NITRIC OXIDE DONATING CARNOSINE COMPOUNDS

Abstract

The present invention relates to nitric oxide donor carnosine derivatives having a great efficacy in reducing elevated intraocular pressure, to processes for their preparation and to their use in the treatment and/or prophylaxis of glaucoma and ocular hypertension.

##STR00001##

Claims

1. A carnosine analogue of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof: ##STR00037## wherein R is:
—(Y)—(CH2)n-[CH(ONO2)]p-CH2-ONO2  1)
—(Y)—(CH2)n-X—(CH2)n1-[CH(ONO2)]p-CH2-ONO2  2) wherein: Y is O, NH or a covalent single bond; n is an integer from 1 to 10, preferably 1 to 4; with the proviso that in formula 2) when Y is O or NH, then n is not 1; n1 is an integer from 1 to 10, preferably 1 to 4; p is 0 or 1; X is O, NH or S; R1 is OH, —OR2, —NH2, —NHR2, wherein R2 is (C1-C10) linear or branched alkyl.

2. A carnosine analogue of formula (I) according to claim 1 wherein Y is a covalent single bond and X is O.

3. A carnosine analogue of formula (I) according to claim 1 wherein Y is a covalent single bond and R1 is OH.

4. A carnosine analogue of formula (I) according to claim 3 wherein R is
—(Y)—(CH2)n-[CH(ONO2)]p-CH2-ONO2  1)

5. A carnosine analogue of formula (I) according to claim 3 wherein R is
—(Y)—(CH2)n-X—(CH2)n1-[CH(ONO2)]p-CH2-ONO2  2) and X is O.

6. A carnosine analogue of formula (I) according to claim 1 wherein Y is a covalent single bond and R1 is —OCH3.

7. A carnosine analogue of formula (I) according to claim 6 wherein R is
—(Y)—(CH2)n-[CH(ONO2)]p-CH2-ONO2  1)

8. A carnosine analogue of formula (I) according to claim 6 wherein R is
—(Y)—(CH2)n-X—(CH2)n1-[CH(ONO2)]p-CH2-ONO2  2) and X is O.

9. A carnosine analogue of formula (I) according to claim 2 wherein p is 0.

10. A carnosine analogue of formula (I) according to claim 2 wherein p is 1.

11. A carnosine analogue according to claim 1 wherein carnosine is L-carnosine.

12. A carnosine analogue of formula (I) according to claim 1 or a pharmaceutically acceptable salt selected from the group consisting of: (S)-methyl-3-(1H-imidazol-4-yl)-2-(3-(5-(nitrooxy)pentanamido)propanamido) propanoate (compound (1)); (S)-methyl-3-(1H-imidazol-4-yl)-2-(3-(5-(nitrooxy)pentanamido)propanamido) propanoate hydrochloride (compound 1a); (S)-3-(1H-imidazol-4-yl)-2-(3-(5-(nitrooxy)pentanamido)propanamido)propanoic acid (compound (2)); (S)-3-(1H-imidazol-4-yl)-2-(3-(5-(nitrooxy)pentanamido)propanamido)propanoic acid 2,2,2-trifluoroacetate (compound (2a)); (S)-methyl-3-(1H-imidazol-4-yl)-2-(3-(6-(nitrooxy)hexanamido)propanamido) (compound (3)); (S)-methyl-3-(1H-imidazol-4-yl)-2-(3-(6-(nitrooxy)hexanamido)propanamido) 2,2,2-trifluoroacetate (compound (3a)); (S)-3-(1H-imidazol-4-yl)-2-(3-(6-(nitrooxy)hexanamido)propanamido)propanoic acid (to compound (4)); (S)-3-(1H-imidazol-4-yl)-2-(3-(6-(nitrooxy)hexanamido)propanamido)propanoic acid 2,2,2-trifluoroacetate (compound (4a)); (S)-methyl-3-(1H-imidazol-4-yl)-2-(3-(2-(2-(nitrooxy)ethoxy)acetamido) propanamido)propanoate (compound (5)); (S)-methyl-3-(1H-imidazol-4-yl)-2-(3-(2-(2-(nitrooxy)ethoxy)acetamido) propanamido)propanoate 2,2,2-trifluoroacetate (compound (5a)); (S)-3-(1H-imidazol-4-yl)-2-(3-(2-(2-(nitrooxy)ethoxy)acetamido)propanamido) propanoic acid (compound (6)); (S)-3-(1H-imidazol-4-yl)-2-(3-(2-(2-(nitrooxy)ethoxy)acetamido)propanamido) propanoic acid 2,2,2-trifluoroacetate (compound (6a)); (S)-methyl 2-(3-(2-((S)-2,3-bis(nitrooxy)propoxy)acetamido)propanamido)-3-(1H-imidazol-4-yl)propanoate (compound 7) (S)-methyl 2-(3-(2-((S)-2,3-bis(nitrooxy)propoxy)acetamido)propanamido)-3-(1H-imidazol-4-yl)propanoate 2,2,2-trifluoroacetate (compound 7a) (S)-2-(3-(2-((S)-2,3-bis(nitrooxy)propoxy)acetamido)propanamido)-3-(1H-imidazol-4-yl)propanoic acid (compound 8) (S)-2-(3-(2-((S)-2,3-bis(nitrooxy)propoxy)acetamido)propanamido)-3-(1H-imidazol-4-yl)propanoic acid 2,2,2-trifluoroacetate (compound 8a) (S)-methyl 2-(3-((5,6-bis(nitrooxy)hexanamido)propanamido)-3-(1H-imidazol-4-yl)propanoate (compound (9)); (S)-methyl 2-(3-((S)-5,6-bis(nitrooxy)hexanamido)propanamido)-3-(1H-imidazol-4-yl)propanoate 2,2,2-trifluoroacetate (compound (9a)); (S)-2-(3-((5,6-bis(nitrooxy)hexanamido)propanamido)-3-(1H-imidazol-4-yl)propanoic acid (compound (10)); (S)-2-(3-((S)-5,6-bis(nitrooxy)hexanamido)propanamido)-3-(1H-imidazol-4-yl)propanoic acid 2,2,2-trifluoroacetate (compound (10a)), (S)-2-(3-((S)-5,6-bis(nitrooxy)hexanamido)propanamido)-3-(1H-imidazol-4-yl)propanoic acid acetate (compound (10b)).

