O O N H 26 Ki = 19 nM (CB1) 459 OH N H OH 27 Ki = 11 nM (CB1) Fig. 5. Side chain modified analogues of anandamide (data with PMSF). AEA (3) shares the biological activities exhibited by D9-THC (2), however it has a faster onset and shorter duration of action than D9-THC (2), as it is hydrolyzed by the enzyme fatty acid amide hydrolase (FAAH). The presence of FAAH in many biochemical bioassay systems required the use of the FAAH inhibitor phenylmethylsulfonyl fluoride (PMSF) while determining the affinities of these endocannabinoid ligands and their analogues (Abadji et al., 1994). 460 G.A. Thakur et al. / Life Sciences 78 (2005) 454 – 466 OH OH OH Br O O 28 AM087 Ki = 0.43 nM (CB1) CN O CN 29 O-774 Ki = 0.2 nM (CB1) = 2.9 nM (CB2) 30 O-581 Ki = 1.75 nM (CB1) = 1.1 nM (CB2) OH OH O O O CN 31 O N N H 32 O-856 Ki = 4.5 nM (CB1) = 3.2 nM (CB2) O-704 Ki = 1.5 nM (CB1) = 1.1 nM (CB2) Fig. 6. D8-THC analogues bearing different substituents at the terminus of the side chain. experiments aimed at studying localization of cannabinoid receptors in the brains of primates (Charalambous et al., 1991b). Incorporation of cyano and other groups at the terminus of the side chain (Fig. 6) was found to dramatically affect pharmacological potency in the D8-THC series with relatively little affect on binding affinity. Furthermore, it was observed that the presence of a cyano group doesn’t affect all pharmacological potency to the same degree. Thus, cyano substituted D8-THC analog (O-774, 29) exhibited similar affinity as 19, but substantial enhancement of in vivo potency {SA (12-fold); TF (4-fold); RT (8-fold)}(Crocker et al., 1999; Griffin et al., 2001; Singer et al., 1998). Shortening the side chain of O-774 (29) by one carbon led to 30, which retained both high affinity and high pharmacological potency. It was also observed that addition of a p-cyanophenoxy moiety (O-704, 31) was well tolerated with respect to receptor affinity, however it showed reduced potency compared to O-581 (29). Piperidinylamido substitution (O-856, 32) showed slightly reduced affinity and potency compared to D8-THC-DMH (19) (Singer et al., 1998). A similar trend was observed with the endocannabinoids (Fig. 7). These studies were carried out with the FAAH-stable anandamide analogue R-(+)-methanandamide (AM356, 33), a metabolically stable template developed by Makriyannis (Abadji et al., 1994; Goutopoulos et al., 2001; Lin et al., 1998). The incorporation of a bromo (O-1860, 34) or a cyano (O1812, 35) group at the C-20 along with geminal dimethyls at C16 dramatically improved CB1 receptor affinities, whereas the corresponding hydroxyl analogue (O-1811, 36) exhibited diminished affinity (Di Marzo et al., 2001). Conformationally restricted side chain analogues The pharmacophoric conformation of the side chain of D8THC (17) and its congeners represented in the cannabinoid O O N H OH 33 N H 34 Br O-1860 Ki = 2.2 nM (CB1) AM356, R-(+)-methanandamide Ki = 17.9 nM (CB1) = 868 nM (CB2) O O N H 35 OH N H 36 CN O-1812 Ki= 4.6 nM (CB1) OH OH O-1811 Ki = 116 nM (CB1) Fig. 7. Tail modified analogues of R-(+)-methanandamide. OH G.A. Thakur et al. / Life Sciences 78 (2005) 454 – 466 461 OH OH O O 37 AMG-1 Ki = 0.65 nM (CB1) = 3.1 nM (CB2) 38 Ki = 0.86 nM (CB1) = 1.80 nM (CB2) OH O 39 Ki = 4.0 nM (CB1) OH O OH O 40 AM855 Ki = 22.3 nM (CB1) = 58.6 nM (CB2) 41 AM856 Ki = 402.4 nM (CB1) = 161.5 nM (CB2) Fig. 8. Side chain conformationally restricted analogues of D8-THC. bicyclic non-classical cannabinoid CP-47,497 which was determined using 2D NMR (Xie et al., 1994). A significant degree of conformational restriction can be imposed upon the side chain (Fig. 8) either by the introduction of unsaturation (37 – 39) or with a new cyclic ring fused to the aromatic A ring (40, 41), thereby restricting the conformations the side chain could attain