NITRIC OXIDE DONATING DERIVATIVES OF FLUPROSTENOL

Abstract

The present invention relates to 15-nitrooxyderivatives of fluprostenol, their use for the treatment of glaucoma and ocular hypertension and formulation containing 15-nitrooxy derivatives of fluprostenol.

Claims

1. A compounds of formula (I) or salts thereof ##STR00047## wherein R is CH(CH.sub.3).sub.2 or H; Ra is selected from A1): (CHR.sup.1)NH(CO)(CH.sub.2).sub.m[O(CH.sub.2).sub.n].sub.p(CHONO.sub.2).sub.qCH.sub.2ONO.sub.2 A2): (CH.sub.2).sub.2NH(CO)(CH.sub.2).sub.m[O(CH.sub.2).sub.n].sub.p(CHONO.sub.2).sub.qCH.sub.2ONO.sub.2 A3): (CH.sub.2).sub.m[O(CH.sub.2).sub.n].sub.p(CHONO.sub.2).sub.qCH.sub.2ONO.sub.2 wherein R.sup.1 is H or CH.sub.3, p is 1 or 0, q is 1 or 0, m is an integer ranging from 1 to 10; n is an integer ranging from 1 to 6.

2. A compound of formula (I) according to claim 1, wherein Ra is selected from A1): (CHR.sup.1)NH(CO)(CH.sub.2).sub.m[O(CH.sub.2).sub.n].sub.p(CHONO.sub.2).sub.qCH.sub.2ONO.sub.2, and A3): (CH.sub.2).sub.m[O(CH.sub.2).sub.n].sub.p(CHONO.sub.2).sub.qCH.sub.2ONO.sub.2.

3. A compound of formula (I) according to claim 2, wherein R is CH(CH.sub.3).sub.2 and Ra is A1): (CHR.sup.1)NH(CO)(CH.sub.2).sub.m[O(CH.sub.2).sub.n].sub.p(CHONO.sub.2).sub.qCH.sub.2ONO.sub.2 wherein R.sup.1 is H or CH.sub.3, p is 1 or 0, q is 1 or 0, m is an integer ranging from 1 to 10; n is an integer ranging from 1 to 6.

4. A compound of formula (I) according to claim 3, wherein Ra is selected from the following group of linkers: ##STR00048##

5. A compound of formula (I) according to claim 2, wherein R is CH(CH.sub.3).sub.2and Ra is A3): (CH.sub.2).sub.m[O(CH.sub.2).sub.n].sub.p(CHONO.sub.2).sub.qCH.sub.2ONO.sub.2 wherein p is 1 or 0, q is 1 or 0, m is an integer ranging from 1 to 10; n is an integer ranging from 1 to 6.

6. A compound of fonnula (I) according to claim 5, wherein Ra is selected from the following group of linkers: ##STR00049##

7. A compound of formula (I) according to claim 3, wherein R is H.

8. A compound of formula (I) according to claim 2 selected from the following group of compounds: (Z)-isopropyl 7-((1R,2R,3R,5S)-3,5-dihydroxy-2-((R,E)-3-(2-(6-(nitrooxy) hexanamido) acetoxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)cyclopentyl)hept-5-enoate (Compound (5)); (Z)-isopropyl 7-((1R,2R,3R,5S)-2-((3R,E)-3-(2-(5,6-bis(nitrooxy)hexanamido)acetoxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)-3,5-dihydroxycyclopentyl)hept-5-enoate (Compound (6)); (Z)-isopropyl 7-((1R,2R,3R,5S)-3,5-dihydroxy-2-((R,E)-3-(2-(2-(2-(nitrooxy)ethoxy) acetamido)acetoxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)cyclopentyl)hept-5-enoate (Compound (7)); (Z)-isopropyl 7-((1R,2R,3R,5S)-2-((3R,E)-3-(2-(3-(2,3-bis(nitrooxy)propoxy) propanamido)acetoxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)-3,5-dihydroxycyclopentyl)hept-5-enoate (Compound (8)); (Z)-isopropyl 7-((1R,2R,3R,5S)-3,5-dihydroxy-2-((R,E)-3-(6-(nitrooxy)hexanoyloxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)cyclopentyl)hept-5-enoate (Compound (9)); (Z)-isopropyl 7-((1R,2R,3R,5S)-2-((3R,E)-3-(5,6-bis(nitrooxy)hexanoyloxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)-3,5-dihydroxycyclopentyl)hept-5-enoate (Compound (10)); (Z)-isopropyl 7-((1R,2R,3R,5S)-3,5-dihydroxy-2-((R,E)-3-(2-(2-(nitrooxy)ethoxy) acetoxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)cyclopentyl)hept-5-enoate (Compound (11)); (Z)-isopropyl 7-((1R,2R,3R,5S)-2-((3R,E)-3-(3-(2,3-bis(nitrooxy)propoxy) propanoyloxy)-4-(3-(trifluoromethyl)phenox y)but-1-enyl)-3 ,5-dihydroxycyclopentyl) hept-5-enoate (Compound (12)). (Z)-7-((1R,2R,3R,5S)-2-((R,E)-3-(5,6-bis(nitrooxy)hexanoyloxy)-4-(3-(trifluoromethyl) phenoxy)but-1-enyl)-3,5-dihydroxycyclopentyl)hept-5-enoic acid (Compound (13)).

9. A compound of formula (I) according to claim 1, wherein R is CH(CH.sub.3).sub.2 and Ra is A2): (CH.sub.2).sub.2NH(CO)(CH.sub.2).sub.m[O(CH.sub.2).sub.n].sub.p(CHONO.sub.2).sub.qCH.sub.2ONO.sub.2 wherein p is 1 or 0, q is 1 or 0, m is an integer ranging from 1 to 10; n is an integer ranging from 1 to 6.

10. A compound of formula (I) according to claim 9, wherein Ra is selected from the following group of linkers: ##STR00050##

11. A compound of formula (I) according to claim 9, wherein R is H.

12. A compound of formula (I) according to claim 9 selected from the following group of compounds: (Z)-isopropyl 7-((1R,2R,3R,5S)-3,5-dihydroxy-2-((R,E)-3-(3-(6-(nitrooxy) hexanamido) propanoyloxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)cyclopentyl)hept-5-enoate (Compound (1)); (Z)-isopropyl 7-((1R,2R,3R,5 S)-2-((3R,E)-3-(3-(5,6-bis(nitrooxy) hexanamido) propanoyloxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)-3,5-dihydroxycyclopentyl) hept-5-enoate (Compound (2)); (Z)-isopropyl 7-((1R,2R,3R,5S)-3,5-dihydroxy-2-((R,E)-3-(3-(2-(2-(nitrooxy) ethoxy) acetamido)propanoyloxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)cyclopentyl)hept-5-enoate (Compound (3)); (Z)-isopropyl 7-((1R,2R,3R,5 S)-2-((3R,E)-3-(3-(3-(2,3-bis(nitrooxy)propoxy) propanamido)propanoyloxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)-3,5-dihydroxycyclopentyl)hept-5-enoate (Compound (4));

13. A compound of formula (I) according to claim 1 for use as medicament.

14. A compound of formula (I) according to claim 1 for use in the treatment of ocular hypertension.

15. A compound of formula (I) according to claim 1 for use in the treatment of ocular hypertension or glaucoma.

16. A compound of formula (I) for the use according to claim 15, wherein glaucoma is primary open angle glaucoma, normal intraocular tension glaucoma, pseudoexfoliation glaucoma, acute angle-closure glaucoma or chronic closed angle glaucoma.

