Use of sGC stimulators, sGC activators, alone and combinations with PDE5 inhibitors for the treatment of systemic sclerosis (SSc)

Abstract

The use of sGC stimulators, sGC activators alone, or in combination with PDE5 inhibitors for the prevention and treatment of fibrotic diseases, such as systemic sclerosis, scleroderma, and the concomitant fibrosis of internal organs.

Claims

1. A method for reducing collagen production in dermal fibroblasts, reducing myofibroblast differentiation and/or for triggering redifferentiation of myofibroblast in a patient suffering from a fibrotic disease selected from the group consisting of systemic sclerosis (SSc), diffuse systemic sclerosis (dSSc), limited systemic sclerosis (lSSc), overlap type of systemic sclerosis, undifferentiated type of systemic sclerosis, systemic sclerosis sine scleroderma, skin fibrosis, nephrogenic fibrosing dermopathy (NFD), nephrogenic systemic fibrosis (NSF), systemic sclerosis (SSc) concomitant fibrosis of internal organs, and keloid formation, comprising administering an effective amount of a compound according to one of formulae (3), (6) or (27) ##STR00023## ##STR00024## ##STR00025## to the patient in need thereof; wherein reduction of collagen production in dermal fibroblasts, reduction of myofibroblast differentiation and/or triggering redifferentiation of myofibroblast occurs without vasodilation.

2. The method of claim 1, comprising administering the compound of one of formulae (3), (6) or (27) in combination with at least one PDE5 inhibitor selected from the group consisting of: tadalafil ((6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylene-dioxyphenyl) pyrazino(1,2:1,6) pyrido(3,4-b)indole-1,4-dione), vardenafil (2-(2-ethoxy-5-(4-ethylpiperazin-1-yl-1-sulfonyl)phenyl)-5-methyl-7-propyl-3H-imidazo (5,1-f) (1,2,4)triazin-4-one), sildenafil (3-[2-ethoxy-5-(4-methylpiperazin-1-yl)sulfonyl-phenyl]-7-methyl-9-propyl-2,4,7,8-tetrazabicyclo [4.3.0]nona-3,8,10-trien-5-one), udenafil (5-[2-propyloxy-5-(1-methyl-2-pyrrolidinylethylamidosulfonyl)phenyl]-methyl-3-propyl-1,6-dihydro-7H-pyrazolo(4,3-d)pyrimidine-7-one), dasantafil (7-(3-bromo-4-methoxybenzyl)-1-ethyl-8-[[(1,2)-2-hydroxycyclopentyl]amino]-3-(2-hydroxyethyl)-3,7-dihydro-1-purine-2,6-dione), avanafil (4-{[(3-chloro-4-methoxyphenyl)methyl]amino}-2-[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]-N-(pyrimidin-2-ylmethyl)pyrimidine-5-carboxamide), mirodenafil, lodenafil, LAS 34179 (triazolo[1,2-]xanthine,6-methyl-4-propyl-2-[2-propoxy-5-(4-methylpiperazino)sulfonyl]phenyl) or a salt, hydrate or hydrate of a salt thereof.

3. The method of claim 2, wherein the PDE5 inhibitor is sildenafil or vardenafil.

4. The method of claim 1, wherein the fibrotic disease is systemic sclerosis (SSc) concomitant fibrosis of internal organs.

5. The method of claim 1, wherein the fibrotic disease is diffuse systemic sclerosis (dSSc).

6. A method for reducing collagen production in dermal fibroblasts, reducing myofibroblast differentiation and for triggering redifferentiation of myofibroblast in a patient suffering from a fibrotic disease selected from the group consisting of systemic sclerosis (SSc), diffuse systemic sclerosis (dSSc), limited systemic sclerosis (lSSc), overlap type of systemic sclerosis, undifferentiated type of systemic sclerosis, systemic sclerosis sine scleroderma, skin fibrosis, nephrogenic fibrosing dermopathy (NFD), nephrogenic systemic fibrosis (NSF), keloid formation, and systemic sclerosis (SSc) concomitant fibrosis of internal organs, comprising administering an effective amount of a compound having the formula (3) ##STR00026## to the patient in need thereof; wherein reduction of collagen production in dermal fibroblasts, reduction of myofibroblast differentiation and/or triggering redifferentiation of myofibroblast occurs without vasodilation.

