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, formula (1), 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.

##STR00001##

Claims

1. A compound of Formula 1: ##STR00070## wherein: R1 is H or —CH.sub.3; R is selected from: ##STR00071## and G is selected from: ##STR00072## or a pharmaceutically acceptable salt thereof.

2. A compound according to Formula 2: ##STR00073## wherein: R1 is H or —CH.sub.3; R is selected from: ##STR00074## and G is selected from: ##STR00075## or a pharmaceutically acceptable salt thereof.

3. A compound according to claim 1: wherein: R1 is H or —CH.sub.3; R is selected from: ##STR00076## and G is selected from: ##STR00077## or a pharmaceutically acceptable salt thereof

4. A compound according to claim 3, or a pharmaceutically acceptable salt thereof, wherein R is selected from: ##STR00078##

5. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R is selected from: ##STR00079##

6. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein G is: ##STR00080##

7. A compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R1 is —CH.sub.3.

8. A compound which is: ##STR00081## or a pharmaceutically acceptable salt thereof.

9. A compound according to claim 8, which is: ##STR00082##

10. A compound according to claim 1 wherein the pharmaceutically acceptable salt is a hydrochloride salt.

11. A pharmaceutically acceptable composition comprising a compound according to claim 1 and at least one of a pharmaceutically acceptable carrier, diluent, or excipient.

12. A method of treating a patient in need of treatment for pain associated with arthritis or osteoarthritis said method comprising administering to the patient an effective amount of a pharmaceutically acceptable composition according to claim 11.

13. A method of treating a patient in need of treatment for inflammation associated with arthritis or osteoarthritis, said method comprising administering to the patient an effective amount of a pharmaceutically acceptable composition according to claim 11.

14. A method of treating a patient in need of treatment for arthritis or osteoarthritis, said method comprising administering to the patient an effective amount of a compound according to claim 1.

15. A method of treating a patient in need of treatment for pain associated with arthritis, said method comprising administering to the patient an effective amount of a compound according to claim 1.

16. A method of treating a patient in need of treatment for pain associated with osteoarthritis, said method comprising administering to the patient an effective amount of a compound according to claim 1.

17. A method of treating a patient in need of treatment for inflammation associated with arthritis, said method comprising administering to the patient an effective amount of a compound according to claim 1.

18. A method of treating a patient in need of treatment for inflammation associated with osteoarthritis, said method comprising administering to the patient an effective amount of a compound according claim 1.

19-23. (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 a pharmaceutically acceptable composition according to claim 9.

Description

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] ##STR00050##

[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 stifling it. Extract the mixture with DCM (2×600 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 (2×250 mL). Set this first batch of isolated solid aside. Collect the aqueous washings; extract with DCM (2×800 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, 2×800 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 (2×300 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 μm 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 (2×500 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): 412 [M+H].sup.+.

[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 [00051] (4S)-N-[4-(Hydroxymethyl)-cis- cyclohexyl]-3,3-dimethyl-1-(8- methyl-2-quinolyl)piperidine-4- carboxamide 410 A  3 [00052] (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 [00053] (4S)-1-(8-Chloro-2-quinolyl)- N-[4-(hydroxymethyl)-cis- cyclohexyl]-3,3-dimethyl- piperidine-4-carboxamide 430 A  5 [00054] (4S)-N-[(1S,3S)-3- (Hydroxymethyl)cyclohexyl]- 3,3-dimethyl-1-[4- (trifluoromethyl)phenyl] piperidine-4-carboxamide 413 C  6 [00055] (4S)-1-(5,8-Dimethyl-2- quinolyl)-N-[4-(hydroxymethyl)- cis-cyclohexyl]-3,3-dimethyl- piperidine-4-carboxamide 424 B  7 [00056] (4S)-N-Cyclopentyl-3,3- dimethyl-1-(8-methyl-2- quinolyl)piperidine-4- carboxamide 366 B  8 [00057] (4S)-N-[(1R)-1,2- Dimethylpropyl]-3,3-dimethyl- 1-(8-methyl-2-quinolyl) piperidine-4-carboxamide 368 B  9 [00058] (4S)-N-[3-(1S,3S)- (Methanesulfonamido) cyclohexyl]-3,3-dimethyl-1-(8- methyl-2-quinolyl)piperidine-4- carboxamide 473 B 10 [00059] (4S)-N-[3-(1S,3S)- (Hydroxymethyl)cyclohexyl]-3, 3-dimethyl-1-(8-methyl-2- quinolyl)piperidine-4- carboxamide 410 A 11 [00060] (4S)-3,3-Dimethyl-1-(8-methyl- 2-quinolyl)-N-[4-cis- [(sulfamoylamino)methyl] cyclohexyl]piperidine-4- carboxamide 488 A 12 [00061] (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, 21×150 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] ##STR00062##

