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Enantiomeric resolution of a novel chiral cannabinoid receptor ligand
J. Biochem. Biophys. Methods 54 (2002) 415 – 422 www.elsevier.com/locate/jbbm
Enantiomeric resolution of a novel chiral cannabinoid receptor ligand Ganesh A. Thakur a, Sonya L. Palmer a, Paul E. Harrington b, Ioanna A. Stergiades b, Marcus A. Tius b, Alexandros Makriyannis a,* a
Departments of Pharmaceutical Sciences and Molecular and Cell Biology, and the Center for Drug Discovery, University of Connecticut, 372 Fairfield Road, Storrs, CT 06269, USA b Department of Chemistry, 2545 The Mall, University of Hawaii, Honolulu, HI 96822, USA
Abstract The enantiomeric resolution of a racemic novel cannabinoid receptor ligand conformationally restricted at the southern aliphatic chain was accomplished using a ChiralPak AD column. Both enantiomers were tested for their competitive binding to the rat brain CB1, mouse spleen CB2 and human CB2 receptors. The levorotatory isomer showed exceptionally high affinity for the CB1 receptor with a seven-fold selectivity over CB2. D 2002 Elsevier Science B.V. All rights reserved. Keywords: D9-THC; Hybrid cannabinoids; ChiralPak AD column; Cannabinoid receptors
1. Introduction The broad spectrum of pharmacological properties exhibited by cannabis is well known, and a great deal of effort has gone into the search for therapeutically useful agents based on the parent structure of D9-THC (Fig. 1, 1) [1]. The recognition of two distinct cannabinoids receptors, one first identified in the central nervous system (CB1) [2] and another exclusively in periphery (CB2) [3,4], as well as the discovery of an endogenous ligand [5] arachidonyl ethanolamide (anandamide), has led to efforts to define the structural requirements for receptor affinity and specificity. Structure –activity relationship studies have focused on the optimization of four principal pharmacophores within the cannabinoid structures [6].
* Corresponding author. Fax: +1-860-486-3089. E-mail address: [email protected] (A. Makriyannis). 0165-022X/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 2 2 X ( 0 2 ) 0 0 1 4 4 - 6
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Fig. 1. Classical, nonclassical, and hybrid cannabinoids.
These include a phenolic hydroxy at C-1 and an aliphatic side chain attached to the phenolic ring at C-3, both of which are present in the natural tetrahydrocannabinol constituent 1. The addition of a northern aliphatic hydroxy (NAH) at the C-9 or C-11 positions leads to increased activity [7]. The fourth pharmacophore was identified from the work with nonclassical cannabinoids, the best known representative of which is CP55,940 (Fig. 1, 2) [6c]. The analogs in this class of cannabimimetic agents do not contain the pyran ring of classical cannabinoids but possess a second aliphatic hydroxy group in the southern portion of the molecule. Earlier work from our group [8] examined the stereochemical preference of the southern aliphatic hydroxy (SAH) pharmacophore by restricting its spatial orientation through reintroduction of the pyran ring (Fig. 1, 3) into the nonclassical cannabinoids skeleton. This led to a novel group of hybrid structures encompassing all the key features of classical and nonclassical cannabinoids. A systematic study, in which the C-6 stereochemistry and the chain length were varied, revealed that C6h hydroxypropyl analogs had high affinity for both the CB1 and CB2 receptors [9]. To further refine our understanding of the stereochemical preferences of CB receptors for hybrid cannabinoids, we sought to restrict the conformation of the SAH group by introducing a double or a triple bond at the C-2W position of the 6-h-hyroxypropyl chain, which led to a series of novel cannabinoid receptor probes (Fig. 2, 4 –6) [10]. Out of these analogs, racemic 5 bearing a trans double bond displayed exceptionally high affinity for the CB1 receptor with 13-fold selectivity over CB2. These results refined our understanding of the effect of a trans double bond at C-2W position. Indeed, in the absence of
Fig. 2. Hybrid cannabinoids conformationally restricted at the SAH. The structures represent the chiral enantiomer expected to retain most of the biological activities.
