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Endocannabinoid structure–activity relationships for interaction at the cannabinoid receptors
Prostaglandins, Leukotrienes and Essential FattyAcids (2002) 66(2&3),143^160 & 2002 Elsevier Science Ltd. All rights reserved. doi:10.1054/plef.2001.0343, available online at http://www.idealibrary.com on
Endocannabinoid structure–activity relationships for interaction at the cannabinoid receptors P. H. Reggio Department of Chemistry, Kennesaw State University, Kennesaw, GA USA
Summary Anandamide (N-arachidonoylethanolamine) was the first ligand to be identified as an endogenous ligand of the G-protein coupled cannabinoid CB1receptor.Subsequently, two other fattyacid ethanolamides, N-homo-g-linolenylethanolamine and N-7,10,13,16-docosatetraenylethanolamine were identified as endogenous cannabinoid ligands. A fatty acid ester, 2-arachidonoylglycerol (2-AG), and a fatty acid ether, 2-arachidonyl glyceryl ether also have been isolated and shown to be endogenous cannabinoidligands.Recent studies have postulated the existence of carrier-mediated anandamide transport that is essential for termination of the biological effects of anandamide. A membrane bound amidohydrolase (fatty acid amide hydrolase, FAAH), located intracellularly, hydrolyzes and inactivates anandamide and other endogenous cannabinoids such as 2-AG. 2-AG has also been proposed to be an endogenous CB2 ligand.Structure^activity relationships (SARs) forendocannabinoidinteraction with the CB receptors are currently emerging in the literature.This review considers cannabinoid receptor SAR developed to date for the endocannabinoids with emphasis upon the conformational implications for endocannabinoid recognition at the cannabinoid receptors. & 2002 Elsevier Science Ltd. All rights reserved.
INTRODUCTION
Endogenous cannabinoids Anandamide and other fatty acid ethanolamides Anandamide (N-arachidonoylethanolamine, 1, AEA; Chart 1), isolated from porcine brain, was the first to be identified as an endogenous ligand of the G-protein coupled cannabinoid CB1 receptor.1 Like other cannabinoid agonists, AEA produces a concentration-dependent inhibition of the electrically evoked twitch response of the mouse vas deferens1 as well as, antinociception, hypothermia, hypomotility and catalepsy in mice.2 AEA exhibits higher affinity for the cannabinoid CB1 receptor (Ki CB1=89710 nM) than for the CB2 receptor (Ki CB2=3717102 nM).3 Two other polyunsaturated fatty acid ethanolamides, homo-g-linolenylethanolamide (2) and 7,10,13,16-docosatetraenylethanolamide (3) were Received 30 October 2001 Accepted 10 November 2001 Correspondence to: Patricia H. Reggio, Department of Chemistry, Kennesaw State University,1000 Chastain Rd., Kennesaw, GA 30144, USA, Tel.: þ770-423-6170; Fax: þ770-423-6744; E-mail: [email protected]
& 2002 Elsevier Science Ltd. All rights reserved.
subsequently isolated from porcine brain and shown to bind to the cannabinoid CB1 receptor with high affinity.4 Increases in intracellular calcium concentrations have been shown to stimulate the formation of anandamide from a membrane phospholipid precursor, N-arachidonoyl phosphatidyl ethanolamine.5–8 Once released, anandamide can activate cannabinoid receptors. These agonist–receptor interactions result in activation of G proteins, particularly of the Gi/o family.9,10 Signal transduction pathways that are regulated by these G proteins include inhibition of adenylyl cyclase;10–15 regulation of ion currents (inhibition of voltage-gated L, N, and P/Q Ca2+ currents;13,16,17 activation of K + currents;17,18 activation of focal adhesion Kinase (FAK);19 mitogen-activated protein Kinase (MAPK)20–22 and induction of immediate early genes;20,21,23,24 and, stimulation of nitric oxide synthase (NOS).25–29 Termination of anandamide signaling at the cannabinoid receptors occurs through an uptake mechanism that transports anandamide into the cell30–32 where it subsequently undergoes rapid degradation. Current evidence suggests that anandamide uptake is a carrier-mediated process that is time- and temperature-dependent, saturable and inhibited with a unique pharmacological
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O 9
8
6
5
7
4
3
2
1
10 11
12
13
14
15
16
17
18
19
NHCH2 CH2 OH 20
Anandamide 1 NHCH2 CH2OH O
Homo-γ -linolenylethanolamide 2 O
NHCH2 CH2 OH
Docosatetraenylethanolamide 3 O
OCH(CH2 OH)2
2-AG 4 CH2 -O-CH(CH2OH)2
Noladin ether 5 O
NHCH 2CH2OH
Palmitoyl-EA 6
Chart 1
profile.5,31,33 In brain and liver, AEA is hydrolyzed enzymatically to yield arachidonic acid and ethanolamine.34 The reaction is catalyzed by a membrane-bound amidohydrolase (called anandamide amidohydrolase or fatty acid amide hydrolase, FAAH) which has been cloned.35 Anandamide amidohydrolase appears to be located intracellularly, as suggested by both cell fractionation studies and sequence analysis of the cloned enzyme. A close correspondence between the distribution of FAAH and CB1 in rat brain has been reported. The complementary pattern of FAAH and CB1 expressions at the cellular level has suggested that FAAH participates in cannabinoid signalling mechanisms in the brain.36–38 FAAH has been shown recently to work in conjunction with other membrane proteins to facilitate anandamide transport by creating an inward concentration gradient as a result of its metabolism of anandamide.39
2-AG: sn-2-Arachidonoylglycerol (2-AG; 4) was isolated by Mechoulam and co-workers40 from canine intestinal tissue and by Sugiura and co-workers41 from rat brain and shown also to be an endogenous CB ligand (CB1 Ki =472755 nM; CB2 Ki =14007172 nM).40 2-AG has been found present in the brain at concentrations 170 times42 to 800 times41 greater than anandamide. Interaction of 2-AG with the cannabinoid receptors results in activation of G proteins,15,43 particularly of the Gi/o family and the inhibition of adenylyl cyclase.15 2-AG induces a rapid, transient elevation of intracellular free [Ca2þ] in neuroblastoma glioma hybrid NG108-15 cells which express the CB1 receptor 44 and in N18TG2 cells.45 This action of 2-AG is blocked by pretreatment with pertussis toxin, indicating that Gi/o is involved in the response. This action of 2-AG also is blocked by treatment with the CB1 antagonist SR141716A, suggesting that this effect may be CB1 receptor mediated. The elevation of intracellular [Ca2þ] effect is also produced by cannabinoid agonists, WIN55,212-2, HU210, CP55940, anandamide and R-methanandamide; however, these agonists produce much lower maximal responses than do 2-AG, and its congeners. The relative efficacy profile for CB agonists differs from that described for [35S]GTPgS binding and inhibition of adenylyl cyclase, suggesting that the signal transduction pathway for this intracellular [Ca2þ] regulation must differ, perhaps in the type of G protein that tranduces the response.44–46 A similar effect in HL-60 cells which express the CB2 receptor has also been demonstrated.47 2-AG has also been found to induce the activation of p42/44 MAP Kinase in HL-60 cells.48 2-AG has been shown to compete with AEA for uptake by the anandamide transporter.32,49 The FAAH enzyme has been reported to hydrolyze 2-AG four times faster than its hydrolysis of anandamide.50 It now appears that signalling by the endocannabinoid system represents a mechanism by which neurons can communicate backwards across synapses to modulate their inputs. Recently, it has been suggested that both endocannabinoids mediate retrograde signalling at hippocampal synapses by their ability to downregulate GABA release at pre-synaptic terminals.51 Noladin ether : An ether-type endocannabinoid, 2arachidonyl glyceryl ether (noladin ether, 5) was recently isolated from porcine brain.52 2-arachidonyl glyceryl ether was found to bind to the CB1 receptor (Ki =21.270.5 nM) and cause sedation, hypothermia, intestinal immobility and mild antinociception in mice, effects typically produced by cannabinoid agonists. Compound 5 was found to bind weakly to CB2 (Ki43 mM). This compound had been synthesized previously by the Mechoulam lab in a program to develop compounds less prone to enzymatic hydrolysis.53 Independently, Sugiura and co-workers 46 prepared this
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Endocannabinoid structure^activity relationships
compound and examined its effects on Ca2þ levels in cells.54 Compound 5 was found by these investigators to exhibit appreciable agonistic activity, although its activity was significantly lower than that of 2-AG. PEA: Facci and co-workers55 reported that N-palmitoylethanolamide (PEA, 6), a C16 saturated fatty acid ethanolamide, behaves as an endogenous agonist of the CB2 receptor on rat mast cells (RBL-2H3 cells). However, PEA was found to displace only 10% of [3H]CP-55 940 from human cloned CB2 receptor at concentrations up to 10 mM.3 For this reason, the identification of PEA as an endogenous cannabinoid remains controversial. PEA has been reported to be orally active in reducing edema formation and inflammatory hyperalgesia by downmodulating mast cell activation.56 PEA has also been shown to act in synergy with anandamide. Recently, Di Marzo and co-workers showed that PEA potently enhances the anti-proliferative effect of AEA on human breast cancer cells (HBCCs), in part by inhibiting the expression of the FAAH enzyme.57 A substantial structure–activity relationship (SAR) literature now exists for fatty acid ethanolamide (e.g. anandamide) interaction with the CB1 receptor. This literature has been used to propose models for the interaction of anandamide at the CB1 receptor. Currently emerging in the literature is the SAR for fatty acid glycerol ester (e.g. 2-AG) interaction at both the CB1 and CB2 receptors. Studies have focused on both head group and acyl chain requirements for interaction with each of these biological targets. One important question that is addressed in these studies is whether the structural requirements for endocannabinoid interaction with CB1 and CB2 are the same or are different. In this paper, endocannabinoid SAR for interaction with CB receptors will be reviewed. This SAR, of course, has been based on measured receptor affinities and activities. Because the inactivation of anandamide and 2-AG has been found to take place very rapidly, it is important that any study of endocannabinoid interaction with the CB receptors pay particular attention to enzymatic inactivation as a confounding problem in assays for affinity or activity. We begin here by discussing this important problem.
Fatty acid amide hydrolase Fatty acid amide hydrolase (FAAH, oleamide hydrolase, anandamide amidohydrolase) is an integral membrane protein that hydrolyzes and inactivates fatty acid primary amides and ethanolamides including oleamide and anandamide.35 The distribution of FAAH in the CNS suggests that it is posed to degrade neuromodulating fatty acid amides near their sites of action. This localization suggests that FAAH is intimately related to the regulation of the effects evoked by these fatty acid & 2002 Elsevier Science Ltd. All rights reserved.
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amides.34,36,58 In addition, FAAH has been shown to hydrolyze another endogenous cannabinoid, 2-AG four times faster than AEA, as well as the related 1-AG and 1oleylglycerol, and the corresponding simple ester, methyl arachidonate.50,59–61 Human and rat FAAH share a similar qualitative substrate selectivity with anandamide, oleamide (18:1 D9) and myristic (14:0) amide serving as the preferred substrates and palmitic (16:0) and stearic (18:0) amides being hydrolyzed at significantly lower rates. However, human FAAH was less selective than rat FAAH as it hydrolyzed myristic amide at a rate comparable to oleamide (18:1 D9) and palmitic (16:0) amide at a rate only 2-fold slower than oleamide.35,62
FAAH Inhibitors Given the plurality of fatty acid amides and esters that FAAH hydrolyzes, it is interesting that a relatively small set of inhibitors of this enzyme has been reported in the literature. Linoleoylethanolamide has been reported to be a natural inhibitor of FAAH.63 Several other fatty acid ethanolamides have been reported by Desarnaud34 to inhibit AEA hydrolysis. Arachidonoylserotonin has been reported to be the first FAAH inhibitor that is inactive both against cPLA2 and at CB1 receptors.64 The endogenous potent FAAH inhibitor, 2-octyl-g-bromo acetoacetate, has been reported to be an endogenous sleep compound65 and its analogs are among the set of FAAH inhibitors. Other classes of inhibitors are electrophilic carbonyl inhibitors (trifluoromethyl ketones, a-halo Ketones, a-Keto esters and amides and aldehydes)66–68 or irreversible inhibitors (sulfonyl fluorides and fluorophosphonates) incorporated into fatty acid structures.69–72
Considerations for the interpretation of endocannabinoid binding data The development of structure–activity relationships for endocannabinoid interaction with the CB receptors hinges upon comparisons of receptor affinities for a large number of compounds. This inevitably entails comparisons across laboratories. In no other structural class of cannabinoid agonists is such a comparison process fraught with more peril than in the endocannabinoids. The rapid degradation of anandamide in cultured cells and crude homogenates of other cultured cells or of rat tissues was recognized early in the literature.73 At that time, phenylmethylsulfonyl fluoride (PMSF) came into standard use in filtration-based CB1 binding assays to inhibit the breakdown of anandamide. In very early literature, no PMSF was used in filtration-based binding assays of anandamide and its analogs. These studies reported very high Ki values for anandamide as a
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consequence. In fact, a common method used in the literature today to test for the susceptibility of analogs to enzymatic breakdown has been to compare the receptor affinities of the analogue with and without PMSF in the assay.74 However, in other binding assay protocols, PMSF has not been found to be necessary. Sheskin and coworkers75 reported that in a centrifugation-based ligand binding assay, PMSF was not necessary, as these investigators found that the addition of an amidase inhibitor to their rat synaptosomal membrane assay did not lower the Ki. Another concern for cross-laboratory comparisons is whether the binding assay is performed using a cloned cell line or tissue homogenates. The use of a cloned cell line presumably assures a single CB sub-type receptor population for the binding assay. Tissue-based assays may not, on the other hand, guarantee a single-receptor sub-type population. For many CB1 binding assays, rat brain membranes have been commonly used with the assumption that only CB1 receptors are present in this preparation. Munro and co-workers76 found no CB2 transcripts in brain tissue by either Northern analysis or in situ hybridization studies. However, Skaper and coworkers77 have reported that cerebellar granule cells and cerebellum express genes encoding both the CB1 and CB2 receptors. In CB2 binding assays, mouse spleen membranes or human tonsils have been used. Spleen has been reported to exhibit much less CB2 mRNA than human tonsils, and CB1 mRNA is present in spleen, but not in tonsils.78 Because CB1 binding assays of anandamide analogs can be performed either using a filtration- or centrifugation-based assay on either tissue homogenates or cloned cells with and without PMSF or other inhibitor, there is variation in reported Ki values for endocannabinoids between laboratories. For these reasons, in the sections that follow here, an attempt has been made to provide the Ki value for AEA obtained in a given laboratory as a benchmark from which to compare relative affinities of other analogs.
