Novel Methyl-Piperidine Compounds Useful for Inhibiting Microsomal Prostaglandin E2 Synthase-1

Abstract

The present invention provides compounds of Formula 1, or a pharmaceutically acceptable salt thereof,

##STR00001##

where R, R1, and G are as described herein; methods of preparing the compounds; and use of the compounds to treat pain and/or inflammation associated with arthritis or osteoarthritis.

Claims

1-23. (canceled)

24. A method of treating a patient in need of treatment for pain or inflammation associated with arthritis or osteoarthritis said method comprising administering to the patient an effective amount of ##STR00071## or a pharmaceutically acceptable salt thereof, in combination with an effective amount of a second agent selected from the group consisting of ibuprofen, aspirin, acetaminophen, celecoxib, naproxen, and ketoprofen.

Description

[0046] The following Preparations and Examples further illustrate the invention.

Preparation 1

tert-Butyl 3,3-dimethyl-4-(((trifluoromethyl)sulfonyl)oxy)-3,6-dihydropyridine-1(2H)-carboxylate

[0047] ##STR00017##

[0048] Under a nitrogen atmosphere, cool a solution of diisopropylamine (260 mL, 1.85 mol) in THF (1.0 L) to 20 C. then add a solution of n-butyllithium (2.50 M in hexanes, 650 mL, 1.60 mol) drop-wise over 30 minutes. Allow the mixture to warm to 10 C. and stir for 1 hour. Cool the mixture to 74 C. and add a solution of tert-butyl 3,3-dimethyl-4-oxopiperidine-1-carboxylate (260 g, 1.14 mol) in THF (1.0 L mL) drop-wise over 60 minutes. Stir the mixture at 74 C. for 2 hours, and then add a solution of N-phenylbis(trifluoromethanesulfonimide) (430 g, 1.20 mol) in THF (1.0 L). Warm the mixture to 0 C. and stir for 2 hours. Allow the mixture to warm to room temperature and stir overnight. Quench the reaction with saturated aqueous NH.sub.4Cl (1.0 L); dilute with water (2.0 L); separate the layers; and extract the aqueous layer with EtOAc (22 L). Combine the organic extracts; dry over Na.sub.2SO.sub.4; filter; collect the filtrate; and concentrate the filtrate under reduced pressure. Subject the resulting crude material to silica gel flash chromatography, eluting with a gradient of 0% to 15% TBME in hexanes, to provide the title compound as an orange oil in about 75% purity by mass as estimated by .sup.1H NMR (430 g, 78%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) 1.05 (s, 6H), 1.40 (s, 9H), 3.36 (s, 2H), 4.02 (d, J=3.4 Hz, 2H), 5.82 (br s, 1H).

Preparation 2

O1-tert-Butyl-O4-methyl 3,3-dimethyl-2,6-dihydropyridine-1,4-dicarboxylate

[0049] ##STR00018##

[0050] Combine palladium(II) acetate (4.40 g, 20.0 mmol), 1,1-bis(diphenylphosphino)ferrocene (13.3 g, 22.8 mmol), tert-butyl 3,3-dimethyl-4-(((trifluoromethyl)sulfonyl)oxy)-3,6-dihydropyridine-1 (2H)-carboxylate (72.0 g, 150 mmol), anhydrous acetonitrile (850 mL), anhydrous MeOH (570 mL), and triethylamine (36.0 mL, 245 mmol) in a 2-L PARR autoclave fitted with a mechanical stirrer. Seal the autoclave. Purge and then pressurize the autoclave with carbon monoxide to 689 kPa. Heat the mixture to 65 C. for 2.25 hours; cool the mixture to room temperature; and carefully vent the autoclave. (Caution! Poison gas!). Concentrate under reduced pressure to give crude material. Combine this material with five other batches of material prepared by an analogous procedure on similar scales. Subject the combined material to silica gel flash chromatography, eluting with a gradient of 0% to 20% TBME in hexanes, to give the title compound as a yellow oil in about 83% purity by mass (260 g, 88%). MS (m/z): 214 (Mt-Bu+2H).sup.+.

Preparation 3

()-O1-tert-Butyl-O4-methyl 3,3-dimethylpiperidine-1,4-dicarboxylate

[0051] ##STR00019##

[0052] Suspend palladium (10 wt % on carbon, 5.4 g, 5.1 mmol) in MeOH (700 mL) then add a solution of O1-tert-butyl-O4-methyl 3,3-dimethyl-2,6-dihydropyridine-1,4-dicarboxylate (130 g, 396 mmol) dissolved in MeOH (700 mL) in a 2.25 L PARR reactor. Seal the reactor and purge it first with nitrogen gas then with hydrogen gas. Pressurize the reactor to 414 kPa with hydrogen and stir the mixture at room temperature for 1.5 hours. Release the pressure and filter the mixture to remove the catalyst. Combine the filtrate with that obtained from another essentially identical reaction and concentrate under reduced pressure to give the title compound as a yellow oil in about 85% purity by mass (240 g, 95%). MS (m/:): 216 (Mt-Bu+2H).sup.+.

Preparation 4

()-Methyl 3,3-dimethylpiperidine-4-carboxylate hydrochloride

[0053] ##STR00020##

[0054] Add HCl (4.0 M solution in 1,4-dioxane, 2.0 L, 8.0 mol) to a solution of O1-tert-butyl-O4-methyl 3,3-dimethylpiperidine-1,4-dicarboxylate (240 g, 752 mmol) in 1,4-dioxane (500 mL). Stir the resulting mixture at room temperature overnight and concentrate under reduced pressure. Dilute the residue with TBME (500 mL) and collect the resulting solids by filtration. Rinse the filter cake with TBME (2400 mL) and dry the solid in a vacuum oven at 35 C. overnight to give the title compound as a white solid (144 g, 92%). MS (m/z): 172 (M+H).sup.+.

Preparation 5

()-Methyl (4S)-3,3-dimethyl-1-(8-methyl-2-quinolyl)piperidine-4-carboxylate

[0055] ##STR00021##

[0056] Add K.sub.2CO.sub.3 (210 g, 1.52 mol) to a mixture of methyl 3,3-dimethylpiperidine-4-carboxylate hydrochloride (144 g, 693 mmol) and 2-chloro-8-methylquinoline (125 g, 704 mmol) in DMSO (1.4 L). Stir the resulting mixture over night at 1311 C. Cool the mixture to room temperature; filter to remove the solids; collect the filtrate; dilute the filtrate with water (2 L); then extract with EtOAc (23 L). Wash the combined organic extracts with water (31.5 L); dry over Na.sub.2SO.sub.4; filter; collect and concentrate the filtrate under reduced pressure. Subject the resulting crude material to silica gel flash chromatography, eluting with a gradient of 25% to 30% (10% TBME in DCM) in hexanes, to provide the racemate of the title compound. Dissolve this material in MeOH (7.5 L) and filter. Subject the material to chiral SFC (Chiralpak OJ-H, 50 mm250 mm5 m) using 15% (0.2% dimethylethylamine in i-PrOH) in CO.sub.2(scf) as the mobile phase at a flow rate of 400 g/min, by injecting 5 mL of solution every 95 seconds until all of the material has been subjected. For each injection, collect the first fraction to elute (t.sub.R=2.57 min by SFC Method 1). Combine the collected fractions with those from a previous reaction prepared similarly to provide 98 g of crude methyl 3,3-dimethylpiperidine-4-carboxylate hydrochloride. Concentrate mixture under reduced pressure and recrystallize the material from hot EtOH (1.38 L). Collect the crystals and dry the crystalline material in a vacuum oven at 40 C. overnight to give the title compound as a white crystalline solid (156 g, 43% yield on a batch-proportional basis). MS (m/z): 313 (M+H).sup.+, [].sup.20.sub.D 45 (c 0.21, DCM). ee=>99% as determined by SFC Method 1.

