Condensed thiophene derivatives useful as NaPi-IIb inhibitors

Abstract

The invention provides compounds of the formula: (A), pharmaceutically acceptable salts, pharmaceutical compositions thereof and methods of using these compounds, salts, or compositions to treat hyperphosphatemia, chronic kidney disease, and/or the cardiovascular disease associated with chronic kidney disease. ##STR00001##

Claims

1. A compound of the formula: ##STR00093## wherein Y is a fused cyclohexane ring or a fused phenyl ring, wherein A is ##STR00094## wherein the crossed lines indicate bonds for the point of attachment to the core of Formula II, and the dashed lines indicate bonds for the point of attachment to R.sup.2, wherein R.sup.2 is selected from the group consisting of CH.sub.3, (CH.sub.2).sub.3OH, (CH.sub.2).sub.3OCH.sub.3, (CH.sub.2).sub.3CO.sub.2H, COOCH.sub.3, COCH.sub.3, CO(CH.sub.2).sub.3CH.sub.3, COCH(CH.sub.3).sub.2, CO(CH.sub.2).sub.2CO.sub.2H, COCH.sub.2NH.sub.2, COCH.sub.2N(CH.sub.3).sub.2, SO.sub.2N[(CH.sub.2).sub.2OCH.sub.3].sub.2, SO.sub.2NHCH.sub.3, SO.sub.2(CH.sub.2).sub.2OCH.sub.3, CONH(CH.sub.2).sub.4OH, CONH(CH.sub.2).sub.4OCH.sub.3, CONHCH.sub.3, CONH(CH.sub.2).sub.2CO.sub.2H, CONH(CH.sub.2).sub.2OCH.sub.3, CON(CH.sub.2CH.sub.2OCH.sub.3).sub.2, CSNHCH.sub.3, ##STR00095## wherein the dashed lines represent the point of attachment, wherein R is CO.sub.2H or CONH.sub.2, or a pharmaceutically acceptable salt thereof.

2. The compound of C claim 1, wherein Y is a fused cyclohexane ring, A is ##STR00096## and R is CO.sub.2H, or a pharmaceutically acceptable salt thereof.

3. The compound of claim 1, wherein Y is a fused cyclohexane ring, A is ##STR00097## and R is CO.sub.2H, or a pharmaceutically acceptable salt thereof.

4. The compound of claim 1 which is of the formula: ##STR00098## wherein R.sup.2 is selected from the group consisting of (CH.sub.2).sub.3OH, (CH.sub.2).sub.3OCH.sub.3, (CH.sub.2).sub.3CO.sub.2H, CONH(CH.sub.2).sub.4OH, COCH.sub.2NH.sub.2, SO.sub.2N[(CH.sub.2).sub.2OCH.sub.3].sub.2, CONH(CH.sub.2).sub.4OCH.sub.3, and CO(CH.sub.2).sub.2CO.sub.2H, wherein R is CO.sub.2H or CONH.sub.2, or a pharmaceutically acceptable salt thereof.

5. The compound of claim 4 wherein R is CO.sub.2H, or a pharmaceutically acceptable salt thereof.

6. The compound of claim 1 which is 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid, which can be structurally represented as: ##STR00099## or a pharmaceutically acceptable salt thereof.

7. The compound claim 1 which is 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(3-hydroxypropyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid, which can be structurally represented as: ##STR00100## or a pharmaceutically acceptable salt thereof.

8. The compound claim 1 which is 4-[2-[4-[[2-[[3-[[4-[bis(2-methoxyethyl)sulfamoyl]-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid, which can be structurally represented as: ##STR00101## or a pharmaceutically acceptable salt thereof.

9. The compound claim 1 which is 4-[2-[4-[[2-[[3-[[4-(3-carboxypropyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid, which can be structurally represented as: ##STR00102## or a pharmaceutically acceptable salt thereof.

10. The compound claim 1 which is 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(3-methoxypropyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid, which can be structurally represented as: ##STR00103## or a pharmaceutically acceptable salt thereof.

11. The compound G claim 1 which is 4-[2-[4-[[2-[[3-[[4-(2-aminoacetyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid, which can be structurally represented as: ##STR00104## or a pharmaceutically acceptable salt thereof.

12. The compound claim 1 which is 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-methoxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid, which can be structurally represented as: ##STR00105## or a pharmaceutically acceptable salt thereof.

13. The compound claim 1 which is 4-[4-[[3-[[3-[[4-[2-(4-carbamoylphenyl)ethyl]-3,5-difluoro-phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen-2-yl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazin-1-yl]-4-oxo-butanoic acid, which can be structurally represented as: ##STR00106## or a pharmaceutically acceptable salt thereof.

14. A compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from the group consisting of: 4-[2-[2,6-difluoro-4-[[2-[[3-[(2,2,4-trimethylpiperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid formic acid salt; 4-[2-[4-[[2-[[2,2-dimethyl-4-(methylcarbamoyl)piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid; 4-[2-[4-[[2-[[3-[[2,2-dimethyl-4-(methylcarbamothioyl)piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid; 4-[2-[4-[[2-[[3-[[4-(2-carboxyethylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid; 4-[2-[2,6-difluoro-4-[[2-[[3-[[8-(methylcarbamoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]benzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid, formic acid salt; 4-[4-[2,6-difluoro-4-[[2-[[3-[(4-methoxycarbonyl-2,2-dimethyl-piperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid; 4-(2,6-difluoro-4-(2-(3-(((1R,5S)-8-pentanoyl-3,8-diazabicyclo[3.2.1]octan-3-yl)methyl)benzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamido)phenethyl)benzoic acid; 4-[2-[4-[[2-[[3-[[2,2-dimethyl-4-(methylsulfamoyl)piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid; 4-[2-[2,6-difluoro-4-[[2-[[3-[[8-(tetrahydropyran-4-ylcarbamoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid; 4-[2-[4-[[2-[[3-[(8-acetyl-3,8-diazabicyclo[3.2.1]octan-3-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate; 4-[2-[2,6-difluoro-4-[[2-[[3-[[8-(2-methoxyethylsulfonyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate; 4-[2-[2,6-difluoro-4-[[2-[[3-[[8-(2-methoxyethylcarbamoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate; 4-[2-[2,6-difluoro-4-[[2-[[3-[[8-(4-methoxybutylcarbamoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate; 4-[2-[4-[[2-[[3-[[8-[bis(2-methoxyethyl)carbamoyl]-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate; 4-[2-[2,6-difluoro-4-[[2-[[3-[[8-(2-methylpropanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate; 4-[2-[4-[[2-[[3-[[8-[2-(dimethylamino)acetyl]-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid; 4-[2-[2,6-difluoro-4-[[2-[[3-[[8-[1-(methoxymethyl)cyclopropanecarbonyl]-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid; 4-[2-[2,6-difluoro-4-[[2-[[3-[[8-[2-(4-methylpiperazin-1-yl)acetyl]-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid; 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]benzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid; and 4-[2-[4-[[2-[[3-[(4-acetyl-2,2-dimethyl-piperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid.

15. The compound of claim 6 which is 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid, disodium.

16. A pharmaceutical composition comprising a compound or salt according to claim 1, and a pharmaceutically acceptable carrier, diluent or excipient.

17. A solid dispersion formulation comprising 30% of 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid, disodium and 70% polyvinylpyrrolidone-vinyl acetate.

18. A method of treating hyperphosphatemia comprising administrating to a patient in need thereof an effective amount of a compound or salt according to claim 1.

19. A method of treating chronic kidney disease comprising administrating to a patient in need thereof an effective amount of a compound or salt according to claim 1.

20. A method of treating cardiovascular disease associated with chronic kidney disease comprising administrating to a patient in need thereof an effective amount of a compound or salt according to claim 1.

21. A method of treating cardiovascular disease associated with chronic kidney disease comprising administrating to a patient in need thereof an effective amount of 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid, disodium.

Description

PREPARATIONS AND EXAMPLES

(1) The following Preparations and Examples further illustrate the invention and represent typical synthesis of the compound of the invention. The reagents and starting materials are readily available or may be readily synthesized by one of ordinary skill in the art. It should be understood that the Preparations and Examples are set forth by way of illustration and not limitation, and that various modifications may be made by one of ordinary skill in the art.

(2) LC-ES/MS is performed on an AGILENT HP1100 liquid chromatography system. Electrospray mass spectrometry measurements (acquired in positive and/or negative mode) are performed on a Mass Selective Detector quadrupole mass spectrometer interfaced to the HP1100 HPLC. LC-MS conditions (low pH): column: PHENOMENEX GEMINI NX C18 2.150 mm 3.5 m; gradient: 5-100% B in 3 min, then 100% B for 0.75 min, or 5-95% B in 1.5 min, then 95% B for 0.25 min; column temperature: 50 C.+/10 C.; flow rate: 1.2 mL/min; Solvent A: deionized water with 0.1% HCOOH; Solvent B: ACN with 0.10% formic acid; wavelength 214 nm. Alternate LC-MS conditions (high pH): column: XTERRA MS C18 columns 2.150 mm, 3.5 m; gradient: 5% of solvent A for 0.25 min, gradient from 5% to 100% of solvent B in 3 min and 100% of solvent B for 0.5 min or 10% to 100% of solvent B in 3 min and at 100% of solvent B for 0.75 min or 5-95% B in 1.5 min, then 95% B for 0.25 min; column temperature: 50 C.+/10 C.; flow rate: 1.2 mL/min; Solvent A: 10 mM NH.sub.4HCO.sub.3 pH 9; Solvent B: ACN; wavelength: 214 nm.

(3) NMR spectra are performed on a Bruker AVIII HD 400 MHz NMR Spectrometer or a Varian VNMRS 300 or 400 MHz NMR Spectrometer, obtained as CDCl.sub.3 or DMSO-d.sub.6 solutions reported in ppm, using residual solvent [CDCl.sub.3, 7.26 ppm; DMSO-d.sub.6, 2.50 ppm] as reference standard. When peak multiplicities are reported, the following abbreviations may be used: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br s (broad singlet), dd (doublet of doublets), dt (doublet of triplets). Coupling constants (J), when reported, are reported in hertz (Hz).

Preparation 1

methyl 4-[2-(4-amino-2,6-difluoro-phenyl)ethynyl]benzoate

(4) ##STR00018##

(5) A suspension of 3,5-difluoro-4-iodoaniline (14.7 g, 55.9 mmol), CuI (0.745 g, 3.91 mmol), bis(triphenylphosphine)palladium(II) dichloride (1.59 g, 2.24 mmol), methyl 4-ethynylbenzoate (9.05 g, 55.9 mmol), TEA (114 mL) and THF (44.1 mL) is stirred at 60 C. for 3 hr. The mixture is cooled to RT and the solvent evaporated to dryness under reduced pressure. EtOAc (100 mL) and H.sub.2O (100 mL) are added, and the resulting solid is filtered over diatomaceous earth. The organic layer from the filtrate is separated, dried over MgSO.sub.4, and evaporated to dryness under reduced pressure. A 1:1 mixture of DCM:heptane (400 mL) is added to the resulting residue and the mixture is stirred at RT overnight. The resulting solid is collected by filtration and dried under vacuum to obtain the title compound (8.0 g, 45.8% yield) as a brown solid. .sup.1H NMR (300 MHz, DMSO-d.sub.6) 3.86 (s, 3H), 6.26-6.37 (m, 4H), 7.59 (d, J=8.2 Hz, 2H), 7.96 (d, J=8.5 Hz, 2H).

Preparation 2

methyl 4-[2-(4-amino-2,6-difluoro-phenyl)ethyl]benzoate

(6) ##STR00019##

(7) A 500 mL Parr shaker is charged with Pd black (0.53 g, 5.0 mmol) under N.sub.2. A degassed 4:1 solution of MeOH/THF (25 mL) is added followed by a degassed solution of methyl 4-[2-(4-amino-2,6-difluoro-phenyl)ethynyl]benzoate (1.17 g, 3.38 mmol) in a 4:1 mixture of MeOH/THF (25 mL) under N.sub.2. The resulting mixture is purged with N.sub.2 and pressurized with H.sub.2 to 60 psi. The sealed vessel is heated at 40 C. for 14 hr. The resulting suspension is filtered through a pad of diatomaceous earth under N.sub.2 and evaporated to dryness in vacuo. The resulting residue is purified by chromatography over silica, eluting with a gradient of 25-35% hexanes/THF, to afford the title compound as a white solid (404 mg, 400/% yield) after solvent evaporation and drying under vacuum. .sup.1H NMR (400.13 MHz, DMSO-d.sub.6) , 2.71-2.83 (m, 4H), 3.84 (s, 3H), 5.52 (s, 2H), 6.10-6.15 (m, 2H), 7.28 (d, J=8.3 Hz, 2H), 7.85 (d, J=8.2 Hz, 2H). LC-ES/MS (m/z) 292 [M+1].

Preparation 3

tert-butyl 4-[(4-methoxy-4-oxo-butyl)carbamoyl]-2,2-dimethyl-piperazine-1-carboxylate

(8) ##STR00020##

(9) A 30 mL scintillation vial is charged with t-butyl 2,2-dimethylpiperazine-1-carboxylate (500 mg, 2.28 mmol) and DCM (12 mL). The resulting solution is cooled in an ice/water bath and DIPEA (1.20 mL, 6.85 mmol) is added in one portion. A solution of methyl 4-isocyanatobutanoate (447 mg, 2.97 mmol) in DCM (3 mL) is added drop wise over 5 min. The reaction mixture is slowly warmed to RT and stirred for a further 15 min. The mixture is partitioned between 5% aqueous citric acid (100 mL) and DCM (20 mL). The organic layer is separated, and the aqueous layer is extracted twice more with DCM (20 mL each). The combined organic extracts are washed sequentially with saturated aqueous NaHCO.sub.3 (30 mL) and saturated aqueous NaCl (30 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and evaporated under reduced pressure. The resulting residue is purified by chromatography over silica, eluting with a gradient of 30-50% hexanes/acetone, to obtain the title compound as colorless viscous oil (848 mg, 95% yield) after solvent removal and drying under vacuum. .sup.1H NMR (399.8 MHz, CDCl.sub.3) 1.34 (s, 6H), 1.45 (s, 9H), 1.83 (t, J=6.9 Hz, 2H), 2.37 (t, J=7.1 Hz, 2H), 3.25-3.30 (m, 2H), 3.37 (t, J=5.7 Hz, 2H), 3.47 (s, 2H), 3.65 (s, 3H), 3.71 (t, J=5.7 Hz, 2H), 4.65-4.68 (m, 1H). LC-ES/MS (m/z) 358 [M+1].

