Urea compounds and their use as enzyme inhibitors

Abstract

A compound having the following structure: ##STR00001##
or a pharmaceutically acceptable salt or derivative thereof. The compound may be used in the treatment or prevention of a disorder selected from appetite regulation, obesity, metabolic disorders, cachexia, anorexia, pain, inflammation, neurotoxicity, neurotrauma, stroke, multiple sclerosis, spinal cord injury, Parkinson's disease, levodopa-induced dyskinesia, Huntington's disease, Gilles de la Tourette's syndrome, tardive dyskinesia, dystonia, amyotrophic lateral sclerosis, Alzheimer's disease, epilepsy, schizophrenia, anxiety, depression, insomnia, nausea, emesis, alcohol disorders, drug addictions such as opiates, nicotine, cocaine, alcohol and psychostimulants, hypertension, circulatory shock, myocardial reperfusion injury, atherosclerosis, asthma, glaucoma, retinopathy, cancer, inflammatory bowel disease, acute and chronic liver disease such as hepatitis and liver cirrhosis, arthritis and osteoporosis.

Claims

1. A compound having the following structure: ##STR00013## or a derivative thereof in which the -NH-(C═O)-NH.sub.2 group of Formula A is -NH-(C═O)-NHR or -NH-(C═O)-NR.sub.2, and wherein each R group is selected from C.sub.1-6 alkyl, aryl, heteroaryl, and C.sub.3-8 cycloalkyl, or a pharmaceutically acceptable salt of the compound or the derivative.

2. The compound according to claim 1, wherein the compound has the structure of Formula A: ##STR00014## or is a pharmaceutically acceptable salt thereof.

3. The compound or salt according to claim 2, wherein the salt is selected from the group consisting of a hydrochloride salt, acetate salt, trifluoroacetate salt, methanesulfonate salt, 2-hydroxypropane-1,2,3-tricarboxylate salt, (2R,3R)-2,3-dihydroxysuccinate salt, phosphate salt, sulphate salt, benzoate salt, 2-hydroxy-benzoate salt, S-(+)-mandelate salt, S-(−)-malate salt, S-(−) pyroglutamate salt, pyruvate salt, p-toluenesulfonate salt, 1-R-(−)-camphorsulfonate salt, fumarate salt, and oxalate salt.

4. The compound according to claim 1, wherein the compound has the structure of Formula A: ##STR00015##

5. A pharmaceutical composition comprising a compound, derivative, or salt according to claim 1, together with one or more pharmaceutically acceptable excipients.

6. The pharmaceutical composition of claim 5, further comprising one or more additional active pharmaceutical ingredients.

7. The pharmaceutical composition of claim 6, wherein the one or more additional active pharmaceutical ingredients is/are selected from the group consisting of anandamide, oleoyl ethanolamide, and palmitoyl ethanolamide.

8. A method of treatment of a condition wherein development or symptoms of the condition are linked to a substrate of the FAAH enzyme, the method comprising the administration, to a subject in need of such treatment, of a therapeutically effective amount of a compound, derivative, or salt according to claim 1, wherein the treatment is effected by increasing a level of anandamide, N-oleoylethanolamine, N-palmitoylethanolamine, and/or oleamide.

9. A method according to claim 8, wherein the condition is a disorder associated with the endocannabinoid system.

10. A method according to claim 9, wherein the disorder is selected from appetite regulation, obesity, metabolic disorders, cachexia, anorexia, pain, inflammation, neurotoxicity, neurotrauma, stroke, multiple sclerosis, spinal cord injury, Parkinson's disease, levodopa-induced dyskinesia, Huntington's disease, Gilles de la Tourette's syndrome, tardive dyskinesia, dystonia, amyotrophic lateral sclerosis, Alzheimer's disease, epilepsy, schizophrenia, anxiety, depression, insomnia, nausea, emesis, alcohol disorders, drug addictions, hypertension, circulatory shock, myocardial reperfusion injury, atherosclerosis, asthma, glaucoma, retinopathy, cancer, inflammatory bowel disease, acute liver disease, chronic liver disease, arthritis, and osteoporosis.

11. The method of claim 10, wherein the disorder is a drug addiction to an opiate, nicotine, cocaine, alcohol, or a psychostimulant.

12. The method of claim 10, wherein the disorder is an acute or a chronic liver disease, wherein the liver disease is hepatitis or liver cirrhosis.

13. The method of claim 9, wherein the disorder is ocular hypertension.

14. The method of claim 9, wherein the disorder is glaucoma.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The invention will now be described in more detail by way of example only:

(2) 1. Synthetic Methodologies

(3) The methods used for synthesis of the compounds of the invention are illustrated by the general schemes below. All compounds and intermediates were characterised by nuclear magnetic resonance (NMR). The starting materials and reagents used in preparing these compounds are available from commercial suppliers or can be prepared by methods obvious to those skilled in the art. These general schemes are merely illustrative of methods by which the compounds of this invention can be synthesised, and various modifications to these schemes can be made and will be suggested to one skilled in the art having referred to this disclosure.

