Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

At least five distinct classes of cannabinoids have been identified: traditional tricyclic tetrahydrocannabinols [e.g., ⁹-tetrahydrocannabinol (THC)], synthetic bicyclic cannabinoids (e.g., CP 55,940; Little et al., 1988), aminoalkylindoles (e.g., WIN 55,212; D'Ambra et al., 1992), endocannabinoids (e.g., anandamide; Devane et al., 1992), and pyrazole antagonists (e.g., SR141716A; Rinaldi-Carmona et al., 1994). Although the chemical structures of these cannabinoids differ markedly, all of them contain at least one oxygen that is hypothesized to be involved in the binding of these drugs to brain cannabinoid (CB₁) receptors. ⁹-THC, the primary psychoactive constituent of the marijuana plant, and other tetrahydrocannabinols contain two oxygens: a phenolic hydroxyl at position 1 and an oxygen in a pyran ring on the opposite side of the molecule (Fig. 1). The phenolic hydroxyl group at position 1 interacts with the CB₁ receptor through hydrogen bonding with a lysine residue (Lys-192) (Song and Bonner, 1996). The role of the oxygen of the benzopyran substituent of ⁹-THC is less clear; however, it is known that opening the pyran ring (as in CP 55,940) does not eliminate binding or in vivo activity (Little et al., 1988). Furthermore, in the absence of a phenolic hydroxyl, as in 1-deoxy analogs of ⁸-THC, orientation of the cannabinoid molecule with respect to the CB₁ receptor may be inverted, and the pyran oxygen may substitute as a substrate for hydrogen bonding with Lys 192 (Huffman et al., 1996, 1999).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Chemical structures of 9-THC, CP 55,940, and cannabidiol.

In contrast to the high binding affinity of CP 55,940 and other similar pyran ring open analogs, the natural product cannabidiol is also a pyran ring open compound, but it does not bind to CB₁ or CB₂ receptors nor does it have a cannabinoid profile of effects in vivo. Even the 1',1'-dimethylheptyl analog of cannabidiol binds very poorly to the CB₁ receptor (R. K. Razdan, unpublished observations). This intriguing feature of cannabidiol prompted us to examine the structure-activity relationship of resorcinol derivatives, which could be considered as cannabidiol analogs.

After our work on the resorcinol series was initiated, Hanu et al. (1999) published the synthesis and activity of HU-308, a dimethoxyresorcinol derivative that is a CB₂-selective agonist. The transmembrane regions of CB₂ receptors (areas involved in ligand recognition) exhibit 68% homology with those of CB₁ receptors (Munro et al., 1993). Showalter et al. (1996) reported a high positive correlation (r = 0.82) between binding affinities at these two cannabinoid receptors for cannabinoids in various classes. Given these findings, it is not surprising that some of the structural features of the tetrahydrocannabinols that enhance affinity for CB₁ receptors also increase binding to CB₂ receptors. For example, addition of a 1',1'-dimethyl group to the lipophilic C3 side chain of ⁸-THC results in higher affinities for both types of cannabinoid receptors compared with a nonbranched chain of identical length (Showalter et al., 1996). Several previous studies have explored the role of oxygen in CB₂ binding. Synthesis of a series of ⁸-THC analogs in which the phenolic hydroxyl at position 1 was removed (deoxy-⁸-THC analogs) or replaced with a methoxyl resulted in analogs with selectivity for CB₂ receptors (Gareau et al., 1996; Huffman et al., 1996, 1999). Incorporation of an oxygen into a fourth ring attached at C1 also increased CB₂ selectivity, suggesting possible differences in the interaction of oxygen in the binding pockets of CB₁ and CB₂ receptors (Reggio et al., 1997). In the present study, we examined structure-activity relationships of a series of bicyclic resorcinols in which the core chemical structure contained two hydroxyl substituents positioned with a single intervening carbon on a benzene ring. For most of the bicyclic resorcinols presented here, the second cyclic substituent is attached at the intermediate carbon.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Subjects. Male Institute for Cancer Research (ICR) mice (25-32 g), obtained from Harlan (Indianapolis, IN), were housed in groups of five. All animals were kept in a temperature-controlled (20-22°C) environment with a 12-h light/dark cycle (lights on at 7 AM). Separate mice were used for testing each dose of each experimental compound in the in vivo behavioral procedures. Brain tissue for binding studies was obtained from male Sprague-Dawley rats (150-200 g) purchased from Harlan.