ENDOCANNABIOID INTERACTION WITH CANNABINOID RECEPTORS
SAR of fatty acid ethanolamides Cannabinoid SAR literature has primarily focused on receptor affinities of fatty acid ethanolamides, such as anandamide, and its analogs. Recently, however, Sugiura has begun to develop an SAR for 2-AG and its analogs based on a functional assay of Ca2þ mobilization discussed earlier here.46,48 Because of their difference in basis, each of these SARs will be presented separately here.
Endocannabinoid SAR at the CB receptors can be divided into studies of head group requirements for high-affinity binding and studies of acyl chain requirements for high-affinity binding.
Amide head group SAR Arachidonamide and simple alkyl esters of arachidonic acid do not show significant CB1 affinity.79 Cyclization of the head group into an oxazoline ring diminishes affinity.74 Methylation at the C-1’ position in the AEA head group results in an 1’-R-methyl isomer (AM356, Rmethanandamide, 7) which has 4-fold higher CB1 affinity than AEA, while the 1’-S-methyl isomer has 2-fold lower CB1 affinity than AEA. AM356 also was found to be resistant to enzymatic breakdown (Ki =2071.6 nM with PMSF; Ki =2873 nM without PMSF).80 Methylation at the 2’ position also produces some stereoselectivity, as the S(þ) isomer was found to have 2–5-fold higher CB1 affinity than the R()-isomer.43,80 Goutopoulos and coworkers81 reported that introduction of a single methyl at the 2 positions (R or S ) on the acyl chain of anandamide leads to compounds that show moderately improved affinities for CB1 relative to anandamide, but limited enantioselectivity (AEA, Ki =7871.6 nM; (R)-N-(2-hydroxyethyl)-2-methyl-arachidonamide Ki =54.175.2 nM; (S )N-(2-hydroxyethyl)-2-methyl-arachidonamide Ki =35.37 4.3 nM) and only modestly improved metabolic stability. The CB1 affinity of the 2,2-dimethyl analog of anandamide (Ki =72.276.3 nM) is unchanged from that of anandamide. This compound did show somewhat enhanced metabolic stability, however. Introduction of larger alkyl groups at the 2 position has a detrimental effect on CB1 affinity.82 A high degree of enantio- and diastereoselectivity was observed for the 2, 1’-dimethyl analogs. (R)-N-(1-methyl-2-hydroxyethyl)-2-(R)-methylarachidonamide81 exhibits the highest CB1 affinity in this series with a Ki =7.4270.86 nM , a 10-fold improvement on anandamide (Ki =7871.6 nM ). Enlargement of the ethanolamine head group by insertion of methylene groups revealed that the Npropanol analog has slightly higher CB1 affinity than AEA, while higher homologs have reduced CB1 affinity.75,79 Alkyl branching of the alcoholic head group led to lower affinity analogs,75 as did the incorporation of hydroxyphenyl groups such as in AM404.84 N-(propyl) arachidonylamide (8), in which the hydroxyl group is replaced with a methyl group, possesses higher CB1 affinity (Ki =7.3 nM) than anandamide itself (Ki=22 nM).75,79 However, when this same substitution was performed in R-methanandamide (7, Ki =17.9 nM, Chart 2), a lower affinity analog resulted (Ki =73.6 nM).74 Substitution of an N-cyclopropyl group for the ethanolamine head group of AEA leads to a very high-affinity CB1 compound, arachidonylcyclopropylamide, ACPA
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Endocannabinoid structure^activity relationships
O
Me OH NH
R - methanandamide 7
O
NHCH2CH2CH3
8 O
147
methylene group between the amide nitrogen and a 4phenol group results in an analog with modest CB1 affinity (Ki=21773 nM vs AEA Ki =7872 nM).86 Replacement with a 3,4-dihydroxy-ethyl-phenyl results in an analog with greater CB1 affinity than AEA.88 Taken together, all of these results suggest that the hydroxyl in the anandamide head group is not essential for receptor interaction, but that the cannabinoid receptor can accomodate both hydrophobic and hydrophilic head groups, possibly in two different subsites.83 While the hydrophilic site accomodates slightly larger head groups, the sizes of the cavities in which the head group binds still appear to be relatively small, as only conservative variations on the head group permit the retention of high-affinity binding.
Amido group modifications NH
ACPA 9
NHCH2CH2F
NH O 10
Chart 2
(9; Ki =2.270.4 nM), while methylene insertion between the amide nitrogen and the cyclopropyl ring leads to reduced affinity (Ki4100 nM).10,49 Substitution of an Nallylarachidonamide (Ki =9.9 nM) and N-propargylarachidonamide (Ki=10.8 nM) shows approximately 6-fold higher affinity for CB1 than anandamide (Ki =61.0 nM).74 Substitution of an N-phenyl group for the ethanolamine head group of AEA results in a compound that retains CB1 affinity (Ki =109 nM) similar to AEA (Ki =143737 nM), while substitution of an N-adamantyl group, results in an extreme loss of affinity (Ki41000 nM).49 These results suggest that there may exist a hydrophobic sub-site for the AEA head group such that the hydroxyl of AEA may not be necessary for receptor interaction.74,83 These results also suggest that this hydrophobic sub-site is of limited size, as large hydrophobic groups are not well tolerated. Replacement of the hydroxyl group of AEA with a halogen such as F or CI increases CB1 affinity as well.10,74,85 Replacement of the 2-hydroxyethyl group of AEA with a phenolic group or pyridine group decreases affinity for CB1;49,86,87 however, the insertion of a & 2002 Elsevier Science Ltd. All rights reserved.
In order for high-affinity binding to the CB1 receptor to occur and for agonist binding to activate G-proteins, the carbonyl group of the AEA amide head group cannot be replaced with a methylene group.43 Retro-anandamide, in which the position of the carbonyl and the NH of the amide group in anandamide are reversed, retains receptor affinity and exhibits stability with regard to hydrolysis by FAAH (Ki =115 nM with PMSF and 134 nM without PMSF).74 Arachidonylethers, carbamates and norarachidonlycarbamates have poor CB1 affinity.89 However, norarachidonyl ureas show generally good binding affinities to the CB1 receptor (Ki =55–746 nM; AEA Ki =89710 nM). Norarachidonyl 2fluoroethyl urea (10; Ki =5578 nM) has higher CB1 affinity than AEA (Ki =89710 nM).89 Some of the weaker affinity analogs in this series, produced potent pharmacological activity. These analogs showed hydrolytic stability toward amidase enzymes, as well.89
Acyl chain SAR. Arachidonic acid The overall conformations of the endocannabinoids are controlled largely by the conformations of their fatty acid acyl chains. While no X-ray crystal structure data is available for the endocannabinoids, there is X-ray crystal data for the fatty acid that comprises the acyl chain in anandamide and 2-AG, arachidonic acid (AA, 20:4 D5,8,11,14 ; 11). We begin here by discussing the conformations of arachidonic acid that have been determined both by experiment and by calculation. X-ray crystal structure studies of arachidonic acid (AA, 11) have revealed that AA is a very flexible molecule that can adopt very different conformations depending on its environment. In the X-ray crystal structure of pure arachidonic acid, arachidonic acid exists in an extended (angle-iron) conformation in which double bonds 1 and 3 and double bonds 2 and 4 are coplanar, while the planes of adjacent double bonds are perpendicular to one
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O 9
8
6
5
7
4
2
3
1
10 11
12
13
14
15
17
16
18
Arachidonic Acid 20:4 ∆5,8,11,14 11 10
19
OH 20
O
7 NHCH2CH2OH
13
16 12
Fig. 1 (a) This figure illustrates the crystal structure of arachidonic acid.91,95 (b) This figure illustrates the conformation that arachidonic acid92 (PDB Accession No.1ADL) adopts when complexed with adipocyte lipid binding protein, a fatty acid transporter. (c) This figure illustrates the conformation that arachidonic acid adopts at the active site of prostaglandin synthase-1.93
8
NHCH2CH2OH O
11
14 13
another (see Fig. 1a).91,95 An X-ray crystal study of arachidonate ion complexed with adipocyte-lipid-binding protein revealed that AA binds within the b barrel cavity of this protein. Here the carboxylate group of AA is engaged in a strong electrostatic interaction with Arg126, Tyr128 and Arg106 through an intervening water molecule, while the arachidonate acyl chain assumes a hairpin (i.e. U-shaped) conformation for which the first double bond of arachidonic acid is distorted out of the plane formed by the other three double bonds (see Fig. 1b).90,92 In the 3 A˚ resolution structure of prostaglandin H synthase-1 (Fig. 1c), arachidonic acid adopts an extended L-shaped conformation that positions the 13proS hydrogen of AA for abstraction by Tyr 385, the likely radical donor at this enzyme’s active site.93 One important feature of the arachidonic acid unsaturated acyl chain is the great torsional mobility about the two torsion angles involving each methylene carbon between adjacent pairs of double bonds in the acyl chain (for example, the C8–C9–C10–C11 and C9–C10– C11–C12 torsion angles, see Chart 3 structure 11 for numbering system). Rabinovich and Ripatti94 found that polyunsaturated acyl chains in which double bonds are separated by one methylene group are characterized by the highest equilibrium flexibility compared with other unsaturated acyl chains. Rich95 reports that a broad domain of low-energy conformational freedom exists for these C–C bonds. Conformational analyses of arachidonic acid suggest that the high flexibility built into the acyl chain of AA, permits this fatty acid to assume many conformations. In their Monte Carlo study, Rabinovich and Ripatti94 found that polyunsaturated fatty acids, whose double
O
NHCH2CH2OH 11
14 14 Me 9 OH 1
Me Me
3
O
1′
∆9 -THC 15 NHCH2CH2OH O 16
O
NHCH2CH2OH O H
OH PGB2-EA 17
Chart 3
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Endocannabinoid structure^activity relationships
bonds are separated by one methylene carbon, assume an extended (angle-iron) conformation (see Fig. 1a) when all molecules are efficiently packed below the phase-transition temperature. Molecular dynamics (MD) or molecular dynamics/simulated annealing (MD/SA) studies (in vacuo) of arachidonic acid (11, AA) and of other polyunsaturated fatty acids related to AA, have been published by several groups.94–98,100,101,103 These computational studies of AA have primarily found looped or back-folded conformations to be low-energy conformers of AA. Rich conducted a quenched molecular dynamics study of AA in vacuo.95 The two lowest enthalpy conformers found for AA were J-shaped conformers in which the carboxylic acid group is in close proximity to the C14–C15 p bond. This same J-shape was reported by Corey and co-workers101 as one type of low-energy minimum identified in their conformational analysis of arachidonic acid. The authors suggested that such a Jshaped conformation in solution would be energetically favorable and would be consistent with the chemistry of peroxyarachidonic acid for which an internal epoxidation leads to 14, 15-epoxyarachidonic acid.102 Using the A* Algorithim followed by minimization in the ‘Batchmin’ module of Macromodel, Leach and Prout98 found rightand left-handed looped or ‘helical-like’ conformations of AA to be its lowest energy conformations. A simulated annealing study of AA identified a looped conformation,103 while a molecular dynamics study in vacuo found low-energy structures of AA to be hairpin-, helical- and crown-shaped (i.e. U-shaped conformers in which the ends of the chain come close to one another).96 Lopez and co-workers also employed molecular dynamics simulations in their study of AA, finding two low-energy conformations, one of which was U-shaped and was proposed as the bioactive conformation at its cyclooxygenase binding site.97 A recent modeling study of the binding of arachidonic acid in the cyclo-oxygenase active site of mouse prostaglandin endoperoxide synthase-2(COX-2) predicts that AA will orient in a kinked or L-shaped conformation. In this conformation, the carboxylate moiety binds to Arg,120 while the o-end is positioned above Ser530 in a region termed the top channel.104 In their theoretical conformational analysis of the complex of arachidonic acid (AA) with a-tocopherol, Shamovsky and Yarovskaya105 found that AA assumes an extended conformation. A new conformational analysis method, Conformational Memories (CM)106,107 was employed by Barnett-Norris and co-workers108 to study the conformations of arachidonic acid. Results of the biased sampling phase from CM calculations of arachidonic acid were consistent with Rich’s and with Rabinovich and Ripatti’s results108 as they revealed, for example, a relatively broad distribution of populated torsional space about the classic & 2002 Elsevier Science Ltd. All rights reserved.
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skew angles of 1191(s) and 1191(s’) for the C8–C9–C10– C11 torsion angle in anandamide. CM calculations identified extended conformers and folded or U-shaped conformers to be the major conformational families of arachidonic acid. The more compact (U-shaped) structure was found to predominate in water, while the extended shape was found to predominate in CHCl3 (and in vacuo). The existence of J-shaped conformers as suggested in the literature 95,101,102 was confirmed by CM. In this case, the J-shaped family was found to comprise a smaller, but still significant conformational family of arachidonic acid. These results illustrate that the CM method resulted in a much broader sampling of the conformational space of arachidonic acid, than had been performed previously using molecular dynamics or molecular dynamics/simulated annealing techniques.95–98,100,101,103 These previous studies of arachidonic acid found primarily only compact structures (U’s, J’s, helical) as energy minima. While no other theoretical studies of arachidonic acid in solvent have been reported, the trends in the CM results reported by Barnett-Norris et al.108 for AA in CHCl3 and in H2O are consistent with the idea that in H2O, arachidonic acid would minimize the exposure of its hydrophobic portions by forming a more compact U-shape. While Rich reported that methylene carbons attached to the sp2 hybridized carbon of a C=C double bond demonstrate a broad region of conformational space (i.e. for Er 1.0 kcal/mol) available to them, the same was not true for saturated carbon chains such as the acyl chain of palmitic acid. Rich reported that for these chains, freedom of motion was restricted to trans (71801) or gauche (7601) configurations. As a result, saturated fatty acids are much less flexible and favor more extended conformations.95,99 In summary, the fatty acid literature indicates that saturated fatty acids, such as palmitic acid, tend to adopt extended conformations, while unsaturated fatty acids, such as arachidonic acid which possesses cis double bonds separated by methylene carbons, exhibit a high degree of flexibility that allows them to adopt folded, as well as extended conformations quite easily.