(4) Room temperature in the following schemes means the temperature ranging from 20° C. to 25° C.

General Scheme for Synthesis of N-cyclopentyl-N-methyl-4-(3-ureidophenyl-1H-imidazole-1-carboxamide (Compound 1)

(5) ##STR00004##

2-Bromo-1-(3-nitrophenyl)ethanone

(6) ##STR00005##

(7) A solution of phenyltrimethylammonium tribromide (50.1 g, 133 mmol) in THF (200 mL) was added dropwise to a stirred solution of 1-(3-nitrophenyl)ethanone (20 g, 121 mmol) in THF (200 mL) at room temperature. The reaction mixture was allowed to stir at room temperature for 1 h. The white suspension was filtered and the filter cake was washed with THF, the filtrate was evaporated in vacuum to give a yellow oil. The residue was then dissolved in EtOAc and was washed with water. The organic layer was dried (MgSO.sub.4) and evaporated in vacuum to give a yellow oil that solidified into an yellow solid. Solid recrystallised from propan-2-ol and final product was isolated as an off-white solid. 2-Bromo-1-(3-nitrophenyl)ethanone (20.5 g, 70% yield).

(8) (.sup.1H, 600 MHz, 20° C., CDCl.sub.3) δ: 8.83 (1H, t, J=2 Hz), 8.49 (1H, ddd, J=1.0, 2.3, 8.2 Hz), 8.34 (1H, ddd, J=1.0, 1.7, 7.8 Hz), 7.75 (1H, t, J=8.1 Hz), 4.49 (2H, s).

(9) (.sup.13C, 150 MHz, 20° C., CDCl.sub.3) δ: 189.3, 148.5, 135.1, 134.4, 130.2, 128.1, 123.8, 29.9

(10) Melting point (mp): 90-91° C.

(11) An alternative route for the bromination reaction is as follows:

(12) To a solution of 3-Nitroacetophenone (1 wt, 1 eq) in Acetic acid (10 vol) is charged over a period of not less than 2 hours, maintaining the temperature below 30° C., a solution of bromine (0.34 vol, 1.08 eq). After stirring for 1 hour at a temperature between 25° C. and 30° C., the reaction is checked for completeness. After reaction completeness cold water (12 vol) is charged, forming a white precipitate. The suspension is stirred for an additional hour at 15° C. and then filtered. The cake is washed with water (4.5 vol). The product is dried under vacuum at a temperature not more than 45° C. until loss on drying<1.0%. The isolated yield of the brominated product was around 66%. This alternative approach may lend itself better to scaling-up.

4-(3-Nitrophenyl)-1H-imidazole

(13) ##STR00006##

(14) Water (8 mL) was added to a stirred suspension of 2-bromo-1-(3-nitrophenyl)ethanone (57.1 g, 234 mmol) and formamide (116 mL, 2.9 mol). The mixture was allowed to stir at 140° C. for 5 h. The brown residue was poured into 300 mL of water and the resulting precipitate was separated by filtration and was washed with a 1M HCl solution. The filtrate was basified with 50% NaOH and the resulting yellow precipitate was separated by filtration and was washed with water. The solid was dried and then recrystallised from propan-2-ol. 4-(3-Nitrophenyl)-1H-imidazole (7.05 g, 44% yield).

(15) (.sup.1H, 600 MHz, 20° C., DMSO) δ: 12.37 (1H, s, br), 8.58 (1H, mt, J=2.0 Hz), 8.21 (1H, ddd, J=1.0, 1.6, 7.8 Hz), 8.02 (1H, ddd, J=1.0, 2.5, 8.2 Hz), 7.88 (1H, dd, J=1.2 Hz), 7.79 (1H, dd, J=1.1 Hz), 7.64 (1H, J=8.1 Hz)

(16) (.sup.13C, 150 MHz, 20° C. DMSO) δ: 148.4, 137.9, 136,8, 136,6, 130.5, 130.0, 120.5, 118.3, 114.6

(17) Melting point: 221° C. (dec.)

(18) In terms of enhancements to this step of the process, it has been found that the use of formamide alone (i.e. without water) as suspension medium leads to increased yield, as does increasing the temperature from 140 to 170° C. (up to 80%). An enhanced protocol is thus as follows:

(19) A solution of 2-bromo-1-(3-nitrophenyl)ethanone (1 wt, 1 eq) in Formamide (10 vol) is heated to 170° C. and stirred over a period of not more than 4 hours. After stirring for 4 hours the reaction is checked for completeness. After reaction completeness the mixture is cooled to r.t., and water (15 vol) is charged. The suspension is filtered and the cake is washed with 3N HCl (2 vol) and the mother liquor filtered again. Adjust the solution pH to 14 by addition of 50% NaOH (2 vol), maintaining the mixture temperature between 0° C. and 5° C. After stirring the suspension at 0/5° C. for NLT 30 minutes, filter and wash the cake with water (5 vol). The product is dried under vacuum at a temperature not more than 45° C. until loss on drying<1.0%.

