Patent Application
20070270400
It should be emphasised that the compounds of the present invention most often show highly complex NMR spectra due to the existence of conformational isomers. This is believed to be a result from slow rotation about the amide and/or aryl bond. The following abbreviations are used in the presentation of the NMR data of the compounds: s-singlet; d-doublet; t-triplet; qt-quartet; qn-quintet; m-multiplet; b-broad; cm-complex multiplet, which may include broad peaks.
The following examples will describe, but not limit, the invention.
The following abbreviations are used in the experimental: DMSO (dimethylsulfoxide), THF (tetrahydrofuran), MTBE (metyl-tertbutyleter) and RT (room temperature).
3-Chloro-N-[(2S)-2-(4-fluorophenyl)-4-oxobutyl]-N-methyl-5-(trifluoromethyl)benzamide (see Method 1; 0.25 g, 0.62 mmol) and 1-acetyl-4-azetidin-3-ylpiperazine hydrochloride (see WO 96/05193; 0.20 g, 0.93 mmol) together with triethylamine (0.17 mL, 1.24 mmol) were mixed with methanol (7 mL). The mixture was stirred at RT for 30 min and then sodium triacetoxyborohydride (0.26 g, 1.24 mmol) was added. The reaction mixture was stirred at RT for 1 h. The solvent was removed by evaporation and the residue was partitioned between methylene chloride (5 mL) and an aqueous solution of NaHCO3 (5 mL 10%). The organic phase was separated by means of a phase separator column, which then was washed with more of methylene chloride. The solvent was removed by evaporation. The product was purified by chromatography on silica gel (ammonia saturated methanol-methylene chloride: 2%-15% methanol). There was obtained 0.21 g (60%) of the title compound as a colorless oil. 1H NMR (500 MHz, CDCl3): 1.3-1.8 (cm, 2H), 2.0 (s, 3H), 2.1-3.7 (cm, 21H), 6.7-7.3 (cm, 6H), 7.5 (s, 1H): m/z 569 (M+1)+.
3-Chloro-N-[(2S)-2-(4-fluorophenyl)-4-oxobutyl]-N-methyl-5-(trifluoromethyl)benzamide (see Method 1; 1.57 g, 3.90 mmol) and triethylamine (0.79 g, 7.82 mmol) were mixed with methanol (7 mL). 1-Azetidin-3-yl-4-isobutyrylpiperazine hydrochloride (see Method 2; 1.26 g, 5.08 mmol) was added and the mixture was stirred at RT for 30 min. Sodium triacetoxyborohydride (1.24 g, 5.86 mmol) was added over a period of 15 min. The reaction mixture was stirred at RT for 30 min. The solvent was removed by evaporation and the residue was partitioned between methylene chloride (5 mL) and an aqueous solution of NaHCO3 (5 mL 10%). The organic phase was separated by means of a phase separator column, which then was further washed with methylene chloride. The solvent was removed by evaporation. The product was purified by chromatography on silica gel (ammonia saturated methanol-methylene chloride: 2%-15% methanol). There was obtained 0.21 g (60%) of the title compound as a colorless oil. 1H NMR (500 MHz, CDCl3): 1.1 (d, 6H), 1.3-1.8 (cm, 2H), 2.2-3.8 (cm, 22H), 6.8-7.3 (cm, 6H), 7.6 (s, 1H): m/z 597 (M+1)+.
A mixture of 3-chloro-N-[(2S)-2-(4-fluorophenyl)-4-oxobutyl]-N-methyl-5-(trifluoromethyl)benzamide (see Method 1; 0.54 g, 1.8 mmol), triethylamine (0.5 mL, 3.6 mmol) and (8aR)-2-azetidin-3-ylhexahydropyrrolo[1,2-a]pyrazin-6(2H)-one (see Method 3; 0.70 g, 1.8 mmol) in methanol was stirred for 1 h at RT. Triacetoxy sodium borohydride (0.52 g, 3.6 mmol) was added while cooling to 0° C. and then the mixture was stirred overnight at RT. Water was added and the mixture was extracted with dichloromethane. The organic solution was washed with brine and dried over Na2SO4. The solvent was removed by evaporation and the residue was purified by chromatography on silica gel (methanol-methylene chloride 3:97). There was obtained 0.31 g (29%) of the title compound as a pale yellow gummy oil. 1H NMR (400 MHz, CDCl3): 1.6-3.6 (cm, 24H), 4.0 (d, 2H), 6.8-7.6 (cm, 7H): m/z 582 (M+1)+.
A mixture of 3-chloro-N-[(2S)-2-(4-fluorophenyl)-4-oxobutyl]-N-methyl-5-(trifluoromethyl)benzamide (see Method 1; 0.54 g, 1.8 mmol), triethylamine (0.5 mL, 3.6 mmol) and (8aS)-2-azetidin-3-ylhexahydropyrrolo[1,2-a]pyrazin-6(2H)-one (see Method 4; 0.70 g, 1.8 mmol) in methanol was stirred for 1 h at RT. Triacetoxy sodium borohydride (0.52 g, 3.6 mmol) was added while cooling to 0° C. and then the mixture was stirred overnight at RT. Water was added and the mixture was extracted with dichloromethane. The organic solution was washed with brine and dried over Na2SO4. The solvent was removed by evaporation and the residue was purified by chromatography on silica gel (methanol-methylene chloride 3:97). There was obtained 0.32 g (30%) of the title compound as a pale yellow gummy oil. 1H NMR (400 MHz, CDCl3): 1.6-3.6 (cm, 24H), 4.0 (d, 2H), 6.8-7.2 (cm, 6H), 7.6 (s, 1H): m/z 582 (M+1)+.
