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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 (D

9-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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454Biosynthesis of cannabinoids and endocannabinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456

Side chain SAR of cannabinoids and endocannabinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458

Side chain length and branching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458

Effects of side chain substituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459

Conformationally restricted side chain analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460

Cyclic substituents at C-1 V for probing a novel pharmacophoric subsite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462

Effects of other cyclic and bulky side chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463

Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464

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

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 cannabichro-

menes (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

0024-3205/$ - see front matter D

2005 Elsevier Inc. All rights reserved.doi:10.1016/j.lfs.2005.09.014

* Corresponding author. Tel.: +1 617 373 4200; fax: +1 617 373 7493.

E-mail address: [email protected] (A. Makriyannis).

Life Sciences 78 (2005) 454 – 466

www.elsevier.com/locate/lifescie



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 non-

polar organic solvents. These phenolic derivatives are morewater 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. D9-

THC (2) is formed by the decarboxylation of its non-

psychoactive 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 endocanna-

binoid 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 andCravatt, 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 neuro-

modulator is supported by its pharmacological profile as well

as its mechanisms of biosynthesis and bioinactivation.

A second important endocannabinoid, 2-arachidonoylgly-

cerol (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).

OH

OR3R2

Cannabidiols (CBDs)

OH

R2O

∆9-Tetrahydrocannabinols (THCs)

OH

R2O

Cannabichromenes (CBCs)

R1

R3

R1 or R3 = H or COOHR2 = C1, C3, C4, C5 side chain

R1 = H or COOHR2 = C1, C3, C4, C5 side chainR3 = H or CH3

R1

R1 = H or COOH

R2 = C3, C5 side chain

R1

R3O

R2

OR1

Cannabinols (CBNs)

R1 = H or CH3R2 = H or COOHR3 = C1, C3, C4, C5 side chain

OH

R1

R3O R2

R1 = H or COOH

R2 = C3, C5 R3 = H, CH3

Cannabigerols (CBGs)

OH

HOR

Cannabinodiols (CBNDs)

R = C3, C5 side chain

Fig. 1. Subclasses of cannabinoids present in Cannabis sativa (ElSohly, 2002).

O

OH

COOH

O

OHAction of light, heatduring storage or smoking

-CO2

1 2

(-)-∆9-Tetrahydrocannabinol (THC)(-)-∆9-Tetrahydrocannabinolic acid (THCA)

Polar Head

Hydrophobic side chain

AB

C 1

31' 5'

6a

9

11

10a

6

Fig. 2. Non-enzymatic formation of D9-THC from its precursor.

G.A. Thakur et al. / Life Sciences 78 (2005) 454– 466 455



The common structural features between the plant-derived

cannabinoid receptor agonist D9-THC (2) and the endocanna-

binoid 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 n-

hexanoyl-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 geranylpyropho-

sphate: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 biosynthe-

sized from cannabigerolic acid (CBGA, 13) and toa lesser extent

from cannabinerolic acid (CBNA, 14).

The key cannabinoid precursor D9-tetrahydrocannabinolic

acid (THCA, 1) is formed from CBGA (13) through stereo-

selective oxidocyclization involving tetrahydrocannabinolic

acid synthase (THCA synthase) (Sirikantaramas et al., 2004;

Taura et al., 1995). THCA synthase is a monomeric flaviny-

lated 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 co-enzymes, 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-arachido-

noylglycerol (2-AG, 4) in neural cells has been well-reviewed

(De Petrocellis et al., 2004; Piomelli, 2004; Ueda et al., 2005).

NH

O

OHO

O

OH

OH

Anandamide (AEA) 2-Arachidonoylglycerol (2-AG)

K i = 61 nM (CB1)

= 1930 nM (CB2)

K i = 472 nM (CB1)

= 1400 nM (CB2)

1'58

11 14

2

3 4

Polar Head

Tail

Fig. 3. Endogenous cannabinoid receptor ligands.




The biosynthesis of AEA (3) involves the enzymatic transfer of

an arachidonoyl group from the sn -1 position of phosphatidyl-

choline (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 -acylphosphatidy-

lethanolamine-specific phospholipase D (NAPE-PLD) (Ok a-

moto 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 biosyn-

thesis 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.

