MODULATORS OF TMEM16A FOR TREATING RESPIRATORY DISEASE

Abstract

Compounds of general formula (I):

##STR00001##

wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, A, Z.sup.1, Z.sup.2 and Y are as defined herein are useful for treating respiratory disease and other diseases and conditions modulated by TMEM16A.

Claims

1.-26. (canceled)

27. A compound of formula (I): ##STR00094## or a tautomer, a stereoisomer, isotopic variants, a salt, or solvate thereof, wherein A is ##STR00095## where one of X.sup.1, X.sup.2, X.sup.3 is S and the other two of X.sup.1, X.sup.2 and X.sup.3 are CH; represents a single or a double bond such that the ring A is aromatic; each of X.sup.4 and X.sup.5 is independently CH or N; *.sup.1 indicates the point of attachment to Z.sup.1 and *.sup.2 indicates the point of attachment to Z.sup.2; each of Z.sup.1 and Z.sup.2 is independently selected from *—C(O)NH— and *—NHC(O)—, where * indicates the point of attachment to the ring A; Y is —CH.sub.2— or —CH(CH.sub.3)—; R.sup.4 is a 6- to 14-membered aryl, 5- to 14-membered heteroaryl or a 5- to 10-membered carbocyclic ring system, any of which is optionally substituted with one or more substituents selected from: halo, CN, nitro, R.sup.19, OR.sup.19, OR.sup.6, SR.sup.6, NR.sup.6R.sup.7, C(O)R.sup.6, C(O)R.sup.19, C(O)OR.sup.6, C(O)N(R.sup.6)(R.sup.7), N(R.sup.7)C(O)R.sup.6; C.sub.1-6 alkyl or O(C.sub.1-6 alkyl) either of which is optionally substituted with one or more substituents selected from halo, CN, nitro, R.sup.19, OR.sup.6, SR.sup.6, NR.sup.6R.sup.7, C(O)R.sup.6 C(O)OR.sup.6, C(O)N(R.sup.6)(R.sup.7) and N(R.sup.7)C(O)R.sup.6; and when R.sup.4 is not fully aromatic in character, oxo; wherein R.sup.19 is a 5- or 6-membered aryl or heteroaryl ring system or a 3- to 7-membered carbocyclic or heterocyclic ring system, any of which aryl, heteroaryl, carbocyclic or heterocyclic ring systems are optionally substituted with one or more substituents selected from halo, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl, OH, O(C.sub.1-4 alkyl), O(C.sub.1-4 haloalkyl); R.sup.6 is H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, benzyl, 3- to 7-membered carbocyclyl or 3- to 7-membered heterocyclyl; R.sup.7 is H, C.sub.1-6 alkyl or C.sub.1-6 haloalkyl; or R.sup.6 and R.sup.7 together with the nitrogen atom to which they are attached form a 4 to 7-membered heterocyclic ring optionally containing one or more further heteroatoms and optionally substituted with one or more substituents selected from C.sub.1-4 alkyl, oxo and halo;

28. The compound of claim 27 having formula (Ia), (Ib) or (Ic): ##STR00096## wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, Z.sup.1, Z.sup.2 and Y are as defined in claim 27.

29. The compound of claim 27 having formula (Id), (Ie), (If) or (Ig): ##STR00097## wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, Z.sup.1, Z.sup.2 and Y are as defined in claim 27.

30. The compound according to claim 27, wherein Y is —CH.sub.2—.

31. The compound according to claim 27, wherein: R.sup.4 is a 6- to 11-membered aryl group selected from phenyl, naphthyl, indanyl, 1,2,3,4-tetrahydronaphthyl and benzocycloheptanyl, any of which is unsubstituted or substituted as defined in claim 27; or R.sup.4 is a 5- to 10-membered heteroaryl group selected from pyridyl, quinolinyl, quinoxalinyl, indazolyl, indolyl, benzoxazolyl, dihydrobenzofuranyl, furyl and thienyl, any of which is unsubstituted or substituted as defined in claim 27; or R.sup.4 is a carbocyclyl group selected from cyclohexyl and adamantyl, any of which is unsubstituted or substituted as defined in claim 27.

32. The compound of claim 27 having formula (Ii): ##STR00098## wherein R.sup.1, R.sup.2, R.sup.3, A, Z.sup.1 and Z.sup.2 are as defined in claim 27; R.sup.10 is H, OH, halo, C.sub.1-6 alkyl, —O(C.sub.1-6 alkyl); each R.sup.11 is independently H, halo, OH, CN, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, —O(C.sub.1-6 alkyl) or C(O)O—(C.sub.1-6 alkyl); and n is 1 or 2; or a compound of formula (Iii) or (liii): ##STR00099## wherein R.sup.1, R.sup.2, R.sup.3, A, Z.sup.1 and Z.sup.2 are as defined in claim 27; R.sup.11a is H, halo, C.sub.1-4 alkyl, C.sub.1-4 haloalkyl or C(O)O(C.sub.1-4 alkyl); R.sup.11b is H, halo, C.sub.1-4 alkyl or C.sub.1-4 haloalkyl; and R.sup.11c is H, halo, CN, C.sub.1-4 alkyl or C.sub.1-4 haloalkyl; or

33. The compound of claim 32 having formula (Iai), (Ibi) or (Ici): ##STR00100## a compound of formula (Idi), (Iei), (Ifi) or (Igi): ##STR00101## or a compound of formula (Iaii), (Ibii) or (Icii): ##STR00102## or a compound of formula (Idii), (Ieii), (Ifii) or (Igii): ##STR00103## wherein R.sup.11a, R.sup.11b and R.sup.11c are as defined in claim 32.

34. The compound according to claim 32, wherein: the compound is a compound of formula (Iai), (Ibi), (Ici), (Idi), (Iei), (Ifi) or (Igi) and either R.sup.11a is H, halo, C.sub.1-4 alkyl or C(O)O(C.sub.1-4 alkyl) and R.sup.11b is H; or both R.sup.11a and R.sup.11b are halo; or the compound is a compound of formula (Iaii), (Ibii), (Icii), (Idii), (Ieii), (Ifii) or (Igii) and R.sup.11a is H; and R.sup.11b is C.sub.1-4 alkyl, C.sub.1-4 alkyl substituted with OH or C.sub.1-4 haloalkyl; and R.sup.11c is H, halo, methyl or ethyl.

35. A compound selected from: N-tert-Butyl-5-[[2-(5-chloro-2-hydroxy-phenyl)acetyl] amino] thiophene-2-carboxamide (Compound 1); N-tert-Butyl-5-[[2-(5-chloro-2-hydroxy-phenyl)acetyl]amino]thiophene-3-carboxamide (Compound 1.2); N-tert-Butyl-4-[[2-(5-chloro-2-hydroxy-phenyl)acetyl] amino] thiophene-2-carboxamide (Compound 1.3); N-[(5-Chloro-2-hydroxy-phenyl)methyl]-3-(2,2-dimethylpropanoylamino)benzamide (Compound 2); N-[(5-Chloro-2-hydroxy-phenyl)methyl]-4-(2,2-dimethylpropanoylamino)pyridine-2-carboxamide (Compound 3); N2-tert-Butyl-N4-[(2-hydroxyphenyl)methyl]pyridine-2,4-dicarboxamide (Compound 4); N1-tert-Butyl-N3-[(2-hydroxyphenyl)methyl]benzene-1,3-dicarboxamide (Compound 5); N-[3-[[2-(5-Chloro-2-hydroxy-phenyl)acetyl]amino]phenyl]-2,2-dimethyl-propanamide (Compound 6); N-[4-[[2-(5-Chloro-2-hydroxy-phenyl)acetyl]amino]-2-pyridyl]-2,2-dimethyl-propanamide (Compound 7); N-[4-[[2-(5-Chloro-2-hydroxy-phenyl)acetyl]amino]-2-pyridyl]cyclohexanecarboxamide (Compound 7.1); 5-[[2-(4-tert-Butyl-2-fluoro-5-hydroxy-phenyl)acetyl]amino]-N-(1-cyano-1-methyl-ethyl)thiophene-2-carboxamide (Compound 8); 5-[[2-[2-Fluoro-5-hydroxy-4-(2-hydroxy-1,1-dimethyl-ethyl)phenyl]acetyl]amino]-N-[1-(trifluoromethyl)cyclopropyl]thiophene-2-carboxamide (Compound 9); and salts and solvates thereof.

36. A pharmaceutical composition comprising a compound according to claim 27 and a pharmaceutically acceptable excipient.

37. A method for the treatment or prophylaxis of diseases and conditions affected by modulation of TMEM16A, the method comprising administering to a patient in need of such treatment an effective amount of a compound according to claim 27.

38. The method of claim 37, wherein the diseases and conditions affected by modulation of TMEM16A are selected from respiratory diseases and conditions, dry mouth (xerostomia), intestinal hypermobility, cholestasis and ocular conditions.

