MUSCARINIC AGONISTS

Abstract

This invention relates to compounds that are agonists of the muscarinic M.sub.1 receptor and/or M.sub.4 receptor and which are useful in the treatment of muscarinic M.sub.1/M.sub.4 receptor mediated diseases. Also provided are pharmaceutical compositions containing the compounds and the therapeutic uses of the compounds. Compounds include those according to formula 1

##STR00001##

or a salt thereof, wherein q, r, s, Q, R.sup.1, R.sup.2′, R.sup.2″, R.sup.3 and R.sup.4 are as defined herein.

Claims

1. A method for the treatment of a cognitive disorder or psychotic disorder in a subject or for the treatment or lessening the severity of acute, chronic, neuropathic, or inflammatory pain in a subject or for the treatment of addiction in a subject, or for the treatment of movement disorders in a subject, comprising administering to the subject an effective amount of a compound of the formula (1): ##STR00029## or a salt thereof, wherein q is 1; r is 1 or 2; s is 0 or 1 where the total of r and s is 1 or 2; Q is an azetidine linked to the adjacent six-membered ring by the nitrogen atom of the azetidine ring; R.sup.1 is selected from hydrogen; methyl; ethyl; fluorine oxo; and COOMe; R.sup.2′ and R.sup.2″ are independently selected from hydrogen; fluorine; and a C.sub.1-6 non-aromatic hydrocarbon group; R.sup.3 is hydrogen; R.sup.4 is hydrogen or a C.sub.1-6 non-aromatic hydrocarbon group which is optionally substituted with one to six fluorine atoms and the dotted line indicates a single bond.

2. The method according to claim 1, wherein R.sup.4 is selected from hydrogen and methyl.

3. The method according to claim 1, wherein the compound is selected from the group consisting of: Ethyl 2-[4-(3,3-difluoroazetidin-1-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate; Ethyl 2-{4-[(2R)-2-(methoxycarbonyl)azetidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate; Ethyl 2-[4-(2-oxoazetidin-1-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate; Ethyl 2-{4-[(2S)-2-methyl-4-oxoazetidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate; Ethyl 2-{4-[(2S)-2-ethyl-4-oxoazetidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate; Ethyl 2-{4-[(2R)-2-methyl-4-oxoazetidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate; Ethyl 2-{4-[(2R)-2-ethyl-4-oxoazetidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate; or a salt thereof.

4. The method according to claim 1, wherein the compound is a pharmaceutically acceptable salt of a compound as defined in claim 1.

5. The method according to claim 1, wherein the compound is administered to the subject as in the form of a pharmaceutical composition comprising the compound as defined in claim 1 and a pharmaceutically acceptable excipient.

6. The method according to claim 1 wherein the moiety: ##STR00030## is selected from: ##STR00031##

7. The method according to claim 1 wherein the moiety: ##STR00032## is selected from: ##STR00033##

8. The method according to claim 1, wherein the disorder is Alzheimer's Disease, dementia with Lewy bodies or schizophrenia.

9. The method according to claim 1, wherein the disorder is Alzheimer's Disease.

10. The method according to claim 1, wherein the disorder is dementia with Lewy bodies.

11. The method according to claim 1, wherein the disorder is schizophrenia.

Description

EXAMPLES

[0270] The invention will now be illustrated, but not limited, by reference to the specific embodiments described in the following examples.

Examples 1-1 to 1-9

[0271] The compounds of Examples 1-1 to 1-9 shown in Table 1 below have been prepared. Their NMR and LCMS properties and the methods used to prepare them are set out in Table 3.

TABLE-US-00001 TABLE 1 [00015] Example 1-1 [00016] Example 1-2 [00017] Example 1-3 [00018] Example 1-4 [00019] Example 1-5 [00020] Example 1-6 [00021] Example 1-7

General Procedures

[0272] Where no preparative routes are included, the relevant intermediate is commercially available. Commercial reagents were utilized without further purification. Room temperature (rt) refers to approximately 20-27° C. .sup.1H NMR spectra were recorded at 400 MHz on either a Bruker or Jeol instrument. Chemical shift values are expressed in parts per million (ppm), i.e. (δ:)-values. The following abbreviations are used for the multiplicity of the NMR signals: s=singlet, br=broad, d=doublet, t=triplet, q=quartet, quint=quintet, td=triplet of doublets, tt=triplet of triplets, qd=quartet of doublets, ddd=doublet of doublet of doublets, ddt=doublet of doublet of triplets, m=multiplet. Coupling constants are listed as J values, measured in Hz. NMR and mass spectroscopy results were corrected to account for background peaks. Chromatography refers to column chromatography performed using 60-120 mesh silica gel and executed under nitrogen pressure (flash chromatography) conditions. TLC for monitoring reactions refers to TLC run using the specified mobile phase and Silica gel F254 (Merck) as a stationary phase. Microwave-mediated reactions were performed in Biotage Initiator or CEM Discover microwave reactors.