Apparatus. Measurement of spontaneous activity in mice occurred in standard activity chambers interfaced with a Digiscan animal activity monitor (Omnitech Electronics, Inc., Columbus, OH). A standard tail-flick apparatus and a digital thermometer (Fisher Scientific, Pittsburgh, PA) were used to measure antinociception and rectal temperature, respectively.

Compounds. Resorcinols were synthesized in our laboratories (Organix, Inc., Woburn, MA) according to the procedure specified below and were suspended in a vehicle of absolute ethanol, Emulphor-620 (Rhone-Poulenc, Inc., Princeton, NJ), and saline in a ratio of 1:1:18. Experimental compounds were administered to the mice i.v. in the tail vein at a volume of 0.1 ml/10 g.

View this table: [in this window] [in a new window]   TABLE 1 CB1 and CB2 binding affinities and pharmacological effects of phenols The Ki values are presented as means ± S.E.M. All ED50 values are expressed as micromoles per kilogram (with 95% confidence limits in parentheses). For compounds that failed to produce either maximal or dose-related effects, the percent effect at the highest dose a(milligrams per kilogram, in parentheses) is provided.

View this table: [in this window] [in a new window]   TABLE 2 Pharmacological effects and cannabinoid receptor binding affinities of bicyclic resorcinols The Ki values are presented as means ± S.E.M. All ED50 values are expressed as micromoles per kilogram (with 95% confidence limits in parentheses). For compounds that failed to produce either maximal or dose-related effects, the percent effect at the highest dose (milligrams per kilogram, in parentheses) is provided.

View this table: [in this window] [in a new window]   TABLE 3 In vitro and in vivo cannabinoid effects of bicyclic resorcinols with methylated cyclohexane The Ki values are presented as means ± S.E.M. All ED50 values are expressed as micromole per kilogram (with 95% confidence limits in parentheses). For compounds that failed to produce either maximal or dose-related effects, the percent effect at the highest dose (mg/kg; in parentheses) is provided.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Scheme for synthesis of resorcinol analogs. a, n-BuLi, hexane/THF, 0°C, 1 h; ketone, THF, 0°C, 0.5 h to 23°C, 18 h, 80 to 90%. b, CF3COOH, ET3SiH, CH2Cl2, 23°C, 1 h. c, BBr3/CH2Cl2, 0°C to 23°C, 18 h. Overall yield (a to c), ~50 to 80%. d, 10% HCl, ether/THF (5:4), 23°C, 0.5 h; yield O-2115 (41%) and O-2114 (19%). e, m-chloroperbenzoic acid, CH2Cl2/H2O (4:3), 23°C, 10 min. f, NaBH4, CH2Cl2/MeOH (1:1), 23°C, 18 h, ~90%. Overall yield (e and f), ~50%.

View this table: [in this window] [in a new window]   TABLE 4 CB1 and CB2 binding affinities of dimethoxy-dimethylheptyl resorcinol analogs The Ki values are presented as means ± S.E.M.

View this table: [in this window] [in a new window]   TABLE 5 CB1 and CB2 binding affinities of hydroxylated dimethoxy-dimethylheptyl resorcinols The Ki values are presented as means ± S.E.M.