39. A process for the preparation of a compound according to claim 27 comprising: A. for compound of formula (I) in which Z.sup.1 is *—C(O)NH—, Z.sup.2 is *—NHC(O)— and A is: ##STR00104## reacting a compound of formula (II): ##STR00105## wherein X.sup.1, X.sup.2, X.sup.3, Y and R.sup.4 are as defined in claim 27; with a compound of formula (III): ##STR00106## wherein R.sup.1, R.sup.2 and R.sup.3 are as defined in formula (I); in the presence of a coupling reagent and under basic conditions; or B. for a compound of formula (I) in which Z.sup.1 is *—NHC(O)— and Z.sup.2 is *—C(O)NH—: reacting a compound of formula (X): ##STR00107## wherein R.sup.1, R.sup.2, R.sup.3 and A are as defined in claim 27; with a compound of formula (XI):
H.sub.2N—Y—R.sup.4  (XI) wherein Y and R.sup.4 are as defined in claim 27; using a coupling agent under basic conditions; or C. for a compound of general formula (I) in which Z.sup.1 is *—NHC(O)— and Z.sup.2 is *—C(O)NH—: reacting a compound of formula (XV) as defined above with a compound of formula (XVI): ##STR00108## wherein A, Y and R.sup.4 are as defined in claim 27; under basic conditions; or D. for a compound of formula (I) in which Z.sup.1 is *—C(O)NH— and Z.sup.2 is *—C(O)NH—: reacting a compound of formula (XX): ##STR00109## wherein A, Y and R.sup.4 are as defined in claim 27; with a compound of formula (III) as defined above; in the presence of a coupling agent under basic conditions; or E. for a compound of formula (I) in which Z.sup.1 is *—C(O)NH— and Z.sup.2 is *—C(O)NH—: reacting a compound of formula (XXV): ##STR00110## wherein R.sup.1, R.sup.2, R.sup.3 and A are as defined in claim 27; with a compound of formula (XI) as defined above; in the presence of a coupling agent under basic conditions; or F. for a compound of formula (I) in which Z.sup.1 is *—NHC(O)— and Z.sup.2 is *—NHC(O)—: reacting a compound of formula (XXX): ##STR00111## wherein A, Y and R.sup.4 are as defined in claim 27 and R.sup.32 is halo; with a compound of formula (XXXI) ##STR00112## wherein R.sup.1, R.sup.2 and R.sup.3 are as defined in claim 27; in the presence of a phosphorus ligand, a palladium catalyst and a base; or G. for a compound of formula (I) in which Z.sup.1 is *—NHC(O)—, Z.sup.2 is *—NHC(O)—, Y is —CH.sub.2— and R.sup.4 is 2-hydroxy-5-chlorophenyl: reacting a compound of formula (XXXV): ##STR00113## wherein R.sup.1, R.sup.2, R.sup.3 and A are as defined in claim 27; with 5-chloro-3H-benzofuran-2-one, which has the structure: ##STR00114## or H. converting a compound of formula (I) as defined in claim 27 in which either R.sup.3; R.sup.1, R.sup.2 and R.sup.3 together; or R.sup.4 includes a phenyl group substituted with alkoxy to a compound of general formula (I) as defined in claim 27 in which either R.sup.3; R.sup.1, R.sup.2 and R.sup.3 together; or R.sup.4 includes a phenyl group substituted with OH by treatment with boron tribromide.

40. A compound of formula (II), (X), (XVI), (XX), (XXV), (XXX) or (XXXV) as defined in claim 39.

Description

[0262] The invention is illustrated by the following non-limiting Examples and the drawing in which:

[0263] FIG. 1 is an example trace from a whole-cell patch clamp (Qpatch) TMEM16A potentiator assay as used in Biological Example 95 and illustrates the methodology used in the assay.

EXAMPLES

[0264] General Conditions:

[0265] Mass spectra were run on LC-MS systems using electrospray ionization. These were run using either a Waters Acquity uPLC system with Waters PDA and ELS detectors or Shimadzu LCMS-2010EV systems. [M+H]+ refers to mono-isotopic molecular weights.

[0266] NMR spectra were recorded on a Bruker Avance III HD 500 MHz with a 5 mm Broad Band Inverse probe, a Bruker Avance III HD 250 MHz or a 400 MHz Avance III HD Nanobay fitted with a 5 mm Broad Band Observed SmartProbe using the solvent as internal deuterium lock. Spectra were recorded at room temperature unless otherwise stated and were referenced using the solvent peak.

[0267] Referring to the examples that follow, compounds of the preferred embodiments were synthesized using the methods described herein, or other methods, which are known in the art.

[0268] The various starting materials, intermediates, and compounds of the preferred embodiments may be isolated and purified, where appropriate, using conventional techniques such as precipitation, filtration, crystallization, evaporation, distillation, and chromatography. Unless otherwise stated, all starting materials are obtained from commercial suppliers and used without further purification. Salts may be prepared from compounds by known salt-forming procedures.

[0269] Compounds were purified by flash column chromatography on normal phase silica on Biotage® Isolera systems using the appropriate SNAP cartridge and gradient. Alternatively, compounds were purified on reverse phase silica using Biotage® Isolera systems with the appropriate SNAP C18 cartridges and reverse phase eluent or by preparative HPLC (if stated otherwise).

[0270] Preparative HPLC Using Acidic pH, Early Elution Method

[0271] Purifications by were performed on a Gilson LC system using Waters Sunfire C18 columns (30 mm×100 mm, 10 μM; temperature: RT) and a gradient of 10-95% B (A=0.1% formic acid in water; B=0.1% formic acid in acetonitrile) over 14.44 min then 95% B for 2.11 min, with an injection volume of 1500 μL and a flow rate of 40 mL/min. UV spectra were recorded at 215 nm using a Gilson detector.

[0272] Preparative HPLC Using Acidic pH, Standard Elution Method

[0273] Purifications by preparative HPLC (acidic pH, standard elution method) were performed on a Gilson LC system using Waters Sunfire C18 columns (30 mm×100 mm, 10 μM; temperature: RT) and a gradient of 30-95% B (A=0.1% formic acid in water; B=0.1% formic acid in acetonitrile) over 11 min then 95% B for 2.11 min, with an injection volume of 1500 μL and a flow rate of 40 mL/min. UV spectra were recorded at 215 nm using a Gilson detector.

[0274] Preparative HPLC Using Basic pH, Early Elution Method

[0275] Purifications by preparative HPLC (basic pH, early elution method) were performed on a Gilson LC system using Waters Xbridge C18 columns (30 mm×100 mm, 10 μM; temperature: RT) and a gradient of 10-95% (A=0.2% ammonium hydroxide in water; B=0.2% ammonium hydroxide in acetonitrile) over 14.44 min then 95% B for 2.11 min, with an injection volume of 1500 μL and a flow rate of 40 mL/min. UV spectra were recorded at 215 nm using a Gilson detector.

[0276] Preparative HPLC Using Basic pH, Standard Elution Method

[0277] Purifications by preparative HPLC (basic pH, standard elution method) were performed on a Gilson LC system using Waters Xbridge C18 columns (30 mm×100 mm, 10 μM; temperature: RT) and a gradient of 30-95% (A=0.2% ammonium hydroxide in water; B=0.2% ammonium hydroxide in acetonitrile) over 11 min then 95% B for 2.11 min, with an injection volume of 1500 μL and a flow rate of 40 mL/min. UV spectra were recorded at 215 nm using a Gilson detector.

[0278] If not indicated otherwise, the analytical HPLC conditions are as follows:

[0279] Method A [0280] Column: Phenomenex Kinetix-XB 018 2.1×100 mm, 1.7 μm [0281] Column Temp: 40° C. [0282] Eluents: A: H.sub.2O+0.1% formic acid, B: acetonitrile+0.1% formic acid [0283] Flow Rate: 0.6 mL/min [0284] Gradient: 0-5.3 min 5-100% B, 5.3-5.8 min 100% B, 5.8-5.82 min 100-5% B, 5.82-7.00 min 5% B

[0285] Method B [0286] Column: Waters UPLC® BEH™ C18 2.1×100 mm, 1.7 μm [0287] Column Temp: 40° C. [0288] Eluents: A: 2 mM ammonium bicarbonate, buffered to pH10, B: acetonitrile [0289] Flow Rate: 0.6 mL/min [0290] Gradient: 0-5.3 min 5-100% B, 5.3-5.8 min 100% B, 5.8-5.82 min 100-5% B, 5.82-7.00 min 5% B

[0291] Method C [0292] Column: Kinetex Core-Shell C18 2.1×50 mm, 5 μm [0293] Column Temp: 40° C. [0294] Eluents: A: H.sub.2O+0.1% formic acid, B: acetonitrile+0.1% formic acid [0295] Flow Rate: 1.2 mL/min [0296] Gradient: 0-1.20 min 5-100% B, 1.20-1.30 min 100% B, 1.30-1.31 min 100-5% B, 1.31-1.7 min 5% B

[0297] Method G [0298] Column: Waters UPLC® BEH™ C18, 2.1 mm×50 mm, 1.7 μm, [0299] Column Temp: 40° C. [0300] Eluents: A: H.sub.2O+0.1% formic acid, B: acetonitrile+0.1% formic acid [0301] Flow Rate: 0.9 mL/min [0302] Gradient: 0-1.10 mins 5-100% B, 1.10-1.35 mins 100% B, 1.35-1.40 mins 100-1% B

[0303] Method H [0304] Column: Kinetex Core-Shell C18 2.1×50 mm, 5 μm [0305] Column Temp: 40° C. [0306] Eluents: A: H.sub.2O+0.1% formic acid, B: acetonitrile+0.1% formic acid [0307] Flow Rate: 1.2 mL/min [0308] Gradient: 0-1.83 min 5-100% B, 1.83-2.25 min 100% B, 2.25-2.26 min

[0309] The following example are intended to illustrate the invention and are not to be construed as being limitations thereon. Temperatures are given in degrees centigrade. If not mentioned otherwise, all evaporations are performed in vacuo, preferably between about 15 mm Hg and 100 mm Hg (=20-133 mbar). The structure of final products, intermediates and starting materials is confirmed by standard analytical methods, e.g., microanalysis and spectroscopic characteristics, e.g., MS, IR, and NMR. Abbreviations used are those conventional in the art. If not defined, the terms have their generally accepted meanings.

ABBREVIATION

[0310] aq. aqueous
br broad
d doublet
dd doublet of doublets
DCM dichloromethane
DIPEA diisopropylethylamine

DMF N,N-dimethylformamide

[0311] EtOAc ethyl acetate
HATU 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
HPLC high pressure liquid chromatography
MeCN acetonitrile
MS mass spectrometry
m multiplet
min minute(s)
mL milliliter(s)
m/z mass to charge ratio
NMR nuclear magnetic resonance
Rt retention time
s singlet
t triplet
TBTU N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate
TEA triethylamine
TFA trifluoroacetic acid
THF tetrahydrofuran

PREPARATION EXAMPLES

Example 1

N-tert-Butyl-5-[[2-(5-chloro-2-hydroxy-phenyl)acetyl]amino]thiophene-2-carboxamide

[0312] ##STR00054##

Step 1: Methyl 5-[[2-(5-chloro-2-methoxy-phenyl)acetyl]amino]thiophene-2-carboxylate

[0313] ##STR00055##

[0314] To a solution of 2-(5-chloro-2-methoxy-phenyl)acetic acid (351 mg, 1.75 mmol) and DIPEA (306 μL, 1.75 mmol) in DMF (8 mL) was added HATU (665 mg, 1.75 mmol) followed by methyl 5-aminothiophene-2-carboxylate (250 mg, 1.59 mmol) and the mixture stirred at room temperature overnight. The resulting mixture was diluted with EtOAc (30 mL) and washed with 1M HCl (30 mL) solution and brine (30 mL). The organic portion was separated, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was absorbed onto silica and purification by chromatography on silica eluting with 0-50% EtOAc in heptanes afforded the title compound as a colourless waxy solid.