17. A topical ocular pharmaceutical composition comprising a compound of formula (I) according to claim 1 as active principle and a pharmaceutically acceptable excipient or a combination of excipients.

18. A composition comprising a compound of formula (I) according to claim 1 and at least another active agent selected from the following classes of drugs: Beta-adrenergic antagonists, Adrenergic agonists, Alpha.sub.2-selective adrenergic agonists, Carbonic Anhydrase Inhibitors, Cholinergic agonists, Cholinesterase inhibitors.

19. A composition according to claim 18 for use in the treatment of ocular hypertension or glaucoma.

Description

EXAMPLE 1

Synthesis of (Z)-isopropyl 7-((1R,2R,3R,5S)-3,5-dihydroxy-2-((R,E)-3-(6-(nitrooxy) hexanoyloxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)cyclopentyl)hept-5-enoate (corresponding to compound (9))

[0216] ##STR00034##

Step 1: Synthesis of 4-nitrophenyl 6-bromoexanoate

[0217] To a solution of 6-bromohexanoic acid (1.0 g, 5.12 mmol) and DCC (1.1 g, 5.33 mmol) in DCM (15 ml), cooled to 0 C., p-nitrophenol (0.713 g, 5.12 mmol) was added portionwise. The mixture was stirred overnight at room temperature. The mixture was then filtered, evaporated and purified by flash chromatography (Gradient: Cy/AcOEt 5% to 50% in 12 CV), yielding 4-nitrophenyl 6-bromoexanoate (1.301 g, 80.3%).

[0218] .sup.1H NMR (300 MHz, CDCl.sub.3) 8.33-8.21 (m, 2H), 7.33-7.23 (m, 2H), 3.45 (t, J=6.6 Hz, 2H), 2.64 (t, J=7.4 Hz, 2H), 1.93 (dt, J=14.6, 6.8 Hz, 2H), 1.86-1.74 (m, 2H), 1.65-1.54 (m, 2H).

Step 2: Synthesis of 4-nitrophenyl 6-(nitrooxy)hexanoate

[0219] To a solution of 4-nitrophenyl 6-bromohexanoate (1.301 g, 4.12 mmol) in CH.sub.3CN (30 ml), kept in the dark, AgNO.sub.3 (840.85 mg, 4.95 mmol) was added. The mixture was refluxed overnight. Then, the salts were filtered off and the solvent concentrated. EtOAc was added to the residue and the salts filtered off again. The solution was concentrated and the residue purified by flash chromatography (DCM 100%) affording 1.2 g of the 4-nitrophenyl 6-(nitrooxy)hexanoate (Yield: 97.6%).

[0220] .sup.1H NMR (300 MHz, CDCl.sub.3) 8.28 (d, J=9.1 Hz, 2H), 7.28 (d, J=9.5 Hz, 2H), 4.49 (t, J=6.4 Hz, 2H), 2.64 (t, J=7.3 Hz, 2H), 1.82 (q, J=7.7 Hz, 4H), 1.56 (td, J=8.7, 4.1 Hz, 2H).

Step 3: Synthesis of (Z)-isopropyl 7-((1R,5S,6R,7R)-3-butyl-7-((R,E)-3-hydroxy-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)-2,4-dioxa-3-borabicyclo[3.2.11octan-6-yl)hept-5-enoate

[0221] ##STR00035##

[0222] The compound was prepared following a procedure reported in Organic Syntheses, Coll. Vol. 10, p. 613 (2004).

[0223] To a solution of fluprostenol isopropyl ester (0.5 g, 1.0 mmol) in Et.sub.2O (3 ml) and DCM (1 ml), butylboronic acid (0.1 g, 1.0 mmol) was added. After stirring for a few minutes, some molecular sieves (4 ) were added. The mixture was stirred 5 h at room temperature. The reaction was controlled in TLC (Cy/AcOEt 7:3) using alumina. Because the reaction was not finished, a second aliquot of butylboronic acid (0.05 g, 0.5 mmol) was added and the reaction was stirred overnight. Then the mixture was filtered and concentrated affording the title compound (0.55 mg, Yield: 97%).

[0224] .sup.1H NMR (300 MHz, Chloroform-d) 7.49-7.35 (m, 1H), 7.32-7.00 (m, 3H), 5.81-5.53 (m, 2H), 5.53-5.33 (m, 2H), 5.10-4.90 (m, 1H), 4.61-4.46 (m, 1H), 4.34 (s, 1H), 4.16 (s, 1H), 4.08-3.82 (m, 2H), 2.58-2.38 (m, 1H), 2.38-2.19 (m, 3H), 2.18-2.04 (m, 2H), 2.06-1.93 (m, 1H), 1.92-1.56 (m, 4H), 1.50-1.06 (m, 9H), 1.03-0.75 (m, 4H), 0.74-0.65 (m, 2H).

Step 4: Synthesis of (Z)-isopropyl 7-((1R,2R,3R,5S)-3,5-dihydroxy-2-4R,E)-3-(6-(nitrooxy)hexanoyl oxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)cyclopentyl)hept-5-enoate (corresponding to compound (9))

[0225] (Z)-isopropyl 7-((1R,5S,6R,7R)-3-butyl-7-((R,E)-3-hydroxy-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)-2,4-di oxa-3-borabicyclo[3.2.1]octan-6-yl)hept-5-enoate (prepared in Step 3) (0.50 g, 0.88 mmol) and 4-nitrophenyl 6-(nitrooxy)hexanoate (prepared in Step 2) (0.316 g, 1.01 mmol) and DMAP (0.107 g, 0.88 mmol) were dissolved in DCM (15 ml). The mixture was stirred for 6 hrs at room temperature. Then DMAP (0.53 g, 0.44 mmol) was added and the mixture was stirred overnight. The mixture was then evaporated and redissolved in MeOH and stirred for 4 hrs at room temperature. Then the mixture was evaporated and purified by flash chromatography (Cyclohexane:EtOAc from 8:2 to 4:6) yielding the title compound as a clear oil (0.35 g, 71.4% yield).

EXAMPLE 2

Synthesis of (Z)-isopropyl 7-((1R,2R,3R,5S)-2-((R,E)-3-((S)-5,6-bis(nitrooxy) hexanoyloxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)-3,5-dihydroxycyclopentyphept-5-enoate (corresponding to compound (10), (S)-isomer)

[0226] ##STR00036##

Step 1: Synthesis of Hex-5-enyl-4-nitrobenzoate

[0227] To a stirred solution of hex-5-enol (21.25 g, 200 mmol) and 4-nitrobenzoyl chloride (37.11 g, 200 mmol) in DCM (300 mL) at 0 C., triethylamine (28 mL, 200 mmol) was added. The reaction was stirred at room temperature for 4 h and washed with water, HCl 1M, water and brine. The solvent was removed under reduced pressure to give a crude oil which was treated with n-hexane to give a solid that was filtered off. The mother liquor was evaporated to give the title compound as yellow oil (41 g, 82%).