13. A composition comprising a carnosine analogue of formula (I) according to claim 1 and at least a further active ingredient selected from the group consisting of alpha adrenergic agonist, beta blocker, carbonic anhydrase inhibitor, prostaglandin analogs, non-steroidal anti-inflammatory drugs, steroidal anti-inflammatory drugs.

14. A carnosine analogue of formula (I) according to claim 1 for use as medicament.

15. A carnosine analogue of formula (I) according to claim 1 for use in the treatment and/or prophylaxis of hypertensive glaucoma, normotensive glaucoma and ocular hypertension.

16. A carnosine analogue according to claim 12 for use as medicament.

17. A carnosine analogue according to claim 12 for use in the treatment and/or prophylaxis of hypertensive glaucoma, normotensive glaucoma and ocular hypertension.

18. Pharmaceutical formulation comprising at least a carnosine analogue of formula (I) according to claim 1 and at least an ophthalmically acceptable component and/or ophthalmically acceptable vehicle.

19. Pharmaceutical formulation comprising at least a composition according to claim 13 and at least an ophthalmically acceptable component and/or ophthalmically acceptable vehicle.

Description

EXAMPLES

Glossary

[0132] ACN Acetonitrile [0133] Cy Cyclohexane [0134] DCC Dicyclohexylcarbodiimide [0135] DCM dichloromethane [0136] DMAP 4-dimethylaminopyridine [0137] DMF N,N-Dimethylformamide [0138] DMSO Dimethyl sulfoxide [0139] EDC*HCl N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride [0140] EtOAc Ethyl Acetate [0141] iPrOH 2-Propanol [0142] MTBE tert-Butyl methyl ether [0143] RT room temperature [0144] TEA Triethylamine [0145] TFA Trifluoroacetic acid

Example 1

Synthesis of (S)-methyl 3-(1H-imidazol-4-yl)-2-(3-(5-(nitrooxy)pentanamido) propanamido)propanoate (compound (1))

[0146] ##STR00017##

Step 1: Synthesis of 4-nitrophenyl 5-bromopentanoate

[0147] ##STR00018##

[0148] To a solution of 5-bromovaleric acid (5 g, 27.6 mmol) and DCC (5.7 g, 27.6 mmol) in DCM (200 ml), p-nitrophenol (4.23 g, 30.4 mmol) was added portionwise. The mixture was stirred overnight at RT, then precipitate was filtered off and the solvent was removed under reduced pressure. The crude was purified by flash chromatography (EtOAc in cyclohexane from 5% to 50%) affording 7.1 g of desired compound (Yield: 85%)

[0149] MS: m/z=303 [M+H].sup.+

[0150] TLC: (Cy/EtOAc 9:1) R.sub.f=0.40

Step 2: Synthesis of 4-nitrophenyl 5-(nitrooxy)pentanoate

[0151] ##STR00019##

[0152] To a solution of 4-nitrophenyl 5-bromopentanoate (5 g, 16.6 mmol) in CH.sub.3CN (145 ml), kept in the dark, AgNO.sub.3 (2 g, 12 mmol) was added. The mixture was heated at 80° C. After 15 minutes, AgNO.sub.3 (1 g, 6 mmol) was added and after 1 hour, the last portion of AgNO.sub.3 (1.25 g, 7.3 mmol) was added. The reaction was left under stirring for 2 hours.

[0153] 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 4.12 g of the desired compound (Yield: 87%).

[0154] MS: m/z=285 [M+H]+

[0155] TLC: (DCM 100%) Rf=0.30

Step 3: Synthesis of methyl (2S)-2-[(3-aminopropanoyl)amino]-3-(4H-imidazol-4-yl)propanoate hydrochloride

[0156] ##STR00020##

[0157] To a suspension of L-Camosine (3 g, 13.2 mmol) in dry MeOH (100 ml) cooled at 0° C., SOCl.sub.2 (1.13 ml, 15.6 mmol) was added dropwise and the mixture was stirred 10 minutes. The solution was then refluxed for 1 h and, after cooling to RT and the solvent was removed under reduced pressure. The product was triturated in DCM/MTBE 1/1 for 2 hours at rt, filtered and dried in vacuum to give the desired product in quantitative yield.