Cyclopentyl(methyl)carbamic chloride

(20) ##STR00007##

(21) A solution of N-methylcyclopentanamine (10 g, 101 mmol) in THF (126 mL) was added dropwise to phosgene solution (63.7 mL, 121 mmol, 20% in toluene) at 0° C. to give a white suspension. The reaction mixture was allowed to stir at room temperature for 1 h. The solution was poured into ice. The organic layer was diluted with EtOAc, was separated washed with 1M HCl, dried (MgSO.sub.4) and evaporated in vacuum to give a clear mobile oil. Cyclopentyl(methyl)carbamic chloride (13.1 g, 80% yield).

(22) (.sup.1H, 600 MHz, 20° C., CDCl.sub.3) δ: 4.65 (1H, m), 3.0, 2.93 (3H, 2 singlets), 1.92 (2H, m), 1.73 (2H, m), 1.59 (4H, m)

(23) (.sup.13C, 150 MHz, 20° C., CDCl.sub.3) δ: 149.7, 149.3, 61.1, 59.5, 33.1, 31.1, 28.8, 28.5, 24.0

(24) The carbamoylation step can also be carried out using triphosgene/DCM and sodium carbonate, as follows:

(25) A solution of Triphosgene (1.2 wt, 0.4 eq) in DCM (10 vol) is cooled to 0/5° C. and stirred over a period of not more than 10 minutes. A solution of N-Methylcyclopentylamine (1 wt, 1 eq) in DCM (5 vol) is charged maintaining the reaction temperature below 10° C. After the amine solution addition charge Na2CO3 (2.14 wt, 2 eq) and allow to warm to r.t. After stirring for 2 hours the reaction mixture is filtered and the cake is washed with DCM (1 vol). After concentration to dryness yellow oil is obtained and used as-is without further treatment.

N-Cyclopentyl-N-methyl-4-(3-nitrophenyl)-1H-imidazole-1-carboxamide

(26) ##STR00008##

(27) Sodium hydride (5.1 g, 127 mmol, 60% dispersion in mineral oil) was added portionwise to a stirred suspension of 4-(3-nitrophenyl)-1H-imidazole (20 g, 106 mmol) in THF (500 mL) at 0° C. The yellow suspension turned into a deep red suspension. The mixture was allowed to stir at room temperature for 30 minutes before adding a solution of cyclopentyl(methyl)carbamic chloride (25.6 g, 159 mmol) in THF (26 mL). The suspension was then allowed to stir at room temperature for 2 h. Water was added at 0° C. and the THF was evaporated. The organic residue was extracted with DCM, the organic layer was separated, dried (MgSO.sub.4) and evaporated in vacuum to give a beige solid. The solid was triturated with propan-2-ol. N-Cyclopentyl-N-methyl-4-(3-nitrophenyl)-1H-imidazole-1-carboxamide (25.18 g, 76% yield).

(28) (.sup.1H, 600 MHz, 20° C., CDCl3) δ: 8.63 (1H, mt, J=2.0 Hz), 8.16 (1H, ddd, J=1.0, 1.6, 7.8 Hz), 8.14 (1H, ddd, J=1.0, 2.3, 8.2 Hz), 7.96 (1H, d, J=1.3 Hz), 7.65 (1H, dd, J=1.3 Hz), 7.58 (1H, t, J=8.1 Hz), 4.45 (1H, m), 3.03 (3H, s), 1.98 (2H, m), 1.80 (2H, m), 1.73 (2H, m), 1.66 (2H, m)

(29) (.sup.13C, 150 MHz, 20° C., CDCl3) δ: 151.3, 148.7, 140.1, 137.3, 134.9, 130,9, 129.7, 122.1, 119.9, 114.6, 59.4, 31.3, 28.9, 24.4

(30) Melting point: 121-122° C.

4-(3-Aminophenyl)-N-cyclopentyl-N-methyl-1H-imidazole-1-carboxamide

(31) ##STR00009##

(32) A mixture of Ethyl acetate (160 mL) and EtOH (160 mL) was added to wet Pd/C (0.846 g, 0.795 mmol, 10%) under an atmosphere of argon. To this was added N-cyclopentyl-N-methyl-4-(3-nitrophenyl)-1H-imidazole-1-carboxamide (5 g, 15.91 mmol) portion wise and the suspension was allowed to stir at room temperature overnight under an atmosphere of hydrogen. The mixture was flushed with argon and filtered through celite and the celite was washed with DCM. The filtrate was evaporated in vacuum to give a clear oil that solidified into a colourless solid. The solid was recrystallised from propan-2-ol. 4-(3-Aminophenyl)-N-cyclopentyl-N-methyl-1H-imidazole-1-carboxamide (3.62 g, 80% yield).