[0315] LC-MS (Method C): Rt 1.15 min; MS m/z 340.0/341.9=[M+H]+ (81%@215 nm)

[0316] 1H NMR (500 MHz, DMSO-d6) δ 11.79 (s, 1H), 7.60 (d, J=4.2 Hz, 1H), 7.33-7.26 (m, 2H), 7.02-6.98 (m, 1H), 6.72 (d, J=4.2 Hz, 1H), 3.76 (s, 3H), 3.75 (s, 3H), 3.72 (s, 2H).

Step 2: 5-[[2-(5-Chloro-2-methoxy-phenyl)acetyl]amino]thiophene-2-carboxylic acid

[0317] ##STR00056##

[0318] To a solution of methyl 5-[[2-(5-chloro-2-methoxy-phenyl)acetyl]amino]thiophene-2-carboxylate (step 1) (95%, 268 mg, 0.75 mmol) in EtOAc (1.33 mL), THF (1.33 mL) and water (1.33 mL) was added LiOH.H.sub.2O (63 mg, 1.5 mmol) and the mixture was stirred at room temperature for 4 h. Further LiOH.H.sub.2O (95 mg, 2.25 mmol) was added and the mixture stirred at room temperature for 2 days followed by heating to 50° C. for 2 h. After cooling to room temperature, the mixture was acidified to pH 2 using 1M HCl (4 mL). The resulting precipitate was filtered, washed with water and dried in a vacuum oven to afford the title compound.

[0319] LC-MS (Method C): Rt 1.06 min; MS m/z 323.9/325.8=[M−H] (96%@215 nm)

[0320] 1H NMR (500 MHz, DMSO-d6) δ 11.70 (s, 1H), 7.51 (d, J=4.1 Hz, 1H), 7.33-7.28 (m, 2H), 7.01 (d, J=9.4 Hz, 1H), 6.69 (d, J=4.1 Hz, 1H), 3.75 (s, 3H), 3.71 (s, 2H).

Step 3: N-tert-Butyl-5-[[2-(5-chloro-2-methoxy-phenyl)acetyl]amino]thiophene-2-carboxamide

[0321] ##STR00057##

[0322] To a solution of 5-[[2-(5-chloro-2-methoxy-phenyl)acetyl]amino]thiophene-2-carboxylic acid (step 2) (130 mg, 0.40 mmol) and DIPEA (84 μL, 0.48 mmol) in DMF (2 mL) was added HATU (182 mg, 0.48 mmol) followed by 2-methylpropan-2-amine (50 μL, 0.48 mmol) and the mixture was stirred at room temperature overnight. The resulting mixture was diluted with EtOAc (10 mL) and washed with 1M HCl (10 mL) and brine (10 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was absorbed onto silica and purified by chromatography on silica eluting with 0-50% EtOAc in heptanes to afford the title compound as a colourless powder.

[0323] LC-MS (Method C): Rt 1.17 min; MS m/z 381.0/383.0=[M+H]+ (100%@215 nm) 1H NMR (500 MHz, DMSO-d6) δ 11.44 (s, 1H), 7.54 (d, J=4.1 Hz, 1H), 7.49 (s, 1H), 7.32-7.29 (m, 2H), 7.01 (d, J=8.4 Hz, 1H), 6.61 (d, J=4.1 Hz, 1H), 3.75 (s, 3H), 3.68 (s, 2H), 1.34 (s, 9H).

Step 4: N-tert-Butyl-5-[[2-(5-chloro-2-hydroxy-phenyl)acetyl]amino]thiophene-2-carboxamide

[0324] To a solution of N-tert-butyl-5-[[2-(5-chloro-2-methoxy-phenyl)acetyl]amino]thio phene-2-carboxamide (step 3) (100%, 130 mg, 0.34 mmol) in DCM (1.5 mL) at 0° C. was added 1M BBr.sub.3 in DCM (0.51 mL, 0.51 mmol) and the mixture was stirred at room temperature for 1 h. The reaction was quenched by addition of sat. NaHCO.sub.3 solution (10 mL) and the resulting mixture extracted with CHCl.sub.3/IPA (3:1, 2×10 mL). The combined organic extracts were washed with brine (10 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was absorbed onto silica and purification by chromatography on silica eluting with 0-50% EtOAc in heptanes afforded the title compound as a colourless powder.

[0325] LC-MS (Method A): Rt 2.97 min; MS m/z 367.1/369.1=[M+H]+ (98%@215 nm)

[0326] 1H NMR (500 MHz, DMSO-d6) δ 11.41 (s (br), J=14.4 Hz, 1H), 9.83 (s (br), 1H), 7.55 (d, J=4.1 Hz, 1H), 7.50 (s, 1H), 7.21 (d, J=2.7 Hz, 1H), 7.13 (dd, J=8.6, 2.7 Hz, 1H), 6.81 (d, J=8.6 Hz, 1H), 6.61 (d, J=4.1 Hz, 1H), 3.64 (s, 2H), 1.35 (s, 9H).

Example 1.1

N-tert-Butyl-5-[[2-(5-chloro-2-hydroxy-phenyl)acetyl]amino]thiophene-3-carboxamide

[0327] ##STR00058##

[0328] The title compound was prepared analogously to Example 1 by replacing methyl 5-aminothiophene-2-carboxylate (step 1) with methyl 5-aminothiophene-3-carboxylate.

[0329] LC-MS (Method A): Rt 2.97 min; MS m/z 367.2/369.1=[M+H]+ (97%@215 nm)

[0330] 1H NMR (500 MHz, DMSO-d6) δ 11.29 (s (br), 1H), 9.84 (s (br), 1H), 7.53 (d, J=1.7 Hz, 1H), 7.46 (s, 1H), 7.20 (d, J=2.7 Hz, 1H), 7.12 (dd, J=8.6, 2.7 Hz, 1H), 6.95 (d, J=1.7 Hz, 1H), 6.80 (d, J=8.6 Hz, 1H), 3.62 (s, 2H), 1.34 (s, 9H).

Example 1.2

N-tert-Butyl-4-[[2-(5-chloro-2-hydroxy-phenyl)acetyl]amino]thiophene-2-carboxamide

[0331] ##STR00059##

Step 1: Methyl 4-[[2-(5-chloro-2-methoxy-phenyl)acetyl]amino]thiophene-2-carboxylate

[0332] ##STR00060##

[0333] 2-(5-Chloro-2-methoxy-phenyl)acetic acid (0.16 g, 0.82 mmol) was dissolved in thionyl chloride (0.73 mL, 8.24 mmol) and the mixture heated to 70° C. for 1 h. The resulting mixture was concentrated in vacuo and the residue azeotroped with toluene (2×2 ml). The crude acid chloride was dissolved in DCM (1 mL) and added to a cooled (0° C.) solution of methyl 4-aminothiophene-2-carboxylate (125 mg, 0.80 mmol) and DIPEA (0.28 mL, 1.59 mmol) in DCM (2 mL). The resulting mixture was stirred and allowed to warm to room temperature. After 1 hour the mixture was diluted with DCM (4 ml) and water (5 mL), the phases separated and the aqueous portion extracted with DCM (5 mL). The combined organic extracts were concentrated in vacuo to give a pale brown solid. The product was by purified by chromatography on silica eluting with 0-80% EtOAc in heptanes to afford the title compound as a white waxy solid.

[0334] LC-MS (Method C): Rt 1.16 min; MS m/z 339.9/341.9=[M+H]+ (100%@215 nm)

[0335] 1H NMR (500 MHz, DMSO-d6) δ 10.57 (s, 1H), 7.79 (d, J=1.6 Hz, 1H), 7.78 (d, J=1.6 Hz, 1H), 7.31-7.27 (m, 2H), 7.02-6.98 (m, 1H), 3.81 (s, 3H), 3.76 (s, 3H), 3.62 (s, 2H).

Step 2-4: N-tert-Butyl-4-[[2-(5-chloro-2-hydroxy-phenyl)acetyl]amino]thiophene-2-carboxamide

[0336] The title compound was prepared from methyl 4-[[2-(5-chloro-2-methoxy-phenyl)acetyl]amino]thiophene-2-carboxylate (step 1) analogously to Example 1 steps 2-4.

[0337] LC-MS (Method A): Rt 3.09 min; MS m/z 367.2/369.1=[M+H]+ (97% @215 nm)

[0338] 1H NMR (500 MHz, DMSO-d6) δ 10.44 (s, 1H), 9.78 (s, 1H), 7.85 (s, 1H), 7.80 (d, J=1.4 Hz, 1H), 7.49 (d, J=1.4 Hz, 1H), 7.19 (d, J=2.6 Hz, 1H), 7.10 (dd, J=8.6, 2.7 Hz, 1H), 6.79 (d, J=8.6 Hz, 1H), 3.56 (s, 2H), 1.35 (s, 9H).

Example 2

N-[(5-Chloro-2-hydroxy-phenyl)methyl]-3-(2,2-dimethylpropanoylamino)benzamide

[0339] ##STR00061##

Step 1: Methyl 3-(2,2-dimethylpropanoylamino)benzoate

[0340] ##STR00062##

[0341] To a cooled (0° C.) mixture of methyl 3-aminobenzoate (500 mg, 3.31 mmol) and TEA (1.73 mL, 9.92 mmol) in DCM (5.5 mL) was added 2,2-dimethylpropanoyl chloride (0.61 mL, 4.96 mmol) and the resulting mixture stirred for 2 h. The reaction mixture was diluted with DCM (5 mL) and the organic portion was washed with sat. aqueous sodium hydrogen carbonate solution (5 mL), brine (5 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford the title compound as a light brown solid.

[0342] LC-MS (Method C): Rt 1.08 min; MS m/z 236.1=[M+H]+ (99%@215 nm)

[0343] 1H NMR (250 MHz, DMSO-d6) δ 9.43 (s, 1H), 8.30 (t, J=1.9 Hz, 1H), 7.95 (ddd, J=8.1, 2.2, 1.1 Hz, 1H), 7.63 (dt, J=7.7, 1.2 Hz, 1H), 7.43 (t, J=7.9 Hz, 1H), 3.85 (s, 3H), 1.23 (s, 9H).