COO

O

H

O

O

OH

P

O

O

O

+

Deoxyxylulose phosphatepathway

O P

O

O

O

P

O

O

O

IPP (5)OP

O

O

O

P

O

O

O

+

O P

O

O

O

P

O

O

O

O

O

S CoA +

HO O

O O

S CoA

3

n-Hexanoyl-CoA (9) Malonyl-CoA (10)

O

O

O

O

O S CoA

OH

HO

COOH

Olivetolic acid (12)

Geranyl pyrophosphate(GPP, 7)

DMAPP (6)

+

OH

HO

COOH

Cannabigerolic acid (CBGA, 13)

OH

HO

COOH

Cannabinerolic acid (CBNA, 14)

OH

HO

COOH

Cannabidiolic acid

(CBDA, 16)

OH

COOH

O

∆9-Tetrahydrocannabinolic acid

(THCA, 1)

OH

COOH

O

Cannabichromenicacid synthase

Cannabidiolicacid synthaseTetrahydrocannabinolic

acid synthase

Cannabichromenic acid(CBCA, 15)

O P

O

O

O

P

O

O

Oisomerase

Neryl pyrophosphate(NPP, 8)

Polyketide synthase(PKS)

Polyketide intermediate (11)

Geranylpyrophosphate:olivetolategeranyltransferase

Scheme 1. Biosynthesis of D9-THCA and other cannabinoids.




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 1 V,1 V-dimethyl-

heptyl 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 C-

3 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 D8-

THC 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

1 V,1 V-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 equipo-

tent and thermodynamically more stable isomer of D9-THC (2).

Thus, 1 V,1 V-dimethylethyl-D8-THC (CB1, K i=14.0 nM), 1 V,1 V-

dimethylbutyl-D8-THC (CB1, K i=10.9 nM), 1 V,1 V-dimethylpen-

tyl-D8-THC (18) (CB1, K i=3.9 nM), 1 V,1 V-dimethylhexyl-D8-

THC (CB1, K i=2.7 nM), and 1 V,1 V-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 1 V,1 V-dimethyloctyl-D8-

THC, 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 1 V,1 V-dimethylheptyl side chainincreased 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

1 V,1 V-dimethyl branching in the side chain brings about

significant improvements in affinity and potency of cannabi-

noids. 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 1 V,2

V-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-(1 V,2 V-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

1 V,2 V-dimethylheptylresorcinol and their corresponding D8-

THC analogues was carried out by Huffman et al. (1997a),

Liddle and Huffman (2001) and Liddle et al. (1998), who

showed that the 1 VS ,2 V R 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-1 V (21, 22),

C-2 V, C-3 V or C-4 V (23) positions of the side chain (Huf fman et al.

O

OH

O

OH

O

OH

O

OH

O

OH

O

OH

(-)-∆8-THC

K i = 47.6 nM (CB1)

= 39.3 nM (CB2)K i = 0.77 nM (CB1)

K i = 141 nM (CB1)K i = 0.46 nM (CB1)

1718 19

20 21, 1' R; K i = 7.6 nM (CB1)

22, 1' S ; K i = 20 nM (CB1)

23

(-)-∆8-THC-DMHK i = 3.9 nM (CB1)

Fig. 4. D8-THC analogues with variations in side chain length and branching.




1997b). Analogues possessing C-1 V (21, 22) and C-2 V methyl

groups exhibited greater affinities for the CB1 receptor than D8-

THC (17), whereas two other analogues, 3 V-methyl-D8-THC

(CB1, K i=54 nM for racemic, K i=38 nM for 3 V R, K i=53 nM

for 3 VS ) and 4 V-methyl-D8-THC (23, K i= 141 nM), showed

decreased affinities. Of the two C-1 V methyl analogues, the 1 V R

isomer 21 exhibited higher CB1 affinity ( K i=7.6 nM) and potency (TF and RT tests) compared with the 1 VS analogue 22

( K i=20 nM). The in vivo pharmacological data (SA, TF and RT

test) for 1 V-methyl, 2 V-methyl, and 4 V-methyl isomers were found

to be consistent with their receptor affinities. In another study it

was observed that the 3 VS epimer of 3 V-hydroxy-D8-THC was

significantly more potent than the 3 V R 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 t he

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 exhibit ed

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 classicalcannabinoids. The corresponding monomethyl substituted

analog 27 showed a smaller decrease in CB1 binding affinity.