(33) (.sup.1H, 600 MHz, 20° C., DMSO) δ: 8.06 (1H, d, J=1.3 Hz), 7.77 (1H, d, J=1.1 Hz). 7.08 (1H, t, J=1.9 Hz), 7.0 (1H, t, J=7.8 Hz), 6.98 (1H, md, J=7.7 Hz), 6.45 (1H, ddd, J=1.2, 2.3, 7.7 Hz), 5.07 (2H, s), 4.37 (1H, m), 2.92 (3H, s), 1.87 (2H, m), 1.68 (4H, m), 1.53 (2H, m)

(34) (.sup.13C, 150 MHz, 20° C., DMSO) δ: 151.2, 148.8, 141.4, 137.3, 133.8, 129.0, 113.7, 112.9, 112.8, 110.4, 58.4, 31,2, 28.2, 24.0

(35) Melting point: 108-109° C.

(36) In an alternative embodiment, the aniline derivative product of this step can be used in the subsequent step without purification, i.e. such that this and the subsequent step can be telescoped.

N-Cyclopentyl-N-methyl-4-(3-ureidophenyl)-1H-imidazole-1-carboxamide (Compound 1)

(37) ##STR00010##

(38) Potassium cyanate (0.445 g, 5.49 mmol) was added portionwise to a stirred solution of 4-(3-aminophenyl)-N-cyclopentyl-N-methyl-1H-imidazole-1-carboxamide (1.3 g, 4.57 mmol) in a mixture of 2N hydrogen chloride (2.286 mL, 4.57 mmol) in Water (4 mL) at 0° C. The mixture was allowed to stir at room temperature for 24 h. Potassium cyanate (0.220 g, 2.74 mmol) was added and the mixture was allowed to stir at room temperature for another night. Water was added and the organic layer was diluted with a mixture of DCM/propan-2-ol 7:3. The organic layer was separated and was washed with a 1N HCl aqueous solution. The organic layer was separated, dried (MgSO.sub.4) and evaporated in vacuum to give a colourless foam. The product was purified by column chromatography (silica, DCM/MeOH 5%, 10%) and was isolated as a colourless solid. The solid was recrystallised from EtOH at 0° C. N-Cyclopentyl-N-methyl-4-(3-ureidophenyl)-1H-imidazole-1-carboxamide (0.403 g, 26% yield).

(39) The compounds of the invention above were characterised by melting point and NMR as detailed below. NMR spectra were recorded on a Bruker 600 MHz Avance IIIspectrometer with solvent used as internal standard. 13C spectra were recorded at 150 MHz and 1H spectra were recorded at 600 MHz. Data are reported in the following order: approximate chemical shift (ppm), number of protons, multiplicity (br, broad; d, doublet; m, multiplet, s, singlet, t; triplet) and coupling constant (Hz).

(40) Compound no. 1 (melting point: 204° C.).

(41) (.sup.13C, 150 MHz, 20° C., DMSO) δ: 156, 151.1, 140.9, 140.8, 137.5, 133.7, 128.9, 117.9, 116.6, 114.2, 114.2, 58.4, 31.2, 28.2, 24.

(42) (.sup.1H, 600 MHz, 20° C., DMSO) δ: 8.55 (1H, s), 8.09 (1H, d, J=1.2 Hz), 7.86 (1H, d, J=1.2 Hz), 7.85 (1H, t, J=1.8 Hz), 7.35 (1H, md), 7.34 (1H, md), 7.22 (1H, t, J=7.8 Hz), 5.84 (2H, s), 4.36 (1H, m), 2.93 (3H, s), 1.87 (2H, m), 1.69 (4H, m), 1.54 (2H, m).

(43) An enhancement to this final step (urea formation on the phenyl ring) consists of using acetic acid as solvent for the 4-(3-aminophenyl)-N-cyclopentyl-N-methyl-1H-imidazole-1-carboxamide instead of water. This leads to an improvement in yield (around 78%), and an improved isolation protocol. The enhanced step may be described as follows: 4-(3-aminophenyl)-N-cyclopentyl-N-methyl-1H-imidazole-1-carboxamide is dissolved in AcOH (8.8 vol) at room temperature. To the resulting solution at room temperature is added a solution of potassium cyanate (0.65 wt, 2.5 eq) in water (0.9 vol). The resulting solution is stirred at room temperature until reaction completion (starting material<0.1%). Within 1 h, the precipitation of the urea product occurred. To the resulting slurry is added water (5 vol), and more solid crashes out. The beige suspension is then aged for 1 h at room temperature, and filtered. The beige solid is washed with water (10 vol), dried under vacuum oven until loss on drying<1.5%.

(44) In case this enhanced step (using acetic acid) leads to an N-acetylated aniline impurity, a recrystallisation may be performed. This may be as follows:

(45) To a solution of the urea product (1 wt) in acetic acid (5 vol) at room temperature was added drop wise water (5 vol) over 30 minutes. After having seeded, water (2 vol) was added and the slurry was aged at room temperature for 1 h. The slurry is cooled to 10° C., stirred at 10° C. for at least 1 h and filtered. The off white solid is washed with a 9:1 mixture of water/acetic acid (2 vol), water (10 vol), dried in a vacuum oven at 55° C. The off white solid (0.82 wt) is then dissolved in acetic acid (3.96 vol) at room temperature and water (4.1 vol) was added drop wise over 30 minutes. To the solution was then added seed, followed by water (1.6 vol). The resulting slurry was stirred at room temperature for at least 1 h and then cooled to 10° C. After aging the slurry at 10° C. for at least 1 h, the solid is filtered, washed with a 9:1 mixture of water/acetic acid (1.6 vol), water (10 vol), dried in a vacuum oven at 55° C. until loss on drying is<1.5%.