Step 2: 3-(2,2-Dimethylpropanoylamino)benzoic acid

[0344] ##STR00063##

[0345] To a solution of methyl 3-(2,2-dimethylpropanoylamino)benzoate (step 1) (99%, 706 mg, 2.97 mmol) in 2:1 THF/water (11 mL) was added 1M LiOH (14.85 mL, 14.85 mmol) and the mixture was stirred for 1 h. The resulting mixture was diluted with water (20 mL) and acidified to pH 1 by the dropwise addition of 6M HCl. The mixture was extracted with EtOAc (3×15 mL) and the combined organic extracts were washed with brine (30 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford the title compound as a light brown solid.

[0346] LC-MS (Method C): Rt 0.97 min; MS m/z 222.0=[M+H]+ (100%@215 nm)

[0347] 1H NMR (500 MHz, DMSO-d6) δ 9.38 (s, 1H), 8.25 (t, J=1.8 Hz, 1H), 7.91 (ddd, J=8.1, 2.2, 1.0 Hz, 1H), 7.61 (dt, J=7.7, 1.3 Hz, 1H), 7.40 (t, J=7.9 Hz, 1H), 1.23 (s, 9H).

Step 3: (5-Chloro-2-methoxy-phenyl)methanamine

[0348] ##STR00064##

[0349] A cooled (0° C.) solution of 5-chloro-2-methoxy-benzonitrile (500 mg, 2.98 mmol) in THF (7.5 mL) was added dropwise to a cooled solution of 2.4M LiAlH.sub.4 solution in THF (1.86 mL, 4.48 mmol) in THF (5.5 mL). The mixture was allowed to warm to room temperature and stirred for 3 h. The reaction was quenched by slow addition of water (0.25 mL) followed by 4M NaOH (0.25 mL). The mixture was filtered through Celite® (filter material) rinsing through with with EtOAc (2×5 mL). The filtrate was concentrated in vacuo to afford the title compound as a yellow oil.

[0350] LC-MS (Method B): Rt 2.43 min; MS m/z 171.7/173.7=[M+H]+ (89%@215 nm)

[0351] 1H NMR (500 MHz, DMSO-d6) δ 7.38 (d, J=2.7 Hz, 1H), 7.21 (dd, J=8.7, 2.7 Hz, 1H), 6.94 (d, J=8.7 Hz, 1H), 3.77 (s, 3H), 3.64 (s, 2H), 1.89 (s (br), 2H).

Step 4: N-[(5-Chloro-2-methoxy-phenyl)methyl]-3-(2,2-dimethylpropanoylamino) benzamide

[0352] ##STR00065##

[0353] To a solution of 3-(2,2-dimethylpropanoylamino)benzoic acid (step 2) (100 mg, 0.45 mmol) and (5-chloro-2-methoxy-phenyl)methanamine (step 3)(89%, 95.9 mg, 0.50 mmol) in 1,4-dioxane (3 mL) was added TEA (0.20 mL, 1.13 mmol) and 50% T3P® solution in EtOAc (0.27 mL, 0.45 mmol) and the mixture stirred at room temperature for 2 h. The reaction mixture was diluted with water (5 mL) and extracted into EtOAc (3×5 mL). The combined organic extracts were washed with sat. aqueous sodium hydrogen carbonate (10 mL), brine (10 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford the title compound as a yellow oil.

[0354] LC-MS (Method C): Rt 1.18 min; MS m/z 375.1/377.0=[M+H]+ (95%@215 nm) 1H NMR (500 MHz, DMSO-d6) δ 9.37 (s, 1H), 8.86 (t, J=5.9 Hz, 1H), 8.11 (t, J=1.8 Hz, 1H), 7.88-7.85 (m, 1H), 7.60-7.56 (m, 1H), 7.40 (t, J=7.9 Hz, 1H), 7.28 (dd, J=8.7, 2.7 Hz, 1H), 7.13 (d, J=2.7 Hz, 1H), 7.03 (d, J=8.8 Hz, 1H), 4.41 (d, J=5.9 Hz, 2H), 3.83 (s, 3H), 1.23 (s, 9H).

Step 5: N-[(5-Chloro-2-hydroxy-phenyl)methyl]-3-(2,2-dimethylpropanoylamino) benzamide

[0355] To a cooled (0° C.) solution N-[(5-chloro-2-methoxy-phenyl)methyl]-3-(2,2-dimethyl propanoylamino)benzamide (step 4) (95%, 150 mg, 0.38 mmol) in DCM (3.23 mL) was added 1M BBr.sub.3 in DCM (1.14 mL, 1.14 mmol) dropwise and allowed to warm to room temperature, stirring for 100 min. The reaction was quenched with sat. aqueous sodium hydrogen carbonate (5 mL) and the mixture extracted into EtOAc (3×5 mL). The combined organic extracts were dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Purification of the crude residue by preparative HPLC (acidic pH, early elution method) afforded the title compound as a white solid.

[0356] LC-MS (Method A): Rt 3.20 min; MS m/z 361.1/363.2=[M+H]+ (98%@215 nm)

[0357] 1H NMR (500 MHz, DMSO-d6) δ 9.91 (s, 1H), 9.37 (s, 1H), 8.90 (t, J=5.9 Hz, 1H), 8.11 (t, J=1.8 Hz, 1H), 7.88-7.84 (m, 1H), 7.57 (d, J=7.9 Hz, 1H), 7.39 (t, J=7.9 Hz, 1H), 7.11 (dd, J=8.5, 2.7 Hz, 1H), 7.08 (d, J=2.6 Hz, 1H), 6.83 (d, J=8.5 Hz, 1H), 4.38 (d, J=5.9 Hz, 2H), 1.23 (s, 9H).

Example 3

N-[(5-Chloro-2-hydroxy-phenyl)methyl]-4-(2,2-dimethylpropanoylamino)pyridine-2-carboxamide

[0358] ##STR00066##

Step 1: 4-Amino-N-[(5-chloro-2-methoxy-phenyl)methyl]pyridine-2-carboxamide

[0359] ##STR00067##

[0360] A mixture of (5-chloro-2-methoxy-phenyl)methanamine (0.34 mL, 2.72 mmol) and 4-aminopyridine-2-carboxylic acid (250 mg, 1.81 mmol) in DMF (5 mL) was treated with DIPEA (0.63 mL, 3.62 mmol) followed by HATU (1.03 g, 2.72 mmol) and stirred at room temperature for 16 hours. The resulting mixture was diluted with EtOAc (25 mL) and water (25 mL) and the phases separated. The organic portion was washed with water (2×25 mL), brine (25 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Purification of the crude residue by chromatography on silica (Biotage® SNAP KP-NH) eluting with 0-100% EtOAc in heptanes afforded the title compound as a yellow oil,

[0361] LC-MS (Method C): Rt 0.89 min; MS m/z 292.0/294.0=[M+H]+ (89%@215 nm)

[0362] 1H NMR (500 MHz, DMSO-d6) δ 8.95 (t, J=6.4 Hz, 1H), 8.04 (d, J=5.5 Hz, 1H), 7.27 (dd, J=8.7, 2.7 Hz, 1H), 7.22 (d, J=2.3 Hz, 1H), 7.07 (d, J=2.7 Hz, 1H), 7.02 (d, J=8.8 Hz, 1H), 6.61 (dd, J=5.6, 2.4 Hz, 1H), 6.33 (s, 2H), 4.39 (d, J=6.4 Hz, 2H), 3.83 (s, 3H).

Step 2: N-[(5-Chloro-2-methoxy-phenyl)methyl]-4-(2,2-dimethylpropanoylamino)pyridine-2-carboxamide

[0363] ##STR00068##

[0364] A cooled (0° C.) mixture of 4-amino-N-[(5-chloro-2-methoxy-phenyl)methyl]pyridine-2-carboxamide (step 1) (89%, 58 mg, 0.18 mmol) and DIPEA (47 μL, 0.27 mmol) in DCM (4 mL) was treated with a solution of pivaloyl chloride (26 μL, 0.21 mmol) in DCM (1 mL). The mixture was allowed to warm to room temperature and stirred for 24 h. Additional DIPEA (47 μL, 0.27 mmol) was added followed by a solution of pivaloyl chloride (26 μL, 0.21 mmol) in DCM (1 mL). The resulting mixture was stirred for a further 6 hours and then allowed to stand at room temperature for 2 days. The mixture was concentrated in vacuo and purification of the crude residue by chromatography on silica eluting with 0-80% EtOAc in heptanes afforded the title compound as a yellow film.

[0365] LC-MS (Method C): Rt 1.23 min; MS m/z 376.1, 378.1=[M+H]+ (95%@215 nm)

[0366] 1H NMR (500 MHz, DMSO-d6) δ 9.80 (s, 1H), 9.14 (t, J=6.4 Hz, 1H), 8.51 (d, J=5.5 Hz, 1H), 8.34 (d, J=2.1 Hz, 1H), 8.01 (dd, J=5.6, 2.2 Hz, 1H), 7.28 (dd, J=8.7, 2.7 Hz, 1H), 7.09 (d, J=2.7 Hz, 1H), 7.03 (d, J=8.8 Hz, 1H), 4.44 (d, J=6.3 Hz, 2H), 3.84 (s, 3H), 1.24 (s, 9H).

Step 3: N-[(5-Chloro-2-hydroxy-phenyl)methyl]-4-(2,2-dimethylpropanoylamino)pyridine-2-carboxamide

[0367] The title compound was prepared from N-[(5-chloro-2-methoxy-phenyl)methyl]-4-(2,2-dimethylpropanoylamino)pyridine-2-carboxamide (step 2) and 1M BBr.sub.3 in DCM analogously to Example 1 step 4.

[0368] LC-MS (Method A): Rt 3.28 min; MS m/z 362.2, 364.2=[M+H]+ (100%@215 nm) 1H NMR (500 MHz, DMSO-d6) δ 9.99 (s, 1H), 9.81 (s, 1H), 9.14 (t, J=6.4 Hz, 1H), 8.50 (d, J=5.6 Hz, 1H), 8.34 (d, J=2.1 Hz, 1H), 8.01 (dd, J=5.6, 2.2 Hz, 1H), 7.11 (dd, J=8.5, 2.7 Hz, 1H), 7.07 (d, J=2.7 Hz, 1H), 6.83 (d, J=8.5 Hz, 1H), 4.42 (d, J=6.3 Hz, 2H), 1.24 (s, 9H).