[0228] MS: m/z=250 [M+H].sup.+

[0229] TLC: DCM 100% R.sub.f=0.4

Step 2: Synthesis of (5S)-5,6-dihydroxyhexyl 4-nitrobenzoate

[0230] A stirred solution of AD-Mix (50 g) in a mixture tBuOH /H.sub.2O (227 mL each) was stirred for 10 min at room temperature and then cooled to 4 C. After 15 min, hex-5-enyl 4-nitrobenzoate (Step 1) (8.8 g, 35.5 mmol) was added and the reaction stirred overnight at 4 C. Then EtOAc (200 mL) was added and followed by careful addition of sodium metabisulfite (12 g). The reaction was left for 30 min at 4 C. and then treated with water (200 mL). The two layers were separated and the aqueous phase extracted twice with ethyl acetate (2100 mL). The combined organic phases were washed with water and brine, dried over sodium sulfate, evaporated to give a white solid (9.7 g, 97%).

[0231] The residue was dissolved in diethylether (100 mL) and stirred overnight to give the title compound as white solid (8.1 g, 84%).

[0232] MS: 284 [M+H].sup.+

[0233] TLC: (DCM/MeOH-0.5%) R.sub.f=0.36

Step 3: Synthesis of (5S)-5,6-bis(nitrooxy)hexyl 4-nitrobenzoate

[0234] To a stirred solution of fuming HNO.sub.3 (3.6 mL, 88 mmol) in DCM (3 mL) at 0 C., was added acetic anhydride (13.7 mL) and after 10 minutes of stirring, a solution of (5S)-5,6-dihydroxyhexyl 4-nitrobenzoate (Step 2) (5 g, 17.6 mmol) in dichloromethane (2 mL) was added and the reaction stirred at this temperature for 60 min. The crude mixture was then poured on ice and the organic layer extracted, washed with water, brine, dried over sodium sulfate, evaporated to give the title compound as pale yellow oil (6.4 g, 99%). The residue obtained was used in the next step without further purification.

[0235] MS: 374 [M+H].sup.+

[0236] TLC: (DCM 100%) R.sub.f=0.37

Step 4: Synthesis of (2S)-6-hydroxy-2-(nitrooxy)hexyl nitrate

[0237] To a stirred solution of (5S)-5,6-bis(nitrooxy)hexyl 4-nitrobenzoate (Step 3) (7.4 g, 19.82 mmol) in a 1/1 mixture of ethanol/THF (33 mL of each) at 0 C., a 2 M NaOH solution (19.8 mL, 2 eq) was added and the reaction was stirred for 2 h. The reaction was diluted with ethyl acetate and water (100 mL of each) and extracted. The organic layer was successively washed with water and brine, dried over sodium sulfate and evaporated. The oily residue was purified by column chromatography (gradient system from 4/6 ethyl acetate/Cy to 60/40 ethyl acetate/Cy) to give the title compound as colorless oil (4.1 g, 92%).

[0238] TLC: (EtOAc/Cy-50%) R.sub.f=0.31

Step 5: Synthesis of (5S)-5,6-bis(nitrooxy)hexanoic acid

[0239] To a solution of (2S)-6-hydroxy-2-(nitrooxy)hexyl nitrate (Step 4) (3 g, 13.4 mmol) and Sodium periodate (8.4 g, 40.2 mmol) in CHCl.sub.3, Acetonitrile, H.sub.2O (1:1:1), ruthenium (IV) oxide (180 mg, 1.34 mmol) was added. The mixture was stirred overnight at RT, the precipitate was filtered off and the solvent was removed under reduced pressure. The residue was dissolved in DCM, washed with water, dried with MgSO.sub.4, filtered and concentrated under reduced pressure. The crude was purified by flash chromatography (EtOAc in cyclohexane from 5% to 50%) affording 2.4 g of (5S)-5,6-bis(nitrooxy)hexanoic acid (Yield: 75%)

[0240] TLC: (DCM/MeOH-0.5%) R.sub.f=0.34

Step 6: Synthesis of (S)-4-nitrophenyl 5,6-bis(nitrooxy)hexanoate

[0241] To a solution of (5S)-5,6-bis(nitrooxy)hexanoic acid (1.7 g, 6.76 mmol) and DCC (1.4 g, 6.76 mmol) in DCM (65 ml), p-nitrophenol (0.94 g, 6.76 mmol) was added portion wise. The mixture was stirred overnight at RT. Progress was checked by TLC (Cy/AcOEt 4:6). Once the reaction was complete, the mixture was filtered, evaporated and purified by flash chromatography (Gradient: Cy/AcOEt 10% to 60% in 12 CV), yielding 1.51 (S)-4-nitrophenyl 5,6-bis(nitrooxy)hexanoate (84.3%).

[0242] TLC: (Cy/EtOAc-4/6) R.sub.f=0.36

Step 7: Synthesis of (Z)-isopropyl 7-((1R,2R,3R,5S)-2-((R,E)-3-((S)-5,6-bis(nitrooxy) hexanoyloxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)-3,5-dihydroxycyclopentyl)hept-5-enoate (corresponding to compound (10) (S)-isomer)

[0243] ##STR00037##

[0244] (Z)-isopropyl 7-((1R,5S,6R,7R)-3-butyl-7-4R,E)-3-hydroxy-4-(3-(trifluoromethyl) phenoxy)but-1-enyl)-2,4-dioxa-3-borabicyclo[3.2.1]octan-6-yl)hept-5-enoate (prepared in Example 1, Step 3) (0.50 g, 0.88 mmol) and (S)-4-nitrophenyl 5,6-bis(nitrooxy)hexanoate (prepared in Step 6) (0.37 g, 1.01 mmol) and DMAP (0.110 g, 0.88 mmol) were dissolved in DCM (15 ml). The mixture was stirred for 6 hrs at room temperature. Then DMAP (0.53 g, 0.44 mmol) was added and the mixture was stirred overnight. The mixture was then evaporated and redissolved in MeOH and stirred for 4 hrs at room temperature. Then the mixture was evaporated and purified by flash chromatography (Cyclohexane:EtOAc from 9:1 to 1:1) yielding the title compound as a clear oil (0.310 g, 49.6% yield).

EXAMPLE 3

Synthesis of (Z)-isopropyl 7-((1R,2R,3R,5S)-3,5-dihydroxy-2-((R,E)-3-(2-(2-(nitrooxy)ethoxy)acetoxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)cyclopentyphept-5-enoate (corresponding to compound (11))

[0245] ##STR00038##

Step 1: Synthesis of 2-(2-hydroxyethoxy)ethyl nitrate

[0246] A solution of ethylene glycol (4.0 g, 37.7 mmol) in DCM (200 ml) was cooled to 30 C. and a mixture of HNO.sub.3 (1.6 ml, 37.7 mmol) in Ac.sub.2O (10.7 ml, 113 mmol) was added dropwise under vigorous stirring. The mixture was stirred 2 hours at room temperature and then poured into ice. The organic phase was evaporated. The aqueous phase was neutralized to pH 7 with NaHCO.sub.3 and then extracted 3 times with cyclohexane and 3 times with DCM. The DCM layer only was dried over Na.sub.2SO.sub.4 and evaporated to give the desired product (2.9 g, Yield: 50%) that was used in the next step without further purification.