and leading to variations in biological responses. For all the analogues bearing unsaturation, it was observed that their CB1 affinities and efficacies were differentially altered. The analogue which receptor:ligand complex is not known. Based on a quantitive structure –activity relationship (QSAR) analysis of the side chain conformation in a variety of D8-THC analogues, Keimowitz et al. (2000) postulated that the length of the cannabinoid side chain as well as its ability to fold back placing the terminus close to ring structure are critical in determining the ligand’s affinity for CB1. This is consistent with the compact conformation of D8-THC (17) in a model membrane system determined using neutron diffraction (Martel et al., 1993) as well as the conformation of the 1V,1V-dimethylheptyl side chain of Pfizer’s OH O S OH S 42 AMG-9 Ki = 1.8 nM (CB1) = 3.6 nM (CB2) O S OH S 43 AMG-3 Ki = 0.3 nM (CB1) = 0.5 nM (CB2) O 45 AMG-41 Ki = 0.44 nM (CB1) = 0.86 nM (CB2) 46 AMG-36 Ki = 0.45 nM (CB1) = 1.92 nM (CB2) O O 44 AMG-14 Ki = 0.52 nM (CB1) = 0.22 nM (CB2) OH OH OH O O O Cl 47 Cl AMG-32 Ki = 1.27 nM (CB1) = 0.29 nM (CB2) Fig. 9. D8-THC analogues bearing different cyclic moieties at the C-1V position of the side chain. 462 G.A. Thakur et al. / Life Sciences 78 (2005) 454 – 466 01 OH C13 C9 C1 C10 C9’ C2 C3 C10A C8 C10B C1’ C10’ C7’ C2’ C3’ O C4 C6A C7 48 C4A C11 AM411 Ki = 6.8 nM (CB1) = 52.0 nM (CB2) C6 C5’ C6’ C4’ C8’ 05 C12 Fig. 10. CB1 selective ligand AM411 (48) and its crystal structure. has the triple bond in the C-1V position (1V-heptynyl-D8-THC, 37) and its cis double bond analogue 38 bound to both CB1 and CB2 receptors with high affinity (Martin et al., 2002; Papahatjis et al., 1998). It was observed that the efficacy of these compounds increased with heterosubstitution at the terminus of the unsaturated side chain. Analogues generated by the incorporation of a triple bond at the C-2V position of hexyl, octyl and nonyl (39) side chains of D8-THC exhibited 5- to 10-fold improved affinities compared to D8-THC (17). However, the in vivo potencies of these compounds varied greatly (Ryan et al., 1995). Analogues bearing unsaturation at the C-2V position and a polar moiety such as a CN or azido group at the terminal carbon of the side chain exhibited high affinities for cannabinoid receptors; however, they behaved either as partial agonists or antagonists compared to D8-THC (17) which is a full agonist (Martin et al., 1999). The conformationally constrained tetracyclic analogues 40 and 41 were synthesized with a fourth ring in the tricyclic cannabinoid skeleton fused to the phenolic A ring (Khanolkar et al., 1999). Analogue 40, in which the side chain is fixed at a distance of 1– 1.5 Å from the phenolic ring, has its side chain pointing downwards and has an 18-fold higher CB1 affinity compared to analogue 41 in which the side chain is fixed at a distance of at least 3– 3.5 Å and has a lateral orientation. Analogue 40 also exhibits 3-fold higher affinity than analogue 41 for the CB2 receptor. These results suggest that the cannabinoid receptor affinities decreased significantly when the side chain is forced into a lateral orientation and further away from the phenolic ring. 49 AM744 Ki = 34.9 nM (CB1) = 14.0 nM (CB2) As discussed earlier, a comparison of the binding data of nheptyl-D8-THC and its 1V,1V-dimethylheptyl analogue 19 suggested that the presence of the two C-1V-methyl groups enhanced the ligand’s affinity for CB receptors. To optimize the steric contribution due to C-1V substitution, Papahatjis et al. synthesized novel analogues (Fig. 9, 42– 47) in which the 1V,1V-dimethyl group was replaced with 3- to 6-membered heterocyclic and carbocyclic rings (Papahatjis et al., 1998, 2001, 2002, 2003). The most interesting compounds which resulted from this work were the