Example 4

N2-tert-Butyl-N4-[(2-hydroxyphenyl)methyl]pyridine-2,4-dicarboxamide

[0369] ##STR00069##

Step 1: Methyl 4-[(2-methoxyphenyl)methylcarbamoyl]pyridine-2-carboxylate

[0370] ##STR00070##

[0371] The title compound was prepared from 2-methoxycarbonylpyridine-4-carboxylic acid and (2-methoxyphenyl)methanamine analogously to Example 3 step 1.

[0372] LC-MS (Method C): Rt 1.01 min; MS m/z 301.1=[M+H]+ (97%@215 nm)

[0373] 1H NMR (500 MHz, DMSO-d6) δ 9.34 (t, J=5.7 Hz, 1H), 8.87 (dd, J=4.9, 0.7 Hz, 1H), 8.48 (dd, J=1.7, 0.8 Hz, 1H), 8.06 (dd, J=5.0, 1.7 Hz, 1H), 7.26 (td, J=8.2, 1.7 Hz, 1H), 7.21 (dd, J=7.5, 1.5 Hz, 1H), 7.01 (dd, J=8.2, 0.7 Hz, 1H), 6.91 (td, J=7.4, 1.0 Hz, 1H), 4.48 (d, J=5.8 Hz, 2H), 3.92 (s, 3H), 3.83 (s, 3H).

Step 2: 4-[(2-Methoxyphenyl)methylcarbamoyl]pyridine-2-carboxylic acid

[0374] ##STR00071##

[0375] 1M LiOH (0.85 mL, 0.85 mmol) was added to a mixture of methyl 4-[(2-methoxyphenyl)methylcarbamoyl]pyridine-2-carboxylate (step 1) (97%, 220 mg, 0.71 mmol) in THF (2 mL) and water (1 mL). After stirring at room temperature for 1 hour, the mixture was acidified to pH 1 using 1M HCl and diluted with EtOAc (15 ml) and water (15 ml). The phases were separated and the organic layer was washed with brine (15 ml), dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford the title compound as a beige foamy solid.

[0376] LC-MS (Method C): Rt 0.89 min; MS m/z 287.0=[M+H]+ (98%@215 nm)

[0377] 1H NMR (500 MHz, DMSO-d6) δ 9.32 (t, J=5.8 Hz, 1H), 8.85 (d, J=4.9 Hz, 1H), 8.50-8.45 (m, 1H), 8.02 (dd, J=5.0, 1.7 Hz, 1H), 7.28-7.23 (m, 1H), 7.22-7.19 (m, 1H), 7.01 (d, J=7.8 Hz, 1H), 6.91 (td, J=7.4, 0.9 Hz, 1H), 4.47 (d, J=5.8 Hz, 2H), 3.83 (s, 3H).

Step 3: N2-tert-Butyl-N4-[(2-methoxyphenyl)methyl]pyridine-2,4-dicarboxamide

[0378] ##STR00072##

[0379] TEA (0.18 mL, 1.03 mmol) was added to a mixture of 4-[(2-methoxyphenyl)methylcarbamoyl]pyridine-2-carboxylic acid (step 2) (98%, 100 mg, 0.34 mmol) and TBTU (131.9 mg, 0.41 mmol) in DMF (2 mL). The resulting mixture was stirred for 5 min then treated with 2-methylpropan-2-amine (0.04 mL, 0.41 mmol) and stirring continued at room temperature for 1 hour. The resulting mixture was diluted with EtOAc (20 ml) and water (20 ml) and the phases separated. The organic portion was washed with water (2×20 ml), brine (20 ml), dried over Na.sub.2SO.sub.4 and concentrated in vacuo to yield a yellow oil. Purification of the crude product by chromatography on silica eluting with 0-60% EtOAc in heptanes afforded the title compound as a thick yellow gum.

[0380] LC-MS (Method C): Rt 1.18 min; MS m/z 342.1=[M+H]+ (100%@215 nm)

[0381] 1H NMR (500 MHz, DMSO-d6) δ 9.33 (t, J=5.7 Hz, 1H), 8.76 (d, J=4.9 Hz, 1H), 8.49-8.44 (m, 1H), 8.08 (s, 1H), 7.98 (dd, J=5.0, 1.7 Hz, 1H), 7.26 (dd, J=7.6, 1.6 Hz, 1H), 7.20 (dd, J=7.5, 1.3 Hz, 1H), 7.01 (d, J=7.8 Hz, 1H), 6.91 (dd, J=7.4, 0.7 Hz, 1H), 4.47 (d, J=5.8 Hz, 2H), 3.83 (s, 3H), 1.43 (s, 9H).

Step 4: N2-tert-Butyl-N4-[(2-hydroxyphenyl)methyl]pyridine-2,4-dicarboxamide

[0382] The title compound was prepared from N2-tert-butyl-N4-[(2-methoxyphenyl)methyl]pyridine-2,4-dicarboxamide (step 3) and 1M BBr.sub.3 in DCM analogously to Example 1 step 4.

[0383] LC-MS (Method A): Rt 2.94 min; MS m/z 328.1=[M+H]+ (100%@215 nm)

[0384] 1H NMR (500 MHz, DMSO-d6) δ 9.56 (s, 1H), 9.35 (t, J=5.8 Hz, 1H), 8.76 (dd, J=5.0, 0.7 Hz, 1H), 8.46 (dd, J=1.7, 0.7 Hz, 1H), 8.08 (s, 1H), 7.98 (dd, J=5.0, 1.8 Hz, 1H), 7.14 (dd, J=7.5, 1.4 Hz, 1H), 7.08 (td, J=7.9, 1.7 Hz, 1H), 6.82 (dd, J=8.0, 1.0 Hz, 1H), 6.76 (td, J=7.4, 1.1 Hz, 1H), 4.44 (d, J=5.8 Hz, 2H), 1.42 (s, 9H).

Example 5

N1-tert-Butyl-N3-[(2-hydroxyphenyl)methyl]benzene-1,3-dicarboxamide

[0385] ##STR00073##

Step 1: Methyl 3-(tert-butylcarbamoyl)benzoate

[0386] ##STR00074##

[0387] The title compound was prepared from 3-methoxycarbonylbenzoic acid and 2-methylpropan-2-amine analogously to Example 1 step 3.

[0388] LC-MS (Method C): Rt 1.09 min; MS m/z 236.1=[M+H]+ (64%@215 nm)

[0389] 1H NMR (500 MHz, Chloroform-d) δ 8.30 (t, J=1.7 Hz, 1H), 8.13 (dt, J=7.8, 1.3 Hz, 1H), 7.99 (dt, J=7.8, 1.4 Hz, 1H), 7.51 (t, J=7.8 Hz, 1H), 6.01 (s, 1H), 3.94 (s, 3H), 1.48 (s, 9H).

Step 2: 3-(tert-Butylcarbamoyl)benzoic acid

[0390] ##STR00075##

[0391] To a solution of methyl 3-(tert-butylcarbamoyl)benzoate (step 1) (219 mg, 0.93 mmol) in 1,4-dioxane (3 mL) was added 2M LiOH (0.93 mL, 1.86 mmol) and the mixture was shaken at room temperature for 80 min. The solvent was removed in vacuo and the residue was acidified to pH1 with 1M HCl (2 mL). The mixture was extracted with DCM (2×5 mL) and the organic extracts were passed through a hydrophobic frit. The filtrate was concentrated in vacuo to afford the title compound as a white solid.

[0392] LC-MS (Method C): Rt 0.93 min; MS m/z 222.2=[M+H]+ (97%@215 nm)

[0393] 1H NMR (500 MHz, Methylene Chloride-d2) δ 8.33 (t, J=1.6 Hz, 1H), 8.06-7.96 (m, 3H), 7.55 (t, J=7.7 Hz, 1H), 1.39 (s, 9H).

Step 3: N1-tert-Butyl-N3-[(2-hydroxyphenyl)methyl]benzene-1,3-dicarboxamide

[0394] To a solution of 3-(tert-butylcarbamoyl)benzoic acid (step 2) (70 mg, 0.32 mmol) and 2-(aminomethyl)phenol (0.13 mL, 0.35 mmol) in DCM (5 mL) was added DIPEA (0.06 mL, 0.32 mmol) followed by HATU (120.3 mg, 0.32 mmol) and the mixture was shaken at room temperature overnight. The resulting mixture was washed with water (3 mL) and passed through a hydrophobic frit. The filtrate was concentrated in vacuo to afford the title compound as an off white solid,

[0395] LC-MS (Method A): Rt 2.79 min; MS m/z 327.2=[M+H]+ (95%@215 nm)

[0396] 1H NMR (500 MHz, Chloroform-d) δ 8.10 (d, J=1.6 Hz, 1H), 7.88 (d, J=7.8 Hz, 1H), 7.83 (d, J=7.8 Hz, 1H), 7.46 (t, J=7.8 Hz, 1H), 7.23 (td, J=8.1, 1.7 Hz, 1H), 7.20-7.13 (m, 2H), 6.97 (dd, J=8.1, 1.0 Hz, 1H), 6.85 (td, J=7.4, 1.1 Hz, 1H), 6.06 (s, 1H), 4.56 (d, J=6.5 Hz, 2H), 1.47 (s, 9H).

Example 6

N-[3-[[2-(5-Chloro-2-hydroxy-phenyl)acetyl]amino]phenyl]-2,2-di methyl-propanamide

[0397] ##STR00076##

Step 1: N-(3-Aminophenyl)-2,2-dimethyl-propanamide

[0398] ##STR00077##

[0399] A cooled (0° C.) solution of benzene-1,3-diamine (1.0 g, 9.25 mmol) in DCM (5 mL) was treated with DIPEA (4.85 mL, 27.74 mmol) and 2,2-dimethylpropanoyl 2,2-dimethylpropanoate (2.06 mL, 10.17 mmol) and stirred at 0° C. for 2 h. The resulting mixture was allowed to warm to room temperature and stirred for 20 h. The mixture was diluted with water (12.5 mL), saturated NaHCO.sub.3 (25 mL) and DCM (30 mL) and passed through a hydrophobic frit. The organic portion was further washed with saturated brine (25 mL) and collected using a hydrophobic frit. Purification of the crude product by chromatography on silica eluting with 25-100% EtOAc in heptanes afforded the title compound as a light red brown solid.

[0400] LC-MS (Method C): Rt 0.69 min; MS m/z 193=[M+H]+ (100%@215 nm) 1H NMR (500 MHz, DMSO-d6) δ 8.84 (s, 1H), 6.93 (t, J=2.1 Hz, 1H), 6.88 (t, J=7.9 Hz, 1H), 6.71 (ddd, J=8.0, 1.9, 0.9 Hz, 1H), 6.24 (ddd, J=7.9, 2.2, 0.9 Hz, 1H), 4.97 (s, 2H), 1.19 (s, 9H).

