Radiolabeled cannabinoid receptor 2 ligand

Abstract

The present invention relates to a compound of formula (I) ##STR00001##
wherein R.sup.1 is defined as in the description and in the claims. The compound of formula (I) can be used as radiolabeled ligand.

Claims

1. A compound of formula (I) ##STR00010## wherein R.sup.1 is a methyl group and wherein at least one of the atoms of the methyl group of R.sup.1 is replaced with a radionuclide independently selected from the group consisting of [.sup.3H], [.sup.11C] and [.sup.14C].

2. The compound according to claim 1, wherein R.sup.1 is C[.sup.3H].sub.3, [.sup.11C]H.sub.3 or [.sup.14C]H.sub.3.

3. A pharmaceutical composition comprising a compound according to claim 1.

4. A process for the manufacture of a compound according to claim 1 comprising reacting a compound of formula (A) ##STR00011## in the presence of [.sup.3H]-methyl 4-nitrobenzenesulfonate, [.sup.3H]-methyl iodide, [.sup.11C]-methyl triflate, [.sup.11C]-methyl iodide, [.sup.11C]-methyl 4-nitrobenzenesulfonate, [.sup.14C]-methyl triflate, [.sup.14C]-methyl iodide or [.sup.14C]-methyl 4-nitrobenzenesulfonate.

5. The compound according to claim 1, wherein at least one of the hydrogen atoms of the methyl group of R.sup.1 is replaced with [.sup.3H].

6. The compound according to claim 1, wherein the compound is (NE)-N-[5-tert-butyl-2-(cyclobutylmethyl)-1-(tritritiomethyl)pyrazol-3-ylidene]-2-(2-hydroxy-2-methyl-propoxy)-5-(trifluoromethyl)benzamide, having the formula: ##STR00012##

7. The compound according to claim 1, wherein the compound is (NE)-N-[5-tert-butyl-2-(cyclobutylmethyl)-1-([.sup.14C]methyl)pyrazol-3-ylidene]-2-(2-hydroxy-2-methyl-propoxy)-5-(trifluoromethyl)benzamide, having the formula: ##STR00013##

8. The compound according to claim 1, wherein the compound is (NE)-N-[5-tert-butyl-2-(cyclobutylmethyl)-1-([.sup.11C]methyl)pyrazol-3-ylidene]-2-(2-hydroxy-2-methyl-propoxy)-5-(trifluoromethyl)benzamide, having the formula: ##STR00014##

Description

EXAMPLES

(1) Abbreviations

(2) CAN=CAS Registry Number; LC=liquid chromatography; MS=mass spectrometry; NMR=nuclear magnetic resonance; SEM=standard error of the mean; THF=tetrahydrofuran; WT=wild type.

Experimental

(3) All reactions were undertaken in flame dried glassware under an atmosphere of argon. Analytical grade solvents were used for reactions and when required, dry solvents were used without further purification. Reagents were purchased from reputable commercial suppliers and used without further purification, unless otherwise stated. All .sup.1H NMR spectra were recorded on a Bruker Advance Ultra Shield 300 MHz spectrometer. Chemical shifts are reported relevant to the stated deuterated solvent. Mass spectra were recorded on PE Sciex API 150EX LC/MS Turbo Spray System. Flash chromatography was conducted using an Isco Combi Flash companion, using prepacked silica columns (230-400 mesh, 40-63 m) of various sizes from various commercial suppliers. Thin layer chromatography was carried on pre-coated plates (2020 cm, silica gel F254) purchased from Merck KgaA and was visualized using a 254 nm CAMAG UV lamp or using a basic potassium permanganate solution. All reactions were monitored using a combination of thin layer chromatography, LCMS and .sup.1H NMR.

Example 1 (NE)-N-[5-tert-butyl-2-(cyclobutylmethyl)-1-(tritritiomethyl)pyrazol-3-ylidene]-2-(2-hydroxy-2-methyl-propoxy)-5-(trifluoromethyl)benzamide

(4) ##STR00005##

a) N-[5-tert-butyl-2-(cyclobutylmethyl)pyrazol-3-yl]-2-fluoro-5-(trifluoromethyl)benzamide

