Quinone based nitric oxide donating compounds for ophthalmic use

Abstract

The present invention relates to nitric oxide donor compounds having a quinone based structure, to processes for their preparation and to their use in the treatment and/or prophylaxis of glaucoma and ocular hypertension.

Claims

1. A compound of formula (I) ##STR00061## or stereoisomers thereof, wherein: R.sub.1 is selected from H, methyl, methoxy; R.sub.2 is H or methyl; R.sub.3 is selected from H, methyl, methoxy; or R.sub.1 and R.sub.3 together form CHCHCHCH; R.sub.4 and R.sub.5 are methyl and n is 1, or R.sub.4 is H, R.sub.5 is selected from phenyl, para-fluorophenyl, para-methoxyphenyl, para-isopropylphenyl, para-trifluoromethyl phenyl or para-methylphenyl and n is 2; m is an integer from 1 to 10; p is 0 or 1; R.sub.6 is H or methyl.

2. A compound of formula (I) according to claim 1 wherein R.sub.2 is methyl; R.sub.4 and R.sub.5 are methyl and n is 1.

3. A compound of formula (I) according to claim 1 wherein R.sub.2 is methyl; R.sub.4 is H, R.sub.5 is selected from phenyl, para-fluorophenyl, para-methoxyphenyl, para-isopropylphenyl, para-trifluoromethylphenyl or para-methylphenyl and n is 2.

4. A compound of formula (I) according to claim 2 wherein R.sub.1 and R.sub.3 are methyl.

5. A compound of formula (I) according to claim 2 wherein R.sub.1 and R.sub.3 are methoxy.

6. A compound of formula (I) according to claim 2 wherein R.sub.1 and R.sub.3 together form CHCHCHCH.

7. A compound of formula (I) according to claim 2 wherein R.sub.1 is methyl and R.sub.3 is methoxy.

8. A compound of formula (I) according to claim 2 wherein R.sub.1 is methoxy and R.sub.3 is methyl.

9. A compound of formula (I) according to claim 4 wherein p is 0 and R.sub.6 is H.

10. A compound of formula (I) according to claim 4 wherein p is 1.

11. A compound of formula (I) according to claim 3 wherein R.sub.1 and R.sub.3 are methyl.

12. A compound of formula (I) according to claim 3 wherein R.sub.1 and R.sub.3 are methoxy.

13. A compound of formula (I) according to claim 3 wherein R.sub.1 and R.sub.3 together form CHCHCHCH.

14. A compound of formula (I) according to claim 3 wherein R.sub.1 is methyl and R.sub.3 is methoxy.

15. A compound of formula (I) according to claim 3 wherein R.sub.1 is methoxy and R.sub.3 is methyl.

16. A compound of formula (I) according to claim 11 wherein p is 0 and R.sub.6 is H.

17. A compound of formula (I) according to claim 11 wherein p is 1.

18. A compound of formula (I) according to claim 1 selected from: 4-(nitrooxy)butyl 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (Compound 1); 6-(nitrooxy)hexyl 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (Compound 2); 6-(nitrooxy)hexyl 4-phenyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (Compound 3); 4-(nitrooxy)butyl 3-methyl-3-(3-methyl-1,4-dioxo-1,4-dihydro naphthalene-2-yl)butanoate (Compound 4); 6-(nitrooxy)hexyl 4-(4-fluorophenyl)-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (Compound 5); 4-(nitrooxy)butyl 4-phenyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (Compound 6); 4-(nitrooxy)butyl 4-(4-fluorophenyl)-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (Compound 7); (5S,6R)-5,6-bis(nitrooxy)heptyl 3-methyl-3-(2,4,5-trimethyl-3,6-dioxo cyclohexa-1,4-dienyl)butanoate (Compound 8); 4-(nitrooxy)butyl 4-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dienyl)-4-phenylbutanoate (Compound 9); 5,6-bis(nitrooxy)hexyl-3-methyl 3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (Compound 10) 5,6-bis(nitrooxy)hexyl 3-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dienyl)-3-methylbutanoate (Compound 11) 4-(nitrooxy)butyl 3-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dienyl)-3-methylbutanoate (Compound 12) 5,6-bis(nitrooxy)hexyl 4-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dienyl)-4-phenylbutanoate (Compound 13) 5,6-bis(nitrooxy)hexyl 4-phenyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (Compound 14) (S)-5,6-bis(nitrooxy)hexyl 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (Compound 15) (R)-5, 6-bis(nitrooxy)hexyl 3-methyl-3-(2, 4, 5-trimethyl-3, 6-dioxocyclohexa-1,4-dienyl)butanoate (Compound 16) (S)-5,6-bis(nitrooxy)hexyl-3-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dienyl)-3-methylbutanoate (Compound 17), and stereoisomers thereof.

19. A method for treating hypertensive glaucoma, normotensive glaucoma, secondary glaucoma and/or ocular hypertension comprising administering to a subject in need thereof the compound of formula (I).

20. A method for treating age related macular degeneration, diabetic retinopathy, macular degeneration, inflammatory retinal disease, and/or uveitis comprising administering to a subject in need thereof the compound according to claim 1.

21. An ophthalmic composition comprising a compound of formula (I) according to claim 1 and at least an ophthalmically acceptable component and/or ophthalmically acceptable vehicle.

22. A composition comprising a compound of formula (I) according to claim 1 and one or more further active ingredients selected from alpha adrenergic agonists, beta blockers, carbonic anhydrase inhibitors, prostaglandin analogs, non-steroidal anti-inflammatory drugs, or a steroidal anti-inflammatory drugs.

23. A method for treating hypertensive glaucoma, normotensive glaucoma, secondary glaucoma and/or ocular hypertension comprising administering to a subject in need thereof of composition according to claim 22.

24. A method for treating age related macular degeneration, diabetic retinopathy, macular degeneration, inflammatory retinal disease, and/or uveitis comprising administering to a subject in need thereof the composition according to claim 21.

25. An ophthalmic composition comprising a composition according to claim 22 and at least an ophthalmically acceptable component and/or ophthalmically acceptable vehicle.

Description

Example 1

Synthesis of 4-(nitrooxy)butyl 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (Compound 1)

(1) ##STR00016##

Step 1: Synthesis of 6-hydroxy-4,4,5,7,8-pentamethylchroman-2-one

(2) ##STR00017##

(3) A synthetic procedure similar to the one described by Carpino et al., J. Org. Chem., 1989, 54, 3303-3310 was used.

(4) Methanesulfonic acid (20 mL) was heated at 70 C. In parallel, 2,3,5-trimethylbenzene-1,4-diol (2.0 g, 13.14 mmol) and methyl 3-methylbut-2-enoate (1.94 mL, 13.14 mmol, 1 eq) were added quickly and the reaction was heated for 2 h at this temperature. The reaction was then poured in water and, after cooling, was extracted with EtOAc (3100 mL). The combined organic layers were washed successively with water, saturated NaHCO.sub.3, water and brine, dried (Na.sub.2SO.sub.4), filtered and evaporated. The residue was crystallized from 30% CHCl.sub.3 in n-Hexane to give the title compound as a pale grey solid (1.86 g, Yield: 60%). Melting point: 185 C.

(5) .sup.1H NMR (300 MHz, CDCl.sub.3) 4.63 (s, 1H), 2.54 (s, 2H), 2.36 (s, 3H), 2.22 (s, 3H), 2.18 (s, 3H), 1.45 (s, 6H).

Step 2: Synthesis of 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanoic acid (Intermediate 2)

(6) ##STR00018##

(7) The reaction was performed according to conditions described by Borchardt et al., J. Am. Chem. Soc., 1972, 94, 9175.

(8) A stirred solution of 6-hydroxy-4,4,5,7,8-pentamethylchroman-2-one (2.0 g, 0.853 mmol) in 10% aqueous acetonitrile (100 mL) was added with a solution of freshly recrystallised NBS (1.6 g, 0.853 mmol, 1 eq) in acetonitrile (20 mL). The reaction was stirred for 1 h and then diluted with water (100 mL) and extracted with Et.sub.2O (3100 mL). The combined organic layers were washed with water and brine, dried (Na.sub.2SO.sub.4), filtered and evaporated. The residue was crystallized from Et.sub.2O/n-Hexane to give the title compound as a yellow solid (1.64 g, Yield: 77%).

(9) .sup.1H NMR (300 MHz, CDCl.sub.3) 11.08-8.78 (m, 1H), 3.02 (s, 2H), 2.14 (s, 3H), 1.95 (s, 3H), 1.93 (s, 3H), 1.44 (s, 6H).

Step 3: Synthesis of 4-hydroxybutyl 4-nitrobenzoate

(10) ##STR00019##

(11) A stirred solution of 1,4-butandiol (3.0 g, 33.29 mmol, 1.1 eq) and 4-nitrobenzoyl chloride (5.56 g, 29.96 mmol) in EtOAc (100 mL) cooled to 0 C. was added dropwise with triethylamine (4.6 mL, 33.3 mmol, 1.1 eq) and the reaction was stirred vigorously for 6 h. The reaction was diluted with water and the organic layer separated, washed with HCl 0.1 M, water and brine. The organic phase was dried (Na.sub.2SO.sub.4), filtered and evaporated. The residue was triturated in cold Et.sub.2O and the solid filtered off. The filtrate was evaporated to give the title compound as a viscous oil which solidified upon standing (2.86 g, Yield: 40%).

(12) .sup.1H NMR (300 MHz, CDCl.sub.3) 8.30 (d, J=8.8, 2H), 8.22 (d, J=8.8, 2H), 4.44 (t, J=6.5, 2H), 3.76 (t, J=6.3, 2H), 1.93 (dt, J=14.4, 6.7, 2H), 1.75 (dt, J=13.2, 6.4, 2H).

Step 4: Synthesis of 4-(nitrooxy)butyl 4-nitrobenzoate

(13) ##STR00020##

(14) Concentrated nitric acid (1.9 mL, 45.15 mmol, 3 eq) was added dropwise to acetic anhydride (20 mL) cooled to 0 C. Then solid 4-hydroxybutyl 4-nitrobenzoate was added and the reaction was stirred at this temperature for 30 min then poured on ice. After melting, the organic oil was separated from the aqueous liquid and diluted with EtOAc. The organic layer was washed with NaHCO3 (230 mL), water and brine, dried (Na2SO4), filtered and evaporated. The residue was purified by flash chromatography (Biotage System, SNAP Cartridge silica 100 g, eluent: n-hexane/ethyl acetate 85/15 to n-hexane/ethyl acetate 75/25 during 8 CV) affording the title compound as a yellow oil (3.73 g, Yield: 87%).