Step 2: N-[3-[[2-(5-Chloro-2-hydroxy-phenyl)acetyl]amino]phenyl]-2,2-dimethyl-propanamide

[0401] A solution of N-(3-aminophenyl)-2,2-dimethyl-propanamide (step 1) (48 mg, 0.25 mmol) and 5-chloro-3H-benzofuran-2-one (prepared according to the literature procedure of Journal of Fluorine Chemistry 99 (1999) 189-195) (40 mg, 0.24 mmol) in toluene (2 mL) was stirred at 120° C. for 2 h. The resulting mixture was concentrated in vacuo and purification of the residue by preparative HPLC (acidic pH, early elution method) afforded the title compound as an off-white powder.

[0402] LC-MS (Method A): Rt 3.24 min; MS m/z 361/363=[M+H]+ (98%@215 nm)

[0403] 1H NMR (500 MHz, DMSO-d6) δ 10.04 (s, 1H), 9.80 (s, 1H), 9.18 (s, 1H), 7.97 (t, J=1.9 Hz, 1H), 7.30-7.24 (m, 2H), 7.21-7.14 (m, 2H), 7.10 (dd, J=8.6, 2.7 Hz, 1H), 6.80 (d, J=8.6 Hz, 1H), 3.59 (s, 2H), 1.21 (s, 9H).

Example 7

N-[4-[[2-(5-Chloro-2-hydroxy-phenyl)acetyl]amino]-2-pyridyl]-2,2-dimethyl-propanamide

[0404] ##STR00078##

Step 1: 2-(5-Chloro-2-methoxy-phenyl)-N-(2-chloro-4-pyridyl)acetamide

[0405] ##STR00079##

[0406] The title compound was prepared from 2-(5-chloro-2-methoxy-phenyl)acetic acid and 2-chloropyridin-4-amine analogously to Example 1.2 step 1.

[0407] LC-MS (Method C): Rt 1.12 min; MS m/z 311.1=[M+H]+ (100%@215 nm)

[0408] 1H NMR (500 MHz, Chloroform-d) δ 8.23 (d, J=5.6 Hz, 1H), 7.91 (s, 1H), 7.49 (d, J=1.7 Hz, 1H), 7.32-7.27 (m, 3H), 6.91 (d, J=8.7 Hz, 1H), 3.95 (s, 3H), 3.69 (s, 2H).

Step 2: N-[4-[[2-(5-Chloro-2-methoxy-phenyl)acetyl]amino]-2-pyridyl]-2,2-dimethyl-propanamide

[0409] ##STR00080##

[0410] A suspension of 2-(5-chloro-2-methoxy-phenyl)-N-(2-chloro-4-pyridyl)acetamide (step 1) (100%, 50 mg, 0.16 mmol), 2,2-dimethylpropanamide (33 mg, 0.32 mmol) and potassium carbonate (44 mg, 0.32 mmol) in 1,4-dioxane (1 mL) and DMF (0.2 mL) was degassed with N.sub.2 for 5 min. XantPhos Pd-G3 (15 mg, 0.02 mmol) added and the sealed tube was heated to 120° C. using microwave radiation for 45 min followed by 130° C. for 30 min. The resulting mixture was concentrated in vacuo and purification of the crude residue by chromatography on silica eluting with EtOAc in heptanes afforded the title compound as a colourless glass.

[0411] LC-MS (Method C): Rt 1.06 min; MS m/z 376.0=[M+H]+ (54%@215 nm) 1H NMR (500 MHz, Chloroform-d) δ 8.16 (d, J=6.1 Hz, 1H), 7.90 (t, J=6.4 Hz, 2H), 7.29-7.27 (m, 1H), 7.24 (d, J=2.5 Hz, 1H), 6.89 (d, J=8.8 Hz, 1H), 3.95 (s, 3H), 3.81 (s, 2H), 1.39 (s, 9H).

Step 3: N-[4-[[2-(5-Chloro-2-hydroxy-phenyl)acetyl]amino]-2-pyridyl]-2,2-dimethyl-propanamide

[0412] The title compound was prepared from N-[4-[[2-(5-chloro-2-methoxy-phenyl)acetyl]amino]-2-pyridyl]-2,2-dimethyl-propanamide (step 2) and 1M BBr.sub.3 in DCM analogously to Example 1 step 4.

[0413] LC-MS (Method A): Rt 2.17 min; MS m/z 362.1=[M+H]+ (100%@215 nm)

[0414] 1H NMR (500 MHz, Methanol-d4) δ 8.25 (d, J=1.8 Hz, 1H), 8.15 (d, J=6.3 Hz, 1H), 7.50 (dd, J=6.3, 2.0 Hz, 1H), 7.18 (d, J=2.6 Hz, 1H), 7.09 (dd, J=8.6, 2.6 Hz, 1H), 6.78 (d, J=8.6 Hz, 1H), 3.73 (s, 2H), 1.32 (s, 9H).

Example 7.1

N-[4-[[2-(5-Chloro-2-hydroxy-phenyl)acetyl]amino]-2-pyridyl]cyclohexane carboxamide

[0415] ##STR00081##

[0416] The title compound was prepared from 2-(5-chloro-2-methoxy-phenyl)-N-(2-chloro-4-pyridyl)acetamide (Example 7 step 1) and cyclohexanecarboxamide analogously to Example 7 step 2 and 3.

[0417] LC-MS (Method A): Rt 2.51 min; MS m/z 388.3=[M+H]+ (99%@215 nm)

[0418] 1H NMR (500 MHz, Methanol-d4) δ 8.14 (d, J=6.7 Hz, 1H), 8.11 (d, J=2.0 Hz, 1H), 7.43 (dd, J=6.7, 2.0 Hz, 1H), 7.18 (d, J=2.6 Hz, 1H), 7.10 (dd, J=8.6, 2.6 Hz, 1H), 6.78 (d, J=8.6 Hz, 1H), 3.75 (s, 2H), 2.47 (tt, J=11.6, 3.5 Hz, 1H), 1.97-1.89 (m, 2H), 1.85 (dt, J=12.5, 3.1 Hz, 2H), 1.73 (d, J=12.5 Hz, 1H), 1.53 (qd, J=12.5, 3.1 Hz, 2H), 1.43-1.22 (m, 3H).

Example 8

5-[[2-(4-tert-Butyl-2-fluoro-5-hydroxy-phenyl)acetyl]amino]-N-(1-cyano-1-methyl-ethyl)thiophene-2-carboxamide

[0419] ##STR00082##

Step 1: 2-(4-tert-Butyl-2-fluoro-5-methoxy-phenyl)acetic acid

[0420] ##STR00083##

[0421] A solution of 2-(2-fluoro-5-methoxy-phenyl)acetic acid (5.0 g, 27.15 mmol) in DCE (181.01 mL) was treated with tert-butanol (31.16 mL, 325.8 mmol) and concentrated sulfuric acid (17.37 mL, 325.8 mmol). After stirring for 1 h, additional tert-butanol (10.0 mL, 105 mmol) and concentrated sulfuric acid (5.8 mL, 109 mmol) were added and the mixture was stirred overnight. The resulting mixture was diluted with water (150 mL) and the phases were separated. The aqueous layer was extracted with DCM (3×150 mL). The combined organic extracts were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The residue was diluted with MeOH (100 mL) and treated with 2M HCl in MeOH (100 mL, freshly prepared from thionyl chloride) and the mixture was heated at reflux for 40 min. The resulting mixture was concentrated in vacuo and the residue was dissolved in DCM (150 mL), washing with saturated aqueous sodium bicarbonate solution (150 mL). The aqueous washes were further extracted with DCM (3×100 mL) and the combined organic extracts were dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Purification by chromatography on silica eluting with 0-10% EtOAc in heptanes afforded the methyl ester intermediate. The material was dissolved in 1M LiOH (80 mL) and THF (80 mL) and stirred for 1 h. The volatiles were removed in vacuo and the aqueous solution was acidified with HCl resulting in precipitation of a solid. The solid was washed with excess water and dried to afford the title compound as a pale-yellow solid.

[0422] LC-MS (Method C): Rt 1.16 min; no ionisation (98%@215 nm)

[0423] 1H NMR (500 MHz, DMSO-d6) δ 6.93 (d, J=6.1 Hz, 1H), 6.92 (s, 1H), 3.77 (s, 3H), 3.54 (s, 2H), 1.31 (s, 9H).

Step 2: 2-(4-tert-Butyl-2-fluoro-5-hydroxy-phenyl)acetic acid

[0424] ##STR00084##

[0425] A cooled (0° C.) solution of 2-(4-tert-butyl-2-fluoro-5-methoxy-phenyl)acetic acid (step 1) (600 mg, 2.5 mmol) in DCM (12 mL) and treated with 1M BBr.sub.3 in DCM (7.49 mL, 7.49 mmol) and the mixture was warmed to room temperature and stirred for 3 days. The resulting mixture was diluted with water (20 mL), stirred for 5 min then diluted with DCM (30 mL) and water (20 mL). The phases were separated and the aqueous further extracted with DCM (30 mL). The combined organic extracts were dried over Na.sub.2SO.sub.4 and concentrated in vacuo to give a brown oil. The oil was dissolved in THF (5 mL) and treated with 2M aqueous LiOH (2 mL), stirring at room temperature for 20 min. The resulting mixture was acidified to pH 1-2 by the dropwise addition of 1M HCl then diluted with EtOAc (40 mL) and water (30 mL). The phases were separated and the organic portion was washed with brine (30 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford the title compound as a brown oil

[0426] LC-MS (Method C): Rt 1.07 min; no ionisation (97%@215 nm)

[0427] 1H NMR (500 MHz, DMSO-d6) δ 12.30 (s (br), 1H), 9.27 (s, 1H), 6.85 (d, J=11.9 Hz, 1H), 6.67 (d, J=7.0 Hz, 1H), 3.45 (s, 2H), 1.32 (s, 9H).