7. The method of claim 6, wherein the fibrotic disease is systemic sclerosis (SSc) concomitant fibrosis of internal organs.

Description

DRAWINGS

(1) FIG. 1: Effects of the compound of formula 27 on mean arterial blood pressure (left) and heart rate (right).

(2) FIG. 2A and FIG. 2B: Effects of the compound of formula 3 on systolic blood pressure (2A) and heart rate (2B)

(3) FIG. 3A and FIG. 3B: Effects of the compound of formula 4 on systolic blood pressure (3A) and heart rate (3B)

(4) FIG. 4A and FIG. 4B: Effects of the compound of formula 7 on systolic blood pressure (4A) and heart rate (4B)

EXPERIMENTAL PART

A. Examples

Abbreviations and Acronyms

(5) aq. aqueous solution calc. calculated DCI direct chemical ionization (in MS) DMF dimethylformamide DMSO dimethyl sulfoxide of th. of theory (in yield) eq. equivalent(s) ESI electrospray ionization (in MS) Et ethyl fnd. found h hour(s) HPLC high-pressure, high-performance liquid chromatography HRMS high-resolution mass spectrometry conc. concentrated LC/MS liquid chromatography-coupled mass spectrometry LiHMDS lithium hexamethyldisilazide Me methyl min minute(s) MS mass spectrometry NMR nuclear magnetic resonance spectrometry Pd.sub.2dba.sub.3 tris(dibenzylideneacetone)dipalladium Ph phenyl RT room temperature R.sub.t retention time (in HPLC) THF tetrahydrofuran UV ultraviolet spectrometry v/v volume to volume ratio (of a solution) XPHOS dicyclohexyl(2,4,6-triisopropylbiphenyl-2-yl)phosphine

(6) LC/MS-Methods:

(7) Method 1: MS instrument: Waters ZQ; HPLC instrument: Agilent 1100 Series; UV DAD; Column: Thermo Hypersil GOLD 3 20 mm4 mm; Eluent A: 1 l water+0.5 ml 50% formic acid, Eluent B: 1 l acetonitrile+0.5 ml of 50% formic acid; Gradient: 0.0 min 100% A.fwdarw.3.0 min 10% A.fwdarw.4.0 min 10% A.fwdarw.4.1 min 100% A (flow rate 2.5 ml/min); Oven: 55 C.; Flow rate: 2 ml/min; UV detection: 210 nm.

(8) Method 2: Instrument: Waters ACQUITY SQD UPLC System; Column: Waters Acquity UPLC HSS T3 1.8 501 mm; Eluent A: 1 l water+0.25 ml 99% formic acid, Eluent B: 1 l acetonitrile+0.25 ml 99% formic acid; Gradient: 0.0 min 90% A.fwdarw.1.2 min 5% A.fwdarw.2.0 min 5% A; Oven: 50 C.; Flow rate: 0.40 ml/min; UV detection: 210-400 nm.

Starting Compounds and Intermediates

Example 1A

2,6-Dichloro-5-fluoronicotinamide

(9) ##STR00009##

(10) A suspension of 25 g (130.90 mmol) of 2,6-dichloro-5-fluoro-3-cyanopyridine in conc. sulphuric acid (125 ml) was stirred at 60-65 C. for 1 h. After cooling to RT, the contents of the flask were poured into ice-water and extracted three times with ethyl acetate (100 ml each time). The combined organic phases were washed with water (100 ml) and then with saturated aqueous sodium hydrogen carbonate solution (100 ml), dried and concentrated on a rotary evaporator. The material obtained was dried under a high vacuum.

(11) Yield: 24.5 g (90% of theory)

(12) .sup.1H NMR (400 MHz, DMSO-d.sub.6): =7.95 (br s, 1H), 8.11 (br s, 1H), 8.24 (d, 1H).