(15) .sup.1H NMR (300 MHz, CDCl.sub.3) 8.34-8.29 (m, 2H), 8.24-8.19 (m, 2H), 4.56 (t, J=5.9, 2H), 4.44 (t, J=6.0, 2H), 1.99-1.90 (m, 4H).

Step 5: Synthesis of 4-hydroxybutyl nitrate

(16) ##STR00021##

(17) A stirred solution of 4-(nitrooxy)butyl 4-nitrobenzoate (2.13 g, 7.49 mmol) in a 3/1 THF/EtOH mixture (40 mL) cooled to 0 C. was added with NaOH 1M (7.5 mL, 1 eq). The reaction was stirred at this temperature for 3 h then diluted with EtOAc and water. The organic layer was separated, washed with water and brine, dried (Na.sub.2SO.sub.4), filtered and evaporated. The residue was purified flash chromatography (Biotage System, SNAP Cartridge silica 100 g, eluent: n-hexane/ethyl acetate 85/15 to n-hexane/ethyl acetate 75/25 during 8 CV) affording the title compound as a colourless oil (0.48 g, Yield: 49%).

(18) .sup.1H NMR (300 MHz, CDCl.sub.3) 4.52 (t, J=6.5, 2H), 3.72 (t, J=6.2, 2H), 1.87 (dt, J=14.2, 6.5, 2H), 1.75-1.63 (m, 1H).

Step 6: Synthesis of 4-(nitrooxy)butyl 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (Compound 1)

(19) A stirred solution of 4-hydroxybutyl nitrate (prepared in Step 5) (2.0 g, 14.8 mmol) and 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanoic acid (prepared in Step 2) (3.70 g, 14.8 mmol) in dry CH.sub.2Cl.sub.2 cooled to 0 C. was added with EDC (3.12 g, 16.28 mmol, 1.1 eq) and a catalytic amount of DMAP (0.05 g). The reaction was stirred for 5 h at this temperature and then washed with water, HCl 1M, water and brine, dried (Na.sub.2SO.sub.4), filtered and evaporated. The residue was purified by flash chromatography (Biotage System, 2 SNAP Cartridge silica 340 g, eluent: n-hexane/ethyl acetate 85/15 to n-hexane/ethyl acetate 70/30 during 8 CV) affording the title compound as a yellow oil (5.02 g, Yield: 92%).

(20) .sup.1H NMR (300 MHz, CDCl.sub.3) 4.45 (t, J=6.2, 2H), 4.01 (t, J=6.1, 2H), 2.98 (s, 2H), 2.14 (s, 3H), 1.94 (s, 6H), 1.82-1.62 (m, 4H), 1.42 (s, 6H).

Example 2

Synthesis of 6-(nitrooxy)hexyl 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (Compound 2)

(21) ##STR00022##

Step 1: Synthesis of 6-Nitrooxy-hexan-1-ol

(22) ##STR00023##

(23) A solution of 6-bromohexan-1-ol (2.2 mL, 16.6 mmol) in CH.sub.3CN (100 mL) was added with silver nitrate (5.95 g, 35 mmol, 2 eq). The reaction was stirred at room temperature for 3 days. The reaction was quenched by addition of a solution of brine. After 15 min of stirring, the solution was filtered, extracted with ethyl acetate, washed with H.sub.2O, brine, dried over sodium sulfate, filtered and evaporated. The residue was purified by column chromatography (Biotage System, SNAP Cartridge silica 100 g, eluent: n-hexane/ethyl acetate 80/20 to n-hexane/ethyl acetate 50/50 during 12 CV) to give the desired product as a colorless oil (2.34 g, Yield: 86%).

(24) .sup.1H NMR (300 MHz, CDCl.sub.3) 4.47 (t, J=6.6 Hz, 2H), 3.68 (t, J=6.1 Hz, 2H), 1.77 (m, 2H), 1.62 (m, 2H), 1.48 (m, 4H), 1.27 (s, 1H).

Step 2: Synthesis of 6-(nitrooxy)hexyl 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate

(25) A stirred solution of 6-hydroxyhexyl nitrate (164 mg, 1.0 mmol) and 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanoic acid (prepared in Example 1, Step 2) (250 mg, 1.0 mmol) in dry CH2C12 cooled to 0 C. was added with EDC (202 mg, 1.1 mmol, 1.1 eq) and a catalytic amount of DMAP (0.02 g). The reaction was stirred for 16 h from 0 C. to rt. The reaction was washed with water, HCl 1M, water and brine, dried (Na.sub.2SO.sub.4), filtered and evaporated. The residue was purified by flash chromatography (Biotage System, SNAP Cartridge silica 100 g, eluent: n-hexane/ethyl acetate 85/15 to n-hexane/ethyl acetate 70/30 during 8 CV) affording the title compound as a yellow oil (286 mg, Yield: 72%).

(26) .sup.1H NMR (300 MHz, CDCl.sub.3) 4.44 (t, J=6.6, 2H), 3.97 (t, J=6.6, 2H), 2.97 (s, 2H), 2.12 (s, 3H), 1.94 (d, J=10.4, 6H), 1.77-1.65 (m, 2H), 1.63-1.50 (m, 2H), 1.47-1.41 (m, 6H), 1.41-1.30 (m, 4H).

Example 3

Synthesis of 6-(nitrooxy)hexyl 4-phenyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (Compound 3)

(27) ##STR00024##

Step 1: Synthesis of 4-(2,5-dihydroxy-3,4,6-trimethylphenyl)-4-phenylbutanoic acid

(28) ##STR00025##

(29) The reaction was performed according to conditions described by Mitsuru et al., J. Med. Chem. Soc., 1989, 32, 2214-2221.

(30) Boron trifluoride etherate (0.25 ml; 1.99 mmol) was added dropwise to a mixture of trimethylhydroquinone (1.0 g; 6.57 mmol) and -phenyl--butyrolactone (1.1 g; 6.57 mmol) in toluene (70 ml) at 60 C. during 10 minutes. The mixture was stirred for further 2 hours and then the solvent was evaporated under reduced pressure. The residue was purified by flash chromatography (Biotage system, SNAP Cartridge silica 100 g, EtOAc in n-hexane from 9% to 60% in 10 CV) affording the title compound (0.74 g; Yield: 36%) as an orange solid.

(31) .sup.1H NMR (300 MHz, CDCl.sub.3) 7.39-7.07 (m, 5H), 4.72-4.25 (m, 3H), 2.74-2.25 (m, 4H), 2.25 (s, 3H), 2.08 (s, 3H), 1.98 (m, 2H).

Step 2: Synthesis of 4-phenyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanoic acid

(32) ##STR00026##

(33) To a solution of 4-(2,5-dihydroxy-3,4,6-trimethylphenyl)-4-phenylbutanoic acid (0.74 g; 2.33 mmol) in CH.sub.3CN:H.sub.2O 1:1 (50 ml), Ammonium cerium nitrate (3.3 g; 5.87 mmol) was added. The mixture was stirred 3 hours at room temperature then was poured into H.sub.2O (30 ml). Et.sub.2O (20 ml) was added, the two phases were separated and the organic layer was extracted with Et.sub.2O (220 ml). The combined organic layers were washed with brine, dried on Na.sub.2SO.sub.4 and concentrated affording 560 mg of the title compound without any further purification.

(34) .sup.1H NMR (300 MHz, CDCl.sub.3) 7.40-7.06 (m, 5H), 4.35 (t, J=7.6, 1H), 2.77-2.25 (m, 4H), 2.15-2.03 (m, 3H), 1.97 (m, 6H).

Step 3: Synthesis of 6-(nitrooxy)hexyl 4-phenyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (compound (3))

(35) To a solution of 4-phenyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanoic acid (0.29 g; 0.92 mmol) and 6-Nitrooxy-hexan-1-ol (synthesised in Example 2, step 1) (0.17 mg; 0.92 mmol) in CH.sub.2Cl.sub.2 (5 ml), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (0.29 g; 1.38 mmol) and DMAP cat. were added. The solution was stirred 30 minutes at 0 C. and 4 hours at room temperature then washed with a solution of NaH.sub.2PO.sub.4 5% (5 ml), H.sub.2O (5 ml) and brine. The organic layer was dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by flash chromatography (Biotage SP1 instrument, SNAP cartridge silica 50 g, Hex/EtOAc 9:1, 10 CV) affording the title compound (0.35 g; Yield: 83%)

(36) .sup.1H NMR (300 MHz, CDCl.sub.3) 7.37-7.08 (m, 5H), 4.44 (t, 2H), 4.38-4.27 (m, 1H), 4.06 (t, 2H), 2.68-2.52 (m, 1H), 2.52-2.35 (m, 1H), 2.35-2.26 (m, 2H), 2.07 (s, 3H), 1.97 (m, 6H), 1.80-1.66 (m, 2H), 1.66-1.52 (m, 2H), 1.50-1.29 (m, 4H).

Example 4

Synthesis of 4-(nitrooxy)butyl 3-methyl-3-(3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)butanoate (Compound 4)

(37) ##STR00027##

Step 1: Synthesis of 6-hydroxy-4,4,5-trimethyl-3,4-dihydro-2H-benzo[h]chromen-2-one

(38) ##STR00028##

(39) Methanesulfonic acid (30 mL) was heated at 70 C. In parallel, 2-methylnaphthalene-1,4-diol (4.75 g, 25.0 mmol) and methyl 3-methylbut-2-enoate (2.85 g, 25.0 mmol, 1 eq) were added quickly and the reaction was heated for 2 h at this temperature. The reaction was then poured in water and, after cooling, was extracted with EtOAc (3100 mL). The combined organic layers were washed successively with water, saturated NaHCO.sub.3, water and brine, dried (Na.sub.2SO.sub.4), filtered and evaporated. The residue was purified flash chromatography (Biotage System, 2 SNAP Cartridge silica 100 g, eluent: n-hexane/ethyl acetate 90/10 to n-hexane/ethyl acetate 70/30 during 10 CV) affording the title compound as a pale yellowish solid (2.26 g, Yield: 35%).