Preparation 4

tert-butyl 4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazine-1-carboxylate

(10) ##STR00021##

(11) A 2 M solution of LiBH.sub.4 in THF (3.02 mL, 6.04 mmol) is added drop wise to a 100 mL round bottom flask containing tert-butyl 4-[(4-methoxy-4-oxo-butyl)carbamoyl]-2,2-dimethyl-piperazine-1-carboxylate (783 mg, 2.01 mmol) and THF (2 mL) at RT. The resulting mixture is stirred for 12 hr at RT. The reaction mixture is quenched with 0.5 mL of MeOH, stirred at RT for 20 min, and partitioned between 5% aqueous NaHCO.sub.3 (150 mL) and DCM (50 mL). The organic layer is separated, and the aqueous layer is extracted twice more with DCM (50 mL each). The combined organic layers are washed with saturated aqueous NaCl (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and evaporated to dryness under reduced pressure to afford the title compound as a white solid (634 mg, 96% yield). .sup.1H NMR (399.8 MHz, CDCl.sub.3) 1.36 (s, 6H), 1.46 (s, 9H), 1.58-1.63 (m, 4H), 3.31-3.28 (m, 2H), 3.49 (s, 2H), 3.66-3.69 (m, 2H), 3.71-3.74 (m, 2H), 3.37-3.39 (m, 2H). LC-ES/MS (m/z) 330 [M+1].

Preparation 5

N-(4-hydroxybutyl)-3,3-dimethyl-piperazine-1-carboxamide hydrochloride

(12) ##STR00022##

(13) A 30 mL scintillation vial is charged with a solution of tert-butyl 4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazine-1-carboxylate (631 mg, 1.92 mmol) in DCM (20 mL). A 4 N solution of HCl in dioxane (2.4 mL, 9.58 mmol) is added drop wise over 5 min and the resulting solution is stirred at RT for 2 hr. The volatiles are removed in vacuo and the residue is dried under vacuum to afford the title compound as a hygroscopic white oily solid (100% yield, quantitative), suitable for use in the next step without further purification. LC-ES/MS (m/z) 230 [M+1].

Preparation 6

methyl 4-[2-[4-[[2-(tert-butoxycarbonylamino)-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate

(14) ##STR00023##

(15) A 100 mL round bottom flask is charged with methyl 4-[2-(4-amino-2,6-difluoro-phenyl)ethyl]benzoate (2.11 g, 7.24 mmol), 3-(tert-butoxycarbonylamino)-4,5,6,7-tetrahydrobenzothiophene-2-carboxylic acid (2.37 g, 7.97 mmol), and DCM (60 mL). The resulting suspension is cooled in an ice/water bath, and DIPEA (5.05 mL, 29.0 mmol) is added drop wise to afford a yellow-brown turbid solution. Solid bis-(2-oxo-3-oxazolidinyl)phosphinic chloride (2.30 g, 9.05 mmol) is added in small portions over 30 min at 0 C. The reaction mixture is then warmed to RT and stirred for 24 hr. Additional solid 3-(tert-butoxycarbonylamino)-4,5,6,7-tetrahydrobenzothiophene-2-carboxylic acid (0.6 g, 2.0 mmol) is added followed by additional DIPEA (1.26 mL, 7.25 mmol) and solid bis-(2-oxo-3-oxazolidinyl)phosphinic chloride (0.58 g, 2.26 mmol) in small portions over 5 min. The resulting turbid brown reaction mixture is stirred at RT for 12 hr. The reaction mixture is partitioned between 5% aqueous citric acid (150 mL) and DCM (25 mL), the organic layer is separated, and the aqueous layer is extracted twice more with DCM (50 mL each). The combined organic extracts are washed sequentially with 10% aqueous NaHCO.sub.3 (50 mL), saturated aqueous NaCl (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The resulting brown oily solid is purified by chromatography over silica, eluting with a gradient of 15-40% hexanes/(10% MTBE in DCM). The collected fractions containing desired product are combined and concentrated under reduced pressure, and the resulting residue is triturated with MTBE (15 mL). The resulting solid is collected by filtration and dried under vacuum to afford the title compound (2.75 g, 66% yield). .sup.1H NMR (400.1 MHz, DMSO-d.sub.6) 1.44 (s, 9H), 1.69-1.77 (m, 4H), 2.53-2.65 (m, 4H), 2.91 (br s, 4H), 3.84 (s, 3H), 7.30-7.34 (m, 4H), 7.86 (d, J=8.3 Hz, 2H), 9.80 (br s, 1H), 9.94 (s, 1H). LC-ES/MS (m/z) 569 [M1].

Preparation 7

methyl 4-[2-[4-[(2-amino-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl)amino]-2,6-difluoro-phenyl]ethyl]benzoate hydrochloride

(16) ##STR00024##

(17) A 30 mL scintillation vial is charged with methyl 4-[2-[4-[[2-(tert-butoxycarbonylamino)-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate (351 mg, 0.61 mmol) and DCM (6 mL). The resulting light yellow solution is degased by passing through a gentle stream of N.sub.2. A solution of 4 N HCl in dioxane (155 mL, 6.1 mmol) is added drop wise over 5 min and the resulting solution is stirred at RT for 12 hr. The reaction mixture is concentrated in vacuo and the resulting pale yellow residue is triturated with a minimal amount of DCM. The resulting solid is collected by filtration and dried under vacuum to afford the title compound as an off-white powder (337 mg, 99% yield). .sup.1H NMR (399.8 MHz, DMSO-d.sub.6) 1.73-1.75 (m, 4H), 2.40-2.44 (m, 2H), 2.56-2.59 (m, 2H), 2.85 (s, 4H), 3.80 (s, 3H), 7.26-7.28 (m, 4H), 7.81-7.83 (m, 2H), 9.21 (s, 1H). LC-ES/MS (m/z) 571 [M+1].

Preparation 8

methyl 4-[2-[4-[[2-[[3-(chloromethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate

(18) ##STR00025##

(19) A suspension of methyl 4-[2-[4-[(2-amino-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl)amino]-2,6-difluoro-phenyl]ethyl]benzoate hydrochloride (2.43 g, 4.55 mmol) in DCM (80 mL) in a 250 mL round bottom flask is cooled to 0 C. with an ice/water bath. Pyridine (0.92 mL, 11 mmol) is added drop wise with stirring over 5 min. The resulting pale yellowish solution is stirred for an additional 5 min at 0 C., and a solution of 3-(chloromethyl)benzoyl chloride (0.71 mL, 5.0 mmol) in DCM (20 mL) is added drop wise over 5 min. The reaction mixture is stirred for additional 30 min at 0 C. The reaction mixture is diluted with 150 mL of 10% aqueous citric acid and stirred at RT for 1 hr. The organic layer is separated and the aqueous layer is extracted twice more with DCM (50 mL each). The combined organic extracts are washed sequentially with 5% aqueous NaHCO.sub.4, (250 mL) and saturated aqueous NaCl (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and evaporated to dryness under reduced pressure. The resulting residue is triturated with EtOH (30 mL), and the resulting solid is collected by filtration, washed with EtOH (15 mL), and dried under vacuum to yield the title compound as a tan solid (2.61 g, 92% yield). .sup.1H NMR (400.1 MHz, DMSO-d.sub.6) ), 1.73-1.81 (m, 4H), 2.68 (m, 4H), 2.92 (s, 4H), 3.84 (s, 3H), 4.83 (s, 2H), 7.32 (d, J=8.3 Hz, 2H) 7.38-7.344 (m, 2H), 7.56 (t, J=7.7 Hz, 1H), 7.69 (d, J=7.8 Hz, 1H), 7.82 (d, J=7.9 Hz, 1H), 7.87 (d, J=8.2 Hz, 2H), 7.96 (br s, 1H), 10.10 (s, 1H), 11.34 (s, 1H). LC-ES/MS (m/z) 621 [M1].

Preparation 9

methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-hydroxybutyl carbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate

(20) ##STR00026##

(21) A 2 mL microwave vial is charged with methyl 4-[2-[4-[[2-[[3-(chloromethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate (50 mg, 0.08 mmol), N-(4-hydroxybutyl)-3,3-dimethyl-piperazine-1-carboxamide hydrochloride (42.7 mg, 0.16 mmol) and DIPEA (0.056 mL, 0.32 mmol) in a mixture of ACN (1.5 mL) and MeOH (50 L). The resulting yellow suspension is heated in a BIOTAGE Initiator microwave synthesizer at 110 C. for 4 hr. The reaction mixture is concentrated in vacuo and the residue is partitioned between 5% aqueous NaHCO.sub.3 (75 mL) and DCM (25 mL). The organic layer is separated, the aqueous layer is extracted with twice more with DCM (25 mL each), and the combined organic extracts are washed with saturated aqueous NaCl (25 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and evaporated to dryness under reduced pressure. The resulting residue is purified by reverse phase chromatography over C-18 silica, eluting with a gradient of 0-100% of a mixture of 5% HCOOH in H.sub.2O/ACN, to afford the title compound as a light yellow foamy solid (30.2 mg, 46% yield) after solvent evaporation. .sup.1H NMR (400.1 MHz, DMSO-d.sub.6) 1.05 (s, 6H), 1.37-1.44 (m, 4H), 1.80-1.85 (m, 4H), 2.25-2.27 (m, 2H), 2.65-2.677 (m, 4H), 2.91 (br s, 4H), 2.96-3.05 (m, 2H), 3.12 (s, 2H), 3.17-3.26 (m, 2H), 3.35-3.41 (m, 2H), 3.51 (s, 2H), 3.84 (s, 3H), 4.36 (t, J=5.1 Hz, 1H), 6.35-6.39 (m, 1H), 7.32 (d, J=8.3 Hz, 2H), 7.51-7.56 (m, 4H), 7.70-7.80 (m, 1H), 7.87 (m, 3H), 10.01 (br s, 1H), 11.45 (br s, 1H). LC-ES/MS (m/z) 816 [M+1].

Preparation 10

tert-butyl 4-[[3-[[3-[[3,5-difluoro-4-[2-(4-methoxycarbonylphenyl)ethyl]phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen-2-yl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazine-1-carboxylate

(22) ##STR00027##

(23) A 20 mL microwave reaction vessel is charged with methyl 4-[2-[4-[[2-[[3-(chloromethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate (1.85 g, 2.97 mmol), tert-butyl 3,3-dimethylpiperazine-1-carboxylate (0.91 g, 4.16 mmol), and DIPEA (2.07 mL, 11.9 mmol) in 15 mL of ACN. The resulting yellow suspension is heated in a BIOTAGE Initiator microwave synthesizer at 110 C. for 4 hr. The reaction mixture is concentrated in vacuo and the residue is partitioned between 5% aqueous NaHCO.sub.3 (150 mL) and DCM (50 mL). The organic layer is separated, the aqueous layer is extracted with DCM (225 mL). The combined organic layers are washed with saturated aqueous NaCl (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to dryness under reduced pressure. The resulting residue is purified by chromatography over silica, eluting with a gradient of 0-100% of a mixture of 9:1 DCM/acetone in hexane, to afford the title compound as a yellow solid (1.63 g, 69% yield) after solvent evaporation. .sup.1H NMR (400.1 MHz, DMSO-ds) 1.04 (s, 6H), 1.38 (s, 9H), 1.81-1.74 (m, 4H), 2.28 (t, J=5.0 Hz, 2H), 2.70 (br s, 4H), 2.91 (s, 4H), 3.13 (s, 2H), 3.26 (s, 2H), 3.52 (s, 2H), 3.84 (s, 3H), 7.31 (d, J=8.3 Hz, 2H), 7.44-7.39 (m, 2H), 7.55-7.46 (m, 2H), 7.75 (d, J=7.6 Hz, 1H), 7.87 (m, 3H), 9.99 (s, 1H), 11.49 (s, 1H). LC-ES/MS (m/z) 801 [M+1].

Preparation 11

methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate dihydrochloride

(24) ##STR00028##

(25) A solution of tert-butyl 4-[[3-[[3-[[3,5-difluoro-4-[2-(4-methoxycarbonylphenyl)ethyl]phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen-2-yl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazine-1-carboxylate (1.13 g, 1.41 mmol) in DCM (14.1 mL) is stirred at RT and 4 N HCl in dioxane (3.5 mL, 14.1 mmol) is added via syringe. Upon complete addition, the reaction is stirred at RT overnight and concentrated to dryness under reduced pressure. The solid is triturated with DCM/Et.sub.2O, the resulting precipitate is collected via vacuum filtration, and the filter cake is dried in a vacuum oven at 50 C. to afford the title compound as a white solid (0.93 g, 1.20 mmol, 85% yield). LC-ES/MS (m/z) 701 [M+1].

Preparation 12

methyl 4-[2-[4-[[2-[[3-[[4-[bis(2-methoxyethyl)sulfamoyl]-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate

(26) ##STR00029##

(27) To a solution of methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate dihydrochloride (190.8 mg, 0.24 mmol) and TEA (0.165 mL, 1.18 mmol) in 4 mL of THF, bis(2-methoxyethyl)sulfamoyl chloride (87 mg, 0.36 mmol) in 4 mL of THF is added drop wise. The resulting mixture is heated to 50 C. for 18 hr. After cooling to RT, the reaction mixture is diluted with a mixture of 5% aqueous NaHCO.sub.3 (75 mL) and DCM (25 mL). The layers are separated, and the aqueous layer is washed with additional DCM (225 mL). The organic extracts are combined, washed with saturated aqueous NaCl (25 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The resulting residue is purified by chromatography over silica, eluting with a gradient of 10-30% of a mixture of acetone in hexanes, to afford the desired product as a light yellow solid (150.1 mg, 70.8% yield) after solvent evaporation. .sup.1H NMR (400.1 MHz, DMSO-d.sub.6) 1.13 (s, 6H), 1.85-1.70 (m, 4H), 2.43-2.36 (m, 2H), 2.75-2.65 (m, 4H), 3.03-2.86 (m, 8H), 3.24 (s, 6H), 3.36-3.32 (m, 4H), 3.44 (t, J=5.8 Hz, 4H), 3.54 (s, 2H), 3.84 (s, 3H), 7.32 (d, J=8.3 Hz, 2H), 7.52-7.38 (m, 3H), 7.56 (d, J=7.6 Hz, 1H), 7.74 (d, J=7.6 Hz, 1H), 7.91-7.84 (m, 3H), 10.00 (s, 1H), 11.47 (s, 1H). LC-ES/MS (m/z) 896 [M+1].