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the trans double bond, the C6h hydroxypropyl pharmacophore does not discriminate between CB1 and CB2 receptors (racemic 3; Ki for CB1 = 2.2 nM and CB2 = 3.4 nM) [9]. Finally, to understand the stereochemical preferences of the receptor sites toward each enantiomer, it was essential to isolate both enantiomers in pure form. We now describe the HPLC resolution of the enantiomers of compound 5 using commercial Daicel ChiralPak AD column and the affinities of each enantiomer for the CB1 and CB2 receptors.
2. Experimental 2.1. Instrumentation A high-performance liquid chromatograph equipped with a constant flow pump (SYSTEM GOLD Programmable Solvent Module 126P (binary pump), Beckman Instruments, San Ramon, USA) was used, with a diode-array UV detector (SYSTEM GOLD Programmable Detector Module 168, Beckman). A Rheodyne (Cotati, CA, USA) injection valve was used, equipped with 20 Al loop. The chiral column was a ChiralPak AD column (Daicel Chemicals Industries, Tokyo, Japan) (250 4.6 mm I.D., 10 Am film thickness). Optical rotations were measured on a Jasco DP-1000 digital polarimeter at 25 jC. 2.2. Materials and methods HPLC grade ethanol, hexane and 2-propanol were purchased from Aldrich (Milwaukee, WI, USA). Spectroscopic grade chloroform (Aldrich) was used for measuring optical rotations. All the HPLC experiments were performed at a flow rate of 1.0 ml/min at room temperature and monitored simultaneously at wavelengths 220 and 260 nm. The sample solutions were prepared by dissolving the analyte in ethanol. Initial experiments were carried out using 20 Al of the analyte solution (1 mg/ml) per injection. After standardizing the HPLC parameters and mobile phase conditions for complete separation of both enantiomers, semipreparative separations were performed by injecting 20 Al of 10 mg/ml solution of the analyte. The elution order for the enantiomers was (+)/( ) in all of the cases. 2.3. Pharmacological methods The pharmacological studies were carried out as discussed earlier [9]. Rat forebrain synaptosomal membranes were prepared by the method of Dodd et al. [11] and were used to assess the affinity of 5A and 5B for the CB1 binding sites. The displacement of specifically bound [3H] CP-55,940 from these membranes using a standard filtration assay was used to determine the IC50 for the test compounds. Briefly, 40 Ag of protein was incubated for 1 h at 30 jC in the presence of 0.76 nM [3H] CP-55,940 and various concentrations of test compounds; final volume is 200 Al. Nonspecific binding was defined by 100 nM cold CP-55,940. The incubation was terminated by rapid filtration and washing, and the amount of specifically bound [3H] CP-55,940 was determined. Data normalized between 0% and 100% specific binding were plotted against log concentration
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of test compound and the IC50 values were determined from the average of at least three experiments ran in duplicate, by fitting to a four variable nonparametric equation, holding the maximum to 100 and the minimum to 0. IC50 values were converted to Ki values according to the methods of Cheng and Prusoff [12]. Mouse spleen membranes or CB2 HEK cells were used as the source materials for CB2 receptors and prepared essentially according to the procedure of Dodd et al. [11]. The CB2 binding assays were conducted in a similar manner as CB1.
3. Results and discussion The relative stereochemistry of racemic 5 was determined previously [9] using NOESY experiments. On this basis, the absolute configuration of each of the two enantiomers are 6R, 6aS, 9S, 10aS, and 6S, 6aR, 9R, 10aR, respectively. Earlier work in our laboratory has utilized cellulose-derived Chiralcel OD (cellulose tris(3,5-dimethylphenylcarbamate)) and Chiralcel OJ (cellulose tris(4-methylbenzoate)) columns for chiral resolution of 1,3-dimethyl-4-phenylpiperidine derivatives [13] and 4(hydroxymethyl)-2-pentadecyl-1,3-dioxolanes [14]. For the enantiomeric resolution of compound 5, we chose ChiralPak column packed with amylose tris-(dimethylphenylcarbamate) supported on macroporous silica based on the earlier literature reports [15] where separation of enantiomeric pairs of cannabinoids was achieved with 2-propanol and
Fig. 3. Chromatograms showing the separation of (+) and ( ) isomer of 5 in ethanol as mobile phase modifier. Mobile phase: (I) ethanol/n-hexane (15:85 v/v); (II) ethanol/n-hexane (10:90 v/v); (III) ethanol/n-hexane (5:95 v/v).