(46) 2. Biological Efficacy

(47) In vivo testing was performed according to the protocol described below. BRh (brain homogenate) indicates inhibition in central nervous tissue, in this case, brain, and LVh (liver homogenate) indicates inhibition in peripheral tissue, in this case, liver. The controls were the reaction mix minus the test compounds. Therefore, a low value for the test compound indicates a strong inhibitor. A value of 100 indicates that no measurable inhibition took place.

(48) In Vivo Protocol

(49) Animal Treatment

(50) The animals used for experiments were male NMRI mice (weighing 27-44 g) obtained from Interfauna Ibérica (Spain). Mice were kept 5 per cage, under controlled environmental conditions (12 hr light/dark cycle and room temperature 22±1° C.). Food and tap water were allowed ad libitum and the experiments were all carried out during daylight hours.

(51) Animals were administered 30 mg/kg or 3 mg/kg compound of the invention or comparator compounds via oral route (8 ml/kg; compound suspended in 0.5% carboxymethylcellulose (CMC) or solubilized in water) or vehicle (controls) using animal feeding stainless steel curve needles (Perfectum, U.S.A.). Fifteen minutes before sacrifice animal were anesthetized with pentobarbital 60 mg/kg administered intraperitoneally. A fragment of liver, left lung lobe and brain without cerebellum were removed and put in plastic vials containing membrane buffer (3 mM MgCl.sub.2, 1 mM EDTA, 50 mM Tris HCl pH 7.4). Tissues were stored at −30° C. until analysis.

(52) Animals were always fasted overnight before administration of compounds except for time points of >18 h, where food was removed on the morning of day of administration and the compound was administered in the afternoon of the same day. Animals were then given water but nothing else.

(53) All animal procedures were conducted in strict adherence to the European Directive for Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (86/609CEE) and Portuguese legislation (Decreto-Lei 129/92, Portarias 1005/92 e 1131/97). The number of animals used was the minimum possible in compliance with current regulations and scientific integrity

(54) Reagents and Solutions

(55) Anandamide [ethanolamine-1-.sup.3H-] (40-60 Ci/mmol) was obtained from American Radiochemicals. All other reagents were obtained from Sigma-Aldrich. Optiphase Supermix was obtained from Perkin Elmer and activated charcoal were obtained from Sigma-Aldrich.

(56) Tissue Preparation

(57) Tissues were thawed on ice and were homogenized in 10 volumes of membrane buffer (3 mM MgCl.sub.2, 1 mM EDTA, 50 mM Tris HCl pH 7.4) with either Potter-Elvejhem (brains—8 strokes at 500 rpm) or Heidolph Diax (livers—2 strokes at position 5 for 20 sec with 30 sec pauses).

(58) Total protein in tissues was determined with the BioRad Protein Assay (BioRad) using a standard curve of BSA (50-250 μg/ml).

(59) Enzymatic Assay

(60) Reaction mix (total volume of 200 μl) contained: 2 μM AEA (2 μM AEA+5 nM .sup.3H-AEA), 0.1% fatty acid free BSA, 15 μg (brain), 5 μg (liver) or 50 μg (lung) protein, in 1 mM EDTA, 10 mM Tris pH 7.6. After a 15 mM pre-incubation period at 37° C., reaction was started by the addition of the substrate solution (cold AEA+radiolabelled AEA+BSA). Reaction was carried out for 10 min (brain and lung) or 7 mM (liver) before termination by the addition of 400 μl activated charcoal suspension (8 g charcoal in 32 ml 0.5 M HCl in continuous agitation). After a 30 mM incubation period at room temperature with agitation, charcoal was sedimented by centrifugation in microfuge (10 min at 13000 rpm). 200 μl of the supernatant were added to 800 μl Optiphase Supermix scintillation cocktail previously distributed in 24-well plates. Counts per minute (cpm) were determined in a MicrobetaTriLux scintillation counter.

(61) In each assay blanks (without protein) were prepared.

(62) The percentage of remaining enzymatic activity was calculated with respect to controls and after blank subtraction.

(63) ED.sub.50 Determination

(64) The test compounds were given in increasing doses (10, 3, 1, 0.3, 0.03 and 0.01 mg/kg) to the animals and at 8 h post-administration FAAH activity was determined according to the aforementioned in-vivo protocol, then ED.sub.50 values were calculated by “Prisma” software with 95% of confidence intervals.

(65) CYPs Metabolic Stability Assay

(66) Stability of the test compounds was performed in MLM (mouse liver microsomes) or HLM (human liver microsomes) in the presence and in the absence of NADPH.