Preparation 13

methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-methoxy-4-oxo-butyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate

(28) ##STR00030##

(29) To a solution of methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate dihydrochloride (148.4 mg, 0.1688 mmol), DIPEA (60 L, 0.34 mmol) and 4-oxobutanoic acid methyl ester (40 mg, 0.34 mmol) in 4 mL of DCM is added AcOH (0.01 mL), followed by sodium triacetoxyborohydride (0.073 g, 0.34 mmol). The resulting reaction mixture is allowed to stir at RT for 12 hr. The reaction mixture is diluted with a mixture of 5% aqueous NaHCO.sub.3 (75 mL) and DCM (25 mL). The layers are separated, and the aqueous layer is washed with additional DCM (225 mL). The organic extracts are combined, washed with saturated aqueous NaCl (25 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The resulting residue is purified by chromatography over silica, using a gradient of 35-40% of a mixture of 9:1 EtOH/DCM in hexane, to afford the desired product as a light yellow solid (132.1 mg, 91.8% yield) after solvent evaporation. .sup.1H NMR (400.1 MHz, DMSO-d.sub.6) 1.09 (s, 6H), 1.70-1.58 (m, 2H), 1.87-1.70 (m, 4H), 2.20-2.09 (m, 2H), 2.36-2.27 (m, 4H), 2.63-2.54 (m, 2H), 2.75-2.64 (m, 4H), 2.92 (s, 4H), 3.30-3.28 (m, 2H), 3.65-3.53 (m, 5H), 3.84 (s, 3H), 7.32 (d, J=8.3 Hz, 2H), 7.58-7.37 (m, 4H), 7.72-7.69 (m, 1H), 7.91-7.83 (m, 3H), 9.99 (s, 1H), 11.45 (s, 1H). LC-ES/MS (m/z) 801 [M+1].

Preparation 14

methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(3-methoxypropyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate

(30) ##STR00031##

(31) To a solution of methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate dihydrochloride (0.108 g, 0.13 mmol), 3-methoxypropionaldehyde (0.023 g, 0.25 mmol), and DIPEA (0.043 mL, 0.25 mmol) in 4 mL of DCM, AcOH (0.01 mL) is added, followed by sodium triacetoxyborohydride (0.053 g, 0.25 mmol). The resulting reaction mixture is stirred at RT for 12 hr. The reaction mixture is diluted with of 5% aqueous NaHCO.sub.3 (75 mL) and of DCM (25 mL). The layers are separated, and the aqueous layer is washed with additional DCM (225 mL). The organic extracts are combined, washed with saturated aqueous NaCl (25 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The resulting residue is purified by chromatography over silica, using a gradient of 25-80% acetone in hexane, to afford the desired product as a light yellow solid (26.2 mg, 27.6% yield) after solvent evaporation. .sup.1H NMR (400.1 MHz, DMSO-d.sub.6) 11.44 (m, 1H), 9.98 (m, 1H), 7.92-7.82 (m, 3H), 7.76-7.70 (m, 1H), 7.57-7.38 (m, 4H), 7.36-7.27 (m, 2H), 3.84 (s, 4H), 3.27-3.16 (m, 4H), 2.97-2.87 (m, 4H), 2.76-2.64 (m, 4H), 2.37-2.28 (m, 2H), 2.25-2.15 (m, 2H), 1.90-1.71 (m, 4H), 1.67-1.54 (m, 2H), 1.09 (s, 6H). LC-ES/MS (m/z) 773 [M+1].

Preparation 15

methyl 4-[2-[4-[[2-[[3-[[4-[2-(tert-butoxycarbonylamino)acetyl]-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate

(32) ##STR00032##

(33) To a round bottom flask is added methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate dihydrochloride (500 mg, 1.4 mmol), DMF (25 mL), and 1,8-diazabicyclo [5.4.0]undec-7-ene (0.19 mL, 1.27 mmol). The mixture is stirred briefly and treated with HOBT (103 mg, 0.67 mmol), EDCI (250 mg, 1.6 mmol), and N-(tert-butoxycarbonyl)glycine (132 mg, 0.75 mmol). The mixture is stirred at RT for 18 hours. The mixture is diluted with water (25 mL) and the resulting slurry is stirred at RT for 3 hr. The resulting light yellow solid is collected by filtration, washed with water, and air-dried. The resulting powder is dissolved in DCM, dried over anhydrous MgSO.sub.4, filtered, and concentrated under reduced pressure. The residue is purified by chromatography over silica, eluting with 15% EtOAc in DCM for 20 min then 25% EtOAc in DCM, to afford the title compound (451 mg, 38% yield) after solvent evaporation. .sup.1H NMR (400.13 MHz, DMSO) 11.52-11.46 (m, 1H), 9.99 (s, 1H), 7.90-7.86 (m, 3H), 7.76-7.74 (m, 1H), 7.57-7.55 (m, 1H), 7.52-7.47 (m, 1H), 7.44-7.39 (m, 2H), 7.33 (d, J=7.6 Hz, 2H), 6.76-6.71 (m, 1H), 3.84 (s, 3H), 3.81-3.76 (m, 2H), 3.57-3.54 (m, 2H), 3.43-3.38 (m, 2H), 3.32 (s, 12H, water), 3.28-3.22 (m, 2H), 2.91 (s, 4H), 2.70 (s, 4H), 2.51-2.50 (m, 16H, DMSO), 2.39-2.35 (m, 2H), 1.87-1.81 (m, 4H), 1.40-1.35 (m, 10H), 1.29-1.25 (m, 1H), 1.07 (d, J=21.1 Hz, 6H). LC-ES/MS (m/z) 858 [M+1].

Preparation 16

methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-methoxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate

(34) ##STR00033##

(35) In a 30 mL scintillation vial, a solution of methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate dihydrochloride (120 mg, 0.155 mmol) and TEA (63 mg, 0.62 mmol) in DCM (3 mL) is stirred at RT as a solution of 1-isocyanato-4-methoxy-butane (30 mg, 0.23 mmol) in DCM (1 mL) is added via syringe. The resulting reaction mixture is allowed to stir at RT for 4 hours. The reaction mixture is concentrated under reduced pressure and purified by chromatography over silica, eluting with a gradient of 0-100% EtOAc/hexanes, to afford the title compound as a tan foam (102 mg, 790/% yield) after solvent evaporation. .sup.1H NMR (400.1 MHz, DMSO-d.sub.6) 1.03 (s, 6H), 1.35-1.45 (m, 4H), 1.68-1.82 (m, 4H), 2.21-2.25 (m, 2H), 2.63-2.70 (m, 4H), 2.89 (s, 4H), 2.95-3.02 (m, 2H), 3.095 (s, 2H), 3.18 (s, 3H); 3.15-3.22 (m, 2H), 3.25-3.30 (m, 2H), 3.48 (s, 2H), 3.815 (s, 3H), 6.334 (t, J=5.5 Hz, 1H), 7.30 (d, J=7.9 Hz, 2H), 7.35-7.42 (m, 2H), 7.42-7.50 (m, 1H), 7.50-7.55 (m, 1H), 7.71 (d, J=7.6 Hz, 1H), 7.83-7.89 (m, 3H), 9.981 (s, 1H), 11.44 (s, 1H). LC-ES/MS (m/z) 830 [M+1].

Preparation 17

4-[2-[4-[[2-[[3-[(4-tert-butoxycarbonyl-2,2-dimethyl-piperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic Acid

(36) ##STR00034##

(37) A 60 mL scintillation vial is charged with tert-butyl 4-[[3-[[3-[[3,5-difluoro-4-[2-(4-methoxycarbonylphenyl)ethyl]phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen-2-yl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazine-1-carboxylate (3.92 g, 4.8 mmol), lithium hydroxide (570 mg, 24 mmol), THF (20 mL), MeOH (10 mL) and H.sub.2O (10 mL) and the resulting suspension is stirred at RT for 12 hr. The reaction mixture is diluted with H.sub.2O (40 mL) and concentrated in vacuo to 2 volume. The pH of the resulting mixture is adjusted to 5-6 with 10% aqueous citric acid and the resulting colorless suspension is partitioned between 150 mL of water and 50 mL of 4:1 chloroform/isopropanol. The organic layer is separated, the pH of the aqueous layer is adjusted again to pH5 with 10% aqueous citric acid, and the mixture is extracted twice with additional 4:1 chloroform/isopropanol (250 mL). The organic layers are combined, washed with saturated aqueous NaCl, dried over anhydrous Na.sub.2SO.sub.4, filtered, and the filtrate is concentrated under reduced pressure to give the title compound (3.7 g, >99% yield) as an off-white solid that may be used in the subsequent step without additional purification. LC-ES/MS (m/z) 787 [M+1].

Preparation 18

tert-butyl 4-[[3-[[3-[[4-[2-(4-carbamoylphenyl)ethyl]-3,5-difluoro-phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen-2-yl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazine-1-carboxylate

(38) ##STR00035##

(39) A 500 mL round bottom flask is charged with a solution of 4-[2-[4-[[2-[[3-[(4-tert-butoxycarbonyl-2,2-dimethyl-piperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid (3.50 g, 4.45 mmol) and DIPEA (3.1 mL, 17.8 mmol) in DCM (75 mL). To this solution solid bis(2-oxo-3-oxazolidinyl)phosphinic chloride (1.4 g, 5.3 mmol) is added in small portions over 10 min and the resulting suspension is stirred at RT for 10 min. A 0.5 M solution of NH.sub.3 in 1,4-dioxane (40 mL, 22.25 mmol) is added in one portion and the resulting suspension is stirred for 2 h at RT. Additional bis(2-oxo-3-oxazolidinyl)phosphinic chloride (0.7 g, 2.7 mmol) is added in small portions over 5 min and the resulting suspension is left to stir for 12 h at RT. Additional bis(2-oxo-3-oxazolidinyl)phosphinic chloride (0.7 g, 2.7 mmol) is again added in small portions over 5 min and the resulting suspension is left to stir for 12 h at RT. The resulting reaction mixture is partitioned between 300 mL of 5% aqueous NaHCO.sub.3 and 50 mL of DCM. The layers are separated, and the aqueous layer is extracted twice with additional DCM (2100 mL). The combined organic layers are washed with saturated aqueous NaCl, dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to dryness in vacuo. The resulting yellow foamy residue is purified by chromatography over silica, eluting with a gradient of 10-12% acetone/DCM; the mobile phase is then switched to 35% MeOH/DCM. The collected fractions containing desired product are combined and concentrated under reduced pressure and the residue is triturated with DCM. The resulting solid is collected by filtration and dried under vacuum to afford the title compound. The trituration filtrate is recovered and evaporated to dryness in vacuo. The resulting residue is purified by chromatography over silica, eluting with a gradient of 5-10% MeOH in DCM and the collected fractions containing desired product are combined and evaporated and added to the material obtained from the first purification to give the title compound as a light yellow solid (2.43 g, 70% yield). .sup.1H NMR (400.1 MHz, DMSO-d.sub.6) 1.05 (s, 6H), 1.39 (s, 9H), 1.68-1.90 (m, 4H), 2.29 (m, 2H), 2.70 (m, 4H), 2.89 (m, 4H), 3.14 (s, 2H), 3.28 (s, 2H), 3.51 (s, 2H), 7.16-7.34 (m, 3H), 7.36-7.58 (m, 4H), 7.68-7.73 (m, 3H), 7.90 (m, 2H), 10.00 (s, 1H), 11.49 (s, 1H). LC-ES/MS (m/z) 786 [M+1].

Preparation 19

N-[4-[2-(4-carbamoylphenyl)ethyl]-3,5-difluoro-phenyl]-2-[[3-[(2,2-dimethylpiperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carboxamide dihydrochloride

(40) ##STR00036##

(41) A solution of tert-butyl 4-[[3-[[3-[[4-[2-(4-carbamoylphenyl)ethyl]-3,5-difluoro-phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen-2-yl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazine-1-carboxylate (2.43 g, 3.1 mmol) in a mixture of DCM (80 mL) and MeOH (8 mL) in a 250 mL round-bottom flask is thoroughly degassed under nitrogen, and a 4 N solution of HCl in 1,4-dioxane (16 mL, 62 mmol) is added drop wise over 10 min. The resulting solution is stirred for 12 h at RT. Volatiles are removed in vacuo to give the title compound as a yellow, hygroscopic solid (2.45 g, >99%) which may be used in the next step without further purification.

Preparation 20

methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[(2,2,4-trimethylpiperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate

(42) ##STR00037##

(43) A 5 mL microwave reaction vessel is charged with methyl 4-[2-[4-[[2-[[3-(chloromethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate (225 mg, 361 mmol) and a solution of 1,3,3-trimethylpiperazine (65 mg, 0.488 mmol) and DIPEA (0.25 mL, 1.4 mmol) in 3 mL of ACN. The resulting yellow suspension is heated in a BIOTAGE Initiator microwave synthesizer at 110 C. for 3 hr. The reaction mixture is concentrated in vacuo and the residue is partitioned between 5% aqueous NaHCO.sub.3 (75 mL) and DCM (25 mL). The organic layer is separated, the aqueous layer is extracted with DCM (225 mL). The combined organic layers are washed with saturated aqueous NaCl (25 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to dryness under reduced pressure. The resulting residue is purified by chromatography over silica, eluting with a gradient of 20-50% of a mixture of 10% 7N NH.sub.3/MeOH in dcm in MTBE, to afford the title compound as a light yellow solid (195 mg, 760/% yield) after solvent evaporation. .sup.1H NMR (400.1 MHz, DMSO-d.sub.6): 1.11 (two s, 6H). 1.90-1.64 (m, 4H), 2.25-1.94 (m, 4H), 2.40-2.26 (m, 2H), 2.50 (under residual dmso resonance, 3H), 2.79-2.60 (br s, 4H), 2.92 (s, 4H), 3.77-3.76 (br, 2H), 3.84 (s, 3H), 7.32 (m, 2H), 7.60-7.36 (m, 4H), 7.79-7.67 (m, 1H), 7.87 (m, 3H), 9.99 (s, 1H), 11.46 (s, 1H). LC-ES/MS (m/z) 715 [M+l].