(40) .sup.1H NMR (300 MHz, CDCl.sub.3) 7.90 (d, J=8.3, 2H), 7.45 (d, J=8.0, 2H), 3.88-3.67 (m, 2H), 2.45 (s, 3H), 1.56 (s, 6H).

Step 2: Synthesis of 3-methyl-3-(3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)butanoic acid

(41) ##STR00029##

(42) A stirred solution of 6-hydroxy-4,4,5-trimethyl-3,4-dihydro-2H-benzo[h]chromen-2-one (1.2 g, 4.44 mmol) in 10% aqueous acetonitrile (100 mL) was added with a solution of freshly recrystallised NBS (0.8 g, 4.44 mmol, 1 eq) in acetonitrile (20 mL). The reaction was stirred for 1 h and then diluted with water (100 mL) and extracted with Et.sub.2O (3100 mL). The combined organic layers were washed with water and brine, dried (Na.sub.2SO.sub.4), filtered and evaporated. The residue was purified flash chromatography (Biotage System, SNAP Cartridge silica 100 g, eluent: n-hexane/ethyl acetate 70/30 to n-hexane/ethyl acetate 50/50 during 8 CV) affording the title compound as a yellow oil (0.86 g, Yield: 68%).

(43) .sup.1H NMR (300 MHz, CDCl.sub.3) 12.11 (m, 1H), 8.06-7.99 (m, 1H), 7.87-7.78 (m, 1H), 7.70-7.58 (m, 2H), 3.02 (s, 2H), 2.14 (s, 3H), 1.44 (s, 6H).

Step 3: 4-(nitrooxy)butyl 3-methyl-3-(3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)butanoate (Compound 4)

(44) A solution of 4-hydroxybutyl nitrate (synthesized in Example 1, steps 1, 2 and 3) (150 mg, 1.11 mmol) and 3-methyl-3-(3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)butanoic acid (303 mg, 1.11 mmol, 1 eq) in dry CH.sub.2Cl.sub.2 cooled to 0 C. was added with EDC (234 mg, 1.22 mmol, 1.1 eq) and a catalytic amount of DMAP. The reaction was stirred for 6 h at 0 C. and then washed with water, HCl 0.1 M, water and brine, dried (Na.sub.2SO.sub.4), filtered and evaporated. The residue was purified flash chromatography (Biotage System, SNAP Cartridge silica 100 g, eluent: n-hexane/ethyl acetate 80/20 to n-hexane/ethyl acetate 70/30 during 8 CV) affording the title compound as a yellow oil (268 mg, yield: 62%).

(45) .sup.1H NMR (300 MHz, CDCl.sub.3) 8.02 (dd, J=6.1, 2.9, 1H), 7.89-7.80 (m, 1H), 7.70-7.58 (m, 2H), 4.36 (t, J=6.3, 2H), 3.94 (t, J=6.2, 2H), 3.10 (s, 2H), 2.32 (s, 3H), 1.67 (dt, J=10.8, 6.1, 2H), 1.65-1.45 (m, 14H).

Example 5

Synthesis of 6-(nitrooxy)hexyl 4-(4-fluorophenyl)-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (Compound 5)

(46) ##STR00030##

Step 1: Synthesis of 4-(2,5-dihydroxy-3,4,6-trimethylphenyl)-4-(4-fluorophenyl)butanoic acid

(47) ##STR00031##

(48) The reaction was performed according to conditions described by Mitsuru et al., J. Med. Chem. Soc., 1989, 32, 2214-2221.

(49) Boron trifluoride etherate (0.21 ml; 1.65 mmol) was added dropwise to a mixture of trimethylhydroquinone (0.50 g; 3.30 mmol) and -(4-fluorophenyl)--butyrolactone (0.59 g; 3.30 mmol) in toluene (10 ml) at 60 C. during 10 minutes. The mixture was stirred for further 2 hours and then the solvent was evaporated under reduced pressure. The residue was purified by flash chromatography (Biotage system, SNAP Cartridge silica 50 g, EtOAc in n-hexane from 9% to 60% in 10 CV) affording the title compound (0.48 g; Yield: 43%) as an orange solid.

Step 2: Synthesis of 4-(4-fluorophenyl)-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanoic acid

(50) ##STR00032##

(51) To a solution of 4-(2,5-dihydroxy-3,4,6-trimethylphenyl)-4-(4-fluorophenyl)butanoic acid (0.48 g; 1.44 mmol) in CH.sub.3CN:H.sub.2O 1:1 (40 ml), ammonium cerium nitrate (2.04 g; 3.60 mmol) was added. The mixture was stirred 3 hours at room temperature then was poured into H.sub.2O (30 ml). Et.sub.2O (20 ml) was added, the two phases were separated and the organic layer was extracted with Et.sub.2O (220 ml). The combined organic layers were washed with brine, dried on Na.sub.2SO.sub.4 and concentrated affording 430 mg of the title compound without any further purification.

(52) .sup.1H NMR (300 MHz, CDCl.sub.3) 7.32-7.19 (m, 3H), 6.97 (m, 2H), 4.29 (t, J=7.6, 1H), 2.70-2.25 (m, 4H), 2.10 (s, 3H), 2.03-1.89 (m, 6H).

Step 3: 6-(nitrooxy)hexyl 4-(4-fluorophenyl)-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (Compound 5)

(53) To a solution of 4-(4-fluorophenyl)-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanoic acid (0.22 g; 0.66 mmol) and 6-Nitrooxy-hexan-1-ol (synthesised in Example 2, step 1) (0.12 mg; 0.66 mmol) in CH.sub.2Cl.sub.2 (5 ml), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (0.19 g; 0.98 mmol) and DMAP cat. were added. The solution was stirred 30 minutes at 0 C. and 4 hours at room temperature then washed with a solution of NaH.sub.2PO.sub.4 5% (5 ml), H.sub.20 (5 ml) and brine. The organic layer was dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by flash chromatography (Biotage SP1 instrument, SNAP cartridge silica 50 g, Hex/EtOAc 9:1, 10 CV) affording the title compound (0.18 g; Yield: 58%).

(54) .sup.1H NMR (300 MHz, CDCl.sub.3) 7.36-7.17 (m, 3H), 7.05-6.88 (m, 2H), 4.44 (t, 2H), 4.28 (t, 1H), 4.06 (t, 2H), 2.67-2.33 (m, 2H), 2.33-2.22 (m, 2H), 2.11 (s, 3H), 2.00 (s, 3H), 1.95 (s, 3H), 1.80-1.55 (m, 4H), 1.49-1.30 (m, 4H).

Example 6

Synthesis of 4-(nitrooxy)butyl 4-phenyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (Compound 6)

(55) ##STR00033##

Step 1: Synthesis of 4-chlorobutyl 4-nitrobenzoate

(56) ##STR00034##

(57) To a solution of 4-Chlorobutanol (1.09 g; 10.04 mmol) and TEA (1.7 ml; 12.05 mmol) in CH.sub.2Cl.sub.2 (25 ml) cooled at 0 C., 4-Nitrobenzoyl chloride (2.23 g; 12.05 mmol) was added portionwise. The mixture was stirred 2 hours at room temperature then was washed with NaH2PO4 (25 ml), H.sub.2O and brine. The residue was purified by flash chromatography (Biotage SP1 instrument, SNAP cartridge silica 100 g, n-Hexane/EtOAc 9:1, 10 CV) affording the title compound (2.48 g; Yield: 96%)

(58) .sup.1H NMR (300 MHz, CDCl.sub.3) 8.38-8.25 (m, 2H), 8.25-8.14 (m, 2H), 4.55-4.33 (m, 2H), 3.73-3.53 (m, 2H), 2.13-1.85 (m, 4H).

Step 2: Synthesis of 4-(nitrooxy)butyl 4-nitrobenzoate

(59) ##STR00035##

(60) To a solution of 4-chlorobutyl 4-nitrobenzoate (2.48 g; 9.62 mmol) in CH.sub.3CN (40 ml), NaI (5.77 g, 38.30 mmol) was added. The mixture was heated in a microwave apparatus (40 minutes; 120 C.) then the salts were filtered off and the solvent evaporated under reduced pressure. EtOAc (50 ml) was added and the solution was washed with a solution of Na.sub.2S.sub.2O.sub.5 5% (50 ml), H.sub.2O and brine. The organic layer was dried on Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was dissolved in CH.sub.3CN (40 ml) and AgNO3 (1.97 g; 11.54 mmol) was added. The mixture was heated at the mw for 15 minutes at 120 C. then the salts were filtered off and the solvent evaporated under reduced pressure. EtOAc (30 ml) was added, the precipitate was removed again by filtration and the solvent was evaporated. This procedure was repeated three times then the organic layer was washed with brine, dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by flash chromatography (Biotage SP1 instrument, SNAP cartridge silica 100 g, EtOAc in n-hexane from 5% to 40% in 10 CV) affording the title compound (2.50 g; Yield: 93%) as a clear oil.

Step 3: Synthesis of 4-hydroxybutyl nitrate

(61) ##STR00036##

(62) To a solution of 4-(nitrooxy)butyl 4-nitrobenzoate (2.5 g; 8.76 mmol) in THF (30 ml) cooled at 0 C., NaOH 2M (8.7 ml; 17.53 mmol) was added dropwise. The solution was stirred 4 hours at room temperature then was diluted with NaHCO3 sutured solution (20 ml) and extracted with CH.sub.2CL.sub.2 (330 ml). The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by flash chromatography (Biotage SP1 instrument, SNAP cartridge silica 50 g, EtOAc in n-hexane from 10% to 100% in 10 CV) affording the title compound (1.0 g; Yield: 85%).

(63) .sup.1H NMR (300 MHz, CDCl.sub.3) 4.50 (td, J=6.5, 2H), 3.70 (t, J=6.2, 2H), 1.95-1.76 (m, 2H), 1.76-1.59 (m, 2H).

