ANALOGUES OF 3-(5-METHYL-1,3-THIAZOL-2-YL)-N-{(1R)-1-[2-(TRIFLUORO-METHYL)PYRIMIDIN-5-YL]ETHYL}BENZAMIDE

Abstract

The present invention covers P2X3 inhibitor compounds of general formula (I) in which R.sup.1 and R.sup.2 are as defined herein, methods of preparing said compounds, intermediate compounds useful for preparing said compounds, pharmaceutical compositions and combinations comprising said compounds and the use of said compounds for manufacturing pharmaceutical compositions for the treatment or prophylaxis of diseases, in particular of neurogenic disorders, as a sole agent or in combination with other active ingredients.

##STR00001##

Claims

1. A compound of formula (I): ##STR00008## wherein R.sup.1 is methyl, R.sup.2 is C.sub.3-C.sub.4-alkyl substituted with two substituents which are the same or different and independently selected from the group consisting of OH and —COOH, or 5-membered heterocycloalkyl having one O atom and substituted at any carbon atom with one or two substituents which are the same or different, and independently selected from the group consisting of oxo and OH, a stereoisomer, a hydrate, a solvate, or a salt thereof, or any mixture thereof.

2. (canceled)

3. The compound of claim 1, wherein R.sup.2 is C.sub.3-alkyl optionally substituted with OH and COOH, or a stereoisomer, a hydrate, a solvate, or a salt thereof, or any mixture thereof.

4. The compound of claim 1, wherein R.sup.2 is C.sub.4-alkyl optionally substituted with two OH, or a stereoisomer, a hydrate, a solvate, or a salt thereof, or any mixture thereof.

5. The compound of claim 1, wherein R.sup.2 is C(CH.sub.2OH)(CH.sub.2).sub.2OH, or a stereoisomer, a hydrate, a solvate, or a salt thereof, or any mixture thereof.

6. The compound of claim 1, wherein R.sup.2 is tetrahydrofuranyl substituted at any carbon atom with OH, or a stereoisomer, a hydrate, a solvate, or a salt thereof, or any mixture thereof.

7. The compound claim 1, wherein R.sup.2 is C(CH.sub.2OH)(CH.sub.2).sub.2OH, or a stereoisomer, a hydrate, a solvate, or a salt thereof, or any mixture thereof.

8. A compound selected from the group consisting of: 3-{[(2R)-1,4-dihydroxybutan-2-yl]oxy}-5-(5-methyl-1,3-thiazol-2-yl)-N-{(1R)-1-[2-(trifluoromethyl)pyrimidin-5-yl]ethyl}benzamide, rel-3-{[(3R,5R)-5-Hydroxytetrahydrofuran-3-yl]oxy}-5-(5-methyl-1,3-thiazol-2-yl)-N-{(1R)-1-[2-(trifluoromethyl)pyrimidin-5-yl]ethyl}benzamide, {[(3R,5R)-5-Hydroxytetrahydrofuran-3-yl]oxy}-5-(5-methyl-1,3-thiazol-2-yl)-N-{(1R)-1-[2-(trifluoromethyl)pyrimidin-5-yl]ethyl}benzamide, {[(3R,5S)-5-hydroxytetrahydrofuran-3-yl]oxy}-5-(5-methyl-1,3-thiazol-2-yl)-N-{(1R)-1-[2-(trifluoromethyl)pyrimidin-5-yl]ethyl}benzamide, (3R)-4-hydroxy-3-[3-(5-methyl-1,3-thiazol-2-yl)-5-({(1R)-1-[2-(trifluoromethyl)pyrimidin-5-yl]ethyl}carbamoyl)phenoxy]butanoic acid, and 2-{3-[(3R)-tetrahydrofuran-3-yloxy]-5-({(1R)-1-[2-(trifluoromethyl)pyrimidin-5-yl]ethyl}carbamoyl)phenyl}-1,3-thiazole-5-carboxylic acid, or a stereoisomer, a hydrate, a solvate, or a salt thereof, or a mixture of same.

9. A method for treatment of a disease in a human in need thereof, comprising administering to the human an effective amount of a compound of formula (I) according to claim 1, or an enantiomer, diastereomer, racemate, hydrate, solvate, or a pharmaceutically acceptable salt thereof, or a mixture thereof.

10. The method of claim 9, wherein the disease is a neurogenic disorder, such as genitourinary, gastrointestinal, respiratory, cardiovascular disease associated with autonomic imbalance caused by increased chemoreceptor sensitivity, and pain-related diseases.

11. (canceled)

12. The method of claim 10, wherein the genitourinary disease is selected from the group consisting of dysmenorrhea, dyspareunia, endometriosis, adenomyosis, endometriosis-associated pain, endometriosis-associated proliferation, pelvic hypersensitivity, dysuria, and dyschezia.

13. The method of claim 10, wherein the genitourinary disease is selected from the group consisting of bladder outlet obstruction, urinary incontinence conditions, reduced bladder capacity, increased frequency of micturition, urge incontinence, stress incontinence, bladder hyperreactivity, benign prostatic hypertrophy, prostatic hyperplasia, prostatitis, detrusor hyperreflexia, pelvic hypersensitivity, urethritis, prostatitis, prostatodynia, cystitis, Interstitial cystitis, idiopathic bladder hypersensitivity, overactive bladder, and symptoms related to overactive bladder wherein said symptoms are increased urinary frequency, nocturia, urinary urgency or urge incontinence.