[0273] LCMS experiments were typically carried out using electrospray conditions as specified for each compound under the following conditions:

LCMS Method A

[0274] Instruments: Waters 2695, Photo Diode Array, ZQ-2000 Detector; Column: X-Bridge C18, 3.5 micron, 150×4.6 mm; Gradient [time (min)/solvent B in A (%)]: 0.00/5, 5.00/90, 5.80/95, 10/95; Solvents: solvent A=0.1% ammonia in H.sub.2O; solvent B=0.1% ammonia in MeCN; Injection volume 10 μL; UV detection 200 to 400 nM; Mass detection 60 to 1000 AMU (+ve electrospray); column at ambient temperature; Flow rate 1.0 mL/min.

LCMS Method B

[0275] Instruments: Waters 2695, Photo Diode Array, ZQ-2000 Detector; Column: X-Bridge C18, 3.5 micron, 50×4.6mm; Gradient [time (min)/solvent B in A (%)]: 0.01/0, 0.20/0, 5.00/90, 5.80/95, 7.20/95, 7.21/100, 10.00/100; Solvents: solvent A=0.1% ammonia in H.sub.2O; solvent B=0.1% ammonia in MeCN; Injection volume 10 μL; UV detection 200 to 400 nM; Mass detection 60 to 1000 AMU (+ve electrospray); column at ambient temperature; Flow rate 1.0 mL/min.

LCMS Method C

[0276] Instruments: Agilent 1260 Infinity LC with Diode Array Detector, Agilent 6120B Single Quadrupole MS with API-ES Source; Column: Phenomenex Gemini-NX C-18, 3 micron, 2.0×30 mm; Gradient [time (min)/solvent B in A (%)]: Method: 0.00/5, 2.00/95, 2.50/95, 2.60/5, 3.00/5; Solvents: solvent A=2.5 L H.sub.2O+2.5 mL of (28% NH.sub.3 in H.sub.2O); solvent B=2.5 L MeCN+129 mL H.sub.2O+2.7 mL of (28% NH.sub.3 in H.sub.2O); Injection volume 0.5 μL; UV detection 190 to 400 nM; column temperature 40° C.; Flow rate 1.5 mL/min.

LCMS Methods D and E

[0277] Instruments: HP 1100 with G1315A DAD, Micromass ZQ; Column: Waters X-Bridge C-18, 2.5 micron, 2.1×20 mm or Phenomenex Gemini-NX C-18, 3 micron, 2.0×30 mm; Gradient [time (min)/solvent D in C (%)]: Method D: 0.00/2, 0.10/2, 2.50/95, 3.50/95, 3.55/2, 4.00/2 or Method E: 0.00/2, 0.10/2, 8.40/95, 9.40/95, 9.50/2, 10.00/2; Solvents: solvent C=2.5 L H.sub.2O+2.5 mL 28% ammonia in H.sub.2O solution; solvent D=2.5 L MeCN+135 mL H.sub.2O+2.5 mL 28% ammonia in H.sub.2O solution); Injection volume 1 μL; UV detection 230 to 400 nM; Mass detection 130 to 800 AMU (+ve and −ve electrospray); column temperature 45° C.; Flow rate 1.5 mL/min.

[0278] LCMS data in the experimental section are given in the format: Mass ion, retention time, UV activity.

Abbreviations

[0279] AcOH=acetic acid [0280] CDI=1,1′-Carbonyldiimidazole [0281] d=day(s) [0282] DAST=diethylaminosulfur trifluoride [0283] DCE=dichloroethane [0284] DCM=dichloromethane [0285] DIPEA=diisopropylethylamine [0286] DIAD=diisopropyl azodicarboxylate [0287] DMF=dimethylformamide [0288] DMP=Dess-Martin periodinane [0289] DMSO=dimethylsulfoxide [0290] ES=electro spray ionisation [0291] EtOAc=ethyl acetate [0292] h=hour(s) [0293] HATU=1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate [0294] HPLC=high performance liquid chromatography [0295] LC=liquid chromatography [0296] LiAlH.sub.4/LAH=Lithium aluminium hydride [0297] MeCN=acetonitrile [0298] MeOH=methanol [0299] min=minute(s) [0300] MS=mass spectrometry [0301] Et.sub.3N=triethylamine [0302] NMR=nuclear magnetic resonance [0303] rt=room temperature [0304] sat.=saturated [0305] sol.=solution [0306] STAB=sodium triacetoxyborohydride [0307] THF=tetrahydrofuran [0308] TLC=thin layer chromatography [0309] Prefixes n-, s-, i-, t- and tert- have their usual meanings: normal, secondary, iso, and tertiary.