Preparation of Starting Materials
The starting materials for the examples above are either commercially available or are readily prepared by standard methods from known materials. For example, the following reactions are an illustration, but not a limitation, of some of the starting materials.
Method 1
Lithium diisopropylamide (LDA, 52 L, 1.8 M, 93.6 mol) in a solution of THF/heptane and ethylbenzene was charged to a reactor under a nitrogen atmosphere, and THF (52 L) was then added. The temperature was adjusted to an inner temperature (the temperature of the reaction solution) of −48° C. 4-Fluorophenylacetonitrile (13.0 kg, 96.2 mol) in a THF-solution (25 L) was charged during 1 h and 50 min to the solution comprising LDA, while the temperature of the reaction mixture was kept below −30° C. The temperature was increased to −6° C. over 1 h, during that time the yellow slurry transformed into a dark purple solution. THF (5 L) followed by tert-butylbromoacetate (20.25 kg, 104 mol) and finally THF (25 L) were charged to a second reactor. The temperature was lowered to an inner temperature of −48° C. The dark purple solution above was charged to the tert-butyl-bromoacetate-solution over 7.5 h, while the inner temperature was kept below −34° C. The inner temperature was adjusted to −5° C. and the reaction mixture was quenched by adding a solution of ammonium chloride (12.7 kg) and water (55 L) over 15 min. Methyl tert-butyl ether (43 L) was charged and the obtained mixture was stirred for 5 min. After phase separation, the aqueous phase was discarded. Brine (7.6 kg sodium chloride in 25 L of water) was charged to the remaining organic phase and the mixture was stirred for 5 min. The aqueous phase was discarded and the remaining solution was concentrated by distillation at reduced pressure to a volume of 150 L. Isooctane (43 L) was charged and the distillation was continued until the resulting volume was 60 L at which point crystallization started. MTBE (25 L) was charged and the jacket temperature was set to 0° C. After 2 h the batch was filtered (inner temperature 2° C.) and washed with isooctane (2×20 L). After drying 16.8 Kg (72%) of tert-butyl 3-cyano-3-(4-fluorophenyl)propanoate was obtained. 1H NMR (DMSO-d6) δ 7.51 (app d, J=8 Hz, 1 H), 7.50 (app d, J=8 Hz, 1 H), 7.24 (app t, J=8 Hz, 2H), 4.50 (app dd, J1=6 Hz, J2=8 Hz, 1 H), 3.02 (app dd, J1=8 Hz, J2=16 Hz, 1 H), 2.86 (app dd, J1=6 Hz, J2=16 Hz, 1 H), 1.36 (s, 9H); 13C NMR (DMSO-d6) δ 168.4, 161.7 (d, JC,F=244 Hz), 131.3 (d, JC,F=3 Hz), 129.8 (d, JC,F=9 Hz), 120.6, 115.7 (d, JC,F=22 Hz), 81.0, 39.1, 31.4, 27.6.
tert-Butyl 3-cyano-3-(4-fluorophenyl)propanoate (16.7 kg, 67.0 mol) was charged under nitrogen atmosphere to a reactor and THF (50 L) was then added. The temperature was adjusted to an inner temperature of 65° C. Borane-dimethylsulfide complex (16.6 L, 166 mol) in a THF solution (5 L) was charged to the reaction mixture over a period of 43 minutes. The mixture was then refluxed for 2 hours. The reaction mixture was cooled to 10° C. Water (75 L) and hydrochloric acid (25.5 L) was charged to a second vessel and the reaction solution above was charged to this aqueous phase accompanied by gas evolution (H2 is formed). When the addition was complete (after 1.5 h), the jacket temperature was increased to 105° C. and the solvents were distilled off until the temperature of the reaction mixture reached 85° C. The reaction mixture was refluxed for 12.5 h and then cooled to 24° C. Aqueous sodium hydroxide (50% solution, 32.4 kg) was charged followed by toluene (55 L) and THF (18 L). After phase separation, the aqueous phase was extracted with a mixture of toluene (30 L) and THF (13 L). The organic phases were combined and approximately 65L of solvent mixture was removed by distillation under reduced pressure. Toluene (40 L) and THF (5 L) was charged to the organic phase and the resulting mixture was clear filtered and returned to the reactor. The solvents were distilled off at reduced pressure until 50 L remained. Toluene (20 L) was charged and the distillation was continued until approximately 35 L remained. The inner temperature was lowered from 59° C. to 12° C. over 1 h and seeding crystals (0.2 g) were added, which started the crystallization. Heptane (12 L) was charged and the slurry was cooled down to 6° C. over 2 h. The slurry was filtered and the solid was washed with heptane (2×10 L) and dried. There was obtained 6.13 kg (50 %) of 4-amino-3-(4-fluorophenyl)butan-1-ol. 1H NMR (DMSO-d6) δ 7.21 (app d, J=8 Hz, 1 H), 7.19 (app d, J=8 Hz, 1 H), 7.10 (app t, J=8 Hz, 2H), 3.13-3.35 (m, 2 H), 2.59-2.81 (m, 2 H), 1.77-1.94 (m, 2 H), 1.50-1.68 (m, 2 H); 13C NMR (CDCl3) δ 161.7 (d, JC,F=244 Hz), 139.9 (d, JC,F=3 Hz), 129.0 (d, JC,F=8 Hz), 115.6 (d, JC,F=21 Hz), 61.1, 48.2, 46.7, 38.6.
