The present invention relates to a nitric oxide releasing naproxen derivative, its use for the treatment of diseases associated with pain and inflammation. The present invention also relates to pharmaceutical formulation comprising it.

Naproxen is a non steroidal anti-inflammatory drug (NSAID) used for the treatment of a variety of conditions associated with pain and inflammation, including osteoarthritis, rheumatoid arthritis, gout and ankylosing spondylitis. However, long-term use of naproxen is significantly limited by the risk of gastrointestinal side effects such as erosions, ulcers with perforation and bleeding of the stomach or intestine (Lane ME, Kim MJ. (2006), Assessment and prevention of gastrointestinal toxicity of non-steroidal antiinflammatory drugs. J Pharm Pharmacol. 58:1295-1304). Naproxen can also increase cardiovascular or cerebrovascular events (Farkouh ME, Greenberg BP. (2009). An evidence-based review of the cardiovascular risks of nonsteroidal anti-inflammatory drugs. Am J Cardiol. 103:1227-1237).

Over the past years the search for new NSAIDs having good efficacy as anti-inflammatory agents but reduced side effects has led to the design and testing of a variety of new chemical classes .

In the 1990' s, selective inhibitors of Cycloxygenase isoform - 2 inhibitors (COX-2) were developded as an advance on conventional NSAIDs, as they appeared to cause less gastrointestinal injury. However, concerns have been raised regarding their cardiovascular toxicity.

EP 722434 discloses nitrooxyalkylester of NSAIDs. This publication discloses nitrooxybutyl ester of naproxen that has a good anti-inflammatory activity and a safer gastrointestinal profile . WO 2004/004648 discloses mononitrate and dinitrate naproxen derivatives. This publication discloses that these compounds have anti-inflammatory activities comparable to naproxen but with decreased gastric lesion liability.

WO 00/51988 discloses salts of nitrooxy-derivatives of NSAID in which the nitrate group is linked to the drug molecule through a heterocyclic linker. These compounds show an improved solubility in water and a good anti-inflammatory activity. This publication discloses two naproxen derivatives namely (S) -6-methoxy-α-methylnaphthalenacetic acid 6- [ (nitrooxy) methyl] -2-methylpyridyl ester nitrate salts and (S) -6-methoxy-α-methylnaphthalenacetic acid 6- [ (nitrooxy) methyl ] -2-methylpyridyl ester hydrochloride which are soluble in water.

The publication by J. L. Ellis et al. (Inflammopharmacology, Vol. 12, No 5-6, pp 521-534 (2005)) compares the antiinflammatory effect and the gastric tolerability of (2R) -2,3- bis (nitrooxy) propyl-2 ( 6-methoxy (2-naphthyl) propanoate (NMI- 1182), a prototypic dinitrate nitric oxide donating (NO) naproxen, with nitrooxybutyl ester of naproxen (AZ3582), a mononitrate nitric oxide donating (NO) naproxen, and naproxen itself. This publication shows that the NO-donating naproxen derivatives are gastro-protective compared to naproxen and that they retain the ability to act as effective anti- inflammatory agents.

Notwithstanding the great effort already put forth to find naproxen derivatives which have reduced side effects, there remains a continuing need to identify new compounds that are effective for treating inflammation and inflammatory diseases and devoid of side effects.

The present invention relates to a Nitric Oxide (NO) releasing naproxen having a chiral dinitrate ester chain chemically linked to the carboxylic function of naproxen. This compound has formula (I) and it corresponds to [ (S) - ( (S) -5, 6- bis (nitrooxy) hexyl) 2- ( 6-methoxynaphthalen-2-yl) propanoate] , hereafter called (5S) -5, 6-dinitroxy hexanoate ester of (S)- naproxen .

:D

(5S) -5, 6-dinitroxy hexanoate ester of (S) -naproxen is effective as anti-inflammatory agent, it shows a surprisingly improved gastrointestinal safety and it has the following additional characteristics: elicits significantly less of an increase in intestinal damage (ulcers) and possesses cardioprotective benefits.

Thus (5S) -5, 6-dinitroxy hexanoate ester of (S) -naproxen can be used for the treatment of inflammation and other inflammation- associated disorders, for the treatment of pain, with the additional benefit of having significantly less harmful side effects .

For example, inflammation-associated disorders include osteoarthritis, rheumatoid arthritis, spondyloarthopathies, gouty arthritis, systemic lupus erythematosus and juvenile arthritis .

(5S) -5, 6-dinitroxy hexanoate ester of (S) -naproxen can be used in the treatment of asthma, bronchitis, menstrual cramps, tendinitis, bursitis, skin-related conditions such as psoriasis, eczema, burns and dermatitis.