General Synthetic Procedures for Intermediates

Route 1

Procedure for the Preparation of Intermediate 2, ethyl 2-oxo-6-azaspiro[3.4]octane-6-carboxylate

[0310] ##STR00022##

[0311] 6-Boc-2-oxo-6-azaspiro[3.4]octane (3.37 g, 15 mmol) was added portionwise to hydrogen chloride (4 M dioxane solution, 50 mL, 210 mmol). Caution: effervescence. After 24 h, the reaction was concentrated in vacuo and the residual solid was dissolved in a mixture of Et.sub.3N (4.18 ml, 30 mmol) and DCM (66 mL). On completion of dissolution, the solution was immediately cooled to 0° C., then ethyl chloroformate (1.57 mL, 16.5 mmol) was added dropwise. After 18 h, the mixture was poured into dichloromethane (100 mL) and NaHCO.sub.3 (aq) (100 mL) and extracted (2×100 mL). The organic layers were collected, washed with brine (20 mL), dried over MgSO.sub.4, then the residue after evaporation was purified by column chromatography (normal phase, [Biotage SNAP cartridge KP-sil 100 g, 40-63 μm, 60 Å, 50 mL per min, gradient 0% to 4% MeOH in DCM]) to give Intermediate 2, ethyl 2-oxo-6-azaspiro[3.4]octane-6-carboxylate (2.47 g, 83%) as a colourless oil. The data for the title compound are in Table 2.

Route 2

Procedure for the Preparation of Intermediate 8, (4S)-4-ethyl-1-(piperidin-4-yl)azetidin-2-one

[0312] ##STR00023##

[0313] To a solution of MeOH (60 mL) at −10° C. was added thionyl chloride (1.25 mL, 17.1 mmol) dropwise, and the resulting mixture stirred at −10° C. for 1 h. (3S)-3-Aminopentanoic acid (1.0 g, 8.5 mmol) was then added in one portion and the resulting mixture stirred at rt for 20 h. The solvent was removed in vacuo to yield methyl (3S)-3-aminopentanoate.HCl (1.69 g) as a light brown oil, which was used crude in the next step.

[0314] LC/MS (method C): m/z 132 (M+H)+(ES+), at 0.56 min, UV inactive.

[0315] To methyl (3S)-3-aminopentanoate.HCl (250 mg, assumed 1.3 mmol) in DMF (15 mL) was added DIPEA (1.30 mL, 7.5 mmol), AcOH (0.13 mL, 2.3 mmol), tert-butyl 4-oxopiperidine-1-carboxylate (297 mg, 1.5 mmol) and sodium triacetoxyborohydride (632 mg, 3.0 mmol) and the resulting mixture stirred at rt for 24 h. The solvent was then removed in vacuo and the residue purified on silica (Biotage Isolera, SNAP 25 g cartridge, 0-10% 0.7 M NH.sub.3 in MeOH/DCM) and relevant fractions combined to yield tert-butyl 4-{[(3S)-1-methoxy-1-oxopentan-3-yl]amino}piperidine-1-carboxylate (1.14 g) as a yellow oil, which crystallized upon standing at rt to a white solid, and was used crude in the next step.

[0316] LC/MS (method C): m/z 315 (M+H)+(ES+), at 1.40 min, UV active.

[0317] ME1 33921713v.1

[0318] To a solution of crude tert-butyl 4-{[(3S)-1-methoxy-1-oxopentan-3-yl]amino}piperidine-1-carboxylate (1.14 g, assumed 1.3 mmol) in THF (30 mL) was added LiOH (313 mg, 7.5 mmol) and H.sub.2O (7.5 mL) and the mixture stirred at rt for 65 h. The mixture was then heated to reflux for 2 h before addition of further LiOH (313 mg, 7.5 mmol) and heating at reflux for an additional 2 h. Volatiles were then removed in vacuo, and the aqueous layer acidified to pH 3 with 1 M aq. HCl and extracted with EtOAc. The aqueous layer was concentrated in vacuo to a colourless oil which was purified on silica (Biotage Isolera, SNAP 50 g cartridge, 0.7 M NH.sub.3 in MeOH/DCM, 4:1) to yield (3S)-3-{[1-(tert-butoxycarbonyl)piperidin-4-yl]amino}pentanoic acid (0.91 g) as a white powder, which was used crude in the next step.

[0319] LC/MS (method C): m/z 301 (M+H)+(ES+), at 0.62 min, UV active.

[0320] Tert-butyl 4-{[(3S)-1-methoxy-1-oxopentan-3-yl]amino}piperidine-1-carboxylate (624 mg, assumed 0.9 mmol), 1-methyl-2-chloropyridinium iodide (584 mg, 2.3 mmol) and Et.sub.3N (0.64 mL, 4.6 mmol) in MeCN (40 mL) were heated to reflux for 22 h. The mixture was purified on silica (Biotage isolera, SNAP 50 g cartridge, EtOAc/isohexane, 1;1 to 100% EtOAc over 5 CV and then 100% EtOAc over 10 CV) to yield tert-butyl 4-[(2S)-2-ethyl-4-oxoazetidin-1-yl]piperidine-1-carboxylate (194 mg, 80%) as a colourless oil.

