Protease inhibitors

Abstract

Compounds of the formula II: ##STR00001##
wherein R.sup.1 and R.sup.2 are independently H, F or CH.sub.3; or R.sup.1 forms an ethynyl bond and R.sup.2 is H or C.sub.3-C.sub.6 cycloalkyl which is optionally substituted with one or two substituents independently selected from methyl, CF.sub.3, OMe or halo; R.sup.3 is C.sub.1-C.sub.3 alkyl or C.sub.3-C.sub.6 cycloalkyl, either of which is optionally substituted with one or two methyl and/or a fluoro, trifluoromethyl or methoxy, when R.sup.3 is C.sub.3-C.sub.6 cycloalkyl it may alternatively be gem substituted with fluoro; R.sup.4 is methyl or fluoro; m is 0, 1 or 2; E is a bond, or thiazolyl, optionally substituted with methyl or fluoro; A.sub.1 is CH or N, A.sub.2 is CR.sup.6R7 or NR.sup.6, provided at least one of A.sub.1 and A.sub.2 comprises N; R.sup.6 is H, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.3 alkyl-O—C.sub.1-C.sub.3 alkyl, or when A.sub.2 is C, R.sup.6 can also be C.sub.1-C.sub.4 alkoxy or F; R.sup.7 is H, C.sub.1-C.sub.4 alkyl or F
or a pharmaceutically acceptable salt, N-oxide or hydrate thereof, have utility in the treatment of disorders characterized by inappropriate expression or activation of cathepsin K, such as osteoporosis, osteoarthritis, rheumatoid arthritis or bone metastases.

Claims

1. A compound of the formula Ih′″: ##STR00075## wherein the depicted N-Boc is optionally replaced by a conventional N-protecting group selected from the group consisting of formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, benzyl, and benzoxycarbonyl, and/or the depicted dimethylacetal protecting group is optionally replaced by 1,3-dioxolane protecting group.

2. The compound according to claim 1, consisting of the N-Boc and dimethylacetal protecting groups depicted in Formula Ih′″.

3. A method for the treatment of osteoarthritis, comprising the administration to a patient in need thereof a safe and effective amount of a compound of the formula II: ##STR00076## wherein: A.sub.1 is CH or N; A.sub.2 is CR.sup.6R.sup.7 or NR.sup.6, provided at least one of A.sub.1 and A.sub.2 comprises N; E is thiazolyl, optionally substituted with methyl or fluoro; m is 0, 1 or 2; n is 0 or 1, such that the ring containing A.sub.1 and A.sub.2 is a saturated, nitrogen-containing ring of 5 or 6 ring atoms; R is H, methyl, CF.sub.3, OMe or halo, located at any of the 1, 2 or 3 positions on the depicted cyclopropyl in Formula II; R.sub.3 is C.sub.1-C.sub.3 alkyl or C.sub.3-C.sub.6 cycloalkyl, either of which is optionally substituted with one or two methyl and/or a fluoro, trifluoromethyl or methoxy; R.sup.4 is methyl or fluoro; R.sup.6 is H, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 haloalkyl, C.sub.1-C.sub.3 alkyl-O—C.sub.1-C.sub.3 alkyl, or when A.sub.2 is C, R.sup.6 can also be C.sub.1-C.sub.4 alkoxy or F; R.sup.7 is H, C.sub.1-C.sub.4 alkyl or F; or a pharmaceutically acceptable salt, N-oxide or hydrate thereof, to a subject in need thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 depicts plasma concentration against time for male C57BI mice orally administered a compound of the invention or a compound of the prior art.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(2) Various embodiments of the invention will now be described by way of illustration only with reference to the following Examples and FIG. 1 which depicts plasma concentration against time for male C57BI mice orally administered a compound of the invention or a compound of the prior art.

Reference Example 1

(3) A P3 Building Block

Step a) 4-Cyanopropiophenone

(4) ##STR00023##

(5) As described for the preparation of 4-cyanoacetophenone (Synth. Commun 1994, 887-890), a mixture of 4-bromopropiophenone (5.65 g, 26.4 mmol), Zn(CN).sub.2 (1.80 g, 15.3 mmol), and Pd(PPh.sub.3).sub.4 (2.95 g, 2.6 mmol) was refluxed at 80° C. in deoxygenated DMF (35 mL, stored over 4 Å molecular sieves, bubbled with Ar before use) for 18 h. The mixture was partitioned between toluene (100 mL) and 2N NH.sub.4OH (100 mL). The organic phase was extracted with 2N NH.sub.4OH (100 mL), washed with saturated aqueous NaCl (2×100 mL), dried, and evaporated. A 10 mmol scale reaction was done similarly and the crude products were combined. Flash chromatography (330 g silica, 6/1 petroleum ether-EtOAc) gave white solids (5.17 g, 89%).

(6) 1H NMR (CDCl.sub.3) δ ppm: 1.22 (t, 3H, J=7.2 Hz), 3.00 (q, 2H, J=7.3 Hz), 7.75 (d, 2H, J=8.8 Hz), 8.03 (d, 2H, J=8.4 Hz)

(7) 13C NMR (CDCl.sub.3) δ ppm: 7.8, 32.1, 116.1, 117.9, 128.3, 132.4, 139.7, 199.2

Step b) 4-Propionylbenzoic Acid

(8) ##STR00024##

(9) 4-Cyanopropiophenone (4.67 g, 29.3 mmol) was refluxed with 2N NaOH (90 mL, 180 mmol) and dioxane (90 mL) at 95° C. overnight. The mixture was diluted with water (150 mL), washed with ether (75 mL), acidified to pH 2 with concentrated HCl, and extracted with ether (3×75 mL). The organic phase was washed with saturated aqueous NaCl (3×75 mL), dried, and evaporated to give yellow solids (5.12 g, 98%).

(10) 1H NMR (CDCl.sub.3+CD.sub.3OD) δ ppm: 1.18 (t, 3H, J=7.2 Hz), 2.99, (q, 2H, J=7.1 Hz), 7.95 (d, 2H, J=8.4 Hz), 8.08 (d, 2H, J=8.8 Hz)

(11) 13C NMR (CDCl.sub.3) δ ppm: 7.9, 32.1, 127.7, 130.0, 134.0, 140.0, 168.0, 200.8

Step c) Methyl 4-propionylbenzoate

(12) ##STR00025##

(13) The benzoic acid above (890 mg, 5 mmol), NaHCO.sub.3 (1.26 g, 15 mmol) and iodomethane (935 μL, 15 mmol) in DMF (10 mL) were stirred at RT overnight. The mixture was diluted with saturated aqueous NaCl (50 mL) and extracted with ether (3×50 mL). The organic phase was washed with water (50 mL), dried, and evaporated. Flash chromatography (90 g silica, 2/1 petroleum ether-EtOAc) gave white solids (744 mg, 77%).

(14) 1H NMR (CDCl.sub.3) δ ppm: 1.24 (t, 3H, J=7 Hz), 3.03 (q, 2H, J=7 Hz), 3.95 (s, 3H), 8.0 and 8.12 (ABq, 4H)

Step d) Methyl 4-(2-bromopropionyl)benzoate

(15) ##STR00026##

(16) Methyl 4-propionylbenzoate (744 mg, 3.87 mmol), pyrrolidone hydrotribromide (1.98 g), and 2-pyrrolidinone (380 mg, 4.5 mmol) in THF (38 mL) were heated at 50° C. under nitrogen for 3 h. The mixture was cooled, filtered, concentrated, and then redissolved in ether (50 mL). The ether solution was washed successively with water (20 mL), saturated aqueous Na.sub.2S.sub.2O.sub.5 (20 mL), saturated aqueous NaCl (20 mL), and water (20 mL), dried and evaporated to give a yellow oil (1.025 g) that was used directly in the Hantzsch coupling. This material contained 91% of the desired bromoketone, 5% starting ketone, and 4% 4-bromo-1-butanol, as determined by 1H NMR.

(17) 1H NMR (CDCl.sub.3) δ ppm: 1.92 (d, 3H, J=7 Hz), 3.96 (s, 3H), 5.28 (q, 1H, J=7 Hz), 8.07 and 8.14 (ABq, 4H)

Step e) 4-[2-(4-tert-Butoxycarbonylpiperazin-1-yl)-5-methylthiazol-4-yl]benzoic Acid Methyl Ester

(18) ##STR00027##

(19) All of the α-bromoketone above and 4-thionocarbonylpiperazine-1-carboxylic acid tert-butyl ester (J. Med. Chem., 1998, 5037-5054, 917 mg, 3.73 mmol) were refluxed in 36 mL THF at 70° C. for 2 h, under N.sub.2. The precipitate was filtered and the filtrate evaporated to give yellow solids. Flash column chromatography (silica, 5/1 petroleum ether-EtOAc) gave 624 mg of light yellow solids. Chromatography of the precipitate (silica, 2/1 petroleum ether-EtOAc) gave 32 mg more of compound. Total yield is 44%.

(20) 1H NMR (CDCl.sub.3) δ ppm: 1.46 (s, 9H), 2.43 (s, 3H), 3.42, (m, 4H), 3.54 (m, 4H), 3.90 (s, 3H), 7.68 and 8.04 (ABq, 4H).

Step f) 4-[2-(4-tert-Butoxycarbonylpiperazin-1-yl)-5-methylthiazol-4-yl]benzoic Acid

(21) ##STR00028##

(22) The above methyl ester (564 mg, 1.35 mmol) was heated with 1.35 mL 2N NaOH, 5 mL THF, and 3.65 mL water at 60° C. for 4 h. The reaction mixture was evaporated, poured into 20 mL saturated aqueous NaCl and 20 mL CH.sub.2Cl.sub.2, and then acidified to pH 3 with 5% citric acid, in an ice bath. The layers were separated and the organic phase was extracted further with 2×10 mL CH.sub.2Cl.sub.2. The organic phases were combined, washed with water (10 mL), dried, and evaporated to give light yellow solids (537 mg, 98%).

(23) 1H NMR (CDCl.sub.3) δ ppm: 1.48 (s, 9H), 2.47 (s, 3H), 3.47 (m, 4H), 3.57 (m, 4H), 7.74 and 8.12 (ABq, 4H).