(5) ##STR00006##

(6) To a solution of 3-tert-butyl-1-(cyclobutylmethyl)-1H-pyrazol-5-amine (458 mg, 2.21 mmol, CAN 1018679-74-7) and triethylamine (670 mg, 923 L, 6.62 mmol) in dry THF (9 mL) was added dropwise a solution of 2-fluoro-5-(trifluoromethyl)benzoyl chloride (500 mg, 333 L, 2.21 mmol, CAN 207981-46-2) in dry THF (9 mL). The pink suspension was stirred at ambient temperature for 30 minutes and extracted with 1M aqueous sodium hydrogen carbonate solution (70 mL). The aqueous layer was extracted with ethyl acetate (370 mL). The organic layers were combined and dried using sodium sulfate, before concentrating in vacuo. The crude material was purified by flash chromatography (10% to 20% ethyl acetate in heptane) to give 794.4 mg (2.0 mmol, 91% yield) of the title compound as an orange powder.

(7) .sup.1H NMR (300 MHz, CHLOROFORM-d) ppm 1.32 (s, 9H), 1.72-2.20 (m, 6H), 2.81 (dt, J=14.73, 7.37 Hz, 1H), 4.04 (d, J=7.06 Hz, 2H), 6.37 (s, 1H) 7.38 (dd, J=11.61, 8.58 Hz, 1H), 7.80-7.91 (m, 1H), 8.27 (d, J=15.95 Hz, 1H), 8.53 (dd, J=6.96, 2.32 Hz, 1H). MS m/z 398.2 [M+H].sup.+.

b) N-[5-tert-butyl-2-(cyclobutylmethyl)pyrazol-3-yl]-2-(2-hydroxy-2-methyl-propoxy)-5-(trifluoromethyl)benzamide

(8) ##STR00007##

(9) Potassium tert-butoxide (203 mg, 1.81 mmol) was added to an ice-cold solution of 2-methylpropane-1,2-diol (136 mg, 138 L, 1.51 mmol, CAN 558-43-0) in dry THF (7 mL). The colorless suspension was stirred at 0 C. for 5 minutes, allowed to warm to room temperature and stirred for 1 hour. At 0 C. (N-[5-tert-butyl-2-(cyclobutylmethyl)pyrazol-3-yl]-2-fluoro-5-(trifluoromethyl)benzamide (400 mg, 1.01 mmol) was added. The resulting yellow suspension was stirred at room temperature for 72 h. Potassium tert-butoxide (203 mg, 1.81 mmol) was added and stirring was continued at 70 C. for 2 hours. Aqueous 1M sodium hydrogen carbonate solution (50 mL) and ethyl acetate (50 mL) were added and the layers were separated. The aqueous phase was extracted with ethyl acetate (250 mL). The organic layers were combined and dried using sodium sulfate, before concentrating in vacuo. The crude material was purified by flash chromatography (30 to 50% ethyl acetate in heptane), giving 334 mg (0.714 mmol, 71% yield) of the title compound as a white powder.

(10) .sup.1H NMR (300 MHz, CHLOROFORM-d) ppm 1.30 (s, 9H), 1.43 (s, 6H), 1.69-2.05 (m, 6H), 2.77 (dt, J=14.63, 7.22 Hz, 1H), 4.02-4.19 (m, 4H), 6.19 (s, 1H), 7.13 (d, J=8.88 Hz, 1H), 7.75 (dd, J=8.68, 2.42 Hz, 1H), 8.55 (d, J=2.22 Hz, 1H), 9.69 (s, 1H). MS m/z 468.3 [M+H].sup.+.

c) (NE)-N-[5-tert-butyl-2-(cyclobutylmethyl)-1-(tritritiomethyl)pyrazol-3-ylidene]-2-(2-hydroxy-2-methyl-propoxy)-5-(trifluoromethyl)benzamide

(11) In a 1-ml screw cap vial N-[5-tert-butyl-2-(cyclobutylmethyl)pyrazol-3-yl]-2-(2-hydroxy-2-methyl-propoxy)-5-(trifluoromethyl)benzamide (1.0 mg, 2.14 mmol) was added to a solution of [.sup.3H]methyl 4-nitrobenzenesulfonate (50 mCi, 0.16 mg, 0.71 mmol) in dry toluene (50 L). The vial was closed and the reaction mixture was stirred for 2 h at 120 C. Afterwards the crude material was purified by flash chromatography (silica, dichloromethane/methanol 95:5) to give 21.4 mCi (43%) of the title compound in >99% radiochemical purity (HPLC: X-Bridge C18, acetonitrile/formate buffer pH 3.0) and in a specific activity of 85 Ci/mmol.