Mouse Behavioral Procedures. Prior to testing in the behavioral procedures, mice were acclimated to the experimental setting (ambient temperature 22-24°C) overnight. Preinjection control values were determined for rectal temperature and tail-flick latency (in seconds). Five min after i.v. injection with an experimental compound or vehicle, mice were placed in individual activity chambers, and spontaneous activity was measured for 10 min. Activity was measured as total number of interruptions of 16 photocell beams per chamber during the 10-min test and expressed as percent inhibition of activity of the vehicle group. Tail-flick latency was measured at 20 min postinjection. Maximum latency of 10 s was used. Antinociception was calculated as the percent maximal possible effect {%MPE = [(test-control latency)/(10-control)] × 100}. Control latencies typically ranged from 1.5 to 4.0 s. At 30 min postinjection, rectal temperature was measured. This value was expressed as the difference between control temperature (before injection) and temperatures following drug administration (^oC). Different mice (n = 5-6 per dose) were tested for each dose of each compound. Each mouse was tested in each of the three procedures.

CB₁ Binding Procedure. The methods used for tissue preparation and binding have been described previously (Compton et al., 1993) and are similar to those described by Devane et al. (1988). All assays, as described briefly below, were performed in triplicate, and the results represent the combined data from three to six individual experiments.

CB₂ Binding Procedure. Human CB₂ cDNA was provided by Dr. Sean Munro (MRC Laboratory of Molecular Biology, Cambridge, England) and was expressed in Chinese hamster ovary cells as previously described (Showalter et al., 1996). Briefly, transfected CB₂ Chinese hamster ovary cell lines were maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) to maintain selective pressure of stable transformants and 10% fetal clone II (Hyclone Laboratories, Inc., Logan, UT) plus 0.3 to 0.5 mg/ml G418 (to maintain selective pressure) under 5% CO₂ at 37°C. When confluent, cells were harvested with 1 mM EDTA in phosphate-buffered saline and centrifuged at 1000g for 5 min at 4°C. The supernatant was saved, and the P₁ pellet was resuspended in centrifugation buffer. Homogenization and centrifugation were repeated twice, and the combined supernatant fractions were centrifuged at 40,000g for 30 min at 4°C. The P₂ pellet was resuspended in centrifugation buffer 2 (50 mM Tris HCl, 1 mM EDTA, and 3 mM MgCl₂, pH 7.4) to a protein concentration of approximately 2 mg/ml. Protein concentrations were determined by the method of Bradford (1976) using Bio-Rad protein assay (Bio-Rad, Hercules, CA) and bovine serum albumin standards (fatty acid free; Sigma-Aldrich). The membrane preparation was divided into amounts that were convenient for binding assays, frozen rapidly in dry ice, and stored at 80°C.

Data Analysis. Based on data obtained from numerous previous studies with cannabinoids, maximal cannabinoid effects in each procedure were estimated as follows: 90% inhibition of spontaneous activity, 100% MPE in the tail-flick procedure, and 6°C change in rectal temperature. ED₅₀ was defined as the dose at which half-maximal effect occurred. For compounds that produced one or more cannabinoid effect, ED₅₀ was calculated separately using least-squares linear regression on the linear part of the dose-effect curve for each measure in the mouse tetrad, plotted against log₁₀ transformation of the dose. For the purposes of potency comparison, potencies were expressed as millimoles per kilogram.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

The CB₁ and CB₂ binding affinities for substituted biphenyl analogs are shown in Table 1. These compounds contain a phenolic hydroxyl and lipophilic side chain in the same orientation as in cannabinol. In addition, the pyran oxygen is absent, and the analogs have substituents in the phenyl ring (ring C) of cannabinol. Two of the analogs (O-1376 and O-1601) have a dimethylheptyl side chain; each possess good CB₁ and CB₂ binding affinities and in vivo activity. O-1601, the more potent of the two active compounds, had a hydroxymethyl group in the phenyl ring. This substitution increased CB₁ affinity and in vivo potencies compared with O-1376 but did not affect affinity for CB₂ receptors. A similar effect was observed in the cannabinol series, where the substitution of a hydroxymethyl group for a methyl at C-9 in cannabinol increased binding affinity and potency (Mahadevan et al., 2000). Shortening the side chain of O-1376 to dimethylbutyl (O-1532) markedly decreased affinity for both receptors and resulted in loss of in vivo activity.