Acyl chain SAR. Fatty acid ethanolamides F effect of branching Mono- (Ki =53711 nM) or di- (Ki =4772 nM) methylation at C2 on the acyl chain of AEA produced analogs with slightly higher CB1 affinities than AEA (Ki =89710 nM); however, larger or branched alkyl substituents at C2 lead to low CB1 affinities.85 Effect of unsaturation: Sheskin et al.75 reported that N-docosatetraenoyl-ethanolamine 22:4, D7,10,13,16 (12, Ki =34.473.2 nM) and N-homo-g-linolenoyl-ethanolamine 20:3, D8,11,14 (13, Ki =53.475.5 nM) had CB1 affinities comparable to anandamide 20:4, D5,8,11,14
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Fig. 2 Presented here are Conformational Memories results for a series of ethanolamides that show the effect of increasing degrees of unsaturation on the fatty acid acyl chain.110 The left column illustrates one major family of conformers.These are extended conformers.The right column illustrates the second major family identified for each compound.This family shows increasing curvature as the degree of unsaturation increases.
(1, Ki =39.275.7 nM). As unsaturation was decreased to two cis double bonds in N-11,14 eicosadienoyl ethanolamine (20:2, D11,14 analog), CB1 affinity was greatly diminished (14, Ki =1500 nM). A saturated fluoro-derivative of AEA (20:0), O-586 was found not to bind to CB1 receptors (Ki410 000 nM with or without PMSF). This compound also did not produce cannabimimetic discriminative stimulus effects.109 Figure 2 illustrates the conformational differences between ethanolamides with varying degrees of unsaturation in their acyl chains as revealed by Conformational Memories calculations.110 Two major families of conformers were identified for each of the following ethanolamides: 18:0, 20:1 D,11 20:2 D,11,14 20:3 D,8,11,14 20:4 D5,8,11,14 (anandamide). The percentages next to each family indicate the percentage population of each family. There are two important points illustrated in Figure 2. (1) Each of these ethanolamides have a family of extended conformers. These are illustrated in the left column of Figure 2. (2) The curvature of the acyl chain increases with increasing unsaturation. The second column in
Figure 2 (on the right) contains curved or U-shaped families of conformers. It is clear in going from the 18:0 ethanolamide to the 20:4 D5,8,11,14 ethanolamide (anandamide) that curvature in the acyl chain increases and, as a consequence, the distance between the head group and acyl tail decreases as the degree of unsaturation increases. Alkyl tail length: The degree of unsaturation is not the only acyl chain requirement for recognition at the CB1 receptor. Sheskin and co-workers75 found that in an n-3 series, highly unsaturated analogs had poor to no CB1 affinity as revealed by the following series of n-3 ethanolamide derivatives: 20:5, D5,8,11,14,17 (Ki =162.37 13.6 nM) and 22:6, D4,7,10,13,16,19 (Ki =324.179.2 nM), 18:4, D6,9,12,15 (Ki41000 nM), 20:3, D11,14,17 (Ki410 000 nM), 18:3, D9,12,15 (no activity). The fact that this n-3 series shows low to no CB1 affinity reveals that flexibility cannot be the only acyl chain requirement for CB1 recognition as all of these n-3 analogs will have great flexibility due to their levels of unsaturation. However, these analogs may lack an essential part of the cannabinoid pharmacophore: a saturated alkyl tail of sufficient length. An analogy has been drawn by several groups in the literature between the C16–C20 saturated portion of AEA and the C-3 pentyl side chain of the classical cannabinoid, D9-THC (15).111–114 In classical cannabinoid SAR, the C-3 pentyl side chain (see D9-THC, 15) is considered a minimum length to produce cannabinoid activity; while a 1’, 1’-dimethylheptyl side chain appears to be optimum.115 Replacement of the pentyl side chain of a classical or non-classical cannabinoid with a 1’,1’dimethylheptyl side chain has been reported to result in as much as a 75-fold enhancement in CB1 affinity.116 While the replacement of the C16–C20 saturated portion of anandamide with a 1’,1’-dimethylheptyl chain did not result in as great an enhancement as in the classical cannabinoids, it did produce a 13-fold enhancement in CB1 affinity.112,113 This enhancement has been taken as support for the hypothesis that the C16–C20 saturated region of AEA corresponds to the C-3 pentyl chain of D9THC for interaction at the CB1 receptor. Therefore, from this correspondence, one might conclude that the drop in affinity which accompanies the shortening of the saturated tail of anandamide in the n-3 series may be related to SAR C-3 side chain requirements for classical cannabinoid binding at CB1. Acyl chain length: Sheskin and co-workers75 also found that the 18:3 D6,9,12 (n-6) analog of AEA had low CB1 affinity (16, Ki =46007300 nM). This result suggests that a C18 acyl chain is too short to be recognized by CB1, because this analog has both a long enough saturated tail (five carbons) and three double bonds separated by methylene carbons.
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Endocannabinoid structure^activity relationships
Conjugated acyl chain: While most acyl chain SAR studies have focused on unsaturated cis polyenes, AEA analogs with altered double bond patterns have also been reported. All of these analogs possess regions in which some double bond conjugation is present. Edgemond and co-workers have described two oxygenated derivatives of AEA that are products of lipoxygenase activity on AEA, 12(S)–hydroxyeicosatetraenoylethanolamide (12(S)-HAEA) and 12(R) hydroxyeicosatetraenoylethanolamide (12(R)HAEA). While anandamide has a Ki of 107754 nM at CB1, the S stereoisomer (Ki =207752 nM) has one-half the CB1 affinity of AEA, and the R stereoisomer (Ki =416714 nM) has one-quarter the CB1 affinity of AEA.117 Conjugated triene anandamide (CTA) has been reported to have 6fold lower CB1 affinity (Ki =607744 nM) than AEA (Ki =9777 nM).118 Pinto et al.79 investigated a series of arachidonyl amides and esters in addition to a series of ‘rigid hairpin’ conformations typified by N-(2-hydroxyethyl)-prostaglandin amides to determine the structural requirements for binding to the CB1 receptor. Two-dimensional drawings of anandamide and PGB2-EA (17) make the shapes of these two compounds look similar and, therefore, it was possible that PGB2-EA (17) may be a rigid (hairpin/Ushaped) analog of AEA. However, all of the rigid prostaglandin analogs synthesized by Pinto et al.79 failed to alter [3H]CP-55 940 binding to CB1 in concentrations as great as 100 mM. a-Alkylamide derivatives of PGE2 and PGF2a showed enhanced binding affinity relative to these prostaglandin ethanolamides; however, none of the affinities of the a-alkylamide derivatives were comparable to that of AEA. In addition, although the PGE2 amides were able to activate G proteins, including Gs, these signal transduction events were not likely mediated by the CB receptors.119 Barnett-Norris and co-workers108 reported CM results for 17 which showed an attenuated ability to adopt extended or tightly folded conformations like AEA and 2AG. Instead, PGB2-EA (17) in CHCI3 adopts a more folded L-shaped structure. The CM results show that the conjugation of the acyl chain with the ring double bond introduces ‘stiffness’ into this part of the molecule. The authors suggest that the attenuated ability of PGB2-EA to adopt an extended conformation may be the reason why PGB2-EA does not bind to CB1 (i.e. PGB2EA lacks the flexibility of AEA). Alternatively, the steric bulk introduced by the five-membered ring in 17 may produce a steric clash at CB1 that prevents ligand binding. Another series of conformationally restricted analogs that contained ortho and meta disubstituted benzene derivatives with alkylamide and alkyl substituents displayed CB1 affinity in the range of low-to-high micromolar in rat membranes.120 These analogs were also & 2002 Elsevier Science Ltd. All rights reserved.
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designed to test if an endocannabinoid hair-pin conformation was recognized at the CB1 receptor. Several of the analogs inhibited basal binding of GTPgS in rat brain membranes at micromolar concentrations, but none of the analogs affected either basal or stimulated adenylate cyclase activity in N18TG2 membranes. In summary, these results for acyl chain variations suggest that high-affinity receptor binding requires high flexibility of the acyl chain because only analogs with three or more double bonds show high CB1 affinity. The acyl chain may need to adopt a folded conformation to interact with CB1 or the analog may need to adopt several different conformations in order to reach, be recognized by and trigger the receptor. Only those analogs with high degrees of equilibrium flexibility may be able to achieve these conformations easily.
Endocannabinoid interaction with the cannabinoid CB1 receptor The hypothesis that AEA adopts a folded conformation in order to interact with the CB1 receptor has been explored by several research groups. Thomas and co-workers 111 were first to report a COMFA QSAR pharmacophore model for anandamide and its analogs. These authors used molecular dynamics studies to explore conformations of 1 that present pharmacophoric similarities with the classical cannabinoid, D9-THC (15). A J-shaped or looped conformation of AEA was identified that had good molecular volume overlap with D9 -THC (15) when (1) the carboxyamide of AEA was overlaid with the pyran oxygen (O-5) in 15; (2) the head group hydroxyl of AEA was overlaid with the C-1 phenolic hydroxyl group of 15, (3) the five terminal carbons of the AEA fatty acid acyl chain were overlaid with the C-3 pentyl side chain of 15; and (4) the polyolefin loop of AEA was overlaid with the tricyclic ring system of 15. These authors supported their use of a J-shaped conformation for AEA by citing synthetic results for the internal epoxidation undergone by peroxyarachidonic acid which point to the J-shape as necessary for such a reaction.102 Eight pharmacologically active anandamide analogs were shown to have similar conformational mobility and pharmacophore alignments that are conformationally accessible. When these compounds were aligned with 15, their potencies were predicted by a quantitative model of cannabinoid SARs based solely on classical and non-classical cannabinoids with a reasonable degree of accuracy. The ability to incorporate the pharmacological potency of these anandamides into the classical/non-classical cannabinoid pharmacophore model was also proposed to support the relevance of the Thomas pharmacophore model. Tong and co-workers114 reported a different pharmacophore model for AEA using constrained conformational searching and COMFA. 9-nor-9b-OH-HHC (18, Chart 4)
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was used as the template to which AEA and its analogs were fitted. The conformation identified for AEA was a helical conformation in which (1) the oxygen of the carboxyamide overlaid the C-1 phenolic hydroxyl group of 18; (2) the head group hydroxyl overlaid the C-9 hydroxyl of 18; (3) the alkyl tail of AEA overlaid the C-3 alkyl side chain of 18; and, (4) the polyolefin loop overlaid the tricyclic ring structure of 18. These authors supported their use of a helical-shaped AEA by citing an X-ray crystallographic structure which shows that arachidonic acid adopts a helical conformation when it is a substrate for cyclooxygenase.121 The authors cite the close matching of common pharmacophoric elements of AEA and 9nor-9b-OH-HHC as persuasive evidence of the biological relevance of this helical conformer. A 3D–QSAR model was derived using COMFA for a training set of 29 classical and non-classical cannabinoids which rationalized the binding affinity in terms of steric and electrostatic properties. Reggio and co-workers have developed an endocannabinoid pharmacophore based upon endocannabinoid SAR only. These investigators recently proposed a hypothesis about endocannabinoid interaction with CB1 based upon their CM results.122 CM calculations of AEA in CHCI3 reported by Barnett-Norris and co-workers108 revealed that AEA adopts predominantly extended and U-shaped conformers. (J-shaped conformers of AEA were found in lower number (10 out of 150)). For 2-AG, both extended and U-shapes were found, with the extended shape predominating in CHCl3 (71 out of 150 structures). Based upon these results, Reggio and co-workers122 have proposed a model that requires anandamide to assume more than one conformation in its interaction with CB1. In this model, anandamide approaches the CB1 receptor in an extended conformation from lipid. Its initial interaction site is a groove on Helix 6 formed by Val6.43(351) and Ile6.46(354) into which the pentyl tail of anandamide fits. This groove serves as a filter for the acyl chain variants of anandamide, as only those with a sufficient number of carbons (20–22) and degree of saturation (3–4 cis double bonds separated by methylene carbons) are at the correct depth in the membrane and the correct angle to interact with this groove. In addition, at least five saturated carbons at the end of the acyl chain are needed for optimal interaction with this groove. CM results for Helix 6 in CB1 suggest that alkyl chain interaction with this groove induces a conformational change in Helix 6 that would move the intracellular end of Helix 6 away from Helix 3.123 Such a change has been proposed to be the beginning of the R (inactive state) to R* (active state) transition for the b2-adrenergic receptor124,125 and for rhodopsin,126 both G-protein-coupled receptors. Once in the protein interior, anandamide folds into a U-shape with the amide oxygen interacting with
OH 9
H OH 1
Me Me
1′
3
O
9-nor-9β-OH-HHC 18 O
NH OH Olvanil 19
OCH3 OH
O
NH
AM 404 20 O
NH OH
Arvanil 21
OCH3 O
Me OH
Me
NH
Me
CN O-1812 22 O
Me
Me
NH Br
OH
23 OCH3
Chart 4
Lys3.28(192). In this conformation, the ligand acyl chain has hydrophobic interactions with several residues in the Helix 2–3–7 region, including Phe3.25. This model is consistent with CM results that identify both extended
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and folded conformations of AEA as low-energy conformations. It is consistent with the cannabinoid mutation literature that suggests that Lys3.28(192) is the primary interaction site for anandamide.127 The model is consistent with all of the acyl chain SAR discussed above (including chain length and degree of unsaturation requirements) and may explain the low affinity seen for all rigid analogs of anandamide synthesized to date.
CB2 SAR for fatty acid ethanolamides AEA exhibits higher affinity for the cannabinoid CB1 receptor (Ki CB1=89710 nM) than for the CB2 receptor (Ki CB2=3717102 nM).3 Anandamide analogs tend to be CB1 selective with CB2 affinities comparable to or lower than the CB2 affinity of AEA.10,74,81,86 Berglund and coworkers119 found that a-alkylamide derivatives of PGE2 and PGF2a showed higher binding affinity for the CB2 receptor than for the CB1 receptor; however, the affinity was reduced relative to anandamide’s affinity for CB2.119 Palmitoylethanolamide: Facci et al.55 reported that palmitoylethanolamide (16:0, PEA, 6) a C16 saturated derivative, behaves as an endogenous agonist of the CB2 receptor on rat mast cells (RBL-2H3 cells). The IC50 for competitive inhibition of the aminoalkylindole (AAI) cannabinoid agonist [3H]WIN55 212-2 binding to RBL2H3 cell membranes by PEA was reported to be 1.070.6 nM, while that of AEA was reported to be 3372.9 nM.55 However, PEA was found to displace only 10% of non-classical cannabinoid agonist [3H]CP-55 940 binding from human cloned CB2 receptor at concentrations up to 10 mM.3 PEA differs from AEA not only in its lack of unsaturation, but also in its shorter acyl chain (C16 vs C20 for AEA). Using the same protocol as described in our previous paper,108 we recently studied the conformational families of PEA in CHCl3 using the CM method.106,107 The results of our CM calculations revealed that PEA adopts predominantly only one conformation, an extended conformation (83 out of 100 conformers in CHCl3). Figure 3 illustrates this predominant cluster of like conformations. Lambert and co-workers synthesized a series of 10 Npalmitoylethanolamide (PEA, 6) homologs and analogs, varying by the elongation of the fatty acid chain from caproyl to stearoyl and by the nature of the amide substituent, respectively, and evaluated the affinity of these compounds for cannabinoid receptors in the rat spleen; in RBL-2H3 cells and in CHO-CB1 and CHO-CB2 receptor transfected cells.128 No displacement of [3H]CP55 940 or [3H]WIN 55 212-2 by PEA derivatives was observed in rat spleen slices. In RBL-2H3 cells, no binding of [3H]CP55 940 or [3H]WIN55 212-2 could be observed and conversely, no inhibitory activity of PEA derivatives and analogs was measurable. These investigators con& 2002 Elsevier Science Ltd. All rights reserved.