Step 4: Synthesis of 4-(nitrooxy)butyl 4-phenyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (Compound 6)

(64) To a solution of 4-phenyl-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanoic acid (synthesized in Example 3, steps 1 and 2) (0.27 g; 0.86 mmol) and 4-hydroxybutyl nitrate (0.15 mg; 0.86 mmol) in CH.sub.2Cl.sub.2 (4 ml), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (0.25 g; 1.30 mmol) and DMAP cat. were added. The solution was stirred 30 minutes at 0 C. and 4 hours at room temperature then washed with a solution of NaH.sub.2PO.sub.4 5% (5 ml), H.sub.20 (5 ml) and brine. The organic layer was dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by flash chromatography (Biotage SP1 instrument, SNAP cartridge silica 50 g, Hex/EtOAc 9:1, 10 CV) affording the title compound (0.23 g; Yield: 62%) as an orange oil.

(65) .sup.1H NMR (300 MHz, CDCl.sub.3) 7.38-7.11 (m, 5H), 4.47 (m, 2H), 4.34 (t, J=7.7, 1H), 4.09 (t, J=6.0, 2H), 2.73-2.52 (m, 1H), 2.52-2.23 (m, 3H), 2.07 (s, 3H), 1.97 (m, 6H), 1.89-1.65 (m, 4H).

Example 7

Synthesis of 4-(nitrooxy)butyl 4-(4-fluorophenyl)-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (Compound 7)

(66) ##STR00037##

(67) To a solution of 4-(4-fluorophenyl)-4-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanoic acid (synthesized in Example 5, steps 1 and 2) (0.19 g; 0.57 mmol) and 4-hydroxybutyl nitrate (synthesized in Example 6, steps 1, 2 and 3) (0.10 mg; 0.57 mmol) in CH.sub.2Cl.sub.2 (4 ml), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (0.16 g; 0.86 mmol) and DMAP cat. were added. The solution was stirred 30 minutes at 0 C. and 4 hours at room temperature then washed with a solution of NaH.sub.2PO.sub.4 5% (5 ml), H.sub.2O (5 ml) and brine. The organic layer was dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by flash chromatography (Biotage SP1 instrument, SNAP cartridge silica 50 g, n-Hexane/EtOAc 9:1, 10 CV) affording the title compound (0.15 g; Yield: 59%) as an orange oil.

(68) .sup.1H NMR (300 MHz, CDCl.sub.3) 7.35-7.15 (m, 2H), 7.06-6.87 (m, 2H), 4.47 (m, 2H), 4.28 (t, J=7.7, 1H), 4.09 (t, J=6.0, 2H), 2.68-2.22 (m, 4H), 2.08 (s, 3H), 1.97 (m, 6H), 1.86-1.65 (m, 4H).

Example 8

Synthesis of (5S,6R)-5,6-bis(nitrooxy)heptyl 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate ((5S,6R)-isomer of Compound 8)

(69) ##STR00038##

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

(70) ##STR00039##

(71) At 0 C., a solution of 5-hexen-1-ol (19.4 mL; 161.54 mmol) in dichloromethane (513 mL), was added with p-nitrobenzoyl chloride (35.97 g, 193.85 mmol) followed by a solution of triethylamine (27.0 mL, 193.85 mmol) in dichloromethane (150 mL) dropwise. The mixture was stirred at ambient temperature for 21 hours, then washed with water, 1M aqueous HCl, brine. The organic layer was dried (Na.sub.2SO.sub.4) and the solvent removed under reduced pressure. The residue was purified by flash chromatography (Biotage System, two SNAP Cartridge silica 340 g, eluent: n-hexane/ethyl acetate 90/10 to n-hexane/ethyl acetate 50/50 during 12 CV) to give the title compound as a yellow oil (40.00 g, 99%).

(72) .sup.1H NMR (300 MHz, CDCl.sub.3) 8.30 (dt, J=9.0, 3.0 Hz, 2H), 8.22 (dt, J=9.0, 3.0 Hz, 2H), 5.83 (1H, ddt, J=16.9, 10.2, 6.7 Hz), 4.95-5.11 (2H, m), 4.39 (2H, t, J=6.6 Hz), 2.15 (2H, m), 1.84 (2H, m) 1.50-1.66 (2H, m).

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

(73) ##STR00040##

(74) To a vigorously stirred solution of commercially available AD mix (112.5 g) in 1:1 water/t-butanol (822 mL), at 0 C., hex-5-enyl 4-nitrobenzoate was added (20.00 g, 80.23 mmol). The mixture was stirred vigorously at 4 C. (cold room) for 21 hours. The mixture was cooled to 0 C. and ethyl acetate (450 mL) was added, followed by slow portionwise addition of sodium metabisulfite (33.1 g). The mixture was stirred at 0 C. for 30 minutes, then at ambient temperature for 1 hour. The organic phase was separated and the aqueous extracted with ethyl acetate. The combined organic extracts were washed with brine, dried (Na.sub.2SO.sub.4) and the solvent removed under reduced pressure. Purification by filtration over a short pad of silica gel, eluting with ethyl acetate, gave the title compound as an off-white solid (21.90 g, 96%).

(75) .sup.1H NMR (300 MHz, CDCl.sub.3) 8.30 (dt, J=9.0, 2.0 Hz, 2H), 8.21 (dt, J=9.0, 2.0 Hz, 2H), 4.39 (t, J=6.6 Hz, 2H), 3.80-3.62 (2H, m), 3.47 (1H, m), 2.59 (bs, 1H), 2.42 (bs, 1H), 1.90-1.75 (2H, m), 1.73-1.45 (4H, m).

Step 3: Synthesis of (5S)-6-triphenylmethyloxy-5-hydroxyhexyl 4-nitrobenzoate

(76) ##STR00041##

(77) A solution of (5S)-5,6-dihydroxyhexyl 4-nitrobenzoate (13.46 g, 47.53 mmol) in anhydrous N,N-dimethylformamide (123 mL), under N.sub.2, was added with triphenylchloromethane (14.57 g, 52.28 mmol), followed by triethylamine (7.29 mL, 52.28 mmol) and 4-dimethylaminopyridine (581 mg, 4.75 mmol). The resulting solution was stirred at ambient temperature for 23 hours. The mixture was poured into water and extracted with diethyl ether (3). The combined organic extracts were washed with saturated aqueous NH.sub.4Cl and water, then dried (Na.sub.2SO.sub.4) and the solvent removed under reduced pressure. Purification by flash chromatography, eluting with 20% ethyl acetate/hexane to 50% ethyl acetate/n-hexane gave the title compound as pale yellow oil (20.90 g, 84%).

(78) .sup.1H NMR (300 MHz, CDCl.sub.3) 8.28 (d, J=8.8 Hz, 2H), 8.20 (d, J=8.8 Hz, 2H), 7.49-7.42 (m, 6H), 7.36-7.23 (m, 9H), 4.36 (t, J=6.5 Hz, 2H), 3.82 (dp, J=10.9, 3.1 Hz, 1H), 3.21 (dd, J=9.3, 3.3 Hz, 1H), 3.07 (dd, J=9.2, 7.7 Hz, 1H), 2.36 (d, J=2.9 Hz, 1H), 1.86-1.72 (m, 2H), 1.56-1.38 (m, 4H).

Step 4: Synthesis of (5S)-6-triphenylmethyloxy-5-tert-butyldiphenylsilyloxyhexyl 4-nitrobenzoate

(79) ##STR00042##

(80) A solution of (5S)-6-triphenylmethyloxy-5-hydroxyhexyl 4-nitrobenzoate (7.10 g, 13.51 mmol) in anhydrous N,N-dimethylformamide (65 mL), under N.sub.2, was added with imidazole (1.84 g, 27.02 mmol) and the solution cooled to 0 C. tert-Butyldiphenylsilyl chloride (7.03 mL, 27.02 mmol) was added and the solution stirred at 0 C. for 10 minutes, then at ambient temperature for 15 hours. The mixture was poured into water and extracted with diethyl ether. The combined organics were dried (Na.sub.2SO.sub.4) and the solvent removed under reduced pressure. Purification by flash chromatography, eluting with 5% ethyl acetate/n-hexane gave the title compound as off-white foam (5.29 g, 51%).

(81) .sup.1H NMR (300 MHz, CDCl.sub.3) 8.28-8.20 (m, 2H), 8.19-8.09 (m, 2H), 7.67-7.53 (m, 4H), 7.48-7.11 (m, 21H), 4.22 (t, J=6.4 Hz, 2H), 3.97-3.87 (m, 1H), 3.15 (dd, J=9.3, 4.8 Hz, 1H), 3.03 (dd, J=9.2, 6.4 Hz, 1H), 1.78-1.44 (m, 4H), 1.27 (m, 2H), 1.02 (s, 9H).

Step 5: Synthesis of (5S)-5-tert-butyldiphenyloxy-6-hydroxyhexyl 4-nitrobenzoate

(82) ##STR00043##

(83) A solution of (5S)-6-triphenylmethyloxy-5-tert-butyldiphenyl silyloxyhexyl-4-nitrobenzoate (4.06 g, 5.32 mmol) in dichloromethane (15 mL) was added with methanol (157 mL) and p-toluenesulfonic acid monohydrate (202 mg, 1.06 mmol). The solution was stirred at ambient temperature for 17 hours. The solvent was removed under reduced pressure and the residue dissolved in ethyl acetate and washed with saturated aqueous NaHCO.sub.3, water, then brine. The organic layer was dried (Na.sub.2SO.sub.4) and the solvent removed under reduced pressure. Purification by flash chromatography (Biotage System, SNAP Cartridge silica 340 g, eluent: n-hexane/ethyl acetate 90/10 to n-hexane/ethyl acetate 70/30 during 12 CV) gave the title compound as a pale yellow oil (1.33 g, 48%).

(84) .sup.1H NMR (300 MHz, CDCl.sub.3) 8.32-8.26 (m, 2H), 8.20-8.15 (m, 2H), 7.73-7.66 (m, 5H), 7.48-7.35 (m, 5H), 4.25 (t, J=6.5 Hz, 2H), 3.83 (dt, J=10.3, 5.3 Hz, 1H), 3.52 (ddd, J=11.4, 5.9, 3.7 Hz, 1H), 3.58 (ddd, J=11.4, 4.8, 3.1 Hz, 1H), 1.79 (bs, 1H), 1.70-1.46 (m, 4H), 1.36 (dd, J=15.0, 7.4 Hz, 2H), 1.09 (s, 9H).