Preparation 21

tert-butyl 4-[(3-methoxy-3-oxo-propyl)carbamoyl]-2,2-dimethyl-piperazine-1-carboxylate

(44) ##STR00038##

(45) 20 mL scintillation vial is charged with a solution of tert-butyl 2,2-dimethylpiperazine-1-carboxylate (500 mg, 2.29 mmol) in DCM (12 mL) and cooled down in ice bath. While cooling DIPEA (1.20 mL, 6.86 mmol) is added followed by a solution of methyl 3-isocyanatopropanoate (404 mg, 2.97 mmol) in DCM (3 mL) added dropwise over 5 min. The resulting mixture is allowed to warm up to rt and stirred at rt for 15 min. The rxn mixture is diluted with 5% aqueous citric acid (100 mL) and DCM (25 mL), and the layers are separated. The aqueous layer is extracted with DCM (225 mL). The organic extracts are combined, washed with saturated aqueous NaCl (25 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The resulting residue is purified by chromatography over silica, eluting with a gradient of 30-53% of acetone in hexanes, to afford the title compound as a colorless thick oil (726 mg, material contains 10% of residual DCM, 83% yield) after solvent evaporation. .sup.1H NMR (399.8 MHz, CDCl.sub.3): 1.34 (s, 6H), 1.45 (s, 9H), 2.58-2.48 (m, 2H), 3.37 (t, J=5.7 Hz, 2H), 3.54-3.43 (m, 4H), 3.75-3.62 (m, 5H), 5.08-4.99 (m, 1H). LC-ES/MS (m/z) 344 [M+1].

Preparation 22

methyl 3-[(3,3-dimethylpiperazine-1-carbonyl)amino]propanoate hydrochloride

(46) ##STR00039##

(47) To a 20 mL scintillation vial containing a solution of tert-butyl 4-[(3-methoxy-3-oxo-propyl)carbamoyl]-2,2-dimethyl-piperazine-1-carboxylate (250 mg, 0.65 mmol) in DCM (7 mL), 4 N hydrochloric acid in dioxane (1.63 mL, 6.54 mmol) is added dropwise with stirring. The resulting suspension is stirred at rt for 1 hr, concentrated in vacuo and the residue is dried under vacuum to obtain the title compound as white hygroscopic solid which is used in the next step without as it is. LC-ES/MS (m/z) 244 [M+1].

Preparation 23

methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-[(3-methoxy-3-oxo-propyl)carbamoyl]-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate

(48) ##STR00040##

(49) A 5 mL microwave reaction vessel is charged with methyl 4-[2-[4-[[2-[[3-(chloromethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate (180 mg, 0.29 mmol), a solution of methyl 3-[(3,3-dimethylpiperazine-1-carbonyl)amino]propanoate hydrochloride (162 mg, 0.58 mmol) and N,N-DIPEA (0.202 mL, 1.16 mmol) in a mixture of 3 mL of ACN and 0.5 mL of MeOH. The resulting yellow suspension is heated in a BIOTAGE Initiator microwave synthesizer at 110 C. for 3 hr. The reaction mixture is concentrated in vacuo and the residue is partitioned between 5% aqueous NaHCO.sub.3 (75 mL) and DCM (25 mL). The organic layer is separated; the aqueous layer is extracted with DCM (225 mL). The combined organic layers are washed with saturated aqueous NaCl (25 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to dryness under reduced pressure. The resulting residue is purified by chromatography over C-18 silica, eluting with a gradient of 15-80% of a mixture of 0.1% formic acid/ACN in 0.1% formic acid/H.sub.2O for 15 minutes, to obtain the title compound as a pale orange solid (81 mg, 34% yield) after solvent evaporation. LC-ES/MS (m/z) 830 [M+1].

Preparation 24

tert-butyl 3-[[3-[[3-[[3,5-difluoro-4-[2-(4-methoxycarbonylphenyl)ethyl]phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen-2-yl]carbamoyl]phenyl]methyl]-3,8-diazabicyclo[3.2.1]octane-8-carboxylate

(50) ##STR00041##

(51) A 20 mL microwave reaction vessel is charged with methyl 4-[2-[4-[[2-[[3-(chloromethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate (1.80 g, 2.89 mmol) and a solution of tert-butyl 3,8-diazabicyclo [3.2.1]octane-8-carboxylate (760 mg, 3.47 mmol) and DIPEA (1.01 mL, 5.78 mmol) in ACN (12 mL). The resulting yellow suspension is heated in a BIOTAGE Initiator microwave synthesizer at 110 C. for 1 hr. The reaction mixture is concentrated in vacuo and the residue is partitioned between 5% aqueous NaHCO.sub.3 (150 mL) and DCM (50 mL). The organic layer is separated, the aqueous layer is extracted with DCM (250 mL). The combined organic layers are washed with saturated aqueous NaCl (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to dryness under reduced pressure. The resulting residue is purified by chromatography over silica, eluting with a gradient of 15-45% of EtOAc in hexanes to afford the title compound as a light yellow solid (2.23 g, 97% yield) after solvent evaporation. .sup.1H NMR (400.1 MHz, DMSO-d.sub.6): 1.38 (s, 9H), 1.95-1.60 (m, 8H), 2.16 (d, J=10.0 Hz, 2H), 2.57 (d, J=10.0 Hz, 1H), 2.70 (br s, 4H), 2.92 (s, 4H), 3.51 (s, 2H), 3.84 (s, 3H), 4.08-3.96 (m, 2H), 7.35-7.26 (m, 2H), 7.45-7.35 (m, 2H), 7.58-7.45 (m, 2H), 7.79-7.72 (m, 1H), 7.92-7.79 (m, 3H), 10.00 (s, 1H), 11.41 (s, 1H). LC-ES/MS (m/z) 799 [M+1].

Preparation 25

methyl 4-[2-[4-[[2-[[3-(3,8-diazabicyclo[3.2.1]octan-3-ylmethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate dihydrochloride

(52) ##STR00042##

(53) Under N.sub.2, to a 250 mL RBF containing a solution tert-butyl 3-[[3-[[3-[[3,5-difluoro-4-[2-(4-methoxycarbonylphenyl)ethyl]phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen-2-yl]carbamoyl]phenyl]methyl]-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (2.23 g, 2.79 mmol) in DCM (45 mL), 4 N hydrochloric acid in dioxane (7.0 mL, 28 mmol) is added dropwise with stirring. The resulting suspension is stirred at rt for 12 hr, concentrated in vacuo and the residue is dried under vacuum to afford the title compound as light yellowish solid which is used in the next step without further purification. LC-ES/MS (m/z) 699 [M+1].

Preparation 26

methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[[8-(methylcarbamoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]benzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate

(54) ##STR00043##

(55) 20 mL scintillation vial is charged with methyl 4-[2-[4-[[2-[[3-(3,8-diazabicyclo[3.2.1]octan-3-ylmethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate dihydrochloride (1.04 g, 1.27 mmol), DCM (8 mL) and TEA (0.89 mL, 6.4 mmol). The resulting suspension is agitated at rt until all solids dissolved. A solution of methylaminoformyl chloride (144 mg, 1.46 mmol) in 2 mL of DCM is added dropwise with stirring and stirring is continued for additional 15 min. The rxn mixture is diluted with 5% aqueous NaHCO.sub.3 (150 mL) and DCM (50 mL), and the layers are separated. The aqueous layer is extracted with DCM (250 mL). The organic extracts are combined, washed with saturated aqueous NaCl (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The resulting residue is purified by chromatography over C-18 silica, eluting with a gradient of 10-20% of a mixture of 5% MeOH in 10 mM ammonium bicarbonate in ACN over 5 min and 20-80% of a mixture of 5% MeOH in 10 mM ammonium bicarbonate in ACN over 15 minutes, to obtain the title compound as an off-white solid (53 mg, 68% yield) after solvent evaporation. The title compound was isolated as a minor component of the mixture as a yellowish green solid (177 mg, 17% yield). The material is used in the next step without further purification. LC-ES/MS (m/z) 752 [M+1].

Preparation 27

methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate

(56) ##STR00044##

(57) A solution of tert-butyl 4-[[3-[[3-[[3,5-difluoro-4-[2-(4-methoxycarbonyl phenyl)ethyl]phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen-2-yl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazine-1-carboxylate (480 mg, 0.599 mmol) in DCM (5 mL) is stirred at RT and 4 N HCl in dioxane (4 mL, 16 mmol) is added via syringe. Upon complete addition, the reaction is stirred at RT for 2 hours, during which time a precipitate formed. The resulting mixture is concentrated in vacuo to a light yellow solid then further dried under high vacuum at ambient temperature for 16 h to yield 464 mg of the intermediate hydrochloride salt as a light yellow solid. This solid is partitioned between saturated sodium bicarbonate and ethyl acetate. The layers are separated and the aqueous portion washed with an additional portion of ethyl acetate. The combined organic extracts are dried over sodium sulfate, decanted, then concentrated in vacuo and dried under vacuum at 55 C. for 16 h to yield the title product as a yellow-brown solid (323 mg, 0.461 mmol, 77% yield). LC-ES/MS (m/z) 701 [M+1].

Preparation 28

methyl 4-[2-[4-[[2-[[3-[(4-acetyl-2,2-dimethyl-piperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate

(58) ##STR00045##

(59) A solution of methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate (226 mg, 0.322 mmol) in dichloromethane (10 mL) is stirred at RT and treated with acetyl chloride (46 L, 0.645 mmol). To the resulting mixture is slowly added saturated aqueous sodium bicarbonate solution (5 mL). The mixture is stirred rapidly at r.t. for 1 h, and then diluted with saturated aqueous sodium bicarbonate solution (10 mL) and dichloromethane (15 mL). The layers are separated and the organic layer dried with magnesium sulfate. The mixture is filtered and the filter cake with dichloromethane. The resulting filtrate is dried in vacuo. The crude product is purified via normal phase flash chromatography, eluting with 3:1 ethyl acetate/hexanes under isocratic conditions, collecting fractions at 240 nM. Product containing fractions are pooled and concentrated in vacuo to afford the title product as light yellow solids (169 mg, 227 mmol, 71% yield). LC-ES/MS (m/z) 741 [M1].

Preparation 29

methyl 4-(2,6-difluoro-4-(2-(3-(((I R,5S)-8-pentanoyl-3,8-diazabicyclo[3.2.1]octan-3-yl)methyl)benzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamido)phenethyl)benzoate

(60) ##STR00046##

(61) To a solution methyl 4-(4-(2-(3-(((1R,5S)-3,8-diazabicyclo[3.2.1]octan-3-yl)methyl)benzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamido)-2,6-difluorophenethyl)benzoate dihydrochloride (200 mg, 0.24 mmol) and TEA (0.145 mL, 4.0 eq., 1.04 mmol) in 2.60 mL of DCM, is added pentanoyl chloride dissolved in 0.5 mL DCM (87 mg, 1.5 eq., 0.39 mmol) via syringe. The resulting mixture is stirred at rt for 6 hr. The reaction mixture is concentrated in vacuo and the residue is partitioned between water (15 mL) and EtOAc (10 mL). The organic layer is separated, the aqueous layer is extracted with EtOAc (210 mL). The combined organic layers are washed with saturated aqueous NaCl (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to dryness under reduced pressure to afford the desired product as a light yellow solid (214 mg, 100% yield). LC-ES/MS (m/z) 783 [M+1].

Preparation 30

methyl 4-(4-(2-(3-(((1R,5S)-8-(dimethylglycyl)-3,8-diazabicyclo[3.2.1]octan-3-yl)methyl)benzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamido)-2,6-difluorophenethyl)benzoate

(62) ##STR00047##

(63) A solution of methyl 4-[2-[4-[[2-[[3-(3,8-diazabicyclo[3.2.1]octan-3-ylmethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate; dihydrochloride (200 mg, 0.2592 mmol), 2-(dimethylamino)acetic acid (40 mg, 0.39 mmol), triethylamine (4 equiv., 1.037 mmol), EDCI (74 mg, 1.5 equiv., 0.39 mmol) and 1-hydroxy-7-azobenzotriazole (53 mg, 1.5 equiv., 0.39 mmol) in DCM (2.6 mL) was stirred at it overnight. The reaction mixture is concentrated in vacuo and the residue is partitioned between water (15 mL) and EtOAc (10 mL). The organic layer is separated, the aqueous layer is extracted with EtOAc (210 mL). The combined organic layers are washed with saturated aqueous NaCl (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to dryness under reduced pressure. The residue was purified by column chromatography (0-5% MeOH/DCM) to afford the desired product as a pale yellow solid (135 mg, 66% yield). LC-ES/MS (m/z) 785 [M+1].

Preparation 31

methyl 4-[[3-[[3-[[3,5-difluoro-4-[2-(4-methoxycarbonylphenyl)ethyl]phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen-2-yl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazine-1-carboxylate

(64) ##STR00048##

(65) To a solution of methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate dihydrochloride (200 mg, 0.26 mmol) and TEA (0.146 mL, 1.03 mmol) in 3 mL of DCM, methyl chloroformate (36 mg, 0.39 mmol) in 1 mL of DCM is added drop wise. The resulting mixture was stirred at room temperature overnight. The reaction mixture is diluted with a diluted with EtOAc and partitioned with water. The product was extracted with EtOAc. All organics were combined, washed with brine, dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The resulting residue is purified by chromatography over silica, eluting with a gradient of 0-30% of a mixture of EtOAc in hexanes, to afford the desired product as a tan foam (157 mg, 80% yield) after solvent evaporation. LC-ES/MS (m/z) 757 [M1].

Preparation 32

methyl 4-[2-[4-[[2-[[3-[[2,2-dimethyl-4-(methyl sulfamoyl)piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate

(66) ##STR00049##

(67) To a solution of methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate dihydrochloride (201 mg, 0.26 mmol) and TEA (0.146 mL, 1.03 mmol) in 3 mL of DCM, N-methylsulfamoyl chloride (50 mg, 0.39 mmol) in 1 mL of DCM is added drop wise. The resulting mixture was stirred at room temperature overnight. The reaction mixture is diluted with a diluted with EtOAc and partitioned with water. The product was extracted with EtOAc. All organics were combined, washed with brine, dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The resulting residue is purified by chromatography over silica, eluting with a gradient of 0-50% of a mixture of EtOAc in hexanes, to afford the desired product as a tan foam (55 mg, 27% yield) after solvent evaporation. LC-ES/MS (m/z) 792 [M1].

Preparation 33

methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[[8-[1-(methoxymethyl)cyclopropanecarbonyl]-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate

(68) ##STR00050##

(69) A solution of methyl 4-[2-[4-[[2-[[3-(3,8-diazabicyclo[3.2.1]octan-3-ylmethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate; dihydrochloride (150 mg, 0.194 mmol), 1-(methoxymethyl)cyclopropanecarboxylic acid (30 mg, 0.233 mmol), triethylamine (4 equiv., 0.972 mmol), EDCI (56 mg, 1.5 equiv., 0.29 mmol) and 1-hydroxy-7-azobenzotriazole (39 mg, 1.5 equiv., 0.29 mmol) in DCM (2.6 mL) was stirred at rt overnight. The reaction mixture is concentrated in vacuo and the residue is partitioned between water and EtOAc. The organic layer is separated, the aqueous layer is extracted with EtOAc. The combined organic layers are washed with saturated aqueous NaCl, dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to dryness under reduced pressure. The residue was purified by column chromatography (0-100% EtOAc/hexanes) to afford the desired product as a white foam (128 mg, 81% yield). LC-ES/MS (m/z) 810 [M1].