Example 2A

2-Chloro-5-fluoronicotinamide

(13) ##STR00010##

(14) A suspension of 21.9 g (335.35 mmol) of zinc in methanol (207 ml) was admixed at RT with 44 g (210.58 mmol) of 2,6-dichloro-5-fluoronicotinamide. Then acetic acid (18.5 ml) was added and the mixture was heated with stirring at reflux for 24 h. Thereafter the contents of the flask were decanted from the zinc, and ethyl acetate (414 ml) and saturated aqueous sodium hydrogen carbonate solution (414 ml) were added, followed by intense extractive stirring. Subsequently the reaction mixture was filtered with suction over kieselguhr and the filter product was washed three times with ethyl acetate (517 ml each time). The organic phase was separated off and the aqueous phase was washed with ethyl acetate (258 ml). The combined organic phases were washed once with saturated aqueous sodium hydrogen carbonate solution (414 ml), dried and concentrated under reduced pressure. The resulting crystals were admixed with dichloromethane (388 ml) and extractively stirred for 20 min. Filtration with suction was carried out again, and the filter product was washed with diethyl ether and sucked dry.

(15) Yield: 20.2 g (53% of theory)

(16) .sup.1H NMR (400 MHz, DMSO-d.sub.6): =7.87 (br s, 1H), 7.99 (dd, 1H), 8.10 (br s, 1H), 8.52 (d, 1H).

Example 3A

2-Chloro-5-fluornicotinonitrile

(17) ##STR00011##

(18) A suspension of 46.2 g (264.66 mmol) of 2-chloro-5-fluoronicotinamide in dichloromethane (783 ml) was admixed with 81.2 ml (582.25 mmol) of triethylamine and cooled to 0 C. Then, with stirring, 41.12 ml (291.13 mmol) of trifluoroacetic anhydride were added slowly dropwise and the mixture was stirred at 0 C. for 1.5 h. The reaction solution was subsequently washed twice with saturated aqueous sodium hydrogen carbon solution (391 ml each time), dried and concentrated under reduced pressure.

(19) Yield: 42.1 g (90% of theory).

(20) .sup.1H NMR (400 MHz, DMSO-d.sub.6): =8.66 (dd, 1H), 8.82 (d, 1H).

Example 4A

5-Fluoro-1H-pyrazolo[3,4-b]pyridin-3-amine

(21) ##STR00012##

(22) A suspension of 38.5 g (245.93 mmol) of 2-chloro-5-fluoronicotinonitrile was introduced in 1,2-ethanediol (380 ml) and subsequently admixed with hydrazine hydrate (119.6 ml, 2.459 mol). The mixture was heated at reflux with stirring for 4 h. On cooling, the product precipitated. The yellow crystals were admixed with water (380 ml) and subjected to extractive stirring at RT for 10 min. Then the suspension was filtered with suction over a frit, and the filter product was washed with water (200 ml) and with 10 C. cold THF (200 ml). The residue was dried under a high vacuum over phosphorus pentoxide.

(23) Yield: 22.8 g (61% of theory)

(24) .sup.1H NMR (400 MHz, DMSO-d.sub.6): =5.54 (s, 2H), 7.96 (dd, 1H), 8.38 (m, 1H), 12.07 (m, 1H).

Example 5A

5-Fluoro-3-iodo-1H-pyrazolo[3,4-b]pyridine

(25) ##STR00013##

(26) In THF (329 ml), 10 g (65.75 mmol) of 5-fluoro-1H-pyrazolo[3,4-b]pyridin-3-amine were introduced and cooled to 0 C. Then 16.65 ml (131.46 mmol) of boron trifluoride diethyl ether complex were slowly added. The reaction mixture was cooled further to 10 C. Then a solution of 10.01 g (85.45 mmol) of isopentyl nitrite in THF (24.39 ml) was added slowly, followed by stirring for a further 30 min. The mixture was diluted with cold diethyl ether (329 ml) and the resulting solid was isolated by filtration. The diazonium salt thus prepared was added in portions to a 0 C. cold solution of 12.81 g (85.45 mmol) of sodium iodide in acetone (329 ml), and the mixture was stirred at RT for 30 min. The reaction mixture was poured into ice-water (1.8 l) and extracted twice with ethyl acetate (487 ml each time). The collected organic phases were washed with saturated aqueous sodium chloride solution (244 ml), dried, filtered and concentrated. This gave 12.1 g (86% purity, 60% of th.) of the desired compound in the form of a brown solid. The crude product was reacted without further purification.