[0158] MS: m/z=241 [M+H].sup.+

Step 4: Synthesis of (S)-methyl 3-(1H-imidazol-4-yl)-2-(3-(5-(nitrooxy)pentanamido) propanamido)propanoate (corresponding to compound (1))

[0159] To a solution of 4-nitrophenyl 5-(nitrooxy)pentanoate (2 g, 7.04 mmol) and methyl (2S)-2-[(3-aminopropanoyl)amino]-3-(4H-imidazol-4-yl)propanoate hydrochloride (2.2 g, 7.04 mmol) in DMF (16 ml) cooled at 0° C., TEA (3.2 ml, 21.12 mmol) was added dropwise. The solution was stirred for 15 minutes and DMAP (84 mg, 0.7 mmol) was added. The solution was stirred at RT overnight, the solvent removed under reduced pressure. The residue was purified by flash chromatography (85:15 DCM:MeOH-1% NH.sub.4OH) affording 1.8 g of (S)-methyl 3-(1H-imidazol-4-yl)-2-(3-(5-(nitrooxy)pentanamido) propanamido)propanoate (Yield: 66%).

[0160] MS: m/z=386 [M+H].sup.+

[0161] TLC: (DCM/MeOH/NH.sub.4OH 79:20:1) R.sub.f=0.55

[0162] .sup.1H NMR (300 MHz, DMSO) δ 8.96 (s, 1H), 8.41 (d, J=7.9, 1H), 7.82 (t, J=5.5, 1H), 7.38 (s, 1H), 4.67-4.53 (m, 1H), 4.49 (t, J=6.2, 2H), 3.63 (s, 3H), 3.22-2.90 (m, 4H), 2.31-2.18 (m, 2H), 2.06 (t, J=7.0, 2H), 1.70-1.43 (m, 4H).

Example 2

Synthesis of (S)-3-(1H-imidazol-4-yl)-2-(3-(5-(nitrooxy)pentanamido) propanamido)propanoic acid 2,2,2-trifluoroacetate (compound (2a) that corresponds to compound (2) as 2,2,2-trifluoroacetate) and (S)-3-(1H-imidazol-4-yl)-2-(3-(5-(nitrooxy)pentanamido) propanamido)propanoic acid acetate (compound (2b) that corresponds to compound (2) as acetate)

[0163] ##STR00021##

[0164] To a solution of compounds (1) (described in Example 1) (0.3 g, 0.78 mmol) in H.sub.2O/Dioxane (1:3) cooled at 0° C., KOH 2.5M solution (0.9 ml, 1.44 mmol) was added dropwise. The solution was stirred for 1 hour. When the reaction was completed the solvent was removed under reduce pressure. The residue was dissolved in DCM and iPrOH (1%) and was purified by flash chromatography (60:40 DCM:iPrOH-2% Acetic acid) affording 89 mg of the title compound as acetate (2b).

[0165] MS: m/z=372 [M+H]+

[0166] TLC: (DCM/iPrOH/AceticAcid 60:38:2) Rf=0.20

[0167] In order to obtain the trifluoro acetate derivative (2a), a solution of compounds (1) (described in Example 1) (0.3 g, 0.78 mmol) in H.sub.2O/Dioxane (1:3) cooled at 0° C., was treated with KOH 2.5M solution (0.9 ml, 1.44 mmol), added dropwise. The solution was stirred for 1 hour. When the reaction was completed the solvent was removed under reduce pressure. The residue was dissolved in DCM and iPrOH (1%) and was purified by preparative HPLC:

[0168] Column: Kinetex AXIA 21.2×250 mm 5 micron C18

[0169] A: H2O 0.05% TFA

[0170] B: ACN 0.05% TFA

[0171] Flow: 16 ml/min

[0172] Gradient:

[0173] 95% A to 50% A in 10 min.

[0174] 50% A to 95% A in 2 min.

[0175] Detection: UV @ 210 nm

[0176] Affording 0.100 mg of the title compound (2a)

[0177] .sup.1H NMR (300 MHz, DMSO) δ 8.96 (s, 1H), 8.26 (m, 1H), 7.82 (m, 1H), 7.33 (s, 1H), 4.71-4.35 (m, 3H), 3.32-2.81 (m, 4H), 2.36-2.12 (m, 2H), 2.12-1.90 (m, 2H), 1.81-1.28 (m, 4H).

Example 3

Synthesis of (S)-methyl 3-(1H-imidazol-4-yl)-2-(3-(6-(nitrooxy)hexanamido) propanamido) 2,2,2-trifluoroacetate (compound (3a) that corresponds to compound (3) as 2,2,2-trifluoroacetate)

[0178] ##STR00022##

Step 1: Synthesis of 4-nitrophenyl 6-bromoexanoate

[0179] ##STR00023##

[0180] To a solution of 6-Bromohexanoic acid and DCC in DCM, p-nitrophenol was added portion wise. The mixture was stirred overnight at RT. Progress was checked by TLC (Cy/AcOEt 5:5). Once the reaction was complete, the mixture was filtered, evaporated and purified by flash chromatography (Gradient: Cy/AcOEt 5% to 50%), yielding 4-nitrophenyl 6-bromoexanoate (1.301 g, 80.3%).

[0181] .sup.1H NMR (300 MHz, CDCl.sub.3) δ 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

[0182] ##STR00024##

[0183] 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%).