Thus, a comparison of affinities and potencies of AEAanalogues

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 halogensubstitution 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 eff ects

(Charalambous et al., 1991a; Martin et al., 1993; Nikas et al.,

2004). Thus, the 5 V-Br-D8-THC and 5 V-Br-1 V,1 V-dimethylpentyl-

D8-THC (AM087, 28, Fig. 6) exhibited 7.6 and 0.43 nM

affinities, respectively, for the CB1 receptor, whereas 5 V-Br-

1 V,1 V-dimethylheptyl-D8-THC showed a slight reduction in

affinity (1.27 nM) compared to 28. 5 V-I-D8-THC has high

affinity (CB1, K i=7.8 nM) as compared to ()-1 V-iodoheptyl-

D8-THC (CB1, K i= 328 nM) which exhibits an 8-fold reduction

in affinity compared to D8-THC (17) ( Nikas et al. 2004).

Although (

)-5 V-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

NH

O

OH

24

K i = 18 nM (CB1)

NH

O

OH

25

K i = 7.0 nM (CB1)

NH

O

OH

26

K i = 19 nM (CB1)

NH

O

OH

27K i = 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 theseendocannabinoid ligands and their analogues (Abadji et al., 1994).




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 pharmaco-

logical 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) byone 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)oracyano(O-

1812, 35) group at the C-20 along with geminal dimethyls at C-

16 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 D8-

THC (17) and its congeners represented in the cannabinoid

NH

O

OH

NH

O

OHNH

O

OH

NH

O

OH

AM356, R-(+)-methanandamide

K i = 17.9 nM (CB1)

= 868 nM (CB2)

K i = 116 nM (CB1)K i= 4.6 nM (CB1)

O-1811O-1812

O-1860

OHCN

Br

33

3635

34

K i = 2.2 nM (CB1)

Fig. 7. Tail modified analogues of R -(+)-methanandamide.

O

OH

O

OH

O

OH

CN

O

OH

O

OH

O

NH

N

Br CN

O CN

AM087K i = 0.43 nM (CB1)

O-704

K i = 1.5 nM (CB1)

= 1.1 nM (CB2)

K i = 4.5 nM (CB1)

= 3.2 nM (CB2)

O-856

O-774K i = 1.75 nM (CB1)

= 1.1 nM (CB2)

28 29 30

31 32

O-581K i = 0.2 nM (CB1)

= 2.9 nM (CB2)

Fig. 6. D8

-THC analogues bearing different substituents at the terminus of the side chain.




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, Keimo-

witz 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 conforma-

tion of D8-THC (17) in a model membrane system determined

using neutron diffraction (Martel et al., 1993) as well as theconformation of the 1 V,1 V-dimethylheptyl side chain of Pfizer’s

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 affinitiesand efficacies were differentially altered. The analogue which

O

OH

O

OH

O

OH

O

OH

O

OH

K i = 0.65 nM (CB1)

= 3.1 nM (CB2)

K i = 22.3 nM (CB1) = 58.6 nM (CB2)

K i = 402.4 nM (CB1) = 161.5 nM (CB2)

K i = 4.0 nM (CB1)

39

37 38

40 41

K i = 0.86 nM (CB1) = 1.80 nM (CB2)

AMG-1

AM855 AM856

Fig. 8. Side chain conformationally restricted analogues of D8-THC.

O

OH

S S

O

OH

O

OH

O O

O

OH

K i = 1.8 nM (CB1)

= 3.6 nM (CB2)

K i = 0.52 nM (CB1)

= 0.22 nM (CB2)

K i = 0.45 nM (CB1)

= 1.92 nM (CB2)

42 44

45 46

O

OH

K i = 0.3 nM (CB1)

= 0.5 nM (CB2)

43

S S

O

OH

Cl

Cl47

K i = 1.27 nM (CB1)

= 0.29 nM (CB2)

AMG-3AMG-9 AMG-14

AMG-41 AMG-36 AMG-32

K i = 0.44 nM (CB1)

= 0.86 nM (CB2)

Fig. 9. D8-THC analogues bearing different cyclic moieties at the C-1 V position of the side chain.




has the triple bond in the C-1 V position (1 V-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-2 V 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-2 V 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 A 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 A 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.