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Table 1 Chromatographic retention parameters for enantiomers 5A and 5B Compound
Retention time (tR) (min) 15% ethanol/hexane
10% ethanol/hexane
5% ethanol/hexane
5A (+ isomer) 5B ( isomer)
19.6 20.6
25.1 27.3
59.9 73.6
ethanol as modifiers of hexane in mobile phase. The novel cannabinoid 5 has a total of four chiral centers and additional southern aliphatic hydroxyl functionality as compared to the cannabinoids separated earlier [15]. The initial mobile phase used was 15% ethanol in hexane. The separation observed was not adequate for obtaining baseline resolution of the enantiomers (Fig. 3, I). The use of 10% ethanol/hexane as the mobile phase showed some improvement in resolution (Fig. 1, II); however, it is not sufficient enough to isolate each isomer in pure form. Decreasing the polarity of the mobile phase by using 5% ethanol/hexane system gave excellent baseline separation (Fig. 1, III). Although the retention times (Table 1) were much longer relative to the 15% and 10% ethanol systems, both isomers could be isolated in pure form. The suggested use of 2-propanol as an alternative to ethanol to improve resolution [15] did not give better separation in our case. Repeated purification by loading f 0.25 mg of racemate 5 each time and using 5% ethanol/hexane afforded a total of f 3.0 –4.0 mg of each isomer in high enantiomeric purity. The first isomer eluted, 5A, was found to be dextrorotatory {[a]D25= + 43.3 (c 0.19, CHCl3)}whereas the second isomer, 5B, was levorotatory {[a]D25 = 45.1 (c 0.13, CHCl3)}. The pure isomers were tested for their abilities to bind to the cannabinoid receptors CB1 and CB2, and the results are listed in Table 2. Enantiomer 5B showed high affinity for both CB1 and CB2 with a seven-fold selectivity for CB1 as illustrated by the binding experiments depicted in Fig. 4A. The high affinity of 5B with rat CB receptors prompted further testing in human cells. The binding studies of 5B with CB2 receptors expressed in human embryonic kidney cells (CB2-HEK293, Fig. 4B) showed good correlation with the results obtained from mouse CB2 receptors as shown in Table 2. The log concentration displacement curves for 5A and 5B are depicted in Fig. 4. The known correlation between absolute stereochemistry, optical rotation sign and the pharmacological properties of the chiral cannabinoids [1,6,15,16] supports our postulate
Table 2 Ki values for both enantiomers of compound 5 competing with radiolabeled CP-55,940 for the CB1 and CB2 receptor Compound
5A (+ isomer) 5B ( isomer)
Ki (nM) CB1
CB2
Human CB2
94.82 (87.51 – 102.70) 0.16 (0.13 – 0.19)
124.80 (109.00 – 142.80) 1.15 (1.02 – 1.31)
183.7 (162.5 – 207.7) 1.33 (1.17 – 1.51)
Values in brackets indicate 95% confidence limits.
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Fig. 4. Log concentration displacement curves for hybrid cannabinoids 5A and 5B competing with radiolabeled CP-55,940 for (A) rat brain CB1 and (B) human CB2 receptors.
that the (+) isomer 5A has a 6R, 6aS, 9S, 10aS absolute configuration, while the ( isomer 5B has 6S, 6aR, 9R, 10aR configuration.
)
4. Conclusion In summary, the enantiomeric resolution of a potent racemic cannabinoid is reported. The ( ) isomer was found to be a novel, high-affinity CB1 selective ligand of potential therapeutic usefulness.
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Acknowledgements The NIDA, DA-07215, DA-03801 and P01 DA9158 are acknowledged for generous support of this work.