14. The method of claim 10, wherein the respiratory disease is selected from the group consisting of chronic obstructive pulmonary disorder (COPD), asthma, bronchospasm, pulmonary fibrosis, acute cough, and chronic cough including chronic idiopathic and chronic refractory cough.

15. A pharmaceutical composition comprising a compound of formula (I) according to claim 1, or an enantiomer, diastereomer, racemate, hydrate, solvate, a pharmaceutically acceptable salt thereof, or a mixture thereof, and one or more pharmaceutically acceptable excipients.

16. A pharmaceutical combination comprising a compound of formula (I) according to claim 1, or an enantiomer, diastereomer, racemate, hydrate, solvate, a pharmaceutically acceptable salt thereof, or a mixture thereof, and one or more further active ingredients, wherein said further active ingredients are selected from the group consisting of cough suppressants, NSAIDS (Non-Steroidal Antiinflammatory Drug), Combined Oral Contraceptives (COC), GnRAH antagonists, Selective Progesterone Receptor Modulators (SPRMs), Progesterone antagonists, P2X3 inhibitors, NK1 inhibitors and nicotinic Acetylcholine modulators.

17. A method for treatment of a disease in a human in need thereof, comprising administering to the human an effective amount of a compound according to claim 8, or an enantiomer, diastereomer, racemate, hydrate, solvate, or a pharmaceutically acceptable salt thereof, or a mixture thereof.

18. The method of claim 17, wherein the disease is a neurogenic disorder, such as genitourinary, gastrointestinal, respiratory, cardiovascular disease associated with autonomic imbalance caused by increased chemoreceptor sensitivity, and pain-related diseases.

19. The method of claim 17, wherein the genitourinary disease is selected from the group consisting of dysmenorrhea, dyspareunia, endometriosis, adenomyosis, endometriosis-associated pain, endometriosis-associated proliferation, pelvic hypersensitivity, dysuria, and dyschezia.

20. The method of claim 17, wherein the genitourinary disease is selected from the group consisting of bladder outlet obstruction, urinary incontinence conditions, reduced bladder capacity, increased frequency of micturition, urge incontinence, stress incontinence, bladder hyperreactivity, benign prostatic hypertrophy, prostatic hyperplasia, prostatitis, detrusor hyperreflexia, pelvic hypersensitivity, urethritis, prostatitis, prostatodynia, cystitis, Interstitial cystitis, idiopathic bladder hypersensitivity, overactive bladder, and symptoms related to overactive bladder, wherein said symptoms are increased urinary frequency, nocturia, urinary urgency or urge incontinence.

21. The method of claim 17, wherein the respiratory disease is selected from the group consisting of chronic obstructive pulmonary disorder (COPD), asthma, bronchospasm, pulmonary fibrosis, acute cough, and chronic cough including chronic idiopathic and chronic refractory cough.

22. A pharmaceutical composition, comprising a compound of according to claim 8, or an enantiomer, diastereomer, racemate, hydrate, solvate, a pharmaceutically acceptable salt thereof, or a mixture thereof, and one or more pharmaceutically acceptable excipients.

23. A pharmaceutical combination, comprising a compound according to claim 8, or an enantiomer, diastereomer, racemate, hydrate, solvate, a pharmaceutically acceptable salt thereof, or a mixture thereof, and one or more further active ingredients, wherein said further active ingredients are selected from the group consisting of cough suppressants, NSAIDS (Non-Steroidal Antiinflammatory Drug), Combined Oral Contraceptives (COC), GnRAH antagonists, Selective Progesterone Receptor Modulators (SPRMs), Progesterone antagonists, P2X3 inhibitors, NK1 inhibitors and nicotinic Acetylcholine modulators.

Description

EXPERIMENTAL SECTION—EXAMPLES

Example 1

{[(3R,5R)-5-Hydroxytetrahydrofuran-3-yl]oxy}-5-(5-methyl-1,3-thiazol-2-yl)-N-{(1R)-1-[2-(trifluoromethyl)pyrimidin-5-yl]ethyl}benzamide and {[(3R,5S)-5-hydroxytetrahydrofuran-3-yl]oxy}-5-(5-methyl-1,3-thiazol-2-yl)-N-{(1R)-1-[2-(trifluoromethyl)pyrimidin-5-yl]ethyl}benzamide

[0249] ##STR00005##

[0250] A mixture of 3-(5-methyl-1,3-thiazol-2-yl)-5-[(3R)-tetrahydrofuran-3-yloxy]-N-{(1R)-1-[2-(trifluoromethyl)pyrimidin-5-yl]ethyl}benzamide (200 mg, 0.42 mmol), and Iron(III) chloride (56 mg, 0.21 mmol) were added to a reaction vessel, pyridine was added (2 ml), and tert-butylhydroperoxide 70% mixture in water, 239 μl, 1.67 mmol) was added dropwise. The reaction vessel was sealed, and the reaction left to stir for 48 hours at RT. Saturated Ethylenediaminetetraacetic acid solution (mono sodium salt, 15 ml) was added and the mixture stirred at RT for 10 minutes. Brine was added, and the aqueous phase was extracted with dichloromethane (100 ml), the organic phase was dried by passing it through a water repellent filter and the solvent removed under reduced pressure. The title compounds (2.4 mg, 1,2% yield) were obtained by purification using a Labomatic HD5000, Labocord-5000; Gilson GX-241, Labcol Vario 4000 system, with a Chiralpak ID 5μ 250×30 mm column; A mobile phase of hexane:ethanol was used with a gradient of 25-50% (ethanol) over 15 min with a flow rate of 40.0 ml/min with detection using a UV wavelength of 325 nm.