(R)-O-Acetylmandelic acid (18.79 kg, 96.76 mol) was charged to a reactor followed by water (845 g) and ethyl acetate (100 L). The solution was stirred at an inner temperature of 17-20° C. for 0.5 h. The clear solution was collected in a drum and the reactor was rinsed with ethyl acetate (20 L). The rinsing solution was then combined with the above clear (R)-O-acetylmandelic acid solution. 4-Amino-3-(4-fluoro-phenyl)-butan-1-ol (20.64 kg, 112.65 mol) was charged to a reactor followed by absolute ethanol (99.7% w/w, 19 L) and ethyl acetate (43 L). Stirring was started and the inner temperature was raised to 59° C. The (R)-O-acetylmandelic acid solution was charged to the solution of 4-amino-3-(4-fluoro-phenyl)-butan-1-ol over 24 min. The dark yellow solution thus obtained started to crystallize at an inner temperature of 53° C. about 5 min after complete addition of (R)-O-acetylmandelic acid. The inner temperature was kept at 52-53° C. for 20 min, and the slurry was then cooled down to 25° C. over 1 h and 20 min. The white slurry was filtered and the solid was washed with ethyl acetate (2×37.5 L) to give, after drying on the filter, 15.33 kg of needle like white crystals having an optical purity of 83% enantiomeric excess (ee). The ee corrected yield is 66%. The obtained product (15.33 kg, 40.62 mol) was charged to a reactor followed by absolute 99.5% ethanol (27.5 L) and ethyl acetate (22.5 L). Stirring was started and the mixture was heated to an inner temperature of 70° C. Ethyl acetate (105 L) was charged to the mixture over 44 min. The inner temperature was kept between 67-70° C. during the addition. The crystallization started 8 min after the last addition of ethyl acetate (inner temperature 69° C.). The slurry was cooled to an inner temperature of 25° C. over 1 h and 50 min and then filtered. The solid was washed with ethyl acetate (2×37.5 L) and dried giving 11.65 kg (82% ee corrected yield) of (3S)-4-amino-3-(4-fluorophenyl)butan-1-ol as white crystals. The optical purity was 98% ee according to chiral HPLC. 1H NMR (DMSO-d6) δ 7.41 (app dd, J1=7 Hz, J2=1 Hz, 2 H), 7.16-7.34 (m, 5 H), 7.12 (app t, J=9 Hz, 2H), 5.53 (app s, 1 H), 3.08-3.33 (m, 2 H), 2.92-3.08 (m, 2 H), 2.78-2.92 (m, 1 H), 2.04 (s, 3 H), 1.77-1.94 (m, 1 H), 1.50-1.69 (m, 1 H); 13C NMR (DMSO-d6) δ 170.6, 169.7, 168.4, 161.1 (d, JC,F=242 Hz), 138.3, 137.7 (d, JC,F=3 Hz), 129.7 (d, JC,F=8 Hz), 127.9, 127.4, 127.3, 115.2 (d, JC,F=21 Hz), 77.2, 58.2, 44.0, 38.7, 36.3, 21.1. [α]D (c 1.0 in methanol, 25° C.) −60.4°.
(S)-4-Amino-3-(4-fluorophenyl)-butan-1-ol (R)-O-acetylmandelic acid salt (11.61 kg, 30.76 mol) was charged to a stirred solution of aqueous sodium hydroxide (11.30 kg of 50% sodium hydroxide in water, 141.3 mol, diluted to approximately 70 L) at 16° C. inner temperature under nitrogen atmosphere. THF (7.5 L) and toluene (74 L) was charged resulting in a clear two-phase system. The solution was cooled to −1° C. and ethyl chloroformate (3.60 kg, 33.2 mol) in a mixture of THF (1.1 L) and toluene (10 L) was charged to the mixture over 18 min. During the addition the inner temperature rose to 9° C. The reaction mixture was heated to 18° C. over 1 h and 48 min at which point HPLC analysis indicated that the reaction was complete. Toluene (17.5 L) was charged and good mixing was achieved followed by phase separation. The resulting two phases were separated and the aqueous phase was discarded. The organic phase was washed with water (3×8 L) and concentrated to approximately 50 L by distillation at reduced pressure. Toluene (25 L) was charged and the distillation was continued until approximately 30 L of the solvents had been distilled off. Toluene (25 L) was charged and the distillation continued until approximately 40 L remained in. The toluene solution containing ethyl [(2S)-2-(4-fluorophenyl)-4-hydroxybutyl]carbamate was taken straight into the next step.