Table 2 presents binding and in vivo data for a series of two cyclic ring-substituted-5-dimethylheptyl resorcinols. Manipulation of the size of the cyclic structure attached at position 2 of the resorcinol ring resulted in changes in binding affinities and potencies. Substitution of a cyclopentane ring (O-1424) resulted in moderate affinity for the CB₁ receptor, with excellent affinity for the CB₂ receptor. Although this compound was active in all three in vivo assays, potency was relatively poor. In addition, potencies across the measures were not equal; i.e., potency for reducing spontaneous activity was approximately half that for producing antinociceptive and hypothermic effects. Increasing ring size to a cyclohexane (O-1422), cycloheptane (O-1656), or adamantyl (O-1660) improved affinity 5- to 14-fold for both cannabinoid receptors and greatly increased potencies in vivo. Substitution of a sulfur for a carbon in a cyclohexane ring (O-1425) decreased CB₁ affinity by 14-fold and CB₂ affinity by 8-fold (compared with O-1422) as well as reducing in vivo potencies. Similarly, sulfur substitution in a cyclopentane ring (O-1661) also attenuated binding to both cannabinoid receptors. When a methylated nitrogen (O-1662) was inserted into the cyclohexane ring in the same position as the sulfur of O-1425, binding to CB₁ receptors did not occur. In addition, CB₂ binding was drastically decreased, and the compound was not fully active in vivo. In contrast, placing a double bond in the cyclohexane ring (O-1423) decreased affinities and potencies, but the compound remained active. However, moving the lipophilic side chain of O-1422 from C-5 to C-4 and replacing the dimethylheptyl with an n-hexyl chain (O-2010) produced a 865-fold decrease in CB₁ affinity and a loss of activity in vivo.

Table 3 shows results of tests with cyclohexane-substituted resorcinols in which the position of the substituent at the cyclohexane ring attached to the core resorcinol was varied. All compounds were diastereomeric mixtures. All of these analogs had high (K_i = 2 nM) to moderate (K_i = 144 nM) affinity for CB₁ receptors and were CB₂-selective (K_i range = 0.3-13 nM). Methylation at the 2-position of the cyclohexane ring (O-1658) did not dramatically alter affinity for either cannabinoid receptor or in vivo potencies compared with the corresponding cannabinoid with a nonmethylated cyclohexane (O-1422 in Table 2). Moving the methyl to position 4 of the cyclohexane ring (O-1659) decreased affinity for both cannabinoid receptors by about 5-fold and produced an even greater decrease (11- to 24-fold) in potencies in vivo. Substituting a phenyl group for the methyl at this same position (O-1663) resulted in 2- to 3-fold decreases in CB₂ and CB₁ affinities, respectively, and a loss of activity in vivo. In the next five analogs shown in Table 3, the methyl was attached at position 3 of the cyclohexane ring. O-1657 exhibited CB₁ and CB₂ affinities that were similar to those of O-1658; however, the profiles of in vivo potencies differed. Whereas the two analogs showed approximately equal potencies in suppressing spontaneous activity, O-1658 was twice as potent in producing antinociception and three times as potent in reducing body temperature. As described under Materials and Methods, compound O-1657 was separated into two distinct entities, which were designated O-1797A and O-1798B. These analogs were still mixtures. Affinities of O-1797A and O-1798B were two to three times greater than those of O-1657. Although potencies of these isomers for suppression of locomotor activity and hypothermia were not notably different from those of O-1657, antinociceptive potencies were reduced by about half. The 3S isomer of this series (O-1826) showed decreased affinity for CB₁ receptors compared with O-1657; however, affinity for CB₂ receptors was identical for both compounds. Not surprisingly given its decreased CB₁ affinity, O-1826 was less potent than O-1657 in vivo. Substitution of a dimethylbutyl for the dimethylheptyl side chain at C5 of the resorcinol component (O-1890) decreased affinities for both cannabinoid receptors. This compound was active in vivo, although potency was notably low for all measures. In contrast, addition of a gem-dimethyl group at the 3-position of the cyclohexane ring, with retention of the dimethylheptyl side chain of the resorcinol component (O-1871), resulted in the best CB₁ and CB₂ affinities of this series. Given its higher CB₁ binding affinity, in vivo potencies for this compound were lower than expected, although the lack of pharmacokinetics assessments tempers this conclusion somewhat.