(24) 13C NMR (CDCl.sub.3) δ ppm: 12.6, 28.3, 42.8, 48.1, 80.3, 119.1, 127.8, 128.2, 130.1, 140.5, 145.6, 154.6, 167.2, 171.4.

(25) LCMS: (M+H).sup.+ 404, (M−H).sup.− 402.

Step g) 4-[5-methyl-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]benzoic Acid

(26) ##STR00029##

(27) 4-[4-(4-Carboxy-phenyl)-5-methyl-thiazol-2-yl]-piperazine-1-carboxylic acid tert-butyl ester (0.421 mmol) was dissolved in 4M HCl in 1,4-dioxane, and stirred at room temperature for 1 h. The solvent was then removed under vacuum, and the residue 4-(5-methyl-2-piperazin-1-yl-thiazol-4-yl)-benzoic acid was suspended in methanol (10 ml) and treated with AcOH/AcONa buffer (pH ˜5.5, 5 ml), and formaldehyde (0.547 mmol). The reaction mixture was stirred at room temperature for 1 h, then treated with NaCNBH.sub.3 (0.547 mmol) and stirred at room temperature overnight. The solvent was then removed under vacuum, and the residue was purified by column chromatography to afford the title compound (0.403 mmol, 95%).

(28) MS(ES) m/z 318 (100%, [M+H].sup.+).

Reference Example 2

(29) An Alternative P3 Building Block

3-Fluoro-4-[2-(4-methylpiperazin-1-yl)-thiazol-4-yl]benzoic Acid HCl Salt

(30) ##STR00030##

Step a) Methyl 4-bromo-3-fluorobenzoate

(31) 4-Bromo-3-fluorobenzoic acid (2.46 g, 11.2 mmol) was dissolved in MeOH (9 mL) and toluene (4 mL) and cooled in an ice bath. (Trimethylsilyl)diazomethane (11 mL, 2.0 M in hexanes, 22 mmol) was added dropwise until the yellow color persisted. The solution was stirred at room temperature for 40 mins and then concentrated in vacuo. A second batch of carboxylic acid (2.43 g) was treated similarly. The crude product from both batches were combined and subjected to flash chromatography (silica, 5/1 pentane-EtOAc) to give the methyl ester as white solids (4.92 g, 95% yield).

(32) .sup.1H NMR (400 MHz, CDCl.sub.3) delta ppm 7.77 (m, 1H), 7.71 (m, 1H), 7.64 (m, 1H), 3.93 (s, 3H).

Step b) Methyl 4-acetoxy-3-fluorobenzoate

(33) Allyl chloride (105 μL, 1.28 mmol) and TFA (20 μL, 0.26 mmol) were added to a suspension of zinc dust (480 mg, 7.34 mmol) and anhydrous cobalt(II) bromide (96.6 mg, 0.44 mmol) in MeCN (4 mL), under inert gas. After stirring at room temperature for 10 min, the aryl bromide (1.003 g, 4.30 mmol dissolved in 5 mL MeCN) from (a) was added, followed by acetic anhydride (0.45 mL, 4.79 mmol) and more MeCN (1 mL). The mixture was stirred overnight, quenched with 1M HCl (20 mL), and then extracted with EtOAc (3×20 mL). The organic phase was washed successively with saturated aqueous NaHCO.sub.3 (20 mL) and saturated NaCl (2×20 mL), dried (Na.sub.2SO.sub.4), and concentrated. Flash chromatography (silica, 6/1 to 4/1 petroleum ether-EtOAc gave recovered bromide (161.1 mg, 16%) and the desired ketone (white solids, 305.5 mg, 36%).

(34) NMR (CDCl.sub.3) δ ppm: .sup.1H (400 MHz) 7.94-7.86 (m, 2H), 7.80 (dd, 1H, J=11.2, 1.6 Hz), 3.95 (s, 3H), 2.67 (d, 3H, J=4.4 Hz); .sup.19F (376 MHz) −109.2 (m); .sup.13C (100 MHz) 195.4 (d, J=3.7 Hz), 165.1 (d, J=2.2 Hz), 161.6 (d, J=255 Hz), 135.8 (d, J=8.1 Hz), 130.7 (d, J=2.9 Hz), 129.0 d, J=14 Hz), 125.2 (d, J=3.6 Hz), 117.9 (d, J=26 Hz), 52.7 (s), 31.4 (d, J=7.3 Hz).

Step c) Methyl 4-(2-bromoacetoxy)-3-fluorobenzoate

(35) THF (10 mL) and 2-pyrrolidinone (91 μL, 1.20 mmol) were added to a mixture of the ketone from b) (198 mg, 1.01 mmol) and pyrrolidone hydrotribromide (532 mg, 1.07 mmol). After heating at 60-65° C. for 2 h, the mixture was concentrated under vacuum and then partitioned between EtOAc (20 mL) and saturated Na.sub.2S.sub.2O.sub.3 (10 mL). The aqueous phase was extracted with EtOAc (10 mL). The organic phases were combined, washed with saturated NaCl (2×10 mL), dried (Na.sub.2SO.sub.4), and concentrated. Flash chromatography (silica, 7/1 petroleum ether-EtOAc) gave white solids (0.2634 g) containing 84% of the desired bromide (as determined by integration of .sup.19F NMR peaks).

(36) NMR (CDCl.sub.3) δ ppm: .sup.1H (400 MHz) 7.93 (m, 1H), 7.88 (m, 1H), 7.79 (dd, 1H, J=11.2, 1.6 Hz), 4.50 (d, 2H, J=2.4 Hz), 3.94 (s, 3H); .sup.19F (376 MHz) −108.4 (m).

Step d) Methyl 3-fluoro-4-[2-(4-methylpiperazin-1-yl)-thiazol-4-yl]benzoate

(37) EtOH (5.0 mL) was added to the bromoketone above (193 mg, 0.70 mmol) and 4-methyl-piperazine-1-carbothioic acid amide (113 mg, 0.71 mmol) and the mixture was heated at 70° C. for 2 h 15 min. The precipitates were filtered, washed with cold EtOH, and dried under vacuum and characterized. The procedure was repeated in a larger scale for 1.75 g bromoketone (6.36 mmol).

(38) NMR (1/1 CDCl.sub.3—CD.sub.3OD) δ ppm: .sup.1H (400 MHz) 8.20 (m, 1H), 7.86 (dd, 1H, J=8.4, 1.6 Hz), 7.76 (dd, 1H, J=11.4, 1.8 Hz), 7.38 (d, 1H, J=2.4 Hz), 4.23 (br, 2H), 3.95, (s, 3H), 3.65 (br, 4H), 3.32 (br, 2H), 2.98 (s, 3H); .sup.19F (376 MHz) −114.0 (m). LCMS [M+H].sup.+=336.

(39) The precipitates from both preparations were combined and suspended in saturated NaHCO.sub.3 (50 mL). The mixture was extracted with EtOAc. The organic phase was washed with water, dried (Na.sub.2SO.sub.4), and evaporated to give the title compound as cream solids (1.76 g).

Step e) 3-fluoro-4-[2-(4-methylpiperazin-1-yl)-thiazol-4-yl]benzoic Acid HCl Salt

(40) The methyl ester (1.76 g, 5.25 mmol) from (d) was heated at 80° C. with 6M HCl (40 mL) for 5.5 h. More 6M HCl (10 mL) was added and the mixture was heated at 90° C. for 1 h 15 min. After cooling, the mixture was then evaporated under vacuum and freeze-dried from water to give the final product as cream solids in quantitative yield.

(41) NMR (DMSO-d6) δ ppm: .sup.1H (400 MHz) 11.60 (br, 1H), 8.18 (t, 1H, J=8.0 Hz), 7.82 (dd, 1H, J=8.4, 1.6 Hz), 7.72 (dd, 1H, J=12.0, 1.6 Hz), 7.48 (d, 1H, J=2.8 Hz), 4.11 (m, 2H), 3.58 (m, 2H), 3.49 (m, 2H), 3.19 (m, 2H), 2.80 (d, 3H, J=4.4 Hz); .sup.19F (376 MHz) −113.5 (m); .sup.13C (100 MHz) 168.9, 166.0, 159.0 (d, J=250 Hz), 143.4, 131.4 (d, J=8 Hz), 129.8, 125.8 (d, J=11 Hz), 125.6, 116.6 (d, J=24 Hz), 111.1 (J=15 Hz), 51.1, 45.0, 41.9. LCMS [M+H].sup.+=322.

Reference Example 3

6-Aldehyde—Intermediate

(42) ##STR00031##

6-Formyl-3,3-dimethoxy-hexahydro-furo[3,2-b]pyrrole-4-carboxylic Acid Tert-Butyl Ester

Step a

(43) ##STR00032##

(3as, 6aS)-6R-benzyloxy-3-oxo-hexahydro-furo[3,2-b]pyrrole-4-carboxylic Acid Benzyl Ester (1a)

(44) Dess-Martin reagent (12.5 g, 30 mmol) was dissolved in DCM (250 mL). 6-Benzyloxy-3-hydroxy-hexahydro-furo[3,2-b]pyrrole-4-carboxylic acid benzyl ester (prepared as described in WO05/066180) (7.4 g, 20 mmol) in DCM (50 mL) was added to a stirred solution of oxidant at rt under a nitrogen atmosphere over 45 min. After an additional 90 min stirring the reaction was deemed to be complete by TLC. Aqueous 10% Na.sub.2S.sub.2O.sub.3 (200 mL) was added and the mixture was stirred at rt for another 15 minutes. The two phase system was transferred into a separation funnel and extracted twice with EtOAc (200 mL and 100 mL respectively). The combined organic phases were washed once with aqueous saturated NaHCO.sub.3 (100 mL) and brine (100 mL), dried over Na.sub.2SO.sub.4, filtered and the solvent removed in vacuo, yielding the crude product title compound as a clear oil (7.69 g); ESI+, m/z: 368 (M++1).