(67) The stability was measured using the incubation mixture (100 μl total volume) contained 1 mg/ml total protein, MgCl.sub.2 5 mM and 50 mM K-phosphate buffer. Samples were incubated in the presence and in the absence of NADPH 1 mM. Reactions were pre-incubated 5 min and the reaction initiated with the compound under test (5 μM for HLM and 50 μM for MLM). Samples were incubated for 60 min in a shaking water bath at 37° C. The reaction was stopped by adding 100 μl of acetonitrile. Samples were then centrifuged, filtered and supernatant injected in HLPC-MSD. Test compounds were dissolved in DMSO and the final concentration of DMSO in the reaction was below 0.5% (v/v). At T0 acetonitrile was added before adding the compound. All experiments were performed with samples in duplicate.

(68) Compound 1 (N-cyclopentyl-N-methyl-4-(3-ureidophenyl)-1H-imidazole-1-carboxamide; also referred to as the compound of Formula A, above) was tested. Also, two comparator compounds were tested which are disclosed in WO 2010/074588. These are as follows:

(69) Comparator compound 1—N-cyclohexyl-4-(3-guanidinophenyl)-N-methyl-1H-imidazole-1-carboxamide.

(70) Comparator compound 2—N-cyclopentyl-4-(4-fluoro-3-hydroxyphenyl)-N-methyl-1H-imidazole-1-carboxamide.

(71) Comparator compound 1 is structurally similar to compound 1, although there are clear differences between these compounds. Comparator compound 2 is also structurally similar to compound 1 but, again, there are clear differences between these two compounds.

(72) TABLE-US-00001 FAAH Activity (%) FAAH Activity (%) Br. h. 3 mg/kg. Lv. h. 3 mg/kg. 8 h. po 8 h. po Compound 1 85.1 0.7 Comparator Compound 1 86.2 20.3 Comparator Compound 2 121.1 2.1

(73) As can be seen from the above table, compound 1 is the most potent compound in terms of FAAH inhibition in the liver. In particular, compound 1 is much more potent than comparator compound 1.

(74) Peripheral selectivity can be calculated by dividing the FAAH activity in the liver by the FAAH activity in the brain. When doing this, a lower number shows a compound is peripherally more selective. The results are given in the table below:

(75) TABLE-US-00002 Peripheral Selectivity Compound 1 0.008 Comparator Compound 1 0.235 Comparator Compound 2 0.017

(76) These results show that compound 1 is the most peripherally selective compound by more than a factor of 2. Further, compound 1 is much more peripherally selective than comparator compound 1.

(77) Additional data relating to the activity of FAAH at various concentrations for compound 1 and comparator compound 2 are given in the table below:

(78) TABLE-US-00003 FAAH Activity (%) mouse Liver 1 h 8 h 3 mg/kg 10 mg/kg 3 mg/kg 1 mg/kg 0.1 mg/kg Compound 1 1.9 0.4 0.7 1.6 6.4 Comparator 17.0 1.2 2.1 4.9 73.8 Compound 2

(79) As can be seen, at all doses, the FAAH activity is much lower following administration of compound 1 compared to comparator compound 2. In particular, at 0.1 mg/kg at 8 hours post-dose, the FAAH activity is significantly lower for compound 1 compared to comparator compound 2. This shows that compound 1 is significantly more potent than comparator compound 2. At 0.1 mg/kg, compound 1 is more than 10-fold more potent than comparator compound 2. This is a surprisingly big difference in potency. This data is also evidence that compound 1 is metabolically stable since, when conducting inhibition experiments in vivo, the metabolic stability of the compound will also play a role in the level of inhibition and the length of time over which inhibition takes place.

(80) The table below shows FAAH inhibition ED.sub.50 data (median effective dose, the dose of compound required to produce 50% inhibition of FAAH in liver) of the compounds after p.o. administration in mouse. Confidence intervals (95%) are included.

(81) TABLE-US-00004 Compound Liver ED.sub.50 (95CI) (mg/kg) Time (h) Compound 1 0.03 (0.02; 0.04) 1 Comparator Compound 2 0.17 (0.13; 0.23) 8

(82) The below table shows the metabolic stability of compound 1 and comparator compound 2. The stability data are given as % of remaining compound after 1 h exposure to MLM or HLM. 100% means no metabolic reaction at all and 0% corresponds to full enzymatic degradation. “CYP−” refers to the absence of cofactor (NADPH) which is essential for CYP metabolic reactions. Therefore “CYP−” can be regarded as control value. “CYP+” refers to the presence of cofactor and the enzymatic degradation may take place according to the stability of the test compound. As can be seen, compound 1 is more stable than comparator compound 2 in both MLM and HLM.

(83) TABLE-US-00005 Metabolic Stability (% of Remaining) Mouse Human CYP+ CYP− CYP+ CYP− Compound 1 96 95 82 99 Comparator Compound 2 53 92 70 92

(84) 3. Determination of IC.sub.50 of Compound 1

(85) 3.1 Materials and Methods

(86) a) Reagents and Solutions

(87) Anandamide [ethanolamine-1-.sup.3H-] was obtained from American Radiochemicals—with a specific activity of 60 Ci/mmol. All other reagents were obtained from Sigma-Aldrich. Optiphase Supermix was obtained from Perkin Elmer and activated charcoal was obtained from Sigma.