[0184] .sup.1H NMR (300 MHz, CDCl.sub.3) δ 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 (S)-methyl 3-(1H-imidazol-4-yl)-2-(3-(6-(nitrooxy)hexanamido) propanamido)propanoate 2,2,2-trifluoroacetate

[0185] To a solution of 4-nitrophenyl 6-(nitrooxy)hexanoate (Step 2) (656.1 g, 2.2 mmol) and methyl-(2S)-2-[(3-aminopropanoyl)amino]-3-(4H-imidazol-4-yl)propanoate hydrochloride (obtained in Example 1, Step 3) (0.538.7, 2.2 mmol) in DMF (16 ml) cooled at 0° C., TEA (0.306 ml, 2.2 mmol) was added dropwise. The solution was stirred at RT overnight, the solvent removed under reduce pressure. The crude was then solubilized in water and purified by reverse phase HPLC. (Phase A: H.sub.2O+TFA 0.1%; Phase B: MeOH+TFA 0.1%; linear gradient t=0: A 95%, B 5%, t=20′: 100% B, t=25′ 100% B; flow: 14 ml/min) affording 399 mg of the title compound (Yield: 36.5%).

[0186] .sup.1H NMR (300 MHz, DMSO) δ 8.98 (s, 1H), 8.42 (d, J=7.7, 1H), 7.80 (t, J=5.7, 1H), 7.39 (s, 1H), 4.68-4.53 (m, 1H), 4.48 (t, J=6.6, 2H), 3.62 (s, 3H), 3.20-2.89 (m, 6H), 2.33-2.13 (m, 2H), 2.02 (t, J=7.3, 2H), 1.72-1.55 (m, 2H), 1.55-1.38 (m, 2H), 1.36-1.18 (m, 2H).

Example 4

Synthesis of (S)-3-(1H-imidazol-4-yl)-2-(3-(6-(nitrooxy)hexanamido)propanamido) propanoic acid 2,2,2-trifluoroacetate (compound (4a) that corresponds to compound (4) as 2,2,2-trifluoroacetate)

[0187] ##STR00025##

[0188] To a solution of (S)-methyl 3-(1H-imidazol-4-yl)-2-(3-(6-(nitrooxy)hexanamido) propanamido)propanoate (Example 3) (150 mg, 0.302 mmol) in Dioxane/H.sub.2O (3:1) cooled to 0° C., a solution of KOH (2.5N, 041 ml)) was added dropwise. The solution was left reacting for 3.5 h at room temperature. The solution was then cooled to 0° C. acidified with HCl conc. to pH=2 and the dioxane evaporated at reduced pressure. The crude was then purified with reverse phase HPLC. The crude was then purified with reverse phase HPLC. (Phase A: H.sub.2O+TFA 0.1%; Phase B: MeOH+TFA 0.1%; linear gradient t=−0: A 95%, B 5%, t=20′: 100% B, t=−25′ 100% B; flow: 14 ml/min) yielding the title compound (76 mg, 52%).

[0189] .sup.1H NMR (300 MHz, DMSO) δ 8.97 (s, 1H), 8.29 (d, J=8.1, 1H), 7.77 (t, J=5.7, 1H), 7.36 (s, 1H), 4.65-4.40 (m, 3H), 3.26-2.83 (m, 4H), 2.35-2.11 (m, 2H), 2.02 (t, J=7.3, 2H), 1.73-1.54 (m, 2H), 1.54-1.37 (m, 2H), 1.37-1.16 (m, 2H).

Example 5

Synthesis of (S)-methyl 3-(1H-imidazol-4-yl)-2-(3-(2-(2-(nitrooxy)ethoxy)acetamido) propanamido)propanoate 2,2,2-trifluoroacetate (compound (5a) that corresponds to compound (5) as 2,2,2-trifluoroacetate)

[0190] ##STR00026##

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

[0191] ##STR00027##

[0192] Ethylene glycol (4 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 Acetic anhydride (10.7 ml) was added dropwise under vigorous stirring. The mixture was left reacting at −30° C. for 2 h. The crude was poured on ice and diluted with AcOEt. The two phases were separated, and the organic phase washed with NaHCO.sub.3, H.sub.2O and brine. The organic phase was dried over Na.sub.2SO.sub.4 anhydrous, filtered and concentrated to afford reddish oil, containing 2-(2-hydroxyethoxy)ethyl nitrate (2.28 g, 40%) that was used without any further purification for the next step.

[0193] .sup.1H NMR (300 MHz, CDCl.sub.3) δ 4.69-4.59 (m, 2H), 3.85-3.66 (m, 4H), 3.61 (dd, J=5.2, 3.8 Hz, 2H).

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

[0194] ##STR00028##

[0195] To a solution of 2-(2-hydroxyethoxy)ethyl nitrate (Step 1) (1 g, 6.6 mmol) in acetone (25 ml), cooled at 0° C., a saturated solution of NaHCO.sub.3 (8 ml), NaBr (276.9 mg, 2.64 mmol) and TEMPO (206 mg, 1.32 mmol) were added. Trichloroisocyanuric acid (3.1 g, 13.2 mmol) was added portion wise. The reaction was allowed to reach RT and stirred for 3 h The mixture was then cooled to 0° C. and 10 ml of isopropanol were slowly added. The mixture was stirred at 0° C. for 30 minutes and the precipitate was filtered off and the solvent concentrated. The residue was basified with NaOH 2M (pH=12) and washed twice with EtOAc. To the aqueous phase HCl conc. was added until pH=2-3 and then extracted with EtOAc (5×). The combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated to afford the title compound (875 mg, 80.3%) as pale yellow oil.