Fig. 3 The major family of palmitoylethanolamide (PEA, 6) conformers (83 out of100) obtained using Conformational Memories.110 Here it is clear that the lack of unsaturation in the PEA acyl chain results in PEA’s preference for an extended conformation.
cluded that it seems unlikely that PEA is an endogenous agonist of the CB2 receptor.128 Most recently, Jonnson and co-workers129 studied the effects of homologs and analogs of palmitoylthanolamide upon the inactivation of the endocannabinoid anandamide. Oleoylethanolamide at 100 mM showed 64.671.5% inhibition of [3H]-WIN 55 212-2 binding to human CB2 receptors expressed on CHO cells. Other compounds had lesser effects at CB2. PEA, palmitoylisopropylamide and R-palmitoyl-(2-methyl)ethanolamide had modest effects upon [3H]-CP 55 940 binding to human CB1 receptors expressed on CHO cells. Oleamide: Oleamide (18:1, D9 cis) was isolated from the cerebro-spinal fluid (CSF) of sleep-deprived cats and shown to induce physiological sleep in rats.71 Cheer and co-workers130 reported that oleamide occupied CB1 receptors on rat brain membranes labeled with the nonclassical cannabinoid agonist [3H]CP-55 940 with an IC50 of 10 mM. However, oleamide has been reported previously by several other groups not to bind to recombinant CB1 or CB2 receptors overexpressed in host
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cells.75,131,132 Oleamide, at a 50 mM concentration, potentiates AEA binding to CB1 receptors by an order of magnitude.132 This effect was not observed in the presence of the FAAH inhibitor PMSF (200 mM), which alone also potentiates AEA binding, thus suggesting that oleamide facillatory action is due to inhibition of AEA hydrolysis in membrane preparations. This is not suprising as oleamide and anandamide have both been shown to be substrates for the FAAH enzyme.35 Summary: In summary, the CB1 receptor does not tolerate very large endocannabinoid head groups; however, it does recognize both polar and non-polar moieties in the head group region. The receptor recognizes ethanolamides whose fatty acid acyl chains have 20 or 22 carbons, with at least three cis double bonds separated by methylene carbons and saturation in at least the last five carbons of the acyl chain. Double bond conjugation within the acyl chain results in diminished CB1 affinity. From a conformational standpoint, for ligands of sufficient length and tail saturation, these results suggest either that only acyl chains that can assume a tightly folded (U-shaped) conformation can bind to CB1 or that ligand flexibility (i.e. ability to extend and to fold) is very important for binding to and activating the CB1 receptor.
2-AG SAR at CB1 and CB2
ities of 2-eicosatrienoyl(20:3 D8,11,14, n-6)glycerol and 2eicosatrienoyl(20:3 D11,14,17, n-3)glycerol are lower than those of 2-eicosatrienoyl(20:3 D5,8,11, n-9) and 2-eicosapentaenoyl(20:5 D5,8,11,14,17, n-3)glycerol, it appears that the presence of a double bond at the D5 position, rather than the structure near the methyl end, is crucially important, probably for some characteristic conformation of the agonistic molecules.
CB2 SAR Sugiura and co-workers have also found that 2-AG can induce rapid transient increases in [Ca2þ] in HL-60 cells.47 It was evident in this case that the response was mediated by the CB2 receptor, but not the CB1 receptor, because the CB2 antagonist, SR144528, but not the CB1 antagonist, SR141716A, blocked the response. 2-AG was found to have more activity than its 1(3)isomer. Ester and ether analogs, including nolandin ether (5, Chart 1) showed appreciable activity, albeit less than that of 2-AG. Anandamide was found to be a weak partial agonist toward the CB2 receptor. The cannabinoid agonist D9 –THC (15, Chart 3) exhibited weak agonistic activity for the CB2 receptor in the transient [Ca2+] assay. 2-Glycerol esters of 20–22 carbon fatty acids showed the strongest CB2 agonist activity. For C20 fatty acids, activity was best for the 20:3 D5,8,11 and 20:4 D5,8,11,14 (2-AG, 4, Chart 1), suggesting that like the 2-AG SAR generated for CB1, a double bond at D5 was important.
CB1 SAR Sugiura and co-workers have reported that 2-AG (4) and other cannabinoid ligands such as anandamide (1) and D9-THC (15) induce rapid transient increases in [Ca2+] in NG108-15 cells through a cannabinoid CB1 receptordependent mechanism.44–46 2-AG was the most potent compound for inducing these transient increases, as its activity was detectable from as low as 0.3 nM. The maximal response induced by 2-AG exceeded responses induced by other CB1 agonists. Activities of the CB1 agonists, HU-210 and CP55,940 were also detectable from as low as 0.3 nM, whereas, the maximal responses induced by these compounds were low compared with 2AG. Anandamide was also found to act as a partial agonist in this system. Arachidonic acid (11) failed to elicit a response, while noladin ether (5) possessed appreciable activity, although its activity was apparently lower than that of 2-AG.46 Sugiura and co-workers46 have begun to generate an SAR based on this transient increase in [Ca2þ]. Glycerol has been found to be the most suitable head group, and the 2-isomer is preferable over the 1(3)-isomer. Arachidonic acid is the most preferred fatty acid moiety, although the activity of eicosatrienoic acid (n-9)-containing species was almost comparable to that of the arachidonic acid containing species. Because the activ-
Divergence between CB1and CB2 SAR While much of the SAR for 2-AG at CB2 is similar to that previously discussed for CB1, there is one divergence. 2Glycerol esters of C22 fatty acids do not show appreciable activity in the CB1 assay, but do show appreciable activity in the CB2 assay.
Endocannabinoid connection withVanilloids The chemical similarity between some synthetic agonists of the vanilloid receptors, such as olvanil (19, N-vanillylcis-9-octadecenoamide) and AEA prompted Di Marzo and co-workers136 to study possible interactions between the cannabinoid and vanilloid signaling systems. Zygmunt and co-workers133 reported that anandamide, but not 2AG or PEA, induces vasodilation by activating vanilloid receptors on perivascular sensory nerves causing release of calcitonin-gene-related peptide (CGRP). The vanilloid receptor may thus be another molecular target for endogenous anandamide, besides cannabinoid receptors in the nervous and cardiovascular system. Subsequently, Ross and co-workers134 reported that the mouse vas deferens (MVD) expresses both CB1 and VR1 receptors and that both receptor types appear to contribute to anandamide-invoked contractions of the MVD.
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AM404 (20), the AEA transport inhibitor, has very low CB1 affinity (Ki =17607142 nM),86 but has been shown to activate vanilloid receptors.135 Indeed, structural similarities between AM404 and the synthetic vanilloid agonist olvanil (19) prompted Di Marzo and colleagues136 to investigate the possibility that olvanil (19) may interfere with anandamide transport. Their results indicated that olvanil blocked both the uptake and the hydrolysis of [14C]AEA by intact RBL-2H3 cells (IC50 =9 mM), while capsaicin and pseudocapsaicin (N-vanillyl-nonanamide) were much less active. Olvanil was more potent than previously reported inhibitors of AEA facilitated transport, i.e. phloretin (IC50=80 mM), AM404 (12.9% inhibition at 10 mM) or oleoylethanolamide (27.5% inhibition at 10 mM). Olvanil was a poor inhibitor of [14C]AEA hydrolysis by RBL-2H3 and N18TG2 cell membranes, suggesting that the inhibitory effect on [14C]AEA breakdown observed in intact cells was due to inhibition of [14C]AEA uptake. Olvanil was stable to enzymatic hydrolysis, and (i) displaced the binding of high-affinity cannabinoid receptor ligands to membrane preparations from N18TG2 cells and guinea pig forebrain (Ki =1.64–7.08 mM), but not from cells expressing the CB2 cannabinoid receptor subtype; (ii) inhibited forskolin-induced cAMP formation in intact N18TG2 cells (IC50 =1.60 mM), this effect being reversed by the selective CB1 antagonist SR141716A. These data suggest that some of the analgesic actions of olvanil may be due to its interactions with the endogenous cannabinoid system. Melck and co-workers found that arvanil (21) was a potent inhibitor of AEA accumulation (IC50=3.6 mM) and was 4-fold more potent than AEA on CB1 receptors (Ki =0.25–0.52 mM), whereas its 18:3 D9,12,15 congener was more selective than arvanil for the transporter (IC50=8.0 mM) vs CB1 receptors (Ki =3.4 mM). None of the compounds efficiently inhibited [14C] AEA hydrolysis or bound to CB2 receptors.137 In a separate study, Arvanil was found to exhibit a CB1 affinity similar to that of AEA, but to be less efficacious at inducing CB1 receptormediated GTPgS binding.138 The Ki for arvanil at the VR1 receptor was 0.28 mM. Arvanil exhibited a pharmacological profile that was more similar to anandamide than capsaicin. However, arvanil was much more potent in vivo than could be expected from its CB1 affinity and efficacy, suggesting that arvanil may act through novel sites for anandamide.138 De Petrocellis and co-workers139 found that four known anandamide transport inhibitors, AM404, arvanil, olvanil and linvanil, activate VR1 receptors at concentrations 400–10 000-fold lower than those necessary to inhibit the anandamide transporter. However, these investigators also found three novel AEA derivatives, named VDM11, VDM12 and VDM13, which inhibit the anandamide transporter as potently as AM404, but & 2002 Elsevier Science Ltd. All rights reserved.
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exhibit little or no agonist activity at hVR1. These compounds are weak inhibitors of AEA enzymatic hydrolysis and poor CB1/CB2 receptor ligands. These investigators showed that, despite the overlap between the chemical moieties of anandamide transport inhibitors and VR1 agonists, selective inhibitors of AEA uptake that do not activate VR1 (e.g. VDM11) can be developed. Di Marzo and co-workers140 recently reported the synthesis of a highly selective CB1 ligand and novel CB1/VR1 vanilloid receptor hybrid ligands. O-1812 (22) was found to be highly (500- to 1000-fold) selective for CB1 vs both VR1 and CB2 receptors. O-1861 (23) bound with high affinity to both the CB1 (Ki =24.372.6 nM) and VR1 (EC50=3.070.2 nM), but had essentially no CB2 affinity (Ki410,000 nM).
ACKNOWLEDGEMENTS The author wishes to thank Dow P. Hurst and Beverly Brookshire for their technical assistance. Financial support from the National Institute on Drug Abuse (grants DA03934 and kO2 DA00489) is gratefully acknowledged.
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receptors and pathways of inactivation. Mol Pharmacol 1998; 54: 180–188. Wise M. L., Soderstrom K., Murray T. F., Gerwick W. H. Synthesis and cannabinoid receptor binding activity of conjugated triene anandamide, a novel eicosanoid. Experientia 1996; 52: 88–92. Berglund B. A., Boring D. L., Howlett A. C. Investigation of structural analogs of prostaglandin amides for binding to and activation of CB1 and CB2 cannabinoid receptors in rat brain and human tonsils. Adv Exp Med Biol 1999; 469: 527–533. Berglund B., Fleming P., Rice K., Boring D., Howlett A. Investigation of structural analogs of anandamide for binding and activation of cannabinoid receptors. 1997 Symposium on the Cannabinoids, International Cannabinoid Research Society, Burlington, VT, p. 6. Stegeman R., Pawlitz J., Stevens A., Gierse J., Stallings W., Kurumbail R. Abstract, American Crystallographic Association, 1998. Hurst D., Norris J., Guarnieri F., Reggio P. Conformational requirements for CB1/endocannabinoid interaction. 2001 Symposium on the Cannabinoids (ISBN: 0-9658053-5-3), International Cannabinoid Research Society, Burlington, VT, 2001; p. 11. Barnett-Norris J., Hurst D., Buehner K., Reggio P., Ballesteros J., Guarnieri F. Agonist alkyl tail interaction with cannabinoid CB1 receptor V6.43/I6.46 groove induces a helix 6 active conformation. Int J Quant Chem 2002, in press. Ballesteros J. A., Jensen A. D., Liapakis G., Rasmussen S. G. F., Shi L., Gether U., Javitch J. A. Activation of the b2 adrenergic receptor involves disruption of an ionic lock between the cytoplasmic ends of transmembrane segments 3 and 6. J Biol Chem 2001; 276: 29 171–29 177. Jensen A. D., Guarnieri F., Rasmussen S. G. F., Asmar F., Ballesteros J. A., Gether G. Agonist-induced conformational changes at the cytoplasmic side of TM 6 in the b2 adrenergic receptor mapped by site-selective fluorescent labeling. J Biol Chem 2001; 276: 9279–9290. Farrens D. L., Altenbach C., Yang K., Hubbell W. L., Khorana H. G. Requirement of rigid-body motion of transmembrane helicies for light activation of rhodopsin. Science 1996; 274: 768–770. Song Z. H., Bonner T. I. A lysine residue of the cannabinoid receptor is critical for receptor recognition by several agonist, but not WIN-55,212. Mol Pharmacol 1996; 49: 891–896. Lambert D. M., Di Paolo F. G., Sonveaux P., Kanyonyo M., Govaerts S. J., Hermans E., Bueb J.-L., Delzenne N. M., Tschirhart E. J. Analogues and homologues of N-palmitoylethanolamide, a putative endogenous CB2 cannabinoid, as potential ligands for the cannabinoid receptors. Biochem Biophys Acta 1999; 1440: 266–274. Jonsson K.-O., Vandevoorde S., Lambert D. M., Tiger G., Fowler C. J. Effects of homologues and analogues of palmitoylethanolamide upon the inactivation of the endocannabinoid anandamide. Br J Pharmacol 2001; 133: 1263–1275. Cheer J. F., Cadogan A. K., Marsden C. A., Fone, K. C. F., Kendall D. A. Modification of 5-HT2 receptor mediated behavior in the rat by oleamide and the role of cannabinoid receptors. Neuropharmacolgy 1999; 38: 533–541. Boring D. L., Berglund B. A., Howlett A. C. Cerebrodiene, arachidonyl-ethanolamide, and hybrid structures: potential for interaction with brain cannabinoid receptors. Prostaglandins Leukot Essent Fatty Acids 1996; 55: 207–210.