[0321] LC/MS (method C): m/z 305 (M+Na)+(ES+), at 1.26 min, UV active.

[0322] To tert-butyl 4-[(2S)-2-ethyl-4-oxoazetidin-1-yl]piperidine-1-carboxylate (194 mg, 0.7 mmol) in DCM (3 mL) was added TFA (3 mL) and the mixture stirred at rt for 30 min. The solvent was removed in vacuo to yield intermediate 8, (4S)-4-ethyl-1-(piperidin-4-yl)azetidin-2-one TFA salt (374 mg) as a brown oil, which was used crude in the next step. The data for the title compound are in table 2.

Route 3

Procedure for the Preparation of Intermediate 9, 4-(3,3-difluoroazetidin-1-yl)piperidine

[0323] ##STR00024##

[0324] A solution of 3,3-difluoroazetidine hydrochloride (300 mg, 2.3 mmol), tert-butyl 4-oxopiperidine-1-carboxylate (461 mg, 2.3 mmol) and DIPEA (0.40 mL, 2.3 mmol) in DMF (6 mL) was heated to 50° C. for 16 h before cooling to rt, addition of sodium triacetoxyborohydride (1.23 g, 5.8 mmol) and AcOH (0.13 mL, 2.3 mmol) and further heating at 40° C. for 16 h. The mixture was cooled to rt, quenched by addition of sat. aq. NaHCO.sub.3 (3 mL) and concentrated in vacuo. The residue was diluted with DCM (15 mL) and washed with sat. aq. NaHCO.sub.3 (15 mL) and brine (15 mL). Organics were dried (Biotage phase separator) and concentrated in vacuo. The crude residue was purified on silica (Biotage Isolera, SNAP 25 g cartridge, 0-10% MeOH/DCM and subsequently SNAP 25 g cartridge, 0-5% MeOH/DCM) to yield tert-butyl 4-(3,3-difluoroazetidin-1-yl)piperidine-1-carboxylate (273 mg, 43%) as a white crystalline solid, which was used directly in the next step.

[0325] To tert-butyl 4-(3,3-difluoroazetidin-1-yl)piperidine-1-carboxylate (273 mg, 1.0 mmol) in DCM (8 mL) at 0° C. was added TFA (2 mL) and the mixture stirred at rt for 16 h. The reaction mixture was then concentrated in vacuo to yield intermediate 9, 4-(3,3-difluoroazetidin-1-yl)piperidine.2TFA as a pink oil, which was used directly in the next step. The data for the title compound are in table 2.

Step 1: Alternative Work Up Procedure

[0326] The reaction was treated with H.sub.2O (0.5 mL) and concentrated in vacuo. The crude residue was applied directly to a 25 g SNAP chromatography cartridge and eluted with a gradient 0-10% MeOH/DCM (Biotage Isolera).

Route 4

Procedure for the Preparation of Intermediate 15, 1-(piperidin-4-yl)azetidin-2-one.TFA

[0327] ##STR00025##

[0328] To tert-butyl 4-oxopiperidine-1-carboxylate (5.0 g, 25.1 mmol) in MeOH (50 mL) was added methyl 3-aminopropanoate (3.5 g, 25.1 mmol) and Et.sub.3N (10.7 mL, 75.3 mmol) and the reaction mixture stirred at 50° C. for 7 h. The mixture was then cooled to rt, followed by addition of NaBH.sub.3CN (4.75 g, 75.1 mmol) portionwise and further stirring at rt for 17 h. The solvent was then removed in vacuo and the residue partitioned between H.sub.2O (250 mL) and EtOAc (200 mL). The layers were separated and the aqueous layer extracted with EtOAc (2×200 mL). Combined organics were dried (Na.sub.2SO.sub.4), the solvent was removed in vacuo and the residue was purified by column chromatography (normal basic activated alumina, 10-13% EtOAc/hexane) to give tert-butyl 4-((3-methoxy-3-oxopropyl)amino)piperidine-1-carboxylate (3.30 g, 47%) as a yellow gum.

[0329] LC/MS (method A): m/z 287 (M+H)+(ES+), at 3.82 min, UV active.

[0330] To a solution of tert-butyl 4-((3-methoxy-3-oxopropyl) amino) piperidine-1-carboxylate (2.0 g, 7.0 mmol) in THF (20 mL) was added methyl magnesium bromide (3.5 mL, 10.5 mmol) dropwise at 0° C., and the resulting mixture stirred at rt for 50 h. The reaction was quenched with sat. aq. NH.sub.4Cl, the solvent removed in vacuo and the residue partitioned between H.sub.2O (150 mL) and EtOAc (120 mL). The layers were separated and the aqueous layer further extracted with EtOAc (2×120 mL). Combined organics were dried (Na.sub.2SO.sub.4), the solvent removed in vacuo and the residue purified by column chromatography (normal basic activated alumina, 8-10% EtOAc/hexane) to give tert-butyl 4-(2-oxoazetidin-1-yl)piperidine-1-carboxylate (600 mg, 35%) as a yellow gum.