Preparation 6

()-(4S)-3,3-Dimethyl-1-(8-methyl-2-quinolyl)piperidine-4-carboxylic acid

[0057] ##STR00022##

[0058] Treat methyl (4S)-3,3-dimethyl-1-(8-methyl-2-quinolyl)piperidine-4-carboxylate (154 g, 493 mmol) with sulfuric acid (10% v/v in water, 2.31 L, 2.75 mol) and reflux the mixture overnight. Cool the mixture to room temperature and add NaOH (50 wt % in water) until the pH reaches 13. Add TBME (500 mL) to provide a triphasic mixture. Label the layers from top to bottom as: Layer A, Layer B, and Layer C. Remove Layer C (the bottom layer). Add water (600 mL) to Layers A and B and separate the aqueous layer from the organic layers. Set aside the organic layers (A and B). Combine the aqueous layer with Layer C. Extract the combined aqueous layers with TBME (500 mL); separate the layers; and set aside the organic extracts. Add HCl (5.0 M) to the aqueous layer until the pH is 6.5. Extract the resulting aqueous mixture with TBME (2400 mL). Combine all of the organic extracts (including Layers A and B); dry over MgSO.sub.4; filter; collect and concentrate the filtrate under reduced pressure to give the title compound as a white solid in 96% purity (145 g, 95%). MS (m/z): 299 (M+H).sup.+. [].sup.20.sub.D59.60 (c 3.12, CH.sub.3OH).

Preparation 7

()-2-((Benzyloxy)methyl)-2,3-dihydro-4H-pyran-4-one

[0059] ##STR00023##

[0060] Add a solution of ZnCl.sub.2 in THF (0.5 M, 1.21 L, 607 mmol) to a cold (0 C.) solution of (E)-1-methoxy-3-(trimethylsilyl)oxy-1,3-butadiene (95% pure, 100 g, 551 mmol) and (benzyloxy)acetaldehyde (97% pure, 80 mL, 551.370 mmol) in toluene (551 mL) over 90 minutes while maintaining the internal temperature of the reaction mixture below 10 C. Allow the mixture to warm to room temperature and stir overnight. Divide the reaction mixture into two equal portions; perform the following procedures on each portion. Add trifluoroacetic acid (35 mL, 457 mmol) in four portions. After 20 minutes, concentrate the resulting mixture under reduced pressure; dilute with EtOAc; add excess saturated NaHCO.sub.3; filter to remove the solids; collect the filtrate; separate and collect the organic layer. Wash the organic solution with saturated aqueous NaCl; isolate the organic extracts; dry over MgSO.sub.4; filter; collect the filtrate; and concentrate under reduced pressure. Combine the resulting material from each of the previously separated portions. Subject the resulting material to silica gel flash chromatography, eluting with a gradient of 20% to 50% EtOAc in hexanes, to give the title compound as an orange oil in 92% purity (96 g, 73%). .sup.1H NMR (400 MHz, CDCl.sub.3) 7.40-7.28 (m, 6H), 5.42 (d, J=6.0 Hz, 1H), 4.65-4.56 (m, 3H), 3.74-3.67 (m, 2H), 2.75 (dd, J=16.8, 14.3 Hz, 1H), 2.42 (dd, J=16.9, 3.2 Hz, 1H).

Preparation 8

()-2-(Benzyloxymethyl)tetrahydropyran-4-one

[0061] ##STR00024##

[0062] Stir a mixture of EtOAc (880 mL), ()-2-((benzyloxy)methyl)-2,3-dihydro-4H-pyran-4-one (96 g, 0.440 mol), triethylamine (123 mL, 0.882 mol), and palladium (10% on carbon, 4.68 g, 4.40 mmol) under an atmosphere of hydrogen at room temperature for 73 hours. Filter the mixture through diatomaceous earth; rinse the filter cake with EtOAc (250 mL); and sequentially wash the filtrate with aqueous HCl (0.1 M), saturated aqueous NaHCO.sub.3, and saturated aqueous NaCl. Dry the organic layer over MgSO.sub.4; filter; collect the filtrate; and concentrate under reduced pressure to give the title compound as a light yellow material (81.4 g, 84%). LC/MS (ESI.sup.+): 221 [M+H].sup.+, 238 [M+NH.sub.4].sup.+, 243 [M+Na].sup.+.

Preparation 9

(2S,4R)-2-(Benzyloxymethyl)tetrahydropyran-4-ol

[0063] ##STR00025##

[0064] Cool a solution of ()-2-(benzyloxymethyl)tetrahydropyran-4-one (40.6 g, 184 mmol) in THF (1.08 L) to 78 C. Add a solution of LiAlH.sub.4 (1.0 M in THF, 240 mL, 240 mmol) drop-wise over 15 minutes. Allow the mixture to warm to 0 C. after the addition is complete and slowly add water (8.4 mL) drop-wise. After stirring the mixture for 5 minutes, add a solution of NaOH (15 mass % in water, 8.4 mL) and stir for an additional 5 minutes. Then add water (38.4 mL) and allow the mixture to warm to room temperature. After 30 minutes, filter the suspension to remove the solids and concentrate the filtrate under reduced pressure to provide the title compound as a colorless oil. Rinse the solids once with THF; filter; and concentrate the filtrate under reduced pressure. Combine this material with those obtained from previously reactions performed essentially according to the same procedure starting with 40 g, 22 g, and 45 g of ()-2-(benzyloxymethyl)tetrahydropyran-4-one. Dissolve the combined material in i-PrOH (637 mL). Subject the material to chiral SFC (Chiralpak AS-H, 50 mm150 mm5 m) using 25% i-PrOH in CO.sub.2(scf) as the mobile phase at a flow rate of 300 g/min, by injecting 1.35 g of solution every 114 seconds until all of the material has been injected. For each injection, collect the first fraction to elute (major isomer). Combine all of the collected fractions to give the title compound in >99% ee as determined by SFC Method 2 (62.8 g, 42% yield on a batch proportional basis). LC/MS (ESI.sup.+): 223 [M+H].sup.+, 240 [M+NH.sub.4]+, 245 [M+Na].sup.+, 467 [2M+Na].sup.+.

Preparation 10

((2S,4R)-2-(Benzyloxymethyl)tetrahydropyran-4-yl)methanesulfonate

[0065] ##STR00026##

[0066] Drop-wise add a solution of methanesulfonyl chloride (11.7 mL, 150.6 mmol) in DCM (70 mL) to a mixture of (2S,4R)-2-(benzyloxymethyl)tetrahydropyran-4-ol (31 g, 139.5 mmol), DCM (1.40 L), and N,N-diisopropylethylamine (73.0 mL, 418.4 mmol) at 0 C. Allow the mixture to warm to room temperature and stir it overnight. Thereafter add a solution of methanesulfonyl chloride (0.537 mL, 6.98 mmol) in DCM (5 mL) and stir the mixture for 5 minutes at room temperature. Cool the mixture to 0 C., pour it into water; separate; and collect the organic layer. Wash the organic layer with water (500 mL); combine the organic layer with organic layers/extracts obtained from other, essentially identical reactions. Dry the combined organic solutions over MgSO.sub.4; filter; collect the filtrate; and concentrate the filtrate under reduced pressure to give the title compound as a light orange oil (84.7 g, 97% yield on a batch proportional basis). .sup.1H NMR (400 MHz, d.sub.6-DMSO) 7.36-7.24 (m, 5H), 4.86-4.77 (m, 1H), 4.47 (s, 2H), 3.96 (dd, J=7.6, 4.4 Hz, 1H), 3.60-3.54 (m, 1H), 3.48-3.36 (m, 3H), 3.18 (s, 3H) 2.05 (d, J=7.8 Hz, 1H), 1.98 (d, J=7.8 Hz, 1H), 1.58 (app qd, J=12.0, 4.8 Hz, 1H), 1.40 (app q, J=11.8 Hz, 1H).