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132. Mechoulam R., Fride E., Hanus L., Sheskin T., Bisogno T., Di Marzo V., Bayewitch M., Vogel Z. Anandamide may mediate sleep induction. Nature 1997; 389: 25–26. 133. Zygmunt P. M., Petersson J., Andersson D. A., Chuang H. H., Sorgard M., Di Marzo V., Julius D., Hogestatt E. D. Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide. Nature 1999; 400: 452–457. 134. Ross R. A., Gibson T. M., Brockie H. C., Leslie M., Pashmi G., Craib S. J., Di Marzo V., Pertwee R. G. Structure–activity relationship for the endogenous cannabinoid, anandamide, and certain of its analogues at vanilloid receptors in transfected cells and vas deferens. Br J Pharmacol 2001; 132: 631–640. 135. Zygmunt P. M., Chuang H.-H., Movahed P., Julius D., Hogestatt E. D. The anandamide transport inhibitor AM404 activates vanilloid receptors. Eur J Pharmacol 2000; 396: 39–42. 136. Di Marzo V., Bisogno T., Melck D., Ross R., Brockie H., Stevenson L., Pertwee R., De Petrocellis L. Interactions between synthetic vanilloids and the endogenous cannabinoid system. FEBS Lett 1998; 436: 449–454.
137. Melck D., Bisogno T., DePetrocellis L., Chuang H.-H., Julius D., Bifulco M., Di Marzo V. Unsaturated long-chain N-acyl-vanillylamides (N-AVAMs): vanilloid receptor ligands that inhibit anandamide-facilitated transport and bind to CB1 cannabinoid receptors. Biochem Biophys Res Commun 1999; 262: 275–284. 138. Di Marzo V., Breivogel C., Bisogno T., Melck D., Patrick G., Tao Q., Szallasi A., Razdan R. K., Martin B. R. Neurobehavioral activity in mice of N-vanillyl-arachidonyl-amide. Eur J Pharmacol 2000; 406: 363–374. 139. De Petrocellis L., Bisogno T., Davis J. B., Pertwee R. G., Di Marzo V. Overlap between the ligand recognition properties of the anandamide transporter and the VR1 vanilloid receptor: inhibitors of anandamide uptake with negligible capsaicin-like activity. FEBS Lett 2000; 483: 52–56. 140. Di Marzo V., Bisogno T., De Petrocellis L., Brandi I., Jefferson R. G., Winckler R. L., Davis J. B., Dasse O., Mahadevan, A., Razdan R. K., Martin B. R. Highly selective CB1 cannabinoid receptor ligands and novel CB1/VR1 vanilloid receptor ‘hybrid’ ligands. Biochem Biophys Res Commun 2001; 281: 444–451.
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Endocannabinoid structure–activity relationships for interaction at the cannabinoid receptors P. H. Reggio Department of Chemistry, Kennesaw State University, Kennesaw, GA USA
Summary Anandamide (N-arachidonoylethanolamine) was the first ligand to be identified as an endogenous ligand of the G-protein coupled cannabinoid CB1receptor.Subsequently, two other fattyacid ethanolamides, N-homo-g-linolenylethanolamine and N-7,10,13,16-docosatetraenylethanolamine were identified as endogenous cannabinoid ligands. A fatty acid ester, 2-arachidonoylglycerol (2-AG), and a fatty acid ether, 2-arachidonyl glyceryl ether also have been isolated and shown to be endogenous cannabinoidligands.Recent studies have postulated the existence of carrier-mediated anandamide transport that is essential for termination of the biological effects of anandamide. A membrane bound amidohydrolase (fatty acid amide hydrolase, FAAH), located intracellularly, hydrolyzes and inactivates anandamide and other endogenous cannabinoids such as 2-AG. 2-AG has also been proposed to be an endogenous CB2 ligand.Structure^activity relationships (SARs) forendocannabinoidinteraction with the CB receptors are currently emerging in the literature.This review considers cannabinoid receptor SAR developed to date for the endocannabinoids with emphasis upon the conformational implications for endocannabinoid recognition at the cannabinoid receptors. & 2002 Elsevier Science Ltd. All rights reserved.
INTRODUCTION
Endogenous cannabinoids Anandamide and other fatty acid ethanolamides Anandamide (N-arachidonoylethanolamine, 1, AEA; Chart 1), isolated from porcine brain, was the first to be identified as an endogenous ligand of the G-protein coupled cannabinoid CB1 receptor.1 Like other cannabinoid agonists, AEA produces a concentration-dependent inhibition of the electrically evoked twitch response of the mouse vas deferens1 as well as, antinociception, hypothermia, hypomotility and catalepsy in mice.2 AEA exhibits higher affinity for the cannabinoid CB1 receptor (Ki CB1=89710 nM) than for the CB2 receptor (Ki CB2=3717102 nM).3 Two other polyunsaturated fatty acid ethanolamides, homo-g-linolenylethanolamide (2) and 7,10,13,16-docosatetraenylethanolamide (3) were Received 30 October 2001 Accepted 10 November 2001 Correspondence to: Patricia H. Reggio, Department of Chemistry, Kennesaw State University,1000 Chastain Rd., Kennesaw, GA 30144, USA, Tel.: þ770-423-6170; Fax: þ770-423-6744; E-mail: [email protected]
& 2002 Elsevier Science Ltd. All rights reserved.
subsequently isolated from porcine brain and shown to bind to the cannabinoid CB1 receptor with high affinity.4 Increases in intracellular calcium concentrations have been shown to stimulate the formation of anandamide from a membrane phospholipid precursor, N-arachidonoyl phosphatidyl ethanolamine.5–8 Once released, anandamide can activate cannabinoid receptors. These agonist–receptor interactions result in activation of G proteins, particularly of the Gi/o family.9,10 Signal transduction pathways that are regulated by these G proteins include inhibition of adenylyl cyclase;10–15 regulation of ion currents (inhibition of voltage-gated L, N, and P/Q Ca2+ currents;13,16,17 activation of K + currents;17,18 activation of focal adhesion Kinase (FAK);19 mitogen-activated protein Kinase (MAPK)20–22 and induction of immediate early genes;20,21,23,24 and, stimulation of nitric oxide synthase (NOS).25–29 Termination of anandamide signaling at the cannabinoid receptors occurs through an uptake mechanism that transports anandamide into the cell30–32 where it subsequently undergoes rapid degradation. Current evidence suggests that anandamide uptake is a carrier-mediated process that is time- and temperature-dependent, saturable and inhibited with a unique pharmacological
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O 9
8
6
5
7
4
3
2
1
10 11
12
13
14
15
16
17
18
19
NHCH2 CH2 OH 20
Anandamide 1 NHCH2 CH2OH O
Homo-γ -linolenylethanolamide 2 O
NHCH2 CH2 OH
Docosatetraenylethanolamide 3 O
OCH(CH2 OH)2
2-AG 4 CH2 -O-CH(CH2OH)2
Noladin ether 5 O
NHCH 2CH2OH
Palmitoyl-EA 6
Chart 1
profile.5,31,33 In brain and liver, AEA is hydrolyzed enzymatically to yield arachidonic acid and ethanolamine.34 The reaction is catalyzed by a membrane-bound amidohydrolase (called anandamide amidohydrolase or fatty acid amide hydrolase, FAAH) which has been cloned.35 Anandamide amidohydrolase appears to be located intracellularly, as suggested by both cell fractionation studies and sequence analysis of the cloned enzyme. A close correspondence between the distribution of FAAH and CB1 in rat brain has been reported. The complementary pattern of FAAH and CB1 expressions at the cellular level has suggested that FAAH participates in cannabinoid signalling mechanisms in the brain.36–38 FAAH has been shown recently to work in conjunction with other membrane proteins to facilitate anandamide transport by creating an inward concentration gradient as a result of its metabolism of anandamide.39
2-AG: sn-2-Arachidonoylglycerol (2-AG; 4) was isolated by Mechoulam and co-workers40 from canine intestinal tissue and by Sugiura and co-workers41 from rat brain and shown also to be an endogenous CB ligand (CB1 Ki =472755 nM; CB2 Ki =14007172 nM).40 2-AG has been found present in the brain at concentrations 170 times42 to 800 times41 greater than anandamide. Interaction of 2-AG with the cannabinoid receptors results in activation of G proteins,15,43 particularly of the Gi/o family and the inhibition of adenylyl cyclase.15 2-AG induces a rapid, transient elevation of intracellular free [Ca2þ] in neuroblastoma glioma hybrid NG108-15 cells which express the CB1 receptor 44 and in N18TG2 cells.45 This action of 2-AG is blocked by pretreatment with pertussis toxin, indicating that Gi/o is involved in the response. This action of 2-AG also is blocked by treatment with the CB1 antagonist SR141716A, suggesting that this effect may be CB1 receptor mediated. The elevation of intracellular [Ca2þ] effect is also produced by cannabinoid agonists, WIN55,212-2, HU210, CP55940, anandamide and R-methanandamide; however, these agonists produce much lower maximal responses than do 2-AG, and its congeners. The relative efficacy profile for CB agonists differs from that described for [35S]GTPgS binding and inhibition of adenylyl cyclase, suggesting that the signal transduction pathway for this intracellular [Ca2þ] regulation must differ, perhaps in the type of G protein that tranduces the response.44–46 A similar effect in HL-60 cells which express the CB2 receptor has also been demonstrated.47 2-AG has also been found to induce the activation of p42/44 MAP Kinase in HL-60 cells.48 2-AG has been shown to compete with AEA for uptake by the anandamide transporter.32,49 The FAAH enzyme has been reported to hydrolyze 2-AG four times faster than its hydrolysis of anandamide.50 It now appears that signalling by the endocannabinoid system represents a mechanism by which neurons can communicate backwards across synapses to modulate their inputs. Recently, it has been suggested that both endocannabinoids mediate retrograde signalling at hippocampal synapses by their ability to downregulate GABA release at pre-synaptic terminals.51 Noladin ether : An ether-type endocannabinoid, 2arachidonyl glyceryl ether (noladin ether, 5) was recently isolated from porcine brain.52 2-arachidonyl glyceryl ether was found to bind to the CB1 receptor (Ki =21.270.5 nM) and cause sedation, hypothermia, intestinal immobility and mild antinociception in mice, effects typically produced by cannabinoid agonists. Compound 5 was found to bind weakly to CB2 (Ki43 mM). This compound had been synthesized previously by the Mechoulam lab in a program to develop compounds less prone to enzymatic hydrolysis.53 Independently, Sugiura and co-workers 46 prepared this
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Endocannabinoid structure^activity relationships
compound and examined its effects on Ca2þ levels in cells.54 Compound 5 was found by these investigators to exhibit appreciable agonistic activity, although its activity was significantly lower than that of 2-AG. PEA: Facci and co-workers55 reported that N-palmitoylethanolamide (PEA, 6), a C16 saturated fatty acid ethanolamide, behaves as an endogenous agonist of the CB2 receptor on rat mast cells (RBL-2H3 cells). However, PEA was found to displace only 10% of [3H]CP-55 940 from human cloned CB2 receptor at concentrations up to 10 mM.3 For this reason, the identification of PEA as an endogenous cannabinoid remains controversial. PEA has been reported to be orally active in reducing edema formation and inflammatory hyperalgesia by downmodulating mast cell activation.56 PEA has also been shown to act in synergy with anandamide. Recently, Di Marzo and co-workers showed that PEA potently enhances the anti-proliferative effect of AEA on human breast cancer cells (HBCCs), in part by inhibiting the expression of the FAAH enzyme.57 A substantial structure–activity relationship (SAR) literature now exists for fatty acid ethanolamide (e.g. anandamide) interaction with the CB1 receptor. This literature has been used to propose models for the interaction of anandamide at the CB1 receptor. Currently emerging in the literature is the SAR for fatty acid glycerol ester (e.g. 2-AG) interaction at both the CB1 and CB2 receptors. Studies have focused on both head group and acyl chain requirements for interaction with each of these biological targets. One important question that is addressed in these studies is whether the structural requirements for endocannabinoid interaction with CB1 and CB2 are the same or are different. In this paper, endocannabinoid SAR for interaction with CB receptors will be reviewed. This SAR, of course, has been based on measured receptor affinities and activities. Because the inactivation of anandamide and 2-AG has been found to take place very rapidly, it is important that any study of endocannabinoid interaction with the CB receptors pay particular attention to enzymatic inactivation as a confounding problem in assays for affinity or activity. We begin here by discussing this important problem.
Fatty acid amide hydrolase Fatty acid amide hydrolase (FAAH, oleamide hydrolase, anandamide amidohydrolase) is an integral membrane protein that hydrolyzes and inactivates fatty acid primary amides and ethanolamides including oleamide and anandamide.35 The distribution of FAAH in the CNS suggests that it is posed to degrade neuromodulating fatty acid amides near their sites of action. This localization suggests that FAAH is intimately related to the regulation of the effects evoked by these fatty acid & 2002 Elsevier Science Ltd. All rights reserved.