Step 3: Methyl 5-[[2-(4-tert-butyl-2-fluoro-5-hydroxy-phenyl)acetyl]amino]thiophene-2-carboxylate

[0428] ##STR00085##

[0429] To a solution of methyl 5-aminothiophene-2-carboxylate (219 mg, 1.39 mmol), 2-(4-tert-butyl-2-fluoro-5-hydroxy-phenyl)acetic acid (step 2)(75%, 350 mg, 1.16 mmol) and DIPEA (0.26 mL, 1.51 mmol) in DMF (4 mL) was added HATU (485 mg, 1.28 mmol) and the mixture was stirred at room temperature for 4 h. The resulting mixture was diluted with EtOAc (30 mL) and water (30 mL) and the phases were separated. The organic portion was washed with water (30 mL), brine (30 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Purification of the crude material by chromatography on silica eluting with 0-100% EtOAc in heptanes afforded the title compound as a orange solid.

[0430] LC-MS (Method C): Rt 1.21 min; MS m/z 366.1=[M+H]+ (84%@215 nm)

[0431] 1H NMR (400 MHz, DMSO-d6) δ 11.87 (s, 1H), 9.33 (s, 1H), 7.60 (d, J=4.2 Hz, 1H), 6.88 (d, J=11.9 Hz, 1H), 6.74-6.69 (m, 2H), 3.76 (s, 3H), 3.65 (s, 2H), 1.32 (s, 9H).

Step 4: 5-[[2-(4-tert-Butyl-2-fluoro-5-hydroxy-phenyl)acetyl]amino]thiophene-2-carboxylic acid

[0432] ##STR00086##

[0433] 2M LiOH (0.72 mL, 1.44 mmol) was added to a solution of methyl 5-[[2-(4-tert-butyl-2-fluoro-5-hydroxy-phenyl)acetyl]amino]thiophene-2-carboxylate (step 3) (84%, 314 mg, 0.72 mmol) in THF (2 mL) and water (2 mL) and the mixture was heated to 50° C. overnight. The resulting mixture was concentrated in vacuo to remove the majority of the THF and then acidified to pH 1-2 by the dropwise addition of 1M HCl. The mixture was partitioned between EtOAc (15 mL) and water (15 mL) and the phases were separated. The organic portion was washed with brine (15 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo to afford the title compound as an orange oil.

[0434] LC-MS (Method C): Rt 1.11 min; MS m/z 352.0=[M+H]+ (98%@215 nm) 1H NMR (400 MHz, DMSO-d6) δ 11.78 (s, 1H), 9.33 (s, 1H), 7.52 (d, J=4.1 Hz, 1H), 6.87 (d, J=11.9 Hz, 1H), 6.73 (d, J=7.0 Hz, 1H), 6.70 (d, J=4.2 Hz, 1H), 3.64 (s, 2H), 1.32 (s, 9H).

Step 5: 5-[[2-(4-tert-Butyl-2-fluoro-5-hydroxy-phenyl)acetyl]amino]-N-(1-cyano-1-methyl-ethyl)thiophene-2-carboxamide

[0435] HATU (133 mg, 0.35 mmol) was added to a solution of 5-[[2-(4-tert-butyl-2-fluoro-5-hydroxy-phenyl)acetyl]amino]thiophene-2-carboxylic acid (step 4) (70%, 175 mg, 0.35 mmol), 2-amino-2-methyl-propanenitrile hydrochloride (63 mg, 0.52 mmol) and DIPEA (0.15 mL, 0.87 mmol) in DMF (3 mL) and the mixture was stirred at room temperature overnight. The resulting mixture was partitioned between EtOAc (20 mL) and water (20 mL) and the phases were separated. The organic portion was washed with washed with brine (15 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Purification of the crude material by preparative HPLC (acidic pH, standard elution method) afforded the title compound as an off-white solid.

[0436] LC-MS (Method A): Rt 3.30 min; MS m/z 418.2=[M+H]+ (99%@215 nm)

[0437] 1H NMR (500 MHz, DMSO-d6) δ 11.69 (s, 1H), 9.33 (s, 1H), 8.49 (s, 1H), 7.65 (d, J=4.2 Hz, 1H), 6.87 (d, J=11.9 Hz, 1H), 6.74 (d, J=6.9 Hz, 1H), 6.68 (d, J=4.2 Hz, 1H), 3.64 (s, 2H), 1.66 (s, 6H), 1.32 (s, 9H).

Example 9

5-[[2-[2-Fluoro-5-hydroxy-4-(2-hydroxy-1,1-dimethyl-ethyl)phenyl]acetyl]amino]-N-[1-(trifluoromethyl)cyclopropyl]thiophene-2-carboxamide

[0438] ##STR00087##

Intermediate A: 2-(5-Fluoro-3,3-dimethyl-2-oxo-benzofuran-6-yl)acetic acid

Step A1: 2-(4-Bromo-2-fluoro-5-methoxy-phenyl)acetic acid

[0439] ##STR00088##

[0440] To a cooled (0° C.) solution of 2-(2-fluoro-5-methoxy-phenyl)acetic acid (45 g, 244.4 mmol) in MeCN (1.2 L) was added dropwise a solution of bromine (12.63 mL, 219.9 mmol) in MeCN (100 mL) over a period of 10 min. The resulting mixture was allowed to warm to room temperature gradually without removing the ice bath (˜1.5 h). Additional bromine (4.21 mL, 73.3 mmol) in MeCN (50 mL) was added dropwise to the mixture at 0° C. which was stirred at room temperature for a further 3.5 h. Further bromine (4.21 mL, 73.3 mmol) in MeCN (50 mL) was added at room temperature and mixture was stirred at room temperature for 30 min. The reaction was quenched carefully with saturated aqueous sodium sulfite (˜700 mL) until the bright orange colour had disappeared. The colourless solution was diluted with brine (200 mL) and EtOAc (200 mL), stirred vigorously for 10 min. The organic layer was separated and the aqueous layer was extracted further with EtOAc (200 mL). The organic layers were combined, dried over Na.sub.2SO.sub.4 and concentrated in vacuo to obtain the crude product as a white solid. The crude product was recrystallised by dissolving the solid in AcOH (700 mL), then treated with water (4 L). The mixture was stirred to mix the solvents whereupon crystals gradually appeared. The mixture was kept at room temperature for 1 h and then at 0° C. for 3 h. Filtration followed by vacuum drying at 40° C. afforded the title compound as a fluffy white solid.

[0441] LC-MS (Method C): Rt 1.07 min; (99%@215 nm)

[0442] .sup.1H NMR (500 MHz, DMSO-d6) δ 12.55 (br s, 1H), 7.50 (d, J=8.9 Hz, 1H), 7.13 (d, J=6.6 Hz, 1H), 3.81 (s, 3H), 3.61 (d, J=1.3 Hz, 2H).

Step A2: Benzyl 2-(4-bromo-2-fluoro-5-methoxy-phenyl)acetate

[0443] ##STR00089##

[0444] A mixture of 2-(4-bromo-2-fluoro-5-methoxy-phenyl)acetic acid (step A1)(15 g, 57.02 mmol) and K.sub.2CO.sub.3 (15.76 g, 114.0 mmol) in DMF (140 mL) was treated with benzylbromide (7.45 mL, 62.7 mmol) and stirred at room temperature for 18 h. The resulting mixture was filtered and concentrated in vacuo. The residue was dissolved in EtOAc (300 mL) and sequentially washed with brine (200 mL) and saturated aqueous sodium bicarbonate (2×200 mL). The organic layer was dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Purification by chromatography on silica eluting with 0-20% EtOAc in heptanes afforded the title compound as a colourless solid.

[0445] LC-MS (Method G): Rt 1.13 min; (95%@215 nm)

[0446] .sup.1H NMR (500 MHz, DMSO-d6) δ 7.53 (d, J=8.9 Hz, 1H), 7.40-7.31 (m, 5H), 7.15 (d, J=6.6 Hz, 1H), 5.14 (s, 2H), 3.81-3.78 (m, 5H).

Step A3: Methyl 2-[4-(2-benzyloxy-2-oxo-ethyl)-5-fluoro-2-methoxy-phenyl]-2-methyl-propanoate

[0447] ##STR00090##

[0448] Benzyl 2-(4-bromo-2-fluoro-5-methoxy-phenyl)acetate (step A2) (5.0 g, 14.16 mmol), ZnF.sub.2 (1.1 g, 10.62 mmol) and Pd(PtBu.sub.3).sub.2 (0.36 g, 0.71 mmol) were added to a reaction vessel and placed under a nitrogen atmosphere. A solution of (1-methoxy-2-methyl-prop-1-enoxy)-trimethyl-silane (5.75 mL, 28.31 mmol) in degassed DMF (50 mL) was added and the reaction mixture was heated to 80° C. for 18 h. The resulting mixture was filtered and concentrated in vacuo. The residue was dissolved in EtOAc (100 mL) and washed with brine (2×50 mL) and the organic layer was dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Purification by chromatography on silica eluting with 0-30% EtOAc in heptanes the title compound as a pale yellow oil.

[0449] LC-MS (Method A): Rt 3.99 min; MS m/z 375.3=[M+H]+ (92%@215 nm)

[0450] .sup.1H NMR (400 MHz, DMSO-d6) δ 7.40-7.30 (m, 5H), 7.09 (d, J=11.0 Hz, 1H), 6.98 (d, J=6.5 Hz, 1H), 5.14 (s, 2H), 3.76 (s, 2H), 3.66 (s, 3H), 3.53 (s, 3H), 1.40 (s, 6H).

Step A4: 2-(5-Fluoro-3,3-dimethyl-2-oxo-benzofuran-6-yl)acetic acid

[0451] ##STR00091##

[0452] 1M BBr.sub.3 in DCM (34.13 mL, 34.1 mmol) was added to a cooled (0° C.) mixture of methyl 2-[4-(2-benzyloxy-2-oxo-ethyl)-5-fluoro-2-methoxy-phenyl]-2-methyl-propanoate (step A3) (92%, 2777 mg, 6.83 mmol) in dry DCM (60 mL). The resulting mixture was allowed to warm to room temperature and stirred for 4.5 h. The reaction mixture was re-cooled to 0° C. and water (50 mL) was added. Stirring continued whilst gradually warming to room temperature over 30 min. The resulting mixture was diluted with DCM (80 mL) and water (80 mL) and the phases were separated. The aqueous portion was extracted with EtOAc (80 mL) then the combined organic extracts were dried over Na.sub.2SO.sub.4 and concentrated in vacuo to give the crude product as a brown oil. Purification of the crude product by C.sub.18 reverse phase chromatography eluting with 10-100% MeCN/water (+0.1% formic acid) afforded the title compound as a pale yellow solid.