Step 6: Synthesis of (5S)-5-tert-butyldiphenylsilyloxy-6-oxo hexyl 4-nitrobenzoate

(85) ##STR00044##

(86) A 0.4 M solution of (5S)-5-tert-butyldiphenyloxy-6-hydroxyhexyl 4-nitrobenzoate (9.29 g, 17.81 mmol) in dichloromethane (44.5 mL) was added with silica-supported TEMPO (307 mg; 0.178 mmol), followed by a 0.5 M solution aqueous KBr (3.53 mL). The mixture was cooled to 0 C. and stirred vigorously. A 0.37 M solution of NaOCl (10-15% active CO (13.73 mL) in water (46.30 mL) was added and the mixture buffered with solid NaHCO.sub.3. The mixture was stirred vigorously at 0 C. for 3.5 hours. The solids were removed by filtration and washed well with dichloromethane and water. The organic layer was separated and the aqueous extracted with dichloromethane. The combined organics were dried (Na.sub.2SO.sub.4) and the solvent removed under reduced pressure to give the title compound as crude pale yellow oil (9.09 g, 98%) for use directly without further purification.

(87) .sup.1H NMR (300 MHz, CDCl.sub.3) 9.64 (d, J=1.4 Hz, 1H), 8.30 (dt, J=9.0, 2.0 Hz, 2H), 8.19 (dt, J=9.0, 2.0 Hz, 2H), 7.75-7.60 (m, 4H), 7.55-7.35 (m, 6H), 4.30 (t, J=6.4 Hz, 2H), 4.09 (td, J=5.6, 1.4 Hz, 1H), 1.90-1.35 (m, 6H), 1.12 (9H, s).

Step 7: Synthesis of (5S,6R)-6-hydroxy-5-terbutyldiphenyl silyloxyheptyl 4-nitrobenzoate and (5S,6S)-6-hydroxy-5-terbutyldiphenylsilyloxyheptyl 4-nitrobenzoate

(88) ##STR00045##

(89) A 250 mL schlenk flask (dried and flushed with N.sub.2) was added with (1R,2S)-(+)-(dibutylamino)-1-phenyl-1-propanol (3.28 g, 12.45 mmol, 1 eq) followed by a 2 M solution of dimethylzinc in toluene (37.35 mL, 74.7 mmol, 6 eq). The resulting yellow solution was cooled to 0 C. and a solution of (5S)-5-tert-butyldiphenylsilyloxy-6-oxohexyl 4-nitrobenzoate (6.47 g, 12.45 mmol) in anhydrous toluene (40 mL) was added slowly. The solution was stirred at 0 C. for 10 minutes then allowed to warm to ambient temperature and stirred for 18 hours. The solution was cooled to 0 C. and slowly quenched by addition of saturated aqueous NH.sub.4Cl (75 mL). The mixture was allowed to warm to ambient temperature and extracted with ethyl acetate. The combined organics were dried (Na.sub.2SO.sub.4) and the solvent removed under reduced pressure. Purification by flash chromatography, eluting with 15% ethyl acetate/n-hexane to 25% ethyl acetate/n-hexane gave the title compound, a yellow oil (4.31 g, 65%), as an inseparable mixture of the diastereoisomers 5S,6R (major) and 5S,6S (minor).

(90) (5S,6R)-major diastereoisomer

(91) .sup.1H NMR (300 MHz, CDCl.sub.3) 8.29 (dt, J=9.0, 2.0 Hz, 2H). 8.16 (dt, J=9.0, 2.0 Hz, 2H), 7.75-7.65 (4H, m), 7.50-7.30 (6H, m), 4.20 (t, J=6.4 Hz, 2H), 3.83 (m, 1H), 3.72 (m, 1H), 2.09 (d, J=4.9 Hz, 1H), 1.65-1.20 (m, 6H), 1.12 (d, J=6.5 Hz, 3H), 1.09 (s, 9H).

(92) (5S,6S)-minor diastereoisomer:

(93) .sup.1H NMR (300 MHz, CDCl.sub.3) 8.29 (dt, J=9.0, 2.0 Hz, 2H), 8.16 (dt, J=9.0, 2.0 Hz, 2H), 7.75-7.65 (m, 4H), 7.30-7.50 (m, 6H), 4.20 (t, J=6.4 Hz, 2H), 3.71 (1H, m), 3.60 (1H, m), 2.21 (d, J=6.1 Hz, 1H), 1.65-1.20 (m, 6H), 1.16 (d, J=6.3 Hz, 3H), 1.09 (s, 9H).

Step 8: Synthesis of (5S,6R)-5,6-dihydroxyheptyl 4-nitrobenzoate

(94) ##STR00046##

(95) A solution of (5S,6R)-6-hydroxy-5-terbutyldiphenyl silyloxyheptyl 4-nitrobenzoate (805 mg, 1.50 mmol) in diethyl ether (50 mL) was added with a 3% solution of HCl in methanol, dropwise (made from addition of acetyl chloride (2.00 mL) to methanol (50 mL)). The solution was stirred at ambient temperature for 41 hours. Amberlite IRA 400 (OH) resin was added and the mixture stirred for 1 hour, with further addition of the resin until the pH=7/8. The resin was filtered off and washed with ethyl acetate, then methanol. The solvent was removed under reduced pressure and the residue partitioned between ethyl acetate and saturated aqueous NaHCO.sub.3 and the aqueous extracted with ethyl acetate. The combined organics were dried (Na.sub.2SO.sub.4) and the solvent removed under reduced pressure. Purification by flash chromatography, eluting with 10% ethyl acetate/hexane to 90% ethyl acetate/n-hexane, gave the title compound, a pale yellow oil (189 mg, 42%), as a mixture of the diastereoisomers 5S,6R (major) and 5S,6S (minor). Diastereoisomeric excess (5S,6R)=56.4%.

(96) The diastereoisomers were separated by preparative HPLC (conditions: column Phenomenex Gemini phenyl-hexyl 10021.2 mm/5 m

(97) Mobile phase: A: water+0.1% Formic acid; B: methanol+0.1% formic acid.

(98) Flow rate: 25 mL/min.

(99) Gradient profile: time 0 min: 45% A/55% B; 5.5 min: 40% A/60% B; 5.6 min: 0% A/100% B; 7.6 min: 0% A/100% B; 7.7 min: 45% A/55% B. Detector: : 254 nm) to give compound H as a white solid (135 mg). Enantiomeric excess/diastereomeric excess=72.1%.

(100) .sup.1H NMR (300 MHz, CDCl.sub.3) 8.32 (dt, J=9.0, 2.0 Hz, 2H), 8.23 (dt, J=9.0, 2.0 Hz, 2H), 4.42, (t, J=6.5 Hz, 2H), 3.84 (m, 1H), 3.66 (m, 1H), 1.42-2.0 (m, 8H), 1.19 (d, J=6.4 Hz, 3H).

(101) Alternative Deprotection Procedure

(102) A stirred solution of (5S,6R)-6-hydroxy-5-terbutyldiphenyl silyloxyheptyl 4-nitrobenzoate (2.96 g, 5.52 mmol) in acetonitrile (60 mL) was added at 0 C. with borontrifluoride-diethyletherate (3.5 mL, 5 eq) and the reaction was stirred at RT for 6 h. The reaction was cooled to 0 C. before quenching with a saturated solution of sodium bicarbonate. The reaction was diluted with ethyl acetate (50 mL) and the organic layer was separated, washed successively with water and brine (5 mL each), dried (Na.sub.2SO.sub.4), filtered and the solvent removed under reduced pressure. The residue was purified by flash chromatography (Biotage System, 2SNAP Cartridge silica 100 g, eluent: gradient n-hexane/ethyl acetate 35/65 to n-hexane/ethyl acetate 30/70 during 7 CV) to give the title compound as a colourless oil (1.47 g, 90%) as a mixture of the diastereoisomers 5S,6R (major) and 5S,6S (minor).

(103) The diastereoisomers were separated by preparative HPLC (conditions: column Phenomenex Gemini phenyl-hexyl 10021.2 mm/5 m to give the major diastereoisomer as a white solid (1.09 g, 66%).

Step 9: Synthesis of (5S,6R)-5,6-bis(nitrooxy)heptyl 4-nitrobenzoate

(104) ##STR00047##

(105) A stirred solution of (5S,6R)-5,6-dihydroxyheptyl 4-nitrobenzoate (400 mg, 1.34 mmol), tetrabutylammonium nitrate (863 mg, 2.82 mmol, 2.1 eq) and 2,6-di-tert-butyl-4-methylpyridine (580 mg, 2.82 mmol, 2.1 eq) in dry CH.sub.2Cl.sub.2 cooled to 78 C. was added dropwise with triflic anhydride (0.778 g, 2.75 mmol, 2.05 eq) and the reaction was stirred for 1 h at 78 C. and left to turn back to rt. The reaction was then quenched with water and the organic layer was separated, washed with water and brine, dried on sodium sulfate, filtered and evaporated. The residue was purified by flash chromatography (Biotage SP4, SNAP 100 column, EtOAc in n-hexane from 20% to 30% in 10 CV) affording the title compound as an yellow oil (406 mg, Yield: 77%).

(106) .sup.1H NMR (300 MHz, CDCl.sub.3) 8.32 (dt, J=9.0 2.0 Hz, 2H), 8.22 (dt, J=9.0, 2.0 Hz, 2H), 5.30 (m, 1H), 4.42 (td, J=6.4 Hz, 1H), 1.95-1.55 (m, 6H), 1.43 (d, J=6.7 Hz, 3H).

Step 10: Synthesis of (1R,2S)-6-hydroxy-1-methyl-2-(nitrooxy)hexyl nitrate

(107) ##STR00048##

(108) A solution of (5S,6R)-5,6-bis(nitrooxy)heptyl 4-nitrobenzoate (163 mg, 0.42 mmol) in tetrahydrofuran (1.27 mL) and ethanol (1.27 mL), was added with a 1 M aqueous NaOH solution (546 L, 0.546 mmol). The resulting yellow solution was stirred at ambient temperature for 1.5 hours. The solvent was concentrated under reduced pressure and the aqueous residue partitioned between ethyl acetate and saturated aqueous NaHCO.sub.3. The organic layer was washed with saturated aqueous NaHCO.sub.3 and the aqueous back-extracted with ethyl acetate. The combined organics were dried (Na.sub.2SO.sub.4) and the solvent concentrated to a small volume (2 mL). The product was carefully purified by flash chromatography (Biotage System, SNAP Cartridge silica 100 g, eluent: gradient n-hexane/ethyl acetate 75/25 to n-hexane/ethyl acetate 50/50 during 8 CV) to give the separation of the two diastereoisomers and the title compound as a colourless oil (0.147 g, 90%).