Step b

(45) ##STR00033##

(3aS,6aS)-6R-benzyloxy-3,3-dimethoxy-hexahydro-furo[3,2-b]pyrrole-4-carboxylic Acid Benzyl Ester (1b)

(46) The keto derivative (1a) (7.6 g) was dissolved in dry methanol (100 mL). Trimethyl orthoformate (30 mL) and pTsOH (0.2 g) was added at rt under a nitrogen atmosphere. The mixture was heated at 60° C. for 8 hours. When the reaction was deemed to have reached completion according to TLC, it was cooled to rt and concentrated in vacuo. The crude product was purified by column chromatography over silica gel eluting with ethyl acetate-heptane (1:4) which gave the title compound as a clear oil (5.9 g, 71% over 2 steps); ESI+, m/z: 382 (M+-OMe).

Step c

(47) ##STR00034##

(3aS,6aS)-3,3-dimethoxy-hexahydro-furo[3,2-b]pyrrol-6R-ol (1c)

(48) A solution of compound (1b) (2.5 g, 6.4 mmol) in methanol (60 mL) and Pd(OH).sub.2 (0.7 g) was stirred at rt under H.sub.2 atmosphere for 48 hours. When the reaction was deemed to have reached completion according to TLC, the mixture was filtered and concentrated in vacuo to yield the crude title compound as a brownish oil (1.15 g); ESI+, m/z: 190 (M++1).

Step d

(49) ##STR00035##

(3aS, 6aS)-6R-hydroxy-3,3-dimethoxy-hexahydro-furo[3,2-b]pyrrole-4-carboxylic Acid Tert-Butyl Ester (1d)

(50) 3,3-Dimethoxy-hexahydro-furo[3,2-b] pyrrol-6-ol (1c) (2.80 g, 14.8 mmol) was dissolved in 75 mL of a mixture of dioxan/water (2:1). A solution of 10% Na.sub.2CO.sub.3 (25 mL) was added drop wise to pH 9-9.5. The mixture was cooled to 0° C. in an ice-water bath and Boc anhydride was added in one portion. The reaction was stirred at rt overnight and the pH of the mixture was maintained at 9-9.5 by addition of more 10% solution of Na.sub.2CO.sub.3 if necessary. The reaction was monitored by TLC (50:50 ethyl acetate:isohexane). Once completed, the mixture was filtered to eliminate the salts formed and the solvent was evaporated in vacuo. The aqueous mixture was extracted with 3×100 mL EtOAc, the combined organic phases were washed with 100 mL of water and 100 mL brine, dried over Na.sub.2SO.sub.4, filtered and the solvent was evaporated in vacuo to afford 3.79 g of the title carbamate as a clear oil (89%), 94% pure (HPLC), ESI.sup.+, m/z: 312 (M.sup.++Na).

Step e

(51) ##STR00036##

3,3-Dimethoxy-6-oxo-hexahydro-furo[3,2-b]pyrrole-4-carboxylic Acid Tert-Butyl Ester (1e)

(52) To the alcohol (1e) (3.674 g, 12.70 mmol) dissolved in DCM (80 mL) was added Dess-Martin Periodinane (7.00 g, 16.5 mmol) and the solution was stirred for 3 h at room temperature. The reaction was then quenched by the addition of 10% Na.sub.2S.sub.2O.sub.3 (aq) (150 mL) and the resulting slurry was stirred for 15 minutes. The mixture was transferred to a separation funnel and the phases were separated. The aqueous phase was extracted trice with DCM and the combined organic phases were subsequently washed twice with sat. NaHCO.sub.3 solution and were the dried, filtered, and concentrated. The crude material was purified by flash column chromatography (toluene/ethyl acetate 3:1) which gave the title compound (2.882 g, 79%).

Step f

(53) ##STR00037##

3,3-Dimethoxy-6-methylene-hexahydro-furo[3,2-b]pyrrole-4-carboxylic Acid Tert-Butyl Ester (1f)

(54) The keto compound 1e (1.10 g, 3.83 mmol) was dissolved in dry THF (30 mL) and the solution was cooled to 0° C. A solution of methyl triphenylphosphonium bromide (4.0 g, 11.2 mmol) and KOtBu (1.17 g, 10.5 mmol) in dry THF (40 mL) was added in 3 aliquots with 2 hours interval. After 6 hrs the solution was poured into a separatory funnel with diethyl ether (70 mL) and extracted with 10% citric acid .sub.(aq)(2*40 mL). The organic phase was washed with saturated aqueous NaHCO.sub.3 (40 mL), dried with Na.sub.2SO.sub.4, filtered and the solvent was evaporated in vacuo. The crude product was purified by flash chromatography (heptane:ethyl acetate 4:1) which gave the title compound (524 mg, 48%)

(55) .sup.1H NMR (CDCl.sub.3, 400 MHz) δ 1.48 (s, 9H), 3.27 (s, 3H), 3.40 (d, 3H, J=16.6), 3.57-3.64 (m, 1H), 3.84 (d, 1H, J=9.5), 3.92 (d, 1H, J=16.3), 4.07-4.25 (m, 1H), 4.35-4.49 (m, 1H), 4.98 (bs, 1H), 5.22 (d, 1H, J=16.4), 5.34 (s, 1H).

Step g

(56) ##STR00038##

6-Hydroxymethyl-3,3-dimethoxy-hexahydro-furo[3,2-b]pyrrole-4-carboxylic Acid Tert-Butyl Ester (1g)

(57) The olefin 1e (524 mg, 1.84 mmol) was dissolved in dry THF (70 mL). 9-BBN-H (0.5 M in THF) (7.34 mL, 3.67 mmol) was added and the solution was stirred over night. The solvent was removed by rotary evaporation and redissolved in THF (20 mL). MeOH (10 mL) was slowly added to the solution and when the gas evolution had ceased, H.sub.2O (20 mL) was added to the solution followed by NaBO.sub.3. The solution was filtered after it had been stirred for 18 hrs and the filtrate was diluted with EtOAc (70 mL) and washed with brine (2*50 mL). The organic phase was dried with Na.sub.2SO.sub.4, filtered and the solvent was evaporated in vacuo. The crude product was purified by flash chromatography (heptane:ethyl acetate 2:1) which gave the title compound (477 mg, 86%).

(58) .sup.1H NMR (CDCl.sub.3, 400 MHz) δ1.47 (s, 9H), 2.09-2.25 (m, 2H), 3.02-3.20 (m, 1H), 3.29 (s, 3H), 3.39 (s, 3H), 3.65-3.93 (m, 4H), 4.44 (d, 1H, J=5.7), 4.70-4.84 (m, 1H).

Step h

(59) ##STR00039##

6-Formyl-3,3-dimethoxy-hexahydro-furo[3,2-b]pyrrole-4-carboxylic Acid Tert-Butyl Ester (1h)

(60) To a solution of the alcohol 1g (370 mg, 1.22 mmol) dissolved in dry DCM (10 mL) was added Dess Martin periodinane (673 mg, 1.59 mmol). The reaction was stirred for 40 minutes and then quenched by addition of 10 mL of 10% Na.sub.2S.sub.2O.sub.3:NaHCO.sub.3(sat) 1:1. The solution was diluted with DCM (50 mL) and extracted with a 1:1 mixture of 10% Na.sub.2S.sub.2O.sub.3:NaHCO.sub.3(sat) (50 mL). The organic phase was dried with Na.sub.2SO.sub.4, filtered and evaporated. The crude product was purified by flash chromatography (heptane:ethyl acetate (2:1) which gave the title compound (290 mg, 79%).

(61) .sup.1H NMR (CDCl.sub.3, 400 MHz) δ1.47 (s, 9H), 2.90-3.06 (m, 1H), 3.29 (s, 3H), 3.38 (s, 3H), 3.67-3.85 (m, 2H), 3.88-4.55 (m, 3H), 4.93-5.19 (m, 1H), 9.64* and 9.80* (s, 1H). *Two peaks due to rotamers.

Reference Example 4

(62) 6-Acetylene P1 Building Block

(63) ##STR00040##

6-Ethynyl-3,3-dimethoxy-hexahydro-furo(3,2-b)pyrrole-4-carboxylic Acid Tert Butyl Ester

Step a)

(64) ##STR00041##

(65) The aldehyde of Reference Example 3 (318.3 mg, 1.06 mmol) in 6 mL dichloromethane (DCM) was added dropwise to a solution of CBr.sub.4 (700 mg, 2.11 mmol) and PPh.sub.3 (1.10 g, 4.19 mmol) in 10 mL DCM, with cooling in an ice bath. After stirring at 0° C. for 2 h, the mixture was diluted with 30 mL iso-hexane and then filtered through a short Celite column. The column was washed with 20 mL i-hexane, followed by 3/1 i-hexane-DCM. The filtrate was evaporated to give light yellow solids. Flash chromatography (silica, 3/1 i-hexane-EtOAc) gave compound 2 (339.5 mg, 70% yield).

(66) 1H NMR (400 MHz, CDCl.sub.3) δ 6.55 (d, 1H, J=8.8 Hz, HC═CBr.sub.2), 4.64 (br s, 1H), 4.42 (d, 1H, J=5.2 Hz), 4.10-3.90 (br m, 1H), 3.85-3.65 (m, 2H), 3.37 (s, 3H, OMe), 3.29 (s, 3H, OMe), 3.00 (m, 1H), 2.84 (br s, 1H).

Step b)

(67) ##STR00042##

(68) Butyllithium solution (1.6M in hexanes, 1.50 mL) was added dropwise at −78° C. to a solution of the dibromoalkene of step a (327.5 mg, 0.72 mmol) in 13 mL THF. After stirring at −78° C. for 1.75 h, the reaction was quenched with 3 mL saturated aqueous NH.sub.4Cl. The reaction mixture was concentrated, then partitioned between 30 mL saturated aqueous NaCl and 30 mL EtOAc. The aqueous phase was extracted with 2×30 mL EtOAc. The organic phases were combined, dried (Na.sub.2SO.sub.4), and evaporated to give a yellow oil. Flash chromatography (silica, 3/1 i-hexane-EtOAc) gave title compound as white solids (161.7 mg, 67% yield).