(5S) -5, 6-dinitroxy hexanoate ester of (S) -naproxen can be used in the treatment of pain such as postoperative pain, dental pain, muscular pain, pain resulting from post-operative inflammation including from ophthalmic surgery such as cataract surgery and refractive surgery and pain resulting from cancer.

Compound (I) of the invention also can be used to treat gastrointestinal conditions such as inflammatory bowel disease, Crohn's disease, irritable bowel syndrome, and for the prevention and or treatment of cancer, such as colorectal cancer .

(5S) -5, 6-dinitroxy hexanoate ester of (S) -naproxen can be used in treating inflammation in such diseases as vascular diseases, migraine headaches, atherosclerosis, periarteritis nodosa, thyroiditis, aplastic anemia, Hodgkin's disease, scleroderma, Sickle cell anemia, rheumatic fever, type I diabetes, neuromuscular junction disease including myasthenia gravis, fibromyalgia, white matter disease including multiple sclerosis, sarcoidosis, nephrotic syndrome, Behcet's syndrome, polymyositis, gingivitis, nephritis, swelling occurring after injury, myocardial ischemia, and the like.

Compound (I) of the invention also can be used in the treatment of ophthalmic diseases, such as retinitis, retinopathies.

Preferably (5S) -5, 6-dinitroxy hexanoate ester of (S) -naproxen can be used in treating inflammation, inflammation-associated disorders include osteoarthritis, rheumatoid arthritis, juvenile arthritis, tendinitis, scleroderma, Sickle cell anemia, pain, muscular pain

Another object of the present invention is to provide pharmaceutical compositions containing (5S) -5, 6-dinitroxy hexanoate ester of (S) -naproxen in combination with at least a pharmaceutical acceptable excipient. Such pharmaceutical compositions can be prepared by methods and contain excipients which are well known in the art. A generally recognized compendium of such methods and ingredients is Remington's Pharmaceutical Sciences by E. W. Martin (Mark Publ . Co., 15th Ed., 1975). The compounds and compositions of the present invention can be administered orally, parenterally (for example, by intravenous, intraperitoneal or intramuscular injection), topically, or rectally.

The amount of (5S) -5, 6-dinitroxy hexanoate ester of (S)- naproxen which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.

The usual daily doses of (5S) -5, 6-dinitroxy hexanoate ester of (S) -naproxen may be in amount of from 0.01 g to 3 g, preferably from 0.05 g to 2 g. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems and are in the same ranges or less than as described for the commercially available naproxen.

Another embodiment of the invention provides compositions comprising (5S) -5, 6-dinitroxy hexanoate ester of (S) -naproxen and other therapeutic agents, such as NSAIDs, 5-lipoxygenase (5-LO) inhibitors, leukotriene B ₄ (LTB ₄ ) receptor antagonists, leukotriene A ₄ (LTA ₄ ) hydrolase inhibitors, 5-HT agonists, HMG- CoA inhibitors, H ₂ receptor antagonists, antineoplastic agents, antiplatelet agents, thrombin inhibitors, thromboxane inhibitors, decongestants, diuretics, sedating or non-sedating anti-histamines, inducible nitric oxide synthase inhibitors, opiods, analgesics, Helicobacter pylori inhibitors, proton pump inhibitors, isoprostane inhibitors, and mixtures of two or more thereof. Another embodiment of the invention provides the use of the compositions comprising (5S) -5, 6-dinitroxy hexanoate ester of (S) -naproxen and other therapeutic agents, such as NSAIDs, 5- lipoxygenase (5-LO) inhibitors, leukotriene B ₄ (LTB ₄ ) receptor antagonists, leukotriene A ₄ (LTA ₄ ) hydrolase inhibitors, 5-HT agonists, HMG-CoA inhibitors, H ₂ receptor antagonists, antineoplastic agents, antiplatelet agents, thrombin inhibitors, thromboxane inhibitors, decongestants, diuretics, sedating or non-sedating anti-histamines, inducible nitric oxide synthase inhibitors, opiods, analgesics, Helicobacter pylori inhibitors, proton pump inhibitors, isoprostane inhibitors, for the treatment of inflammation, inflammation- associated disorders including osteoarthritis, rheumatoid arthritis, spondyloarthopathies, gouty arthritis, systemic lupus erythematosus and juvenile arthritis.

Another embodiment of the invention provides compositions comprising (5S) -5, 6-dinitroxy hexanoate ester of (S) -naproxen and proton pump inhibitors selected from the group consisting of Omeprazole, Esomeprazole, Lansoprazole, Pantoprazole, Dexlansoprazole, Rabeprazole.

The acid susceptible proton pump inhibitors used in the dosage forms of the invention may be used in their neutral form or in the form of an alkaline salt, such as for instance the Mg ²⁺ , Ca ²⁺ , Na ⁺ , K ⁺ or Li ⁺ salts, preferably the Mg ²⁺ salts.