[0331] LC/MS (method B): m/z 255 (M+H)+(ES+), at 3.63 min, UV active.

[0332] To tert-butyl 4-(2-oxoazetidin-1-yl) piperidine-1-carboxylate (600 mg, 2.4 mmol) in DCM (5 mL) at 0° C. was added trifluoroacetic acid (0.4 mL, 4.7 mmol) dropwise and the resulting mixture stirred at rt for 8 h. The solvent was then removed in vacuo and the residue purified by trituration with ether (3×2 mL) to give Intermediate 15, 1-(piperidin-4-yl) azetidin-2-one.TFA (510 mg, 81%) as a yellow gum. The data for the title compound are in table 2.

General Synthetic Procedures for Examples

Route a

Typical Procedure for the Preparation of Piperidines via Reductive Amination, as Exemplified by the Preparation of Example 1-1 Ethyl 2-[4-(3,3-difluoroazetidin-1-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate

[0333] ##STR00026##

[0334] A solution of crude 4-(3,3-difluoroazetidin-1-yl)piperidine (assumed 1.0 mmol), ethyl 2-oxo-6-azaspiro[3.4]octane-6-carboxylate (183 mg, 0.9 mmol) and DIPEA (0.34 mL, 2.0 mmol) in DMF (6 mL) was stirred at 40° C. for 2 h. The solution was cooled to rt before addition of sodium triacetoxyborohydride (522 mg, 2.5 mmol) and AcOH (60 μL, 1.0 mmol) and further stirring at 40° C. for 16 h. The solvent was removed in vacuo and the residue diluted with sat. aq. NaHCO.sub.3 (15 mL) and extracted with DCM (2×15 mL). Combined organics were dried (Biotage phase separator cartridge) and concentrated in vacuo. The crude residue was purified on silica (Biotage Isolera, SNAP 25 g cartridge, 0-10% MeOH/DCM) and subsequently by prep HPLC [reverse phase (Gemini-NX, C18, 5 μ, 100×30 mm), 30 mL per min, gradient 30% (0.3 min), then 30-60% (over 8.7 min), then 60% (for 0.5 min), then 60-100% (over 0.2 min), then 100% (for 1 min), then 30% (for 0.8 min), MeCN/0.2% NH.sub.3 in water] to yield ethyl 2-[4-(3,3-difluoroazetidin-1-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate isomer 1 (29 mg, 9%) as a colourless oil and Example 1-1, ethyl 2-[4-(3,3-difluoroazetidin-1-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate isomer 2 (30 mg, 9%) as a colourless oil. The data for the title compound are in table 3

Route a Alternative Work Up Procedure

[0335] The reaction was allowed to cool to rt, treated with H.sub.2O (0.5 mL) and concentrated in vacuo. The crude residue was applied directly to a 10 g SNAP chromatography cartridge and eluted with a gradient 0-10% MeOH/DCM (Biotage Isolera). Relevant fractions were combined and further purified by reverse phase prep HPLC.

Route b

Typical Procedure for the Preparation of Piperidines via Reductive Amination, as Exemplified by the Preparation of Example 1-3 ethyl 2-[4-(2-oxoazetidin-1-yl)piperidin-1-yl]-6-azaspiro[3.4]octane-6-carboxylate

[0336] ##STR00027##

[0337] A solution of 1-(piperidin-4-yl) azetidin-2-one.TFA (200 mg, 0.8 mmol), ethyl 2-oxo-6-azaspiro [3.4] octane-6-carboxylate (254 mg, 1.3 mmol) and Et.sub.3N (0.6 mL, 3.9 mmol) in MeOH (10 mL) was stirred at 55° C. for 5 h. The reaction mixture was then cooled to 0° C. before addition of NaBH.sub.3CN (246 mg, 3.9 mmol) portionwise and further stirring at 25° C. for 17 h. The solvent was removed in vacuo, the residue partitioned between H.sub.2O (100 mL) and DCM (80 mL) and the layers separated. The aqueous layer was extracted with DCM (2×80 mL) and combined organics dried (Na.sub.2SO.sub.4) and the solvent removed in vacuo. The residue was purified by prep HPLC [reverse phase (X-BRIDGE C-18, 150×19 mm, 5 μm), 17 mL per min, gradient 20% (over 30.0 min), 100% (over 2.0 min), then 20% (over 2.0 min), 0.1% NH.sub.3 in MeCN/water] to give ethyl 2-(4-(2-oxoazetidin-1-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate isomer-1 (45 mg, 18%) as a yellow gum and Example 1-3, ethyl 2-(4-(2-oxoazetidin-1-yl)piperidin-1-yl)-6-azaspiro[3.4]octane-6-carboxylate isomer-2 (28 mg, 11%) as a yellow gum. The data for the title compound are in table 3.

