Pharmacological evaluation of synthetic cannabinoids identified as constituents of spice

In recent years, many synthetic cannabinoid (CB) receptor agonists have appeared on the market as constituents of herbal incense mixtures known as “spice”. Contrary to the declared use, they are perorally consumed as a replacement for marijuana to get “high”. In many cases, detailed information on the physicochemical and pharmacological properties of the synthetic compounds found in spice preparations is lacking. We have now evaluated a large series of heterocyclic compounds, 1,3-disubstituted indole and 2-azaindole derivatives known or assumed to be CB1 receptor agonists, many of which have previously been identified in forensic samples. The mainly observed structural variations to circumvent restriction by law were bioisosteric exchanges of functional groups in known CB1 agonists. We analyzed the structure-activity relationships of compounds at human CB1 and CB2 receptors based on affinities obtained in radioligand binding studies, and determined their efficacy in cAMP accumulation assays. Moreover, we investigated the activities of the compounds at the orphan G protein-coupled receptors GPR18 and GPR55 both of which are known to interact with cannabinoids. Most of the investigated compounds behaved as potent full agonists of CB1 and CB2 receptors with affinities in the low nanomolar to subnanomolar concentration range. Some compounds were moderately potent GPR55 antagonists, while none interacted with GPR18. Most derivatives were predicted to cross the blood–brain barrier as determined by bioinformatics tools. These data are useful for assessing synthetic cannabinoids and will be helpful for predicting pharmacological properties of novel compounds that appear on the illicit drug market. Electronic supplementary material The online version of this article (doi:10.1007/s11419-016-0320-2) contains supplementary material, which is available to authorized users.

C. Hess and C. T. Schoeder contributed equally to this study.
Indazole-3-carboxylic acid ethyl ester (49) A cooled (~5 o C) solution of indazole-3-carboxylic acid (10 g) in ethanol (50 mL) was treated with concentrated H 2 SO 4 solution (2 mL), warmed to room temperature and stirred for 10 min. The mixture was refluxed for 12 h. The reaction was poured onto ice-water (100 mL) and extracted with ethyl acetate (3x50 mL). The combined organic extracts were washed with saturated NaHCO 3 (2x50 mL), brine solution (2x50 mL), dried over MgSO 4 and the solvent was concentrated under reduce pressure to provide 49.

1-N-(4-Fluorobenzyl)-1H-indazole-3-carboxylic acid (51)
A cooled (~5 o C) solution of 50 (2 g) in ethanol (15 mL) was treated slowly with 2 N NaOH (3 mL), warmed to room temperature and stirred for 10 min. The mixture was refluxed for 12 h and cooled to room temperature. Ethanol was removed under reduced pressure and the residue was dissolved in water (10 mL) and acidified with 6 N HCl.

Procedure for the synthesis of compound A-834,735 (46)
The synthesis of compound A-834,735 (46) was performed by adapting a literature procedure [50].

2,2,3,3-Tetramethylcyclopropanecarbonyl chloride (52)
To a flask containing 2,2,3,3-tetramethylcyclopropane carboxylic acid (8.0 g, 56.0 mmol) was added thionyl chloride (60 mL). The resulting solution was refluxed at 75 o C for 3 h. The mixture was then cooled to room temperature and concentrated under reduced pressure. The residue was diluted three times with 20 mL of dichloromethane (CH 2 Cl 2 ) and concentrated to remove any remaining thionyl chloride. This was repeated two additional times, and the material was used without further purification or characterization.

Tetrahydro-2H-pyran-4-ylmethylmethanesulfonate (53)
To a solution of tetrahydropyran-4-methanol (1.0 g, 8.6 mmol) in 20 mL of tetrahydrofuran (THF) at 0 °C was added triethylamine (4.10 mL, 29.4 mmol) followed by methanesulfonyl chloride (1.06 mL, 13.7 mmol). The mixture was stirred at 0 °C for 10 min and then the reaction mixture was stirred at room temperature for an additional 1.5 h. The reaction mixture was filtered through Celite and washed with THF (100 mL). The solution was concentrated under reduced pressure to give tetrahydro-2H-pyran-4-ylmethyl methanesulfonate, which was used without further purification and characterization.

1-[(Tetrahydro-2H-pyran-4-yl)methyl]-1H-indol-3-yl}-(2,2,3,3-tetramethylcyclopropyl)methanone (A-834,735, 46)
To a solution of 53 (1.2 g, 4.8 mmol) in 15 mL of DMF at 0 °C was added NaH (60% dispersion in mineral oil, 0.545 g, 24 mmol). This mixture was stirred at 0 °C for 10 min, warmed to room temperature, and allowed to stir for 30 min. The solution was again cooled to 0 °C and tetrahydro-2H-pyran-4-ylmethyl methanesulfonate (3.2 g, 16 mmol) in 15 mL of DMF was added via a syringe pump. After the addition was complete, the ice-bath was removed and the reaction mixture was warmed to 50 °C and stirred for 2 h. The mixture was cooled to ambient temperature, diluted with 50 mL of ethyl acetate and quenched with 10 mL of a saturated, aqueous solution of NH 4 Cl. The mixture was poured into water and extracted with ethyl acetate (3 x 30 mL). The combined organic extracts were washed with brine (50 mL), dried with MgSO 4 , and the solvent was removed under reduced pressure. The crude material was purified by flash-column chromatography to give 46. Yield    . Blood-brain barrier category. Two groups of compounds were defined according to their predicted blood-brain barrier penetration. Stardrop 5.5 (Optibrium) defines scores based on established QSAR models. The prediction of the BBB category returns a binary prediction of penetration of the blood-brain barrier (+ : accuracy and specifity is 91%; -: accuracy and specificity is 83%).