[0453] LC-MS (Method H): Rt 1.19 min; (98%@215 nm)

[0454] .sup.1H NMR (500 MHz, DMSO-d6) δ 12.56 (br. s, 1H), 7.42 (d, J=8.9 Hz, 1H), 7.25 (d, J=5.8 Hz, 1H), 3.64 (d, J=1.5 Hz, 2H), 1.44 (s, 6H).

Intermediate B: 5-Amino-N-[1-(trifluoromethyl)cyclopropyl]thiophene-2-carboxamide

Step B1: 5-Nitro-N-[1-(trifluoromethyl)cyclopropyl]thiophene-2-carboxamide

[0455] ##STR00092##

[0456] To a mixture of 5-nitrothiophene-2-carboxylic acid (400 mg, 2.31 mmol) and HATU (1054 mg, 2.77 mmol) in DMF (10 mL) was added 1-(trifluoromethyl)cyclopropanamine hydrochloride (411 mg, 2.54 mmol) followed by DIPEA (0.97 mL, 5.54 mmol) and the reaction mixture was stirred at room temperature for 1 h. The resulting mixture was diluted with EtOAc (40 mL) and water (40 mL) and the phases were separated. The organic portion was washed with water (40 mL), brine (40 mL) then dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Purification of the crude material by chromatography on silica eluting with 0-100% EtOAc in heptanes afforded the title compound as an off-white solid.

[0457] LC-MS (Method C): Rt 1.12 min; MS m/z 280.9=[M+H]+ (100%@215 nm)

[0458] 1H NMR (400 MHz, DMSO-d6) δ 9.66 (s, 1H), 8.14 (d, J=4.4 Hz, 1H), 7.86 (d, J=4.4 Hz, 1H), 1.38-1.29 (m, 2H), 1.23-1.15 (m, 2H).

Step B2: 5-Amino-N-[1-(trifluoromethyl)cyclopropyl]thiophene-2-carboxamide

[0459] ##STR00093##

[0460] A mixture of 5-nitro-N-[1-(trifluoromethyl)cyclopropyl]thiophene-2-carboxamide (step B1) (596 mg, 2.13 mmol) in EtOH (20 mL) under a nitrogen atmosphere was treated with Pd on carbon (10%, 225 mg, 0.21 mmol) and placed under a hydrogen atmosphere. After stirring at room temperature for 2 h, the resulting mixture was filtered through Celite® (filter material) and washed with EtOH. The filtrate was concentrated in vacuo and purification of the crude product by chromatography on KP-NH silica eluting with EtOAc in heptanes afforded the title compound as a yellow oil.

[0461] LC-MS (Method G): Rt 0.67 min; MS m/z 251.1=[M+H]+ (97%@215 nm)

[0462] 1H NMR (400 MHz, DMSO-d6) δ 8.55 (s, 1H), 7.38 (d, J=4.1 Hz, 1H), 6.34 (s, 2H), 5.81 (d, J=4.1 Hz, 1H), 1.27-1.23 (m, 2H), 1.10-1.05 (m, 2H).

Final Step: Intermediate A+Intermediate B

5-[[2-[2-Fluoro-5-hydroxy-4-(2-hydroxy-1,1-dimethyl-ethyl)phenyl]acetyl]amino]-N-[1-(trifluoromethyl)cyclopropyl]thiophene-2-carboxamide

[0463] To a solution of 5-amino-N-[1-(trifluoromethyl)cyclopropyl]thiophene-2-carboxamide (Intermediate B) (80%, 134 mg, 0.43 mmol) and 2-(5-fluoro-3,3-dimethyl-2-oxo-benzofuran-6-yl)acetic acid (Intermediate A) (98%, 114 mg, 0.47 mmol) in DMF (3 mL) was added DIPEA (149 μL, 0.85 mmol) followed by 50% T3P® solution in EtOAc (0.38 mL, 0.64 mmol) and the reaction mixture was stirred at room temperature for 30 min. The resulting mixture was diluted with EtOAc (15 mL) and water (15 mL) and the phases were separated. The organic portion was washed with water (15 mL), brine (15 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo to give the crude intermediate as a yellow/orange oil. The oil was dissolved in THF (3 mL) and cooled to −78° C. 4M LiBH4 in THF (107 μL, 0.43 mmol) was added dropwise and the resulting mixture was allowed to warm to room temperature, stirring for 1 h. The mixture was cooled to 0° C. and the reaction was quenched by the dropwise addition of 1M HCl (5 mL). The resulting mixture was diluted with EtOAc (20 mL) and water (15 mL) and the phases were separated. The aqueous portion was further extracted with EtOAc (20 mL) and the combined organic extracts were dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Purification of the crude material by preparative HPLC (acidic pH, early elution method) afforded the title compound as a white solid.

[0464] LC-MS (Method A): Rt 2.76 min; MS m/z 475.3=[M+H]+ (100%@215 nm)

[0465] 1H NMR (400 MHz, DMSO-d6) δ 11.65 (s, 1H), 9.38 (s, 1H), 8.95 (s, 1H), 7.61 (d, J=4.2 Hz, 1H), 6.89 (d, J=12.1 Hz, 1H), 6.71 (d, J=7.0 Hz, 1H), 6.65 (d, J=4.2 Hz, 1H), 4.77 (s, 1H), 3.63 (s, 2H), 3.60 (s, 2H), 1.31-1.22 (m, 8H), 1.15-1.08 (m, 2H).

Biological Example

[0466] Automated Whole-Cell Patch Clamp Assay to Detect TMEM16A Activity in Recombinant Cells

[0467] Cell Culture and Preparation

[0468] Fisher rat thyroid (FRT) cells stably expressing human TMEM16A (TMEM16Aabc variant; Dr Luis Galietta, Insituto Giannina, Italy) were cultured in T-75 flasks in Hams F-12 media with Coon's modification (Sigma) supplemented with 10% (v/v) foetal bovine serum, penicillin-streptomycin (10,000 U/mL/10000 μg/mL), G-418 (750 μg/mL), L-glutamine (2 mM) and sodium bicarbonate solution (7.5% v/v). At ˜90% confluence cells were harvested for experiments by detachment with a 2:1 (v/v) mixture of Detachin (BMS Biotechnology) and 0.25% (w/v) trypsin-EDTA. Cells were diluted to a density of 3.5-4.5×10.sup.6 cells/mL with media consisting of CHO—S-SFM II (Sigma), 25 mM HEPES (Sigma) and Soy bean trypsin inhibitor (Sigma).

[0469] Whole-Cell Patch Clamp Recording

[0470] FRT-TMEM16A cells were whole-cell patch clamped using an automated planar patch clamp system (Qpatch, Sophion). Briefly, once high resistance (GOhm) seals were established between the cells and the planar recording array the patch was ruptured using suction pulses to establish the whole-cell recording configuration of the patch clamp technique. The assay employed the following solutions (all reagents Sigma):

[0471] Intracellular solution (mM): N-methyl-D-glucamine 130, CaCl.sub.2) 18.2, MgCl.sub.2 1, HEPES 10, EGTA 10, BAPTA 20, Mg-ATP 2, pH 7.25, 325mOsm with sucrose.

[0472] Extracellular solution (mM): N-methyl-D-glucamine 130, CaCl.sub.2) 2, MgCl.sub.2 1, HEPES 10, pH 7.3, 320 mOsm with sucrose.

[0473] The intracellular solution buffers intracellular calcium at levels required to give ˜20% activation of the maximal TMEM16A mediated current (EC.sub.20 for calcium ions). Cells were voltage clamped at a holding potential of −70 mV and a combined voltage step (to +70 mV)/ramp (−90 my to +90 mV) was applied at 0.05 Hz. After a period of current stabilisation test compounds, solubilised in 100% (v/v) DMSO and subsequently diluted into extracellular solution, were applied to generate a cumulative concentration response curve. Each concentration of test compound was incubated for 5 minutes before addition of the next concentration. After the final concentration was tested a supramaximal concentration of either a known active positive modulator or the TMEM16A inhibitor, CaCCinhA01 (Del La Fuente et al, 2008) was added to define the upper and lower limits of the assay.

[0474] Compound activity was quantified by measuring the increase in current upon compound addition and expressing this as a percentage increase of baseline TMEM16A current level. Percentage increases in current were determined for each concentration and the data plotted as a function of concentration using either the Qpatch software or Graphpad Prism v6.05 providing the concentration which gave 50% of its maximal effect (EC.sub.50) and maximum efficacy (percentage of baseline increase).

[0475] The method of calculating the results is illustrated in FIG. 1, which shows an example trace from the Qpatch TMEM16A assay. In FIG. 1, I.sub.BL equals baseline current, I.sub.[#1] equals the peak current during test compound concentration 1 incubation period and so on.

[0476] Peak TMEM16A current at +70 mV was plotted as a function of time over the assay period. Baseline current (I.sub.BL) was measured after a period of stabilisation. The increase in current for each compound addition was determined by taking the peak current during the incubation period and subtracting the current from the previous recording period and then expressing this as a percentage of the baseline current. For test compound concentration 1 in FIG. 1 this is:

[00001]$\left(I_{\left[\mathrm{\#1}\right]}-I_{\mathrm{BL}}\text{/}{I}_{\mathrm{BL}}\right)\times 100$

[0477] For each additional concentration tested the increase in current was determined by subtracting the current from the previous incubation period and normalising the baseline value—for test concentration 2 in FIG. 1 this is:

[00002]$\left(I_{\left[\mathrm{\#2}\right]}-I_{\left\{\mathrm{\#1}\right\}}\text{/}{I}_{\mathrm{BL}}\right)\times 100$

[0478] The values for each test concentration were plotted as a cumulative function of concentration e.g. for test concentration two this would be the sum of the peak changes measured during concentration one plus concentration two.

[0479] The results obtained for the example compounds are shown in Table 1, from which it can be seen that the compounds of the present invention are capable of significantly increasing the TMEM16A current level.

TABLE-US-00001 TABLE 1 % Potentiation shown by 3.33 μM solution of Test Compounds and Calculated EC.sub.50 Values % Potentiation @ Example 3.33 μM Avg EC.sub.50 Avg (μM) 1 106.6 0.042 1.1 184.1 0.44 1.2 236.9 0.4 2 127.4 3 81.3 4 35.2 5 41.2 2.909 6 68.1 0.992 7 156.1 0.615 7.1 120.3 1.113 8 134.2 0.085 9 134.4 0.098

REFERENCES

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