[0247] .sup.1H NMR (300 MHz, Chloroform-d) 4.70-4.56 (m, 2H), 3.84-3.71 (m, 4H), 3.65-3.55 (m, 2H).

Step 2: Synthesis of 2-(2-(nitrooxy)ethoxy)acetic acid

[0248] To a solution of 2-(2-hydroxyethoxy) ethyl nitrate (2.9 g, 18.9 mmol) in acetone (100 ml) cooled at 0 C., NaHCO.sub.3 saturated solution (75 ml), NaBr (0.8 g, 7.6 mmol) and TEMPO (0.6 g, 3.8 mmol) were added. Trichloroisocyanuric acid (8.8 g, 37.9 mmol) was then added portionwise. The reaction was allowed to reach room temperature and stirred for 3hours. The mixture was cooled to 0 C. and 20 ml of isopropanol were added slowly. The mixture was stirred at 0 C. for 30 min. The formation of a white solid was observed. The precipitate was filtered off and the solvent concentrated. The residue was basified with NaOH 2N (pH12) and washed twice with EtOAc. To the aqueous phase HCl 1N was added until pH 2-3 and then extracted five times with EtOAc. The organic phase was dried over Na.sub.2SO.sub.4 and then was evaporated. A white solid was observed. The solid was digested with DCM/MeOH 95:5. The solution was then evaporated affording the title compound as a brown oil. (2.26 g, Yield: 72%)

[0249] .sup.1H NMR (300 MHz, Chloroform-d) 4.70-4.54 (m, 2H), 4.19 (s, 2H), 3.92-3.76 (m, 2H).

Step 3: Synthesis of 4-nitrophenyl 2-(2-(nitrooxy)ethoxy)acetate

[0250] To a solution of 2-(2-(nitrooxy)ethoxy)acetic acid (Step 2) (0.9 g, 5.3 mmol) and DCC (1.093 g, 5.3 mmol) in DCM, p-nitrophenol (0.8 g, 5.83 mmol) was added portionwise. The mixture was stirred overnight at room temperature. The mixture was filtered, evaporated and purified by flash chromatography (Biotage SP4 instrumentation EtOAc in cyclohexane from 5% to 50% in 12 CV) yielding the title compound (1.43 g, 94.3%).

Step 4: Synthesis of (Z)-isopropyl 7-((1R,2R,3R,5S)-3,5-dihydroxy-2-((R,E)-3-(2-(2-(nitrooxy)ethoxy)acetoxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)cyclopentyl)hept-5-enoate (corresponding to compound (11))

[0251] (Z)-isopropyl7-((1R,5S,6R,7R)-3-butyl-7-((R,E)-3-hydroxy-4-(3 (trifluoromethyl) phenoxy)but-1-enyl) -2,4-dioxa-3-borabicyclo[3.2.1]octan-6-yl)hept-5-enoate (Example 1, Step 3) (0.55 g, 1.0 mmol) and 4-nitrophenyl 2-(2-(nitrooxy)ethoxy)acetate (0.81 g, 2.8 mmol) and DMAP (0.72 g, 3.6 mmol) were dissolved in DCM (3 ml). The mixture was stirred for a few minutes and some molecular sieves 4 were added. The mixture was stirred 48 h and then filtered. The filtrate was then purified by preparative HPLC. The product was treated with aqueous NaHCO.sub.3 in order to remove TFA and the aqueous phase extracted 3 times with ethyl acetate, dried over Na.sub.2SO.sub.4 and the solvent removed in vacuo affording 0.12 g of a pale yellow oil (Yield: 20%).

[0252] .sup.1H NMR (300 MHz, DMSO-d6) 7.50 (t, 1H), 7.33-7.19 (m, 3H), 5.75-5.49 (m, 3H), 5.47-5.34 (m, 1H), 5.30-5.14 (m, 1H), 4.93-4.72 (m, 1H), 4.71-4.55 (m, 3H), 4.39 (d, 1H), 4.28-4.09 (m, 4H), 3.94-3.60 (m, 4H), 2.31-1.82 (m, 8H), 1.55-1.18 (m, 4H), 1.12 (d, 6H).

EXAMPLE 4

Synthesis of (Z)-isopropyl 7-((1R,2R,3R,5S)-2-((R,E)-3-(3-((S)-2,3-bis (nitrooxy)propoxy)propanoyloxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)-3,5-dihydroxycyclopentyl)hept-5-enoate (corresponding to compound (12), (S)-isomer)

[0253] ##STR00039##

Step 1: Synthesis of (S)-4-(allyloxymethyl)-2,2-dimethyl-1,3-dioxolane

[0254] NaH (60%) (3.4 g; 85.54 mmol) was suspended in dry THF (140 ml) together with S(+) 1,2-Isopropylidenglycerol (5.65 g; 42.75 mmol) and 15-Crown-5 (0.9 g; 4.28 mmol). The mixture was cooled to 0 C. and allyl bromide (7.2 ml; 85.54 mmol) was added dropwise. The suspension was stirred for 6 h at R.T, than NH.sub.4Cl saturated solution (100 ml) was added dropwise at 0 C. and then the mixture extracted with Et.sub.2O (3100 ml). The combined organic layers were washed once with brine and concentrated under reduced pressure carefully. The residue was purified by flash chromatography (Biotage SP4 instrument, SNAP 100 column, isocratic elution Et.sub.2O/Cyclohexane 1:9), affording 5.8 g, (yield 78.5%) of the title compound.

[0255] .sup.1H NMR (300 MHz, Chloroform-d) 6.00-5.78 (m, 1H), 5.35-5.13 (m, 2H), 4.35-4.21 (m, 1H), 4.11-3.98 (m, 3H), 3.73 (m, 1H), 3.57-3.40 (m, 2H), 1.51-1.32 (m, 6H).

Step 2: Synthesis of (S)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)propan-1-ol

[0256] To a stirred solution of (S)-4-(allyloxymethyl)-2,2-dimethyl-1,3-dioxolane (2.0 g, 10.64 mmol) in dry THF (180 ml) cooled at 0 C., 9BBN 0.5M (241.8 ml, 121 mmol) was added dropwise. The reaction was stirred 30 minutes at 0 C. and overnight at room temperature. The reaction mixture was cooled at 0 C. and NaOH .sub.2N (84 ml) was added together with H.sub.2O.sub.2 30% (58 ml). The mixture was diluted with Et.sub.2O (100 ml) and NaOH 1N (100 ml). The 2 phases were separated and aqueous layer was extracted with Et.sub.2O (3120 ml). The combined organic layers were washed once with brine, dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by flash chromatography (Biotage SP4 instrument, SNAP 100 column, EtOAC in cyclohexane, from 30% to 80% in 10 c.v. affording 6.4 g (yield: 100%) of the title compound.

