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Life Sciences 78 (2005) 454 – 466
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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
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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.
Acknowledgements
Supported by grants from the National Institutes on Drug
Abuse (DA9158, DA03801 and DA07215).
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