Preparation 11

((2S,4S)-4-Aminotetrahydro-2H-pyran-2-yl)methanol hydrochloride

[0067] ##STR00027##

[0068] Add sodium azide (12.85 g, 191.7 mmol) to a solution of ((2S,4R)-2-(benzyloxymethyl)tetrahydropyran-4-yl)methanesulfonate (32.0 g, 106.5 mmol) in DMF (304 mL). Heat the resulting mixture to 100 C. and stir it for 4 hours. Cool the reaction mixture to room temperature. Pour the mixture into water (400 mL) and then extract with EtOAc (2400 mL). Combine organic extracts and wash with saturated aqueous NaCl (3200 mL). Dry the organic extracts over MgSO.sub.4; filter; and collect the filtrate. Concentrate filtrate under reduced pressure to about 100 mL total volume. Dissolve resulting mixture in EtOAc (700 mL) and add it to a PARR vessel containing PtO.sub.2 (2.7 g, 12 mmol) and EtOAc (700 mL). Purge the vessel with nitrogen; then pressurize it with hydrogen to 414 kPa. Agitate the mixture for 4 hours at room temperature. Filter the mixture and concentrate the colorless filtrate under reduced pressure to provide 23.2 g of a colorless oil. Combine with 36.2 g of material from a previous reaction prepared by essentially the same procedure. Dissolve the combined material in EtOH (600 mL) and add HCl (37 wt % in H.sub.2O, 50 mL). Add the resulting mixture to a PARR vessel which contains Pd (10/on carbon, 10.0 g, 9.40 mmol) and EtOH (600 mL). Purge the reactor with nitrogen and pressurize it with hydrogen to 414 kPa. Agitate the mixture at room temperature for 1 hour. Filter the mixture; collect the filtrate; and concentrate the colorless filtrate under reduced pressure to give the title compound as a dark, thick oil in about 72% purity (54.2 g, 85%). LC/MS (ESI.sup.+): 132 [M+H]+.

Preparation 12

()-(Methyl 3,3-dimethyl-1-[4-(trifluoromethyl)phenyl]piperidine-4-carboxylate

[0069] ##STR00028##

[0070] Add K.sub.2CO.sub.3 (460 mg, 3.33 mmol) to a mixture of methyl 3,3-dimethylpiperidine-4-carboxylate hydrochloride (300 mg, 1.44 mmol), 1-fluoro-4-(trifluoromethyl)benzene (475 mg, 2.89 mmol) and DMSO (2 mL). Stir the resulting mixture at 130 C. for 2 days. Cool the mixture to room temperature and stir for 3 days. Subject the resulting material to reverse-phase flash chromatography on C.sub.18 silica gel, eluting with a gradient of 10% to 100% acetonitrile (0.1% formic acid) in water (0.1% formic acid), to give the title compound as a yellow oil (113 mg, 25%). MS (m/z): 316 (M+H).sup.+.

Preparation 13

()-3,3-Dimethyl-1-[4-(trifluoromethyl)phenyl]piperidine-4-carboxylic acid

[0071] ##STR00029##

[0072] Add 2M aqueous NaOH (1 mL, 2 mmol) to a mixture of methyl ()-3,3-dimethyl-1-[4-(trifluoromethyl)phenyl]piperidine-4-carboxylate (113 mg, 0.36 mmol), MeOH (1 mL), and THF (5 mL). Stir the resulting mixture at 50 C. overnight. Cool the mixture to room temperature, and add HCl (33 wt % in water) until the pH of the mixture is 3. Concentrate the mixture under reduced pressure to give the title compound as a yellow solid in 88% purity (123 mg, 99% yield). MS (n/z): 302 (M+H).sup.+.

Preparation 14

()-3,3-Dimethyl-1-[5-(trifluoromethyl)pyrimidin-2-yl]piperidine-4-carboxylic acid

[0073] ##STR00030##

[0074] Add triethylamine (7.65 mL, 54.9 mmol) to a mixture of ()-methyl 3,3-dimethylpiperidine-4-carboxylate hydrochloride (8.55 g, 41.2 mmol), 2-chloro-5-(trifluoromethyl)pyrimidine (5.0 g, 27.4 mmol), and acetonitrile (67 mL). Heat the mixture in a microwave to 180 C. for 1 hour; then cool the mixture to room temperature; and concentrate under reduced pressure. Add MeOH (40 mL), THF (80 mL), and 2 M aqueous NaOH (40 mL, 80 mmol). Stir the resulting mixture for 2 days at 50 C. Add 2 M aqueous NaOH (45 mL, 90 mmol) and stir the resulting mixture for 4 hours at 50 C. Cool the mixture to room temperature, and then add HCl (33 wt % in water) until the pH reaches 3. Concentrate under reduced pressure. Dilute with 1 N aqueous HCl (100 mL) and extract with EtOAc (2100 mL). Wash the combined organic extracts with brine (100 mL); dry over Na.sub.2SO.sub.4; filter; collect the filtrate; and concentrate under reduced pressure to give the title compound (7.60 g, 61%). MS (m/z): 302 (M+H).sup.+.

Preparation 15

5,8-Dimethylquinoline-1-oxide

[0075] ##STR00031##

[0076] Add 3-chloroperoxybenzoic acid (5.96 g, 24.1 mmol) to a mixture of 5,8-dimethylquinoline (2 g, 12.1 mmol) and DCM (80 mL) maintained at 0 C. After 1 hour, add Na.sub.2SO.sub.4 (5 g) and filter the mixture. Collect the filtrate. Stir the resulting mixture at room temperature overnight. Dilute with DCM (100 mL); wash with 1 N aqueous NaOH (350 mL); dry over Na.sub.2SO.sub.4; filter; collect the filtrate; and concentrate to dryness under reduced pressure. Subject the resulting crude material to reverse-phase flash chromatography on C.sub.18 silica gel, eluting with a gradient of 15% to 70% acetonitrile (10 mM ammonium bicarbonate) in water (10 mM ammonium bicarbonate) to give the title compound (372 mg, 18%). MS (m/z): 327 (M+H).sup.+.

Preparation 16

()-Methyl 1-(5,8-dimethyl-2-quinolyl)-3,3-dimethyl-piperidine-4-carboxylate

[0077] ##STR00032##

[0078] Add N,N-diisopropylethylamine (2.02 mL, 11.6 mmol) and PyBroP (1.75 g, 3.75 mmol) to a mixture of ()-methyl 3,3-dimethylpiperidine-4-carboxylate hydrochloride (600 mg, 2.89 mmol), 5,8-dimethylquinoline-1-oxide (678 mg, 3.91 mmol), and DCM (15 mL). Stir the resulting mixture at room temperature for 3 days. Subject the crude reaction to reverse-phase flash chromatography on C.sub.18 silica gel, eluting with a gradient of 10% to 100% acetonitrile (0.1% formic acid) in water (0.1% formic acid). Combine the fractions containing the desired product and concentrate under vacuum to approximately 30 mL volume. Adjust the pH to 6 with addition of a IN aqueous NaOH solution. Extract the aqueous solution with EtOAc (230 mL), wash with a pH 6 aqueous buffer solution (430 mL), dry over MgSO.sub.4; filter; and concentrate under reduced pressure to give the title compound (166 mg, 18%). MS (m/z): 327 (M+H).sup.+.

Preparation 17

()-1-(5,8-Dimethyl-2-quinolyl)-3,3-dimethyl-piperidine-4-carboxylic acid

[0079] ##STR00033##

[0080] Combine a mixture of methyl ()-1-(5,8-dimethyl-2-quinolyl)-3,3-dimethyl-piperidine-4-carboxylate (166 mg, 0.51 mmol), MeOH (0.1 mL), THF (0.5 mL), and 1 M aqueous NaOH (2.5 mL, 2.5 mmol) in a microwave vessel. Heat the resulting mixture at 120 C. for 2 hours in a microwave reactor. Cool the mixture to room temperature and add 1 N aqueous HCl until the pH is 6. Extract with CHCl.sub.3 (225 mL); dry over Na.sub.2SO.sub.4; filter; collect the filtrate; and concentrate under reduced pressure to give the title compound (135 mg, 85%). MS (ml:): 312 (M+H).sup.+.