(69) 1H NMR (500 MHz, CDCl.sub.3) δ 4.67 (br s, 1H), 4.40 (br s, 1H), 4.16-4.0 (br m, 1H), 3.88-3.74 (m, 2H), 3.37 (s, 3H, OMe), 3.29 (s, 3H, OMe), 3.16 (br s, 1H), 2.81 (br s, 1H, HC—C≡CH), 2.20 (d, 1H, C≡CH), 1.47 (s, 9H, tBu).

Reference Example 5

(70) A Typical P1/P2 Coupling & Deprotection

Step a)

(71) ##STR00043##

(72) Acetyl chloride (0.51 mL) was added dropwise to a solution of the terminal alkyne of Reference Example 4 (153 mg, 0.514 mmol) in MeOH (4.6 mL), chilled in an ice bath. The reaction mixture was stirred at RT overnight and then evaporated. Boc-Leu-OH—H.sub.2O (145 mg, 0.58 mmol) was added and the mixture was coevaporated from DMF, then redissolved in 5 mL DMF, and cooled in an ice bath. DIEA (0.36 mL, 2.1 mmol) was added, followed by HATU (220 mg, 0.578 mmol). After stirring at 0° C. for 15 min, the mixture was stirred at RT for 3 h and then concentrated. The mixture was dissolved in 20 mL EtOAc, washed successively with saturated aqueous NaHCO.sub.3 (10 mL) and saturated aqueous NaCl (2×10 mL), dried (Na.sub.2SO.sub.4), and evaporated to give a yellow-brown oil. Flash chromatography (silica, 1/1 i-hexane-EtOAc) gave title compound (213 mg, quantitative).

(73) HPLC-MS: single peak, mass 411 [M+H].sup.+, R.sub.t=3.15 min (gradient 5 to 99% B in 3 min, then 100% B for 1.5 min)

(74) Method-Flow: 0.8 mL/min, UV=210-400 nm, ACE C8 3×50 mm; Mobile phase A: 10 mM NH.sub.4Ac in 90% H.sub.2O, B: 10 mM NH.sub.4Ac in 90% MeCN

Step b)

(75) ##STR00044##

(76) Boc deprotection of the compound of step a) (0.514 mmol) was done as for Reference Example 4 above to give the title HCl salt. The salt was dissolved in CH.sub.2Cl.sub.2 and divided into three portions, evaporated separately to give 0.17 mmol per sample.

Reference Example 6

(77) An Alternative P3 Building Block

(78) ##STR00045##

(79) Availability of Starting Materials—

(80) Methyl 4-acetylbenzoate is available from Aldrich; 4-methyl-piperazine-1-carbothioic acid amide—11 suppliers found in SciFinder (perhaps Chem Pur Products Ltd in Germany most convenient).

Step a) 4-(2-Bromo-acetyl)-benzoic Acid Methyl Ester

(81) ##STR00046##

(82) To a solution of 4-acetyl-benzoic acid methyl ester (8.4 mmol) in acetic acid (20 mL) was added bromine (8.4 mmol). The reaction was stirred at RT for 2 h over which time the red colour disappeared and an off white precipitate formed. The product was collected by filtration and washed with cold methanol/water (200 mL 1:1) to yield a white powder (55%). 1H NMR (400 MHz, CDCl.sub.3) 3.98 (3H, s), 4.20 (2H, s), 8.02 (2H, d, J=8 Hz), 8.18 (2H, d, J=8 Hz).

Step b) 4-(2-Fluoro-acetyl)-benzoic Acid Methyl Ester

(83) ##STR00047##

(84) To a suspension of potassium fluoride (3.11 mmol) in acetonitrile (1 mL) was added 18-crown-6 (0.1 mmol) and the reaction was heated at 90° C. for 30 mins. 4-(2-Bromo-acetyl)-benzoic acid (1.56 mmol) was added and the reaction heated at 90° C. for 16 h. The reaction was diluted with water (10 mL) and extracted with ethyl acetate (3×20 mL). The product was purified on silica eluting with 5-15% ethyl acetate in iso-hexane to yield on concentration in vacuo of the desired fractions, the title product as a white solid (31%). 1H NMR (400 MHz, CDCl.sub.3) 3.98 (3H, s), 5.55 (2H, d, J=50 Hz), 7.95 (2H, d, J=8 Hz), 8.18 (2H, d, J=8 Hz).

Step c) 4-(2-Bromo-2-fluoro-acetyl)-benzoic Acid Methyl Ester

(85) ##STR00048##

(86) To a suspension of 4-(2-fluoro-acetyl)-benzoic acid (1.19 mmol) in acetic acid (5 mL) was added bromine (1.19 mmol). The reaction was heated at 45° C. for 4 h over which time a green solution formed. The reaction was concentrated in vacuo and azeotroped twice with toluene to yield the title compound as a green solid (100%). The product was used crude in the next step. 1H NMR (400 MHz, CDCl.sub.3) 3.98 (3H, s), 7.04 (1H, s), 8.05-8.10 (4H, m).

Step d) 4-[5-Fluoro-2-(4-methyl-piperazin-1-yl)-thiazo-4-yl]-benzoic Acid Methyl Ester

(87) ##STR00049##

(88) 4-(2-Bromo-2-fluoro-acetyl)-benzoic acid methyl ester (1.18 mmol) and 4-methyl-piperazine-1-carbothioic acid amide (1.18 mmol) were dissolved in ethanol (10 mL). The reaction was heated at reflux for 2 h. The reaction was cooled to RT causing the product to precipitate. The product was collected by filtration and washed with cold ethanol. The product was given an aqueous sodium bicarbonate work up to yield the title compound as a colourless oil (74%). MS (ES+) 337 (M+H, 100%).

Step f)

4-[5-Fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzoic Acid Di-Hydrochloride

(89) ##STR00050##

(90) To a solution of 4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzoic acid methyl ester (0.43 mmol) in tetrahydrofuran/water (2.5 mL, 4:1) was added lithium hydroxide (0.5 mmol). The reaction was stirred at RT for 16 h. The reaction was concentrated in vacuo and hydrochloric acid (2N, 3 mL) was added causing the product to precipitate as a white solid. The product was collected by filtration to yield the title product as a white solid (79%). MS (ES+) 322 (M+H, 100%).

Reference Example 7

(91) An Alternative P1 Building Block

(92) ##STR00051##

Step a) Oxidation of Alcohol 7-1

(93) A solution of compound 7-1 (0.85 g, 2.8 mmol) (see reference example 3 step g) in dry CH.sub.2Cl.sub.2 (15 mL) was purged with argon for 30 minutes. Dess-Martin periodinone (1.78 g, 4.2 mmol) was added at 0° C. and the reaction mixture was stirred at room temperature for 2 hours. When the reaction was deemed to have reached completion, the reaction was quenched with 10% Na.sub.2S.sub.2O.sub.3 solution. The reaction mixture was diluted with CH.sub.2Cl.sub.2 (30 mL), and then washed in turn with saturated Na.sub.2S.sub.2O.sub.3 solution (2×50 mL), and saturated sodium bicarbonate solution (2×50 mL). The organic layer was then dried over anhydrous sodium sulfate, filtered and the filtrate conc in vacuo at room temperature. The crude product aldehyde 7-2 (0.75 g, crude) was used in the next step without further purification.

(94) TLC: EtOAc:Pet ether 1:1 R.sub.f=0.5.

Step b) Wittig Reaction with Compound 7-2

(95) To a stirred solution of isopropyltriphenylphosphonium iodide (6.48 g, 0.015 mol) (co-evoparated with dry toluene prior to start of the reaction) in dry THF (20 mL) was added a solution of n-BuLi (10.7 mL, 0.0172 mol, 1.6M in hexane) at −10° C. under nitrogen atmosphere and further stirred at 0° C. for 1 hour. The color of the reaction mixture slowly turned to dark orange color. A solution of compound 2 (0.75 g, 0.0025 mol) and anhydrous lithium chloride (˜1.5 mg) in dry THF (20 mL) was slowly added to the reaction mixture at 0° C. under a nitrogen atmosphere. The reaction mixture was further stirred for 30 minutes maintaining the same temperature and then slowly warmed to rt and stirred for another 30 minutes. The reaction mixture was then quenched with saturated ammonium chloride solution (20 mL). The crude product was extracted with EtOAc (2×30 mL) and the organic layer was washed with brine (50 mL), and dried over anhydrous sodium sulfate. The solvent was removed in vacuo and the crude product was purified by column chromatography (neutral alumina, eluent 1% EtOAc in pet ether) to afford pure compound 7.3 (0.19 g, yield 21% over two steps).

Reference Example 8

(96) An Alternative P1-P2 Building Block

(97) ##STR00052##

(98) The initial reaction was carried out in a similar way as reported for reference example 5 but instead using 4 (0.2 g, 0.67 mmol) in dry MeOH (15 mL) and acetyl chloride (0.66 mL) to get the Boc-deprotected amine compound which was then treated with Boc-cyclopentyl glycine (0.17 g, 0.7 mmol), HATU (0.27 g, 0.7 mmol) and DIEA (0.45 mL.

(99) 2.6 mmol) in dry DMF (7 mL) to get the pure title compound [0.195 g, yield 68%].

(100) TLC system: EtOAc:pet ether 1:1 v/v, R.sub.f=0.4

Reference Example 9

(101) Probe Compound for the Feasibility of Dihalovinyl P1

(102) ##STR00053##

(103) This compound tests the synthetic and biological feasibility of a dihalovinyl P1 end product. It will be appreciated that introduction of 6-difluorovinyl by the corresponding Wittig reaction of reference example 3 is trivial and that preparation of a biologically active end-product provides confidence that the claimed difluorovinyl compounds are also synthetically stable and active.

(104) ##STR00054##

Step a)

(105) ##STR00055##

(106) The aldehyde 9-1 (318.3 mg, 1.06 mmol) (see reference example 3) in 6 mL dichloromethane (DCM) was added dropwise to a solution of CBr.sub.4 (700 mg, 2.11 mmol) and Ph.sub.3P (1.10 g, 4.19 mmol) in 10 mL DCM, with cooling in an ice bath. After stirring at 0° C. for 2 h, the mixture was diluted with 30 mL iso-hexane and then filtered through a short Celite column. The column was washed with 20 mL i-hexane, followed by 3/1 i-hexane-DCM. The filtrate was concentrated in vacuo to give light yellow solids. Flash chromatography (silica, 3/1 i-hexane-EtOAc) gave compound 9-2 (339.5 mg, 70% yield).