(27) LC-MS (Method 1): R.sub.t=1.68 min; MS (ESIpos): m/z=264 (M+H).sup.+

Example 6A

5-Fluoro-1-(2-fluorobenzyl)-3-iodo-1H-pyrazolo[3,4-b]pyridine

(28) ##STR00014##

(29) In DMF (2538 ml), 141 g (462.11 mmol) of the compound from Example 5A were introduced and then 96.09 g (508.32 mmol) of 2-fluorobenzyl bromide and 165.62 g (508.32 mmol) of cesium carbonate were added. The mixture was stirred at RT for two hours. The reaction mixture was then poured into saturated aqueous sodium chloride solution (13 670 ml) and extracted twice with ethyl acetate (5858 ml) The collected organic phases were washed with saturated aqueous sodium chloride solution (3905 ml), dried, filtered and concentrated. The residue was chromatographed on silica gel (eluent: petroleum ether/ethyl acetate 97:3) and the product fractions were concentrated. The resulting solid was dissolved in dichloromethane and washed once with saturated aqueous sodium thiosulphate solution (500 ml) and then with saturated aqueous sodium chloride solution (500 ml). The product was concentrated to dryness and the residue was suspended with diethyl ether, isolated by filtration with suction and dried under a high vacuum. This gave 106.6 g (62% of theory) of the desired compound.

(30) LC-MS (Method 1): R.sub.t=2.57 min

(31) MS (ESIpos): m/z=372 (M+H).sup.+

(32) .sup.1H NMR (400 MHz, DMSO-d.sub.6): =5.73 (s, 2H), 7.13-7.26 (m, 3H), 7.33-7.41 (m, 1H), 7.94 (dd, 1H), 8.69-8.73 (m, 1H).

Example 7A

2-[5-Fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-nitropyrimidine-4,6-diamine

(33) ##STR00015##

(34) In 1,4-dioxane (86 ml), 860 mg (2.32 mmol) of the compound from Example 6A were introduced under argon and the reaction mixture was flushed with argon for 10 min. Then 3.51 ml (6.95 mmol) of hexabutylditin and 483 mg (2.55 mmol) of 2-chloro-5-nitropyrimidine-4,6-diamine (prepared by the method of Helvetica Chimica Acta (1951), 34, 835-40) were added. Subsequently 860 mg (0.744 mmol) of tetrakis(triphenylphosphine)palladium(0) were added and the reaction mixture was heated at reflux overnight. It was then cooled to RT, admixed with water and extracted twice with ethyl acetate. The collected organic phases were dried over sodium sulphate, filtered and concentrated. The residue was subjected to extractive stirring in ethyl acetate, and the solid was isolated by filtration and dried under a high vacuum. This gave 355 mg (62% purity, 24% of th.) of the desired compound. The crude product was reacted without further purification.

(35) LC-MS (Method 2): R.sub.t=1.03 min

(36) MS (ESIpos): m/z=399 (M+H).sup.+

Example 8A

5-Fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-carbonitrile

(37) ##STR00016##

(38) A suspension of 16.03 g (43.19 mmol) of 5-fluoro-1-(2-fluorobenzyl)-3-iodo-1H-pyrazolo[3,4-b]pyridine (Example 6A) and 4.25 g (47.51 mmol) of copper cyanide were introduced in DMSO (120 ml) and stirred at 150 C. for 2 h. After cooling, the contents of the flask were cooled to about 40 C., poured into a solution of conc. aqueous ammonia (90 ml) and water (500 ml), admixed with ethyl acetate (200 ml) and subjected to brief extractive stirring. The aqueous phase was separated off and extracted twice more with ethyl acetate (200 ml each time). The combined organic phases were washed twice with 10% strength aqueous sodium chloride solution (100 ml each time), dried and concentrated under reduced pressure. The crude product was reacted without further purification.

(39) Yield: 11.1 g (91% of theory)

(40) .sup.1H NMR (400 MHz, DMSO-d.sub.6): =5.87 (s, 2H), 7.17-7.42 (m, 4H), 8.52 (dd, 1H), 8.87 (dd, 1H).

Example 9A

5-Fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-carboximidamide acetate

(41) ##STR00017##

(42) To 2.22 g (41.07 mmol) of sodium methoxide in methanol (270 ml) were added 11.1 g (41.07 mmol) of 5-fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-carbonitrile (Example 8A) and the mixture was stirred at RT for 2 h. Then 2.64 g (49.29 mmol) of ammonium chloride and acetic acid (9.17 ml) were added and the mixture was heated at reflux overnight. It was then concentrated to dryness and the residue was taken up in water (100 ml) and ethyl acetate (100 ml) and adjusted to a pH of 10 using 2N aqueous sodium hydroxide solution. It was stirred intensively at RT for about 1 h. The resulting suspension was filtered with suction and the filter product was washed with ethyl acetate (100 ml), with water (100 ml) und again with ethyl acetate (100 ml). The residue was dried under a high vacuum over phosphorus pentoxide.