(109) An optimal separation could be obtained using a Thar Investigator SFC system using the following conditions:

(110) Column: CHIRALPACK IB 25010 mm (5 m)

(111) Cosolvent: n-Hexane/2-Propanol 1/1

(112) Isocratic elution: CO.sub.2/co-solvent 90/10

(113) Flow 10 ml/min T column: 40 C. Injection volume: 80 l Detector wavelength: 210 nm

(114) Run time: 8.5 min Cycle time: 3 min Injected amount: 14-16 mg

(115) (Sample preparation: 800 mg of crude compound were solubilized in 4 ml of MeOH)

(116) .sup.1H NMR (300 MHz, CDCl.sub.3) 5.25-5.34 (m, 2H), 3.70 (t, J=5.9 Hz, 2H), 1.47-1.85 (m, 7H), 1.42 (d, J=6.8 Hz, 3H).

Step 11: Synthesis of (5S,6R)-5,6-bis(nitrooxy)heptyl 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (compound 8)

(117) A stirred solution of 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanoic acid (Prepared in Example 1, Step 2) (146 mg, 0.583 mmol, 1 eq) and (1R,2S)-6-hydroxy-1-methyl-2-(nitrooxy)hexyl nitrate (140 mg, 0.583 mmol) in dry CH.sub.2Cl.sub.2 cooled to 0 C. was added with EDC (117 mg, 0.612 mmol, 1.05 eq) and a catalytic amount of DMAP. The reaction was stirred at this temperature for 5 h and then washed with water, HCl 0.1 M, water and brine. The organic solution was dried (Na.sub.2SO.sub.4), filtered and evaporated. The residue was purified by flash chromatography (Biotage SP4 system, SNAP Cartridge silica 100 g, eluent: gradient n-hexane/ethyl acetate 80/20 to n-hexane/ethyl acetate 70/30 during 8 CV) to give the title compound as a yellow oil (210 mg, 76%).

(118) .sup.1H NMR (300 MHz, CDCl.sub.3) 5.32-5.17 (m, 2H), 3.99 (t, J=6.2, 2H), 2.98 (s, 2H), 2.14 (s, 3H), 1.98-1.94 (m, 6H), 1.76-1.45 (m, 15H), 1.43 (s, 6H), 1.39 (d, J=6.7, 3H).

Example 9

Synthesis of (4-(nitrooxy)butyl 4-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dienyl)-4-phenylbutanoate (Compound 9)

(119) ##STR00049##

Step 1: Synthesis of 4-(2,5-dihydroxy-3,4-dimethoxy-6-methylphenyl)-4-phenylbutanoic acid

(120) ##STR00050##

(121) The reaction was performed according to conditions described by Mitsuru et al., J. Med. Chem. Soc., 1989, 32, 2214-2221.

(122) Boron trifluoride etherate (0.23 ml; 1.59 mmol) was added dropwise to a mixture of 2,3-dimethoxy-5-methylbenzene-1,4-diol (1.00 g; 5.49 mmol) and -phenyl--butyrolactone (0.89 g; 5.49 mmol) in Toluene (55 ml) at 60 C. during 10 minutes. The mixture was stirred for further 3 hours and then the solvent was evaporated under reduced pressure. The residue was purified by flash chromatography (Biotage system, SNAP Cartridge silica 100 g, EtOAc in n-hexane from 9% to 60% in 10 CV) affording the title compound (0.80 g; Yield: 42%) a pale yellow solid.

(123) .sup.1H NMR (300 MHz, CDCl.sub.3) 7.44-7.10 (m, 5H), 5.42 (m, 2H), 4.49 (m, 1H), 3.99-3.81 (m, 6H), 2.77-2.47 (m, 2H), 2.47-2.29 (m, 2H), 2.10 (s, 3H).

Step 2: Synthesis of 4-(4,5-dimethoxy-2-methyl-3,6-dioxo cyclohexa-1,4-dien-1-yl)-4-phenylbutanoic acid

(124) ##STR00051##

(125) To a solution of 4-(2,5-dihydroxy-3,4-dimethoxy-6-methylphenyl)-4-phenylbutanoic acid (0.40 g; 1.15 mmol) in CH.sub.3CN:H.sub.2O 1:1 (40 ml), Ammonium cerium nitrate (1.63 g; 2.89 mmol) was added. The mixture was stirred 3 hours at room temperature then was poured into H.sub.2O (30 ml). Et.sub.2O (20 ml) was added, the two phases were separated and the organic layer was extracted with Et.sub.2O (220 ml). The combined organic layers were washed with brine, dried on Na.sub.2SO.sub.4 and concentrated affording 400 mg of the title compound without any further purification.

Step 3: Synthesis of (4-(nitrooxy)butyl 4-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dienyl)-4-phenylbutanoate (Compound 9)

(126) To a solution of 4-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-4-phenylbutanoic acid (0.40 g; 0.57 mmol) and 4-hydroxybutyl nitrate (synthesised in Example 6, steps 1, 2 and 3) (0.10 mg; 0.57 mmol) in CH.sub.2Cl.sub.2 (4 ml), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (0.16 g; 0.86 mmol) and DMAP cat. were added. The solution was stirred 30 minutes at 0 C. and 4 hours at room temperature then washed with a solution of NaH.sub.2PO.sub.4 5% (5 ml), H.sub.2O (5 ml) and brine. The organic layer was dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by flash chromatography (Biotage SP1 instrument, SNAP cartridge silica 25 g, Hex/EtOAc 8:2, 10 CV) affording the title compound (95 mg; Yield: 36%) as a red oil.

(127) .sup.1H NMR (300 MHz, CDCl.sub.3) 7.38-7.15 (m, 5H), 4.49 (m, 2H), 4.35 (t, J=7.7, 1H), 4.11 (t, J=6.0, 2H), 3.98 (s, 6H), 2.74-2.52 (m, 1H), 2.52-2.25 (m, 3H), 2.09 (s, 3H), 1.89-1.66 (m, 4H).

Example 10

In Vitro Antioxidant Activity (TBARS Test)

(128) The antioxidant properties of compound (1) (example 1), its precursors (3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanoic acid, described in Example 1 Step 2 (Intermediate 2) and reference antioxidant compounds were assessed after NADPH-induced lipidic peroxidation of membrane lipids in rat hepatic microsomes using the detection of 2-thiobarbituric acid reactive substances (TBARS) by visible spectroscopy.

(129) Hepatic microsomal membranes from male Wistar rats (200-250 g) were prepared by differential centrifugation (8000 g, 20 min; 120000 g, 1 h) in a HEPES/sucrose buffer (10 mM, 250 mM, pH 7.4) and stored at 80 C. Incubation was performed at 37 C. in a Tris-HCl/KCl (100 mM/150 mM, pH 7.4) containing microsomal membranes (2 mg prot/mL), sodium ascorbate (100 M), and DMSO solutions of the tested compounds.

(130) Lipid peroxidation was initiated by adding ADP-FeCl.sub.3 and NADPH (Method A) or 2.5 M FeSO.sub.4 (Method B) (as described by Boschi D. et al., J. Med. Chem. 2006, 49:2886-2897). Aliquots were taken from the incubation mixture at 5, 15, and 30 min and treated with trichloroacetic acid (TCA) 10% w/v.

(131) Lipid peroxidation was assessed by spectrophotometric (543 nm) determination of the TBARS consisting mainly of malondialdehyde (MDA). TBARS concentrations (expressed in nmol/mg protein) were obtained by interpolation with a MDA standard curve. The antioxidant activity of tested compounds was evaluated as the percent inhibition of TBARS production with respect to control samples, using the values obtained after 30 min of incubation. IC.sub.50 values were calculated by nonlinear regression analysis.

(132) The results reported in Table 1, showed that compound (1) proved to inhibit in a concentration-dependent manner the generation of TBARS with a potency (IC.sub.50=28 PA) that is comparable to well known antioxidant compounds as ferulic or caffeic acids, edavarone or melatonin.

(133) TABLE-US-00001 TABLE 1 In vitro Antioxidant activity (TBARS test) Antioxidant activity IC50 M Compound (CL 95%) Method Compound (1) 28 (25-31) A Intermediate 2 157 (79-309) A Ferulic acid 50.5 0.4.sup.a B Caffeic acid 33 (32-34) B Edavarone 17 (15-18).sup.b B Melatonin 476 (442-512).sup.c B Results are expressed as IC.sub.50 of inhibition of TBARS production after 30 min incubation at 37 C. Method A: inhibition of rat hepatic lipid peroxidation induced by ADP-FeCl.sub.3 and NADPH. Method B: inhibition of rat hepatic lipid peroxidation induced by FeSO4 and ascorbic acid .sup.atested at 1 mM concentration; .sup.bChegaev, K. et al. J. Med. Chem. 2009, 52: 574-578: .sup.cChegaev, K. et al. J. Pineal Res. 2007, 42: 371-385

Example 11

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

(134) The Intraocular pressure (IOP) lowering activity of compound (1) (Example 1) was assessed in an animal model of elevated IOP.

(135) Adults male New Zealand White rabbits weighting 1.8-2.0 Kg were used in the experiments.

(136) Animals were anesthetized using 20 mg/ml/kg of sodium pentobarbital. The 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).

(137) IOP was measured using a Tono-Pen XL prior to hypertonic saline injection (basal) and at 30, 60, 90, 120, 240 and 360 min thereafter. Vehicle (5% cremophor-EL; 0.3% DMSO; 0.2 mg/ml Benzalkonium chloride (bac) in PBS pH 6.0,) or compound of the invention were instilled as eye drops immediately after hypertonic saline injection. Eyes were randomly assigned to different treatment groups. Vehicle or compounds of the invention were directly instilled into the conjunctiva pocket at the desired doses. One drop of 0.2% oxybuprocaine hydrochloride (Novesine, Sandoz) diluted 1:1 with saline was instilled in each eye immediately before each set of pressure measurements.