[0251] Analytical HPLC Method:

[0252] Instrument: Waters Autopurification MS SingleQuad; Column: Waters XBrigde C18 5μ 100×30 mm; eluent A: water+0.1 vol % formic acid (99%), eluent B: acetonitrile; gradient: 0-5.5 min 5-100% B; flow 70 ml/min; temperature: 25° C.; DAD scan: 210-400 nm. LC-MS: R.sub.t=1.10 min; 495,27 (M+H).sup.+.

[0253] .sup.1H NMR (600 MHz, DMSO-d.sub.6) δ ppm 1.59-1.63 (m, 3H) 2.13-2.21 (m, 1H) 2.39-2.45 (m, 1H) 2.52-2.55 (m, 3H) 2.77 (t, J=6.10 Hz, 1H) 3.32 (s, 1H) 3.38-3.50 (m, 1H) 3.79 (q, J=6.10 Hz, 1H) 3.84-3.99 (m, 1H) 3.88 (d, J=10.30 Hz, 1H) 3.95 (dd, J=9.92, 3.43 Hz, 1H) 4.10-4.15 (m, 1H) 4.96 (t, J=5.34 Hz, 1H) 5.06-5.35 (m, 1H) 5.09-5.17 (m, 1H) 5.24-5.33 (m, 1H) 5.38-5.60 (m, 1H) 5.41-5.44 (m, 1H) 5.55 (q, J=4.58 Hz, 1H) 6.20-6.34 (m, 1H) 6.25 (d, J=4.58 Hz, 1H) 6.30 (d, J=4.96 Hz, 1H) 7.47-7.55 (m, 1H) 7.59-7.71 (m, 1H) 7.81 (m, 1H) 7.91-7.94 (m, 1H) 9.10-9.20 (m, 3H)

[0254] 1H-NMR (600 MHz, DMSO-d6) delta [ppm]: −0.006 (0.83), 0.005 (0.72), 0.785 (0.50), 0.797 (0.89), 0.810 (0.61), 0.825 (0.44), 0.836 (0.44), 0.842 (0.61), 0.854 (1.16), 0.865 (0.78), 1.008 (0.66), 1.032 (2.05), 1.043 (2.33), 1.086 (0.78), 1.146 (0.94), 1.157 (1.77), 1.170 (0.94), 1.182 (0.55), 1.207 (0.61), 1.234 (3.16), 1.259 (0.94), 1.262 (1.44), 1.285 (0.39), 1.296 (0.72), 1.590 (1.38), 1.602 (12.35), 1.614 (11.35), 1.835 (0.55), 1.858 (0.61), 2.163 (1.27), 2.168 (2.44), 2.174 (2.05), 2.177 (2.33), 2.183 (1.38), 2.386 (1.22), 2.389 (1.66), 2.391 (1.27), 2.421 (0.44), 2.520 (4.76), 2.523 (6.59), 2.525 (5.65), 2.544 (1.49), 2.614 (1.27), 2.617 (1.72), 2.619 (1.27), 2.759 (0.61), 2.769 (1.22), 2.779 (0.61), 3.321 (0.44), 3.377 (0.50), 3.784 (0.83), 3.793 (0.78), 3.873 (1.99), 3.890 (2.10), 3.942 (0.50), 3.948 (0.50), 3.959 (0.55), 3.964 (0.55), 4.112 (1.55), 4.119 (1.94), 4.128 (1.72), 4.136 (1.49), 4.142 (0.50), 4.956 (0.78), 5.101 (0.44), 5.110 (0.39), 5.248 (1.22), 5.255 (1.16), 5.261 (0.61), 5.273 (0.55), 5.285 (1.88), 5.297 (2.66), 5.308 (1.66), 5.320 (0.44), 5.426 (0.55), 5.539 (0.83), 5.547 (1.72), 5.553 (1.77), 5.561 (0.83), 6.242 (0.94), 6.250 (0.94), 6.291 (3.27), 6.299 (3.21), 7.334 (0.44), 7.433 (0.44), 7.474 (1.83), 7.478 (3.04), 7.480 (2.66), 7.492 (3.88), 7.495 (4.15), 7.499 (1.88), 7.503 (1.11), 7.505 (1.16), 7.509 (0.61), 7.622 (0.72), 7.624 (0.66), 7.642 (0.55), 7.650 (4.82), 7.652 (4.93), 7.677 (0.72), 7.679 (0.72), 7.707 (0.44), 7.710 (0.66), 7.713 (0.50), 7.793 (0.66), 7.819 (0.44), 7.822 (0.66), 7.927 (4.21), 7.929 (5.31), 7.931 (2.77), 8.243 (0.50), 8.245 (0.83), 8.248 (0.50), 8.321 (0.78), 9.110 (2.38), 9.121 (16.00), 9.130 (2.93), 9.170 (2.10), 9.182 (2.66), 9.194 (0.83), 10.078 (0.94).