(43) Yield: 9.6 g (78% of th.)

(44) MS (ESIpos): m/z=288 (M+H).sup.+

(45) .sup.1H NMR (400 MHz, DMSO-d.sub.6): =1.85 (s, 3H), 5.80 (s, 2H), 7.14-7.25 (m, 3H), 7.36 (m, 1H), 8.42 (dd, 1H), 8.72 (dd, 1H).

Example 10A

2-[5-Fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-[(E)-phenyldiazenyl]pyrimidine-4,6-diamine

(46) ##STR00018##

(47) Water (40 ml) and concentrated hydrochloric acid (7.07 ml) were admixed with stirring with 3.85 g (41.34 mmol) of aniline and this mixture was cooled to 0 C. Then a solution of 2.85 g (41.34 mmol) of sodium nitrite in water (21 ml) was added dropwise at between 0 C. and 5 C., followed by stirring at 0 C. for 15 min. Thereafter, at 0 C., a solution of 4.28 g (52.25 mmol) of sodium acetate in water (19 ml) was added rapidly dropwise, and then, with thorough stirring, a solution of 2.73 g (41.34 mmol) of malononitrile in ethanol (10 ml) was added dropwise. After 2 h at 0 C., the resulting precipitate was isolated by filtration with suction and washed three times with water (50 ml each time) and with petroleum ether (50 ml). The residue, still moist, was dissolved in DMF (46 ml) and added dropwise at precisely 85 C. to a solution of 9.5 g (33.07 mmol) of 5-fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-carboximidamide acetate (Example 9A) in DMF (46 ml) and triethylamine (5.76 ml). The mixture was then stirred at 100 C. for 4 h and left to cool to RT overnight. The mixture was poured into water (480 ml) and subjected to extractive stirring at RT for 1 h. After the precipitate had been isolated by filtration with suction, it was washed twice with water (100 ml each time) and twice with methanol (50 ml each time) and then dried under a high vacuum.

(48) Yield: 9.6 g (59% of theory)

(49) LC-MS (Method 2): R.sub.t=1.21 min

(50) MS (ESIpos): m/z=458 (M+H).sup.+

Example 11A

2-[5-Fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidine-4,5,6-triamine

(51) ##STR00019##

(52) Variant A: Preparation starting from Example 7A:

(53) In pyridine (30 ml), 378 mg (0.949 mmol) of the compound from Example 7A were introduced and then 143 mg (0.135 mmol) of palladium (10% on carbon) were added. The mixture was hydrogenated overnight at RT under standard hydrogen pressure. The suspension was then filtered through kieselguhr and the filtercake was washed with ethanol. The filtrate was concentrated and yielded 233 mg (81% purity, 51% of theory) of the desired compound, which was reacted without further purification.

(54) Variant B: Preparation starting from Example 10A:

(55) In DMF (800 ml), 39.23 g (85.75 mmol) of the compound from Example 10A were introduced and then 4 g of palladium (10% on carbon) were added. The mixture was hydrogenated with stirring overnight under standard hydrogen pressure. The batch was filtered over kieselguhr and the filter product was washed with a little DMF and then with a little methanol, and concentrated to dryness. The residue was admixed with ethyl acetate and stirred vigorously, and the precipitate was filtered off with suction, washed with ethyl acetate and diisopropyl ether and dried under a high vacuum over Sicapent.

(56) Yield: 31.7 g (100% of theory)

(57) LC-MS (Method 2): R.sub.t+=0.81 min

(58) MS (ESIpos): m/z=369 (M+H).sup.+

Working Examples

Example 1

Methyl {4,6-diamino-2-[5-fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-5-yl}carbamate

(59) ##STR00020##

(60) In pyridine (600 ml), 31.75 g (86.20 mmol) of the compound from Example 11A were introduced under argon and cooled to 0 C. Then a solution of 6.66 ml (86.20 mmol) of methyl chloroformate in dichloromethane (10 ml) was added dropwise and the mixture was stirred at 0 C. for 1 h. Thereafter the reaction mixture was brought to RT, concentrated under reduced pressure and co-distilled repeatedly with toluene. The residue was stirred with water/ethanol and then filtered off on a frit, after which it was washed with ethanol and ethyl acetate. Subsequently the residue was again stirred with diethyl ether, isolated by filtration with suction and then dried under a high vacuum.