References [1] (a) Mechoulam R. Cannabinoids as therapeutic agents. Boca Raton (FL): CRC Press; 1986; (b) Nye JS, Snowman AM, Voglmaier S, Snyder SH. High-affinity cannabinoid binding site: regulation by ions, ascorbic acid, and nucleotides. J Neurochem 1989;52:1892; (c) Little PJ, Compton DR, Johnson MR, Melvin LS, Martin BR. Pharmacology and stereoselectivity of structurally novel cannabinoids in mice. J Neurochem 1988;247:1046; (d) Feigenbaum JJ, Richmond SA, Weissman Y, Mechoulam R. Inhibition of cisplatin-induced emesis in the pigeon by a non-psychotropic synthetic cannabinoid. J Neurochem 1989;169:159; (e) Howlett AC, Johnson MR, Melvin LS, Milne GM. Nonclassical cannabinoid analgetics inhibit adenylate cyclase: development of a cannabinoid receptor model. J Neurochem 1988;33:297; (f) Khanolkar AD, Palmer SL, Makriyannis A. Molecular probes for the cannabinoid receptors. J Neuro chem 2000;108:37. [2] (a) Devane WA, Dysarz III FA, Johnson MR, Melvin LS, Howlett AC. Determination and characterization of cannabinoid receptor in rat brain. Mol Pharmacol 1988;34:605; (b) Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Mol Pharmacol 1990;346:561. [3] Munro S, Thomas KL, Abu-Shaar M. Molecular characterization of a peripheral receptor for cannabinoids. Nature 1993;365:61. [4] Kaminski NE, Abood ME, Kessler FK, Martin BR, Schatz AR. Identification of a functionally relevant cannabinoid receptor on mouse spleen cells that is involved in cannabinoid-mediated immune modulation. Mol Pharmacol 1992;42:736. [5] Devane WA, Hanus L, Breuer A, Pertwee RG, Stevenson LA, Griffin G, et al. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 1992;258:1946. [6] (a) Makriyannis A, Rapaka RS. The molecular basis of cannabinoid activity. Life Sci 1990;47:2173; (b) Razdan RK. Structure – activity relationships in cannabinoids. Life Sci 1986;38:75; (c) Johnson MR, Melvin LS. In: Mechoulam R, editor. In cannabinoids as therapeutic agents. Boca Raton, FL: CRC Press; 1986. p. 121. [7] Wilson RS, May EL. Analgesic properties of the tetrahydrocannabinols, their metabolites and analogues. J Med Chem 1975;18:700. [8] Tius MA, Makriyannis A, Zou XL, Abadji V. Conformationally restricted hybrids of CP-55,940 and HHC: stereoselective synthesis and activity. Tetrahedron 1994;50:2671. [9] (a) Tius MA, Hill WAG, Zou XL, Busch-Petersen J, Kawakami JK, Fernandez-Garcia MC, et al. Classical/ non-classical cannabinoid hybrids; stereochemical requirements for the southern hydroxyalkyl chain. Life Sci 1995;56:2007; (b) Drake DJ, Jensen RS, Busch-Petersen J, Kawakami JK, Fernandez-Garcia MC, Fan P, et al. Classical/ nonclassical hybrid cannabinoids: southern aliphatic chain-functionalized C-6h methyl, ethyl, and propyl analogues. Life Sci 1998;41:3596. [10] Harrington PE, Stergiades IA, Erickson J, Makriyannis A, Tius MA. Synthesis of functionalized cannabinoids. J Org Chem 2000;65:6576. [11] Dodd PR, Hardy JA, Oakley AE, Edwardson JA, Perry EK, Delaunoy J-P. A rapid method for preparing synaptosomes: comparison, with alternative procedures. Brain Res 1981;226:107. [12] Cheng YC, Prusoff WH. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50% inhibition (IC50) of any enzymatic reaction. Biochem Pharmacol 1973;22: 3099. [13] Yin D, Khanolkar AD, Makriyannis A, Froimowitz M. Chiral resolution of 1,3-dimethyl-4-phenylpiperidine
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derivatives using high-performance liquid chromatography with a chiral stationary phase. J Chromatogr, A 1994;678:176. [14] Lin S, Duclos RI, Makriyannis A. Preparation of optically pure 4-(hydroxymethyl)-2-pentadecyl-1,3-dioxananes and their corresponding phosphodiester ether lipid derivatives. Chem Phys Lipids 2001;113:111. [15] (a) Shulamit L, Abu-Lafi S, Zahalka J, Mechoulam R. Resolution of chiral cannabinoids on amylose tris(3,5-dimethylphenylcarbmate) chiral stationary phase: effects of structural features and mobile phase additives. J Chromatogr, A 1993;654:53; (b)Levin S, Sterin M, Abu-Lafi S. Structural features affecting chiral resolution of cannabimimetic enantiomers by amylose 3,5-dimethylphenylcarbamate chiral stationary phase. J Chromatogr, A 1995;7:140. [16] Guo Y, Abadji V, Morse KL, Fournier DJ, Li X, Makriyannis A. ( )-11-Hydroxy-7V-isothiocyanato-1V, 1Vdimethylheptyl-D8-THC: a novel, high-affinity irreversible probe for the cannabinoid receptor in the brain. J Med Chem 1994;37:3867.