Preparation 34

methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[[8-[2-(4-methylpiperazin-1-yl)acetyl]-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate

(70) ##STR00051##

(71) A solution of methyl 4-[2-[4-[[2-[[3-(3,8-diazabicyclo[3.2.1]octan-3-ylmethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate; dihydrochloride (200 mg, 0.26 mmol), 2-(4-methylpiperazin-1-yl)acetic acid (62 mg, 0.39 mmol), Hunig's Base (0.17 g, 1.30 mmol) and HATU (127 mg, 0.32 mmol) in DMF (2.6 mL) was stirred at rt overnight. The reaction mixture is concentrated in vacuo and the residue is partitioned between water and EtOAc. The organic layer is separated, the aqueous layer is extracted with EtOAc. The combined organic layers are washed with saturated aqueous NaCl, dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to dryness under reduced pressure. The residue was purified by column chromatography (0-10% MeOH/DCM) to afford the desired product as a white foam (220 mg, 100% yield). LC-ES/MS (m/z) 840 [M+1].

(72) Preparations 35-41 below are prepared in a manner substantially similar to Preparation 29.

Preparation 35

(73) ##STR00052##

methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[[8-(tetrahydropyran-4-ylcarbamoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate

(74) Prepared using 4-isocyantotetrahydropyran, 52% yield, MS (m/z) 824 [M1]

Preparation 36

(75) ##STR00053##

methyl 4-[2-[4-[[2-[[3-[(8-acetyl-3,8-diazabicyclo[3.2.1]octan-3-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate

(76) Prepared using acetyl chloride, 83% yield, MS (m/z) 741 [M+1]

Preparation 37

(77) ##STR00054##

methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[[8-(2-methoxyethylsulfonyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate

(78) Prepared using 2-methoxyethanesulfonyl chloride, 64% yield, MS (m/z) 820 [M1]

Preparation 38

(79) ##STR00055##

methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[[8-(2-methoxyethylcarbamoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate

(80) Prepared using 1-isocyanato-2-methoxy-ethane, 95% yield, MS (m/z) 800 [M+1]

Preparation 39

(81) ##STR00056##

methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[[8-(4-methoxybutylcarbamoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate

(82) Prepared using 1-isocyanato-4-methoxy-butane, 90% yield, MS (m/z) 827 [M1]

Preparation 40

(83) ##STR00057##

methyl 4-[2-[4-[[2-[[3-[[8-[bis(2-methoxyethyl)carbamoyl]-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate

(84) Prepared using N,N-bis(2-methoxyethyl)carbamoyl chloride, 54% yield, MS (m/z) 857 [M1]

Preparation 41

(85) ##STR00058##

methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[[8-(2-methylpropanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate

(86) Prepared using 2-methylpropanoyl chloride, 100% yield, MS (m/z) 767 [M1]

Preparation 42

tert-butyl 4-[[3-[[3-[[3,5-difluoro-4-[2-(4-methoxycarbonylphenyl)ethyl]phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen-2-yl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazine-1-carboxylate

(87) ##STR00059##

(88) A 20 mL microwave reaction vessel is charged with methyl 4-[2-[4-[[2-[[3-(chloromethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate (1.85 g, 2.97 mmol), tert-butyl 3,3-dimethylpiperazine-1-carboxylate (0.91 g, 4.16 mmol), and DIPEA (2.07 mL, 11.9 mmol) in 15 mL of ACN. The resulting yellow suspension is heated in a BIOTAGE Initiator microwave synthesizer at 110 C. for 4 hr. The reaction mixture is concentrated in vacuo and the residue is partitioned between 5% aqueous NaHCO.sub.3 (150 mL) and DCM (50 mL). The organic layer is separated, the aqueous layer is extracted with DCM (225 mL). The combined organic layers are washed with saturated aqueous NaCl (50 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to dryness under reduced pressure. The resulting residue is purified by chromatography over silica, eluting with a gradient of 0-100% of a mixture of 9:1 DCM/acetone in hexane, to afford the title compound as a yellow solid (1.63 g, 69% yield) after solvent evaporation. .sup.1H NMR (400.1 MHz, DMSO-d.sub.6) 1.04 (s, 6H), 1.38 (s, 9H), 1.81-1.74 (m, 4H), 2.28 (t, J=5.0 Hz, 2H), 2.70

(89) (br s, 4H), 2.91 (s, 4H), 3.13 (s, 2H), 3.26 (s, 2H), 3.52 (s, 2H), 3.84 (s, 3H), 7.31 (d, J=8.3 Hz, 2H), 7.44-7.39 (m, 2H), 7.55-7.46 (m, 2H), 7.75 (d, J=7.6 Hz, 1H), 7.87 (m, 3H), 9.99 (s, 1H), 11.49 (s, 1H). LC-ES/MS (m/z) 801 [M+1].

Preparation 43

methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate dihydrochloride

(90) ##STR00060##

(91) A solution of tert-butyl 4-[[3-[[3-[[3,5-difluoro-4-[2-(4-methoxycarbonylphenyl)ethyl]phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen-2-yl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazine-1-carboxylate (1.13 g, 1.41 mmol) in DCM (14.1 mL) is stirred at RT and 4 N HCl in dioxane (3.5 mL, 14.1 mmol) is added via syringe. Upon complete addition, the reaction is stirred at RT overnight and concentrated to dryness under reduced pressure. The solid is triturated with DCM/Et.sub.2O, the resulting precipitate is collected via vacuum filtration, and the filter cake is dried in a vacuum oven at 50 C. to afford the title compound as a white solid (0.93 g, 1.20 mmol, 85% yield). LC-ES/MS (m/z) 701 [M+1].

Preparation 44

methyl 4-[2-[4-[[2-(tert-butoxycarbonylamino)benzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate

(92) ##STR00061##

(93) A round bottom flask is charged with 2-(tert-butoxycarbonylamino)benzothiophene-3-carboxylic acid (2.60 g, 8.87 mmol), methyl 4-[2-(4-amino-2,6-difluoro-phenyl)ethyl]-benzoate (2.35 g, 8.07 mmol) and 30 mL of CH.sub.2Cl.sub.2. The resulting suspension is cooled in an ice/water bath, and DIPEA (5.63 mL, 32.3 mmol) is added drop wise to afford a yellow-brown turbid solution. Solid bis-(2-oxo-3-oxazolidinyl)phosphinic chloride (2.57 g, 10.1 mmol) is added in small portions over 30 min at 0 C. The reaction mixture is then warmed to RT and stirred for 48 hr. Additional solid 2-(tert-butoxycarbonylamino)benzothiophene-3-carboxylic acid (1.3 g, 4.43 mmol) is added followed by additional DIPEA (2.8 mL) and solid bis-(2-oxo-3-oxazolidinyl)phosphinic chloride (1.3 g, 5.1 mmol) in small portions over 5 min. The resulting turbid brown reaction mixture is stirred at RT for 12 hr. The reaction mixture is diluted with 40 mL of methylene chloride and then washed with 5% aqueous citric acid (150 mL), brine (225 mL). The organic layer is separated and dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The resulting brown foamy solid is purified by flash chromatography to yield the title compound (2.2 g, 48% yield). LC-ES/MS (m/z) 565 [M1].

Preparation 45

methyl 4-[2-[4-[(2-aminobenzothiophene-3-carbonyl)amino]-2,6-difluoro-phenyl]ethyl]benzoate hydrochloride

(94) ##STR00062##

(95) A round bottom flask is charged with methyl 4-[2-[4-[[2-(tert-butoxycarbonylamino)-benzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate (2.2 g, 3.9 mmol) in 30 mL of CH.sub.2Cl.sub.2. 4N HCl in dioxane (9.7 mL, 39 mmol) was added dropwise. The resulting mixture is allowed to stand at r.t. for 12 h. The light yellow suspension was concentrated to dryness under vacuum to yield 2.0 g (100%) of the title compound. LC-ES/MS (m/z) 467 [M+1].

Preparation 46

methyl 4-[2-[4-[[2-[[3-(chloromethyl)benzoyl]amino]benzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate

(96) ##STR00063##

(97) A round bottom flask is charged with methyl 4-[2-[4-[(2-aminobenzothiophene-3-carbonyl)amino]-2,6-difluoro-phenyl]ethyl]benzoate; hydrochloride (2 g, 3.976 mmol) in 24 mL of CH.sub.2Cl.sub.2. The resulting suspension is cooled to 0 C. in an ice bath. Pyridine (0.804 mL, 9.940 mmol) was added dropwise. To the resulting yellow suspension, a solution of 3-(chloromethyl)-benzoyl chloride (0.622 mL, 4.374 mmol) in 6 mL of CH.sub.2Cl.sub.2 was added dropwise over 5 min to yield a dark yellow solution which is allowed to warm up to r.t. for 1 h. The reaction mixture is diluted with 75 mL of 10% aq NaHCO.sub.3. The aqueous layer is washed with CH.sub.2Cl.sub.2 (225 mL). The combined organic layer is washed with brine, dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The crude product is triturated using 20 mL of EtOH. The resulting slurry is stirred at r.t. for 30 min and then filtered to yield 2.2 g (89%) of the title compound as light yellow solid. LC-ES/MS (m/z) 617 [M1].

Preparation 47

methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]benzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate

(98) ##STR00064##

(99) A microwave flask is charged with methyl 4-[2-[4-[[2-[[3-(chloromethyl)benzoyl]-amino]benzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate (0.98 g, 1.6 mmol), N-(4-hydroxybutyl)-3,3-dimethyl-piperazine-1-carboxamide; hydrochloride (0.59 g, 2.2 mmol) and a solution of DIPEA (1.1 mL, 6.3 mmol) in 12 mL of CH.sub.3CN. The reaction mixture is heated to 110 C. in microwave for 4 h. The resulting yellow solution is cooled down to r.t. and concentrated to dryness under a vacuum. The residue is partitioned between 25 mL of CH.sub.2Cl.sub.2 and 75 mL of 5% aqueous NaHCO.sub.3. The organic layer is washed with brine, dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. 1.3 g (100%) of the title compound is yielded as a yellow foamy solid. LC-ES/MS (m/z) 812 [M+1].

Example 1

4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic Acid

(100) ##STR00065##

(101) A 30 mL scintillation vial is charged with methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate (196 mg, 0.24 mmol), lithium hydroxide monohydride (17.2 mg, 0.72 mmol), THF (4 mL), MeOH (2 mL) and H.sub.2O (2 mL). The resulting suspension is stirred at RT for 12 hr. The reaction mixture is diluted with 4 mL of H.sub.2O and concentrated under reduced pressure to approximately/z of the volume. An aqueous solution of 1 N HCl is added drop wise to provide a thick off-white suspension which is evaporated to dryness in vacuo. The resulting residue is purified by reverse phase chromatography over C-18 silica, eluting with a gradient of 0-100% of a mixture of 5% NH.sub.4HCO.sub.3 in H.sub.2O/ACN, to afford the title compound as a pale yellow solid (82.5 mg, 41% yield) after solvent evaporation. .sup.1H NMR (400.1 MHz, DMSO-d.sub.6) 1.05 (s, 6H) 1.37-1.42 (m, 4H), 1.74-1.85 (m, 4H), 2.25-2.30 (m, 2H), 2.64-2.76 (m, 4H), 2.91 (s, 4H), 2.96-3.02 (m, 2H), 3.12-3.15 (m, 2H), 3.17-3.25 (m, 2H), 3.35-3.40 (m, 2H), 3.51 (s, 2H), 4.36 (t, J=5.1 Hz, 1H), 6.36 (t, J=5.4 Hz, 1H), 7.30 (d, J=8.3 Hz, 2H), 7.45-7.55 (m, 4H), 7.74-7.79 (m, 1H), 7.84-7.87 (m, 4H), 10.01 (s, 1H), 11.45 (s, 1H), 12.82 (br s, 1H). LC-ES/MS (m/z) 802 [M+1].

(102) A spray-dried powder solid dispersion of 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid is prepared as an amorphous product containing 30% 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid, disodium/70% PVP-VA (Polyvinylpyrrolidone-vinyl acetate). All materials are tested by ion exchange chromatography and are shown to be consistent with the intended stoichiometry. Cation exchange chromatography with evaporative light scattering detection (ELSD) is used to quantitate the levels of sodium in the active pharmaceutical ingredient (API) 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid, and the solid dispersion formulation of this active pharmaceutical ingredient. Cation exchange chromatography (HPLC) is performed under conditions as follows: ELSD: 60 C., Pump: 2.0 mL/minute, Nitrogen: 1.4 L/min, Column Temp: 30 C., Column: PHENOMENEX LUNA 5 SCX 100A (15 cm4.6 mm, 5 um), Injection Volume: 50 uL, Mobile Phase A: 0.1M Ammonium Formate Buffer, pH 4.5, Mobile Phase B: 100% ACN, Run time: 4 minutes.

(103) Scale-up of 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid, disodium, is performed by placing 126 mg of 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid, free base, in 5 mL of acetone at 60 C. while stirring at 1000 rpm, resulting in a slurry of white solid. 18 L of sodium hydroxide (2.17 equivalents) is added. The sample turns yellow, and polarized light microscopy shows a semi-amorphous solid. The yellow solid is isolated by vacuum filtration, giving a cake of canary yellow material. 102 mg is recovered. X-ray powder diffraction (XRD) shows a poorly crystalline solid.

(104) The solid dispersion of 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid, disodium, is formulated as 30% 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid, disodium/70% PVP-VA. The scale-up of a spray dried solid dispersion containing a 30% drug load of 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid is performed by placing 1040.5 mg of 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic acid, free base, and 2426.7 mg of PVP-VA in 50 mL methanol. The material is stirred resulting in a white slurry. 0.519 mL of 5N sodium hydroxide (2.0 mole equivalents) is added to the slurry and bath sonicated until a clear yellow solution is formed. The solution is slowly pumped into a spray dryer with a stream of hot nitrogen resulting in a solid powder that is collected and further dried in a vacuum oven at 50 C. under vacuum overnight to dry.