(61) Yield: 24.24 g (65% of theory)

(62) LC-MS (Method 2): R.sub.t=0.79 min

(63) MS (ESIpos): m/z=427 (M+H)+

(64) .sup.1H NMR (400 MHz, DMSO-d.sub.6): =3.62 (br. s, 3H), 5.79 (s, 2H), 6.22 (br. s, 4H), 7.10-7.19 (m, 2H), 7.19-7.26 (m, 1H), 7.32-7.40 (m, 1H), 7.67 (br. s, 0.2H), 7.99 (br. s, 0.8H), 8.66 (m, 1H), 8.89 (d, 1H).

Example 2

Methyl {4,6-diamino-2-[5-fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-5-yl}methylcarbamate

(65) ##STR00021##

(66) A quantity of 200 mg (0.469 mmol) of methyl {4,6-diamino-2-[5-fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-5-yl}carbamate (Example 1) was introduced in THF (5 ml) at 0 C. Then 0.704 ml (0.704 mmol) of lithium hexamethyldisilazane solution (1M in THF) was added and the mixture was stirred at this temperature for 20 min. Subsequently 43.8 l (0.704 mmol) of iodomethane were added and the mixture was warmed to RT. After 1 h at this temperature, reaction was terminated with water (1 ml) and the reaction mixture was concentrated, the residue being separated by means of preparative RP-HPLC (water (+0.05% formic acid)-acetonitrile gradient).

(67) Yield: 90 mg (44% of theory)

(68) LC-MS (Method 2): R.sub.t=0.85 min

(69) MS (ESIpos): m/z=441 (M+H).sup.+

(70) .sup.1H NMR (400 MHz, DMSO-d.sub.6): =3.00 (s, 3H), 3.53 (s, 2.2H), 3.66 (s, 0.8H), 5.81 (s, 2H), 6.57 (br. s, 4H), 7.13 (m, 2H), 7.22 (m, 1H), 7.35 (m, 1H), 8.67 (m, 1H), 8.87 (dd, 1H).

Example 3

Methyl {4,6-diamino-2-[5-fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-5-yl}(2,2,2-trifluoroethyl)carbamate

(71) ##STR00022##

(72) A quantity of 3.470 g (8.138 mmol) of the compound from Example 1 was suspended in 35 ml of THF, admixed at 0 C. with 358 mg (8.952 mmol) of sodium hydride (60% suspension in mineral oil) and stirred at 0 C. for 90 min, in the course of which a solution was formed. A quantity of 2.519 g (8.952 mmol) of 2,2,2-trifluoroethyl trichloromethanesulphonate was added and the mixture was stirred at RT for 48 h. It was then stirred with water and concentrated on a rotary evaporator. The residue was taken up in ethyl acetate, and the organic phase was washed twice with water and dried over sodium sulphate. This gave 5.005 g of the target compound (79% of th., purity by HPLC 65%). A quantity of 250 mg of the residue was purified by means of preparative HPLC (Eluent: methanol/water, gradient 30:70.fwdarw.90:10).

(73) LC-MS (Method 2): R.sub.t=0.97 min; MS (EIpos): m/z=509 [M+H].sup.+.

(74) .sup.1H NMR (400 MHz, DMSO-d.sub.6): [ppm]=3.63 (s, 3H), 4.06-4.15 (m, 2H), 5.80 (s, 2H), 6.46 (s br, 4H) 7.11-7.15 (m, 2H), 7.20-7.25 (m, 1H), 7.33-7.38 (m, 1H), 8.66 (dd, 1H), 8.91 (dd, 1H).

Example A

Bleomycin-Induced Skin Fibrosis

(75) Local skin fibrosis was induced in 6-week-old, pathogen-free, female DBA/2 mice (Charles River, Sulzfeld, Germany) by repeated (every other day) subcutaneous injections of bleomycin (0.5 mg/ml in saline) in a defined area of the upper back. Control mice were injected in the same manner with saline only and served as reference. For all groups the injection volume was 100 l. Concomitant to bleomycin treatment, the mice were treated orally with test drug or vehicle. Mice were treated a) with vehicle b) with 1 mg/kg compound of formula 27, and c) with 3 mg/kg compound of formula 27, twice a day via gavage for 21 days. After this 3 weeks treatment period, the animals were sacrificed and skin samples were obtained for analysis.