(88) b) Tissue Preparation

(89) Frozen brains from 4 Wistar rats were homogenized in 20 ml 1 mM MgCl.sub.2, 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) pH 7.0 with Potter-Elvejhem (8 strokes at 500 rpm). Homogenates were centrifuged for 20 mM at 36000 g at 4° C. (Beckman, 70Ti rotor). Pellets were resuspended in 15 ml of the same buffer and centrifuged under the same conditions. Pellets were resuspended in 15 ml of the same buffer and incubated for 15 min at 37° C. after which they were centrifuged for 20 min at 36000 g at 4° C. Each pellet was then resuspended in 10 ml 3 mM MgCl.sub.2, 1 mM EDTA (Ethylenediaminetetraacetic acid), 50 mM Tris (2-Amino-2-hydroxymethyl-propane-1,3-diol) pH 7.4 and protein determined with the BioRad Protein Assay (BioRad) using a standard curve of BSA (Bovine Serum Albumin) (50-250 μg/ml).

(90) Membrane suspensions are aliquoted and stored at −30° C.

(91) c) Enzymatic Assay

(92) Reaction mix (total volume of 200 μl) contained: 2 μM AEA (2 μM AEA+5 nM .sup.3H-AEA), 0.1% fatty acid free BSA, 5 or 10 μg protein, in 1 mM EDTA, 10 mM Tris pH 7.6 and compound 1 at various concentrations. Stock solution (10 mM) was prepared in 100% DMSO (dimethyl sulfoxide) and the DMSO concentration in the assay will be 0.1%. After a 15 min preincubation period at 37° C., reaction was started by the addition of the substrate solution (cold AEA+radiolabelled AEA+BSA). Reaction was carried out for 10 mM before termination by the addition of 400 μl activated charcoal suspension (8 g charcoal in 32 ml 0.5 M HCl in continuous agitation). After a 30 min incubation period at room temperature with agitation, charcoal is sedimented by centrifugation in microfuge (10 min at 15000 g). 200 μl of the supernatant was added to 800 μl Optiphase Supermix scintillation cocktail previously distributed in 24-well plates. Counts per minute (CPM) or disintegrations per minute (DPM) were determined in a MicrobetaTriLux scintillation counter. In each assay blanks (no protein) and controls (no compound) were present.

(93) d) Test Systems

(94) Wallac 1450 MicrobetaTriLux scintillation counter.

(95) e) Test Method

(96) Counting conditions were the following:

(97) TABLE-US-00006 Labels: H-3 Cassette type: 24 wells, 4 by 6 Counting mode: CPM or DPM Sample type: Normal Paralux used: No Counting time: 10 min CPM norm.: Norm_H3 (0) for CPM or 3H AEA standardization for DPM Status: n Corrections BGND corr.: Off CLM corr.: Off Autoquench corr.: No for CPM, NA for DPM Counting control Precision: 0.2 Repeats: 1 Cycles: 1 Cycle delay: 0 Plate delay: 0 Plate orientation: Normal Detector setup: Normal Window 1: 5-360

(98) f) Other Equipment

(99) Spectramax Plus—SOFTmax® PRO Software version 3.0

(100) g) Data Acquisition and Analysis

(101) Raw data acquisition was performed with the software “Microbeta TriLux Windows workstation version 4.01”.

(102) Data analysis was performed using Prism 5 for Windows software, version 5.02 (GraphPad Software Inc., San Diego, Calif.). IC.sub.50 value of compound 1 was determined by fitting experimental data to the log(inhibitor) vs normalized response—Variable slope equation:

(103) $Y=\frac{100}{1+{10}^{\left(\left(\mathrm{LogIC}50-X\right).Math.\mathrm{Hill}\mathrm{Slope}\right)}}$

(104) 3.2 Results

(105) Using this protocol, compound 1 was determined to have an IC.sub.50 of 27 nM.

(106) As can be seen from all the results above, compound 1 is significantly more potent, more peripherally selective and/or more metabolically stable than either of comparator compounds 1 and 2.

(107) 4. Synthesis of HCl Salt of N-methylcyclopentylamine

(108) 4.1 Carbamate Reduction Method

(109) ##STR00011##

(110) Step 1: Formation of Ethyl Carbamate

(111) To a solution of cyclopentylamine (3 ml, 30.3 mmol) in THF (20 mL) at 0° C. were added respectively 3M sodium hydroxide (15.15 ml, 45.5 mmol) and ethyl chloroformate (3.47 ml, 36.4 mmol). The resulting biphasic mixture was stirred for 4 h at room temperature. The reaction mixture was diluted with MTBE (30 mL) and ammonium hydroxide (5 mL). The resulting mixture was stirred at room temperature for 10 minutes and then allowed to separate. The organic layer was washed with water, 0.5M HCl, dried over Na.sub.2SO.sub.4, filtered. The filtrate was concentrated under reduced pressure. Ethyl cyclopentylcarbamate (4.35 g) was obtained as colorless oil in 95% yield and was used in the next step without further purification

(112) This reaction proceeds very well. The yield and the quality of the product were high.