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amides.34,36,58 In addition, FAAH has been shown to hydrolyze another endogenous cannabinoid, 2-AG four times faster than AEA, as well as the related 1-AG and 1oleylglycerol, and the corresponding simple ester, methyl arachidonate.50,59–61 Human and rat FAAH share a similar qualitative substrate selectivity with anandamide, oleamide (18:1 D9) and myristic (14:0) amide serving as the preferred substrates and palmitic (16:0) and stearic (18:0) amides being hydrolyzed at significantly lower rates. However, human FAAH was less selective than rat FAAH as it hydrolyzed myristic amide at a rate comparable to oleamide (18:1 D9) and palmitic (16:0) amide at a rate only 2-fold slower than oleamide.35,62
FAAH Inhibitors Given the plurality of fatty acid amides and esters that FAAH hydrolyzes, it is interesting that a relatively small set of inhibitors of this enzyme has been reported in the literature. Linoleoylethanolamide has been reported to be a natural inhibitor of FAAH.63 Several other fatty acid ethanolamides have been reported by Desarnaud34 to inhibit AEA hydrolysis. Arachidonoylserotonin has been reported to be the first FAAH inhibitor that is inactive both against cPLA2 and at CB1 receptors.64 The endogenous potent FAAH inhibitor, 2-octyl-g-bromo acetoacetate, has been reported to be an endogenous sleep compound65 and its analogs are among the set of FAAH inhibitors. Other classes of inhibitors are electrophilic carbonyl inhibitors (trifluoromethyl ketones, a-halo Ketones, a-Keto esters and amides and aldehydes)66–68 or irreversible inhibitors (sulfonyl fluorides and fluorophosphonates) incorporated into fatty acid structures.69–72
Considerations for the interpretation of endocannabinoid binding data The development of structure–activity relationships for endocannabinoid interaction with the CB receptors hinges upon comparisons of receptor affinities for a large number of compounds. This inevitably entails comparisons across laboratories. In no other structural class of cannabinoid agonists is such a comparison process fraught with more peril than in the endocannabinoids. The rapid degradation of anandamide in cultured cells and crude homogenates of other cultured cells or of rat tissues was recognized early in the literature.73 At that time, phenylmethylsulfonyl fluoride (PMSF) came into standard use in filtration-based CB1 binding assays to inhibit the breakdown of anandamide. In very early literature, no PMSF was used in filtration-based binding assays of anandamide and its analogs. These studies reported very high Ki values for anandamide as a
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consequence. In fact, a common method used in the literature today to test for the susceptibility of analogs to enzymatic breakdown has been to compare the receptor affinities of the analogue with and without PMSF in the assay.74 However, in other binding assay protocols, PMSF has not been found to be necessary. Sheskin and coworkers75 reported that in a centrifugation-based ligand binding assay, PMSF was not necessary, as these investigators found that the addition of an amidase inhibitor to their rat synaptosomal membrane assay did not lower the Ki. Another concern for cross-laboratory comparisons is whether the binding assay is performed using a cloned cell line or tissue homogenates. The use of a cloned cell line presumably assures a single CB sub-type receptor population for the binding assay. Tissue-based assays may not, on the other hand, guarantee a single-receptor sub-type population. For many CB1 binding assays, rat brain membranes have been commonly used with the assumption that only CB1 receptors are present in this preparation. Munro and co-workers76 found no CB2 transcripts in brain tissue by either Northern analysis or in situ hybridization studies. However, Skaper and coworkers77 have reported that cerebellar granule cells and cerebellum express genes encoding both the CB1 and CB2 receptors. In CB2 binding assays, mouse spleen membranes or human tonsils have been used. Spleen has been reported to exhibit much less CB2 mRNA than human tonsils, and CB1 mRNA is present in spleen, but not in tonsils.78 Because CB1 binding assays of anandamide analogs can be performed either using a filtration- or centrifugation-based assay on either tissue homogenates or cloned cells with and without PMSF or other inhibitor, there is variation in reported Ki values for endocannabinoids between laboratories. For these reasons, in the sections that follow here, an attempt has been made to provide the Ki value for AEA obtained in a given laboratory as a benchmark from which to compare relative affinities of other analogs.
ENDOCANNABIOID INTERACTION WITH CANNABINOID RECEPTORS
SAR of fatty acid ethanolamides Cannabinoid SAR literature has primarily focused on receptor affinities of fatty acid ethanolamides, such as anandamide, and its analogs. Recently, however, Sugiura has begun to develop an SAR for 2-AG and its analogs based on a functional assay of Ca2þ mobilization discussed earlier here.46,48 Because of their difference in basis, each of these SARs will be presented separately here.
Endocannabinoid SAR at the CB receptors can be divided into studies of head group requirements for high-affinity binding and studies of acyl chain requirements for high-affinity binding.
Amide head group SAR Arachidonamide and simple alkyl esters of arachidonic acid do not show significant CB1 affinity.79 Cyclization of the head group into an oxazoline ring diminishes affinity.74 Methylation at the C-1’ position in the AEA head group results in an 1’-R-methyl isomer (AM356, Rmethanandamide, 7) which has 4-fold higher CB1 affinity than AEA, while the 1’-S-methyl isomer has 2-fold lower CB1 affinity than AEA. AM356 also was found to be resistant to enzymatic breakdown (Ki =2071.6 nM with PMSF; Ki =2873 nM without PMSF).80 Methylation at the 2’ position also produces some stereoselectivity, as the S(þ) isomer was found to have 2–5-fold higher CB1 affinity than the R()-isomer.43,80 Goutopoulos and coworkers81 reported that introduction of a single methyl at the 2 positions (R or S ) on the acyl chain of anandamide leads to compounds that show moderately improved affinities for CB1 relative to anandamide, but limited enantioselectivity (AEA, Ki =7871.6 nM; (R)-N-(2-hydroxyethyl)-2-methyl-arachidonamide Ki =54.175.2 nM; (S )N-(2-hydroxyethyl)-2-methyl-arachidonamide Ki =35.37 4.3 nM) and only modestly improved metabolic stability. The CB1 affinity of the 2,2-dimethyl analog of anandamide (Ki =72.276.3 nM) is unchanged from that of anandamide. This compound did show somewhat enhanced metabolic stability, however. Introduction of larger alkyl groups at the 2 position has a detrimental effect on CB1 affinity.82 A high degree of enantio- and diastereoselectivity was observed for the 2, 1’-dimethyl analogs. (R)-N-(1-methyl-2-hydroxyethyl)-2-(R)-methylarachidonamide81 exhibits the highest CB1 affinity in this series with a Ki =7.4270.86 nM , a 10-fold improvement on anandamide (Ki =7871.6 nM ). Enlargement of the ethanolamine head group by insertion of methylene groups revealed that the Npropanol analog has slightly higher CB1 affinity than AEA, while higher homologs have reduced CB1 affinity.75,79 Alkyl branching of the alcoholic head group led to lower affinity analogs,75 as did the incorporation of hydroxyphenyl groups such as in AM404.84 N-(propyl) arachidonylamide (8), in which the hydroxyl group is replaced with a methyl group, possesses higher CB1 affinity (Ki =7.3 nM) than anandamide itself (Ki=22 nM).75,79 However, when this same substitution was performed in R-methanandamide (7, Ki =17.9 nM, Chart 2), a lower affinity analog resulted (Ki =73.6 nM).74 Substitution of an N-cyclopropyl group for the ethanolamine head group of AEA leads to a very high-affinity CB1 compound, arachidonylcyclopropylamide, ACPA
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Endocannabinoid structure^activity relationships
O
Me OH NH
R - methanandamide 7
O
NHCH2CH2CH3
8 O
147
methylene group between the amide nitrogen and a 4phenol group results in an analog with modest CB1 affinity (Ki=21773 nM vs AEA Ki =7872 nM).86 Replacement with a 3,4-dihydroxy-ethyl-phenyl results in an analog with greater CB1 affinity than AEA.88 Taken together, all of these results suggest that the hydroxyl in the anandamide head group is not essential for receptor interaction, but that the cannabinoid receptor can accomodate both hydrophobic and hydrophilic head groups, possibly in two different subsites.83 While the hydrophilic site accomodates slightly larger head groups, the sizes of the cavities in which the head group binds still appear to be relatively small, as only conservative variations on the head group permit the retention of high-affinity binding.
Amido group modifications NH
ACPA 9
NHCH2CH2F
NH O 10
Chart 2
(9; Ki =2.270.4 nM), while methylene insertion between the amide nitrogen and the cyclopropyl ring leads to reduced affinity (Ki4100 nM).10,49 Substitution of an Nallylarachidonamide (Ki =9.9 nM) and N-propargylarachidonamide (Ki=10.8 nM) shows approximately 6-fold higher affinity for CB1 than anandamide (Ki =61.0 nM).74 Substitution of an N-phenyl group for the ethanolamine head group of AEA results in a compound that retains CB1 affinity (Ki =109 nM) similar to AEA (Ki =143737 nM), while substitution of an N-adamantyl group, results in an extreme loss of affinity (Ki41000 nM).49 These results suggest that there may exist a hydrophobic sub-site for the AEA head group such that the hydroxyl of AEA may not be necessary for receptor interaction.74,83 These results also suggest that this hydrophobic sub-site is of limited size, as large hydrophobic groups are not well tolerated. Replacement of the hydroxyl group of AEA with a halogen such as F or CI increases CB1 affinity as well.10,74,85 Replacement of the 2-hydroxyethyl group of AEA with a phenolic group or pyridine group decreases affinity for CB1;49,86,87 however, the insertion of a & 2002 Elsevier Science Ltd. All rights reserved.
In order for high-affinity binding to the CB1 receptor to occur and for agonist binding to activate G-proteins, the carbonyl group of the AEA amide head group cannot be replaced with a methylene group.43 Retro-anandamide, in which the position of the carbonyl and the NH of the amide group in anandamide are reversed, retains receptor affinity and exhibits stability with regard to hydrolysis by FAAH (Ki =115 nM with PMSF and 134 nM without PMSF).74 Arachidonylethers, carbamates and norarachidonlycarbamates have poor CB1 affinity.89 However, norarachidonyl ureas show generally good binding affinities to the CB1 receptor (Ki =55–746 nM; AEA Ki =89710 nM). Norarachidonyl 2fluoroethyl urea (10; Ki =5578 nM) has higher CB1 affinity than AEA (Ki =89710 nM).89 Some of the weaker affinity analogs in this series, produced potent pharmacological activity. These analogs showed hydrolytic stability toward amidase enzymes, as well.89
Acyl chain SAR. Arachidonic acid The overall conformations of the endocannabinoids are controlled largely by the conformations of their fatty acid acyl chains. While no X-ray crystal structure data is available for the endocannabinoids, there is X-ray crystal data for the fatty acid that comprises the acyl chain in anandamide and 2-AG, arachidonic acid (AA, 20:4 D5,8,11,14 ; 11). We begin here by discussing the conformations of arachidonic acid that have been determined both by experiment and by calculation. X-ray crystal structure studies of arachidonic acid (AA, 11) have revealed that AA is a very flexible molecule that can adopt very different conformations depending on its environment. In the X-ray crystal structure of pure arachidonic acid, arachidonic acid exists in an extended (angle-iron) conformation in which double bonds 1 and 3 and double bonds 2 and 4 are coplanar, while the planes of adjacent double bonds are perpendicular to one
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O 9
8
6
5
7
4
2
3
1
10 11
12
13
14
15
17
16
18
Arachidonic Acid 20:4 ∆5,8,11,14 11 10
19
OH 20
O
7 NHCH2CH2OH
13
16 12
Fig. 1 (a) This figure illustrates the crystal structure of arachidonic acid.91,95 (b) This figure illustrates the conformation that arachidonic acid92 (PDB Accession No.1ADL) adopts when complexed with adipocyte lipid binding protein, a fatty acid transporter. (c) This figure illustrates the conformation that arachidonic acid adopts at the active site of prostaglandin synthase-1.93
8
NHCH2CH2OH O
11
14 13
another (see Fig. 1a).91,95 An X-ray crystal study of arachidonate ion complexed with adipocyte-lipid-binding protein revealed that AA binds within the b barrel cavity of this protein. Here the carboxylate group of AA is engaged in a strong electrostatic interaction with Arg126, Tyr128 and Arg106 through an intervening water molecule, while the arachidonate acyl chain assumes a hairpin (i.e. U-shaped) conformation for which the first double bond of arachidonic acid is distorted out of the plane formed by the other three double bonds (see Fig. 1b).90,92 In the 3 A˚ resolution structure of prostaglandin H synthase-1 (Fig. 1c), arachidonic acid adopts an extended L-shaped conformation that positions the 13proS hydrogen of AA for abstraction by Tyr 385, the likely radical donor at this enzyme’s active site.93 One important feature of the arachidonic acid unsaturated acyl chain is the great torsional mobility about the two torsion angles involving each methylene carbon between adjacent pairs of double bonds in the acyl chain (for example, the C8–C9–C10–C11 and C9–C10– C11–C12 torsion angles, see Chart 3 structure 11 for numbering system). Rabinovich and Ripatti94 found that polyunsaturated acyl chains in which double bonds are separated by one methylene group are characterized by the highest equilibrium flexibility compared with other unsaturated acyl chains. Rich95 reports that a broad domain of low-energy conformational freedom exists for these C–C bonds. Conformational analyses of arachidonic acid suggest that the high flexibility built into the acyl chain of AA, permits this fatty acid to assume many conformations. In their Monte Carlo study, Rabinovich and Ripatti94 found that polyunsaturated fatty acids, whose double
O
NHCH2CH2OH 11
14 14 Me 9 OH 1
Me Me
3
O
1′
∆9 -THC 15 NHCH2CH2OH O 16
O
NHCH2CH2OH O H
OH PGB2-EA 17
Chart 3
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Endocannabinoid structure^activity relationships
bonds are separated by one methylene carbon, assume an extended (angle-iron) conformation (see Fig. 1a) when all molecules are efficiently packed below the phase-transition temperature. Molecular dynamics (MD) or molecular dynamics/simulated annealing (MD/SA) studies (in vacuo) of arachidonic acid (11, AA) and of other polyunsaturated fatty acids related to AA, have been published by several groups.94–98,100,101,103 These computational studies of AA have primarily found looped or back-folded conformations to be low-energy conformers of AA. Rich conducted a quenched molecular dynamics study of AA in vacuo.95 The two lowest enthalpy conformers found for AA were J-shaped conformers in which the carboxylic acid group is in close proximity to the C14–C15 p bond. This same J-shape was reported by Corey and co-workers101 as one type of low-energy minimum identified in their conformational analysis of arachidonic acid. The authors suggested that such a Jshaped conformation in solution would be energetically favorable and would be consistent with the chemistry of peroxyarachidonic acid for which an internal epoxidation leads to 14, 15-epoxyarachidonic acid.102 Using the A* Algorithim followed by minimization in the ‘Batchmin’ module of Macromodel, Leach and Prout98 found rightand left-handed looped or ‘helical-like’ conformations of AA to be its lowest energy conformations. A simulated annealing study of AA identified a looped conformation,103 while a molecular dynamics study in vacuo found low-energy structures of AA to be hairpin-, helical- and crown-shaped (i.e. U-shaped conformers in which the ends of the chain come close to one another).96 Lopez and co-workers also employed molecular dynamics simulations in their study of AA, finding two low-energy conformations, one of which was U-shaped and was proposed as the bioactive conformation at its cyclooxygenase binding site.97 A recent modeling study of the binding of arachidonic acid in the cyclo-oxygenase active site of mouse prostaglandin endoperoxide synthase-2(COX-2) predicts that AA will orient in a kinked or L-shaped conformation. In this conformation, the carboxylate moiety binds to Arg,120 while the o-end is positioned above Ser530 in a region termed the top channel.104 In their theoretical conformational analysis of the complex of arachidonic acid (AA) with a-tocopherol, Shamovsky and Yarovskaya105 found that AA assumes an extended conformation. A new conformational analysis method, Conformational Memories (CM)106,107 was employed by Barnett-Norris and co-workers108 to study the conformations of arachidonic acid. Results of the biased sampling phase from CM calculations of arachidonic acid were consistent with Rich’s and with Rabinovich and Ripatti’s results108 as they revealed, for example, a relatively broad distribution of populated torsional space about the classic & 2002 Elsevier Science Ltd. All rights reserved.