[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 [00063] (4S)-N-[(1S,3S)-3-(1- Hydroxy-1-methyl-ethyl) cyclohexyl]-3,3-dimethyl-1- [4-(trifluoromethyl)phenyl] piperidine-4-carboxamide 441 15 [00064] (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 [00065] (4S)-1-(8-Methyl-2-quinolyl)- 3,3-dimethyl-N-[(3S)- tetrahydropyran-3-yl] piperidine-4-carboxamide 382 A 17 [00066] (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, 21×250 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] ##STR00067##

[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 (3×10 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] ##STR00068##

[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 (2×25 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] ##STR00069##

[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 (2×50 mL). Collect the organic extracts; wash with brine (3×25 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 5×30 seconds on ice. Homogenate is centrifuged at 5,000×g for 10 minutes at 4° C. The supernatant fraction is decanted and loaded into Beckman Quick-Seal® tubes and centrifuged at 150,000×g. 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 μL/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 μL/well are added to the assay plates. Prostaglandin H.sub.2 (PGH.sub.2) is diluted into assay buffer immediately before use and 14 μL/well is added to the assay plates. Final concentrations are 6.55 μg/mL microsomes and 1.67 μM PGH.sub.2. After a 1.5 minute incubation at room temperature, 5 μL/well of 1 mg/mL SnCl.sub.2 in 0.5 N HCl is added to stop the reaction. Five μL of the stopped reaction are transferred to a 384 well plate containing 45 μL 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 (μM). The compounds of the Examples inhibit human mPGES-1 with an IC.sub.50 μM value of less than 100 nM. The exemplified compound of Example 1 inhibits human mPGES-1 with an IC.sub.50 μM 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 μL/well) is added to the cells. The compounds are added at 1 μL/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-1β) diluted in plating medium to 10× final. A 10 μL/well aliquot is added to give a final rhIL-1β 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 μL) is added to each well of a 96 well plate coated with a capture antibody and containing EIA buffer (49 μL) supplied by the manufacturer. The tracer is diluted with the EIA buffer (50 μL). The detection antibody is diluted with the EIA buffer (50 μL). 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 5×350 μL/well, using a plate washer. The substrate (Ellman's reagent) is diluted with MILLIPORE purified water and then added to the plate at 200 μL/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 μM±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 μL/well is distributed into deep well polypropylene plates. Compounds are diluted in DMSO to 100× final and 4.5 μL/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 μL/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 μg/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 1,800×g, 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 μL/well of the MeOH stop reagent and internal standards, d.sub.4-PGE.sub.2, d.sub.4-PGF.sub.2α, and d.sub.4-TXA.sub.2β 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 μL sample is applied along with 0.1% formic acid in water (900 μL) and allowed to bind for 5 minutes; Step 3, the matrix is washed with 0.1% formic acid in water (600 μL), followed by 80/20 water/MeOH (600 μL); Step 4, the products are eluted with 2-500 μL volumes of EtOAc; Step 5 the samples are dried under nitrogen and reconstituted in 75/25 water/acetonitrile with 0.1% formic acid (50 μL). 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 0.00205±0.00082, n=11. This result supports that Example 1 inhibits PGE.sub.2 synthesis in human whole blood.