Preparation 18

()-Methyl 1-(8-chloro-2-quinolyl)-3,3-dimethyl-piperidine-4-carboxylate

[0081] ##STR00034##

[0082] Add K.sub.2CO.sub.3 (1.07 g, 7.75 mmol) to a mixture of methyl ()-3,3-dimethylpiperidine-4-carboxylate hydrochloride (700 mg, 3.37 mmol), 2,8-dichloroquinoline (876 mg, 4.38 mmol), and DMSO (4.5 mL). Stir the resulting mixture at 130 C. overnight. Cool the mixture to room temperature; filter to remove the solids; dilute with water (5 mL); and extract with EtOAc (420 mL). Wash the combined organic extracts with water (20 mL) then brine (20 mL). Dry the organic extracts over MgSO.sub.4; filter; collect the filtrate; and concentrate under reduced pressure. Subject the resulting crude material to flash chromatography on silica gel, eluting with a gradient of 0% to 40% EtOAc in hexanes, to give the title compound (1.1 g, 98%). MS (ml): 332 (M+H).sup.+.

Preparation 19

()-1-(8-Chloro-2-quinolyl)-3,3-dimethyl-piperidine-4-carboxylic acid

[0083] ##STR00035##

[0084] Combine a mixture of ()-methyl 1-(8-chloro-2-quinolyl)-3,3-dimethyl-piperidine-4-carboxylate (1.1 g, 3.3 mmol), MeOH (0.8 mL), THF (3.3 mL), and 1 M aqueous NaOH (3.3 mL, 17 mmol) in a microwave vessel. Heat the resulting mixture at 120 C. for 2 hours in a microwave. Cool the mixture to room temperature and then add 5 N aqueous HCl until the pH is 6. Extract the mixture with EtOAc (310 mL). Combine the organic extracts; dry over Na.sub.2SO.sub.4; filter; collect the filtrate; and concentrate under reduced pressure to give the title compound (815 mg, 77%). MS (m/:): 318 (M+H).sup.+.

Preparation 20

Methyl 4-anilino-2,5-dihydrofuran-3-carboxylate

[0085] ##STR00036##

[0086] Add p-toluenesulfonic acid (500 mg, 3.16 mmol) to a mixture of ()-methyl 4-oxotetrahydrofuran-3-carboxylate (6.6 g, 45.8 mmol), aniline (4.3 mL, 47.2 mmol) and toluene (70 mL). Fit the reaction vessel with a Dean-Stark trap and heat to 140 C. Stir the resulting mixture at room temperature overnight. Heat the reaction to 140 C. and stir for 2 hours. Cool the mixture and add diethyl ether (100 mL) and water (100 mL). Extract the mixture with diethyl ether (350 mL); dry over MgSO.sub.4; filter; collect the filtrate; and concentrate under reduced pressure. Subject the resulting crude material to flash chromatography on silica gel, eluting with a gradient of 0% to 100% EtOAc in heptane, to give the title compound (6.7 g, 67%). MS (m/z): 220 (M+H).sup.+.

Preparation 21

()-Methyl-3-(iodomethyl)-4-phenylimino-tetrahydrofuran-3-carboxylate

[0087] ##STR00037##

[0088] Add a solution of ()-methyl 4-anilino-2,5-dihydrofuran-3-carboxylate (3.6 g, 16.4 mmol) and 18-crown-6 (4.8 g, 18 mmol) in toluene (15 mL) to a mixture of potassium tert-butoxide (2.0 g, 17.8 mmol) and toluene (15 mL). Stir the resulting mixture at room temperature for 30 minutes. Add diiodomethane (4 mL) and stir the reaction at room temperature overnight. Add water (50 mL) and extract with diethyl ether (250 mL). Collect the organic extracts and wash with brine (50 mL). Dry the organic extracts over MgSO.sub.4; filter; collect the filtrate; and concentrate under reduced pressure. Subject the resulting crude material to flash chromatography on silica gel, eluting with a gradient of 0% to 60% EtOAc in heptane, to give the title compound (1.0 g, 17%). MS (m/z): 359 (M+H).sup.+.

Preparation 22

()-Methyl 5-oxotetrahydropyran-3-carboxylate

[0089] ##STR00038##

[0090] Heat a mixture of tri-n-butyltin hydride (1.12 mL, 4.18 mmol), 2,2-azobis(2-methylpropionitrile) (35 mg, 0.21 mmol) and toluene (10 mL) to reflux. Add a solution of ()-methyl-3-(iodomethyl)-4-phenylimino-tetrahydrofuran-3-carboxylate (1.0 g, 2.78 mmol) in toluene (50 mL) drop-wise over three hours. Stir the resulting mixture at reflux for 3 hours. Concentrate the reaction under reduced pressure. Subject the resulting crude material to flash chromatography on silica gel, eluting with a gradient of 0% to 80% EtOAc in heptane, to give the title compound (0.37 g, 84%). .sup.1H NMR (400 MHz, CDCl.sub.3) 4.09-3.99 (m, 4H), 3.72 (s, 3H), 3.16 (m, 1H), 2.82 (dd, J=7.2, 16.9 Hz, 1H), 2.66 (dd, J=6.3, 16.9 Hz, 1H).

Preparation 23

()-Methyl trans-5-(benzylamino)tetrahydropyran-3-carboxylate

[0091] ##STR00039##

[0092] Add sodium triacetoxyborohydride (0.96 g, 4.53 mmol) to a mixture of ()-methyl 5-oxotetrahydropyran-3-carboxylate (0.33 g, 2.08 mmol), benzylamine (0.31 mL, 2.84 mmol) and 1,2-dichloroethane (5 mL). Stir the resulting mixture at room temperature for 2.5 hours. Add a saturated aqueous solution of sodium bicarbonate (10 mL); extract with DCM (310 mL); separate organic layer; wash with brine (10 mL); dry over MgSO.sub.4; filter; collect the filtrate; and concentrate under reduced pressure. Subject the resulting crude material to flash chromatography on silica gel, eluting with a gradient of 0% to 60% EtOAc in heptane, to give the title compound (0.085 g, 16% yield). MS (m/z): 250 (M+H).sup.+.

Preparation 24

()-Methyl trans-5-aminotetrahydropyran-3-carboxylate

[0093] ##STR00040##

[0094] Add 10% palladium on carbon (0.07 g, 0.66 mmol) to a mixture of ()-methyl trans-5-(benzylamino)tetrahydropyran-3-carboxylate (0.085 g, 0.34 mmol) and ethanol (8 mL). Purge the mixture with hydrogen and stir the resulting mixture at room temperature under hydrogen (1 atm) overnight. Filter the mixture and concentrate under reduced pressure to give the title compound (0.045 g, 83% yield). .sup.1H NMR (400 MHz, CDCl.sub.3) 3.82-3.77 (m, 2H), 3.68 (m, 4H), 3.38-3.31 (m, 1H), 3.11-3.08 (m, 1H), 2.85-2.80 (m, 1H), 2.17-2.11 (m, 1H), 1.78-1.71 (m, 3H).