[0196] .sup.1H NMR (300 MHz, CDCl.sub.3) δ 4.71-4.62 (m, 2H), 4.20 (s, 2H), 3.94-3.84 (m, 2H).

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

[0197] ##STR00029##

[0198] To a solution of 2-(2-(nitrooxy)ethoxy)acetic acid (Step 2) (875 mg, 5.3 mmol) and DCC (1.093 g, 5.3 mmol) in DCM, p-nitrophenol (811 mg, 5.83 mmol) was added portionwise. The mixture was stirred overnight at RT. Then the mixture was filtered, evaporated and purified by flash chromatography (Gradient: Cy/AcOEt 5% to 50%) yielding the title compound (1.43 g, 94.3%).

Step 4: Synthesis of (S)-methyl 3-(1H-imidazol-4-yl)-2-(3-(2-(2-(nitrooxy)ethoxy) acetamido)propanamido)propanoate 2,2,2-trifluoroacetate

[0199] A solution of 4-nitrophenyl 2-(2-(nitrooxy)ethoxy)acetate (251.8 mg, 0.88 mmol) (Step 3) and methyl (2S)-2-[(3-aminopropanoyl)amino]-3-(4H-imidazol-4-yl)propanoate hydrochloride (211.42 mg, 0.88 mmol) (prepared as described in Example 1, Step 3) was cooled to 0° C. and N-methyl-morpholine (0.097 ml, 0.88 mmol) was added dropwise until pH 7-8. The solution was stirred overnight at RT. The crude was then evaporated, solubilized in water and purified with reverse phase HPLC (Phase A: H.sub.2O+TFA 0.1%; Phase B: MeOH+TFA 0.1%; linear gradient t=0: A 95%, B 5%, t=20′: 100% B, t=25′ 100% B; flow: 14 ml/min) to afford the title compound (186 mg, 43.5%).

[0200] .sup.1H NMR (300 MHz, DMSO) δ 8.95 (s, 1H), 8.44 (d, J=7.8, 1H), 7.73 (t, J=5.7, 1H), 7.38 (s, 1H), 4.82-4.49 (m, 3H), 3.86 (s, 2H), 3.81-3.69 (m, 2H), 3.58 (s, J=21.8, 3H), 3.30-3.17 (m, 2H), 3.17-2.90 (m, 2H), 2.39-2.16 (m, 2H).

Example 6

Synthesis of (S)-3-(1H-imidazol-4-yl)-2-(3-(2-(2-(nitrooxy)ethoxy)acetamido) propanamido)propanoic acid 2,2,2-trifluoroacetate (compound (6a) that corresponds to compound (6) as 2,2,2-trifluoroacetate)

[0201] ##STR00030##

[0202] The title compound was obtained from (S)-methyl 3-(1H-imidazol-4-yl)-2-(3-(2-(2-(nitrooxy)ethoxy)acetamido) propanamido)propanoate (Example 5) following the same procedure described in Examples 2 and 4.

[0203] The crude was purified with reverse phase HPLC (Phase A: H.sub.2O+TFA 0.1%; Phase B: MeOH+TFA 0.1%; linear gradient t=0: A 95%, B 5%, t=20′: 100% B, t=25′ 100% B; flow: 14 ml/min) affording the title compound (70 mg, 47.6%).

[0204] .sup.1H NMR (300 MHz, DMSO) δ 8.95 (s, 1H), 8.34-8.31 (m, 1H), 7.76-7.72 (s, 1H), 7.38 (s, 1H), 4.67 (m, 2H), 4.53 (m, 1H), 3.86 (s, 2H), 3.73 (m, 2H), 3.23 (m, 2H), 3.12 (m, 1H), 2.95 (m, 1H), 2.27 (m, 2H).

Example 7

Synthesis of (S)-methyl 2-(3-((S)-5,6-bis(nitrooxy)hexanamido)propanamido)-3-(1H-imidazol-4-yl)propanoate (corresponding to compound (9), (S) isomer)

[0205] ##STR00031##

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

[0206] ##STR00032##

[0207] To a stirred solution of hex-5-enol (21.25 g, 200 mmol) and 4-nitrobenzoyl chloride (37.11 g, 200 mmol) in dichloromethane (300 mL) at 0° C., triethylamine (28 mL, 200 mmol) was added. The reaction was stirred at RT 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%).

[0208] MS: m/z=250 [M+H].sup.+

[0209] TLC: DCM 100% R.sub.f=0.4

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

[0210] ##STR00033##

[0211] A stirred solution of AD-Mix α (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 ethyl acetate (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 organic layer was extracted and the aqueous phase extracted twice with ethyl acetate (2×100 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%).

[0212] The residue was dissolved in diethylether (100 mL) and stirred overnight to give the title compound as white solid (8.1 g, 84%).

[0213] MS: 284 [M+H].sup.+

[0214] TLC: (DCM/MeOH-0.5%) R.sub.f=0.36

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

[0215] ##STR00034##

[0216] To a stirred solution of fuming nitric acid (3.6 mL, 88 mmol, 5 eq) in dichloromethane (3 mL) at 0° C., was added acetic anhydride (13.7 mL) and after 10 mins 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.