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skew angles of 1191(s) and 1191(s’) for the C8–C9–C10– C11 torsion angle in anandamide. CM calculations identified extended conformers and folded or U-shaped conformers to be the major conformational families of arachidonic acid. The more compact (U-shaped) structure was found to predominate in water, while the extended shape was found to predominate in CHCl3 (and in vacuo). The existence of J-shaped conformers as suggested in the literature 95,101,102 was confirmed by CM. In this case, the J-shaped family was found to comprise a smaller, but still significant conformational family of arachidonic acid. These results illustrate that the CM method resulted in a much broader sampling of the conformational space of arachidonic acid, than had been performed previously using molecular dynamics or molecular dynamics/simulated annealing techniques.95–98,100,101,103 These previous studies of arachidonic acid found primarily only compact structures (U’s, J’s, helical) as energy minima. While no other theoretical studies of arachidonic acid in solvent have been reported, the trends in the CM results reported by Barnett-Norris et al.108 for AA in CHCl3 and in H2O are consistent with the idea that in H2O, arachidonic acid would minimize the exposure of its hydrophobic portions by forming a more compact U-shape. While Rich reported that methylene carbons attached to the sp2 hybridized carbon of a C=C double bond demonstrate a broad region of conformational space (i.e. for Er 1.0 kcal/mol) available to them, the same was not true for saturated carbon chains such as the acyl chain of palmitic acid. Rich reported that for these chains, freedom of motion was restricted to trans (71801) or gauche (7601) configurations. As a result, saturated fatty acids are much less flexible and favor more extended conformations.95,99 In summary, the fatty acid literature indicates that saturated fatty acids, such as palmitic acid, tend to adopt extended conformations, while unsaturated fatty acids, such as arachidonic acid which possesses cis double bonds separated by methylene carbons, exhibit a high degree of flexibility that allows them to adopt folded, as well as extended conformations quite easily.
Acyl chain SAR. Fatty acid ethanolamides F effect of branching Mono- (Ki =53711 nM) or di- (Ki =4772 nM) methylation at C2 on the acyl chain of AEA produced analogs with slightly higher CB1 affinities than AEA (Ki =89710 nM); however, larger or branched alkyl substituents at C2 lead to low CB1 affinities.85 Effect of unsaturation: Sheskin et al.75 reported that N-docosatetraenoyl-ethanolamine 22:4, D7,10,13,16 (12, Ki =34.473.2 nM) and N-homo-g-linolenoyl-ethanolamine 20:3, D8,11,14 (13, Ki =53.475.5 nM) had CB1 affinities comparable to anandamide 20:4, D5,8,11,14
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Fig. 2 Presented here are Conformational Memories results for a series of ethanolamides that show the effect of increasing degrees of unsaturation on the fatty acid acyl chain.110 The left column illustrates one major family of conformers.These are extended conformers.The right column illustrates the second major family identified for each compound.This family shows increasing curvature as the degree of unsaturation increases.
(1, Ki =39.275.7 nM). As unsaturation was decreased to two cis double bonds in N-11,14 eicosadienoyl ethanolamine (20:2, D11,14 analog), CB1 affinity was greatly diminished (14, Ki =1500 nM). A saturated fluoro-derivative of AEA (20:0), O-586 was found not to bind to CB1 receptors (Ki410 000 nM with or without PMSF). This compound also did not produce cannabimimetic discriminative stimulus effects.109 Figure 2 illustrates the conformational differences between ethanolamides with varying degrees of unsaturation in their acyl chains as revealed by Conformational Memories calculations.110 Two major families of conformers were identified for each of the following ethanolamides: 18:0, 20:1 D,11 20:2 D,11,14 20:3 D,8,11,14 20:4 D5,8,11,14 (anandamide). The percentages next to each family indicate the percentage population of each family. There are two important points illustrated in Figure 2. (1) Each of these ethanolamides have a family of extended conformers. These are illustrated in the left column of Figure 2. (2) The curvature of the acyl chain increases with increasing unsaturation. The second column in
Figure 2 (on the right) contains curved or U-shaped families of conformers. It is clear in going from the 18:0 ethanolamide to the 20:4 D5,8,11,14 ethanolamide (anandamide) that curvature in the acyl chain increases and, as a consequence, the distance between the head group and acyl tail decreases as the degree of unsaturation increases. Alkyl tail length: The degree of unsaturation is not the only acyl chain requirement for recognition at the CB1 receptor. Sheskin and co-workers75 found that in an n-3 series, highly unsaturated analogs had poor to no CB1 affinity as revealed by the following series of n-3 ethanolamide derivatives: 20:5, D5,8,11,14,17 (Ki =162.37 13.6 nM) and 22:6, D4,7,10,13,16,19 (Ki =324.179.2 nM), 18:4, D6,9,12,15 (Ki41000 nM), 20:3, D11,14,17 (Ki410 000 nM), 18:3, D9,12,15 (no activity). The fact that this n-3 series shows low to no CB1 affinity reveals that flexibility cannot be the only acyl chain requirement for CB1 recognition as all of these n-3 analogs will have great flexibility due to their levels of unsaturation. However, these analogs may lack an essential part of the cannabinoid pharmacophore: a saturated alkyl tail of sufficient length. An analogy has been drawn by several groups in the literature between the C16–C20 saturated portion of AEA and the C-3 pentyl side chain of the classical cannabinoid, D9-THC (15).111–114 In classical cannabinoid SAR, the C-3 pentyl side chain (see D9-THC, 15) is considered a minimum length to produce cannabinoid activity; while a 1’, 1’-dimethylheptyl side chain appears to be optimum.115 Replacement of the pentyl side chain of a classical or non-classical cannabinoid with a 1’,1’dimethylheptyl side chain has been reported to result in as much as a 75-fold enhancement in CB1 affinity.116 While the replacement of the C16–C20 saturated portion of anandamide with a 1’,1’-dimethylheptyl chain did not result in as great an enhancement as in the classical cannabinoids, it did produce a 13-fold enhancement in CB1 affinity.112,113 This enhancement has been taken as support for the hypothesis that the C16–C20 saturated region of AEA corresponds to the C-3 pentyl chain of D9THC for interaction at the CB1 receptor. Therefore, from this correspondence, one might conclude that the drop in affinity which accompanies the shortening of the saturated tail of anandamide in the n-3 series may be related to SAR C-3 side chain requirements for classical cannabinoid binding at CB1. Acyl chain length: Sheskin and co-workers75 also found that the 18:3 D6,9,12 (n-6) analog of AEA had low CB1 affinity (16, Ki =46007300 nM). This result suggests that a C18 acyl chain is too short to be recognized by CB1, because this analog has both a long enough saturated tail (five carbons) and three double bonds separated by methylene carbons.
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Endocannabinoid structure^activity relationships
Conjugated acyl chain: While most acyl chain SAR studies have focused on unsaturated cis polyenes, AEA analogs with altered double bond patterns have also been reported. All of these analogs possess regions in which some double bond conjugation is present. Edgemond and co-workers have described two oxygenated derivatives of AEA that are products of lipoxygenase activity on AEA, 12(S)–hydroxyeicosatetraenoylethanolamide (12(S)-HAEA) and 12(R) hydroxyeicosatetraenoylethanolamide (12(R)HAEA). While anandamide has a Ki of 107754 nM at CB1, the S stereoisomer (Ki =207752 nM) has one-half the CB1 affinity of AEA, and the R stereoisomer (Ki =416714 nM) has one-quarter the CB1 affinity of AEA.117 Conjugated triene anandamide (CTA) has been reported to have 6fold lower CB1 affinity (Ki =607744 nM) than AEA (Ki =9777 nM).118 Pinto et al.79 investigated a series of arachidonyl amides and esters in addition to a series of ‘rigid hairpin’ conformations typified by N-(2-hydroxyethyl)-prostaglandin amides to determine the structural requirements for binding to the CB1 receptor. Two-dimensional drawings of anandamide and PGB2-EA (17) make the shapes of these two compounds look similar and, therefore, it was possible that PGB2-EA (17) may be a rigid (hairpin/Ushaped) analog of AEA. However, all of the rigid prostaglandin analogs synthesized by Pinto et al.79 failed to alter [3H]CP-55 940 binding to CB1 in concentrations as great as 100 mM. a-Alkylamide derivatives of PGE2 and PGF2a showed enhanced binding affinity relative to these prostaglandin ethanolamides; however, none of the affinities of the a-alkylamide derivatives were comparable to that of AEA. In addition, although the PGE2 amides were able to activate G proteins, including Gs, these signal transduction events were not likely mediated by the CB receptors.119 Barnett-Norris and co-workers108 reported CM results for 17 which showed an attenuated ability to adopt extended or tightly folded conformations like AEA and 2AG. Instead, PGB2-EA (17) in CHCI3 adopts a more folded L-shaped structure. The CM results show that the conjugation of the acyl chain with the ring double bond introduces ‘stiffness’ into this part of the molecule. The authors suggest that the attenuated ability of PGB2-EA to adopt an extended conformation may be the reason why PGB2-EA does not bind to CB1 (i.e. PGB2EA lacks the flexibility of AEA). Alternatively, the steric bulk introduced by the five-membered ring in 17 may produce a steric clash at CB1 that prevents ligand binding. Another series of conformationally restricted analogs that contained ortho and meta disubstituted benzene derivatives with alkylamide and alkyl substituents displayed CB1 affinity in the range of low-to-high micromolar in rat membranes.120 These analogs were also & 2002 Elsevier Science Ltd. All rights reserved.
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designed to test if an endocannabinoid hair-pin conformation was recognized at the CB1 receptor. Several of the analogs inhibited basal binding of GTPgS in rat brain membranes at micromolar concentrations, but none of the analogs affected either basal or stimulated adenylate cyclase activity in N18TG2 membranes. In summary, these results for acyl chain variations suggest that high-affinity receptor binding requires high flexibility of the acyl chain because only analogs with three or more double bonds show high CB1 affinity. The acyl chain may need to adopt a folded conformation to interact with CB1 or the analog may need to adopt several different conformations in order to reach, be recognized by and trigger the receptor. Only those analogs with high degrees of equilibrium flexibility may be able to achieve these conformations easily.
Endocannabinoid interaction with the cannabinoid CB1 receptor The hypothesis that AEA adopts a folded conformation in order to interact with the CB1 receptor has been explored by several research groups. Thomas and co-workers 111 were first to report a COMFA QSAR pharmacophore model for anandamide and its analogs. These authors used molecular dynamics studies to explore conformations of 1 that present pharmacophoric similarities with the classical cannabinoid, D9-THC (15). A J-shaped or looped conformation of AEA was identified that had good molecular volume overlap with D9 -THC (15) when (1) the carboxyamide of AEA was overlaid with the pyran oxygen (O-5) in 15; (2) the head group hydroxyl of AEA was overlaid with the C-1 phenolic hydroxyl group of 15, (3) the five terminal carbons of the AEA fatty acid acyl chain were overlaid with the C-3 pentyl side chain of 15; and (4) the polyolefin loop of AEA was overlaid with the tricyclic ring system of 15. These authors supported their use of a J-shaped conformation for AEA by citing synthetic results for the internal epoxidation undergone by peroxyarachidonic acid which point to the J-shape as necessary for such a reaction.102 Eight pharmacologically active anandamide analogs were shown to have similar conformational mobility and pharmacophore alignments that are conformationally accessible. When these compounds were aligned with 15, their potencies were predicted by a quantitative model of cannabinoid SARs based solely on classical and non-classical cannabinoids with a reasonable degree of accuracy. The ability to incorporate the pharmacological potency of these anandamides into the classical/non-classical cannabinoid pharmacophore model was also proposed to support the relevance of the Thomas pharmacophore model. Tong and co-workers114 reported a different pharmacophore model for AEA using constrained conformational searching and COMFA. 9-nor-9b-OH-HHC (18, Chart 4)
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was used as the template to which AEA and its analogs were fitted. The conformation identified for AEA was a helical conformation in which (1) the oxygen of the carboxyamide overlaid the C-1 phenolic hydroxyl group of 18; (2) the head group hydroxyl overlaid the C-9 hydroxyl of 18; (3) the alkyl tail of AEA overlaid the C-3 alkyl side chain of 18; and, (4) the polyolefin loop overlaid the tricyclic ring structure of 18. These authors supported their use of a helical-shaped AEA by citing an X-ray crystallographic structure which shows that arachidonic acid adopts a helical conformation when it is a substrate for cyclooxygenase.121 The authors cite the close matching of common pharmacophoric elements of AEA and 9nor-9b-OH-HHC as persuasive evidence of the biological relevance of this helical conformer. A 3D–QSAR model was derived using COMFA for a training set of 29 classical and non-classical cannabinoids which rationalized the binding affinity in terms of steric and electrostatic properties. Reggio and co-workers have developed an endocannabinoid pharmacophore based upon endocannabinoid SAR only. These investigators recently proposed a hypothesis about endocannabinoid interaction with CB1 based upon their CM results.122 CM calculations of AEA in CHCI3 reported by Barnett-Norris and co-workers108 revealed that AEA adopts predominantly extended and U-shaped conformers. (J-shaped conformers of AEA were found in lower number (10 out of 150)). For 2-AG, both extended and U-shapes were found, with the extended shape predominating in CHCl3 (71 out of 150 structures). Based upon these results, Reggio and co-workers122 have proposed a model that requires anandamide to assume more than one conformation in its interaction with CB1. In this model, anandamide approaches the CB1 receptor in an extended conformation from lipid. Its initial interaction site is a groove on Helix 6 formed by Val6.43(351) and Ile6.46(354) into which the pentyl tail of anandamide fits. This groove serves as a filter for the acyl chain variants of anandamide, as only those with a sufficient number of carbons (20–22) and degree of saturation (3–4 cis double bonds separated by methylene carbons) are at the correct depth in the membrane and the correct angle to interact with this groove. In addition, at least five saturated carbons at the end of the acyl chain are needed for optimal interaction with this groove. CM results for Helix 6 in CB1 suggest that alkyl chain interaction with this groove induces a conformational change in Helix 6 that would move the intracellular end of Helix 6 away from Helix 3.123 Such a change has been proposed to be the beginning of the R (inactive state) to R* (active state) transition for the b2-adrenergic receptor124,125 and for rhodopsin,126 both G-protein-coupled receptors. Once in the protein interior, anandamide folds into a U-shape with the amide oxygen interacting with
OH 9
H OH 1
Me Me
1′
3
O
9-nor-9β-OH-HHC 18 O
NH OH Olvanil 19
OCH3 OH
O
NH
AM 404 20 O
NH OH
Arvanil 21
OCH3 O
Me OH
Me
NH
Me
CN O-1812 22 O
Me
Me
NH Br
OH
23 OCH3
Chart 4
Lys3.28(192). In this conformation, the ligand acyl chain has hydrophobic interactions with several residues in the Helix 2–3–7 region, including Phe3.25. This model is consistent with CM results that identify both extended
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and folded conformations of AEA as low-energy conformations. It is consistent with the cannabinoid mutation literature that suggests that Lys3.28(192) is the primary interaction site for anandamide.127 The model is consistent with all of the acyl chain SAR discussed above (including chain length and degree of unsaturation requirements) and may explain the low affinity seen for all rigid analogs of anandamide synthesized to date.