Preparation 25

()-Methyl trans-5-[[(4S)-3,3-dimethyl-1-(8-methyl-2-quinolyl)piperidine-4-carbonyl]amino]tetrahydropyran-3-carboxylate

[0095] ##STR00041##

[0096] Add EDCI (0.087 g, 0.45 mmol) to a mixture of ()-(4S)-3,3-dimethyl-1-(8-methyl-2-quinolyl)piperidine-4-carboxylic acid (0.087 g, 0.29 mmol), ()-methyl-trans-5-aminotetrahydropyran-3-carboxylate (0.045 g, 0.28 mmol), HOBT (9 mg, 0.06 mmol), triethylamine (0.15 mL, 1.08 mmol), in DCM (5 mL). Stir the resulting mixture at room temperature for 3 hours. Subject the resulting mixture to flash chromatography on silica gel, eluting with a gradient of 5% to 100% EtOAc in heptane, to give the title compound (0.085 g, 66%). MS (m/z): 439 (M+H).sup.+.

Preparation 26

()-Methyl trans-3-(dibenzylamino)cyclohexanecarboxylate

[0097] ##STR00042##

[0098] Add potassium carbonate (3.53 g, 25.6 mmol) and benzyl bromide (1.9 mL, 15.9 mmol) to a mixture of ()-methyl-trans-3-aminocyclohexanecarboxylate (1.07 g, 6.8 mmol) in acetonitrile (40 mL). Heat the resulting mixture to 80 C. and stir for 5 hours. Cool the mixture and add acetonitrile (40 mL). Filter through diatomaceous earth and concentrate the filtrate under reduced pressure. Subject the resulting crude material to flash chromatography on silica gel, eluting with a gradient of 0% to 20% EtOAc in hexanes, to give the title compound (1.6 g, 70%). MS (m/z): 338 (M+H).sup.+.

Preparation 27

()-2-[trans-3-(Dibenzylamino)cyclohexyl]propan-2-ol

[0099] ##STR00043##

[0100] Add methylmagnesium bromide (3.0 M solution in diethyl ether, 16 mL, 48 mmol) to a mixture of ()-methyl-trans-3-(dibenzylamino)cyclohexanecarboxylate (1.6 g, 4.7 mmol) and THF (50 mL), maintained at 0 C. Allow the reaction to warm to room temperature and stir overnight. Add water (25 mL) and filter through diatomaceous earth. Collect the aqueous filtrate. Extract the resulting aqueous material with EtOAc (50 mL). Collect the organic extracts; dry over Na.sub.2SO.sub.4; filter; collect the filtrate; and concentrate under reduced pressure. Add EtOAc (50 mL); wash with saturated aqueous NH.sub.4Cl (225 mL); dry over Na.sub.2SO.sub.4; filter; collect the filtrate; and concentrate under reduced pressure to give the title compound (1.1 g, 69%). MS (m/z): 338 (M+H).sup.+.

Preparation 28

2-[(1S,3S)-3-(Dibenzylamino)cyclohexyl]propan-2-ol

[0101] ##STR00044##

[0102] Dissolve ()-2-[trans-3-(dibenzylamino)cyclohexyl]propan-2-ol (1.1 g, 3.26 mmol) in ethanol (13 mg/mL). Subject the material to chiral SFC (Chiralpak IA, 2 cm15 cm) using 12% (0.1% dimethylethylamine in i-PrOH) in CO.sub.2(scf) as the mobile phase at 220 nm and a flow rate of 70 mL/minute. Collect and combine the first fraction to elute and concentrate under reduced pressure, to provide the title compound (0.54 g, 49%), ee=>99%. MS (m/z): 338 (M+H).sup.+. SFC Analytical method (Chiralpak IA, (15 cm0.46 cm) using 10% (0.1% dimethylethylamine in i-PrOH) in CO.sub.2(scf) as the mobile phase at 220 nm and a flow rate of 3 mL/minutes).

Preparation 29

2-[(1S,3S)-3-Aminocyclohexyl]propan-2-ol hydrochloride

[0103] ##STR00045##

[0104] Add palladium hydroxide (20% on carbon, 0.51 g, 3.7 mmol) to a mixture of 2-[(1S,3S)-3-(dibenzylamino)cyclohexyl]propan-2-ol (0.54 g, 1.6 mmol) in MeOH (20 mL) in a Parr shaker vessel. Purge and pressurize the vessel with hydrogen to 414 kPa. Shake mixture for 4 days at room temperature. Filter the mixture through diatomaceous earth and concentrate the filtrate under reduced pressure. Dilute the mixture in diethyl ether (25 mL) and DCM (25 mL). Add HCl (4.0 M solution in 1,4-dioxane, 0.8 mL) and concentrate under reduced pressure to give the title compound (0.24 g, 77%). MS (m/z): 158 (M+H).sup.+.

Preparation 30

tert-Butyl N-[(1RS,2RS,4SR,6RS)-6-(hydroxymethyl)norbornan-2-yl]carbamate

[0105] ##STR00046##

[0106] Combine (acetylacetonato)dicarbonylrhodium (10 mg, 0.03 mmol), 1,1-bis(diphenylphosphino)ferrocene (27 mg, 0.05 mmol), methyl bicyclo[2.2.1]hept-2-ene-5-carboxylate (2.5 g, 11.9 mmol) and ter-butanol (25 mL) to a PARR autoclave with mechanical stirrer. Purge and pressurize the vessel with Syngas (1:1 mix of carbon monoxide and hydrogen, 310 kPa). Stir overnight. Concentrate the mixture under reduced pressure. Dilute in DCM (29 mL) and MeOH (2.9 mL). Add sodium borohydride (0.26 g, 6.9 mmol) and stir for 20 min at room temperature. Add DCM (60 mL) and wash with saturated aqueous sodium carbonate (225 mL) and brine (25 mL). Dry the organic layer over Na.sub.2SO.sub.4; filter; collect the filtrate; and concentrate under reduced pressure. Subject the resulting crude material to flash chromatography on silica gel eluting with a solution of 30% DCM, 30% tert-butyl methyl ether and 40% hexanes, to give the title compound as a mixture of diastereomers (0.58 g, 21%). .sup.1H NMR (400 MHz, CDCl.sub.3) 4.71-4.59 (m, 1H), 3.92-3.81 (m, 1H), 3.46-3.45 (m, 2H), 2.36 (d, J=3.0 Hz, 1H), 2.21-2.05 (m, 3H), 1.55 (br s, 1H), 1.49-1.25 (m, 12H), 1.16-1.09 (m, 1H), 0.68 (ddd, J=12.9, 4.5, 2.4 Hz, 1H).

Preparation 31

[(1RS,2RS,4SR,6RS)-6-Aminonorbornan-2-yl]methanol hydrochloride

[0107] ##STR00047##

[0108] Add HCl (4.0 M solution in 1,4-dioxane, 2.1 mL, 8.3 mmol) to a mixture of tert-butyl N-[(1RS,2RS,4SR,6RS)-6-(hydroxymethyl)norbornan-2-yl]carbamate (0.2 g, 0.83 mmol) in DCM (8 mL) maintained at 0 C. Allow the resulting mixture to warm to room temperature and stir for 3 hours. Concentrate the mixture under reduced pressure to give the title compound (0.15 g, 100%). .sup.1H NMR (400 MHz, CDCl.sub.3) 8.17-8.02 (m, 3H), 4.93-4.90 (br, 1H), 3.47-3.45 (m, 1H), 3.19-3.15 (m, 2H), 2.31 (m, 1H), 2.14-2.12 (m, 1H), 2.01-1.94 (m, 1H), 1.92-1.88 (m, 1H), 1.44-1.35 (m, 2H), 1.22 (m, 1H), 1.04-0.98 (m, 2H).