Lithium aluminium hydride (2.11 kg, 55.6 mol) was charged to a reactor containing THF (50 L) at an inner temperature of 20° C. under a nitrogen atmosphere, while stirring. The mixture was heated to an inner temperature of 51° C. and ethyl [(2S)-2-(4-fluorophenyl)-4-hydroxybutyl]carbamate in toluene (total volume 43 L) from the previous step was charged to the lithium aluminium hydride slurry in THF over 2 h. The temperature was kept between 51-68° C. during the addition. The charging vessel was rinsed with toluene (5 L) and the batch was then held at 56-58° C. for 2 h. The reaction mixture was cooled to an inner temperature of 2° C. and a solution of aqueous sodium bicarbonate (26 L) was charged over 44 min (inner temperature 15° C. and jacket temperature −25° C. at the end of the quench) after which the jacket was adjusted to 20° C. and the batch was left for 15 h. The slurry in the reactor was filtered and the resulting solid was washed with toluene (30 L) in four portions. The filtrate was returned to the reactor (cleaned from aluminium salts) and washed with water (2×10 L) and then clear filtered. The clear filtered solution was returned to the reactor and concentrated to approximately 15 L by distillation under reduced pressure. The distillation was stopped and isooctane (30 L) was charged to the slurry. The slurry was cooled from an inner temperature of 32° C. to 20° C. over 40 min, then filtered and the isolated solid was washed with isooctane (30 L) in four portions. The solid was dried and this resulted in 4.54 kg (75% over two Steps) of (3S)-3-(4-fluorophenyl)-4-(methylamino)butan-1-ol. 1H NMR (DMSO-d6) δ 7.22 (app d, J=8 Hz, 1 H), 7.20 (app d, J=8 Hz, 1 H), 7.08 (app t, J=8 Hz, 2H), 3.11-3.34 (m, 2 H), 3.72-3.88 (m, 1 H), 3.52-3.66 (m, 2 H), 2.21 (s, 3 H), 1.73-1.91 (m, 1 H), 1.48-1.68 (m, 1 H); 13C NMR δ 160.6 (d, JC,F=241 Hz), 140.7 (d, JC,F=3 Hz), 129.3 (d, JC,F=8 Hz), 114.8 (d, JC,F=21 Hz), 58.9, 57.8, 41.3, 37.4, 36.1. [α]D (c 1.0 in methanol, 25° C.) +8.8°.
(3S)-3-(4-Fluorophenyl)-4-(methylamino)butan-1-ol (4.0 g, 20.5 mmol) was mixed with an aqueous solution of NaOH (3.3 g in 16 mL of water, 41 mmol). To the formed suspension was added by drops a toluene solution of 3-chloro-5-(trifluoromethyl)benzoyl chloride (5.0 g in 24 mL of toluene, 20.5 mmol) while vigorously stirring. The addition was completed after 15 min. The mixture was stirred for 1 h at RT. The aqueous phase was separated off and the organic solution was washed twice with water. The solvent was dried and then removed by evaporation. There was obtained 8.6 g (100%) of 3-chloro-N-[(2S)-2-(4-fluorophenyl)-4-hydroxybutyl]-N-methyl-5-(trifluoromethyl)benzamide as a viscous oil. 1H NMR (500 MHz, CDCl3): 1.6-2.0 (cm, 2H), 2.7 (s, 3H), 3.0-3.8 (cm, 5H), 6.8-7.3 (cm, 6H), 7.6 (s, 1H); LCMS: m/z 404 (M+1)+.
3-Chloro-N-[(2S)-2-(4-fluorophenyl)-4-hydroxybutyl]-N-methyl-5-(trifluoromethyl)benzamide (8.5 g, 21.0 mmol) was dissolved in DMSO (30 mL) together with triethylamine (8.5 g, 84.2 mmol). Sulfur trioxide pyridine complex (7.4 g, 46.3 mmol) dissolved in DMSO (30 mL) was added by drops over a period of 20 min. The mixture was stirred at RT for 3 h and then another portion of sulfur trioxide pyridine complex (3.5 g, 21.0 mmol) was added. The mixture was stirred at RT overnight and then concentrated on a rotavapor for 2 h in order to remove formed dimethylsulfide. The mixture was diluted with MTBE (50 mL) and then sulfuric acid (2.0 g, 21.0 mmol) dissolved in water (40 mL) was added by drops. The mixture was stirred vigorously for 25 min, the two phases were separated and then the organic solution was washed twice with water. The solution was dried and the solvent was removed by evaporation. The product was purified by chromatography on silica gel (ethyl acetate-heptane, 10% to 100% ethyl acetate). There was obtained 2.6 g (30%) of the title compound as an oil. 1H NMR (500 MHz, CDCl3): δ 2.6-3.9 (cm, 8H), 6.8-7.4 (cm, 6H), 7.6 (d, 1H), 9.6-9.8 (d, 1H); LCMS: m/z 400 (M−1)+.
Method 2
A mixture of 1-(diphenylmethyl)azetidin-3-yl methanesulfonate (see J. Org. Chem.; 56; 1991; 6729; 25 g, 78.6 mmol), piperazine (67.7 g, 0.79 mol) and dry acetonitrile was stirred at 60° C. overnight under nitrogen. The mixture was cooled and partitioned between water and methylene chloride. The organic layer was washed with water and brine. The solution was dried over Na2SO4 and then the solvent was removed by evaporation. The residue was purified by column chromatography on silica gel (methanol-methylene chloride 5:95). There was obtained 17.5 g (72%) of 1-[1-(diphenylmethyl)azetidin-3-yl]piperazine as a yellow oil. 1H NMR (400 MHz, CDCl3): 2.1-2.4 (m, 4H), 2.8-2.9 (m, 2H), 3.0 (m, 4H), 3.4-3.5 (m, 2H), 3.7-3.9 (m, 1H), 4.4 (s, 1H), 7.2-7.4 (m, 10H); LCMS: m/z 308 (M+1)+.