CB2 SAR for fatty acid ethanolamides AEA exhibits higher affinity for the cannabinoid CB1 receptor (Ki CB1=89710 nM) than for the CB2 receptor (Ki CB2=3717102 nM).3 Anandamide analogs tend to be CB1 selective with CB2 affinities comparable to or lower than the CB2 affinity of AEA.10,74,81,86 Berglund and coworkers119 found that a-alkylamide derivatives of PGE2 and PGF2a showed higher binding affinity for the CB2 receptor than for the CB1 receptor; however, the affinity was reduced relative to anandamide’s affinity for CB2.119 Palmitoylethanolamide: Facci et al.55 reported that palmitoylethanolamide (16:0, PEA, 6) a C16 saturated derivative, behaves as an endogenous agonist of the CB2 receptor on rat mast cells (RBL-2H3 cells). The IC50 for competitive inhibition of the aminoalkylindole (AAI) cannabinoid agonist [3H]WIN55 212-2 binding to RBL2H3 cell membranes by PEA was reported to be 1.070.6 nM, while that of AEA was reported to be 3372.9 nM.55 However, PEA was found to displace only 10% of non-classical cannabinoid agonist [3H]CP-55 940 binding from human cloned CB2 receptor at concentrations up to 10 mM.3 PEA differs from AEA not only in its lack of unsaturation, but also in its shorter acyl chain (C16 vs C20 for AEA). Using the same protocol as described in our previous paper,108 we recently studied the conformational families of PEA in CHCl3 using the CM method.106,107 The results of our CM calculations revealed that PEA adopts predominantly only one conformation, an extended conformation (83 out of 100 conformers in CHCl3). Figure 3 illustrates this predominant cluster of like conformations. Lambert and co-workers synthesized a series of 10 Npalmitoylethanolamide (PEA, 6) homologs and analogs, varying by the elongation of the fatty acid chain from caproyl to stearoyl and by the nature of the amide substituent, respectively, and evaluated the affinity of these compounds for cannabinoid receptors in the rat spleen; in RBL-2H3 cells and in CHO-CB1 and CHO-CB2 receptor transfected cells.128 No displacement of [3H]CP55 940 or [3H]WIN 55 212-2 by PEA derivatives was observed in rat spleen slices. In RBL-2H3 cells, no binding of [3H]CP55 940 or [3H]WIN55 212-2 could be observed and conversely, no inhibitory activity of PEA derivatives and analogs was measurable. These investigators con& 2002 Elsevier Science Ltd. All rights reserved.
Fig. 3 The major family of palmitoylethanolamide (PEA, 6) conformers (83 out of100) obtained using Conformational Memories.110 Here it is clear that the lack of unsaturation in the PEA acyl chain results in PEA’s preference for an extended conformation.
cluded that it seems unlikely that PEA is an endogenous agonist of the CB2 receptor.128 Most recently, Jonnson and co-workers129 studied the effects of homologs and analogs of palmitoylthanolamide upon the inactivation of the endocannabinoid anandamide. Oleoylethanolamide at 100 mM showed 64.671.5% inhibition of [3H]-WIN 55 212-2 binding to human CB2 receptors expressed on CHO cells. Other compounds had lesser effects at CB2. PEA, palmitoylisopropylamide and R-palmitoyl-(2-methyl)ethanolamide had modest effects upon [3H]-CP 55 940 binding to human CB1 receptors expressed on CHO cells. Oleamide: Oleamide (18:1, D9 cis) was isolated from the cerebro-spinal fluid (CSF) of sleep-deprived cats and shown to induce physiological sleep in rats.71 Cheer and co-workers130 reported that oleamide occupied CB1 receptors on rat brain membranes labeled with the nonclassical cannabinoid agonist [3H]CP-55 940 with an IC50 of 10 mM. However, oleamide has been reported previously by several other groups not to bind to recombinant CB1 or CB2 receptors overexpressed in host
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cells.75,131,132 Oleamide, at a 50 mM concentration, potentiates AEA binding to CB1 receptors by an order of magnitude.132 This effect was not observed in the presence of the FAAH inhibitor PMSF (200 mM), which alone also potentiates AEA binding, thus suggesting that oleamide facillatory action is due to inhibition of AEA hydrolysis in membrane preparations. This is not suprising as oleamide and anandamide have both been shown to be substrates for the FAAH enzyme.35 Summary: In summary, the CB1 receptor does not tolerate very large endocannabinoid head groups; however, it does recognize both polar and non-polar moieties in the head group region. The receptor recognizes ethanolamides whose fatty acid acyl chains have 20 or 22 carbons, with at least three cis double bonds separated by methylene carbons and saturation in at least the last five carbons of the acyl chain. Double bond conjugation within the acyl chain results in diminished CB1 affinity. From a conformational standpoint, for ligands of sufficient length and tail saturation, these results suggest either that only acyl chains that can assume a tightly folded (U-shaped) conformation can bind to CB1 or that ligand flexibility (i.e. ability to extend and to fold) is very important for binding to and activating the CB1 receptor.
2-AG SAR at CB1 and CB2
ities of 2-eicosatrienoyl(20:3 D8,11,14, n-6)glycerol and 2eicosatrienoyl(20:3 D11,14,17, n-3)glycerol are lower than those of 2-eicosatrienoyl(20:3 D5,8,11, n-9) and 2-eicosapentaenoyl(20:5 D5,8,11,14,17, n-3)glycerol, it appears that the presence of a double bond at the D5 position, rather than the structure near the methyl end, is crucially important, probably for some characteristic conformation of the agonistic molecules.
CB2 SAR Sugiura and co-workers have also found that 2-AG can induce rapid transient increases in [Ca2þ] in HL-60 cells.47 It was evident in this case that the response was mediated by the CB2 receptor, but not the CB1 receptor, because the CB2 antagonist, SR144528, but not the CB1 antagonist, SR141716A, blocked the response. 2-AG was found to have more activity than its 1(3)isomer. Ester and ether analogs, including nolandin ether (5, Chart 1) showed appreciable activity, albeit less than that of 2-AG. Anandamide was found to be a weak partial agonist toward the CB2 receptor. The cannabinoid agonist D9 –THC (15, Chart 3) exhibited weak agonistic activity for the CB2 receptor in the transient [Ca2+] assay. 2-Glycerol esters of 20–22 carbon fatty acids showed the strongest CB2 agonist activity. For C20 fatty acids, activity was best for the 20:3 D5,8,11 and 20:4 D5,8,11,14 (2-AG, 4, Chart 1), suggesting that like the 2-AG SAR generated for CB1, a double bond at D5 was important.
CB1 SAR Sugiura and co-workers have reported that 2-AG (4) and other cannabinoid ligands such as anandamide (1) and D9-THC (15) induce rapid transient increases in [Ca2+] in NG108-15 cells through a cannabinoid CB1 receptordependent mechanism.44–46 2-AG was the most potent compound for inducing these transient increases, as its activity was detectable from as low as 0.3 nM. The maximal response induced by 2-AG exceeded responses induced by other CB1 agonists. Activities of the CB1 agonists, HU-210 and CP55,940 were also detectable from as low as 0.3 nM, whereas, the maximal responses induced by these compounds were low compared with 2AG. Anandamide was also found to act as a partial agonist in this system. Arachidonic acid (11) failed to elicit a response, while noladin ether (5) possessed appreciable activity, although its activity was apparently lower than that of 2-AG.46 Sugiura and co-workers46 have begun to generate an SAR based on this transient increase in [Ca2þ]. Glycerol has been found to be the most suitable head group, and the 2-isomer is preferable over the 1(3)-isomer. Arachidonic acid is the most preferred fatty acid moiety, although the activity of eicosatrienoic acid (n-9)-containing species was almost comparable to that of the arachidonic acid containing species. Because the activ-
Divergence between CB1and CB2 SAR While much of the SAR for 2-AG at CB2 is similar to that previously discussed for CB1, there is one divergence. 2Glycerol esters of C22 fatty acids do not show appreciable activity in the CB1 assay, but do show appreciable activity in the CB2 assay.
Endocannabinoid connection withVanilloids The chemical similarity between some synthetic agonists of the vanilloid receptors, such as olvanil (19, N-vanillylcis-9-octadecenoamide) and AEA prompted Di Marzo and co-workers136 to study possible interactions between the cannabinoid and vanilloid signaling systems. Zygmunt and co-workers133 reported that anandamide, but not 2AG or PEA, induces vasodilation by activating vanilloid receptors on perivascular sensory nerves causing release of calcitonin-gene-related peptide (CGRP). The vanilloid receptor may thus be another molecular target for endogenous anandamide, besides cannabinoid receptors in the nervous and cardiovascular system. Subsequently, Ross and co-workers134 reported that the mouse vas deferens (MVD) expresses both CB1 and VR1 receptors and that both receptor types appear to contribute to anandamide-invoked contractions of the MVD.
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AM404 (20), the AEA transport inhibitor, has very low CB1 affinity (Ki =17607142 nM),86 but has been shown to activate vanilloid receptors.135 Indeed, structural similarities between AM404 and the synthetic vanilloid agonist olvanil (19) prompted Di Marzo and colleagues136 to investigate the possibility that olvanil (19) may interfere with anandamide transport. Their results indicated that olvanil blocked both the uptake and the hydrolysis of [14C]AEA by intact RBL-2H3 cells (IC50 =9 mM), while capsaicin and pseudocapsaicin (N-vanillyl-nonanamide) were much less active. Olvanil was more potent than previously reported inhibitors of AEA facilitated transport, i.e. phloretin (IC50=80 mM), AM404 (12.9% inhibition at 10 mM) or oleoylethanolamide (27.5% inhibition at 10 mM). Olvanil was a poor inhibitor of [14C]AEA hydrolysis by RBL-2H3 and N18TG2 cell membranes, suggesting that the inhibitory effect on [14C]AEA breakdown observed in intact cells was due to inhibition of [14C]AEA uptake. Olvanil was stable to enzymatic hydrolysis, and (i) displaced the binding of high-affinity cannabinoid receptor ligands to membrane preparations from N18TG2 cells and guinea pig forebrain (Ki =1.64–7.08 mM), but not from cells expressing the CB2 cannabinoid receptor subtype; (ii) inhibited forskolin-induced cAMP formation in intact N18TG2 cells (IC50 =1.60 mM), this effect being reversed by the selective CB1 antagonist SR141716A. These data suggest that some of the analgesic actions of olvanil may be due to its interactions with the endogenous cannabinoid system. Melck and co-workers found that arvanil (21) was a potent inhibitor of AEA accumulation (IC50=3.6 mM) and was 4-fold more potent than AEA on CB1 receptors (Ki =0.25–0.52 mM), whereas its 18:3 D9,12,15 congener was more selective than arvanil for the transporter (IC50=8.0 mM) vs CB1 receptors (Ki =3.4 mM). None of the compounds efficiently inhibited [14C] AEA hydrolysis or bound to CB2 receptors.137 In a separate study, Arvanil was found to exhibit a CB1 affinity similar to that of AEA, but to be less efficacious at inducing CB1 receptormediated GTPgS binding.138 The Ki for arvanil at the VR1 receptor was 0.28 mM. Arvanil exhibited a pharmacological profile that was more similar to anandamide than capsaicin. However, arvanil was much more potent in vivo than could be expected from its CB1 affinity and efficacy, suggesting that arvanil may act through novel sites for anandamide.138 De Petrocellis and co-workers139 found that four known anandamide transport inhibitors, AM404, arvanil, olvanil and linvanil, activate VR1 receptors at concentrations 400–10 000-fold lower than those necessary to inhibit the anandamide transporter. However, these investigators also found three novel AEA derivatives, named VDM11, VDM12 and VDM13, which inhibit the anandamide transporter as potently as AM404, but & 2002 Elsevier Science Ltd. All rights reserved.
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exhibit little or no agonist activity at hVR1. These compounds are weak inhibitors of AEA enzymatic hydrolysis and poor CB1/CB2 receptor ligands. These investigators showed that, despite the overlap between the chemical moieties of anandamide transport inhibitors and VR1 agonists, selective inhibitors of AEA uptake that do not activate VR1 (e.g. VDM11) can be developed. Di Marzo and co-workers140 recently reported the synthesis of a highly selective CB1 ligand and novel CB1/VR1 vanilloid receptor hybrid ligands. O-1812 (22) was found to be highly (500- to 1000-fold) selective for CB1 vs both VR1 and CB2 receptors. O-1861 (23) bound with high affinity to both the CB1 (Ki =24.372.6 nM) and VR1 (EC50=3.070.2 nM), but had essentially no CB2 affinity (Ki410,000 nM).
ACKNOWLEDGEMENTS The author wishes to thank Dow P. Hurst and Beverly Brookshire for their technical assistance. Financial support from the National Institute on Drug Abuse (grants DA03934 and kO2 DA00489) is gratefully acknowledged.
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