[0255] .sup.13C NMR (151 MHz, DMSO-d6) δ ppm 0.18 11.77 20.83 20.88 37.81 38.95 40.08 40.19 40.26 40.59 45.35 56.86 69.83 70.30 76.43 77.55 78.81 79.03 79.24 97.48 97.58 114.66 115.75 117.53 118.82 120.64 134.78 134.84 135.07 135.10 136.21 136.23 140.40 141.88 153.65 153.89 156.81 156.84 157.51 164.27 164.30 165.11 165.15

Example 2

3-{[(2R)-1,4-dihydroxybutan-2-yl]oxy}-5-(5-methyl-1,3-thiazol-2-yl)-N-{(1R)-1-[2-(trifluoromethyl)pyrimidin-5-yl]ethyl}benzamide

[0256] ##STR00006##

[0257] Preculture Generation

[0258] A DMSO cryo culture (0.2 mL) of Streptomyces roseochromogenus (CBS 41563) was added to a 100-mL Erlenmeyer flask containing sterile growth medium (20 mL) consisting of D-(+)-glucose monohydrate (10 g L.sup.−1), yeast extract (1 g L.sup.−1), beef extract (1 g L.sup.−1) and tryptose (2 g L.sup.−1) which had been adjusted to pH 7.2 with sodium hydroxide solution (16% in water) and had been sterilized at 121° C. for 20 minutes. After inoculation, the mixture was shaken on a rotation shaker (165 rpm) at 27° C. for 48 h. This preculture (10 mL per flask) was added to two 500-mL Erlenmeyer flasks containing the same sterile growth medium (100 mL per flask, prepared under the same conditions) and the flasks were shaken on a rotation shaker (rpm 165) at 27° C. for 72 h.

[0259] Biotransformation

[0260] The preculture (50 mL per flask) of the 500-mL Erlenmeyer flasks was added to a 1 L Biostat Q fermenter containing sterile growth medium (1 L) consisting of D-(+)-glucose monohydrate (10 g L.sup.−1), yeast extract (1 g L.sup.−1), beef extract (1 g L.sup.−1) and tryptose (2 g L.sup.−1) which had been adjusted to pH 7.2 with sodium hydroxide solution. Silicon oil (0.05 mL) and Pluronic® PE 8100 (0.05 mL) sterilized at 121° C. for 30 minutes were added. Compound (III), i.e. 3-(5-methyl-1,3-thiazol-2-yl)-5-[(3R)-tetrahydrofuran-3-yloxy]-N-{(1R)-1-[2-(trifluoromethyl)pyrimidin-5-yl]ethyl}benzamide, (25.0 mg, 0.052 mmol) dissolved in DMF (2 mL) was added and the culture was stirred at 220 rpm at 27° C. with an aeration rate of 2.0 L/min. The culture was stirred at an oxygen partial pressure of 15% regulated by the stirring rate of up to 800 rpm. After 72 h the culture was harvested.

[0261] The preculture (50 mL each) of the 500-mL Erlenmeyer flasks was added to two 1 L Biostat Q fermenters each containing sterile growth medium (1 L per fermenter) consisting of D-(+)-glucose monohydrate (10 g L.sup.−1), yeast extract (1 g L.sup.−1), beef extract (1 g L.sup.−1) and tryptose (2 g L.sup.−1) which had been adjusted to pH 7.2 with sodium hydroxide solution. Silicon oil (0.05 mL) and Pluronic® PE 8100 (0.05 mL) sterilized at 121° C. for 30 minutes were added. After 5 h compound (III), i.e. 3-(5-methyl-1,3-thiazol-2-yl)-5-[(3R)-tetrahydrofuran-3-yloxy]-N-{(1R)-1-[2-(trifluoromethyl)pyrimidin-5-yl]ethyl}benz-amide, (25.0 mg, 0.052 mmol) dissolved in DMF (2 mL) was added and the culture was stirred at 220 rpm at 27° C. with an aeration rate of 2.0 L/min. The culture was stirred at an oxygen partial pressure of 15% regulated by the stirring rate of up to 800 rpm. After 59 or 67 h the culture was harvested.

[0262] The three culture broths of the biotransformations were combined and extracted with 4-methyl-2-pentanone. The organic layer was concentrated to give an oil (0.71 g) which was stirred at 50° C. in methanol (10 mL). The resulting solid was filtered off and the filtrate was concentrated to give an oil (0.30 g).

[0263] Another preculture was generated as described before.

[0264] The preculture (50 mL or 100 mL) of the 500-mL Erlenmeyer flasks was added to two 1 L Biostat Q fermenters each containing sterile growth medium (1 L per fermenter) consisting of D-(+)-glucose monohydrate (10 g L.sup.−1), yeast extract (1 g L.sup.−1), beef extract (1 g L.sup.−1) and tryptose (2 g L.sup.−1) which had been adjusted to pH 7.2 with sodium hydroxide solution. Silicon oil (0.05 mL) and Pluronic® PE 8100 (0.05 mL) sterilized at 121° C. for 30 minutes were added. After 5 h compound (III), i.e. 3-(5-methyl-1,3-thiazol-2-yl)-5-[(3R)-tetrahydrofuran-3-yloxy]-N-{(1R)-1-[2-(trifluoromethyl)pyrimidin-5-yl]ethyl}benz-amide (25.0 mg, 0.052 mmol) dissolved in DMF (2 mL) was added and the culture was stirred at 220 rpm at 27° C. with an aeration rate of 2.0 L/min. The culture was stirred at an oxygen partial pressure of 15% regulated by the stirring rate of up to 800 rpm. After 47 h the culture was harvested.