To a mixture of 1-[1-(diphenylmethyl)azetidin-3-yl]piperazine (15.0 g, 48.8 mmol), triethylamine (13.5 mL, 97.6 mmol) and methylene chloride (150 mL) was added 2-methylpropanoyl chloride (6.2 g, 58.5 mmol) dissolved in methylene chloride (50 mL). The addition was performed while stirring and was completed within 15 min. The mixture was stirred at RT for 15 min and then the solution was washed with aqueous NaHCO3. The organic phase was separated, dried over Na2SO4 and then the solvent was removed by evaporation. There was obtained 18.0 g (97%) of 1-[1-(diphenylmethyl)azetidin-3-yl]-4-isobutyrylpiperazine as a yellow semi-solid material. 1H NMR (500 MHz, CDCl3): 1.1 (d, 6H), 2.2-2.3 (m, 4H), 2.7-2.8 (qn, 1H), 2.9 (t, 2H), 3.0 (qn, 1H), 3.4 (t, 2H), 3.5 (b, 2H), 3.6 (b, 2H), 4.4 (s, 1H), 7.2 (t, 2H), 7.3 (t, 4H), 7.4 (d, 4H); LCMS: m/z 377 (M+1)+.
1-[1-(Diphenylmethyl)azetidin-3-yl]-4-isobutyrylpiperazine (17.9 g, 47.4 mmol) was dissolved in methylene chloride (100 mL) and to the resultant solution, which was cooled to 0° C., was added 1-chloroethyl chloroformate (10.2 mL, 94.8 mmol) by drops. The addition was performed while stirring and was completed within 10 min. The mixture was stirred at RT for 1.5 h. Methanol (35 mL) was added and the mixture was heated to reflux for 30 min. The solvent was removed by evaporation and the residue was left in a freezer for three days. 2-Propanol (150 mL) was added and the mixture was heated to reflux while stirring, cooled to RT and then filtered. The filter cake was washed twice with 2-propanol and then dried under reduced pressure. There was obtained 12.2 g (100%) of the title compound as an off white powder. 1H NMR (500 MHz, CD3OD): 1.1 (d, 6H), 2.9-3.0 (m, 1H), 3.2-3.4 (b, 4H), 3.8-4.1 (b, 4H), 4.3-4.4 (m, 3H), 4.7-4.8 (m, 2H).
Method 3
(8aR)-Hexahydropyrrolo[1,2-a]pyrazin-6(2H)-one (see WO 03/066635; 0.17 g, 1.2 mmol), 1-(diphenylmethyl)azetidin-3-yl methanesulfonate (see J. Org. Chem.; 56; 1991; 6729; 0.40 g, 1.3 mmol) and triethylamine (0.20 mL, 1.4 mmol) were dissolved in acetonitrile. The mixture was heated for 15 min at 150° C. using microwave single node heating and then the solvent was removed by evaporation. The residue was partitioned between ethyl acetate and aqueous NaHCO3 and the aqueous phase extracted further with ethyl acetate. The organic phase was dried and then the solvent was removed by evaporation. The product was purified by chromatography on silica gel (methanol-methylene chloride 5:95). There was obtained 0.23 g (54%) of (8aR)-2-[1-(diphenylmethyl)azetidin-3-yl]hexahydropyrrolo[1,2-a]pyrazin-6(2H)-one as a pale yellow oil. 1H NMR (500 MHz, CDCl3): 1.5-1.6 (m, 2H), 1.7-1.8 (m, 1H), 2.1-2.2 (m, 1H), 2.3-2.4 (m, 2H), 2.6-2.7 (d, 1H), 2.8 (m, 1H), 2.8-2.9 (m, 3H), 3.0 (qn, 1H), 3.4 (t, 2H), 3.6 (m, 1H), 4.0 (d, 1H), 4.4 (s, 1H), 7.2 (m, 2H), 7.2-7.3 (m, 4H), 7.4 (m, 4H); LCMS: m/z 362 (M+1)+.
(8aR)-2-[1-(diphenylmethyl)azetidin-3-yl]hexahydropyrrolo[1,2-a]pyrazin-6(2H)-one (0.23 g, 0.64 mmol) was dissolved in acetic acid (20 mL) and to the resultant solution was added palladium hydroxide on carbon (0.33 g). The mixture was stirred under hydrogen (5 bar) at RT for 48 h and then the catalyst was filtered off by means of Celite®. The solvent was removed by evaporation and the residue was dissolved in ethanol. The solution was filtered through a cation exchange column (Isolute SCX-2, 10 g). The column was washed with ethanol and then the product was eluted with ammonia-saturated methanol. The solvent was removed by evaporation and there was obtained 0.10 g (84%) of the title compound. 1H NMR (500 MHz, CDCl3): 1.5-1.6 (m, 2H), 1.8 (m, 1H), 2.1-2.2 (m, 1H), 2.3-2.4 (m, 2H), 2.7 (d, 1H), 2.8-2.9 (m, 2H), 3.2 (qn, 1H), 3.5-3.7 (m, 4H), 4.0 (dd, 1H); LCMS: m/z 196 (M+1)+.