Route c

Typical Procedure for the Preparation of Piperidines via Reductive Amination, as Exemplified by the Preparation of Example 1-5, ethyl 2-{4-[(2S)-2-ethyl-4-oxoazetidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate

[0338] ##STR00028##

[0339] To crude (4S)-4-ethyl-1-(piperidin-4-yl)azetidin-2-one TFA salt (374 mg, assume 0.7 mmol) in DMF (5 mL) was added DIPEA (0.60 mL, 3.4 mmol), AcOH (60 μL, 1.1 mmol), ethyl 2-oxo-6-azaspiro[3.4]octane-6-carboxylate (136 mg, 0.7 mmol) and sodium triacetoxyborohydride (437 mg, 2.1 mmol) and the mixture stirred at rt for 65 h. The reaction mixture was concentrated to remove DMF and the residue purified on silica (Biotage Isolera, SNAP 25 g cartridge, 0-10% 0.7 M NH.sub.3 in MeOH/DCM over 10 CV) to yield a mixture of two isomers as a yellow residue. This was further purified by prep HPLC [reverse phase (Gemini-NX, C18, 5 μ, 100×30 mm), 30 mL per min, gradient 30% (0.3 min), then 30-60% (over 8.7 min), then 60% (for 0.5 min), then 60-100% (over 0.2 min), then 100% (for 1 min), then 30% (for 0.8 min), MeCN/0.2% NH.sub.3 in water] to yield ethyl 2-{4-[(2S)-2-ethyl-4-oxoazetidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate isomer 1 (100 mg, 40%) as a gum and Example 1-5, ethyl 2-{4-[(2S)-2-ethyl-4-oxoazetidin-1-yl]piperidin-1-yl}-6-azaspiro[3.4]octane-6-carboxylate isomer 2 (76 mg, 30%) as a gum. The data for the title compound are in table 3.

TABLE-US-00002 TABLE 2 Characterising data and commercial sources for starting materials and intermediates Table 2 Intermediate Route Name Data 1 6-Boc-2-oxo-6- Commercially available, azaspiro[3.4]octane CAS: 203661-71-6 2 Route 1 and Ethyl 2-oxo-6- .sup.1H NMR: (400 MHz, intermediates azaspiro[3.4]octane- CDCl.sub.3) δ: 1.27 (t, J = 1 and 5 6-carboxylate 7.0 Hz, 3H), 2.08 (t, J = 6.2 Hz, 2H), 2.94-3.17 (m, 4H), 3.49-3.59 (m, 4H), 4.15 (q, J = 7.0 Hz, 2H) 3 Route 1 and Methyl 2-oxo-6- .sup.1H NMR: (400 MHz, intermediates azaspiro[3.4]octane- CD.sub.3OD) δ:: 2.06-2.15 1 and 4 6-carboxylate (m, 2 H), 2.94-3.04 (m, 2 H), 3.05-3.17 (m, 2 H), 3.47 (td, J = 6.8, 2.5 Hz, 2 H), 3.54 (d, J = 2.5 Hz, 2 H), 3.69 (s, 3 H) 4 Methyl Commercially available, chloroformate CAS: 79-22-1 5 Ethyl chloroformate Commercially available, CAS: 541-41-3 6 (3S)-3- Commercially available. Aminopentanoic CAS: 14389-77-6 acid 7 Tert-butyl Commercially available. 4-oxopiperidine- CAS: 79099-07-3 1-carboxylate 8 Route 2 and (4S)-4-Ethyl-1- m/z 183 (M + H) + intermediates (piperidin-4- (ES+) 6 and 7 yl)azetidin-2-one. TFA 9 Route 3 and 4-(3,3- R.sub.f = 0.10 (MeOH/DCM, intermediates Difluoroazetidin-1- 1:9) 7 and 10 yl)piperidine. 2TFA 10 3,3- Commercially available. Difluoroazetidine CAS: 288315-03-7 hydrochloride 11 (R)-Methyl-2- Commercially available azetidine CAS: 647854-63-5 carboxylate. HCl 12 Route 3 with Methyl (2R)-1- R.sub.f = 0 (MeOH/DCM, step 2 HCI (piperidin-4- 1:9) instead of yl)azetidine-2- TFA. carboxylate. 2HCl Intermediates 7 and 11 13 Methyl β-alaninate Commercially available. CAS: 4138-35-6 14 MeMgBr Commercially available. CAS: 75-16-1 15 Route 4 and 1-(piperidin-4- (LC/MS method A) m/z intermediates yl)azetidin-2-one. 155 (M + H).sup.+ (ES.sup.+) at 7 and 13 TFA 1.87 min, UV active 16 Route 2 and (4R)-4-Ethyl-1- (LC/MS method D) m/z intermediates (piperidin-4- 183 (M + H).sup.+ (ES.sup.+) at 7 and 17 yl)azetidin-2-one. 1.29 min, UV active TFA 17 (3R)-3- Commercially available. Aminopentanoic CAS: 131347-76-7 acid 18 Route 2 and (4R)-4-methyl-1- m/z 169 (M + H).sup.+ (ES.sup.+) intermediates (piperidin-4- 7 and 19 yl)azetidin-2-one 19 (3R)-3- Commercially available. aminobutanoic acid CAS: 3775-73-3 20 Route 2 and (4S)-4-methyl-1- m/z 169 (M + H).sup.+ (ES.sup.+) intermediates (piperidin-4- 7 and 21 yl)azetidin-2-one 21 (3S)-3- Commercially available. aminobutanoic acid CAS: 3775-72-2