Enantiomeric resolution of a novel chiral cannabinoid receptor ligand Ganesh A. Thakur a, Sonya L. Palmer a, Paul E. Harrington b, Ioanna A. Stergiades b, Marcus A. Tius b, Alexandros Makriyannis a,* a
Departments of Pharmaceutical Sciences and Molecular and Cell Biology, and the Center for Drug Discovery, University of Connecticut, 372 Fairfield Road, Storrs, CT 06269, USA b Department of Chemistry, 2545 The Mall, University of Hawaii, Honolulu, HI 96822, USA
Abstract The enantiomeric resolution of a racemic novel cannabinoid receptor ligand conformationally restricted at the southern aliphatic chain was accomplished using a ChiralPak AD column. Both enantiomers were tested for their competitive binding to the rat brain CB1, mouse spleen CB2 and human CB2 receptors. The levorotatory isomer showed exceptionally high affinity for the CB1 receptor with a seven-fold selectivity over CB2. D 2002 Elsevier Science B.V. All rights reserved. Keywords: D9-THC; Hybrid cannabinoids; ChiralPak AD column; Cannabinoid receptors
1. Introduction The broad spectrum of pharmacological properties exhibited by cannabis is well known, and a great deal of effort has gone into the search for therapeutically useful agents based on the parent structure of D9-THC (Fig. 1, 1) [1]. The recognition of two distinct cannabinoids receptors, one first identified in the central nervous system (CB1) [2] and another exclusively in periphery (CB2) [3,4], as well as the discovery of an endogenous ligand [5] arachidonyl ethanolamide (anandamide), has led to efforts to define the structural requirements for receptor affinity and specificity. Structure –activity relationship studies have focused on the optimization of four principal pharmacophores within the cannabinoid structures [6].
* Corresponding author. Fax: +1-860-486-3089. E-mail address: [email protected] (A. Makriyannis). 0165-022X/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 2 2 X ( 0 2 ) 0 0 1 4 4 - 6
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Fig. 1. Classical, nonclassical, and hybrid cannabinoids.
These include a phenolic hydroxy at C-1 and an aliphatic side chain attached to the phenolic ring at C-3, both of which are present in the natural tetrahydrocannabinol constituent 1. The addition of a northern aliphatic hydroxy (NAH) at the C-9 or C-11 positions leads to increased activity [7]. The fourth pharmacophore was identified from the work with nonclassical cannabinoids, the best known representative of which is CP55,940 (Fig. 1, 2) [6c]. The analogs in this class of cannabimimetic agents do not contain the pyran ring of classical cannabinoids but possess a second aliphatic hydroxy group in the southern portion of the molecule. Earlier work from our group [8] examined the stereochemical preference of the southern aliphatic hydroxy (SAH) pharmacophore by restricting its spatial orientation through reintroduction of the pyran ring (Fig. 1, 3) into the nonclassical cannabinoids skeleton. This led to a novel group of hybrid structures encompassing all the key features of classical and nonclassical cannabinoids. A systematic study, in which the C-6 stereochemistry and the chain length were varied, revealed that C6h hydroxypropyl analogs had high affinity for both the CB1 and CB2 receptors [9]. To further refine our understanding of the stereochemical preferences of CB receptors for hybrid cannabinoids, we sought to restrict the conformation of the SAH group by introducing a double or a triple bond at the C-2W position of the 6-h-hyroxypropyl chain, which led to a series of novel cannabinoid receptor probes (Fig. 2, 4 –6) [10]. Out of these analogs, racemic 5 bearing a trans double bond displayed exceptionally high affinity for the CB1 receptor with 13-fold selectivity over CB2. These results refined our understanding of the effect of a trans double bond at C-2W position. Indeed, in the absence of
Fig. 2. Hybrid cannabinoids conformationally restricted at the SAH. The structures represent the chiral enantiomer expected to retain most of the biological activities.