[0217] MS: 374 [M+H].sup.+

[0218] TLC: (DCM 100%) R.sub.f.sup.=0.37

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

[0219] ##STR00035##

[0220] 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 sodium hydroxide 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%).

[0221] TLC: (EtOAc/Cy-50%) R.sub.f.sup.=0.31

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

[0222] 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, ACN, 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%)

[0223] TLC: (DCM/MeOH-0.5%) R.sub.f=0.34

Step 6: Synthesis of (S)-methyl 2-(3-((S)-5,6-bis(nitrooxy)hexanamido)propanamido)-3-(1H-imidazol-4-yl)propanoate (corresponding to compound (9), (S) isomer)

[0224] To a solution of (5S)-5,6-bis(nitrooxy)hexanoic acid (2.4 g, 10 mmol) and methyl-(2S)-2-[(3-aminopropanoyl)amino]-3-(4H-imidazol-4-yl)-propanoate (Example 1, Step 3) (3.1 g, 10 mmol) in DMF (40 ml) cooled at 0° C., TEA (5.5 ml, 40 mmol) was added dropwise. The solution was stirred for 15 minutes EDC*HCl (1.91 g, 10 mmol) and DMAP (1.22 g, 10 mmol) were added. The solution was stirred at RT overnight, the solvent removed under reduced pressure. The residue was purified by flash chromatography (80:20 DCM:MeOH-1% NH.sub.4OH) affording 1.9 g of the (S)-methyl 2-(3-((S)-5,6-bis(nitrooxy)hexanamido)propanamido)-3-(1H-imidazol-4-yl)propanoate (Yield: 41%).

[0225] MS: m/z=461 [M+H].sup.+

[0226] TLC: (DCM/MeOH/NH.sub.4OH 79:20:1) R.sub.f=0.40

[0227] .sup.1H NMR (600 MHz, cd3od) δ 7.61 (s, 1H), 6.88 (s, 1H), 5.42 (m, 1H), 4.94-4.85 (m, 1H), 4.72-4.64 (m, 1H), 4.64-4.54 (m, 1H), 3.72 (s, 3H), 3.49-3.35 (m, 2H), 3.16-3.07 (m, 1H), 3.07-2.97 (m, 1H), 2.50-2.35 (m, 2H), 2.30-2.19 (m, 2H), 1.82-1.66 (m, 4H).

Example 8

Synthesis of (S)-2-(3-((S)-5,6-bis(nitrooxy)hexanamido)propanamido)-3-(1H-imidazol-4-yl)propanoic acid 2,2,2-trifluoroacetate (compound (10a) that corresponds to compound (10), (S) isomer, 2,2,2-trifluoroacetate) and (S)-2-(3-((S)-5,6-bis(nitrooxy)hexanamido)propanamido)-3-(1H-imidazol-4-yl)propanoic acid acetate (compound (10b) that corresponds to compound (10), (S) isomer, acetate)

[0228] ##STR00036##

[0229] To a solution of (S)-methyl 2-(3-((S)-5,6-bis(nitrooxy)hexanamido)propanamido)-3-(1H-imidazol-4-yl)propanoate (Example 7) (0.6 g, 1.3 mmol) in H.sub.2O/Dioxane (1:3) cooled at 0° C., KOH 2.5M solution (1.5 ml, 3.9 mmol) was added dropwise. The solution was stirred for 1 hour and the solvent was removed under reduced pressure. The crude was dissolved in DCM/water then HCl was added until pH of 1-2 and the solvent was removed under reduced pressure. The residue was solubilized in DCM and iPrOH (1%) and was purified by flash chromatography (50:40 DCM:iPrOH-2% Acetic acid) affording 400 mg of (S)-2-(3-((S)-5,6-bis(nitrooxy)hexanamido)propanamido)-3-(1H-imidazol-4-yl)propanoic acid acetate (10b) (Yield: 69%).

[0230] From 150 mg of (10b) 110 mg of (10a) were obtained by preparative HPLC according to the following conditions:

[0231] Column: Kinetex AXIA 21.2×250 mm 5 micron C18

[0232] A: H.sub.2O 0.05% TFA

[0233] B: ACN 0.05% TFA

[0234] Flow: 16 ml/min.

[0235] Gradient:

[0236] from 95% A to 50% A in 10 min.

[0237] from 50% A to 95% A in 2 min.

[0238] iso 95% A for 3 min.

[0239] Detection UV @ 210 nm

[0240] MS: m/z=447 [M+H].sup.+

[0241] .sup.1H NMR (600 MHz, d2o) δ 8.49 (s, 1H), 7.18 (s, 1H), 5.42-5.21 (m, 2H), 4.82-4.72 (m, 1H), 4.68-4.58 (m, 1H), 4.57-4.47 (m, 1H), 3.34-3.14 (m, 2H), 3.12-2.96 (m, 1H), 2.43-2.27 (m, 2H), 2.18-2.08 (m, 2H), 1.68-1.51 (m, 4H).

Example 9

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

[0242] The present study evaluated the intraocular pressure lowering effect of single applications of two compounds of the inventions (compound (1a) and compound (4a)) and two reference compounds at the same concentrations and in rabbits with induced IOP increase.