C-1V-dithiolane analogue 43 and C-1V-dioxolane analogue 44. These two compounds exhibited high affinities but no significant selectivity for either of the CB receptors. The corresponding C-1V-carbocyclic congener 46 also maintained a high affinity for CB1 and CB2 with a 3-fold preference for CB1. The C-1V-cyclopropyl analogues 45 and 47 also show high affinity for both receptors. This increase in the affinity of these analogues 42– 47 was attributed to the presence of a hydrophobic subsite in both CB1 and CB2 in the vicinity of the benzylic position of the side chain. Thus, within the CB1 receptor the putative subsite appears to be indifferent to the presence of oxygen, sulfur or methylene groups attached to the C-1V position. Conversely, the CB2 receptor appears to show a preference for the smaller dioxolane five-membered ring compared to the slightly larger dithiolane or more hydrophobic cyclopentyl analogues. OH OH O Cyclic substituents at C-1V for probing a novel pharmacophoric subsite O 50 AM755 Ki = 48.6 nM (CB1) = 8.9 nM (CB2) OH O 51 AM757 Ki = 79.7nM (CB1) = 76.0 nM (CB2) Fig. 11. D8-THC analogues bearing adamantyl side chains. G.A. Thakur et al. / Life Sciences 78 (2005) 454 – 466 463 Fig. 12. The aromatic rings are perpendicular to the plane of the page with the adamantyl substituent closest to the viewer and the carbocyclic ring furthest from the viewer. Global energy minimum conformers are shown in green tube display. (a) This figure illustrates the union of the Van der Waals’ volume (purple grid) of all accessible conformers of the CB1 selective compound AM411 (48). (b) This figure illustrates the Unique Volume Map (yellow grid) calculated using all conformers of the CB1 selective compound AM411 (48) and of the CB2 selective compound AM755 (50). The yellow grid area shows the region of space into which conformers of AM755 (50) protrude that is not shared with the accessible conformers of AM411 (48). (c) This figure illustrates the Unique Volume Map (red grid) calculated using all conformers of the CB2 selective compound AM755 (50) and of the non-selective compound AM757 (51). The red grid area shows the region of space into which conformers of AM755 (50) protrude that is not shared with the accessible conformers of AM757 (51) (Reproduced with permission from J. Med. Chem. 2005, 48, 4576 – 4585. Copyright 2005 Am. Chem. Soc.). Effects of other cyclic and bulky side chains To add to our present understanding of the possible conformations of the side chain adopted during a ligand’s interaction with its active site, novel analogues carrying bulky and/or rigid aliphatic substituents at C-1V were developed (Figs. 10, 11; 48– 51) (Lu et al., 2005). Based on the binding affinities, it was observed that the bulky adamantyl group can easily be accommodated within CB1/CB2 binding site. The results also revealed that variation in adamantyl substituents can lead to higher affinities and selectivities for each of the two receptors depending on the relative orientation of the adamantyl group with respect to the tricyclic cannabinoid structure. Computational modeling suggested that the differences in affinities and selectivities can be explained on the basis of allowable conformational space for the adamantyl substituents. Thus, the 3-(1-adamantyl) group of the CB1 selective analogue AM411 (48), the first pharmacologically active (Jarbe et al., 2004; Luk et al., 2004; McLaughlin et al., 2005a,b) classical cannabinoid to be crystallized (Lu et al., 2005), orients within a spherical space in the direct proximity of the phenolic A ring. Conversely, in the analogues AM744 (49) and AM755 (50) which show CB2 preference, the allowable adamantyl conformations exist within a donut like space that extends beyond that of the spherical conformational space of AM411 (48). Finally, a ligand AM757 (51) capable of occupying both spaces exhibits no CB1/CB2 selectivity (Fig. 12). To better understand the conformational and steric requirements of the hydrophobic pocket of the receptor where the C-3 side chain resides, Nadipuram et al. (2003) synthesized several D8-THC analogues (Fig. 13) in which the cycloalkyl groups of 5, 6 and 7 carbons were attached at the C-1V position. The C-1V position was further substituted with either 1V,1V-dimethyl (52a– 52c) or a 1V,1V-dithiolane functionality. The geminal dimethyl analogues 52a– 52c exhibited subnanomolar affinities. Within the geminal dimethyl analogues, 1V,1V-dimethyl-1Vcyclopentyl 52a had the highest affinity for the CB1 receptor (K i = 0.34 nM) whereas the 1V,1V-dimethyl-1V-cycloheptyl analogue 52c exhibited greater affinity for the CB2 receptor. Ligand 52b exhibited similar affinities for cannabinoid receptors as 1V,1V-dimethylheptyl-D8-THC (19). However, despite their relatively high affinities, none of these ligands exhibited much CB subtype receptor selectivity. The corresponding dithiolane analogues, where the 1V,1V-dimethyl unit was replaced by a 1V,1V-dithiolane ring, exhibited relatively diminished activities (Nadipuram et al., 2003). Later, Krishnamurthy et al. (2003) reported the synthesis of D8-THC analogues in which the cycloalkyl side chain was replaced with a phenyl ring thus retaining steric bulk and conformational constraint. These analogues 53a – 53d incorporated 1V,1V- OH OH n O 52 O X 53 a; n = 1, Ki = 0.34 nM (CB1); 0.39 nM (CB2) b; n = 2, Ki = 0.57 nM (CB1); 0.65 nM (CB2) c; n = 3, Ki = 0.94 nM (CB1); 0.22 nM (CB2) a; X= C(CH3)2, Ki = 12.3 nM (CB1); 0.91 nM (CB2) b; X = C(-S-CH2-)2, Ki = 17.3 nM (CB1); 17.6 nM (CB2) Ki = 67.6 nM (CB1); 85.9 nM (CB2) c; X = CH2, d; X = O, Ki = 297 nM (CB1); 23.6 nM (CB2) Fig. 13. D8-THC analogues bearing different cyclic side chains. 464 G.A. Thakur et al. / Life Sciences 78 (2005) 454 – 466 dimethyl, 1V,1V-dithiolane, methylene, or carbonyl groups at the C-1V position. These compounds exhibited significantly different binding profiles compared to their cycloalkyl congeners. The dimethyl analogue 53a exhibited high CB2 affinity (K i = 0.91 nM) and 13-fold selectivity over CB1. Analogues 53b and 53c both exhibited diminished affinity and selectivity. Analogue 53d, bearing a polar C-1V keto group, showed reduced affinity for both CB1 and CB2 receptors as was previously shown by Papahatjis et al. (1998) in other THC analogues, but exhibited nearly 13-fold selectivity for CB2. Conclusion Over the past 50 years, much research has been carried out directed towards the development of SAR of the classical cannabinoids, primarily using D8-THC (17) as a template. It has been shown that the key pharmacophore, the n-pentyl chain present in tetrahydrocannabinols and other cannabis constituents, is incorporated during the biosynthesis of olivetolic acid (12). Variation of the n-pentyl side chain of the classical cannabinoids or variation of the n-pentyl tail of the endocannabinoids leads to striking variations in affinities and selectivities of these ligands towards the cannabinoid receptors as well as their pharmacological potencies. The SAR of these analogs have added to our understanding about the steric requirements of CB1 and CB2 receptors and suggested the presence of pharmacophoric subsites within the CB1/CB2 binding domains. Moreover, the design and synthesis of later generation ligands has helped in gaining an insight into ligand binding site(s) and structural features required for receptor activation. Such information has been used in the design of high affinity and receptor subtype selective ligands that may serve as useful pharmacological probes as well as drug prototypes of medications for CB receptor mediated pathologies. 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