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417
the trans double bond, the C6h hydroxypropyl pharmacophore does not discriminate between CB1 and CB2 receptors (racemic 3; Ki for CB1 = 2.2 nM and CB2 = 3.4 nM) [9]. Finally, to understand the stereochemical preferences of the receptor sites toward each enantiomer, it was essential to isolate both enantiomers in pure form. We now describe the HPLC resolution of the enantiomers of compound 5 using commercial Daicel ChiralPak AD column and the affinities of each enantiomer for the CB1 and CB2 receptors.
2. Experimental 2.1. Instrumentation A high-performance liquid chromatograph equipped with a constant flow pump (SYSTEM GOLD Programmable Solvent Module 126P (binary pump), Beckman Instruments, San Ramon, USA) was used, with a diode-array UV detector (SYSTEM GOLD Programmable Detector Module 168, Beckman). A Rheodyne (Cotati, CA, USA) injection valve was used, equipped with 20 Al loop. The chiral column was a ChiralPak AD column (Daicel Chemicals Industries, Tokyo, Japan) (250 4.6 mm I.D., 10 Am film thickness). Optical rotations were measured on a Jasco DP-1000 digital polarimeter at 25 jC. 2.2. Materials and methods HPLC grade ethanol, hexane and 2-propanol were purchased from Aldrich (Milwaukee, WI, USA). Spectroscopic grade chloroform (Aldrich) was used for measuring optical rotations. All the HPLC experiments were performed at a flow rate of 1.0 ml/min at room temperature and monitored simultaneously at wavelengths 220 and 260 nm. The sample solutions were prepared by dissolving the analyte in ethanol. Initial experiments were carried out using 20 Al of the analyte solution (1 mg/ml) per injection. After standardizing the HPLC parameters and mobile phase conditions for complete separation of both enantiomers, semipreparative separations were performed by injecting 20 Al of 10 mg/ml solution of the analyte. The elution order for the enantiomers was (+)/( ) in all of the cases. 2.3. Pharmacological methods The pharmacological studies were carried out as discussed earlier [9]. Rat forebrain synaptosomal membranes were prepared by the method of Dodd et al. [11] and were used to assess the affinity of 5A and 5B for the CB1 binding sites. The displacement of specifically bound [3H] CP-55,940 from these membranes using a standard filtration assay was used to determine the IC50 for the test compounds. Briefly, 40 Ag of protein was incubated for 1 h at 30 jC in the presence of 0.76 nM [3H] CP-55,940 and various concentrations of test compounds; final volume is 200 Al. Nonspecific binding was defined by 100 nM cold CP-55,940. The incubation was terminated by rapid filtration and washing, and the amount of specifically bound [3H] CP-55,940 was determined. Data normalized between 0% and 100% specific binding were plotted against log concentration
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of test compound and the IC50 values were determined from the average of at least three experiments ran in duplicate, by fitting to a four variable nonparametric equation, holding the maximum to 100 and the minimum to 0. IC50 values were converted to Ki values according to the methods of Cheng and Prusoff [12]. Mouse spleen membranes or CB2 HEK cells were used as the source materials for CB2 receptors and prepared essentially according to the procedure of Dodd et al. [11]. The CB2 binding assays were conducted in a similar manner as CB1.
3. Results and discussion The relative stereochemistry of racemic 5 was determined previously [9] using NOESY experiments. On this basis, the absolute configuration of each of the two enantiomers are 6R, 6aS, 9S, 10aS, and 6S, 6aR, 9R, 10aR, respectively. Earlier work in our laboratory has utilized cellulose-derived Chiralcel OD (cellulose tris(3,5-dimethylphenylcarbamate)) and Chiralcel OJ (cellulose tris(4-methylbenzoate)) columns for chiral resolution of 1,3-dimethyl-4-phenylpiperidine derivatives [13] and 4(hydroxymethyl)-2-pentadecyl-1,3-dioxolanes [14]. For the enantiomeric resolution of compound 5, we chose ChiralPak column packed with amylose tris-(dimethylphenylcarbamate) supported on macroporous silica based on the earlier literature reports [15] where separation of enantiomeric pairs of cannabinoids was achieved with 2-propanol and
Fig. 3. Chromatograms showing the separation of (+) and ( ) isomer of 5 in ethanol as mobile phase modifier. Mobile phase: (I) ethanol/n-hexane (15:85 v/v); (II) ethanol/n-hexane (10:90 v/v); (III) ethanol/n-hexane (5:95 v/v).