(105) Conditions for spray drying are as follows:

(106) TABLE-US-00001 Equipment Water Bath Oil Bath Nitrogen Starting Temp. Final Temp. Setting 60 C. 200 C. 60 psi 45 C. 50 C.
The recovered spray dried material is observed to be microscopically non-birefringent particles of approximately 2.5 m in diameter.
Observed levels of sodium in the active pharmaceutical ingredient, and the solid dispersion formulation of the active pharmaceutical ingredient are shown below:

(107) TABLE-US-00002 Theoretical Observed Material % Sodium % Sodium (n = 3) Example 1, disodium 5.43 5.59 salt Example 1, disodium 1.63 1.73 salt, solid dispersion

Example 2

4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(3-hydroxypropyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic Acid

(108) ##STR00066##

(109) To a solution of methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate dihydrochloride (214.6 mg, 0.28 mmol), DIPEA (0.097 mL, 0.55 mmol) in DCM (4 mL) in DCM (4 mL) are added 3-[(tert-butyldimethylsilyl)oxy]-1-propanal (55 mg, 0.28 mmol) and AcOH (25 L). The resulting solution is allowed to stir at RT for 30 min. Sodium triacetoxyborohydride (0.12 g, 0.55 mmol) is added in one portion. The mixture is stirred at RT for 12 h, diluted with 10% aqueous NaHCO.sub.3 (75 mL) and DCM (25 mL), and the layers are separated. The aqueous layer is extracted with DCM (225 mL). The organic extracts are combined, washed with saturated aqueous NaCl (25 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The resulting residue is dissolved in DCM (3 mL) and 4 M HCl in 1,4-dioxane is added. The resulting solution is stirred at RT for 24 hr. The organic solvent is removed under reduced pressure to afford a yellow solid. The material is dissolved in MeOH (2 mL) and LiOH (0.36 g, 1.4 mmol) is added. The resulting mixture is stirred at RT for 16 h, diluted with H.sub.2O (4 mL), and concentrated under reduced pressure to ca. 50% of the volume. An aqueous solution of 1 N HCl is added (5 mL), and the mixture is concentrated under reduced pressure. The resulting residue is purified by chromatography over C-18 silica, eluting with a gradient of 15-20% of a mixture of 0.1% formic acid/CH.sub.3CN in 0.1% formic acid/H.sub.2O for 5 minutes, then a gradient of 20-50% N of a mixture of 0.1% formic acid/CH.sub.3CN in 0.1% formic acid/H.sub.2O over 20 minutes, to obtain the title compound as mono-formic acid salt (73 mg, 33% yield) as a yellow solid after solvent evaporation. .sup.1H NMR (400.1 MHz, DMSO-d.sub.6): 1.10 (s, 6H), 1.55 (quintet, J=6.7 Hz, 2H), 1.85-1.69 (m, 4H), 2.41-2.09 (m, 8H), 2.81-2.61 (m, 4H), 2.91 (s, 4H), 3.65-3.36 (m, 4H), 7.29 (d, J=8.2 Hz, 2H), 7.57-7.36 (m, 4H), 7.75-7.72 (m, 1H), 7.94-7.80 (m, 3H), 8.15 (s, 1H), 11.1-9.1 (br, 3H). LC-ES/MS (m/z) 745 [M+1].

Example 3

4-[2-[4-[[2-[[3-[[4-[bis(2-methoxyethyl)sulfamoyl]-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic Acid

(110) ##STR00067##

(111) A solution of methyl 4-[2-[4-[[2-[[3-[[4-[bis(2-methoxyethyl)sulfamoyl]-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate (148.4 mg, 0.17 mmol) and LiOH (0.016 g, 0.66 mmol) in a 2:1:1 mixture of THF:MeOH:H.sub.2O (6 mL) is stirred at RT for 12 hr. The reaction mixture is diluted with H.sub.2O (2 mL), concentrated under reduced pressure to volume, and 1 N HCl (4 mL) is added. The mixture is subsequently concentrated to dryness under reduced pressure, and the resulting residue is purified by chromatography over C-18 silica, eluting with a gradient of 15-20% of a mixture of 0.1% formic acid/CH.sub.3CN in 0.1% formic acid/H.sub.2O for 5 minutes, then a gradient of 20-50% of a mixture of 0.1% formic acid/CH.sub.3CN in 0.1% formic acid/H.sub.2O over 20 minutes, to obtain the title compound as a yellow solid (99.0 mg, 67.8% yield) after solvent evaporation. .sup.1H NMR (400.1 MHz, DMSO-d.sub.6) 1.13 (s, 6H), 1.87-1.70 (m, 4H), 2.44-2.36 (m, 2H), 2.76-2.64 (m, 4H), 3.02-2.91 (m, 8H), 3.24 (s, 6H), 3.36-3.31 (m, 4H), 3.44 (t, J=5.8 Hz, 4H), 3.54-3.49 (br s, 2H), 7.30 (d, J=8.3 Hz, 2H), 7.59-7.39 (m, 4H), 7.75 (d, J=7.6 Hz, 1H), 7.90-7.81 (m, 3H), 10.01 (s, 1H), 11.47 (s, 1H), 12.83 (s, 1H). LC-ES/MS (m/z) 883 [M+1].

Example 4

4-[2-[4-[[2-[[3-[[4-(3-carboxypropyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic Acid

(112) ##STR00068##

(113) A mixture of methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-methoxy-4-oxo-butyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzo-thiophene-3-carbonyl]amino]phenyl]ethyl]benzoate (120.8 mg, 0.14 mmol) and LiOH (0.017 g, 0.71 mmol) in 2:1:1 mixture of THF:MeOH:H.sub.2O (4 mL) is stirred at RT for 12 hr. The resulting solution is diluted with H.sub.2O (2 mL) and then concentrated to of the volume under reduced pressure. An aqueous solution of 1 N HCl (4 mL) is added, and the solvent is removed under reduced pressure. The resulting residue is purified by chromatography over C18 silica, eluting with a gradient of 15-20% of a mixture of 0.1% formic acid/CH.sub.3CN in 0.1% formic acid/H.sub.2O for 5 minutes, then a gradient of 20-50% of a mixture of 0.1% formic acid/CH.sub.3CN in 0.1% formic acid/H.sub.2O over 20 minutes, to obtain the title compound as a yellow solid (72.5 mg, 62.3% yield) after solvent evaporation. .sup.1H NMR (400.1 MHz, DMSO-d.sub.6) 1.11 (s, 6H), 1.68-1.55 (m, 2H), 1.88-1.69 (m, 4H), 2.40-2.03 (m, 10H), 2.79-2.61 (m, 4H), 2.91 (s, 4H), 3.70-3.40 (m, 2H), 7.29 (d, J=8.3 Hz, 2H), 7.58-7.36 (m, 4H), 7.79-7.69 (m, 1H), 7.92-7.79 (m, 3H), 10.00 (br s, 1H), 11.45 (br s, 1H), 12.54 (br, 2H). LC-ES/MS (m/z) 773 [M+1].

Example 5

4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(3-methoxypropyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic Acid, Formic Acid Salt

(114) ##STR00069##

(115) A mixture of methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(3-methoxypropyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate (22.7 mg, 0.029 mmol) and LiOH (7 mg, 0.3 mmol) in a mixture of 2:1:1 THF:MeOH:H.sub.2O (4 mL) is stirred at RT for 16 hr. The resulting mixture is diluted with H.sub.2O (3 mL) and concentrated to volume under reduced pressure. An aqueous solution of 1 N HCl (5 mL) is added, and the solvent is removed under reduced pressure. The resulting residue is purified by chromatography over C-18 silica, eluting with a gradient of 15-70% over 10 minutes, to obtain the title compound as a yellow solid (16.2 mg, 68.3% yield) after solvent evaporation. .sup.1H NMR (400.1 MHz, DMSO-d.sub.6) 1.11 (s, 6H), 1.68-1.55 (m, 2H), 1.88-1.69 (m, 4H), 2.43-2.07 (m, 6H), 2.79-2.62 (m, 4H), 2.91 (s, 4H), 3.20 (s, 3H), 3.62-3.23 (m, 6H), 7.29 (d, J=8.3 Hz, 2H), 7.58-7.36 (m, 4H), 7.79-7.68 (m, 1H), 7.85 (d, J=8.2 Hz, 3H), 8.14 (s, 1H), 10.00 (br s, 1H), 11.45 (br s, 1H), 13.14-12.45 (br, 1H). LC-ES/MS (m/z) 759 [M+1].

Example 6

4-[2-[4-[[2-[[3-[[4-(2-aminoacetyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic Acid

(116) ##STR00070##

(117) To a 25 mL microwave reaction vial is charged methyl 4-[2-[4-[[2-[[3-[[4-[2-(tert-butoxycarbonylamino)acetyl]-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate (0.451 g, 0.53 mmol), THF (12 mL), MeOH (8 mL), and 1 N LiOH in THF (2.5 mL, 2.5 mmol). The mixture is heated via microwave at 100 C. for 30 min. To this mixture is added 1 N aqueous HCl (2.5 mL), and the resulting mixture is stirred under a nitrogen stream to evaporate volatiles. The resulting oily residue is dissolved in EtOAc (25 mL), diluted with saturated aqueous NaCl (10 mL), and the layers separated. The organic layer is dried over MgSO.sub.4, filtered, and the filter cake washed with solvent. The filtrate is concentrated under reduced pressure to afford the crude title compound (0.431 g) as a light yellow powder. To a round-bottomed flask is added the crude title compound (0.422 g, 0.56 mmol), DCM (25 mL), and 4 N HCl in dioxane (1.25 mL, 5 mmol). The mixture is diluted with THF (10 mL) and stirred at RT under nitrogen for 24 hr, then for 4 h at 50 C. At this point, an additional portion of 4 N HCl in dioxane (1.25 mL, 5 mmol) is added. Heating is continued for 2 hr, and the mixture is cooled to RT. The resulting slurry is diluted with hexanes (50 mL) and filtered to collect crude product (364 mg). The crude material is dissolved in MeOH/DMSO and purified by reverse phase HPLC on a Waters XBRIDGE 3075 mm 5 m C-18 OBD column, eluting with a gradient of 27-50% of a mixture of 10 mM aqueous ammonium bicarbonate/ACN in MeOH at 85 mL/min over 6 min while monitoring at 205 and 237 nm. Appropriate fractions are concentrated to dryness under reduced pressure and dried for 18 hr at 40 C. to obtain the title compound (0.16 g, 43% yield). .sup.1H NMR (400.13 MHz, DMSO) 7.93 (s, 1H), 7.88-7.79 (m, 3H), 7.46 (t, J=6.3 Hz, 1H), 7.42-7.36 (m, 3H), 7.31-7.28 (m, 2H), 4.00-3.97 (m, 30H, broad, exchangeable protons), 2.89-2.86 (m, 4H), 2.86-2.78 (m, 3H), 2.58-2.54 (m, 2H), 2.51-2.50 (m, 18H, DMSO), 2.44-2.41 (m, 3H), 1.76-1.72 (m, 4H), 1.06 (d, J=12.3 Hz, 6H). LC-ES/MS (m/z) 744 [M+1].

Example 7

4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-methoxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic Acid

(118) ##STR00071##

(119) A solution of methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-methoxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate (93 mg, 0.11 mmol) in a 3:2:1 mixture of THF:MeOH:water (3 mL) is stirred at RT as LiOH (14 mg, 0.56 mmol) is added in one portion. The resulting reaction mixture is stirred at RT for 6 hours and concentrated in vacuo. The resulting residue is purified by reverse phase chromatography on a PHENOMENEX GEMINI-NX C-18 column, eluting with a gradient of 23-57% of a mixture of 5% MeOH in 10 mM aqueous ammonium bicarbonate (pH10) and ACN over 7 min, to afford the title compound as a light yellow foamy solid (83 mg, 91%) after solvent evaporation. .sup.1H NMR (400.1 MHz, DMSO-d.sub.6) 1.03 (s, 6H), 1.38-1.43 (m, 4H), 1.72-1.76 (m, 4H), 2.22-2.28 (m, 2H), 2.60-2.67 (m, 2H), 2.67-2.75 (m, 2H), 2.88 (s, 4H), 2.95-3.02 (m, 2H), 3.095 (s, 2H), 3.18 (s, 3H); 3.15-3.22 (m, 2H), 3.23-3.28 (m, 2H), 3.49 (s, 2H), 6.34 (t, J=4.8 Hz, 1H), 7.27 (d, J=7.9 Hz, 2H), 7.35-7.42 (m, 2H), 7.40-7.48 (m, 1H), 7.47-7.52 (m, 1H), 7.72-7.77 (m, 1H), 7.81-7.88 (m, 3H), 10.01 (br s, 1H), 11.49 (br s, 1H), 12.75 (br s, 1H). LC-ES/MS (m/z) 816 [M+1].

Example 8

4-[4-[[3-[[3-[[4-[2-(4-carbamoylphenyl)ethyl]-3,5-difluoro-phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen-2-yl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazin-1-yl]-4-oxo-butanoic Acid

(120) ##STR00072##

(121) DIPEA (0.1 mL, 0.55 mmol) is added to a solution of N-[4-[2-(4-carbamoylphenyl)ethyl]-3,5-difluoro-phenyl]-2-[[3-[(2,2-dimethylpiperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carboxamide dihydrochloride (92.0 mg, 0.11 mmol) in DCM (4 mL) followed by succinic anhydride (160 mg, 1.65 mmol) all at once. The resulting solution is stirred at RT for 20 min, additional succinic anhydride (53.4 mg, 0.05 mmol) is added, and the resulting mixture is stirred for an additional 20 min. The reaction mixture is quenched by the addition of MeOH (0.5 mL), volatiles are removed in vacuo, and the resulting yellow oily residue is purified by reverse phase chromatography on a PHENOMENEX GEMINI-NX C-18 column, eluting with a gradient of 20-70% of a mixture of 5% MeOH in 10 mM aqueous ammonium bicarbonate (pH10) and ACN over 6 min, to afford the title compound as a light yellow solid (50.9 mg, 61% yield) after solvent evaporation. .sup.1H NMR (400.1 MHz, DMSO-d.sub.6) 1.03 (s, 3H), 1.09 (s, 3H), 1.62-1.89 (m, 4H), 2.28 (m, 1H), 2.30 (m, 1H), 2.40-2.47 (m, 2H), 2.58-2.81 (br m, 4H), 2.88 (s, 4H), 3.27 (s, 2H), 3.30 (m, 2H, partial overlap with residual water peak), 3.38 (br s, 2H), 3.54 (s, 2H), 7.19-7.31 (m, 3H), 7.31-7.59 (m, 3H), 7.78 (d, J=8.1 Hz, 3H), 7.92-7.85 (m, 2H), 8.36 (br s, 1H), 8.56 (br s, 1H), 9.99 (br s, 1H), 11.51 (br s, 1H), 11.93 (br s, 1H). LC-ES/MS (m/z) 786 [M+1].