To develop CB₂-selective ligands, we examined cyclic ring-substituted dimethoxyresorcinols. The CB₁ and CB₂ binding affinities of these analogs are shown in Tables 4 and 5. Although most of the compounds shown in Tables 4 and 5 possessed a dimethylheptyl side chain, all had poor CB₁ affinity; hence, they were not tested in vivo. The bicyclic structure of O-1999 (Table 4) was almost identical to that of O-1657 (Table 3), an analog with good CB₁ and CB₂ affinities and potent in vivo effects. Both compounds had a dimethylheptyl side chain attached to the 5-position of a resorcinol core that was attached at position 2 to a cyclohexane ring. Each compound had a methyl group at the 3-position of the cyclohexane ring. The major structural difference between the two compounds was that O-1999 was a dimethoxy derivative of the resorcinol O-1657. This structural change from a phenol to a methoxy derivative resulted in complete loss of affinity for CB₁ receptors and an almost 600-fold reduction in affinity for CB₂ receptors. Similarly, the other analogs that were dimethoxy derivatives of the corresponding resorcinols had poor affinity for CB₁ receptors (K_i ranged from 1716 to > 10,000) regardless of the cyclic ring substitution at position 2. In contrast, CB₂ binding affinities for some of these analogs remained high, as described in more detail below.

Table 4 presents binding data for two cyclic ring-substituted dimethoxy-resorcinol-dimethylheptyl analogs that contain at least one oxygen inserted into or attached to the nonresorcinol cyclohexane ring. Compared with O-1999, which did not contain an oxygen in the cyclohexane ring, conversion of the cyclohexane ring to a pyran ring (O-1964) decreased CB₂ affinity almost 2-fold without effect on CB₁ binding. Further addition of a double bond at position 3 of the pyran ring resulted in O-1965, which did not bind to either cannabinoid receptor. In contrast, the introduction of a tertiary hydroxyl group at C-4 of the pyran ring (O-1962) increased CB₂ affinity by 3-fold. Adding additional oxygens, such as a ketol group attached at C-4 to the point of attachment of the dimethoxyresorcinol substituent (O-2092), also increased CB₂ affinity whereas adding an oxygen as an epoxide (O-2122) decreased it. The presence of a ketone group at C-4 of the cyclohexane ring and having unsaturation in the ring (O-2114) resulted in a compound with poor affinity for either cannabinoid receptor; however, if a tertiary hydroxyl group was added at the site of dimethoxyresorcinol attachment (O-2115), CB₂ affinity improved. Retention of the tertiary hydroxyl, methylation at position 5, and the presence of a ketone at position 3 of the cyclohexane ring increased affinity for both receptors and resulted in a compound (O-2123) with the best CB₂ affinity (K_i = 125 nM) in this series.