Preparation 32

tert-Butyl ((cis-4-((tert-butoxycarbonyl)amino)cyclohexyl)methyl)(sulfamoyl)carbamate

[0109] ##STR00048##

[0110] Add diisopropyl azodicarboxylate (1.6 mL, 7.8 mmol) to a mixture of tert-butyl cis-4-(hydroxymethyl)cyclohexylcarbamate (1.5 g, 6.5 mmol), tert-butyl N-sulfamoylcarbamate (1.9 g, 9.8 mmol), triphenylphosphine (2.1 g, 7.8 mmol), and EtOAc (33 mL). Stir overnight at room temperature. Add water (50 mL); extract with EtOAc (250 mL); collect the organic extracts. Wash the organic extracts with brine (25 mL); dry over MgSO.sub.4; filter; collect the filtrate; and concentrate under reduced pressure. Subject the resulting crude material to flash chromatography on silica gel, eluting with a gradient of 10% to 80% EtOAc in hexane, to give the title compound (1.78 g, 67%). MS (m/z): 430 (M+Na).sup.+.

Preparation 33

cis-1-amino-4-[(sulfamoylamino)methyl]cyclohexane hydrochloride

[0111] ##STR00049##

[0112] Add HCl (4.0 M solution in 1,4-dioxane, 15 mL, 60 mmol) to tert-butyl ((cis-4-((tert-butoxycarbonyl)amino)cyclohexyl)methyl)(sulfamoyl)carbamate (1.78 g, 4.37 mmol). Stir the resulting mixture overnight at room temperature. Concentrate under reduced pressure. Dilute in MeOH (10 mL) and add drop-wise diethyl ether (150 mL) while vigorously stirring. Collect the resulting white precipitate by vacuum filtration to give the title compound (0.68 g, 56%). MS (m/z): 208 (M+H).sup.+.

Preparation 34

Methyl (1RS,3RS)-3-((S)-3,3-dimethyl-1-(8-methylquinolin-2-yl)piperidine-4-carboxamido)cyclohexane-1-carboxylate

[0113] ##STR00050##

[0114] Add triethylamine (0.9 mL, 7 mmol) to a mixture of ()-(4S)-3,3-dimethyl-1-(8-methyl-2-quinolyl)piperidine-4-carboxylic acid (0.8 g, 3 mmol), ()-methyl trans-3-aminocyclohexanecarboxylate hydrochloride (0.5 g, 3 mmol), BOP (2.0 g, 3 mmol) and DMF (5 mL). Stir the resulting mixture at room temperature overnight. Add a saturated aqueous solution of sodium bicarbonate (20 mL) and extract with EtOAc (225 mL). Collect the organic extracts; wash with brine (25 mL); dry over MgSO.sub.4; filter; collect the filtrate; and concentrate under reduced pressure. Subject the resulting crude material to flash chromatography on silica gel, eluting with a gradient of 10% to 90% EtOAc in hexanes, to give the title compound (1.00 g, 80%). MS (m/z): 438 (M+H).sup.+.

EXAMPLE 1

(S)N-((2S,4S)-2-(Hydroxymethyl)tetrahydro-2H-pyran-4-yl)-3,3-dimethyl-1-(8-methylquinolin-2-yl)piperidine-4-carboxamide

[0115] ##STR00051##

[0116] Add benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (153 g, 293 mmol) as a slurry in DMF (152 mL) to a cold (10 C.) mixture of ()-(4S)-3,3-dimethyl-1-(8-methyl-2-quinolyl)piperidine-4-carboxylic acid (76.0 g, 245 mmol), ((2S,4S)-4-aminotetrahydro-2H-pyran-2-yl)methanol hydrochloride (72% pure, 57.37 g, 246 mmol), triethylamine (153 mL, 1.10 mol), and DMF (380 mL) while maintaining the internal reaction temperature below 20 C. Thereafter allow the mixture to warm to room temperature and stir it for 30 minutes. Pour the mixture into ice water (1.5 L) while stirring it. Extract the mixture with DCM (2600 mL) and wash the combined organic extracts with a half-saturated aqueous NaCl solution (1.0 L). Dry the organic solution over MgSO.sub.4; filter; collect the filtrate; and concentrate the filtrate under reduced pressure to give a wet solid. Triturate the wet solid with water and isolate the solid by filtration. Triturate the solid again with water (1.5 L) and isolate the solid by filtration. Wash the solid with water (2250 mL). Set this first batch of isolated solid aside. Collect the aqueous washings; extract with DCM (2800 mL); combine the DCM extracts. Add the first batch of isolated solids to the combined DCM extracts and wash with aqueous HCl (2.5 M, 2800 mL). Separate the aqueous layer and add 50% aqueous NaOH solution to the aqueous layer until the pH reaches 12 to induce precipitation. Filter the mixture to collect the resulting solid. Rinse the solid with water (2300 mL). Dry the solid in a vacuum oven at 50 C. for 3 hours. Dissolve the solid in MeOH (1.2 L); add mercaptopropyl mesoporous silica scavenging resin (1.2 mmol/g, 715 m.sup.2/g, 54 m average particle size); and stir at 50 C. overnight. Filter to remove the solids collecting the filtrate. Rinse the solid with 1:1 CH.sub.2Cl.sub.2:MeOH (2500 mL). Combine the filtrates and concentrate under reduced pressure to give a white solid. Triturate the solid with TBME (1.5 L), and collect the solids. Crystallize the solid from hot EtOH (1.8 L). Dry the solid in a vacuum oven at 50 C. for 2.5 hours to give the title compound as a crystalline, white solid (76.7 g, 76%). LC/MS (ESI.sup.+): 412 [M+H]+.

[0117] The following Examples in Table 1 can be prepared essentially by the procedure of Preparation 34 using the appropriate starting amine in place of ()-methyl-trans-3-aminocyclohexanecarboxylate hydrochloride and the appropriate starting carboxylic acid in place of ()-(4S)-3,3-dimethyl-1-(8-methyl-2-quinolyl)piperidine-4-carboxylic acid.

TABLE-US-00001 TABLE 1 MS Ex. (m/z) Purif.- No. Structure Chemical Name (M + H) Meth 2 [00052] (4S)-N-[4-(Hydroxymethyl)-cis- cyclohexyl]-3,3-dimethyl-1-(8- methyl-2-quinolyl)piperidine-4- carboxamide 410 A 3 [00053] (4S)-N-[6-(1S,2S,4R,6S)- (Hydroxymethyl)norbornan-2-yl]- 3,3-dimethyl-1-(8-methyl-2- quinolyl)piperidine-4- carboxamide 422 B 4 [00054] (4S)-1-(8-Chloro-2-quinolyl)-N- [4-(hydroxymethyl)-cis- cyclohexyl]-3,3-dimethyl- piperidine-4-carboxamide 430 A 5 [00055] (4S)-N-[(1S,3S)-3- (Hydroxymethyl)cyclohexyl]-3,3- dimethyl-1-[4- (trifluoromethyl)phenyl]piperidine- 4-carboxamide 413 C 6 [00056] (4S)-1-(5,8-Dimethyl-2- quinolyl)-N-[4-(hydroxymethyl)- cis-cyclohexyl]-3,3-dimethyl- piperidine-4-carboxamide 424 B 7 [00057] (4S)-N-Cyclopentyl-3,3- dimethyl-1-(8-methyl-2- quinolyl)piperidine-4- carboxamide 366 B 8 [00058] (4S)-N-[(1R)-1,2- Dimethylpropyl]-3,3-dimethyl-1- (8-methyl-2-quinolyl)piperidine- 4-carboxamide 368 B 9 [00059] (4S)-N-[3-(1S,3S)- (Methanesulfonamido)cyclohexyl]- 3,3-dimethyl-1-(8-methyl-2- quinolyl)piperidine-4- carboxamide 473 B 10 [00060] (4S)-N-[3-(1S,3S)- (Hydroxymethyl)cyclohexyl]-3,3- dimethyl-1-(8-methyl-2- quinolyl)piperidine-4- carboxamide 410 A 11 [00061] (4S)-3,3-Dimethyl-1-(8-methyl-2- quinolyl)-N-[4-cis- [(sulfamoylamino)methyl]cyclohexyl] piperidine-4-carboxamide 488 A 12 [00062] (4S)-3,3-Dimethyl-1-(8-methyl-2- quinolyl)-N-[(3R)- tetrahydrofuran-3-yl]piperidine-4- carboxamide 368 A

[0118] Purification Methods (Purif Meth): A=Crude product is purified by silica gel normal phase column chromatography eluting with gradient of EtOAc in hexanes. B=Crude product is purified by C18 reverse phase column chromatography eluting with a gradient of acetonitrile and water. C=Crude product is purified by chiral column chromatography with Chiralpak AS-H, 21150 mm) using 15% IPA in CO.sub.2 as the mobile phase at a flow rate of 70 mL/min.