[0257] .sup.1H NMR (300 MHz, Chloroform-d) 4.35-4.20 (m, 1H), 4.11-4.01 (m, 1H), 3.85-3.63 (m, 5H), 3.58-3.46 (m, 2H), 1.97-1.80 (m, 2H), 1.45 (s, 3H), 1.38 (s, 3H).

Step 3: Synthesis of (S)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)propanoic acid

[0258] To a solution of (S)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy)propan-1-ol (2.03 g; 10.51 mmol) in acetone (50 ml) cooled at 0 C., NaHCO.sub.3 sat solution (56 ml), NaBr (0.45 g, 4.20 mmol) and TEMPO (0.34 g, 2.10 mmol) were added. Trichloroisocyanuric acid (4.91 g, 21.02 mmol) was then added portionwise. The mixture was allowed to reach room temperature and stirred for 3 h. The mixture was then cooled at 0 C. and isopropanol (20 ml) was added slowly. The mixture was stirred at 0 C. 30 minutes. The formation of a white solid was observed. The precipitate was filtered off and the solvent concentrated. NaOH 2N was added to the residue (pH12) and the aqueous solution was washed twice with EtOAc. To the aqueous phase HCl 1N was added until pH 2-3 and it was extracted with EtOAc (550 ml). The combined organic phases was dried over Na.sub.2SO.sub.4 and then evaporated affording 0.82 g (Yield: 38%) of the title compound.

[0259] .sup.1H NMR (300 MHz, Chloroform-d) 4.34-4.16 (m, 1H), 4.11-3.97 (m, 1H), 3.85-3.68 (m, 3H), 3.63-3.42 (m, 2H), 2.74-2.55 (m, 2H), 1.41 (s, 3H), 1.35 (s, 3H).

Step 4: Synthesis of (S)-4-nitrophenyl 3-((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) propanoate

[0260] To a solution of (S)-3-((2,2-dimethyl-1,3-dioxolan-4-yl)methoxy) propanoic acid (0.82 g, 4.02 mmol), DCC (0.83 mg, 4.02 mmol) and DMAP (0.1 g, 0.80 mmol) in DCM (15 ml), 4-nitrophenol (0.56 g; 4.02 mmol) was added portionwise. The mixture was stirred overnight at room temperature, then the precipitate was filtered off and the solvent evaporated. The residue was purified by flash chromatography (Biotage SP4 instrument, EtOAc in cyclohexane from 5% to 50% in 12 CV) affording 0.97 g of the title compound (Yield: 74%). .sup.1H NMR (300 MHz, Chloroform-d) 8.33-8.21 (m, 2H), 7.37-7.20 (m, 2H), 4.37-4.20 (m, 1H), 4.09-4.00 (m, 1H), 3.96-3.84 (m, 2H), 3.80-3.66 (m, 1H), 3.63-3.47 (m, 2H), 2.96-2.78 (m, 2H), 1.42 (s, 3H), 1.36 (s, 3H).

Step 5: Synthesis of (R)-4-nitrophenyl 3-(2,3-dihydroxypropoxy) propanoate

[0261] To a stirred solution of (S)-4-nitrophenyl 3-((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) propanoate (0.97 g, 2.97 mmol) in THF (10 ml), HCl 3N (2 ml) was added and the solution stirred for 4 h at room temperature. Then EtOAc (5 ml) and H.sub.2O (5 ml) were added and the two phases were separated. The aqueous phase was extracted with EtOAc (25 ml). The combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure affording 0.89 g of the title compound, which was used in the next step without further purification.

Step 6: Synthesis of (S)-4-nitrophenyl 3-(2,3-bis(nitrooxy)propoxy) propanoate

[0262] To a solution of Ac.sub.2O (0.76 ml, 8.07 mmol) in DCM (5 ml) at 40 C., fuming HNO.sub.3 (0.38 ml, 9.32 mmol) was added dropwise. A solution of (R)-4-nitrophenyl 3-(2,3-dihydroxypropoxy) propanoate (0.89 g, 3.11 mmol) in DCM (7 ml) was then added dropwise. The mixture was allowed to reach 0 C. and stirred for 4 hours. The mixture was then poured into ice and NaHCO.sub.3 was added portionwise. The two phases were separated and the aqueous phase washed twice with DCM. The combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated affording 0.56 g of the title compound (yield of two steps: 38%).

[0263] .sup.1H NMR (300 MHz, Chloroform-d) 8.35-8.22 (m, 2H), 7.35-7.22 (m, 2H), 5.48-5.34 (m, 1H), 4.88-4.73 (m, 1H), 4.73-4.56 (m, 1H), 3.94-3.85 (m, 2H), 3.85-3.77 (m, 2H), 2.88 (t, 2H).

Step 7: Synthesis of (Z)-isopropyl 7-((1R,2R,3R,5S)-2-4R,E)-3-(3-((S)-2,3-bis(nitrooxy)propoxy)propanoyloxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)-3,5-dihydroxycyclopentyl)hept-5-enoate (corresponding to compound (12), (S)-isomer)

[0264] To a solution of (Z)-isopropyl 7-((1R,5S,6R,7R)-3-butyl-7-((R,E)-3-hydroxy-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)-2,4-dioxa-3-borabicyclo[3.2.1]octan-6-yl)hept-5-enoate (step 3, example 1) (0.25 g, 0.44 mmol) and (S)-4-nitrophenyl 3-(2,3-bis(nitrooxy)propoxy) propanoate (0.24 g, 0.66 mmol) in DCM (1 ml), DMAP (0.34 g, 1.67 mmol) and molecular sieves 4 were added. The mixture was stirred 48 h at room temperature and then the molecular sieves filtered off. The solvent was removed under reduced pressure and the residue was purified by HPLC preparative. NaHCO.sub.3 saturated solution was added to the product and the aqueous phase was extracted twice with DCM. The product was treated with aqueous NaHCO.sub.3 in order to remove TFA and the aqueous phase extracted 3 times with ethyl acetate, dried over Na.sub.2SO.sub.4 and the solvent removed in vacuo affording the title compound as a pale yellow oil (0.17 g, Yield: 52%).

[0265] .sup.1H NMR (300 MHz, DMSO-d.sub.6) 7.59-7.41 (m, 1H), 7.37-7.13 (m, 3H), 5.73-5.45 (m, 3H), 5.47-5.32 (m, 1H), 5.28-5.11 (m, 1H), 4.95-4.75 (m, 2H), 4.75-4.62 (m, 1H), 4.62-4.50 (d, 1H), 4.44-4.33 (d, 1H), 4.24-4.07 (m, 2H), 4.94-3.82 (m, 1H), 3.77-3.54 (m, 4H), 2.56 (t, 2H), 2.29-2.09 (m, 3H), 2.09-1.98 (m, 1H), 1.98-1.82 (m, 3H), 1.57-1.36 (m, 3H), 1.36-1.19 (m, 1H), 1.12 (d, 6H).