Table 5 shows CB₁ and CB₂ affinities for two cyclic ring-substituted dimethoxy-resorcinol-dimethylheptyl analogs in which the ring size and the position of the methyl or hydroxyl substituent on the cyclohexane ring are varied. The first analog (O-2072) contains one hydroxyl attached to the cyclohexane at the same position at which the resorcinol core is attached. This compound is CB₂-selective. Although it had poor affinity for CB₁ receptors, it bound with moderate affinity to CB₂ receptors. Introduction of a methyl substituent in the 3-position of the cyclohexane ring gave a diastereomeric mixture from which two distinct entities were separated by careful chromatography. These analogs (O-1966A and O-1967B) were still mixtures. This substitution resulted in a 5-fold increase in affinity for CB₂ receptors with continued poor affinity for CB₁ receptors. However, one of these isomers (O-1966A) showed the best CB₂ selectivity (225-fold) in the series and had high binding affinity for the CB₂ receptor (K_i = 22.5 nM). Addition of an extra hydroxyl group to the cyclohexane ring (O-2121) reduced both selectivity and binding affinity for the CB₂ receptor comparable with those obtained with O-1967B. Removal of the methyl at position 3 and addition of a hydroxyl at position 4 resulted in two diastereomeric mixtures that could be separated, which were designated as O-2116A and O-2117B. Both of these isomers had poor affinity for CB₁ receptors, but although the B isomer also had poor affinity for CB₂ receptors, the A isomer bound to CB₂ receptors with moderate affinity. Attachment of a gem-dimethyl group to position 3 of O-2072 (i.e., O-2068) did not significantly alter affinities for CB₁ or CB₂ receptors; however, replacement of the dimethylheptyl group of O-2068 with a methyl group (O-2139) produced loss of affinity at both receptors. Changing the dimethyoxy groups of the resorcinol by adding diethoxy groups (O-2090) drastically decreased affinities for CB₁ and CB₂ receptors (compare O-2090 with O-1966A or O-1967B). Enlarging the cyclohexane ring in O-2072 to a cycloheptane ring (O-2091) resulted in little change in affinity for CB₁ receptors and an almost 2-fold increase in CB₂ affinity.

Multiple regression analysis of binding affinity (Y = log CB₁ K_i) and potency for each mouse measure (X_1-3 = log ED₅₀ in mmol/kg) confirmed that overall potency at producing the characteristic profile of cannabinoid effects was significantly correlated with binding affinity at CB₁ receptors [r = 0.78; F(3,13) = 6.9; p = 0.005] for all active cannabinoids. Individual correlations between log K_i and log potency for each measure were 0.78, 0.74, and 0.75 for hypomobility, antinociception, and hypothermia, respectively (p < 0.05 for all three correlations). Furthermore, CB₁ binding affinity was highly correlated with CB₂ binding affinity (r = 0.92, p < 0.05) for all compounds for which both binding affinities could be calculated (i.e., K_i < 10,000). Scatterplots for each regression line are presented in Fig. 3.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Scatterplots and regression lines of log CB1 Ki plotted against log CB2 Ki (top left) and log ED50 for each of the three in vivo tests. SA, spontaneous activity (top right); MPE, percent maximal possible antinociceptive effect (bottom right); RT, change in rectal temperature (bottom right).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The lack of CB₁ binding affinity of cannabidiol compared with other pyran ring open analogs such as CP 55,940 prompted us to examine the structure-activity relationships of resorcinol derivatives for cannabinoid activity. Our results show that many of the structural changes that affect CB₁ receptor recognition and activation in traditional cannabinoids similarly alter binding and activity in this resorcinol series. Previous research has shown that the length and branching of a lipophilic substituent is important for CB₁ receptor recognition in all of the major cannabinoid agonist classes, including tetrahydrocannabinols and bicyclic cannabinoids (Compton et al., 1993), indole-derived cannabinoids (Wiley et al., 1998), and anandamides (Ryan et al., 1997; Seltzman et al., 1997). In the tricyclic and bicyclic series, a 1',1'-dimethylheptyl side chain is optimal (Compton et al., 1993) and is contained in most of the resorcinols presented here. Reducing the length of this substituent resulted in a concomitant elimination or decrease in CB₁ receptor recognition, as occurs in other cannabinoid series with similar structural manipulations (see references above).