Example 9

4-[2-[2,6-difluoro-4-[[2-[[3-[(2,2,4-trimethylpiperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic Acid Formic Acid Salt

(122) ##STR00073##

(123) A solution of methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[(2,2,4-trimethylpiperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate (167 mg, 0.233 mmol) and lithium hydroxide (22 mg, 0.93 mmol) in a 2:1:1 mixture of THF:MeOH:H.sub.2O (6 mL) is stirred at RT for 12 hr. The reaction mixture is diluted with water (2 mL), concentrated under reduced pressure to volume, and 1 N HCl (3 mL) is added. The mixture is subsequently concentrated to dryness under reduced pressure, and the resulting residue is purified by chromatography over C-18 silica, eluting with a gradient of 15-20% of a mixture of 0.1% formic acid/ACN in 0.1% formic acid/H.sub.2O for 5 minutes, then a gradient of 20-50%0/of a mixture of 0.1% formic acid/ACN in 0.1% formic acid/H.sub.2O over 20 minutes, to afford the title compound as a yellow solid (122 mg, 68% yield) after solvent evaporation. .sup.1H NMR (400.1 MHz, DMSO-d.sub.6): 1.11 (s, 6H), 1.89-1.67 (m, 4H), 2.15 (s, 3H), 2.41-2.30 (m, 4H), 2.70 (m, 4H), 2.91 (s, 4H), 4.10-3.10 (br, 4H), 7.29 (m, 2H), 7.57-7.36 (m, 4H), 7.75 (m, 1H), 7.90-7.81 (m, 3H), 8.15 (s, 1H), 10.8-9.7 (br, 1H), 12.9-11.1 (br, 1H). LC-ES/MS (m/z) 701 [M+1].

Example 10

4-[2-[4-[[2-[[3-[[2,2-dimethyl-4-(methylcarbamoyl)piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic Acid

(124) ##STR00074##

(125) 20 mL scintillation vial is charged methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate dihydrochloride (145 mg, 0.187 mmol) and a solution of TEA (0.13 mL, 0.94 mmol) in DCM (2 mL). The resulting suspension is stirred until it is clear. A solution of N-methylcarbamoyl chloride (21 mg, 0.22 mmol) in 1 mL of DCM is added dropwise. The resulting solution is allowed to stir at rt for 15 min. The rxn mixture is diluted with 5% aqueous NaHCO.sub.3 (75 mL) and DCM (25 mL), and the layers are separated. The aqueous layer is extracted with DCM (225 mL). The organic extracts are combined, washed with saturated aqueous NaCl (25 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The resulting crude material and lithium hydroxide (23 mg, 0.94 mmol) are suspended in a 2:1:1 mixture of THF:MeOH:H.sub.2O (4 mL) and stirred at rt for 12 hr. The reaction mixture is diluted with water (3 mL), concentrated under reduced pressure to volume, and 1 N HCl (4 mL) is added. The mixture is subsequently concentrated to dryness under reduced pressure, and the resulting residue is purified by chromatography over C-18 silica, eluting with a gradient of 10-15% of a mixture of 0.1% formic acid/ACN in 0.1% formic acid/H.sub.2O for 5 minutes, then a gradient of 15-50% of a mixture of 0.1% formic acid/ACN in 0.1% formic acid/H.sub.2O over 20 minutes, to afford the title compound as a pale yellow solid (108 mg, 77% yield) after solvent evaporation. .sup.1H NMR (400.1 MHz, DMSO-d.sub.6): 1.06 (s, 6H), 1.88-1.65 (m, 4H), 2.27 (s, 2H), 2.55 (d, J=4.3 Hz, 3H), 2.70 (m, 4H), 2.91 (s, 4H), 3.10 (s, 2H), 3.25-3.15 (m, 2H), 3.51 (s, 2H), 6.33 (m, 1H), 7.34-7.24 (m, 2H), 7.59-7.37 (m, 4H), 7.83-7.79 (m, 1H), 7.93-7.80 (m, 3H), 10.02 (br s, 1H), 11.46 (br s, 1H), 12.79 (br s, 1H). LC-ES/MS (m/z) 744 [M+1].

Example 11

4-[2-[4-[[2-[[3-[[2,2-dimethyl-4-(methylcarbamothioyl)piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic Acid

(126) ##STR00075##

(127) 20 mL scintillation vial is charged with methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate dihydrochloride (165 mg, 0.212 mmol) and a solution of TEA (0.15 mL, 1.1 mmol) in DCM (3 mL). The resulting suspension is stirred until it is clear. A solution of methyl isotiocyanate (18 mg, 0.24 mmol) in 1 mL of DCM is added dropwise. The resulting solution is allowed to stir at rt for 15 min. The rxn mixture is diluted with 5% aqueous NaHCO.sub.3 (75 mL) and DCM (25 mL), and the layers are separated. The aqueous layer is extracted with DCM (225 mL). The organic extracts are combined, washed with saturated aqueous NaCl (25 mL), dried over anhydrous Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure. The resulting crude material and lithium hydroxide (26 mg, 1.1 mmol) are suspended in a 2:1:1 mixture of THF:MeOH:H.sub.2O (4 mL) and stirred at rt for 12 hr. The reaction mixture is diluted with water (3 mL), concentrated under reduced pressure to volume, and 1 N HCl (4 mL) is added. The mixture is subsequently concentrated to dryness under reduced pressure, and the resulting residue is purified by chromatography over C-18 silica, eluting with a gradient of 15-20% of a mixture of 0.1% formic acid/ACN in 0.1% formic acid/H.sub.2O for 5 minutes, then a gradient of 20-60% of a mixture of 0.1% formic acid/ACN in 0.1% formic acid/H.sub.2O over 20 minutes, to afford the title compound as an off-white solid (73 mg, 45% yield) after solvent evaporation. .sup.1H NMR (400.1 MHz, DMSO-d.sub.6): 1.06 (s, 6H), 1.89-1.68 (m, 4H), 2.34 (m, 2H), 2.77-2.61 (m, 4H), 2.99-2.82 (m, 7H), 3.52 (s, 2H), 3.60 (s, 2H), 3.71 (br s, 2H), 7.35-7.22 (m, 2H), 7.53-7.35 (m, 3H), 7.68-7.53 (m, 2H), 7.80-7.68 (m, 1H), 7.94-7.80 (m, 3H), 10.02 (br s, 1H), 11.47 (br s, 1H), 12.79 (br s, 1H). LC-ES/MS (m/z) 760 [M+1].

Example 12

4-[2-[4-[[2-[[3-[[4-(2-carboxyethylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic Acid

(128) ##STR00076##

(129) A solution of methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-[(3-methoxy-3-oxo-propyl)carbamoyl]-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate (80 mg, 0.097 mmol) and lithium hydroxide (12 mg, 0.49 mmol) in a 2:1:1 mixture of THF:MeOH:H.sub.2O (6 mL) is stirred at rt for 12 hr. The reaction mixture is diluted with H.sub.2O (3 mL), concentrated under reduced pressure to volume, and 1 N HCl (2 mL) is added. The mixture is subsequently concentrated to dryness under reduced pressure, and the resulting residue is purified by chromatography over C-18 silica, eluting with a gradient of 10-15% of a mixture of 0.1% formic acid/ACN in 0.1% formic acid/H.sub.2O for 1 minutes, then a gradient of 15-50% of a mixture of 0.1% formic acid/ACN in 0.1% formic acid/H.sub.2O over 20 minutes, to obtain the title compound as an off-white solid (53 mg, 68% yield) after solvent evaporation. .sup.1H NMR (400.1 MHz, DMSO-d.sub.6): 1.05 (s, 6H), 1.88-1.66 (m, 4H), 2.31-2.20 (m, 2H), 2.42-2.31 (m, 2H), 2.79-2.60 (m, 4H), 2.91 (s, 4H), 3.11 (m, 2H), 3.27-3.15 (m, 4H), 3.51 (s, 2H), 6.48 (m, 1H), 7.35-7.23 (m, 2H), 7.60-7.36 (m, 4H), 7.80-7.68 (m, 1H), 7.94-7.80 (m, 3H), 10.02 (br s, 1H), 11.46 (br s, 1H), 12.78-12.15 (br, 1H). LC-ES/MS (m/z) 802 [M+1].

Example 13

4-[2-[2,6-difluoro-4-[[2-[[3-[[8-(methyl carbamoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]benzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic Acid, Formic Acid Salt

(130) ##STR00077##

(131) A solution of methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[[8-(methylcarbamoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]benzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate (100 mg, 0.13 mmol) and lithium hydroxide (15 mg, 0.63 mmol) in a 2:1:1 mixture of THF:MeOH:H.sub.2O (4 mL) is stirred at rt for 12 hr. The reaction mixture is diluted with water (3 mL), concentrated under reduced pressure to volume, and 1 N HCl (3 mL) is added. The mixture is subsequently concentrated to dryness under reduced pressure, and the resulting residue is purified by chromatography over C-18 silica, eluting with 10% of a mixture of 0.1% formic acid/ACN in 0.1% formic acid/H.sub.2O for 5 minutes, then a gradient of 10-65% of a mixture of 0.1% formic acid/ACN in 0.1% formic acid/H.sub.2O over 15 minutes, to afford the title compound as a yellowish tan solid (67 mg, 67% yield) after solvent evaporation. .sup.1H NMR (400.1 MHz, DMSO-d.sub.6): 1.77-1.57 (m, 2H), 1.97-1.79 (m, 2H), 2.19 (d, J=10.2 Hz, 2H), 2.52 (overlaps with residual dmso resonance, 2H), 2.56 (m, 3H), 2.94 (s, 4H), 3.53 (s, 2H), 4.13 (br s, 2H), 6.37 (m, 1H), 7.65-7.25 (m, 8H), 8.08-7.78 (m, 6H), 8.14 (s, 1H), 10.63 (s, 1H), 11.93 (s, 1H), 12.79 (br s, 1H). LC-ES/MS (m/z) 738 [M+1].

Example 14

4-[2-[2,6-difluoro-4-[[2-[[3-[(4-methoxycarbonyl-2,2-dimethyl-piperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic Acid

(132) ##STR00078##

(133) A 25 mL scintillation vial is charged with methyl 4-[[3-[[3-[[3,5-difluoro-4-[2-(4-methoxycarbonylphenyl)ethyl]phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen-2-yl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazine-1-carboxylate (152 mg, 0.20 mmol), lithium hydroxide (25 mg, 5 mmol), THF (1.5 mL), MeOH (1.0 mL) and H.sub.2O (0.5 mL) and the resulting suspension is stirred at RT for 8 hr. The reaction mixture is evaporated, reconstituted in DMSO (2 mL) and purified on Phenomenex Kinetex EVO C18 column utilizing a gradient elution. (32-67% Acetonitrile/Aqueous 10 mM Ammonium bicarbonate pH10/5% MeOH). Eluent was concentrated under reduced pressure to give the title compound (124 mg, 83% yield) as an off-white solid. LC-ES/MS (m/z) 746 [M+1].

Example 15

4-[2-[4-[[2-[[3-[[2,2-dimethyl-4-(methylsulfamoyl)piperazin-1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic Acid

(134) ##STR00079##

(135) A 25 mL scintillation vial is charged with methyl 4-[[3-[[3-[[3,5-difluoro-4-[2-(4-methoxycarbonylphenyl)ethyl]phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen-2-yl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazine-1-carboxylate (152 mg, 0.20 mmol), lithium hydroxide (25 mg, 5 mmol), THF (1.5 mL), MeOH (1.0 mL) and H.sub.2O (0.5 mL) and the resulting suspension is stirred at RT for 8 hr. The reaction mixture is evaporated, reconstituted in DMSO (2 mL) and purified on Phenomenex Kinetex C18 column utilizing a gradient elution. (32-67% Acetonitrile/Aqueous 10 mM Ammonium bicarbonate pH10/5% MeOH). Eluent was concentrated under reduced pressure to give the title compound (124 mg, 83% yield) as an off-white solid. LC-ES/MS (m/z) 746 [M+1].

Example 16

4-(2,6-difluoro-4-(2-(3-(((R,5S)-8-pentanoyl-3,8-diazabicyclo[3.2.1]octan-3-yl)methyl)benzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamido)phenethyl)benzoic Acid

(136) ##STR00080##

(137) A 60 mL scintillation vial is charged with methyl 4-(2,6-difluoro-4-(2-(3-(((1R,5S)-8-pentanoyl-3,8-diazabicyclo[3.2.1]octan-3-yl)methyl)benzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamido)phenethyl)benzoate (214 mg, 0.27 mmol), lithium hydroxide (33 mg, 6.0 eq., 1.37 mmol), THF (1.5 mL), MeOH (1.0 mL) and H.sub.2O (0.5 mL) and the resulting suspension is stirred at RT for 12 hr. The reaction mixture is diluted with H.sub.2O (5 mL) and concentrated in vacuo to volume. The pH of the resulting mixture is adjusted to 5-6 with 10% aqueous citric acid and the resulting colorless suspension is partitioned between 15 mL of water and 5 mL of 4:1 chloroform/isopropanol. The organic layer is separated, the pH of the aqueous layer is adjusted again to pH5 with 10% aqueous citric acid, and the mixture is extracted twice with additional 4:1 chloroformisopropanol (215 mL). The organic layers are combined, washed with saturated aqueous NaCl, dried over anhydrous Na.sub.2SO.sub.4, filtered, and the filtrate is concentrated under reduced pressure to give the title compound (114 mg, 54% yield) as an off-white solid. LC-ES/MS (m/z) 769 [M+1]. Examples 17-27 below are prepared in a manner substantially similar to Example 16.