(107) 1H NMR (400 MHz, CDCl.sub.3) δ 6.55 (d, 1H, J=8.8 Hz, HC═CBr.sub.2), 4.64 (br s, 1H), 4.42 (d, 1H, J=5.2 Hz), 4.10-3.90 (br m, 1H), 3.85-3.65 (m, 2H), 3.37 (s, 3H, OMe), 3.29 (s, 3H, OMe), 3.00 (m, 1H), 2.84 (br s, 1H).

(108) Rf (TLC 1/1 isohexane-EtOAc) 0.79.

(109) HPLC-MS: ion cluster [M+Na].sup.+ 478 (8%), 480 (15%), 482 (7%), R.sub.t=3.56 min (gradient 5 to 99% B in 3 min, then 100% B for 1.5 min)

Step b)

(110) ##STR00056##

(111) Acetyl chloride (0.18 mL) was added dropwise to a solution of the dibromoalkene 9-2 (81.7 mg, 0.179 mmol) in MeOH (1.62 mL), chilled in an ice bath. The reaction mixture was stirred at RT overnight and then evaporated. Boc-Leu-OH—H.sub.2O (50 mg, 0.20 mmol), HATU (75 mg, 0.20 mmol), 1.8 mL DMF, and lastly, DIEA (125 μL, 0.72 mmol) were added. After stirring at RT for 5.5 h the mixture was concentrated in vacuo, and then partitioned between EtOAc and saturated aqueous NaHCO.sub.3. The organic phase was washed twice with saturated aqueous NaCl, dried (Na.sub.2SO.sub.4), and concentrated in vacuo. Flash chromatography (silica, 3/1 isohexane-EtOAc) gave compound 9-3 as a white solid (86.3 mg, 85% yield).

(112) Rf (TLC 1/1 isohexane-EtOAc) 0.54.

(113) HPLC-MS: mass 571 [M+H].sup.+; R.sub.t=3.75 min 96% (gradient 5 to 99% B in 3 min, then 100% B for 1.5 min)

(114) Method-Flow: 0.8 mL/min, UV=210-400 nm, ACE C8 3×50 mm; Mobile phase A: 10 mM NH.sub.4Ac in 90% H.sub.2O, B: 10 mM NH.sub.4Ac in 90% MeCN

Step c)

(115) ##STR00057##

(116) Boc deprotection of compound 9-3 (86.3 mg, 0.15 mmol) was done as for 9-2 above to give the amine HCl salt. DMF (1.5 mL) was added to a mixture of the amine salt, 4-[2-(4-methyl-1-piperazinyl)-4-thiazolyl]-benzoic acid hydrobromide (65 mg, 0.17 mmol), and HATU (65 mg, 0.17 mmol) with cooling in an ice bath. DIEA (120 μL, 0.69 mmol) was added. After stirring at RT for 3 h, 20 mL EtOAc was added, and then the mixture was washed with 1M NaHCO.sub.3 (10 mL), followed by saturated aqueous NaCl. The organic phase was dried (Na.sub.2SO.sub.4) and concentrated in vacuo to give crude product. Flash chromatography (silica, CH.sub.2Cl.sub.2-MeOH 100/1 to 100/4) gave the desired product as a pale yellow solid (65.7 mg, 58% yield).

(117) HPLC-MS: mass 756 [M+H].sup.+; R.sub.t=3.83 min (5 to 99% B in 3 min, then 100% B for 1.5 min) and R.sub.t=4.07 min (30 to 80% B in 3 min, then 100% B for 1.5 min) 96% pure in two gradients

Step d)

(118) ##STR00058##

(119) The ketal 9-4 (65.7 mg, 0.087 mmol) was stirred with 1.0 mL of 97.5/2.5 (v/v) TFA-H.sub.2O for 4 h 20 min and then quenched with an aqueous suspension of NaHCO.sub.3. The mixture was extracted with EtOAc. The organic phase was washed with saturated aqueous NaCl, dried (Na.sub.2SO.sub.4), and concentrated in vacuo. Purification by prep HPLC gave the ketone 9-5 as a white solid (12.6 mg).

(120) 1H NMR (500 MHz, CDCl.sub.3) 2 rotomers, major: δ 7.90 and 7.81 (ABq, 4H, phenyl), 6.91-6.87 (2H, thiazole and NH), 6.67 (d, 1H, HC═CBr.sub.2), 5.05 (td, 1H, Leu CHα), 4.85 (m, 1H, bicyclic bridge HCO), 4.74 (d, 1H, bicyclic bridge HCN), 4.32 and 3.42 (1H each, bicyclic NCH.sub.2), 4.18 and 4.00 (ABq, 2H, OCH.sub.2), 3.59 (4H, piperazine CH.sub.2N-thiazole), 3.25 (1H, Br.sub.2C═CH—CH), 2.56 (4H, piperazine CH.sub.2NMe), 2.37 (s, 3H, NMe), 1.80-1.54 (d, 3H, CH.sub.2CHMe.sub.2), 1.04 (d, 3H, CHMe.sub.2), 0.94 (d, 3H, CHMe.sub.2).

(121) LC-UV/MS: monoisotopic molecular mass 709.1 Da, >94% purity

(122) (Column: ACE C.sub.8 50×3.0 mm, 3 μm particles; Mobile phases A: 10 mM NH.sub.4Ac, B: 10 mM NH.sub.4Ac in 90% MeCN; gradient: 30-70% B in 10 min followed by a wash for 2 min at 100% B; Flow: 0.8 mL/min, Detection: UV @ 210-400 nm and ESI-MS)

Example 1

(123) ##STR00059##

N-[1-6-(ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]-4-[2-(4-methyl-piperazin-1-yl)thiazol-4-yl]-benzamide

Step a)

(124) ##STR00060##

(125) DMF (2 mL) was added to a mixture of 4-[2-(4-methyl-1-piperazinyl)-4-thiazolyl]-benzoic acid hydrobromide (73.9 mg, 0.19 mmol), the compound of Reference Example 5 (0.17 mmol), and HATU (73.4 mg, 0.19 mmol) with cooling in an ice bath. DIEA (0.12 mL, 0.69 mmol) was added. After stirring at RT for 2.5 h, the mixture was concentrated, redissolved in 20 mL EtOAc, and then washed with 10 mL saturated aqueous NaHCO.sub.3. The aqueous phase was extracted with 10 mL EtOAc. The organic phases were combined, washed with saturated aqueous NaCl (2×15 mL), dried (Na.sub.2SO.sub.4), and evaporated to give crude product. Initial flash chromatography (silica 40-63 μm, 5-8% MeOH in EtOAc) gave purified material which was subjected to a second chromatography (YMC gel silica 6 nm S—50 μm, 1-5% MeOH in CH.sub.2Cl.sub.2) to give title compound as light yellow solids (50.1 mg, 49% yield).

(126) HPLC-MS: mass 596 [M+H].sup.+, single peak, R.sub.t=3.18 min (gradient 5 to 99% B in 3 min, then 100% B for 1.5 min)

Step b)

(127) ##STR00061##

(128) The compound of step a) (50 mg, 0.0839 mmol) was dissolved in 10 mL of TFA:H.sub.2O (97.5:2.5) and stirred for 4 hours. The solvent was pored into a separatory funnel, extracted with EtOAc and washed with saturated aqueous NaHCO.sub.3. The organic phase was dried with Na.sub.2SO.sub.4, filtered and evaporated. The crude product was purified by semi-prep. HPLC on a XBrideg Phenyl 5 μm column with mobile phases A (90:10 H.sub.2O:acetonitrile, 10 mM NH.sub.4Ac) and B (10:90 H.sub.2O:acetonitrile, 10 mM NH.sub.4Ac) going from 25-60% B. The product was obtained as a white solid in 62% yield (29 mg). LRMS (M+H) 550.

(129) .sup.1H NMR (CDCl.sub.3, 400 MHz): 0.95 (d, J=6.4, 3H), 1.03 (d, J=6.4, 3H), 1.57-2.10 (m 4H), 2.37 (s, 3H), 2.60-2.54 (m, 4H), 3.17-3.26 (m, 1H), 3.55-3.64 (m, 5H), 4.07 (d, J=17.1, 1H), 4.27 (d, J=17.2, 1H), 4.50 (dd, J=7.8, 9.9, 1H), 4.77 (d, J=5.1, 1H), 4.91 (dd, J=4.6, 4.6, 1H), 4.97-5.06 (m, 1H), 6.85-6.91 (m, 2H), 7.79 (d, J=8.3, 2H), 7.89 (d, J=8.4, 3H).

Example 2

(130) ##STR00062##

N-[1-6-(ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)thiazol-4-yl]-benzamide

Step a)

(131) ##STR00063##

(132) DMF (1.7 mL) was added to a mixture of 4-[5-fluoro-2-(4-methyl-1-piperazinyl)-4-thiazolyl]-benzoic acid hydrochloride (68.0 mg, 0.19 mmol), the compound of Reference Example 5 (0.17 mmol), and HATU (73.8 mg, 0.19 mmol) with cooling in an ice bath. DIEA (0.12 mL, 0.69 mmol) was added. After stirring at RT for 2.75 h, the mixture was treated as for Example 1 to give the crude fluorothiazole analogue. Flash chromatography (YMC gel silica, 1-3% MeOH in CH.sub.2Cl.sub.2) gave the title compound as light yellow solids (62.8 mg, 60% yield).