EXAMPLE 5

Synthesis of (Z)-isopropyl 7-((1R,2R,3R,5S)-3,5-dihydroxy -2-((R,E)-3-(3-(6-(nitrooxy) hexanamido) prop anoyl oxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)cyclopentyl)hept-5-enoate (corresponding to compound (1))

[0266] ##STR00040##

Step 1: Synthesis of t-butyl-3-(6-(nitrooxy)hexanamido)propanoate

[0267] To a solution of 4-nitrophenyl 6-(nitrooxy)hexanoate (prepared as described in Example 1, Step 2), (1.4 g, 4.68 mmol) and -ala-OtBu hydrochloride (0.85 g, 4.68 mmol) in DMF (10 ml) cooled at 0 C., TEA (1.3 ml, 9.36 mmol) was added dropwise. The solution was stirred for 15 minutes and DMAP (61 mg, 0.47 mmol) was added. The solution was stirred at rt overnight and the solvent removed under reduce pressure. The crude was dissolved in diethyleter and washed with HC1 1M to remove DMAP and TEA. The organic layer was washed with NaOH 1M to remove p-nitrophenol, washed with water, brine, dried over sodium sulfate an then evaporated to give 1.2 g, of the title compound as a pale yellow oil (Yield: 90%). The obtained residue was used in the next step without any further purification.

[0268] MS: 305 [M+H].sup.+

[0269] TLC: (Cy:EtOAc 7:3) R.sub.f=0.36

Step 2: Synthesis of 3-(6-(nitrooxy)hexanamido)propanoic acid

[0270] To a stirred solution of t-butyl-3-(6-(nitrooxy)hexanamido)propanoate (1 g, 3.3 mmol) in DCM (5 mL) at 0 C., was added BF3.Et2O (0.5 mL, 4 mmol) in 10 min, and the solution was stirred at rt for 3 h. The crude mixture was then poured on brine and the organic layer separated, dried over sodium sulfate and evaporated to give 0.820 g of the title compound as a pale yellow oil (100%). The obtained residue was used in the next step without any further purification.

[0271] MS: 249 [M+H].sup.+

Step 3: Synthesis of 4-nitrophenyl 3-(6-(nitrooxy)hexanamido)propanoate

[0272] To a solution of 3-(6-(nitrooxy)hexanamido)propanoic acid (0.820 g, 3.3 mmol) and DCC (0.681 g, 3.3mmo1) in DCM (30 ml), p-nitrophenol (0.505 g,.3.63 mmol) was added portion wise. The mixture was stirred overnight at rt. Then the mixture was filtered, evaporated and purified by flash chromatography (Gradient: Cy/AcOEt 5% to 70% in 12 CV), giving 1.1 g of the title compound (Yield: 91%).

[0273] MS: 370 [M+H].sup.+

[0274] TLC: (Cy:EtOAc 4:6) R.sub.f=0.35

Step 4: Synthesis of (Z)-isopropyl 7-((1R,2R,3R,5S)-3,5-dihydroxy-2-((R,E)-3-(3-(6-(nitrooxy) hexanamido) propanoyloxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)cyclopentyl)hept-5-enoate (corresponding to compound (1))

[0275] To a solution of (Z)-isopropyl 7-((1R,5S,6R,7R)-3-butyl-7-((R,E)-3-hydroxy-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)-2,4-dioxa-3-borabicyclo[3.2.1]octan-6-yl)hept-5-enoate (Example 1, Step 3) (220 mg, 0.39 mmol) and 4-nitrophenyl 3-(6-(nitrooxy)hexanamido)propanoate (158 mg, 0.43 mmol) in dry DCM (10 ml), DMAP (41 mg, 0.39 mmol) was added dropwise and the mixture was stirred for 72 hrs. The solvent was removed under reduced pressure and the product was dissolved in MeOH and stirred for 4 hrs at rt. The mixture was then evaporated and purified by reverse phase chromatography (H2O:CH3CN 8:2 to 1:1) affording 0.151 g of the title compound as a clear oil (Yield: 53%).

[0276] .sup.1H NMR: (600 MHz, DMSO-d6) 7.87 (t, 1H), 7.52 (t, 1H), 7.30 (d, 1H), 7.25 (d, 2H), 5.67 (dd,1H), 7.59-7.53 (m, 2H), 5.43 (m, 1H), 5.23 (m, 1H), 4.84 (m, 1H), 4.62 (d, 1H), 4.47 (t, 2H), 4.40 (d, 1H), 4.18 (m, 2H), 4.90 (m, 1H), 3.69 (m, 1H), 3.27 (m, 2H), 2.47 (t, 2H), 2.24-1.91 (m, 10H), 1.61 (m, 2H), 1.53-1.41 (m, 5H), 1.36-1.22 (m, 4H), 1.14 (dd, 6H).

[0277] MS: m/z=731 [M+H].sup.+

EXAMPLE 6

Synthesis of (Z)-isopropyl 7-((1R,2R,3R,5S)-2-((3R,E)-3-(3-(5,6-bis(nitrooxy)hexanamido) propanoyloxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)-3,5-dihydroxycyclopentyl) hept-5-enoate (corresponding to compound (2), (S)-isomer)

[0278] ##STR00041##

Step 1: Synthesis of tert-butyl 3-1[5,6-bis(nitrooxy)hexanoyl]amino}propanoate

[0279] To a solution of (5S)-5,6-bis(nitrooxy)hexanoic acid (Example 2, Step 5) (1.4 g, 4.68 mmol) and -ala-OtBu hydrochloride (0.85 g, 4.68 mmol) in DMF (10 ml) cooled at 0 C., TEA (1.3 ml, 9.36 mmol) was added dropwise. The solution was stirred for 15 minutes, then DCC (0.96 g, 4.68mmo1) and DMAP (61 mg, 0.47 mmol) were added. The solution was stirred at rt overnight. Then the solvent was removed under reduce pressure and the crude was dissolved in diethyleter and washed with HCl 1M to remove DMAP and TEA. The organic layer was washed with water, brine, dried over sodium sulfate, then evaporated affording 1.2 g of the title compound as a pale yellow oil (Yield: 90%). It was used in the next step without further purification.

[0280] MS: 366 [M+H].sup.+

[0281] TLC: (Cy:EtOAc 7:3) Rf=0.37

Step 2: Synthesis of 3-{[(5S)-5,6-bis (nitrooxy)hexanoyl]amino}propanoic acid

[0282] To a stirred solution of t-butyl-3-(6-(nitrooxy)hexanamido)propanoate (0.5 g, 1.4 mmol) in DCM (2.5 mL) at 0 C., BF.sub.3.Et.sub.2O (0.3 mL, 1.7 mmol) was added. After 10 min the solution was stirred at rt for 3 hr. The crude mixture was then poured on brine and the organic layer extracted, dried over sodium sulfate, evaporated to give 0.25 g of the desired product as a pale yellow oil (Yield: 58%). The residue obtained was used in the next step without further purification.

[0283] MS: 310 [M+H].sup.+

Step 3: Synthesis of 4-nitrophenyl 3-{[(5S)-5,6-bis(nitrooxy)hexanoyl]amino}propanoate

[0284] To a solution of 3-{[(5S)-5,6-bis(nitrooxy)hexanoyl]amino}propanoic acid (250 mg, 0.81 mmol) and DCC (167 mg, 0.81 mmol) in DCM (8 ml), p-nitrophenol (124 mg, 0.89) was added portion wise and the mixture was stirred overnight at rt. Then the mixture was filtered, evaporated and purified by flash chromatography (Gradient: Cy/AcOEt 5% to 70% in 12 CV), yielding 0.324 g of the desired product. (Yield: 93%).