[0265] The preculture (100 mL) of the 500-mL Erlenmeyer flasks was added to a 1 L Biostat Q fermenter containing sterile growth medium (1 L) consisting of D-(+)-glucose monohydrate (10 g L.sup.−1), yeast extract (1 g L.sup.−1), beef extract (1 g L.sup.−1) and tryptose (2 g L.sup.−1) which had been adjusted to pH 7.2 with sodium hydroxide solution. Silicon oil (0.05 mL) and Pluronic® PE 8100 (0.05 mL) sterilized at 121° C. for 30 minutes were added. Compound (III), i.e. 3-(5-methyl-1,3-thiazol-2-yl)-5-[(3R)-tetrahydrofuran-3-yloxy]-N-{(1R)-1-[2-(trifluoromethyl)pyrimidin-5-yl]ethyl}benzamide (25.0 mg, 0.052 mmol) dissolved in DMF (2 mL) was added and the culture was stirred at 220 rpm at 27° C. with an aeration rate of 2.0 L/min. The culture was stirred at an oxygen partial pressure of 15% regulated by the stirring rate of up to 800 rpm. After 12 h an aqueous glucose solution (20%, 1 g/h) was added. After further 10 h an aqueous glucose solution (20%, 2 g/h) was added. After 52 h the culture was harvested.

[0266] The preculture (100 mL) of the 500-mL Erlenmeyer flasks was added to a 1L Biostat Q fermenter containing sterile growth medium (1 L) consisting of D-(+)-glucose monohydrate (10 g L.sup.−1), yeast extract (1 g L.sup.−1), beef extract (1 g L.sup.−1) and tryptose (2 g L.sup.−1) which had been adjusted to pH 7.2 with sodium hydroxide solution. Silicon oil (0.05 mL) and Pluronic® PE 8100 (0.05 mL) sterilized at 121° C. for 30 minutes were added. After 5 h compound (III), i.e. 3-(5-methyl-1,3-thiazol-2-yl)-5-[(3R)-tetrahydrofuran-3-yloxy]-N-{(1R)-1-[2-(trifluoromethyppyrimidin-5-yl]ethyl}benzamide, (25.0 mg, 0.052 mmol) dissolved in DMF (2 mL) was added and the culture was stirred at 220 rpm at 27° C. with an aeration rate of 2.0 L/min. The culture was stirred at an oxygen partial pressure of 15% regulated by the stirring rate of up to 800 rpm. After 16 h an aqueous glucose solution (20%, 2 g/h) was added. After 47 h the culture was harvested.

[0267] The four culture broths of the biotransformations were combined and extracted with 4-methyl-2-pentanone. The organic layer was concentrated to give an oil (0.91 g) which was stirred at 50° C. in methanol (20 mL). The resulting solid was filtered off and the filtrate was concentrated to give an oil (0.75 g).

[0268] The two crude products were combined and further purified by flash chromatography using silica gel (dichloromethane/methanol gradient) and by preparative HPLC to give the title compound (21.0 mg, 90% purity, 10% yield).

[0269] Preparative Chiral HPLC Method:

[0270] Instrument: Waters Autopurificationsystem; Column: Waters XBrigde C18 5μ 100×30 mm; Eluent A: water+0.2 vol-% aqueous ammonia (32%), Eluent B: acetonitrile; gradient: 0.00-0.50 min 30% B (25-70 mL/min), 0.51-5.50 min 30-40% B (70 mL/min), DAD scan: 210-400 nm.

[0271] Analytical Chiral HPLC Method:

[0272] Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 μm, 50×2.1 mm; eluent A: water+0.1 vol-% formic acid (99%), eluent B: acetonitrile; gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; flow 0.8 mL/min; temperature: 60° C.; DAD scan: 210-400 nm.

[0273] LC-MS: R.sub.t=1.00 min; MS (ESIpos): m/z=497 [M+H].sup.+

[0274] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=1.61 (d, 3H), 1.70-1.91 (m, 2H), 3.43-3.55 (m, 2H), 3.56-3.61 (m, 2H), 4.37-5.15 (m, 2H), 4.55-4.65 (m, 1H), 5.30 (br d, 1H), 7.55 (dd, 1H), 7.62-7.66 (m, 2H), 7.90 (t, 1H), 9.09-9.14 (m, 2H), 9.14-9.22 (m, 1H).

[0275] Screening various wild-type strains the following strains showed a formation of the title compound:

[0276] DSMZ: Deutsche Sammlung von Mikroorganismen and Zellkulturen

[0277] ATCC: American Type Culture Collection

[0278] NRRL: ARS Culture Collection

[0279] IFO=NBRC: Biological Resource Center, National Institute of technology and Evaluation

[0280] CBS: Centraalbureau voor Schimmelculture, Netherlands

TABLE-US-00002 Strain Origin Streptomyces griseus ATCC 10137 Streptomyces albus ATCC 3004 Streptomyces griseocarneus ATCC 12628 Streptomyces viridis ATCC 15732 Streptomyces fulvissimus NRRL B-1453 Streptomyces halstedii ATCC 13449 Streptomyces vinaceus ATCC 11861 Streptomyces hydrogenans ATCC 19631 Streptomyces fradiae IFO 3360 Streptomyces roseochromogenus IFO 3363 Streptomyces roseochromogenus ATCC 13400 Streptomyces roseochromogenus CBS 41563 Streptomyces roseochromogenus NRRL B-1233 Streptomyces phaeochromogenes ATCC 3338 Streptomyces griseus ATCC 13273 Streptomyces toyocaensis DSMZ 40030 Streptomyces roseus DSMZ 40076 Streptomyces sulphureus DSMZ 40104 Streptomyces capreolus DSMZ 40225 Streptomyces catenulae DSMZ 40258 Streptomyces cavourensis DSMZ 40300 Streptomyces polychromogenus DSMZ 40316 Streptomyces flocculus DSMZ 40327 Streptomyces varsoviensis DSMZ 40346 Streptomyces albulus DSMZ 40492 Streptomyces alboflavus DSMZ 40761 Streptomyces griseus subsp. griseus DSMZ 40695 Streptomyces griseus subsp. griseus ATCC 12648 Streptomyces griseus subsp. griseus ATCC 27001 Streptomyces griseus subsp. griseus ATCC 31591 Pseudonocardia autotrophica DSMZ 43085 Lechevalleria aerocolonigenes ATCC 39243 Streptomyces roseochromogenus NBRC 3411 Streptomyces tubercidicus DSMZ 41958 Streptomyces tubercidicus DSMZ 41959 Streptomyces roseochromogenus ATCC 13400 Streptomyces platensis ATCC 13865 Streptomyces griseus IFO 3102