EXAMPLE 13

(4S)N-[(1S,3S)-3-(1-Hydroxy-1-methyl-ethyl)cyclohexyl]-3,3-dimethyl-1-(8-methyl-2-quinolyl)piperidine-4-carboxamide

[0119] ##STR00063##

[0120] Add N,N-diisopropylethylamine (0.7 mL, 4 mmol) and HATU (0.31 g, 0.8 mmol) to a mixture of ()-(4S)-3,3-dimethyl-1-(8-methyl-2-quinolyl)piperidine-4-carboxylic acid (0.2 g, 0.67 mmol), 2-[(1S,3S)-3-aminocyclohexyl]propan-2-ol hydrochloride (0.13 g, 0.67 mmol) and DMF (5 mL). Stir the resulting mixture at room temperature for 45 minutes. Subject the resulting reaction mixture to reverse phase flash chromatography on C18, eluting with a gradient of 5% to 80% ACN in water, to give the title compound (0.26 g, 88%). MS (m/z): 438 (M+H).sup.+.

[0121] Prepare the following Examples in Table 2 essentially by the procedure for Example 13 using the appropriate starting carboxylic acid in place of ()-(4S)-3,3-dimethyl-1-(8-methyl-2-quinolyl)piperidine-4-carboxylic acid.

TABLE-US-00002 TABLE 2 MS Ex. (m/z) No. Structure Chemical Name (M + H) 14 [00064] (4S)-N-[(1S,3S)-3-(1-Hydroxy-1- methyl-ethyl)cyclohexyl]-3,3- dimethyl-1-[4- (trifluoromethyl)phenyl]piperidine- 4-carboxamide 441 15 [00065] (4S)-N-[(1S,3S)-3-(1-Hydroxy-1- methyl-ethyl)cyclohexyl]-3,3- dimethyl-1-[5- (trifluoromethyl)pyrimidin-2- yl]piperidine-4-carboxamide 442

[0122] Prepare the following Examples in Table 3 essentially by the procedure for Preparation 25 using the appropriate starting amine in place of methyl ()-trans-5-aminotetrahydropyran-3-carboxylate and the appropriate starting carboxylic acid in place of ()-(4S)-3,3-dimethyl-1-(8-methyl-2-quinolyl)piperidine-4-carboxylic acid.

TABLE-US-00003 TABLE 3 MS Purifica- Ex. (m/z) tion No. Structure Chemical Name (M + H) Method 16 [00066] (4S)-1-(8-Methyl-2-quinolyl)- 3,3-dimethyl-N-[(3S)- tetrahydropyran-3-yl]piperidine- 4-carboxamide 382 A 17 [00067] (4S)-3,3-Dimethyl-1-(8-methyl- 2-quinolyl)-N-[(1S,3S)-3- methylsulfonylcyclohexyl] piperidine-4-carboxamide 458 C

[0123] Purification Methods: A=Crude product is purified by silica gel normal phase column chromatography, eluting with a gradient of EtOAc in hexanes. C=Crude product is purified by chiral column chromatography using chiral SFC (Chiralcel OD-H, 21250 mm) using 25% EtOH (0.2% IPAm) in CO.sub.2(scf) as the mobile phase at a flow rate of 70 mL/min.

EXAMPLE 18

(S)N-((3RS,5SR)-5-(Hydroxymethyl)tetrahydro-2H-pyran-3-yl)-3,3-dimethyl-1-(8-methylquinolin-2-yl)piperidine-4-carboxamide

[0124] ##STR00068##

[0125] Add a 5 N aqueous NaOH solution (0.5 mL) to a mixture of methyl (3RS,5RS)-5-((4S)-3,3-dimethyl-1-(8-methyl-2-quinolyl)piperidine-4-carbonyl]amino]tetrahydropyran-3-carboxylate (0.085 g, 0.19 mmol), MeOH (2 mL), and THF (2 mL). Stir the resulting mixture at room temperature for 1 hour. Add a 5 N aqueous HCl solution (0.5 mL) and concentrate. Dilute the crude mixture in THF (6 mL) and cool to 0 C. Add a 2 M solution of borane-THF complex in THF (0.15 mL, 0.3 mmol) drop-wise. Slowly warm the mixture to room temperature and stir at room temperature overnight. Add a saturated aqueous solution of sodium bicarbonate (10 mL) and extract the mixture with EtOAc (310 mL). Collect the organic extracts; wash with brine (10 mL); dry over MgSO.sub.4; filter; collect the filtrate; and concentrate under reduced pressure. Subject the resulting crude material to flash chromatography on silica gel eluting with 100% EtOAc to give the title compound (0.046 g, 59% yield over 2 steps). MS (m/z): 411 (M+H).sup.+.

EXAMPLE 19

Methyl (1RS,3RS)-3-((S)-3,3-Dimethyl-1-(8-methyl-2-quinolyl)piperidine-4-carbonyl]amino]cyclohexanecarboxylic acid

[0126] ##STR00069##

[0127] Add a 5 N aqueous NaOH solution (2.2 mL) to a mixture of methyl ()-trans-[[(4S)-3,3-dimethyl-1-(8-methyl-2-quinolyl)piperidine-4-carbonyl]amino]cyclohexane-3-carboxylate (1.0 g, 2.2 mmol), MeOH (2 mL), and THF (8 mL). Stir the resulting mixture at room temperature overnight. Add a 5 N aqueous HCl solution (2.2 mL) to adjust the pH to 6.5. Extract with EtOAc (225 mL) and collect the organic extracts. Wash the organic extracts with brine (25 mL); dry over MgSO.sub.4; filter; collect the filtrate; and concentrate under reduced pressure to give the title compound (0.935 g, 98%). MS (m/z): 424 (M+H).sup.+.

EXAMPLE 20

(S)N-((1RS,3RS)-3-(Ethylcarbamoyl)cyclohexyl)-3,3-dimethyl-1-(8-methylquinolin-2-yl)piperidine-4-carboxamide

[0128] ##STR00070##

[0129] Add triethylamine (0.7 mL, 5 mmol) to a mixture of ()-trans-3-[[(4S)-3,3-dimethyl-1-(8-methyl-2-quinolyl)piperidine-4-carbonyl]amino]cyclohexanecarboxylic acid (0.9 g, 2 mmol), ethanamine (2 mL, 3 mmol), BOP (1.0 g, 3 mmol) and DMF (4 mL). Stir the resulting mixture at room temperature for 3 hours. Add a saturated aqueous solution of sodium bicarbonate (20 mL). Extract the resulting mixture with EtOAc (250 mL). Collect the organic extracts; wash with brine (325 mL); dry over MgSO.sub.4; filter; collect the filtrate; and concentrate under reduced pressure. Subject the resulting material to reverse-phase flash chromatography on C.sub.18 silica gel, eluting with a gradient of 20% to 80% acetonitrile (0.1% formic acid) in water (0.1% formic acid), to give the title compound (0.25 g, 30%). MS (m/z): 450 (M+H).sup.+.