(113) Step 2: Reduction of Ethyl Carbamate

(114) The reduction of carbamates to the corresponding methyl amine is well known in general. This reduction requires usually the use of an excess of lithium aluminum hydride (LAH) in THF at reflux. However, the use of lithium aluminum hydride in large scale may require a more complex work up. Therefore, the Fieser work-up was used (for x g of LAH, use x ml of water, x mL of 15 to 25% NaOH then followed by 3×mL of water) which is safer and easier to handle. A first attempt was successfully performed on the t-Butyl carbamate using LAH, Fieser work up followed by formation of the hydrochloride salt resulting from the addition of concentrated HCl. The N-methyl cyclopentylamine hydrochloride was obtained in 63% after isolation. The quality and the yield resulting from this first attempt led to a repeat using the ethyl carbamate, which led to a Molar yield of 83%, and a Quality range: >98% by NMR.

(115) To a suspension of LAH (2.414 g, 63.6 mmol, 5 eq) in THF (20 mL) at room temperature under nitrogen was added a solution of ethyl cyclopentyl carbamate (2 g, 12.72 mmol) in THF (8 mL) over 20 minutes. Note: gas evolution. The dropping funnel was rinsed with THF (2 mL). The reaction mixture was heated to 65° C. (internal temperature, reflux) during 6 h. The suspension was cooled to 0° C. (water-ice bath). The suspension was diluted with MTBE (30 mL). To the suspension were added dropwise 2.4 mL of water (strong gas evolution and exothermic reaction was observed), dropwise 3.6 mL of 10% NaOH (good stirring is necessary) and finally dropwise 7.2 mL of water. The resulting slurry was warmed to room temperature and stirred for 30 minutes at room temperature. To the white suspension was added MgSO4 (10 g). The resulting slurry was stirred for 10 minutes, then filtered. The solid was washed with MTBE (20 mL).

(116) To the combined filtrates were added conc HCl (1.272 ml, 15.27 mmol, 1.2 eq). The resulting mixture was stirred overnight at room temperature and then concentrated to dryness. The residue was dissolved in propan-2-ol (20 mL) then concentrated to 2 vol (4 mL). To the resulting solution was then added MTBE (12 mL), a white crystalline solid crushed out. The slurry was stirred at room temperature for 1 h and then the solid was collected, washed with MTBE (4 mL), dried in a vacuum oven at 50° C. for 4 h. A first crop of white needles (884 mg) was obtained, the combined mother liquor and washes were concentrated to dryness. Isopropyl acetate (iPrOAc) was added to the residue, white crystals started to appear. More iPrOAc was added but some solids were crusted on the flask wall. Some DCM was added and clear solid was obtained. The DCM was removed and a white solid was crashed out, filtered and washed with iPrOAc. The white crystalline solid was dried in a vacuum oven at 50° C. for 4 h. A second crop of white needles (547 mg) was obtained. The N-methyl cyclopentylamine hydrochloride was obtained as white needles in 83% molar yield.

(117) 4.2 Reductive Amination Method

(118) ##STR00012##

(119) The use of cyclopentanone and N-methylamine hydrochloride in presence of a catalytic amount of triethylamine and Pd/C under hydrogen pressure in methanol at 65° C. was found to give the best results. Under these conditions, the N-methylcyclopentylamine hydrochloride was isolated as a white solid in 49% yield.

(120) The source of palladium and reagent equivalents were tested to improve the yield and the quality of the product (removal of methylamine hydrochloride). Using Pd/C (JM, 5R39 paste) with a slight excess of Methyl amine hydrochloride (1.1 eq) it was possible to improve the yield up to 69%.

(121) Note that the removal of methyl amine hydrochloride is feasible by suspending the N-methylcyclopentylamine hydrochloride in dichloromethane in the presence of sodium carbonate followed by distillation. No methylamine is detected in the final product.

(122) Protocol Description

(123) To palladium 5% on carbon, 5R 39 paste (0.75 g, 0.176 mmol, 0.001 eq) were added successively MeOH (105 ml), methylamine hydrochloride (13.24 g, 196 mmol), cyclopentanone (15.77 ml, 178 mmol) and finally triethylamine (0.621 ml, 4.46 mmol). The resulting slurry was placed into an autoclave and was charged with 5 bar hydrogen. The autoclave was heated at 65° C. and stirred overnight. The reaction mixture was cooled slowly and TLC (eluent PE/ethyl acetate 8:2, dip permanganate) showed no starting material. The black slurry was filtered through celite and washed with MeOH (10 mL). The methanol was removed and replaced by isopropanol (60 mL). The solution was concentrated to 2 vol and isopropyl ether (60 ml) was added. The resulting mixture was stirred at room temperature. A white solid was observed and then the slurry was stirred at 0° C. for 1 h, then filtered. The solid was washed with isopropyl ether/propan-2-ol 9:1 (30 mL), dried in a vacuum oven overnight. A white crystalline solid of N-methylcyclopentylamine HCl (16.9 g, 69.5% yield) was obtained.