Example 17

(138) ##STR00081##

4-[2-[2,6-difluoro-4-[[2-[[3-[[8-(tetrahydropyran-4-ylcarbamoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic Acid

(139) 17% Yield, MS m/z 812 [M+1]

Example 18

(140) ##STR00082##

4-[2-[4-[[2-[[3-[(8-acetyl-3,8-diazabicyclo[3.2.1]octan-3-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate

(141) 85% yield, MS ml/z 727 [M+1]

Example 19

(142) ##STR00083##

4-[2-[2,6-difluoro-4-[[2-[[3-[[8-(2-methoxyethylsulfonyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate

(143) 70% yield, MS m/z 807 [M+1]

Example 20

(144) ##STR00084##

4-[2-[2,6-difluoro-4-[[2-[[3-[[8-(2-methoxyethylcarbamoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate

(145) 28% yield, MS m/z 786 [M+1]

Example 21

(146) ##STR00085##

4-[2-[2,6-difluoro-4-[[2-[[3-[[8-(4-methoxybutylcarbamoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate

(147) 84% yield, MS m/z 815 [M+1]

Example 22

(148) ##STR00086##

4-[2-[4-[[2-[[3-[[8-[bis(2-methoxyethyl)carbamoyl]-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate

(149) 23% yield, MS m/z 844 [M+1]

Example 23

(150) ##STR00087##

4-[2-[2,6-difluoro-4-[[2-[[3-[[8-(2-methylpropanoyl)-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoate

(151) 62% yield, MS m/z 755 [M+1]

Example 24

(152) ##STR00088##

4-[2-[4-[[2-[[3-[[8-[2-(dimethylamino)acetyl]-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid

(153) 73% yield, MS m/z 770 [M+1]

Example 25

(154) ##STR00089##

4-[2-[2,6-difluoro-4-[[2-[[3-[[8-[1-(methoxymethyl)cyclopropanecarbonyl]-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic Acid

(155) 98% yield MS m/z 797 [M+1]

Example 26

(156) ##STR00090##

4-[2-[2,6-difluoro-4-[[2-[[3-[[8-[2-(4-methylpiperazin-1-yl)acetyl]-3,8-diazabicyclo[3.2.1]octan-3-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic Acid

(157) 59% yield, MS m/z 825 [M+1]

Example 27

4-[2-[4-[[2-[[3-[(4-acetyl-2,2-di methyl-piperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid

(158) ##STR00091##

(159) To a 25 mL microwave reaction vial is charged methyl methyl 4-[2-[4-[[2-[[3-[(4-acetyl-2,2-dimethyl-piperazin-1-yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene-3-carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate (169 mg, 0.227 mmol), tetrahydrofuran (12 mL, 148 mmol), methanol (8 mL, 197 mmol), and 1M aqueous lithium hydroxide solution (1.14 mL, 1.14 mmol). The vial is capped and the resulting mixture is stirred and heated via microwave irradiation at 100 C. for 30 minutes. The reaction mixture is then carefully treated with 1M aqueous hydrochloric acid solution (1.14 mL, 1.14 mmol), resulting in a reaction mixture pH of 4. The resulting mixture is concentrated to dryness in vacuo and the product is purified via low pH, reversed phase chromatography. 1H NMR (400.1 MHz, DMSO-d.sub.6) 1.04 (s, 3H), 1.09 (s, 3H), 1.84-1.72 (m, 4H), 1.97 (bs, 3H), 2.29-2.27 (m, 1H), 2.37-2.33 (m, 1H), 2.75-2.65 (m, 4H), 2.91 (s, 4H), 3.25-3.3.24 (m, 2H), 3.38-3.34 (m, 2H), 3.54 (s, 2H), 7.29 (d. J=7.6 Hz, 2H), 7.45-7.37 (m, 2H), 7.51-7.46 (m, 1H), 7.55 (d, J=7.5 Hz, 1H), 7.76 (d, J=7.5 Hz, 1H), 7.85 (d, J=8.0 Hz, 2H), 7.91 (s, 1H), 9.99 (s, 1H), 11.49 (s, 1H), 12.84 (s, 1H). LC-ES/MS (m/z) 729 [M+1].

Example 28

4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]benzothiophene-3-carbonyl]amino]phenyl]ethyl]benzoic Acid

(160) ##STR00092##

(161) To a solution of methyl 4-[2-[2,6-difluoro-4-[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin-1-yl]methyl]benzoyl]amino]benzothiophene-3-carbonyl]amino]phenyl]-ethyl]benzoate (0.20 g, 0.25 mmol) in 8 mL of a mixed solvent (THF/CH.sub.3OH/H.sub.2O in 2:1:1 ratio), LiOH (18 mg, 0.75 mmol) is added. The resulting mixture is allowed to stand at r.t. for 12 h. Additional LiOH (18 mg, 0.75 mmol) is added. And the mixture is allowed to stand at r.t. for 48 h and diluted with 1 mL of 4N HCl in dioxane. The solvent is removed under a vacuum. The crude product is purified by flash chromatography to yield 120 mg (57%) of the title compound as a white solid. .sup.1H NMR (399.80 MHz, DMSO-d.sub.6): 1.06 (s, 6H), 1.31-1.48 (m, 4H), 2.34 (s, 2H), 2.87-2.97 (m, 4H), 3.07-3.09 (m, 2H), 3.09-3.18 (s, 2H), 3.18-3.28 (m, 2H), 3.38 (partially overlaps with res water peak, 1H), 3.54 (s, 2H), 4.38 (m, 2H), 6.33 (m, 2H), 7.25-7.71 (m, 8H), 7.73-8.08 (m, 6H), 10.64 (s, 1H), 11.99 (s, 1H), 12.8 (br s, 1H). LC-ES/MS (m/z) 798 [M+1].

Inhibition of NaPi-IIb In Vitro

(162) Inhibition of .sup.33P uptake is measured in human and mouse NaPi-IIb T-REX-CHO stable cell lines. cDNA for NaPi-IIb is subcloned in plasmid SLC34A2 pcDNA5/TO (human) and SLC34A2 pcDNA5/TO (mouse) and stable cell lines are generated from clonal isolation for both human and mouse respectively. Mouse and human stable lines are maintained in continuous culture in growth media (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (3:1), 10% Heat Inactivated FBS, 1% penicillin/streptomycin/FUNGIEZONE (HYCLONE), 20 mM HEPES, 250 g/mL hygromycin, 5 g/mL blasticidin). Cells are harvested from T225 cell culture flasks (CORNING) using 0.25% Trypsin, and plated in 96 well CYTOSTAR-T scintillating microplates (Amersham Systems) at 40,000 cells/well in 100 L of growth media plus 100 ng/mL of tetracycline. Cell plates are incubated overnight at 37 C. and 5% C02. The next day, compounds are serially diluted using one to three dilutions in 100% DMSO. Cell plates may remain in the incubator until ready to be assayed. A cell plate is removed from the incubator and media removed. Cells are washed 3 times with 200 L assay buffer (137 mM NaCl, 5.4 mM KCl, 2.8 mM CaCl.sub.2), 1.2 mM MgSO.sub.4, and 14 mM Tris-HCl buffer, pH7.5), removing buffer in between washes. Serially diluted compounds in DMSO are further diluted 50 fold in assay buffer and 50 L added to the CYTOSTAR-T assay plate, immediately followed by the addition of 50 L of .sup.33P solution (PERKIN-ELMER, Walton, Mass.; 0.05 Ci/50 L). CYTOSTAR-T assay plates are covered with foil to protect from light and incubated for 60 min at RT. After the 60 min incubation, 100 L of a stop solution (assay buffer+400 M Phloretin) is added to the assay plate to stop .sup.33P uptake. The plate is immediately read on a Wallac MICROBETA Trilux liquid scintillation counter and luminometer after stop solution is added with 1 minute count per well. Each plate may be processed separately and staggered in time so there may be no delay in counting after stop solution is added. Percent inhibition at all concentrations tested (final assay concentrations 100-0.005 M) are calculated relative to 1% DMSO (minimum effect), and the effect of 100 M of a fully efficacious NaPi-IIb inhibitor (maximum effect). IC.sub.50 values were calculated using a 4 parameter logistic curve fitting equation. The numbers presented are the geometric means with standard deviation (SD) calculated where n is the number of runs. Thus. Table 1 describes the relative IC.sub.50 values for Examples 1-28 against human NaPi-IIb and murine NaPi-IIb, respectively.

(163) TABLE-US-00003 TABLE 1 Relative IC.sub.50 (rel IC.sub.50) values for Examples 1-28 against human and murine NaPi-IIb in vitro data in T-REX Chinese Hamster Ovarian-stable cell lines. h NaPi-IIb m NaPi-IIb rel IC50 rel IC50 Example (nM), (SD) n (nM), (SD) n 1 32.4, (23.0) 3 43.9, (24.2) 3 2 13.6, (18.2) 4 17.5, (9.05) 3 3 51.1, (32.3) 2 40.9 1 4 18.6, (9.8) 3 26.0, (37.3) 3 5 76.3, (4.2) 2 23.6, (8.2) 2 6 8.7, (6.7) 6 9.9, (4.2) 3 7 16.2, (2.6) 2 8.56 1 8 32.6, (19.5) 3 35, (7.8) 2 9 197, (162) 5 314, (526) 5 10 3.4, (4.1) 5 5.9, (5.0) 5 11 10.8, (5.6) 4 12.6, (2.2) 4 12 156, (73.6) 3 217, (122) 3 13 4.3, (9.4) 2 9.0 1 14 5.3, (2.5) 3 11.3, (0.8) 4 15 6.5, (4.4) 4 16.8, (8.5) 4 16 60.0, (50.1) 3 31.2, (6.7) 3 17 3.5, (4.6) 3 8.8, (6.8) 3 18 5.1, (9.2) 3 20.3, (37.4) 3 19 60.3, (36.3) 2 20.0, (31.6) 3 20 9.5, (23.1) 3 9.1, (15.5) 3 21 19.6, (8.7) 2 5.3, (11.7) 2 22 9.4, (14.4) 3 27.3, (16.6) 2 23 46.5, (47.8) 3 47.1, (6.5) 3 24 16.6, (12.7) 3 30.2, (9.6) 3 25 22.5, (3.2) 2 8.3, (11.0) 3 26 2.4 1 15.3 1 27 25.9, (1.9) 3 22.6, (6.4) 3 28 6.2, (1.2) 3 7.6, (1.6) 3

Inhibition of NaPi-IIb In Vivo

(164) For test article and vehicle control preparation, add vehicle, 20% hydroxypropyl-beta-cyclodextrin (HPBCD) in water, to the test article. Sonicate to reduce particle size in an ultrasonic water bath as needed. If necessary, use a polytron to break down any visible particles in test article solution. Add 1 N NaOH as indicated in Table 2 below. The pH of the vehicle control is adjusted to 8.0 to 8.5 with 1N NaOH.

(165) TABLE-US-00004 TABLE 2 Amount of 1N NaOH to add to indicated testing compound. 1N NaOH per Example mg of compound No. (L) 1 2.5 2 3.8 3 2.3 4 3.9 5 3.7 6 2.7 7 2.5 8 2.5

(166) Radioactive phosphate preparation: Prepare a 16.25 mM Na.sub.2HPO.sub.4 solution using 0.9% saline as the vehicle. Mix until a clear solution is formed. Adjust pH to approximately 7.4. Filter using a sterile 0.2 m polyethersulfone membrane. For the final preparation of the radioactive phosphate dosing solution, add 0.5 l of stock H.sub.3.sup.3PO.sub.4 per 1 mL of Na.sub.2HPO.sub.4. Mix thoroughly then sterile filter the H.sub.3.sup.33PO4+Na.sub.2HPO.sub.4 solution using a 0.2 m polyethersulfone membrane prior to dosing. Measure the radioactivity of each sample with the scintillation counter. If the DPMs are between 100,000 and 150,000 proceed with dosing.

(167) In-vivo protocol: Male C57B16 male mice at the age of about 8-9 wks old are fasted for 16 hrs the day before the study. They are assigned to treatment groups based on body weight on the day of study. Mice are dosed orally with either the test article, prepared as described above, or vehicle control at a 10 mis/kg dose volume. Fifteen minutes later, the radioactive phosphate dosing solution is given by oral gavage. Fifteen minutes later, blood is collected by orbital bleed. Plasma is prepared and 50 l of EDTA plasma from each mouse is mixed with 10 ml of scintillation fluid and the counts determined by scintillation counting. The effect of the test article is determined by comparing the counts in the plasma from the test article treated animals to counts in the plasma of the vehicle control treated animals [Percent Inhibition=(counts in the plasma of test article treated animals/counts in the plasma of vehicle treated animals)100%]. Percent inhibition is measured at 30 minutes post administration of the compound or vehicle, and at 15 minutes post administration of the labelled phosphate. Percent Inhibition for indicated Examples are illustrated in Table 3 below.

(168) TABLE-US-00005 TABLE 3 In vivo data for Example compounds 4, 6, and 8 Percent Example Treatment n Inhibition SEM 4 5 mg/kg 6 52 5.4 6 5 mg/kg 6 62 2.5 8 5 mg/kg 6 60 5.2

(169) Alternative vehicles, for example poly-1-vinylpyrrolidone-co-vinyl acetate (PVP-VA), at varying concentrations, may be used. Example 1 can be assayed in PVP-VA, and for this study the compound is formulated as described in the solid dispersion preparation of Example 1, followed by dissolving in water then dosing by oral gavage. The vehicle control is water with varying concentrations of PVP-VA, matching the concentration found in the highest dose of Example 1, the lowest dose of Example 1, and an intermediate dose of Example 1. No effect of varying PVP-VA on the uptake of radiolabeled phosphate is observed over several studies, and thus all the vehicles are averaged to calculate percent inhibition.

(170) TABLE-US-00006 TABLE 4 In vivo data for Example 1 in PVP-VA formulation Percent Group Treatment n Inhibition SEM 1 Vehicle 23 0 5.12 2 30 mg/kg Example 1 7 75.83 3.54 3 10 mg/kg Example 1 7 75.64 4.02 4 3 mg/kg Example 1 7 68.71 4.30 5 1 mg/kg Example 1 7 66.90 3.10 6 0.3 mg/kg Example 1 7 51.21 7.71 7 0.1 mg/kg Example 1 7 30.61 7.57 8 0.03 mg/kg Example 1 7 0.75 14.45

(171) The effect of a test compound on gastric emptying can be assessed by dosing the compound and radiolabeled phosphate in a manner similar to that used to assess a compound's ability to inhibit NaPi-IIb in vivo. After bleeding the mice, they were sacrificed and their stomachs harvested. The harvested stomachs are digested in 10 ml of 1N NaOH overnight, and the recovered radiolabeled phosphate DPMs determined by scintillation counting. The compound mediated effect on the rate of gastric emptying is determined by comparing the dpms in the stomach of animals treated with compound, to that of animals treated with vehicle.

(172) Compounds of the invention, for instance Example 1, show advantageous pharmacological properties, such as potency, in vivo distribution, in vivo efficacy, and favorable lack of toxicity in preclinical testing. For instance Example 1 shows surprisingly advantageous margin of NaPi-IIb inhibition with respect to inhibition of in vivo gastric emptying. In addition, Example 1 is generally well tolerated when administered in vivo to normal rats for a period of four days, and shows an advantageous lack of toxicity in this in vivo experiment.