TABLE-US-00003 TABLE 3 Ex. Synthetic LCMS LCMS No. Name Intermediate method .sup.1H NMR Method data 1-1 Isomer 2: Ethyl 2-[4-(3,3- 2 and 9 Route a (400 MHz, DMSO-d.sub.6) δ: 1.13 (t, J = 7.0 Hz, 3 H), 1.57-1.60 E m/z 358 difluoroazetidin-1-yl) (m, 2 H), 1.70-1.82 (m, 6 H), 1.91-1.96 (m, 2 H), 2.53- (M + H).sup.+ piperidin-1-yl]- 2.64 (m, 3 H), 2.03-2.14 (m, 1 H), 3.10-3.26 (m, 6 H), (ES.sup.+) at 6-azaspiro[3.4]octane-6- 3.48 (t, J = 12.5 Hz, 4 H), 3.96 (q, J = 7.0 Hz, 2 H) 3.20 min, carboxylate UV inactive 1-2 Isomer 2: Ethyl 2-{4-[(2R)- 2 and 12 Route a (400 MHz, DMSO-d.sub.6) δ: 0.99-1.15 (m, 5 H), 1.47-2.14 (m, E m/z 380 2-(methoxycarbonyl) with 13 H), 2.53-2.65 (m, 2 H), 2.81 (q, J = 8.0 Hz, 1 H), 3.11- (M + H).sup.+ azetidin-1- alternative 3.27 (m, 6 H), 3.58 (s, 3 H), 3.67 (t, J = 8.0 Hz, 1 H), 3.97 (ES.sup.+) at yl]piperidin-1-yl} work up (q, J = 7.0 Hz, 2 H) 5.41 min, -6-azaspiro[3.4]octane-6- procedure UV active carboxylate 1-3 Isomer 2: Ethyl 2-[4-(2- 2 and 15 Route b (400 MHz, CDCl.sub.3) δ: 1.22-1.30 (m, 3 H), 1.66-2.54 (m, 12 A m/z 336 oxoazetidin-1-yl) H), 2.66-3.03 (m, 5 H), 3.21-3.68 (m, 7 H), 4.08-4.19 (m, (M + H).sup.+ piperidin-1- 2 H) (ES.sup.+) at yl]-6-azaspiro[3.4]octane- 3.30 min, 6-carboxylate UV active 1-4 Isomer 2: Ethyl 2-{4- 2 and 20 Route c (400 MHz, CDCl.sub.3) δ: 1.19-1.22 (m, 3 H), 1.33 (d, J = 6.0 E m/z 350 [(2S)-2-methyl-4- Hz, 3 H), 1.56-1.91 (m, 10 H), 1.97-2.07 (m, 2 H), 2.41 (M + H).sup.+ oxoazetidin-1- (dd, J = 14.5, 2.0 Hz, 1 H), 2.57-2.69 (m, 1 H), 2.76-2.90 (ES.sup.+) at yl]piperidin-1-yl}-6- (m, 2 H), 2.97 (dd, J = 14.5, 5.0 Hz, 1 H), 3.23 (d, J = 20.5 3.38 min, azaspiro[3.4]octane-6- Hz, 2 H), 3.35 (dt, J = 20.0, 6.5 Hz, 2 H), 3.46-3.58 (m, 1 UV active carboxylate H), 3.68-3.75 (m, 1 H), 4.07 (q, J = 7.0 Hz, 2 H) 1-5 Isomer 2: Ethyl 2-{4-[(2S)- 2 and 8 Route c (400 MHz, CDCl.sub.3) δ: 0.89 (t, J = 7.0 Hz, 3 H), 1.24 (t, J = 6.5 E m/z 364 2-ethyl-4-oxoazetidin-1- Hz, 3 H), 1.39-1.50 (m, 1 H), 1.55-2.16 (m, 13 H), 2.48 (d, (M + H).sup.+ yl]piperidin-1-yl}-6- J = 7.0 Hz, 1 H), 2.56-2.71 (m, 1 H), 2.76-2.94 (m, 3 H), (ES.sup.+) at azaspiro[3.4]octane-6- 3.24-3.43 (m, 4 H), 3.48-3.64 (m, 2 H), 4.10 (q, J = 7.0 3.77 min, carboxylate Hz, 2 H) UV active 1-6 Isomer 2: Ethyl 2-{4-[(2R)- 2 and 18 Route c (400 MHz, CDCl.sub.3) δ: 1.20-1.25 (m, 3 H), 1.34 (d, J = 6.0 E m/z 350 2-methyl-4-oxoazetidin-1- Hz, 3H), 1.57-2.09 (m, 10 H), 2.30-3.59 (m, 12 H), 3.66- (M + H).sup.+ yl]piperidin-1-yl}-6- 3.77 (m, 1 H), 4.05-4.17 (m, 2 H) (ES.sup.+) at azaspiro[3.4]octane-6- 3.38 min, carboxylate UV active 1-7 Isomer 2: Ethyl 2-{4-[(2R)- 2 and 16 Route c (400 MHz, CDCl.sub.3) δ: 0.85-0.92 (m, 3 H), 1.19-1.25 (m, 3 E m/z 364 2-ethyl-4-oxoazetidin-1- H), 1.37-1.48 (m, 1 H), 1.56-2.07 (m, 11 H), 2.43-3.55 (M + H).sup.+ yl]piperidin-1-yl}-6- (m, 13 H), 4.05-4.17 (m, 2 H) (ES.sup.+) at azaspiro[3.4]octane-6- 3.73 min, carboxylate UV active