Other structural features affecting CB₁ receptor recognition and activation in this series are related to the size, saturation, substitution, and methylation of the second nonresorcinol ring. In most tricyclic and bicyclic cannabinoids, the ring corresponding to the nonresorcinol ring in the current series is a cyclohexane. In the resorcinol series, reducing this size to a cyclopentane decreases CB₁ affinity and potency whereas increasing it to a cycloheptane has little effect. Similar modifications of other cannabinoids have not been reported; however, degree of saturation of, as well as the position of the double bond in the cyclohexane ring of tricyclic and bicyclic cannabinoids and in the polyolefin loop of the anandamides, has been shown to affect CB₁ receptor recognition and activity. In the resorcinol series, introduction of a single double bond (O-1423) within the ring decreased CB₁ affinity and potency to the same extent as did a reduction in the size of the ring to a cyclopentane. Greatest affinity and potency within the anandamides is achieved with four double bonds, with greater or lesser saturation resulting in a reduction in CB₁ binding and/or in vivo activity (Adams et al., 1995; Thomas et al., 1996; Sheskin et al., 1997). Similarly, the number and position of double bonds within the cyclohexane ring of tetrahydrocannabinols and bicyclic cannabinoids affect activity. For example, moving the double bond of ⁹-THC to position 8 (as in ⁸-THC) decreases CB₁ affinity 3-fold and somewhat reduces potency (Compton et al., 1993). Unsaturation of the cyclohexane ring results in cannabinol with its greatly reduced CB₁ affinity (Showalter et al., 1996). In contrast, CP 55,940, with a completely saturated cyclohexane ring, is severalfold more potent than ⁸-THC-dimethylheptyl, which has a single double bond in the cyclohexane ring; but ⁸-THC, with its single double bond, binds with better CB₁ affinity than does ⁹⁽¹¹⁾-THC, which has a completely saturated cyclohexane ring (Compton et al., 1993).

The most remarkable structural features of the resorcinol series affecting CB₁ affinity, however, are the length of the lipophilic side chain at position 5 and the size of the cyclic ring substituent at position 2 of the resorcinol core. ⁹-THC and CP 55,940 contain two oxygens: one as a phenol (one hydroxyl in the aromatic ring) with a second oxygen incorporated into a separate ring (pyran oxygen in ⁹-THC) or a hydroxyl group attached as a substituent in the cyclohexane ring, as in CP 55,940. Previous research has shown that eliminating the phenolic hydroxyl of ⁸-THC results in deoxy-⁸-THC analogs that are CB₂-selective (Huffman et al., 1999). Although some of these analogs also retain reasonable affinity for CB₁ receptors, orientation of their binding to CB₁ receptors may be inverted such that the pyran oxygen substitutes for the absent phenolic hydroxyl in hydrogen bonding (Huffman et al., 1996). In the absence of a pyran oxygen, the nature of the substituent at position 2 of the resorcinol core is important for maintenance of in vivo activity. An acyclic ring was found to be better than a heterocyclic ring, with a cyclohexane ring being optimal. In addition, the size and the position of the substituent on the cyclic ring is important to maintenance of CB₁ affinity. The presence of a methyl substituent at position 3 enhanced activity in some cases. Furthermore, the 3S analog (O-1826; Table 2) has a poorer CB₁ binding affinity (K_i = 40 nM) compared with the diastereomeric mixture O-1657 (K_i = 14 nM; Table 2), suggesting that CB₁ binding affinity is enhanced when the orientation of the methyl substituent at position 3 in the cyclohexane ring is 3R compared with 3S. Methylation of the phenols of the resorcinols drastically decreased or eliminated CB₁ affinity, perhaps because hydrogen donation is less likely from a methoxy group than from the free hydroxyl group of ⁹-THC (B. R. Martin, unpublished observations). Similarly, methoxy substitution for the phenolic hydroxyl in the methyl esters of ⁸- and ⁹⁽¹¹⁾-THC-dimethylheptyl resulted in analogs that were CB₂-selective and had little CB₁ affinity (Gareau et al., 1996; Huffman et al., 1999; Ross et al., 1999).