[0243] Tested Compounds

[0244] Compound (1a) is (S)-methyl-3-(1H-imidazol-4-yl)-2-(3-(5-(nitrooxy)pentanamido) propanamido)propanoate hydrochloride and was prepared from compound (1) obtained in Example 1 and hydrochloric acid by known methods. Compound (4a): was prepared as described in (Example 4)

[0245] Timolol and 5-ISMN were tested as reference compounds

[0246] Timolol is a drug commonly used for the treatment of glaucoma and ocular hypertension. Isosorbide mononitrate (5-ISMN) is a commonly used nitric oxide donor drug.

[0247] Adult male New Zealand White rabbits weighting 1.8-2.0 Kg were used in the experiments.

[0248] The transient increase in IOP was induced by the injection of 0.1 ml of hypertonic saline solution (5%) into the vitreous bilaterally (Krauss et al., 2011, Orihashi et al., 2005).

[0249] IOP was measured using a Tono-Pen XL prior to hypertonic saline injection (basal) and at 30, 60, 120 and 240 min thereafter. Vehicle (5% cremophor-EL; 0.3% DMSO; 0.2 mg/ml Benzalkonium chloride in PBS pH 6.0) or compound (1.0%) was instilled as eye drops immediately after hypertonic saline injection. Eyes were randomly assigned to different treatment groups. Vehicle and compounds were directly instilled into the conjunctiva pocket at the desired doses. One drop of 0.4% oxybuprocaine hydrochloride (Novesine, Sandoz) was instilled in each eye immediately before each set of pressure measurements.

[0250] Results are reported in Table 1 and they are expressed as IOP change (at 60, 120 and 240 minutes following topical administration) versus vehicle and versus IOP at basal before hypertonic saline injection. Single application of compounds (1a) and (4a) resulted in a significant IOP reduction compared to ISMN and timolol.

[0251] The experiment results showed that, 4 hours after instillation the compounds of the invention maintained their ocular hypotensive activity and the IOP-lowering effect in the groups treated with the compounds of the invention is higher than in the groups treated with timolol and 5-ISMN, demonstrating prolonged IOP-lowering effect of the compounds of the invention with respect to the reference compounds.

[0252] The experimental results revealed that a potent ocular hypotensive effect and a prolonged action were obtained by using the compounds of the invention.

TABLE-US-00001 TABLE 1 Intraocular pressure (IOP) lowering activity in hypertonic saline-induced IOP increase in rabbits IOP change (mmHg) Test Compound 60 120 240 (conc. 1%) minutes minutes minutes Compound (1a) −11.4 ± 0.8  −12.9 ± 0.4  −7.4 ± 0.5 Compound (4a) −7.6 ± 1.6 −9.0 ± 1.2 −5.3 ± 0.6 ISMN −0.7 ± 2.9 −6.6 ± 1.8 −1.2 ± 1.3 Timolol −5.7 ± 1.4 −8.4 ± 1.2  0.2 ± 1.2

Example 10

Intraocular Pressure (IOP) Lowering Activity in Ocular Normotensive New Zealand White Rabbits

[0253] The present study evaluated the long lasting intraocular pressure lowering effect of single application of two compounds of the inventions (compound (1a) and compound (4a)) with respect to a prior art compound, in an ocular normotensive animal model (rabbit) at the same concentrations.

[0254] Tested Compounds

[0255] Compound (1a) is (S)-methyl-3-(1H-imidazol-4-yl)-2-(3-(5-(nitrooxy) pentanamido) propanamido)propanoate hydrochloride and was prepared from compound (1) obtained in Example 1 and hydrochloric acid by known methods.

[0256] Compound (4a): was prepared as described in (Example 4)

[0257] The prior art compound is (R)-3-((S)-2-(3-aminopropanamido)-3-(1H-imidazol-4-yl)propanamido)propane-1,2-diyl dinitrate 2,2,2-trifluoroacetate (reference compound) and it is disclosed in M. Bertinaria et al., European Journal of Medicinal Chemistry, 54(2012) 103-112.

[0258] Adult male New Zealand White (NZW) rabbits weighting 1.8-2.0 Kg were used in the experiments.

[0259] IOP was measured using a pneumatonometer 30 CLASSIC™ before topical application (basal) and at different time points (30, 60, 120, 240 and 300 min) thereafter. Vehicle (5% cremophor-EL; 0.3% DMSO; 0.2 mg/ml bac in PBS pH 6.0) or compound of the invention were instilled as eye drops into the conjunctiva pocket. Eyes were randomly assigned to different treatment groups. One drop of 0.4% oxybuprocaine hydrochloride (Novesine, Sandoz) was instilled in each eye immediately before each set of pressure measurements. Results are reported in Table 2 and they are expressed as IOP change (at 60, 120, 180 and 240 minutes following topical administration) versus vehicle and versus IOP at basal.

[0260] Single application of compound (1a) and compound (4a) results in a significantly longer IOP reduction as compared to the reference compound.

[0261] The experiment results revealed that the compounds of the invention showed a higher and prolonged IOP-lowering effect with respect to the reference compound.