Example 2 (NE)-N-[5-tert-butyl-2-(cyclobutylmethyl)-1-([14C]methyl)pyrazol-3-ylidene]-2-(2-hydroxy-2-methyl-propoxy)-5-(trifluoromethyl)benzamide

(12) ##STR00008##

(13) In a 2-ml screw cap vial N-[5-tert-butyl-2-(cyclobutylmethyl)pyrazol-3-yl]-2-(2-hydroxy-2-methyl-propoxy)-5-(trifluoromethyl)benzamide (209 mg, 0.45 mmol) was added to a solution of [14C]methyl 4-nitrobenzenesulfonate (25 mCi, 98 mg, 0.45 mmol) in dry toluene (1.5 mL). The vial was closed and the reaction mixture was stirred for 20 h at 120 C. After removal of the solvent the crude material was purified by flash chromatography (silica, dichloromethane/methanol/triethylamine 97:3:0.5) to give 8.7 mCi (35%) of the title compound in >99% radiochemical purity (HPLC:X-Bridge C18, water/acetonitrile containing 0.05% of triethylamine) and in a specific activity of 56 mCi/mmol.

Example 3 (NE)-N-[5-tert-butyl-2-(cyclobutylmethyl)-1-([11C]methyl)pyrazol-3-ylidene]-2-(2-hydroxy-2-methyl-propoxy)-5-(trifluoromethyl)benzamide

(14) ##STR00009##

(15) Carbon-11 was produced via the .sup.14N(p,).sup.11C nuclear reaction at a Cyclone 18/9 cyclotron (18-MeV, IBA, Belgium) in the form of [.sup.11C]CO.sub.2. [.sup.11C]Methyl iodide ([.sup.11C]MeI) was generated in a 2-step reaction sequence involving the reduction of [.sup.11C]CO.sub.2 over nickel catalyst to [.sup.11C]methane and subsequent gas phase iodination. After passing through an AgOTf/C column at 190 C., the more reactive [.sup.11C]methyl triflate (ca. 30 GBq) was formed which was bubbled through a 3-mL reactor vial containing N-[5-tert-butyl-2-(cyclobutyl methyl)pyrazol-3-yl]-2-(2-hydroxy-2-methyl-propoxy)-5-(trifluoromethyl) benzamide (3 mg, 6.46 mol) and dry toluene (0.25 mL). The mixture was heated to 150 C. for 5 min. After dilution with 30% acetonitrile in water (1.5 mL), the crude product was purified using semi-preparative HPLC (column: Sunfire C18 5 m; 10150 mm; product peak at ca. 10 min). The collected product was diluted with water (10 mL), trapped on a C18 cartridge (Waters, preconditioned with 5 mL EtOH and 10 mL water), washed with water (5 mL) and eluted with EtOH (0.5 mL). For formulation of the final product, water for injection (9.5 mL) was added to give an ethanol concentration of 5%. For quality control, an aliquot of the formulated solution was injected into an analytical HPLC system (column: ACE column, C18, 3 m). The identity of the .sup.11C-labeled product was confirmed by comparison with the retention time of its nonradioactive reference compound and by co-injection. Specific activity of the product was calculated by comparison of UV peak intensity with a calibration curve of the cold reference compound. In a typical experiment, the specific activity was 500 GBq/mol with a total activity of 21.00 GBq at the end of synthesis (n=30). The total synthesis time from end of bombardment was approximately 40 min.

Example 4 Radioligand Binding in Mice

(16) Cell Culture

(17) CHO-K1 beta-arrestin cells (DiscoveRx Inc., Fremont, Calif.) expressing human CB1 and human CB2 were cultured in F-12 Nutrient Mixture (HAM) supplemented with 10% FBS, 300 g ml-1 hygromycin and 800 g ml-1 geneticin (G418). Cells were incubated in a humidified atmosphere at 37 C. with 5% CO2.

(18) Radioligand Binding Assay

(19) Stably transfected cells or spleen tissue were homogenized in 15 mmol L-1 Hepes, 0.3 mmol L-1 EDTA, 1 mmol L-1 EGTA, 2 mmol L-1 MgCl.sub.2, complete EDTA-free protease inhibitor (Roche Applied Science, Rotkreuz, Switzerland), pH 7.4 using a glass potter and centrifugation at 47,800 g at 4 C. for 30 min. The pellet was then rehomogenized twice in the same buffer and centrifuged (47,800 g, 4 C., 30 min). The final pellet was then resuspended in 75 mmol L-1 Tris, 0.3 mmol L-1 EDTA, 1 mmol L-1 EGTA, 12.5 mmol L-1 MgCl.sub.2, 250 mmol L-1 sucrose, pH 7.4 at a protein concentration of 1 to 3 mg mL-1, aliquoted, frozen on dry ice and stored at 80 C.