Example 3

(3R)-4-hydroxy-3-[3-(5-methyl-1,3-thiazol-2-yl)-5-({(1R)-1-[2-(trifluoromethyl)-pyrimidin-5-yl]ethyl}carbamoyl)phenoxy]butanoic acid

[0281] ##STR00007##

[0282] Preculture Generation

[0283] A DMSO cryo culture (0.2 mL) of Streptomyces albulus (DSMZ 40492) was added to a 100-mL Erlenmeyer flask containing sterile growth medium (20 mL) consisting of D-(+)-glucose monohydrate (10 g/L), yeast extract (1 g/L), beef extract (1 g/L) and tryptose (2 g/L) which had been adjusted to pH 7.2 with sodium hydroxide solution (16% in water) and had been sterilized at 121° C. for 20 minutes. After inoculation, the mixture was shaken on a rotation shaker (165 rpm) at 27° C. for 48 h. This preculture (8 mL per flask) was added to two 2000-mL Erlenmeyer flasks containing the same sterile growth medium (1000 mL per flask, prepared under the same conditions) and the flasks were shaken on a rotation shaker (rpm 165) at 27° C. for 48 h.

[0284] Biotransformation

[0285] The preculture (1000 mL per flask) of the 2000-mL Erlenmeyer flasks was added to a 10 L fermenter containing sterile growth medium (8.3 L) consisting of D-(+)-glucose monohydrate (4 g/L), yeast extract (4 g/L), malt extract (10 g/L) in demineralized water which had been adjusted to pH 7.2 with sodium hydroxide solution (16% in water). Silicon oil (0.5 mL) and Pluronic® PE 8100 (0.5 mL) sterilized at 121° C. for 40 minutes were added. Compound (III), i.e. 3-(5-methyl-1,3-thiazol-2-yl)-5-[(3R)-tetrahydrofuran-3-yloxy]-N-{(1R)-1-[2-(trifluoromethyl)pyrimidin-5-yl]ethyl}benzamide, (250 mg, 522 μmol) dissolved in DMF (10 mL) was added after 8 hours and the culture was stirred at 300 rpm at 27° C. with an aeration rate of 3.0 L/min. The culture was stirred at an oxygen partial pressure of 15% regulated by the stirring rate of up to 800 rpm. After 91 hours the culture was harvested.

[0286] The culture broth was extracted with 4-methyl-2-pentanone for 17 hours and concentrated to give an oil (4.20 g) which was stirred at 50° C. in methanol. The resulting solid was filtered off and the filtrate was concentrated to give an oil (3.64 g). The culture broth was extracted again with 4-methyl-2-pentanone for 20 hours and concentrated to give an oil (1.47 g) which was stirred at 50° C. in methanol. The resulting solid was filtered off and the filtrate was concentrated to give an oil (0.85 g). The combined crude products were purified by flash chromatography using silica gel (dichloromethane/methanol+0.1% ammonia gradient) and by preparative HPLC to give two batches of the title compound (20.0 mg and 17.6 mg).

[0287] Analytical Chiral HPLC Method:

[0288] Instrument: Waters Acquity UPLCMS SingleQuad; Column: Acquity UPLC BEH C18 1.7 μm, 50×2.1 mm; Eluent A: Water+0.2 Vol-% aqueous ammonia (32%), Eluent B: Acetonitrile; Gradient: 0-1.6 min 1-99% B, 1.6-2.0 min 99% B; Flow 0.8 mL/min; Temperature: 60° C.; DAD scan: 210-400 nm.

[0289] Preparative HPLC Method for Batch 1:

[0290] Instrument: Waters Autopurification system; Column: Waters XBrigde C18 5μ 100×30 mm; Eluent A: Water+0.2 Vol-% aqueous ammonia (32%), Eluent B: Acetonitrile; Gradient: 0.00-0.50 min 17% B (25-70 mL/min), 0.51-10.00 min 17-37% B (70 mL/min), DAD scan: 210-400 nm.

[0291] Preparative HPLC Method for Batch 2:

[0292] Instrument: Waters Autopurification system; Column: Waters XBrigde C18 5μ 100×30 mm; Eluent A: Water+0.2 Vol-% aqueous ammonia (32%), Eluent B: Acetonitrile; Gradient: 0.00-0.50 min 7% B (25-70 mL/min), 0.51-10.00 min 7-27% B (70 mL/min), DAD scan: 210-400 nm.

[0293] Additional Preparative HPLC Method for Both Batches:

[0294] Instrument: Waters Autopurification system; Column: XBrigde C18 5μ, 100×30 mm; eluent A: water+0.1 vol % formic acid; eluent B: acetonitrile; gradient: 0.0-0.5 min 25% B (35-70 mL/min), 0.5-5.5 min 25-70% B; flow: 70 mL/min; temperature: 25° C.; DAD scan: 210-400 nm.