TABLE-US-00002 TABLE 2 Intraocular pressure (IOP) lowering activity in ocular normotensive rabbits IOP change (mmHg) Test Compound 60 120 180 240 (1%) minutes minutes minutes minutes Compound (1a) −1.2 ± 1.0 −1.5 ± 0.9 −1.9 ± 0.9 −1.7 ± 1.2 Compound (4a) −1.6 ± 0.4 −1.5 ± 0.6 −1.4 ± 0.6 −0.9 ± 0.6 Ref. compound −0.8 ± 0.6 −0.3 ± 0.4 −0.2 ± 0.4 −0.05 ± 0.6 

Example 11

Intraocular Pressure (IOP) Lowering Activity

[0262] The intraocular pressure lowering effect and the duration of the effect of a single application of the compound (9) were assessed in a transient ocular hypertensive rabbit model and in an ocular normotensive rabbit model respectively.

[0263] Tested Compound

Compound (9): (S)-methyl 2-(3-((S)-5,6-bis(nitrooxy)hexanamido)propanamido)-3-(1H-imidazol-4-yl)propanoate; the compound was prepared as described in Example 7.

Hypertonic Saline-Induced IOP Increase Rabbit Model

[0264] Adult male New Zealand White rabbits weighting 1.8-2.0 Kg were used in the experiments.

[0265] The transient increase in IOP was induced by the injection of 0.1 ml of hypertonic saline solution (5%) into the vitreous bilaterally (Krauss et al., 2011, Orihashi et al., 2005).

[0266] IOP was measured using a Tono-Pen XL prior to hypertonic saline injection (basal) and at 30, 60, 120 and 240 min thereafter. Vehicle (5% cremophor-EL; 0.3% DMSO; 0.2 mg/ml Benzalkonium chloride in PBS pH 6.0) or compound (9) (1.0%) was instilled as eye drops immediately after hypertonic saline injection. Eyes were randomly assigned to different treatment groups. Vehicle and compound (9) were directly instilled into the conjunctiva pocket at the desired doses. One drop of 0.4% oxybuprocaine hydrochloride (Novesine, Sandoz) was instilled in each eye immediately before each set of pressure measurements.

[0267] Experimental data are reported in Table 3 and they are expressed as IOP change (at 60, 120 and 240 minutes following topical administration) versus vehicle and versus IOP at basal before hypertonic saline injection

Ocular Normotensive Rabbit Model

[0268] Adult male New Zealand White (NZW) rabbits weighting 1.8-2.0 Kg were used in the experiments.

[0269] IOP was measured using a pneumatonometer 30 CLASSIC™ before topical application (basal) and at different time points (30, 60, 120, 240 and 300 min) thereafter. Vehicle (5% cremophor-EL; 0.3% DMSO; 0.2 mg/ml bac in PBS pH 6.0) or compound (9) (1%) were instilled as eye drops into the conjunctiva pocket. Eyes were randomly assigned to different treatment groups. One drop of 0.4% oxybuprocaine hydrochloride (Novesine, Sandoz) was instilled in each eye immediately before each set of pressure measurements.

[0270] Experimental data are reported in Table 4 and they are expressed as IOP change (at 60, 120, 180 and 240 minutes following topical administration) versus vehicle and versus IOP at basal.

Results

[0271] The results of Table 3 show that a single application of compound (9) resulted in a significant IOP reduction. When comparing the IOP reduction effects of compound (9) and of the reference compounds ISMN and timolol a higher IOP reduction was observed after topical application of compound (9) with respect to the IOP reduction induced by the reference compounds ISMN and timolol.

[0272] The hypotensive effects of compound (9) and of ISMN and timolol were evaluated using the same transient ocular hypertensive rabbit model, see Example 9, but in different groups of hypertensive rabbits.

[0273] The experiment results of the ocular normotensive rabbit model reported in Table 4 show that compound (9) showed a long lasting IOP-lowering effect. When comparing the duration of the IOP reduction effects induced by topical application of compound (9) and of the prior art compound (R)-3-((S)-2-(3-aminopropanamido)-3-(1H-imidazol-4-yl)propanamido)propane-1,2-diyl dinitrate 2,2,2-trifluoroacetate (Reference compound), a higher and prolonged IOP-lowering effect was observed with compound (9) with respect to the Reference compound.

[0274] The IOP-lowering effects of compound (9) and of the Reference compound were assessed in the same normotensive rabbit model, see Example 10, but using different groups of hypertensive rabbits.

TABLE-US-00003 TABLE 3 Intraocular pressure (IOP) lowering activity of compound (9) in hypertonic saline-induced IOP increase in rabbits IOP change (mmHg) Test compound 60 120 240 (1%) minutes minutes minutes Compound (9) −7.2 ± 1.0 −6.4 ± 1.0 −3.9 ± 0.9 ISMN −0.7 ± 2.9 −6.6 ± 1.8 −1.2 ± 1.3 Timolol −5.7 ± 1.4 −8.4 ± 1.2  0.2 ± 1.2

TABLE-US-00004 TABLE 4 Intraocular pressure (IOP) lowering activity of Compound (9) in ocular normotensive rabbits Test IOP change (mmHg) compound 30 60 120 180 240 300 (1%) minutes minutes minutes minutes minutes minutes Compound (9) −1.6 ± 0.2 −1.0 ± 0.4 −1.3 ± 0.2 −1.3 ± 0.3  −1.5 ± 0.4 −1.5 ± 0.4 Ref. −1.0 ± 0.5 −0.8 ± 0.6 −0.3 ± 0.4 −0.2 ± 0.4 −0.05 ± 0.6 −0.7 ± 0.4 compound