[0285] MS: 431 [M+H].sup.+

[0286] TLC: (Cy:EtOAc 1:1) R.sub.f=0.35

Step 4: Synthesis of (Z)-isopropyl 7-((1R,2R,3R,5S)-2-((3R,E)-3-(3-(5,6-bis(nitrooxy)hexanamido) propanoyloxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)-3,5-dihydroxycyclopentyl) hept-5-enoate (corresponding to compound (2), (S)-isomer)

[0287] ##STR00042##

[0288] To a solution of isopropyl (Z)-7-((1R,5S,6R,7R)-3-butyl-7-((R,E)-3-hydroxy-4-(3-(trifluoromethyl)phenoxy)but-1-en-1-yl)-2,4-di oxa-3-borabicyclo[3.2.1]octan-6-yl)hept-5-enoateacid (Example 1, Step 3) (300 mg, 0.53 mmol) and 4-nitrophenyl 3-{[(5S)-5,6-bis(nitrooxy)hexanoyl]amino} propanoate (250 mg, 0.58 mmol) in dry DCM (15 ml), DMAP (65 mg, 0.53 mmol) was added dropwise and the mixture was stirred for 72 hrs. The solvent was removed under reduce pressure and the product was dissolved in MeOH (15 ml) and stirred for 4 hrs at room temperature. Then the mixture was evaporated and purified by C18 chromatography (H.sub.2O:CH.sub.3CN 8:2 to 1:1) yielding 0.101 g of the title compound as a clear oil (Yield: 24%).

[0289] .sup.1H NMR: (600 MHz, DMSO-d.sub.6) .92 (t, 1H), 7.51 (t, 1H), 7.29 (d, 1H), 7.25 (d, 2H), 5.67 (dd,1H), 5.59-5.52 (m, 2H), 5.45-5.35 (m, 2H), 5.22 (m, 1H), 4.90 (dd, 1H), 4.84 (m, 1H), 4.67 (dd, 1H), 4.60 (d, 1H), 4.39 (d, 1H), 4.18 (m, 2H), 3.90 (m, 1H), 3.68 (m, 1H), 3.26 (m, 2H), 2.47 (t, 2H), 2.24-2.13 (m, 4H), 2.06 (m, 3H), 1.94 (m, 3H), 1.70-1.28 (m, 8H), 1.13 (dd, 6H).

[0290] MS: m/z =792 [M+H].sup.+

[0291] TLC: (H.sub.2O:CH.sub.3CN 7:3) Rf: 0.28

EXAMPLE 7

Synthesis of (Z)-7-((1R,2R,3R,5S)-2-((R,E)-3-((S)-5,6-bis(nitrooxy)hexanoyloxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)-3,5-dihydroxycyclopentyl)hept-5-enoic acid (corresponding to compound (13), (S)-isomer)

[0292] ##STR00043##

Step 1: Synthesis of (Z)-7-((1R,2R,3R,5S)-3,5-dihydroxy-2-((R,E)-3-hydroxy-4-(3-(trifluoromethyl)phenoxy)but-1-en-1-yl)cyclopentyl)hept-5-enoic acid

[0293] ##STR00044##

[0294] To a solution of fluprostenol isopropyl ester (0.5 g, 1.0 mmol) in a mixture of CH.sub.2Cl.sub.2/CH.sub.3OH (25.5 ml, 9:1 v/v), a methanolic solution of NaOH 2M (2 ml, 4 eq.) was added and the mixture was stirred overnight at RT.

[0295] Then water and CH.sub.2Cl.sub.2 were added to the mixture, and the organic phase was separated to eliminate the unreacted ester. The aqueous layer was acidified to pH 2-3 with HCL 2 N and extracted twice with CH.sub.2Cl.sub.2. The combined organic layers were dried over Mg.sub.2SO.sub.4 and the solvent was removed to afford 0.44 g of the title compound (Yield: 97%).

[0296] MS: m/z=457 [MH].sup.

Step 2: Synthesis of (Z)-7-((1R,5S,6R,7R)-3-butyl-7-((R,E)-3-hydroxy-4-(3-(trifluoromethyl)phenoxy)but-1-en-1-yl)-2,4-dioxa-3-borabicyclo[3.2.1]octan-6-yl)hept-5-enoic acid

[0297] ##STR00045##

[0298] To a solution of (Z)-7-((1R,2R,3R,5S)-3,5-dihydroxy-2-((R,E)-3-hydroxy-4-(3-(trifluoromethyl)phenoxy)but-1-en-1-yl)cyclopentyl)hept-5-enoic acid (0.44 g, 0.968 mmol) in a mixture of Et.sub.2O/CH.sub.2Cl.sub.2 (6.9 ml, 2:1 v/v), butylboronic acid (99 mg, 0.968 mmol) was added. The solution was stirred for 2 minutes, then some molecular sieves (3 diameter) were added and the reaction mixture allowed to stir at room temperature for 48 hours. Then the solvent was removed under reduced pressure affording 0.365 g of the title compound (Yield: 72%) that was used in the next step without any further purification.

Step 3: Synthesis of (Z)-7-((1R,2R,3R,5 S)-2-((R,E)-3-((S)-5,6-bis(nitrooxy)hexanoyloxy)-4-(3-(trifluoromethyl)phenoxy)but-1-enyl)-3,5-dihydroxycyclopentyl)hept-5-enoic acid (corresponding to compound (13), (S)-isomer)

[0299] ##STR00046##

[0300] A mixture of (Z)-7-((1R,5S,6R,7R)-3-butyl-7-((R,E)-3-hydroxy-4-(3-(trifluoromethyl)phenoxy)but-1-en-1-yl)-2,4-dioxa-3-borabicyclo[3.2.1]octan-6-yl)hept-5-enoic acid (0.365g, 0.696 mmol), 4-nitrophenyl (S)-5,6-bis(nitrooxy)hexanoate (Example 2, Step 6) (0.30 mg, 0.835 mmol) and 4-dimethylaminopyridine (85 mg, 0.696 mmol) in CH.sub.2Cl.sub.2 (5 ml) was allowed to stir overnight at room temperature. Then other 85 mg (0.696 mmol) of DMAP were added, and the mixture was stirred for further 24 hours. The solvent was then removed under reduced pressure and the residue was dissolved in CH.sub.3CN/H.sub.2O (8:2 v/v) with 0.01% formic acid mixture and stirred at rt for 4 hours.

[0301] The solution was then concentrated and the mixture purified by reversed-phase chromatography (CH.sub.3CN/H.sub.2O from 5:95 to 50:50). The collected product was further purified by flash chromatography (ethyl acetate/cyclohexane 8:2 with 1% acetic acid), affording 71 mg of the target compound (Yield: 15%).