(138) Results are reported in Table 2 in which the ocular hypotensive activity of compound (1) is expressed as mean average of IOP measurements at 60 and 120 minutes following topical administration.

(139) TABLE-US-00002 TABLE 2 Intraocular pressure (IOP) lowering activity in hypertonic saline- induced IOP increase in rabbits IOP (mmHg) 60 minutes 120 minutes Compound (1) 29 7.8 18.9 4.1 Vehicle 34.9 4.3 25.6 4.6

Example 12

Synthesis of (S)-5,6-bis(nitrooxy)hexyl 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (Compound 15; (S)-isomer of compound (10))

(140) ##STR00052##

Step 1: synthesis of (5S)-5,6-bis(nitrooxy)hexyl 4-nitrobenzoate

(141) To a stirred solution of fuming nitric acid (7.7 mL, 91.8 mmol, 10 eq) in dichloromethane (60 mL) at 78 C., was added sulfuric acid (4 mL) and after 5 mins of stirring, a solution of (5S)-5,6-dihydroxyhexyl 4-nitrobenzoate (prepared in Example 8 Step 2) (5.2 g, 9.2 mmol) in dichloromethane (30 mL) was added and the reaction stirred at this temperature for 30 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 (6.8 g, 100%). The residue obtained was used in the next step without further purification.

(142) 1H NMR (300 MHz, CDCl3) 8.32 (d, J=8.9 Hz, 2H), 8.26-8.15 (m, 2H), 5.39-5.25 (m, 1H), 4.78 (dd, J=12.9, 3.1 Hz, 1H), 4.52 (dd, J=12.9, 6.4 Hz, 1H), 4.46-4.35 (m, 2H), 1.97-1.77 (m, 4H), 1.77-1.49 (m, 2H).

Step 2: Synthesis of (2S)-6-hydroxyhexane-1,2-diyl dinitrate

(143) To a stirred solution of (5S)-5,6-bis(nitrooxy)hexyl 4-nitrobenzoate (prepared in Step 1) (6.8 g, 18.2 mmol) in a 1/1 mixture of ethanol/THF (30 mL of each) at 0 C., a 2M sodium hydroxide solution (9.1 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 (SNAP 100, gradient system from 4/6 ethyl acetate/n-hexane to 60/40 ethyl acetate/n-hexane) to give the title compound as colorless oil (3.82 g, 93%).

(144) 1H NMR (300 MHz, CDCl3) 5.32 (qd, J=6.7, 3.0 Hz, 1H), 4.77 (dd, J=12.9, 3.0 Hz, 1H), 4.49 (dd, J=12.9, 6.6 Hz, 1H), 3.68 (d, J=5.5 Hz, 2H), 1.89-1.71 (m, 2H), 1.70-1.48 (m, 5H), 1.46 (s, 1H).

Step 3: Synthesis of (S)-5,6-bis(nitrooxy)hexyl 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate

(145) To a stirred solution of 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanoic acid (201 mg; 0.80 mmol) (prepared as described in Example 1, steps 1 and 2) and (2S)-6-hydroxyhexane-1,2-diyl dinitrate (181 mg; 0.80 mmol) (prepared in Step 2) in DCM (5 ml) cooled to 0 C., EDAC (137 mg; 0.89 mmol) and a catalytic amount of DMAP were added. The reaction was stirred overnight at 0 C. The crude was then washed with water, HCl 1N, water and brine, dried and evaporated under vacuum. The crude was purified by flash chromatography [Cy/EtOAc: 0% to 20% (1CV), 20% to 40% (7CV), 40% to 60% (2CV)] affording 246 mg of the title compound (Yield: 67.1%) as a yellow oil.

(146) .sup.1H NMR (300 MHz, acetone) 5.49 (qd, J=6.6, 2.6 Hz, 1H), 5.01 (dd, J=13.0, 2.6 Hz, 1H), 4.73 (dd, J=13.0, 6.3 Hz, 1H), 4.00 (t, J=6.3 Hz, 2H), 2.95 (s, 2H), 2.13 (s, 3H), 1.94 (s, J=6.3 Hz, 6H), 1.90-1.79 (m, 2H), 1.70-1.58 (m, 2H), 1.52 (dt, J=8.0, 5.7 Hz, 2H), 1.43 (s, 6H). .sub.D.sup.20=+2.2 (0.44% MeOH)

Example 13

Synthesis of (R)-5,6-bis(nitrooxy)hexyl 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate (Compound 16; (R)-isomer of compound 10)

(147) ##STR00053##

Step 1: Synthesis of (5R)-5,6-dihydroxyhexyl 4-nitrobenzoate

(148) To a vigorously stirred solution of commercially available AD mix (112.5 g) in 1:1 water/t-butanol (822 mL), at 0 C., hex-5-enyl 4-nitrobenzoate (prepared in Example 8, Step 1) was added (20.00 g, 80.23 mmol). The mixture was stirred vigorously at 4 C. (cold room) for 21 hours. The mixture was cooled to 0 C. and ethyl acetate (450 mL) was added, followed by slow portionwise addition of sodium metabisulfite (33.1 g). The mixture was stirred at 0 C. for 30 minutes, then at ambient temperature for 1 hour. The organic phase was separated and the aqueous extracted with ethyl acetate. The combined organic extracts were washed with brine, dried (Na2SO4) and the solvent removed under reduced pressure. Purification by filtration over a short pad of silica gel, eluting with ethyl acetate, gave the title compound as an off-white solid (20.5 g, 90.2%).

(149) 1H NMR (300 MHz, CDCl3) 8.30 (dt, J=9.0, 2.0 Hz, 2H), 8.21 (dt, J=9.0, 2.0 Hz, 2H), 4.39 (t, J=6.6 Hz, 2H), 3.80-3.62 (2H, m), 3.47 (1H, m), 2.59 (bs, 1H), 2.42 (bs, 1H), 1.90-1.75 (2H, m), 1.73-1.45 (4H, m).

Step 2: synthesis of (5R)-5,6-bis(nitrooxy)hexyl 4-nitrobenzoate

(150) To a stirred solution of fuming nitric acid (7.7 mL, 91.8 mmol, 10 eq) in dichloromethane (60 mL) at 78 C., was added sulfuric acid (4 mL) and after 5 mins of stirring, a solution of (5R)-5,6-dihydroxyhexyl 4-nitrobenzoate (prepared in Step 1) (5.2 g, 9.2 mmol) in dichloromethane (30 mL) was added and the reaction stirred at this temperature for 30 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 a pale yellow oil (6.8 g, 100%). The residue obtained was used in the next step without further purification.

(151) 1H NMR (300 MHz, CDCl3) 8.32 (d, J=8.9 Hz, 2H), 8.26-8.15 (m, 2H), 5.39-5.25 (m, 1H), 4.78 (dd, J=12.9, 3.1 Hz, 1H), 4.52 (dd, J=12.9, 6.4 Hz, 1H), 4.46-4.35 (m, 2H), 1.97-1.77 (m, 4H), 1.77-1.49 (m, 2H).

Step 3: Synthesis of (2R)-6-hydroxyhexane-1,2-diyl dinitrate

(152) To a stirred solution of (5R)-5,6-bis(nitrooxy)hexyl 4-nitrobenzoate (prepared in Step 2) (6.8 g, 18.2 mmol) in a 1/1 mixture of ethanol/THF (30 mL of each) at 0 C., a 2M sodium hydroxide solution (9.1 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 (SNAP 100, gradient system from 4/6 ethyl acetate/n-hexane to 60/40 ethyl acetate/n-hexane) to give the title compound as colorless oil (3.50 g, 86%).

(153) 1H NMR (300 MHz, CDCl3) 5.32 (qd, J=6.7, 3.0 Hz, 1H), 4.77 (dd, J=12.9, 3.0 Hz, 1H), 4.49 (dd, J=12.9, 6.6 Hz, 1H), 3.68 (d, J=5.5 Hz, 2H), 1.89-1.71 (m, 2H), 1.70-1.48 (m, 5H), 1.46 (s, 1H).

Step 4: Synthesis of (R)-5,6-bis(nitrooxy)hexyl 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dienyl)butanoate

(154) To a stirred solution of 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanoic acid (237 mg; 0.95 mmol) (prepared as described in Example 1, steps 1 and 2) and (2R)-6-hydroxyhexane-1,2-diyl dinitrate (prepared in Step 3) (213 mg; 0.95 mmol) in DCM (5 ml) cooled to 0 C., EDAC (162 mg; 1.04 mmol) and a catalytic amount of DMAP were added. The reaction was stirred overnight at 0 C. The crude was then washed with water, HCl 1N, water and brine, dried and evaporated under vacuum. The crude was purified by flash chromatography [Cy/EtOAc: 0% to 20% (1CV), 20% to 40% (7CV), 40% to 60% (2CV)] affording 338 mg of the title compound (Yield: 78.2%) as a yellow oil.

(155) .sup.1H NMR (300 MHz, CDCl.sub.3) 5.30-5.21 (m, 1H), 4.74 (dd, J=12.9, 3.0 Hz, 1H), 4.47 (dd, J=12.9, 6.5 Hz, 1H), 3.99 (t, J=6.3 Hz, 2H), 2.98 (s, 2H), 2.16 (d, J=7.2 Hz, 3H), 1.96 (s, 6H), 1.81-1.69 (m, 2H), 1.66-1.56 (m, 2H), 1.52-1.31 (m, 8H). .sub.D.sup.20=+2.3 (0.47% MeOH)

Example 14

Synthesis of (S)-5,6-bis(nitrooxy)hexyl 3-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dienyl)-3-methylbutanoate (Compound 17; (S)-isomer of compound 11)

(156) ##STR00054##

(157) Step 1: Synthesis of 2,3-dimethoxy-5-methylbenzene-1,4-diol

(158) ##STR00055##

(159) NaBH.sub.4 (5.2 g; 137.2 mmol) was dissolved in 150 ml of water, and a solution of 2,3-dimethoxy-5-methylcyclohexa-2,5-diene-1,4-dione (Sg; 27.4 mmol) in a mixture of 75 ml of Et.sub.2O and 38 ml of MeOH was added at room temperature with stirring. After 15 min, the mixture was placed in a separatory funnel, and the layers were allowed to separate. The ether phase was removed and the aqueous phase was extracted twice with 50 ml portions of ether. The combined organic extracts were washed with brine and dried over Na.sub.2S0.sub.4. Solvent removal under reduced pressure afforded the title compound (9 g; 88%) as red oil. It was used in the next step without further purification.