Method 4
The title compound was prepared by utilizing the N-alkylation reaction protocol described in Method 1a but using (8aS)-hexahydropyrrolo[1,2-a]pyrazin-6(2H)-one (see WO 03/066635) as the amine (yield, 56%). 1H NMR (500 MHz, CDCl3): 1.5-1.6 (qn, 2H), 1.7-1.8 (m, 2H), 2.1-2.2 (m, 1H), 2.3-2.4 (m, 2H), 2.6-2.7 (d, 1H), 2.8 (d, 1H), 2.8-2.9 (m, 2H), 3.0 (qn, 1H), 3.4 (t, 2H), 3.6 (m, 1H), 4.0 (d, 1H), 4.4 (s, 1H), 7.1-7.2 (t, 2H), 7.2-7.3 (t, 4H), 7.4 (t, 4H); LCMS: m/z 362 (M+1)+.
The title compound was prepared by utilizing the hydrogenation reaction protocol described in Method 1b but using (8aS)-2-[1-(diphenylmethyl)azetidin-3-yl]hexahydropyrrolo[1,2-a]pyrazin-6(2H)-one as the substrate (yield, 73%). 1H NMR (500 MHz, CDCl3): 1.5-1.6 (m, 2H), 1.8 (m, 1H), 2.1-2.2 (m, 1H), 2.3-2.4 (m, 2H), 2.6-2.8 (d, 1H), 2.8-3.0 (m, 2H), 3.2-3.4 (m, 2H), 3.5-3.7 (m, 4H), 4.0 (dd, 1H); LCMS: m/z 196 (M+1)+.
Pharmacology
Transfection and Culturing of Cells Used in FLIPR and Binding Assays
Chinese Hamster Ovary (CHO) K1 cells (obtained from ATCC) were stably transfected with the human NK2 receptor (hNK2R cDNA in pRc/CMV, Invitrogen) or the human NK3 receptor (hNK3R in pcDNA 3.1/Hygro (+)/IRES/CD8, Invitrogen vector modified at AstraZeneca EST-Bio UK, Alderley Park). The cells were transfected with the cationic lipid reagent LIPOFECTAMINE™ (Invitrogen) and selection was performed with Geneticin (G418, Invitrogen) at 1 mg/ml for the hNK2R transfected cells and with Hygromycin (Invitrogen) at 500 μg/ml for the hNK3R transfected cells. Single cell clones were collected by aid of Fluorescence Activated Cell Sorter (FACS), tested for functionality in a FLIPR assay (see below), expanded in culture and cryopreserved for future use. CHO cells stably transfected with human NK, receptors originates from AstraZeneca R&D, Wilmington USA. Human NK1 receptor cDNA (obtained from RNA-PCR from lung tissue) was subcloned into pRcCMV (Invitrogen). Transfection was performed by Calcium Phosphate and selection with 1 mg/ml G418.
The CHO cells stably transfected with hNK1R, hNK2R and hNK3R were cultured in a humidified incubator under 5% CO2, in Nut Mix F12 (HAM) with Glutamax I, 10% Foetal Bovine Serum (FBS), 1% Penicillin/Streptomycin (PEST) supplemented with 200 μg/ml Geneticin for the hNK1R and hNK2R expressing cells and 500 μg/ml Hygromycin for the hNK3R expressing cells. The cells were grown in T175 flasks and routinely passaged when 70-80% confluent for up to 20-25 passages.
Assessing the Activity of Selected Test Compounds to Inhibit Human NK1/NK2/NK3 Receptor Activation (FLIPR Assay)
The activity of a compound of the invention to inhibit NK1/NK2/NK3 receptor activation measured as NK1/NK2/NK3 receptor mediated increase in intracellular Ca2+ was assessed by the following procedure:
CHO cells stably transfected with human NK1, NK2 or NK3 receptors were plated in black walled/clear bottomed 96-well plates (Costar 3904) at 3.5×104 cells per well and grown for approximately 24 h in normal growth media in a 37° C. CO2-incubator. Before the FLIPR assay the cells of each 96-well plate were loaded with the Ca2+ sensitive dye Fluo-3 (TEFLABS 0116) at 4 μM in a loading media consisting of Nut Mix F12 (HAM) with Glutamax I, 22 mM HEPES, 2.5 mM Probenicid (Sigma P-8761) and 0.04% Pluronic F-127 (Sigma P-2443) for 1 h kept dark in a 37° C. CO2-incubator. The cells were then washed three times in assay buffer (Hanks balanced salt solution (HBSS) containing 20 mM HEPES, 2.5 mM Probenicid and 0.1% BSA) using a multi-channel pipette leaving them in 150 μl at the end of the last wash. Serial dilutions of a test compound in assay buffer (final DMSO concentration kept below 1%) were automatically pipetted by FLIPR (Fluorometric Imaging Plate Reader) into each test well and the fluorescence intensity was recorded (excitation 488 nm and emission 530 nm) by the FLIPR CCD camera for a 2 min pre-incubation period. 50 μl of the Substance P (NK1 specific), NKA (NK2 specific), or Pro-7-NKB (NK3 specific) agonist solution (final concentration equivalent to an approximate EC60 concentration) was then added by FLIPR into each well already containing 200 μl assay buffer (containing the test compound or vehicle) and the fluorescence was continuously monitored for another 2 min. The response was measured as the peak relative fluorescence after agonist addition and IC50s were calculated from ten-point concentration-response curves for each compound. The IC50s were then converted to pKB values with the following formula:
K
B
=IC
50/1+(EC60 conc. of agonist used in assay/EC50 agonist)
pKB=−log KB
Determining the Dissociation Constant (Ki) of Compounds for Human NK1/NK2/NK3 Receptors (Binding Assay)
Membranes were prepared from CHO cells stably transfected with human NK1, NK2 or NK3 receptors according to the following method.