Biological Assays

[0130] Human mPGES-1 Enzyme Inhibition Assay

[0131] Human mPGES-1 (Invitrogen (Cat#97002RG, clone ID 6374722)) is subcloned into pcDNA3.1 and transiently expressed in 293E cells. Microsomes are prepared from cell pellets based on published methods (Oullet et al., Purification and characterization of recombinant microsomal prostaglandin E synthase-1, Protein Expression and Purification, 26 pp 489-495 (2002); and Thoren et al., Human Microsomal Prostanglandin E Synthase-1, J. Biol Chem. 278(25) pp 22199-22209 (2003)). Briefly, pellets are brought up in homogenization buffer (15 mM Tris-HCl, pH 8.0; 0.25 M sucrose; 0.1 mM EDTA; 1 mM glutathione) and sonicated 530 seconds on ice. Homogenate is centrifuged at 5,000g for 10 minutes at 4 C. The supernatant fraction is decanted and loaded into Beckman Quick-Seal tubes and centrifuged at 150,000g. for 90 minutes at 4 C. The supernatant fraction is discarded by decantation and the pellets are re-suspended in assay buffer (10 mM sodium phosphate, pH 7.0; 10% glycerol; 2.5 mM glutathione; Complete Protease Inhibitor Cocktail (Roche)). Protein concentration is determined using the Pierce Coomassie Plus reagent.

[0132] For the enzyme assay, the microsomes are diluted into assay buffer and 14 L/well of the resulting solution is added to 384 well assay plates. Compound dilution plates (Nunc Cat#249944) are generated on a Tecan_MC384 and 4 L/well are added to the assay plates. Prostaglandin H.sub.2 (PGH.sub.2) is diluted into assay buffer immediately before use and 14 L/well is added to the assay plates. Final concentrations are 6.55 g/mL microsomes and 1.67 M PGH.sub.2. After a 1.5 minute incubation at room temperature, 5 L/well of 1 mg/mL SnCl.sub.2 in 0.5 N HCl is added to stop the reaction. Five L of the stopped reaction are transferred to a 384 well plate containing 45 L of 0.1% formic acid, and the plates are stored at 80 C. The plates are shipped to Agilent Technologies, formerly Biocius Lifesciences (Wakefield, Mass. 01880) for standard LC/MS analysis for PGE.sub.2. The data are used to calculate the IC.sub.50 (M). The compounds of the Examples inhibit human mPGES-1 with an IC.sub.50 M value of less than 100 nM. The exemplified compound of Example 1 inhibits human mPGES-1 with an IC.sub.50 M value of 0.00193, 0.00064, n=17. This result demonstrates that the exemplified compound of Example 1 is a potent inhibitor of the mPGES-1 enzyme in an isolated enzyme preparation.

Cell Based Assay for Measuring Eicosanoid Selectivity

[0133] Human epithelial lung carcinoma cell line A549 is obtained from ATCC (CCL-185) and maintained in Kaighn's F12 cell culture medium+10% fetal bovine serum (FBS) (plating medium) under standard 5% CO.sub.2 humidified atmosphere at 37 C. The cells are passaged at 1:3 twice per week.

[0134] For the assays, cells are released from flasks by washing once with PBS, then once with Trypsin/EDTA. After 3-5 minutes at 37 C., the cells are suspended in plating medium and centrifuged at 2,000 rpm, 25 C., for 5 minutes. The supernatant is aspirated and the cell pellet is resuspended in F12K plating medium. The cell number is determined by counting an aliquot of cells which has been diluted in PBS and Trypan blue on a hemocytometer. Cells are plated at 40,000/well in 96 well Falcon plates 24 hours prior to treatment. Compounds are diluted in DMSO to 100 of the final concentration in Screen Mates tubes. The medium is removed from the cells and fresh medium (90 L/well) is added to the cells. The compounds are added at 1 L/well, n=2, to give seven concentrations each. Cells are pretreated with compounds for 30 minutes at 37 C., 5% CO.sub.2. Prostaglandin E.sub.2 production is induced by the addition of recombinant human interleukin (rhIL-1) diluted in plating medium to 10final. A 10 L/well aliquot is added to give a final rhIL-1 concentration of 0.1 ng/mL. The treatment period is approximately 18 hours. Conditioned medium is removed to v-bottom polypropylene plates. The conditioned medium is assayed for levels of PGE.sub.2 and prostaglandin I.sub.2 (PGI.sub.2) by specific enzyme immune-assay EIAs, according to the manufacturer's protocols (Cayman). Briefly, conditioned medium (1 L) is added to each well of a 96 well plate coated with a capture antibody and containing EIA buffer (49 L) supplied by the manufacturer. The tracer is diluted with the EIA buffer (50 L). The detection antibody is diluted with the EIA buffer (50 L). The plate is covered with adhesive sealing film and is incubated for 1 hour at room temperature on an orbital shaker at 100 rpm. The wash buffer is diluted into MILLIPORE purified water, and the plate is washed 5350 L/well, using a plate washer. The substrate (Ellman's reagent) is diluted with MILLIPORE purified water and then added to the plate at 200 L/well. After approximately 90-120 minutes at room temperature on an orbital shaker at 100 rpm, the plates are read at A412 on a plate reader. A standard curve of PGE.sub.2 is used to calibrate the unknowns. The exemplified compound of Example 1 inhibits PGE.sub.2 formation with an IC.sub.50 of 0.00471 M+0.00301, n=2.

Human Whole Blood Assay

[0135] Blood is collected from normal volunteer donors into sodium heparin VACUTAINER tubes. Donors are selected, in part, on their confirmation that they have not taken NSAIDs, aspirin, Celebrex, or glucocorticoids within two weeks of the donation. All tubes/donor are pooled into 250 mL Corning conical centrifuge tubes and 436.5 L/well is distributed into deep well polypropylene plates. Compounds are diluted in DMSO to 100 final and 4.5 UL/well in duplicate or triplicate is added to give 7 point curves. The blood is pretreated with compounds at 37 C., 5% CO.sub.2, in a humidified atmosphere, covered with a MicroClime Environmental Microplate lid, for 30 minutes after which 9 L/well of a solution of 5 mg/mL of LPS (Sigma, serotype 0111:B4) in 1 mg/mL BSA/PBS is added to give a final LPS concentration of 100 g/mL. The plates are incubated for 20-24 hours, at 37 C., 5% CO.sub.2, in a humidified atmosphere. The plates are tightly sealed with the aluminum foil lids and are chilled on ice for approximately 1 hour. Then the plates are centrifuged at 1,800g, 10 minutes, 4 C., in an Eppendorf 5810R centrifuge. Plasma is removed from the cell layer using the Rainin L200 with sterile filtered tips and transferred to v-bottom polypropylene plates. One hundred microliters is quantitatively transferred to Costar cluster tubes blocks and 400 L/well of the MeOH stop reagent and internal standards, d.sub.4-PGE.sub.2, d.sub.4-PGF.sub.2a, and d.sub.4-TXA.sub.2p are added. Samples are vortexed for 5 minutes and are placed at 20 C. for at least one hour. Samples are centrifuged for 10 minutes at 4000 rpm in an Eppendorf 5810R.

[0136] Solid phase extraction is performed using Waters HLB 30 mg/bed 96 well plates on a vacuum manifold: Step 1, the matrix is washed with MeOH (1 mL), followed by 0.1% formic acid in water (1 mL); Step 2, 400 L sample is applied along with 0.1% formic acid in water (900 L) and allowed to bind for 5 minutes; Step 3, the matrix is washed with 0.1% formic acid in water (600 L), followed by 80/20 water/MeOH (600 L); Step 4, the products are eluted with 2-500 L volumes of EtOAc; Step 5 the samples are dried under nitrogen and reconstituted in 75/25 water/acetonitrile with 0.1% formic acid (50 L). The products are analyzed by LC/MS/MS. Example 1 inhibits The PGE.sub.2 formation in this assay with an IC.sub.50 of 0.002050.00082, n=11. This result supports that Example 1 inhibits PGE.sub.2 synthesis in human whole blood.