(133) HPLC-MS: mass 614 [M+H].sup.+, single peak on UV, R.sub.t=3.39 min (gradient 5 to 99% B in 3 min, then 100% B for 1.5 min)

Step b)

(134) ##STR00064##

(135) The compound of step a) (63 mg, 0.102 mmol) was dissolved in 10 mL of TFA:H.sub.2O (97.5:2.5) and stirred for 4 hours. The solvent was pored into a separatory funnel, extracted with EtOAc and washed with sat. NaHCO.sub.3(aq). The organic phase was dried with Na.sub.2SO.sub.4, filtered and evaporated. The crude product was purified by semi-prep. HPLC on a XBrideg Phenyl 5 um column with mobile phases A (90:10 H.sub.2O:acetonitrile, 10 mM NH.sub.4Ac) and B (10:90 H.sub.2O:acetonitrile, 10 mM NH.sub.4Ac) going from 25-60% B. The product was obtained as a white solid in 46% yield (26 mg). LRMS (M+H) 568.

(136) .sup.1H NMR (CDCl.sub.3, 400 MHz): 0.95 (d, J=6.4, 3H), 1.03 (d, J=6.4, 3H), 1.58-2.15 (m 4H), 2.37 (s, 3H), 2.61-2.48 (m, 4H), 3.18-3.25 (m, 1H), 3.53-3.42 (m, 4H), 3.60 (t, J=10.5, 1H), 4.07 (d, J=17.1, 1H), 4.27 (d, J=17.2, 1H), 4.56-4.46 (m, 1H), 4.91 (t, J=4.5, 1H), 4.96-5.06 (m, 1H), 6.87 (d, J=8.2, 1H), 7.81 (d, J=8.4, 2H), 7.90 (t, J=9.4, 2H).

Example 3

(137) ##STR00065##

N-[1-6-(ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]-3-fluoro-4-[2-(4-methyl-piperazin-1-yl)thiazol-4-yl]-benzamide

Step a)

(138) ##STR00066##

(139) DMF (1.7 mL) was added to a mixture of 3-fluoro-4-[2-(4-methyl-1-piperazinyl)-4-thiazolyl]-benzoic acid hydrochloride (68.9 mg, 0.19 mmol), the compound of Reference Example 5 (0.17 mmol), and HATU (80 mg, 0.21 mmol) with cooling in an ice bath. DIEA (0.12 mL, 0.69 mmol) was added. After stirring at RT for 3 h, the mixture was concentrated, redissolved in 30 mL EtOAc, washed successively with 15 mL saturated aqueous NaHCO.sub.3 and then 30 mL saturated aqueous NaCl. The organic phase was dried (Na.sub.2SO.sub.4) and then evaporated. Flash chromatography (YMC gel silica, 1-3% MeOH in CH.sub.2Cl.sub.2) gave the title compound as off-white solids (72.4 mg, 70% yield).

(140) HPLC-MS: mass 614 [M+H].sup.+, R.sub.t=3.46 min (gradient 5 to 99% B in 3 min, then 100% B for 1.5 min

Step b)

(141) ##STR00067##

(142) The ketal of step a) (67 mg, 0.11 mmol) was stirred with 1.10 mL of 97.5/2.5 (v/v) TFA-H.sub.2O for 2 h and then concentrated. The mixture was diluted with EtOAc (10 mL), washed with saturated aqueous NaHCO.sub.3 (5 mL), followed by saturated aqueous NaCl (2×5 mL), dried (Na.sub.2SO.sub.4), and evaporated. The crude material was dissolved in 2 ml MeCN and 1 mL H.sub.2O, and 0.9 ml of this solution was purified by prep HPLC to give the ketone of the title I compound as white solids (8.2 mg).

(143) 1H NMR (500 MHz, CDCl.sub.3) 2 rotomers δ 8.22 (m, 1H, Ph), 7.63-7.55 (m, 2H, Ph), 7.21 (m, 1H, thiazol), major 6.91 and minor 6.87 (d, 1H, J=8.0 and 7.5 Hz, NHC═O), minor 5.05 and major 5.00 (m, 1H), 4.93 (m, 1H), 4.79 (d, 1H, J=5.0 Hz), 4.48 (dd, 1H, J=10.2, 7.7 Hz), 4.28 and 4.09 (ABq, 1H each), 3.64-3.59 (m, 5H), 3.23 (m, 1H, HC—C≡CH), 2.58 (m, 4H), 2.38 (s, 3H, NMe), 2.35 (d, 1H, J=2.0 Hz, C≡CH), 1.78-1.59 (m, 3H), 1.04 (d, 3H, J=6.0 Hz), 0.96 (d, 3H, J=6.5 Hz).

(144) LC-UV/MS: monoisotopic molecular mass 567.2 Da, 97.6% purity

(145) (Column: ACE C.sub.8 50×3.0 mm, 3 μm particles; Mobile phases A: 10 mM NH.sub.4Ac, B: 10 mM NH.sub.4Ac in 90% MeCN; gradient: 20-100% B in 10 min followed by a wash for 2 min at 100%; Flow: 0.8 mL/min, Detection: UV @ 210-400 nm and ESI-MS)

Example 4

N-[1-6-(dimethylvinyl-3-oxo-hexahydro-furo[3,2-b]pyrrole-4-carbonyl)-3-methyl-butyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)thiazol-4-yl]-benzamide

(146) ##STR00068##

(147) Reaction Scheme:

(148) ##STR00069##

(149) Acetyl chloride (0.3 mL) was added dropwise to an ice-cooled solution of compound 7-3 (103.4 mg, 0.316 mmol) (see reference example 7) in methanol (2.9 mL). The solution was stirred at rt overnight, and then concentrated in vacuo, coevaporated twice with CH.sub.2Cl.sub.2, and dried under vacuum. Boc-L-leucine-H.sub.2O (96.2 mg, 0.386 mmol) and HATU (143 mg, 0.376 mmol) were added and the mixture was cooled in an ice bath. DMF (3.2 mL), followed by DIEA (220 μL, 1.26 mmol) were added. The resulting solution was stirred at rt for 6 h. The reaction mixture was concentrated in vacuo, redissolved in EtOAc (15 mL), and washed successively with saturated aqueous NaHCO.sub.3 (10 mL) and saturated aqueous NaCl (10 mL). The organic phase was dried (Na.sub.2SO.sub.4) and then concentrated in vacuo to give the crude material. Flash column chromatography (silica, 2/1 isohexane-EtOAc) gave compound 4i as white solids (127.8 mg, 92% yield).

(150) TLC Rf=0.56 (1/1 isohexane-EtOAc)

(151) LC-UV/MS Rt=1.98 min (single peak), mass 441 [M+H].sup.+ (gradient 70 to 99% B in 3 min, then 100% B for 1.5 min (Method-Flow 0.8 mL/min, UV=210-400 nm, Phenomenex Gemini-NX 3 μm C18 110 Å 50×3.0 mm, Mobile phases A: 10 mM NH.sub.4Ac in H.sub.2O, B: 10 mM NH.sub.4Ac in 90/10 MeCN—H.sub.2O)

(152) Compound 4-i (127.8 mg, 0.290 mmol) was deprotected as above using MeOH (2.7 mL) and acetyl chloride (0.30 mL). The coupling of the resulting amine HCl salt (half of material, 0.145 mmol) with 4-[5-fluoro-2-(4-methyl-1-piperazinyl)-4-thiazolyl]-benzoic acid hydrochloride (67.4 mg, 0.19 mmol) was done similarly as the Boc-Leu-OH coupling step, in DMF (2.0 mL) with HATU (69 mg, 0.18 mmol) and DIEA (115 μL, 0.66 mmol) for 3.5 h. After flash column chromatography of the crude product (silica, 2-5% MeOH in CH.sub.2Cl.sub.2), compound 4-ii was obtained as pale yellow solids (48.3 mg, 52% yield).

(153) TLC Rf=0.5 (9/1 CH.sub.2Cl.sub.2-MeOH)

(154) LC-UV/MS Rt=2.41 min, 95q % pure, mass 644 [M+H].sup.+ (gradient 70 to 99% B in 3 min, then 100% B for 1.5 min

(155) To an ice-cooled solution of the ketal 4-ii (41.3 mg, 0.064 mmol) in CH.sub.2Cl.sub.2 (0.3 mL) was added dropwise 0.65 ml of a solution of TFA-water (97.5/2.5 v/v). The reaction mixture was stirred for 30 min at rt, and then replaced in an ice bath and quenched with saturated aqueous NaHCO.sub.3 (10 mL). The mixture was extracted twice with EtOAc (20 mL, 10 mL). The organic phases were combined, washed with saturated aqueous NaCl (10 mL), dried (Na.sub.2SO.sub.4), and concentrated in vacuo. Purification by prep HPLC gave the final title compound.

(156) LC-UV/MS Rt=6.5 and 7.5 min (hydrate and ketone), 98% pure, monoisotopic molecular mass 597.3 Da (Method-Flow 0.8 mL/min; UV=210-400 nm and ESI-MS; Phenomenex Gemini-NX C18 50×3.0 mm, 3 μm particles; Mobile phases A: 5 mM NH.sub.4Ac in H.sub.2O, B: 5 mM NH.sub.4Ac in MeCN, gradient 20-99% B in 10 min followed by a wash for 2 min at 100% B)

Example 5

N-[1-(6-ethynyl-3-oxo-hexahydro-furo[3,2-b]pyrrol-4-yl)-1-cyclopentyl-2-oxo-ethyl]-4-[5-fluoro-2-(4-methyl-piperazin-1-yl)-thiazol-4-yl]-benzamide

Step a)

(157) ##STR00070##

(158) ##STR00071##

(159) The reaction was carried out in a similar way as reported in case of Example 3 but using the P1-P2 building block of reference example 8 (0.14 g, 0.33 mmol) in dry MeOH (15 mL) and acetyl chloride (0.5 mL) was used to get the Boc-deprotected amine which was further treated with the HCl salt (0.132 g, 0.33 mmol), HOBt (0.04 g, 0.3 mmol), EDC.HCl (0.117 g, 0.61 mmol) and NMM (0.11 mL, 1.0 mmol) in dry DMF (10 mL) to get the pure compound 5-a [0.05 g, yield 26%]. TLC system: CHCl.sub.3:MeOH 9.5:0.5 v/v, R.sub.f=0.25.