(20) Saturation Binding

(21) Saturation binding was performed with 0.05 to 2.4 nM compound of formula (I) and 40 g of membrane protein. CP55940 (10 M) was used to define nonspecific binding. Assay buffer consisted of 50 mmol L-1 Tris-HCl, 5 mmol L-1 MgCl.sub.2, 2.5 mmol L-1 EGTA, and 0.1% fatty acid-free BSA, pH 7.4. Assays were initiated by addition of membranes in a final volume of 250 l/well. Assays were incubated for 2 h at room temperature and then vacuum filtered and rinsed with wash buffer (50 mmol L-1 Tris-HCl, 5 mmol L-1 MgCl.sub.2, 2.5 mmol L-1 EGTA, and 0.5% fatty acid-free BSA, pH 7.4) on a Filtermate cell harvester through Packard GF/B filters presoaked in 0.3% polyethylenimine.

(22) Competition Binding

(23) For competition binding, membrane preparations were incubated either with 0.3 nM of [.sup.3H]-CP55940 in the presence or absence of increasing concentrations of unlabeled (R.sup.1CH.sub.3) compound of formula (I) or with 1.5 nM compound of formula (I) and increasing amounts of membranes (2.5-80 g) in the presence or absence of CP55940 (10 M) for 60 min at 30 C. in a final volume of 0.2 mL of 50 mmol L-1 Tris-HCl, 5 mmol L-1 MgCl.sub.2, 2.5 mmol L-1 EGTA, 0.1% fatty acid-free BSA and 1% DMSO, pH 7.4, buffer, gently shaking. All binding reactions were terminated by vacuum filtration onto 0.5% polyethylenimine presoaked GF/B filter plates (Packard) followed by seven brief washes with 2 mL of ice-cold binding buffer containing 0.5% fatty acid-free BSA. Plates were dried at 50 C. for 1 h and liquid scintillation counting was used to determine bound radiolabel. IC50 values and Hill slopes were determined by a four parameter logistic model using ActivityBase (ID Business Solution, Ltd.).

(24) The results are shown in Table 1 below and in FIGS. 1 to 3.

(25) TABLE-US-00001 TABLE 1 Radioligand competition binding of [.sup.3H]-CP55940 using CHO-K1 cell expressing human CB2 receptors Human CB1 Human CB2 pKi 5.41 8.97 SEM 0.018 0.027 n 6 13

(26) Table 1 demonstrates high binding selectivity of the unlabeled (R.sup.1CH.sub.3) compound of formula (I) for human CB2 receptors (pKi 8.97) vs human CB1 receptors (pKi 5.41) in cells that recombinantly express these receptors and using the non-selective CB1/CB2 radioligand [.sup.3H]-CP55940.

(27) FIG. 1: Radioligand binding (1.5 nM [.sup.3H]-compound of formula (I)) using mouse spleen tissue WT

(28) FIG. 1 shows high specific binding in mouse spleen tissues of the compound of formula (I). Using increasing amounts of membranes, non-specific binding remains constant but specific binding increases.

(29) FIG. 2: Radioligand saturation binding ([.sup.3H]-compound of formula (I)) using mouse spleen tissue WT

(30) FIG. 2 shows that specific binding in mouse spleen tissues of compound of formula (I) is saturable. The calculated half maximal affinity (Kd) is 0.58 nM for mouse CB2 receptors and the CB2 receptor expression level (Bmax) is 430 fmol/mg in mouse spleen tissue.

(31) FIG. 3: Radioligand binding (.sup.3[H]-compound of formula (I)) using mouse spleen KO (NIH)

(32) FIG. 3 shows in contrast to FIG. 1 lack of specific binding of compound of formula (I) in mouse spleen tissues that were retrieved from CB2 deficient mice. Using increasing amounts of membranes, specific binding remains absent, indicating the high specific binding to CB2 receptors as demonstrated in mouse spleen tissues that were retrieved from wild type littermates.