Notably, most of the dimethoxyresorcinols tested here were CB₂-selective. As suggested by the high positive correlation between CB₁ and CB₂ binding affinities, most of the structural features that affected recognition at CB₁ receptors also affected CB₂ receptor recognition, although not always to the same degree or in the same manner. These factors included length and branching of the side chain and size and degree of saturation of the nonresorcinol cyclohexane ring. In a structure-activity relationship study on a series of CB₂-selective deoxy-⁸-THC analogs, Huffman et al. (1999) reported that length and branching of the C3 side chain affected CB₂ binding in a manner similar to its effect on CB₁ affinity, as it did in the present study; however, the range of chain lengths for which moderate to good CB₂ affinity was retained for the deoxy-⁸-THC analogs was greater than the range for CB₁ affinity. Similar results were obtained with a series of CB₂-selective indole-derived cannabinoids in which length of the nitrogen substituent was varied (Aung et al., 2000). To date, anandamide analogs appear to be CB₁ selective, with relatively little affinity for CB₂ receptors across several types of manipulations (Showalter et al., 1996). Insufficient research is available to determine the effect of substitution on a cyclohexane ring on CB₂ affinity across cannabinoid classes.

Other structural manipulations that eliminated or drastically reduced CB₁ receptor recognition did not necessarily alter CB₂ receptor binding in an identical manner. CB₂ selectivity was most evident in the dimethoxy analogs, primarily as a consequence of severe reductions in CB₁ affinity. HU-308, the most selective CB₂ agonist to date, has a dimethoxyresorcinol core structure and does not bind to CB₁ receptors at all (Hanu et al., 1999). In addition, greater tolerance in CB₂ (versus CB₁) receptor recognition was observed with other C2 substitutions in the resorcinols. Huffman et al. (2001) recently reported that bicyclic pyridone analogs with carbonyl substitution at C1 and a nitrogen substituent substitution at C2 of ⁸-THC had little affinity for CB₁ receptors. In contrast, moderate CB₂ affinity (K_i ~ 53 nM) was retained. Differences in allosteric regulation of CB₁ and CB₂ receptors by ions and guanine nucleotides have been noted previously (Showalter et al., 1996). Together, the results presented here and elsewhere (see above) suggest incomplete overlap of the pharmacophores for CB₁ and CB₂ receptors.

In summary, structure-activity relationships of the resorcinol series presented here are consistent with the CB₁ and CB₂ pharmacophores of other cannabinoid classes. In this series of resorcinols, several structural features were essential for maintenance of CB₁ receptor recognition and in vivo activity, including the presence of a branched lipophilic side chain at C5, the presence of free phenols, and substitution of a cyclohexane ring at C2. An important structural feature for receptor recognition at CB₂ receptors was side chain length. The CB₂ selectivity observed with some resorcinols was maximized in the dimethoxyresorcinol analogs, and this selectivity was greatly enhanced when a tertiary hydroxyl group was present in the cyclohexane ring in the same position at which the resorcinol core is attached. In contrast, the presence of unsaturation, a ketone group, or an additional hydroxyl substitution in the cyclohexane ring adversely affected the CB₂ selectivity. Methyl ethers were optimal for CB₂ selectivity because ethyl ethers reduced selectivity.

In conclusion, although resorcinol derivatives with cyclic ring substituents at C2 are closely related to the nonactive cannabinoid cannabidiol, many of these analogs have high CB₁ and/or CB₂ binding affinity as well as potent in vivo activity. In addition, because dimethoxyresorcinols are CB₂-selective, they have potential to offer insight into similarities and differences between requirements for receptor recognition at CB₁ versus CB₂ receptors. The results presented here suggest that the resorcinol series represent a novel template for the development of CB₁- and CB₂-selective cannabinoid agonists.