[0302] .sup.1H NMR (600 MHz, d6-DMSO) 7.51 (t, 1H), 7.30 (d, 1H), 7.27-7.23 (m, 2H), 5.67 (dd, 1H), 5.60-5.53 (m, 2H), 5.45-5.38 (m, 2H), 5.26-5.20 (m, 1H), 4.90 (dd, 1H), 4.66 (dd, 1H), 4.58 (br s, 1H), 4.21 (dd, 1H), 4.17 (dd, 1H), 3.92-3.88 (m, 1H), 3.72-3.67 (m, 1H), 2.44-2.34 (m, 2H), 2.24-2.16 (m, 2H), 2.14 (t, 2H), 2.11-2.04 (m, 1H), 1.98-1.89 (m, 5H), 1.77-1.69 (m, 2H), 1.68-1.60 (m, 2H), 1.53-1.41 (m, 3H), 1.35-1.28 (m, 1H).

[0303] MS: m/z=677 [MH].sup.

EXAMPLE F1

[0304] Intraocular Pressure (IOP) Lowering Activity in Hypertonic Saline-Induced IOP Increase in Rabbits

[0305] Compounds (9), (10), (11), (12) and (13) of the invention and compounds (A-1) and (B-1) disclosed in WO 2009/136281 were directly compared with fluprostenol isopropyl ester and bimatoprost, in the same model of ocular hypertensive rabbit. The hypotensive effects of the two groups of compounds were independently evaluated at different time points and in different sets of hypertensive rabbits.

[0306] Treatment Group A

[0307] Test compounds and dose (equimolar to the correspondent parent drug)

[0308] Compound (9); 0.04%

[0309] Compound (10); 0.043%

[0310] Compound (11); 0.039%

[0311] Compound (12); 0.044%

[0312] Compound (13); 0.041%

[0313] Fluprostenol isopropyl ester (parent drug); 0.03%

[0314] Vehicle: buffered aqueous solution pH 6, polyoxyl 40 hydrogenated castor oil 5 mg/ml, tromethamine 10 mg/ml, boric acid 6 mg/ml, mannitol 40 mg/ml, edetate disodium 0.5 mg/ml and benzalkonium chloride 0.15 mg/mL

[0315] Method

[0316] Male New Zealand white rabbits weighing 2.0-2.5 kg were used in the experiments. The transient increase in IOP was induced by the injection of 0.1 ml of hypertonic saline solution (5%) into the vitreous of both eyes.

[0317] Intraocular pressure (IOP) was determined using a Tono-Pen VET prior to hypertonic saline injection (basal) and at 30, 60, 120 and 240 min thereafter. Vehicle or tested compound dissolved in the vehicle was instilled immediately after the injection of hypertonic saline into the conjunctival pocket. Eyes were randomly assigned to different treatment groups.

[0318] One drop of 0.4% oxybuprocaine hydrochloride (Novesine, Sandoz) was instilled in each eye immediately before each set of pressure measurements.

[0319] Treatment Groups B (Prior Art Compounds)

[0320] Test Compounds and Dose

[0321] Compound (A-1): (1S,2E)-3-{(1R,2R,3 S,5R)-2-[(2Z)-7-(Ethyl amino)-7-oxohept-2-en-1-yl]-3,5-dihydroxycyclopentyl}-1-(2-phenylethyl)prop-2-en-1-yl 4-(nitrooxy) butanoate/0.13%

[0322] Compound (B-1): (1S,2E)-3-{(1R,2R,3S,5R)-2-[(2Z)-7-(Ethyl amino)-7-oxohept-2-en-1-yl]-3,5-dihydroxycyclopentyl}-1-(2-phenylethyl)prop-2-en-1-yl 6-(nitrooxy) hexanoate/0.14%

[0323] Bimatoprost (parent drug)/0.1%.

[0324] Vehicle: tween 80 0.5%, 0.2 mg/ml benzalkonium chloride, 0.3% DMSO in PBS pH 6.7

[0325] Method

[0326] Male New Zealand white rabbits weighing 2.0-2.5 kg were used in the experiments. The transient increase in IOP was induced by the injection of 0.1 ml of hypertonic saline solution (5%) into the vitreous of both eyes.

[0327] Intraocular pressure (IOP) was determined using a pneumatonometer prior to hypertonic saline injection (basal) and at 30, 60, 90, 180 and 300 min thereafter. Vehicle or tested compound dissolved in the vehicle was instilled immediately after the injection of hypertonic saline into the conjunctival pocket. Eyes were randomly assigned to different treatment groups.

[0328] One drop of 0.4% oxybuprocaine hydrochloride (Novesine, Sandoz) was instilled in each eye immediately before each set of pressure measurements.

[0329] The intraocular pressure values measured following topical application of the tested compounds are reported in Table 1 expressed as mean IOP change in treated eyes versus vehicle-treated eyes at same time point and baseline; Table 1 also reports the maximal IOP reduction of the tested compounds expressed in percentage.

[0330] Table 2 shows the IOP lowering activity of each tested compound compared to their parent drug at 60 minutes after topical administration.

[0331] As shown in Table 1 the maximal IOP reduction of each compound of the invention is greater than the maximal IOP reduction of the prior art compounds.

[0332] As shown in Table 2 the IOP reduction elicited by the compounds of the invention with respect to their parent drug (fluprostenol isopropyl ester) is greater than the IOP reduction elicited by the compounds of the prior art with respect to their parent drug (bimatoprost).

TABLE-US-00001 TABLE 1 IOP-lowering activity of the compounds at different time points after topical application in transiently ocular hypertensive New Zealand white rabbits Maximal IOP Mean IOP changes vs vehicle and baseline (mmHg) reduc. Compound 60 min 90 min 120 min 180 min 240 min 300 min (%) A-1 0.06 5.2 0.03 5.0 1.1 2.8 1.9 3.2 3 9 0.13% B-1 3.9 2.5 5.6 2.8 5.3 1.5 3.9 1.9 10 3 0.14% Bimatoprost 1.8 2.4 2.7 2.0 0.8 1.5 0.3 2.6 0.1% Comp. (9) 6.6 0.9 8.2 1.2 2.0 0.7 23 3 0.04% Comp. (10) 9.2 1.0 8.8 1.2 1.2 0.8 26 3 0.043% Comp. (11) 9.7 0.8 7.4 0.9 3.2 0.6 37 4 0.039% Comp. (12) 7.1 0.8 9.6 1.0 2.9 0.5 38 3 0.044% Comp. (13) 8.6 1.2 6.9 0.9 0.3 1.0 39 3 0.41% Fluprostenol 0.08 1.2 2.0 1.3 0.4 0.9 isopropyl ester 0.03%

TABLE-US-00002 TABLE 2 IOP lowering activity of the compounds compared to parent drug Delta.sub.60 min vs parent drug IOP.sub.60 min reduction vs vehicle Compound (mmHg) (%) (A-1) Not effective 4 0.13% (B-1) 2.1 7 0.14% Bimatoprost 0 1 0.1% Comp. (9) 6.5 20 0.04% Comp. (10) 9.1 28 0.043% Comp. (11) 9.6 32 0.039% Comp. (12) 7.0 22 0.044% Comp. (13) 8.5 31 0.041% Fluprostenol 0 3 isopropyl ester 0.03%