Step b)

(160) ##STR00072##

(161) Deprotection of ketal 5-a (40.5 mg, 0.065 mmol) to the title ketone was conducted as for compound 3 using 0.65 mL of the TFA-water solution, with reaction at rt for 2 h 15 min. Purification by prep HPLC gave 11 as pale yellow solids (14.75 mg).

(162) LC-UV/MS Rt=5.3 and 6.0 min, (hydrate and ketone), 96.7% pure, monoisotopic molecular mass 579.2 Da (Method-Flow 0.8 mL/min; UV=210-400 nm and ESI-MS; Phenomenex Gemini-NX C18 50×3.0 mm, 3 μm particles; Mobile phases A: 5 mM NH.sub.4Ac, B: 5 mM NH.sub.4Ac in MeCN, gradient 20-99% B in 10 min followed by a wash for 2 min at 100% B)

BIOLOGICAL EXAMPLES

(163) Determination of Cathepsin K Proteolytic Catalytic Activity

(164) Convenient assays for cathepsin K are carried out using human recombinant enzyme, such as that described in PDB.

(165) ID BC016058 standard; mRNA; HUM; 16991BP.

(166) DE Homo sapiens cathepsin K (pycnodysostosis), mRNA (cDNA clone MGC: 23107

(167) RX MEDLINE; RX PUBMED; 12477932.

(168) DR RZPD; IRALp962G1234.

(169) DR SWISS-PROT; P43235;

(170) The recombinant cathepsin K can be expressed in a variety of commercially available expression systems including E coli, Pichia and Baculovirus systems. The purified enzyme is activated by removal of the prosequence by conventional methods.

(171) Standard assay conditions for the determination of kinetic constants used a fluorogenic peptide substrate, typically H-D-Ala-Leu-Lys-AMC, and were determined in either 100 mM Mes/Tris, pH 7.0 containing 1 mM EDTA and 10 mM 2-mercaptoethanol or 100mMNa phosphate, imM EDTA, 0.1% PEG4000 pH 6.5 or 100 mM Na acetate, pH 5.5 containing 5 mM EDTA and 20 mM cysteine, in each case optionally with 1M DTT as stabiliser. The enzyme concentration used was 5 nM. The stock substrate solution was prepared at 10 mM in DMSO. Screens were carried out at a fixed substrate concentration of 60 μM and detailed kinetic studies with doubling dilutions of substrate from 250 μM. The total DMSO concentration in the assay was kept below 3%. All assays were conducted at ambient temperature. Product fluorescence (excitation at 390 nm, emission at 460 nm) was monitored with a Labsystems Fluoroskan Ascent fluorescent plate reader. Product progress curves were generated over 15 minutes following generation of AMC product.

(172) Cathepsin S Ki Determination

(173) The assay uses baculovirus-expressed human cathepsin S and the boc-Val-Leu-Lys-AMC fluorescent substrate available from Bachem in a 384 well plate format, in which 7 test compounds can be tested in parallel with a positive control comprising a known cathepsin S inhibitor comparator.

(174) Substrate Dilutions

(175) 280 μl/well of 12.5% DMSO are added to rows B-H of two columns of a 96 deep well polypropylene plate. 70 μl/well of substrate is added to row A. 2×250 μl/well of assay buffer (100 mM Na phosphate, 100 mM NaCl, pH 6.5) is added to row A, mixed, and double diluted down the plate to row H.

(176) Inhibitor Dilutions

(177) 100 μl/well of assay buffer is added to columns 2-5 and 7-12 of 4 rows of a 96 well V bottom polypropylene plate. 200 μl/well of assay buffer is added to columns 1 and 6.

(178) The first test compound prepared in DMSO is added to column 1 of the top row, typically at a volume to provide between 10 and 30 times the initially determined rough K.sub.i. The rough Ki is calculated from a preliminary run in which 10 μl/well of 1 mM boc-VLK-AMC (1/10 dilution of 10 mM stock in DMSO diluted into assay buffer) is dispensed to rows B to H and 20 μl/well to row A of a 96 well Microfluor™ plate. 2 μl of each 10 mM test compound is added to a separate well on row A, columns 1-10. Add 90 μl assay buffer containing 1 mM DTT and 2 nM cathepsin S to each well of rows B-H and 180 μl to row A. Mix row A using a multichannel pipette and double dilute to row G. Mix row H and read in the fluorescent spectrophotometer. The readings are Prism data fitted to the competitive inhibition equation, setting S=100 μM and K.sub.M=100 μM to obtain an estimate of the K.sub.i, up to a maximum of 100 μM.

(179) The second test compound is added to column 6 of the top row, the third to column 1 of the second row etc. Add 1 μl of comparator to column 6 of the bottom row. Mix column 1 and double dilute to column 5. Mix column 6 and double dilute to column 10.

(180) Using an 8-channel multistepping pipette set to 5×10 μl, distribute 10 μl/well of substrate to the 384 well assay plate. Distribute the first column of the substrate dilution plate to all columns of the assay plate starting at row A. The tip spacing of the multichannel pipette will correctly skip alternate rows. Distribute the second column to all columns starting at row B.

(181) Using a 12-channel multistepping pipette set to 4×10 μl, distribute 10 μl/well of inhibitor to the 384 well assay plate. Distribute the first row of the inhibitor dilution plate to alternate rows of the assay plate starting at A1. The tip spacing of the multichannel pipette will correctly skip alternate columns. Similarly, distribute the second, third and fourth rows to alternate rows and columns starting at A2, B1 and B2 respectively.

(182) Mix 20 ml assay buffer and 20 μl 1M DTT. Add sufficient cathepsin S to give 2 nM final concentration.

(183) Using the a distributor such as a Multidrop 384, add 30 μl/well to all wells of the assay plate and read in fluorescent spectrophotomoter such as an Ascent.

(184) Fluorescent readings, (excitation and emission wavelengths 390 nm and 460 nm respectively, set using bandpass filters) reflecting the extent of enzyme cleavage of the fluorescent substrate, notwithstanding the inhibitor, are linear rate fitted for each well.

(185) Fitted rates for all wells for each inhibitor are fitted to the competitive inhibition equation using SigmaPlot 2000 to determine V, Km and Ki values.

(186) Cathepsin L Ki

(187) The procedure above with the following amendments is used for the determination of Ki for cathepsin L.

(188) The enzyme is commercially available human cathepsin L (for example Calbiochem). The substrate is H-D-Val-Leu-Lys-AMC available from Bahcem. The assay buffer is 100 mM sodium acetate 1 mM EDTA, pH5.5) The DMSO stock (10 mM in 100% DMSO) is diluted to 10% in assay buffer. Enzyme is prepared at 5 nM concentration in assay buffer plus 1 mM dithiothreitol just before use. 2 ul of 10 mM inhibitor made up in 100% DMSO is dispensed into row A. 10 μl of 50 μM substrate (=1/200 dilution of 10 mM stock in DMSO, diluted in assay buffer)

(189) Inhibition Studies

(190) Potential inhibitors are screened using the above assay with variable concentrations of test compound. Reactions were initiated by addition of enzyme to buffered solutions of substrate and inhibitor. K.sub.i values were calculated according to equation 1.

(191) $\begin{array}{cc}{v}_0=\frac{VS}{K_M\left(1+\frac{I}{K_i}\right)+S}& \left(1\right)\end{array}$

(192) where v.sub.0 is the velocity of the reaction, V is the maximal velocity, S is the concentration of substrate with Michaelis constant of K.sub.M, and I is the concentration of inhibitor.

(193) Results are presented as: A under 50 nanomolar B 50-500 nanomolar C 501-1000 nanomolar D 1001-5000 nanomolar E 5001-10 000 nanomolar F in excess of 10 000 nanomolar

(194) TABLE-US-00001 TABLE 1 Test Example Number Ki cathepsin K Ki cathepsin S Ki cathepsin L 1 Test 1 A F D 2 Test 1 A F D 3 Test 1 A F C 1 Test 2 1.6 nM 25 000 nM 2000 nM 2 Test 2 1.1 nM 21 000 nM 1700 nM 3 Test 2 4 nM 22 000 nm 1400 nM 4 — 2.7 nM 14 500 nM 300 nM 5 — 0.6 nM 2 900 nM 1 170 nm Ref Ex 9 — 2.6 nM 6 100 nM NA

(195) The compounds of formula II are thus potent inhibitors of cathepsin K and yet selective over the closely related cathepsin S and L. The compound of reference example 9 provides confidence that the corresponding difluorovinyl compounds are also active and selective

(196) Metabolic Stability

(197) Compounds of the invention and the indicated comparative examples were tested for metabolic stability in a cytosol assay in which the compounds were incubated with commercially available human hepatic cytosol fractions and the disappearance of the compound monitored by HPLC or LC/MS. Pooled human liver cytosol fractions are less likely to represent outlier individuals than blood from a single individual and can be stored frozen, unlike whole blood. The cytosol assay thus provides a consistent assay testbed as a guide to the stability of a compound in the in vivo environment, such as when exposed to whole blood.

(198) In short, test compounds (2 μM) are incubated in pooled human liver cytosol (Xenotech LLC Lenexa US, 1 mg/mL protein in 0.1M phosphate buffer, pH 7.4) at 37° centigrade over a one hour period. The incubations are initiated by the addition of 1 mM NADPH co-factor. Timed sub-samples were taken at 0, 20, 40 and 60 minutes and “crash precipitated” by the addition of 3 volumes of ice-cold acetonitrile. The samples were centrifuged at reduced temperature and the supernatants were separated and analyzed by LC-MS-MS.

(199) Alternatively, an analogous stability assay is carried out in human or monkey whole blood and/or commercially available liver microsomes, such as XEN 025.

(200) TABLE-US-00002 TABLE 2 CLint CLint whole blood HLM Example Structure ul/min/mg ul/min/mg comparative example 9 49 Example 1 3 16

(201) Comparative Example 1 represents a compound bearing a carbon-carbon bond at the 6 position within the scope of WO2008/007107 cited above. It was prepared in a facile manner from compound 1d (scheme 1). Hence with the exocyclic alkene 1d in hand, stereoselective hydrogenation of the alkene with Adams' catalyst (platinum dioxide) in ethyl acetate under a hydrogen atmosphere, proceeded with syn addition of hydrogen. This hydrogenation afforded essentially one product, namely the C-6 methyl isomer (LCMS [M+H]=288 found) with R-stereochemistry in good yield. The facial selectivity seen here for the hydrogenation step, is similar to that reported previously in the literature for a closely related bicyclic structure (Srinivas et al, Synlett, 1999, 555-556). The thus prepared building block was deprotected, elongated and oxidised to the active keto form as for the compounds of the invention exemplified above.