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Table 1 Chromatographic retention parameters for enantiomers 5A and 5B Compound
Retention time (tR) (min) 15% ethanol/hexane
10% ethanol/hexane
5% ethanol/hexane
5A (+ isomer) 5B ( isomer)
19.6 20.6
25.1 27.3
59.9 73.6
ethanol as modifiers of hexane in mobile phase. The novel cannabinoid 5 has a total of four chiral centers and additional southern aliphatic hydroxyl functionality as compared to the cannabinoids separated earlier [15]. The initial mobile phase used was 15% ethanol in hexane. The separation observed was not adequate for obtaining baseline resolution of the enantiomers (Fig. 3, I). The use of 10% ethanol/hexane as the mobile phase showed some improvement in resolution (Fig. 1, II); however, it is not sufficient enough to isolate each isomer in pure form. Decreasing the polarity of the mobile phase by using 5% ethanol/hexane system gave excellent baseline separation (Fig. 1, III). Although the retention times (Table 1) were much longer relative to the 15% and 10% ethanol systems, both isomers could be isolated in pure form. The suggested use of 2-propanol as an alternative to ethanol to improve resolution [15] did not give better separation in our case. Repeated purification by loading f 0.25 mg of racemate 5 each time and using 5% ethanol/hexane afforded a total of f 3.0 –4.0 mg of each isomer in high enantiomeric purity. The first isomer eluted, 5A, was found to be dextrorotatory {[a]D25= + 43.3 (c 0.19, CHCl3)}whereas the second isomer, 5B, was levorotatory {[a]D25 = 45.1 (c 0.13, CHCl3)}. The pure isomers were tested for their abilities to bind to the cannabinoid receptors CB1 and CB2, and the results are listed in Table 2. Enantiomer 5B showed high affinity for both CB1 and CB2 with a seven-fold selectivity for CB1 as illustrated by the binding experiments depicted in Fig. 4A. The high affinity of 5B with rat CB receptors prompted further testing in human cells. The binding studies of 5B with CB2 receptors expressed in human embryonic kidney cells (CB2-HEK293, Fig. 4B) showed good correlation with the results obtained from mouse CB2 receptors as shown in Table 2. The log concentration displacement curves for 5A and 5B are depicted in Fig. 4. The known correlation between absolute stereochemistry, optical rotation sign and the pharmacological properties of the chiral cannabinoids [1,6,15,16] supports our postulate
Table 2 Ki values for both enantiomers of compound 5 competing with radiolabeled CP-55,940 for the CB1 and CB2 receptor Compound
5A (+ isomer) 5B ( isomer)
Ki (nM) CB1
CB2
Human CB2
94.82 (87.51 – 102.70) 0.16 (0.13 – 0.19)
124.80 (109.00 – 142.80) 1.15 (1.02 – 1.31)
183.7 (162.5 – 207.7) 1.33 (1.17 – 1.51)
Values in brackets indicate 95% confidence limits.
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Fig. 4. Log concentration displacement curves for hybrid cannabinoids 5A and 5B competing with radiolabeled CP-55,940 for (A) rat brain CB1 and (B) human CB2 receptors.
that the (+) isomer 5A has a 6R, 6aS, 9S, 10aS absolute configuration, while the ( isomer 5B has 6S, 6aR, 9R, 10aR configuration.
)
4. Conclusion In summary, the enantiomeric resolution of a potent racemic cannabinoid is reported. The ( ) isomer was found to be a novel, high-affinity CB1 selective ligand of potential therapeutic usefulness.
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Acknowledgements The NIDA, DA-07215, DA-03801 and P01 DA9158 are acknowledged for generous support of this work.
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