Step 2: Synthesis of 6-hydroxy-7,8-dimethoxy-4,4-dimethylchroman-2-one

(160) ##STR00056##

(161) 2,3-dimethoxy-5-methylbenzene-1,4-diol (obtained in Step 2) (9 g; 49 mmol), methyl 3-methylbut-2-enoate (7 ml; 58 mmol) and methanesulfonic acid (80 mL) were heated at 70 C. with stirring for 90 min. Then the mixture was poured into ice then diluted to 600 ml with water and extracted with diethyl ether (3150 ml). The combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated to dryness to afford a brown solid, which was crystallized from methanol to provide pure title compound (6.5 g; 50%) as yellow crystals.

Step 3: Synthesis of 4-nitrophenyl 3-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dienyl)-3-methylbutanoate

(162) ##STR00057##

(163) A synthetic procedure similar to the one described by Carpino et al., J. Org. Chem., 1989, 54, 3303-3310 was used, but 3-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dienyl)-3-methylbutanoic acid, even if described in the paper, proved to be high unstable, so it was converted in situ into its 4-nitrophenyl ester.

(164) Lactone obtained in Step 3 (2 g; 7.51 mmol) in DMF (15 mL) was added to a stirred solution of PDC (12.7 g; 33.7 mmol) in DMF (15 mL) at room temperature, and stirring was continued for 4 h. The mixture was diluted to 300 mL with water and extracted quickly with diethyl ether (3150 mL). The combined ether extracts were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated to about 10 mL. This solution was diluted in EtOAc (40 mL) and then p-nitrophenol (1.57 g; 11.2 mmol), DCC (2.31 g; 11.2 mmol; 1.5 eq.) and DMAP (cat) were successively added. The mixture was stirred at room temperature for 16 h, then it was concentrated to dryness. Purification by chromatography on neutral alumina (eluent: cyclohexane/AcOEt 8/2) led to the title compound (800 mg; 26%) as an orange oil.

(165) .sup.1H NMR (300 MHz, CDCl.sub.3) 8.24 (d, 2H), 7.20 (d, 2H), 3.93 (s, 3H), 3.89 (s, 3H), 3.32 (s, 2H), 2.19 (s, 3H), 1.55 (s, 3H), 1.53 (s, 3H).

Step 4: Synthesis of (S)-5,6-bis(nitrooxy)hexyl 3-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dienyl)-3-methylbutanoate

(166) ##STR00058##

(167) In a 50-ml one-necked flask, 178.0 mg (0.45 mmole) of 4-nitrophenyl dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dienyl)-3-methylbutanoate (obtained in Step 4) and 111.0 mg (0.50 mmole) of (S)-6-hydroxyhexane-1,2-diyl dinitrate (obtained in Example 12, Step 2) were added to DCM (1.5 ml). After a 10 minutes 55.0 mg (0.45 mmole) of DMAP were added. The solution was stirred for 18 hours to room temperature. The reaction was washed with water, dried with MgSO.sub.4, filtered and concentrated under reduced pressure to give red oil in a quantitative yield. The obtained red oil was purified by automatic column chromatography using silica gel Cy/DCM/MeOH (50/50/0 to 68/30/2) mixture as eluent to give 200 mg (91% yield) of the title compound as a orange oil.

(168) MS: m/z=489 [M+H].sup.+

(169) TLC: (Cy/DCM/MeOH 68:30:2) R.sub.f=0.33

(170) NMR (CDCl.sub.3): 5.25 (m, 1H), 4.74 (dd, 1H), 4.50 (dd, 1H), 4.0 (t, 2H), 3.97 (s, 3H), 3.93 (s, 3H), 3.0 (s, 2H), 2.14 (s, 3H), 1.8-1.5 (m, 6H), 1.43 (s, 6H).

Example 15

Synthesis of (S)-5,6-bis(nitrooxy)hexyl 3-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dienyl)-3-methylbutanoate (Compound 17)

(171) ##STR00059##
the title compound was prepared as follow:

Step 1: Synthesis of 2,5-dioxopyrrolidin-1-yl 3-(4,5-dimethoxy-3,6-dioxocyclohexa-1,4-dienyl)-3-methylbutanoate

(172) ##STR00060##

(173) A synthetic procedure similar to the one described by Carpino et al., J. Org. Chem., 1989, 54, 3303-3310 was used, but 3-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dienyl)-3-methylbutanoic acid, even if described in the paper, proved to be high unstable, so it was converted in situ into its N-hydroxysuccinimido ester.

(174) Lactone obtained in Example 14 Step 3 (2.5 g; 9.39 mmol) in DMF (20 mL) was added to a stirred solution of PDC (14.8 g; 39.4 mmol) in DMF (20 mL) at room temperature, and stirring was continued for 4 h. The mixture was diluted to 300 mL with water and extracted quickly with diethyl ether (3150 mL). The combined ether extracts were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated to about 10 mL. This solution was diluted in DCM (40 mL) and NHS (1.29 g; 11.2 mmol) as well as EDCl.HCI (2.16 g; 11.2 mmol) were added successively and stirring was continued for 16 h. The mixture was diluted with water and extracted with DCM. The combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated to dryness to afford the title compound (2.74 g; 78%) as an orange oil.

(175) .sup.1H NMR (300 MHz, CDCl.sub.3) 3.97 (s, 3H), 3.88 (s, 3H), 3.28 (s, 2H), 2.88 (s, 4H), 2.16 (s, 3H), 1.55 (s, 3H), 1.57 (s, 3H).

Step 2: Synthesis of (S)-5,6-bis(nitrooxy)hexyl 3-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dienyl)-3-methylbutanoate (Compound 17)

(176) A mixture of 40.0 mg (0.1 mmole) of 2,5-dioxopyrrolidin-1-yl 3-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dienyl)-3-methylbutanoate, 28.0 mg (0.12 mmole) of (S)-6-hydroxyhexane-1,2-diyl dinitrate (prepared in Example 12, Step 2) and DMF (1 ml) were mixed together under stirring. After 10 minutes 19.0 mg (0.1 mmole) of EDC*HCl and 12.0 mg (0.1 mmole) of DMAP were added. The solution was stirred for 5 hours to 50 C. The reaction was washed with water, dried with MgSO4, filtered and concentrated under reduced pressure to give a red oil in a quantitative yield. The obtained red oil was purified by automatic column chromatography using silica gel Versaflash cartridges with Cy/DCM/MeOH (50/50/0 to 68/30/2) mixture as eluent to give 14.6 mg (31% yield) of the title compound as a orange oil.

(177) MS: m/z=489 [M+H].sup.+

(178) TLC: (Cy/DCM/MeOH 68:30:2) R.sub.f=0.33

(179) NMR (CDCl.sub.3): 5.25 (m, 1H), 4.74 (dd, 1H), 4.50 (dd, 1H), 4.0 (t, 2H), 3.97 (s, 3H), 3.93 (s, 3H), 3.0 (s, 2H), 2.14 (s, 3H), 1.8-1.5 (m, 6H), 1.43 (s, 6H).

Example 16

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

(180) The present study evaluated the intraocular pressure lowering effect of single applications of two different concentrations (1% and 0.3%) of compound (1) in rabbits with experimental increase in IOP.

(181) Adults male New Zealand White rabbits weighting 1.8-2.0 Kg were used in the experiments.

(182) 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).

(183) 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 was instilled as eye drops immediately after hypertonic saline injection. Eyes were randomly assigned to different treatment groups. Vehicle and compound 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.

(184) Results are reported in Table 3 and they are expressed as IOP change (at 60 and 120 minutes following topical administration) versus vehicle and versus IOP at basal before hypertonic saline injection. Single application of both doses of compound (1) resulted in a significant IOP reduction.

(185) TABLE-US-00003 TABLE 3 Intraocular pressure (IOP) lowering effect of compound (1) in hypertonic saline-induced IOP increase in rabbits IOP change (mmHg) Dose 60 minutes 120 minutes 1% 9.1 3.4 9.5 2.2 0.3% 5.5 3.1 5.7 2.1

Example 17

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

(186) The present study evaluated the intraocular pressure lowering effect of single application of compound (15) (Example 12) and of ISMN (isosorbide-5-mononitrate) used as reference compound, in an animal model of elevated IOP.

(187) Adults male New Zealand White rabbits weighting 1.8-2.0 Kg were used in the experiments.

(188) Animals were anesthetized using 20 mg/ml/kg of sodium pentobarbital. The 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).

(189) IOP was measured using a Tono-Pen XL prior to hypertonic saline injection (basal) and at 30, 60, 90, 120, 240 and 360 min thereafter. Vehicle (5% cremophor-EL; 0.3% DMSO; 0.2 mg/ml Benzalkonium chloride in PBS pH 6.0,) or tested compound was instilled as eye drops immediately after hypertonic saline injection. Eyes were randomly assigned to different treatment groups. Vehicle or compounds were directly instilled into the conjunctiva pocket at the desired doses. One drop of 0.2% oxybuprocaine hydrochloride (Novesine, Sandoz) diluted 1:1 with saline was instilled in each eye immediately before each set of pressure measurements.

(190) Results are reported in Table 4 and they are expressed as IOP change (at 30 and 60 minutes following topical administration) versus vehicle and versus IOP at basal before hypertonic saline injection.

(191) Single application of compound (15) resulted in a significantly higher IOP reduction as compared to ISMN treated group.

(192) TABLE-US-00004 TABLE 4 Intraocular pressure (IOP) lowering effect in hypertonic saline-induced IOP increase in rabbits IOP change (mmHg) Compound/dose 30 minutes 60 minutes Compound (15) 6.5 2.6 4.9 2.4 (1%) ISMN 2.6 3.5 0.7 2.9 (1%)