BIOLOGICAL ACTIVITY

Example A

Phospho-ERK1/2 assays

[0340] Functional assays were performed using the Alphascreen Surefire phospho-ERK1/2 assay (Crouch & Osmond, Comb. Chem. High Throughput Screen, 2008). ERK1/2 phosphorylation is a downstream consequence of both Gq/11 and Gi/o protein coupled receptor activation, making it highly suitable for the assessment of M.sub.1, M.sub.3 (Gq/11 coupled) and M.sub.2, M.sub.4 receptors (Gi/o coupled), rather than using different assay formats for different receptor subtypes. CHO cells stably expressing the human muscarinic M.sub.1, M.sub.2, M.sub.3 or M.sub.4 receptor were plated (25K/well) onto 96-well tissue culture plates in MEM-alpha+10% dialysed FBS. Once adhered, cells were serum-starved overnight. Agonist stimulation was performed by the addition of 5 μL agonist to the cells for 5 min (37° C.). Media was removed and 50 μL of lysis buffer added. After 15 min, a 4 μL sample was transferred to 384-well plate and 7 μL of detection mixture added. Plates were incubated for 2 h with gentle agitation in the dark and then read on a PHERAstar plate reader.

[0341] pEC.sub.50 and E.sub.max figures were calculated from the resulting data for each receptor subtype.

[0342] The results are set out in Table 4 below.

[0343] For each example with the 4-5 spiro system two diastereomers exist which have been separated, unless stated otherwise, and assigned based on their retention time on LCMS analytical trace. In most examples, isomer 1 is not active. Analytical data for active isomers is reported in Table 3. Data for several weakly active compounds are included in Table 4 to highlight preference of absolute stereochemistry.

TABLE-US-00004 TABLE 4 Muscarinic Activity pEC.sub.50 M.sub.1 pEC.sub.50 M.sub.2 pEC.sub.50 M.sub.3 pEC.sub.50 M.sub.4 (% Emax cf. (% Emax cf. (% Emax cf. (% Emax cf. Ex. No. ACh) ACh) ACh) ACh) ACh    8.3 (102)    7.8 (105)    8.1 (115)  8.1 (110) 1-1 Isomer 2   6.0 (24) NT NT 6.8 (26) 1-2 Isomer 2 <4.7 (37) <4.7 (7)  <4.7 (7) 7.2 (67) 1-3 Isomer 2 <4.7 (20) NT NT 6.7 (23) 1-4 Isomer 2 <4.7 (18) NT NT 6.8 (41) 1-5 Isomer 2   6.5 (69) <4.7 (37) <4.7 (0) 7.0 (56) 1-6 Isomer 2 <4.7 (13) NT NT 6.6 (74) 1-7 Isomer 2   6.1 (94) *6.3 (26)  <4.7 (21)  7.1 (101) *—variable results, NT—Not tested

Example B

Pharmaceutical Formulations

[0344] (i) Tablet Formulation

[0345] A tablet composition containing a compound of the formula (1) is prepared by mixing 50 mg of the compound with 197 mg of lactose (BP) as diluent, and 3 mg magnesium stearate as a lubricant and compressing to form a tablet in known manner.

[0346] (ii) Capsule Formulation

[0347] A capsule formulation is prepared by mixing 100 mg of a compound of the formula (1) with 100 mg lactose and optionally 1% by weight of magnesium stearate and filling the resulting mixture into standard opaque hard gelatin capsules.

Equivalents

[0348] The foregoing examples are presented for the purpose of illustrating the invention and should not be construed as imposing any limitation on the scope of the invention. It will readily be apparent that numerous modifications and alterations may be made to the specific embodiments of the invention described above and illustrated in the examples without departing from the principles underlying the invention. All such modifications and alterations are intended to be embraced by this application.