[0295] LC-MS: R.sub.t=0.98 min; MS (ESIpos): m/z=511 [M+H].sup.+

[0296] .sup.1H-NMR (400 MHz, DMSO-d.sub.6): δ [ppm]=1.60 (d, 3H), 2.53-2.62 (m, 1H), 2.67-2.75 (m, 1H), 3.54-3.64 (m, 2H), 4.77-4.84 (m, 1H), 5.29 (quin, 1H), 7.55 (dd, 1H), 7.60-7.63 (m, 1H), 7.64 (d, 1H), 7.91 (t, 1H), 9.11 (s, 2H), 9.18 (d, 1H), 12.3 (s, 1H).

[0297] .sup.1H-NMR (400 MHz, DMSO-d6) δ [ppm]: 1.594 (6.62), 1.612 (6.62), 2.518 (4.49), 2.522 (2.74), 2.539 (16.00), 2.563 (0.93), 2.583 (1.50), 2.604 (1.45), 2.678 (0.52), 2.684 (1.33), 2.695 (1.52), 2.724 (0.93), 2.736 (0.82), 3.547 (0.41), 3.559 (0.48), 3.576 (1.87), 3.588 (3.23), 3.600 (1.97), 3.616 (0.44), 3.629 (0.41), 4.791 (0.78), 4.803 (0.96), 4.811 (0.89), 4.823 (0.76), 5.276 (0.95), 5.294 (1.45), 5.312 (0.95), 7.542 (1.84), 7.546 (2.37), 7.548 (2.56), 7.552 (2.19), 7.612 (2.26), 7.616 (2.76), 7.622 (1.97), 7.643 (4.86), 7.646 (4.88), 7.914 (2.52), 7.918 (4.39), 7.921 (2.35), 9.114 (12.72), 9.173 (1.80), 9.191 (1.76).

[0298] Experimental Section—Biological Assays

[0299] Examples were tested in selected biological assays one or more times. When tested more than once, data are reported as either average values or as median values, wherein [0300] the average value, also referred to as the arithmetic mean value, represents the sum of the values obtained divided by the number of times tested, and [0301] the median value represents the middle number of the group of values when ranked in ascending or descending order. If the number of values in the data set is odd, the median is the middle value. If the number of values in the data set is even, the median is the arithmetic mean of the two middle values.

[0302] Examples were synthesized one or more times. When synthesized more than once, data from biological assays represent average values or median values calculated utilizing data sets obtained from testing of one or more synthetic batch.

[0303] The in vitro activity of the compounds of the present invention can be demonstrated in the following assay:

[0304] Cellular In Vitro Assay for Determination of P2X3 Receptor Activity

[0305] The determination of antagonistic activity at the P2X3 receptor of the compounds of the invention was performed by use of a recombinant cell line. These cell line derives originally from the Chinese hamster ovary (CHO) cell line (Tjio J. H.; Puck T. T., 1958, J. Exp. Med. 108: 259-271). The cell line is stably transfected with the human P2X3 receptor and a calcium-sensitive photoprotein, mitochondrial photina, which, after reconstitution with the cofactor coelenterazine, emits light in dependence of calcium binding [Bovolenta S, Foti M, Lohmer S, Corazza S., J Biomol Screen. 2007 August; 12(5):694-704]. The strength of the photina luminescence signal corresponds to the level of receptor activation upon agonist binding. An inhibitor would decrease the signal depending on its potency and concentration. Bioluminescence was detected using a suitable luminometer [Milligan G, Marshall F, Rees S, Trends in Pharmacological Sciences 17, 235-237 (1996)].

[0306] Test Procedure:

[0307] On the day before the assay, the cells are plated out in culture medium (DMEM/F12 (PAN, P04-41451), 10% FCS) in 384-well microtiter plates and kept in a cell incubator (96% humidity, 5% v/v CO.sub.2, 30° C.). On the day of the assay medium is replaced by 2 mM Ca-tyrode buffer (20 mM HEPES, 130 mM NaCl, 5 mM KCl, 5 mM NaHCO.sub.3, 2 mM MgCl.sub.2, pH 7.4) containing 5 μg/ml coelenterazine. Plates are incubated for 3 hours at 37° C. (96% humidity, 5% v/v CO2). After incubation the test substances in various concentrations are placed for 10 minutes in the wells of the microtiter plate before the agonist alpha-beta-methylene-ATP at EC.sub.50 concentration is added. The resulting light signal is measured immediately in the luminometer.

[0308] The examples were tested in quadruples per plate and mean values were used to determine IC.sub.50 values at the human P2X3 receptors and percentage of maximal inhibition.

Table 2: IC.SUB.50 .Values of Examples in Cellular Functional Measurement to Assess Antagonist Activity at Human P2X3 Receptors

[0309]

TABLE-US-00003 Target IC.sub.50 Example [nM] example 11 of 6.0 WO2016/091776 A1 1 20 2 5.1 3 12

[0310] Solubilty

[0311] Examples were tested for its solubility in aqueous media (pH 6.5). Examples were synthesized one or more times in accordance with the above-described protocols. When synthesized more than once, data from solubility assays represent average values or median values calculated utilizing data sets obtained from testing of one or more synthetic batch.

[0312] The high throughput screening method to determine aqueous drug solubility is based on Thomas Onofrey and Greg Kazan, Performance and correlation of a 96-well high throughput screening method to determine aqueous drug solubility, Millipore Corporation Application Note, 2003; Lit. No. AN1731EN00.

[0313] The assay was run in a 96-well plate format. Each well was filled with an individual compound.

[0314] All pipetting steps were performed using a robot platform.

