Benzyl-1,2,4-triazoles as CB1 Cannabinoid Receptor Ligands: Preparation and In Vitro Pharmacological Evaluation

In a previous study, we have identified 3-alkyl-1,5-diaryl-1H-1,2,4-triazoles to be a novel class of cannabinoid type 1 receptor (CB1R) antagonists. In order to expand the number of cannabinoid ligands with a central 1,2,4-triazole scaffold, we have synthesized a novel series of 1-benzyl-1H-1,2,4-triazoles, and some of them were evaluated by CB1R radioligand binding assays. Compound 12a showed the most interesting pharmacological properties, possessing a CB1R affinity in the nanomolar range.


Introduction
Due to the potential therapeutic effects of cannabinoids that include antiemetic, analgesic, antiglaucoma, obesity treatment, alcoholism, bronchodilatation, and inflammation, a considerable number of cannabinoid ligands have been reported in recent years [1]. Their effects are mediated through G-protein coupled cannabinoid receptors, which are part of the endocannabinoid system (ECS) [2]. So far, two types of cannabinoid receptors, designated as CB 1 R and CB 2 R, have been well characterized, and three putative cannabinoid receptors, GPR55, GPR18, and GPR119, have been also proposed [3]. CB 1 R has been found in the peripheral and central nervous system, and CB 2 R is mainly present in the immune system. Cannabinoid ligands belong to families of diverse structural classes such as eicosanoids, classical and nonclassical ligands related to Δ 9tetrahydrocannabinol (THC), and heterocycles. Among the heterocycles family, pyrazoles [4] and aminoalkylindoles [5] are the most representative ligands.
We describe herein the synthesis of new benzyl-1,2,4triazoles [12] and present initial results from radioligand binding assays as part of our investigation on cannabinoid active compounds. was carried out by precoated silica-gel 60 F254 plates (Merck) and detection by UV light (254 nm). Flash-column chromatography was carried out by Kieselgel 60 (230-400 mesh; Merck). Medium pressure chromatography (MPLC) was carried out by Flash Master Personal system with prepacked silica-gel cartridges. The purity of the final compounds was determined by elemental analysis or analytical HPLC. Elemental analysis was performed on a Heraeus CHN-O rapid analyzer. Analyses indicated by the symbols of the elements or functions were within ±0.4% of the theoretical values, except compound 6. Analytical HPLC was run on a Waters 6000 with Delta Pak C 18.5 mm, 300 > (3.9 × 150 mm) column, using an eluent Acetonitrile/H 2 O (0.05% H 3 PO 4 + 0.04% TEA) in the proportion indicated in each case; flow rate used was 1 mL/min and the UV absorption was detected at a wavelength of 254 nm. HPLC analyses were within ≥90% of purity, except compound 11b (81% purity). The mass spectra (electrospray positive mode) were determined on a MSD-Series 1100 Hewlett Packard instrument. Melting points (uncorrected) were determined with a Reichert Jung Thermovar apparatus. 1 H and 13 C NMR spectra were recorded on a Gemini 200, Varian 300 and 400 unity spectrometers using TMS as the internal standard. All chemical shifts are reported in ppm. For the assignment of the protons and carbons of the aromatic rings Scheme 1 is used.     Intermediate 6 (1.00 g, 3.6 mmol) in dry EtOH (20 mL) reacted by refluxing with NaOMe (1.03 g, 19.0 mmol) for 4 days. Under this procedure, 5 was obtained in 78% yield (676 mg).
The second step took place with the alkylation of triazoles 3-5 under phase transfer catalysis conditions, using an aqueous sodium hydroxide solution as base and toluene as organic solvent [16]. These conditions were chosen after unsuccessful attempts of alkylation in an organic solvent (tetrahydrofuran) with mild (sodium bicarbonate) or strong (sodium hydride) bases. As depicted in Scheme 3, reaction of 3-5 with different benzyl halides in the presence of tetrabutylammonium bromide yielded two products by alkylation on N2 (7a-15a) or N1 (7b-15b) of the triazole. Alkylation on N4 of the triazole was not detected, since its formation is hindered by steric reasons. Both alkylated isomers were easily isolated by chromatography, being the N2-benzyl derivatives obtained in greater proportion (≈10 : 1). The only N1 isomer that could not be isolated and characterized was 13b; however it was detected by HPLC during the synthesis of 13a. Higher ratio of N2 isomers was obtained by alkylation of 5 with 4chlorobenzyl and 2,4-dichlorobenzyl chlorides that led to a mixture of N2/N1 isomers in proportion of 13 : 1 and 18 : 1, respectively. These results support the fact that alkylation of 1,2,4-triazoles with benzyl halides is governed by steric reasons. Since compounds 7-15 are very lipophilic, pyridinium salts (16-18; Scheme 4) of some of the triazolylpyridines previously obtained were synthesized in order to test if they possessed improved aqueous solubility compared to the parent compounds. Increasing the aqueous solubility was important to perform the radioligand binding assays of the series of benzyl triazoles. Therefore, compounds 13a-15a readily reacted with an excess of methyl iodide (1.5 equiv for 13a, 11 equiv for 14a, and 44 equiv for 15a). Achievement of the triazolyl-1-methyl pyridinium salts needed long reaction times (16 h for 16 and 8 days for 17 and 18), but the products were obtained in good yields.
Qualitative solubility tests of compounds 16-18 did not show any improvement in their solubility in water; therefore they were not assessed by pharmacological assays.

Radioligand Binding Assays.
Competitive radioligand binding assays have been used to evaluate the affinity of selected synthetized triazoles to CB 1 R in rat cerebellar membranes. They have been performed with [ 3 H]-SR141716A and [ 3 H]-WIN552122 as labelled ligands. The results of these preliminary assays are reported in Table 1. Compound 12a showed high CB 1 R affinity versus [ 3 H]-SR141617 ( = 13.9 nM) and moderate affinity versus [ 3 H]-WIN552122 ( = 323 nM). These binding data indicate that 12a displaced better SR141617, an inverse agonist of CB 1 R, than WIN552122, an agonist of CB 1 R. Since both SR141716 and WIN552122 have been reported in the literature to bind to CB 1 R in the same binding pocket [17], the results obtained here suggest that 12a binds to the inactive state of CB 1 R, as the inverse agonists do (e.g., SR141716), and not to the active state of the receptor, as the agonists do (e.g., WIN552122) [18].
The other tested compounds 7a, 8a, 10a, and 11a showed moderate CB 1 R affinity with affinity constant values in the low micromolar range.
In what refers to the binding to CB 2 R, none of the compounds showed significant affinity using [ 3 H]-CP55940 as radioligand in membranes purified from cells transfected with human CB 2 R (data not shown).