(76) Histological Analysis

(77) The injected skin areas were fixed in 4% formalin and embedded in paraffin. Histological sections were stained with hematoxylin and eosin for the determination of dermal thickness. The dermal thickness was determined by measuring the largest distance between the epidermal-dermal junction and the dermal-subcutaneous fat junction. The measurements were performed by an examiner blinded to the treatment of the mice.

(78) Hydroxyproline Assay

(79) To analyze the collagen content in skin samples, hydroxyproline assay was performed. After digestion of punch biopsies ( 3 mm) in 6M HCl for three hours at 120 C., chloramine T (0.06 M) was added and samples were mixed and incubated for 20 min at room temperature. 3.15 M perchloric acid and 20% p-dimethylaminobenzaldehyde were added and samples were incubated for additional 20 min at 60 C. The absorbance was determined at 557 nm.

(80) Immunohistochemistry for -Smooth Muscle Actin

(81) The expression of -smooth muscle actin (SMA) was analyzed in paraffin embedded sections. After deparaffinization, samples were incubated with 3% bovine serum albumin followed by incubation with 3% H.sub.2O.sub.2. SMA positive cells in mouse sections were detected by incubation with monoclonal anti-SMA antibodies (clone 1A4, Sigma-Aldrich, Steinheim, Germany). Irrelevant isotype antibodies in the same concentration were used for control (Santa Cruz Biotechnology, Santa Cruz, Calif., USA). Antibodies labeled with horseradish peroxidase (Dako, Hamburg, Germany) were used as secondary antibodies. The expression of the NICD and SMA was visualized with DAB peroxidase substrate solution (Sigma-Aldrich). The number of myofibroblasts was counted from 4 different sections of lesional skin for each mouse by an examiner blinded to the treatment of the mice.

(82) TABLE-US-00001 TABLE 1 Effects of the compound of formula 27 on development of Bleomycin-induced skin fibrosis. c) Bleomycin + b) Bleomycin + 3 mg/kg a) Bleomycin + 1 mg/kg compound compound of vehicle of formula 27 formula 27 Dermal thickness 1.70 1.37 1.19 Collagen content 1.31 1.19 1.11 Myofibroblast 3.72 3.23 1.90 count

(83) Fibrosis parameters expressed as x-fold change with respect to vehicle-treated control.

(84) These dose dependent and significant effects were seen with other examples i.e. the compound of formula 3, the compound of formula 6 in a similar manner.

(85) Fibrosis parameters expressed as x-fold change with respect to vehicle-treated control

(86) TABLE-US-00002 TABLE 2 Effects of the compound of formula 3 and the compound of formula 6 on development of Bleomycin-induced skin fibrosis. Fibrosis parameters expressed as x-fold change with respect to vehicle-treated control b) Bleomycin + c) Bleomycin + d) Bleomycin + e) Bleomycin + 1.0 mg/kg 0.3 mg/kg 1 mg/kg 3 mg/kg a) Bleomycin + compound of compound of compound of compound of vehicle formula 3 formula 6 formula 6 formula 6 Dermal thickness 1.71 1.38 1.41 1.24 1.19 Collagen content 1.56 1.38 1.41 1.28 1.20 Myofibroblast 3.86 2.55 3.13 2.05 1.49 count

Example B

Bleomycin-Induced Skin Fibrosis

(87) Local skin fibrosis was induced in 6-week-old, pathogen-free, female DBA/2 mice (Charles River, Sulzfeld, Germany) by repeated (every other day) subcutaneous injections of bleomycin (0.5 mg/ml in saline) in a defined area of the upper back. Control mice were injected in the same manner with saline only. For all groups the injection volume was 100 l. The study comprises 4 arms with a) mice receiving saline injection for 6 weeks (serving as reference) b) mice receiving Bleomycin injection for 6 weeks c) mice receiving Bleomycin injection for 6 weeks and additional treatment with the compound of formula 27 (3 mg/kg) twice a day via gavage for the last 3 weeks d) mice receiving the first 3 weeks bleomycin injections and the second 3 weeks saline injection.