[0315] 100 μl of a 10 mmolar solution of drug in DMSO were concentrated by vacuum centrifugation and resolved in 10 μl DMSO. 990 μl phosphate buffer pH 6.5 were added. The content of DMSO amounts to 1%. The multititer plate was put on a shaker and mixed for 24 hrs at room temperature. 150 μl of the suspension were transferred to a filtration plate. After filtration using a vacuum manifold, the filtrate was diluted with a 1:1 mixture of water and DMSO to 1:400 and 1:8000. A second microtiter plate with 20 μl of a 10 mM solution of drug in DMSO served for calibration. Two concentrations (1.25 nM and 2.5 nM) were prepared by dilution in DMSO/water 1:1 and used for calibration. Filtrate and calibration plates were quantified by HPLC-MS/MS.

[0316] Chromatographic conditions were as follows:

[0317] HPLC column: Ascentis Express C18 4.6×30 mm 2.7 μm

[0318] Injection volume: 1 μl

[0319] Flow: 1.5 ml/min

[0320] Mobile phase: A: Water/0.05% HCOOH [0321] B: Acetonitrile/0.05% HCOOH [0322] 0 min.fwdarw.95% A 5% B [0323] 0.75 min.fwdarw.5% A 95% B [0324] 2.75 min.fwdarw.5% A 95% B [0325] 2.76 min.fwdarw.95% A 5% B [0326] 3 min.fwdarw.95% A 5% B

[0327] The areas of sample- and calibration injections were determined by using mass spectrometry software (AB SCIEX: Discovery Quant 2.1.3. and Analyst 1.6.1). The solubility value was obtained by interpolation from the calibration curve.

[0328] The significantly improved solubility of examples 1, 2 and 3 compared to example 11 of WO2016/091776 A1 is shown in table 3.

[0329] Table 3: Aqueous Solubility of Example Compounds

TABLE-US-00004 Aqueous Solubility pH 6.5 Example [mg/L] example 11 of 2.9 WO2016/091776 A1 1 46.0 2 96.5 3 1190

[0330] Metabolic Stability

[0331] Examples were tested for its metabolic stability in human liver microsomes. Examples were measured one or more times. When measured more than once, data from metabolic stability assays represent average values or median values calculated utilizing data sets obtained from testing of one or more synthetic batch.

[0332] In vitro metabolic stability was determined by incubating a solution of test compounds in dimethylsulfoxide (DMSO) and acetonitrile at 1 μM in a suspension of liver microsomes in 100 mM phosphate buffer, pH7.4 (NaH.sub.2PO.sub.4×H.sub.2O+Na.sub.2HPO.sub.4×2H.sub.2O) and at a protein concentration of 0.5 mg/mL at 37° C. The microsomes were activated by adding a co-factor mix containing 8 mM Glucose-6-Phosphat, 4 mM MgCl2; 0.5 mM NADP and 1 IU/ml G-6-P-Dehydrogenase in phosphate buffer, pH 7.4. The metabolic assay was started shortly afterwards by adding the test compound to the incubation at a final volume of 1 mL. Organic solvent was limited to ≤0.01% dimethylsulfoxide (DMSO) and ≤1% acetonitril. During incubation, the microsomal suspensions were continuously shaken at 580 rpm and aliquots were taken at 2, 8, 16, 30, 45 and 60 min, to which equal volumes of cold methanol were immediately added. Samples were frozen at −20° C. over night, subsequently centrifuged for 15 minutes at 3000 rpm and the supernatant was analyzed with an Agilent 1200 HPLC-system with LCMS/MS detection.

[0333] The half-life of a test compound was determined from the concentration-time plot. From the half-life the intrinsic clearances were calculated. Together with the additional parameters liver blood flow, specific liver weight and microsomal protein content the hepatic in vivo blood clearance (CL) and the maximal oral bioavailability (F.sub.max) were calculated for the different species. The hepatic in vivo blood clearance (CL.sub.blood) and the maximal oral bioavailability (F.sub.max) was calculated using the following formulae: CL′.sub.intrinsic [ml/(min*kg)]=k.sub.el [1/min]/((mg protein/volume of incubation [ml])*fu,.sub.inc)*(mg protein/liver weight [g])*(specific liver weight [g liver/kg body weight]); CL.sub.blood well-stirred [L/(h*kg)]=(QH [L/(h*kg)]*fu,.sub.blood*CL′.sub.intrinsic [L/(h*kg)])/(QH [L/(h*kg)]+fu,.sub.blood*CL′.sub.intrinsic [L/(h*kg)]); F.sub.max=1−CL.sub.blood/QH and using the following parameter values: QH (Liver blood flow): 1.32 L/h/kg (human), 2.1 L/h/kg (dog), 4.2 L/h/kg (rat); specific liver weight: 21 g/kg (human), 39 g/kg (dog), 32 g/kg (rat); microsomal protein content: 40 mg/g. fu,.sub.inc and fu,.sub.blood is taken as 1 with fu,.sub.inc being the unbound fraction (ie the non-protein bound fraction) in the incubation experiment, and fu,.sub.blood being the unbound fraction in blood.

[0334] The significantly improved metabolic stability of example 2 and 3 of the present invention compared to example 11 of WO2016/091776 A1 is shown in table 4.

[0335] Table 4: Metabolic Stability of Example Compounds

TABLE-US-00005 CL.sub.blood F.sub.max Example [L/h/kg] [%] example 11 of 0.52/0.27 61/79 WO2016/091776 A1 1 0.87 34 2 0.001 100 3 0.001 100