Cells were detached with Accutase® solution, harvested in PBS containing 5% FBS by centrifugation, washed twice in PBS and resuspended to a concentration of 1×108 cells/ml in Tris-HCl 50 mM, KCl 300 mM, EDTA-N2 10 mM pH 7.4 (4° C.). Cell suspensions were homogenized with an UltraTurrax 30 s 12.000 rpm. The homogenates were centrifuged at 38.000×g (4° C.) and the pellet resuspended in Tris-HCl 50 mM pH 7.4. The homogenization was repeated once and the homogenates were incubated on ice for 45 min. The homogenates were again centrifuged as described above and resuspended in Tris-HCl 50 mM pH 7.4. This centrifugation step was repeated 3 times in total. After the last centrifugation step the pellet was resuspended in Tris-HCl 50 mM and homogenized with Dual Potter, 10 strokes to a homogenous solution, an aliquot was removed for protein determination. Membranes were aliquoted and frozen at −80° C. until use. The radioligand binding assay is performed at room temperature in 96-well microtiter plates (No-binding Surface Plates, Corning 3600) with a final assay volume of 200 μl/well in incubation buffer (50 mM Tris buffer (pH 7.4 RT) containing 0.1% BSA, 40 mg/L Bacitracin, complete EDTA-free protease inhibitor cocktail tablets 20 pills/L (Roche) and 3 mM MnCl2). Competition binding curves were done by adding increasing amounts of the test compound. Test compounds were dissolved and serially diluted in DMSO, final DMSO concentration 1.5% in the assay. 50 μl Non labelled ZD 6021 (a non selective NK-antagonist, 10 μM final conc) was added for measurement of non-specific binding. For total binding, 50 μl of 1.5% DMSO (final conc) in incubation buffer was used.[3H-Sar,Met(O2)-Substance P] (4 nM final conc) was used in binding experiments on hNK1r. [3H-SR48968] (3 nM final conc.) for hNK2r and [3H-SR142801] (3 nM final conc) for binding experiments on hNK3r. 50 μl radioligand, 3 μl test compound diluted in DMSO and 47 μl incubation buffer were mixed with 5-10 μg cell membranes in 100 μl incubation buffer and incubated for 30 min at room temperature on a microplate shaker.
The membranes were then collected by rapid filtration on Filtermat B(Wallac), presoaked in 0.1% BSA and 0.3% Polyethyleneimine (Sigma P-3143), using a Micro 96 Harvester (Skatron Instruments, Norway). Filters were washed by the harvester with ice-cold wash buffer (50 mM Tris-HCl, pH 7.4 at 4° C., containing 3 mM MnCl2) and dried at 50° C. for 30-60 min. Meltilex scintillator sheets were melted on to filters using a Microsealer (Wallac, Finland) and the filters were counted in a β-Liquid Scintillation Counter (1450 Microbeta, Wallac, Finland).
The Ki value for the unlabeled ligand was calculated using the Cheng-Prusoff equation (Biochem. Pharmacol. 22:3099-3108, 1973): where L is the concentration of the radioactive ligand used and Kd is the affinity of the radioactive ligand for the receptor, determined by saturation binding.
Data was fitted to a four-parameter equation using Excel Fit.
K
i
=IC
50/(1+(L/Kd))
Results
In general, the compounds of the invention, which were tested, demonstrated statistically significant antagonistic activity at the NK1 receptor within the range of 8-9 for the pKB. For the NK2 receptor the range for the pKB was 7-9. In general, the antagonistic activity at the NK3 receptor was 7-9 for the pKB.
In general, the compounds of the invention, which were tested, demonstrated statistically significant CYP3A4 inhibition at a low level. The IC50 values tested according to Bapiro et al; Drug Metab. Dispos. 29, 30-35 (2001) were generally greater than 15 μM.
Activity Against hERG
The activity of compounds according to formula I against the hERG-encoded potassium channel can be determined according to Kiss L, et al. Assay Drug Dev Technol. 1 (2003), 127-35: “High throughput ion-channel pharmacology: planar-array-based voltage clamp”.
In general, the compounds of the invention, which were tested, demonstrated statistically significant hERG activity at a low level. The IC50 values tested as described above were generally greater than 6 μM.