Cyclic substituents at C-1 V for probing a novel pharmacopho-

ric subsite

As discussed earlier, a comparison of the binding data of n -heptyl-D8-THC and its 1 V,1 V-dimethylheptyl analogue 19

suggested that the presence of the two C-1 V-methyl groups

enhanced the ligand’s affinity for CB receptors. To optimize

the steric contribution due to C-1 V substitution, Papahatjis et al.

synthesized novel analogues (Fig. 9, 42 – 47) in which the

1 V,1 V-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-1 V-dithiolane analogue 43

and C-1 V-dioxolane analogue 44. These two compounds

exhibited high affinities but no significant selectivity for either

of the CB receptors. The corresponding C-1 V-carbocyclic

congener 46 also maintained a high affinity for CB1 and

CB2 with a 3-fold preference for CB1. The C-1 V-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-1 V

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 hydropho-

bic cyclopentyl analogues.

O

OH

K i = 6.8 nM (CB1)

= 52.0 nM (CB2)

AM411

48

C3 C1’

C7’ C4’

C8’

C3’C2’

C10’

C9’ C5’C6’

C2C1C13

C8

C9 C10

C7 C6A

C10A

C10B

C11

C6

C12

C4A

C4

01

05

Fig. 10. CB1 selective ligand AM411 (48) and its crystal structure.

O

OH

O

OH

K i = 34.9 nM (CB1)

= 14.0 nM (CB2)

K i = 48.6 nM (CB1)

= 8.9 nM (CB2)

AM744 AM755

49 50

O

OH

51

K i = 79.7nM (CB1)

= 76.0 nM (CB2)

AM757

Fig. 11. D8-THC analogues bearing adamantyl side chains.




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-1 V 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. Computa-

tional modeling suggested that the differences in affinities andselectivities 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 require-

ments 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-1 V position. The C-1 V

position was further substituted with either 1 V,1 V-dimethyl

(52a – 52c) o r a 1 V,1 V-dithiolane functionality. The geminal

dimethyl analogues 52a – 52c exhibited subnanomolar affini-

ties. Within the geminal dimethyl analogues, 1 V,1 V-dimethyl-1 V-

cyclopentyl 52a had the highest affinity for the CB1 receptor

( K i=0.34 nM) whereas the 1 V,1 V-dimethyl-1 V-cycloheptyl ana-

logue 52c exhibited greater affinity for the CB2 receptor.Ligand 52b exhibited similar affinities for cannabinoid

receptors as 1 V,1 V-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 1 V,1 V-dimethyl

unit was replaced by a 1 V,1 V-dithiolane ring, exhibited relatively

diminished activities ( Nadipuram et al., 2003). Later, Krishna-

murthy 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 1 V,1 V-

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.).

O

OH

O

OH

X

52 53

n

a; n = 1, K i = 0.34 nM (CB1); 0.39 nM (CB2)

b; n = 2, K i = 0.57 nM (CB1); 0.65 nM (CB2)

c; n = 3, K i = 0.94 nM (CB1); 0.22 nM (CB2)

a; X= C(CH3)2, K i = 12.3 nM (CB1); 0.91 nM (CB2)

b; X = C(-S-CH2-)2, K i = 17.3 nM (CB1); 17.6 nM (CB2)

c; X = CH2, K i = 67.6 nM (CB1); 85.9 nM (CB2)

d; X = O, K i = 297 nM (CB1); 23.6 nM (CB2)

Fig. 13. D8-THC analogues bearing different cyclic side chains.




dimethyl, 1 V,1 V-dithiolane, methylene, or carbonyl groups at the

C-1 V position. These compounds exhibited significantly differ-

ent 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-1 V keto group, showedreduced 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 constitu-

ents, 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 endocanna-

binoids 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 pharmaco-

phoric subsites within the CB1/CB2 binding domains. More-

over, 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 medica-

tions for CB receptor mediated pathologies.

Acknowledgements

Supported by grants from the National Institutes on Drug

Abuse (DA9158, DA03801 and DA07215).

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