(202) It will be apparent from Comparative Example 1 that a methyl group at the 6 position provides a compound with whole blood CLint value of 9 micrograms/minute/mg, representing an estimated whole blood half life of little over an hour. In contrast acetylene provided a Clint value of 3, which represents a calculated whole blood half life approaching 4 hours. Note also that the HLM microsome clearances (representing the contribution of the liver to metabolism of the respective compounds) is significantly higher for the 6-methyl species than for the compound of the invention, which will further accentuate the better stability of the present invention. Improved stability in vivo allows for a better distribution of the compound in the body throughout the day, notwithstanding QD or BID dosing. This is particularly important for indications such as osteoporosis where diurnal variation is significant.

(203) Permeability

(204) This experiment measures transport of inhibitors through the cells of the human gastroenteric canal. The assay uses the well known Caco-2 cells with a passage number between 40 and 60.

(205) Apical to Basolateral Transport

(206) Generally every compound will be tested in 2-4 wells. The basolateral and the apical wells will contain 1.5 mL and 0.4 mL transport buffer (TB), respectively, and the standard concentration of the tested substances is 10 μM. Furthermore all test solutions and buffers will contain 1% DMSO. Prior to the experiment the transport plates are pre-coated with culture medium containing 10% serum for 30 minutes to avoid nonspecific binding to plastic material. After 21 to 28 days in culture on filter supports the cells are ready for permeability experiments.

(207) Transport plate no 1 comprises 3 rows of 4 wells each. Row 1 is denoted Wash, row 2 “30 minutes” and row 3 “60 minutes”. Transport plate no 2 comprises 3 rows of 4 wells, one denoted row 4 “90 minutes”, row 5 “120 minutes and the remaining row unassigned.

(208) The culture medium from the apical wells is removed and the inserts are transferred to a wash row (No. 1) in a transport plate (plate no. 1) out of 2 plates without inserts, which have already been prepared with 1.5 mL transport buffer (HBSS, 25 mM HEPES, pH 7.4) in rows 1 to 5. In A.fwdarw.B screening the TB in basolateral well also contains 1% Bovine Serum Albumin.

(209) 0.5 mL transport buffer (HBSS, 25 mM MES, pH 6.5) is added to the inserts and the cell monolayers equilibrated in the transport buffer system for 30 minutes at 37° C. in a polymix shaker. After being equilibrated to the buffer system the Transepithelial electrical resistance value (TEER) is measured in each well by an EVOM chop stick instrument. The TEER values are usually between 400 to 1000Ω per well (depends on passage number used).

(210) The transport buffer (TB, pH 6.5) is removed from the apical side and the insert is transferred to the 30 minutes row (No. 2) and fresh 425 μL TB (pH 6.5), including the test substance is added to the apical (donor) well. The plates are incubated in a polymix shaker at 37° C. with a low shaking velocity of approximately 150 to 300 rpm.

(211) After 30 minutes incubation in row 2 the inserts will be moved to new pre-warmed basolateral (receiver) wells every 30 minutes; row 3 (60 minutes), 4 (90 minutes) and 5 (120 minutes).

(212) 25 μL samples will be taken from the apical solution after ˜2 minutes and at the end of the experiment. These samples represent donor samples from the start and the end of the experiment.

(213) 300 μL will be taken from the basolateral (receiver) wells at each scheduled time point and the post value of TEER is measured at the end the experiment. To all collected samples acetonitrile will be added to a final concentration of 50% in the samples. The collected samples will be stored at −20° C. until analysis by HPLC or LC-MS.

(214) Basolateral to Apical Transport

(215) Generally every compound will be tested in 2-4 wells. The basolateral and the apical wells will contain 1.55 mL and 0.4 mL TB, respectively, and the standard concentration of the tested substances is 10 μM. Furthermore all test solutions and buffers will contain 1% DMSO. Prior to the experiment the transport plates are precoated with culture medium containing 10% serum for 30 minutes to avoid nonspecific binding to plastic material.

(216) After 21 to 28 days in culture on filter supports the cells are ready for permeability experiments. The culture medium from the apical wells are removed and the inserts are transferred to a wash row (No. 1) in a new plate without inserts (Transport plate).

(217) The transport plate comprises 3 rows of 4 wells. Row 1 is denoted “wash” and row 3 is the “experimental row”. The transport plate has previously been prepared with 1.5 mL TB (pH 7.4) in wash row No. 1 and with 1.55 mL TB (pH 7.4), including the test substance, in experimental row No. 3 (donor side).

(218) 0.5 mL transport buffer (HBSS, 25 mM MES, pH 6.5) is added to the inserts in row No. 1 and the cell monolayers are equilibrated in the transport buffer system for 30 minutes, 37° C. in a polymix shaker. After being equilibrated to the buffer system the TEER value is measured in each well by an EVOM chop stick instrument.

(219) The transport buffer (TB, pH 6.5) is removed from the apical side and the insert is transferred to row 3 and 400 μL fresh TB, pH 6.5 is added to the inserts. After 30 minutes 250 μL is withdrawn from the apical (receiver) well and replaced by fresh transport buffer. Thereafter 250 μL samples will be withdrawn and replaced by fresh transport buffer every 30 minutes until the end of the experiment at 120 minutes, and finally a post value of TEER is measured at the end of the experiment. A 25 μL samples will be taken from the basolateral (donor) compartment after ˜2 minutes and at the end of the experiment. These samples represent donor samples from the start and the end of the experiment.

(220) To all collected samples acetonitrile will be added to a final concentration of 50% in the samples. The collected samples will be stored at −20° C. until analysis by HPLC or LC-MS.

(221) Calculation

(222) Determination of the cumulative fraction absorbed, FA.sub.cum, versus time. FA.sub.cum is calculated from:

(223) $F{A}_{cum}=.Math.\frac{C_{\mathrm{RI}}}{C_{\mathrm{DI}}}$

(224) Where C.sub.Ri is the receiver concentration at the end of the interval i and C.sub.Di is the donor concentration at the beginning of interval i. A linear relationship should be obtained. The determination of permeability coefficients (P.sub.app, cm/s) are calculated from:

(225) $P_{\mathrm{app}}=\frac{\left(k.Math.V_R\right)}{\left(A.Math.60\right)}$

(226) where k is the transport rate (min.sup.−1) defined as the slope obtained by linear regression of cumulative fraction absorbed (FA.sub.cum) as a function of time (min), V.sub.R is the volume in the receiver chamber (mL), and A is the area of the filter (cm.sup.2).

(227) Reference Compounds

(228) TABLE-US-00003 Category of absorption in man Markers % absorption in man PASSIVE TRANSPORT Low (0-20%) Mannitol 16 Methotrexate 20 Moderate (21-75%) Acyclovir 30 High (76-100%) Propranolol 90 Caffeine 100 ACTIVE TRANSPORT Amino acid transporter L-Phenylalanine 100 ACTIVE EFFLUX PGP-MDR1 Digoxin 30

(229) Greater permeability through the gastrointestinal tissue is advantageous in that it allows for the use of a smaller dose to achieve similar levels of exposure to a less permeable compound administered in a higher dose. A low dose is advantageous in that minimises the cost of goods for a daily dose, which is a crucial parameter in a drug which is taken for protracted time periods.

(230) The compound of Example 2 exhibited a p.sub.app value of 9.1×10.sup.−6 cm/sec in the Caco-2 assay, whereas the prior art compound of Example 2 of WO2008 007107 exhibited a p.sub.app value of 2.7×10.sup.−6 cm/sec in a side-by-side assay run. In this assay system the arguably prior art compound N—((S)-1-((3aS, 6R, 6aS)-6-methoxy-oxodihydro-2H-furo[3,2-b]pyrrol-4(5H, 6H, 6aH)-yl)-4-methyl-oxopentan-2-yl)-4-(2-(4-methylpiperazine-1-yl)thiazol-4-yl)benzamide which is recited at page 33 of WO2008/007107 (but whose preparation is not disclosed) exhibits a p.sub.app value of 0.9×10.sup.−6 cm/sec.

(231) As a rule of thumb, a p.sub.app value around 2 represents an in vivo absorption of only 10-30% whereas a p.sub.app value approaching 10 will generally represent complete absorption.

(232) The substantial difference in p.sub.app values between Example 2 and the abovementioned prior art Example 2 of WO2008 007107 correlates well with in vivo mouse PK experiments, as illustrated in FIG. 1. The respective compounds were orally administered (60 μmol/kg in a conventional 1% Methocel A4C vehicle. As seen clearly in FIG. 1, the in vivo exposure (whether measured as C.sub.max or AUC) was very much greater for the compound of the invention than the the prior art compound (Example 2 of WO2008 007107). Note that the graph has a logarithmic scale

(233) Mutagenicity

(234) The mutagenic potential of compounds is conveniently tested in the Ames Test, typically carried out in a variety of bacterial strains such as Salmonella typhimurium TA100, TA102, TA 1535, TA 1537 with and without liver S9 fraction activation, for example at 30, 300 and 3000 ug/plate concentrations.

(235) Ames testing is readily available at a number of CROs around the world.

(236) Abbreviations

(237) TABLE-US-00004 DMF dimethylformamide DCM dichloromethane TBDMS tert-butyldimethylsilyl RT room temperature THF tetrahydrofuran Ac acetyl TLC thin layer chromatography DMAP dimethylaminopyridine EtOAc ethyl acetate uM micromolar

(238) All references referred to in this application, including patents and patent applications, are incorporated herein by reference to the fullest extent possible.

(239) Throughout the specification and the claims which follow, unless the context requires otherwise, the word ‘comprise’, and variations such as ‘comprises’ and ‘comprising’, will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.