Metabolic Stability
The metabolic stability of compounds according to formula I can be determined as described below:
The rate of biotransformation can be measured as either metabolite(s) formation or the rate of disappearance of the parent compound. The experimental design involves incubation of low concentrations of substrate (usually 1.0 μM) with liver microsomes (usually 0.5 mg/ml) and taking out aliquotes at varying time points (usually 0, 5, 10, 15, 20, 30, 40 min.). The test compound is usually dissolved in DMSO. The DMSO concentration in the incubation mixture is usually 0.1% or less since more solvent can drastically reduce the activities of some CYP450s. Incubations are done in 100 mM potassium phosphate buffer, pH 7.4 and at 37° C. Acetonitrile or methanol is used to stop the reaction. The parent compound is analysed by HPLC-MS. From the calculated half-life, t1/2, the intrinsic clearance, Clint, is estimated by taking microsomal protein concentration and liver weight into account.
In general, the compounds of the invention had in vitro metabolic stability at a high level. Intrinsic clearance values tested as above were generally lower than 40 μl/min/mg protein.
The following table illustrates the properties of the compounds of the present invention:
Biological Evaluation
Gerbil Foot Tap (NK1 Specific Test Model)
Male Mongolian gerbils (60-80 g) are purchased from Charles River, Germany. On arrival, they are housed in groups of ten, with food and water ad libitum in temperature and humidity-controlled holding rooms. The animals are allowed at least 7 days to acclimatize to the housing conditions before experiments. Each animal is used only once and euthanized immediately after the experiment by heart punctuation or a lethal overdose of penthobarbital sodium.
Gerbils are anaesthetized with isoflurane. Potential CNS-permeable NK1 receptor antagonists are administered intraperitoneally, intravenously or subcutaneously. The compounds are given at various time points (typically 30-120 minutes) prior to stimulation with agonist.
The gerbils are lightly anaesthetized using isofluorane and a small incision is made in the skin over bregma. 10 pmol of ASMSP, a selective NK1 receptor agonist, is administered icv in a volume of 5 μl using a Hamilton syringe with a needle 4 mm long. The wound is clamped shut and the animal is placed in a small plastic cage and allowed to wake up. The cage is placed on a piece of plastic tubing filled with water and connected to a computer via a pressure transducer. The number of hind feet taps is recorded.
Fecal Pellet Output (NK2 Specific Test Model)
The in vivo effect (NK2) of the compounds of formula I can be determined by measuring NK2 receptor agonist-induced fecal pellet output using gerbil as described in e.g. The Journal of Pharmacology and Experimental Therapeutics (2001), pp. 559-564.
Colorectal Distension Model
Colorectal distension (CRD) in gerbils is performed as previously described in rats and mice (Tammpere A, Brusberg M, Axenborg J, Hirsch I, Larsson H, Lindström E. Evaluation of pseudo-affective responses to noxious colorectal distension in rats by manometric recordings. Pain 2005; 116: 220-226; Arvidsson S, Larsson M, Larsson H, Lindström E, Martinez V. Assessment of visceral pain-related pseudo-affective responses to colorectal distension in mice by intracolonic manometric recordings. J Pain 2006; 7: 108-118) with slight modifications. Briefly, gerbils are habituated to Bollmann cages 30-60 min per day for three consecutive days prior to experiments to reduce motion artefacts due to restraint stress. A 2 cm polyethylene balloon (made in-house) with connecting catheter is inserted in the distal colon, 2 cm from the base of the balloon to the anus, during light isoflurane anaesthesia (Forene®, Abbott Scandinavia AB, Solna, Sweden). The catheter is fixed to the tail with tape. The balloons are connected to pressure transducers (P-602, CFM-k33, 100 mmHg, Bronkhorst HI-TEC, Veenendal, The Netherlands). Gerbils are allowed to recover from sedation in the Bollmann cages for at least 15 min before the start of experiments.
A customized barostat (AstraZeneca, Mölndal, Sweden) is used to manage air inflation and balloon pressure control. A customized computer software (PharmLab on-line 4.0) running on a standard computer is used to control the barostat and to perform data collection. The distension paradigm used consists of 12 repeated phasic distensions at 80 mmHg, with a pulse duration of 30 sec at 5 min intervals. Compounds or their respective vehicle are administered as intraperitoneal (i.p.) injections before the CRD paradigm. Each gerbil receives both vehicle and compound on different occasions with at least two days between experiments. Hence, each gerbil serves as its own vehicle control.
The analog input channels are sampled with individual sampling rates, and digital filtering is performed on the signals. The balloon pressure signals are sampled at 50 samples/s. A highpass filter at 1 Hz is used to separate the contraction-induced pressure changes from the slow varying pressure generated by the barostat. A resistance in the airflow between the pressure generator and the pressure transducer further enhances the pressure variations induced by abdominal contractions of the animal. A customized computer software (PharmLab off-line 4.0) is used to quantify the magnitude of highpass-filtered balloon pressure signals. The average rectified value (ARV) of the highpass-filtered balloon pressure signals is calculated for 30 s before the pulse (i.e baseline reponse) and for the duration of the pulse. When calculating the magnitude of the highpass-filtered balloon pressure signals, the first and last seconds of each pulse are excluded since these reflect artifact signals produced by the barostat during inflation and deflation and do not originate from the animal.
This application claims the priority benefit of U.S. Provisional Application No. 60/801,576, filed May 18, 2006.
| Number | Date | Country | |
|---|---|---|---|
| 60801576 | May 2006 | US |