(88) After 6 weeks the animals were sacrificed and skin samples were obtained for analysis.

(89) Histological analysis, hydroxyproline assay and immunohistochemistry for -smooth muscle actin were performed as described in the Example 1 section.

(90) TABLE-US-00003 TABLE 3 Effects of the compound of formula 27 (3 mg/kg p.o.) on established Bleomycin-induced skin fibrosis Fibrosis parameters expressed as x-fold change with respect to vehicle-treated control (group a) c) Bleomycin 6 weeks + 3 weeks d) Bleomycin b) Bleomycin the compound 3 weeks + 6 weeks of formula 27 3 weeks NaCl Dermal thickness 1.57 1.26 1.40 Myofibroblast 3.87 1.68 3.50 count

Example C

Tight Skin Mouse Model

(91) In addition to the mouse model of bleomycin-induced dermal fibrosis, the tight-skin (Tsk-1) mouse model of systemic sclerosis was used to evaluate the anti-fibrotic potential of test drugs. Due to a dominant mutation in fibrillin-1, the phenotype of Tsk-1 is characterized by an increased hypodermal thickness. Genotyping of Tsk-1 mice was performed by PCR with the following primers: mutated fibrillin-1/Tsk-1 forward primer: 5-GTTGGCAACTATACCTGCAT-3, reverse primer: 5-CCTTTCCTGGTAACATAGGA-3. Tsk-1 mice were treated daily with test drug or vehicle, respectively, by oral gavage. In addition, a group of corresponding wild type(pa/pa) mice was treated with vehicle. The treatment was started at an age of five weeks. After five weeks of treatment, mice were sacrificed by cervical dislocation and skin samples were obtained for analysis.

(92) Histological analysis, hydroxyproline assay and immunohistochemistry for -smooth muscle actin were performed as described in the Example 1 section.

(93) TABLE-US-00004 TABLE 4 Effects of the compound of formula 27 on established skin fibrosis in Tsk-mice Tsk-1 + Tsk-1 + 1 mg/kg the 3 mg/kg the Tsk-1 + compound of compound of vehicle formula 27 formula 27 hypodermal 5.03 3.46 2.88 thickness Collagen content 2.46 1.61 1.67 Myofibroblast 2.64 2.12 1.70 count

(94) Fibrosis parameters expressed as x-fold change with respect to vehicle-treated wild type mice

Example D

(95) The haemodynamic effects of i.e. example, the compound of formula 3, the compound of formula 4, the compound of formula 6 were analyzed in conscious mice. Telemetric implants (DSI) were used. Signals were received with RMC1-DSI receiver plates, compiled and analyzed with PONEMAH physiology platform software.

(96) The mice received either placebo (tylose), 0.3 mg/kg the compound of formula 27, 1 mg/kg the compound of formula 27, 3 mg/kg the compound of formula 27 (FIG. 1), 1.0 mg/kg the compound of formula 3, 3.0 mg/kg the compound of formula 3, 10.0 mg/kg the compound of formula 3 (FIG. 2A,2B), 1.0 mg/kg the compound of formula 4, 3.0 mg/kg the compound of formula 4, 10.0 mg/kg the compound of formula 4 (FIG. 3A/3B), 0.3 mg/kg the compound of formula 6, 1.0 mg/kg the compound of formula 6, 3.0 mg/kg, 10.0 mg/kg the compound of formula 6 (FIG. 4A/4B). The blood pressure and heart rate was monitored before and after application of placebo or the compounds. FIG. 1 shows effects of the compound of formula 27 on blood pressure (left) and heart rate (right), FIG. 2 shows the effect of the compound of formula 3 on blood pressure (FIG. 2A) and heart rate (FIG. 2B), FIG. 3 shows the effects of the compound of formula 4 on blood pressure (FIG. 3A) and heart rate (FIG. 3B), FIG. 4 shows the effect of the compound of formula 6 on blood pressure (FIG. 4A) and heart rate (FIG. 4B).

Example E

(97) The effects of the compound of formula 27 and vardenafil as stand alone and in combination were analyzed in vitro in human dermal fibroblasts in vitro. The compound of formula 27, vardenafil and combinations thereof significantly blocked the TGFbeta-induced Collagen gene expression and Hydroxyproline (HP) deposition.

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