A further embodiment provides the use of the compositions comprising (5S) -5, 6-dinitroxy hexanoate ester of (S) -naproxen and proton pump inhibitors selected from the group consisting of Omeprazole, Esomeprazole, Lansoprazole, Pantoprazole, Dexlansoprazole, Rabeprazole, for the treatment of inflammation, inflammation-associated disorders including osteoarthritis, rheumatoid arthritis, spondyloarthopathies, gouty arthritis, systemic lupus erythematosus and juvenile arthritis . Example 1

Synthesis of (5S) -5, 6-dinitroxy hexanoate ester of (S)- naproxen (Compound (I) )

Step 1

Hex-5-enyl 4-nitrobenzoate

Triethylamine (9.1 mL, 66 mmole, 1 eq) was added to a stirred solution of hex-5-enol (6.6 g, 66 mmole) and 4-nitrobenzoyl chloride (12.3 g, 66 mmole, 1 eq.) in dichloromethane (100 mL) at 0 ⁰ C. The reaction was stirred at room temperature for 4 h and extracted with water, HCl IM, 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 hex-5-enyl 4- nitrobenzoate as yellow oil (15.8 g, 96%).

Step 2

(5S) -5, 6-dihydroxyhexyl 4-nitrobenzoate

A stirred solution of ADMix alpha (40.5 g) in a mixture tBuOH /H2O (187 mL each) was stirred for 10 mins at room temperature and then transferred to a cold room at 4°C. After 15 mins,

Hex-5-enyl 4-nitrobenzoate (3) (7.15 g, 28.7 mmole) was added and the reaction stirred overnight at 4 ⁰ 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 extracted twice with ethyl acetate

(2 x 100 mL) . The combined organic phases were washed with water and brine, dried over sodium sulfate, evaporated. The residue was dissolved in diethylether (100 mL) and left under stirring overnight to give compound (5S) -5, 6-dihydroxyhexyl 4- nitrobenzoate as white solid (5.2 g, 64%). S tep 3

(5S) -5,6-bis (nitrooxy) hexyl 4-nitrobenzoate

To a stirred solution of fuming nitric acid (7.7 mL, 91.8 mmole, 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 (5.2 g, 9.2 mmole) in dichloromethane (30 mL) was added and the reaction stirred at this temperature for 30 mins. The crude mixture was then poured on ice and the organic layer extracted, washed with water, brine, dried over sodium sulfate, evaporated to give a yellow oil (6.8 g, 100%) . The residue obtained was used in the next step without further purification.

Step 4

(2S) -6-hydroxy-2- (nitrooxy) hexyl nitrate

To a stirred solution of (5S) -5, 6-bis (nitrooxy) hexyl 4- nitrobenzoate (6.8 g, 18.2 mmole) in a 1/1 mixture of ethanol

/THF (30 mL of each) at 0 ⁰ C, a 2M sodium hydroxide solution

(9.1 mL, 2 eq) was added and the reaction was stirred for 2 hrs . 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 (2S) -6-hydroxy-2- (nitrooxy) hexyl nitrate as colorless oil (3.82 g, 93%) .

Step 5

(S) - (5S) -5,6-bis (nitrooxy) hexyl) 2- (6-methoxynaphthalen-2-yl) propanoate (Compound (I) )

To a stirred solution of (2S) -6-hydroxy-2- (nitrooxy) hexyl nitrate (6) (2.0 g, 8.92 mmole), (S) -2- ( 6-methoxy-2-naphthyl) - propionic acid (2.05 g, 8.92 mmole, 1 eq) and N, N- dimethylaminopyridine (32 mg, 0.27 mmole, 0.03 eq) at 0 ⁰ C, N- (3-dimethylaminopropyl) -N' -ethyl-carbodiimide hydrochloride

(EDAC, 1.88 g, 9.81 mmole, 1.1 eq) was added and the reaction was stirred for 5 h at RT. The solution was sequentially washed with water, HCl IM, water and brine. The residue was purified by column chromatography in silica (SNAP 100, in a gradient system from 15% to 25 % of ethyl acetate in n-hexane) to give a colorless oil (3.02g, 78%), that was stirred overnight in a 1/5 n-hexane / diisopropylether mixture, to give a white solid that was collected by filtration (2.73 g, 70%) .

Comparative examples

Example 2

Synthesis of (5R) -5, 6-bis (nitrooxy) hexanoate ester of (S)- naproxen (Compound (II) )

(H)

compound (II) is the diastereoisomer of the compound (I) because the chiral carbon atom of the dinitrate ester chain bound to the carboxylic function of naproxen and has configuration (R) .

Step 1

(5R) -5 , 6-dihydroxyhexyl 4-nitrobenzoate

A stirred solution of ADMix beta (56.1 g, 1.4 g/mmol of substrate) in a 1/1 mixture tBuOH /H ₂ O (200 mL each) was stirred for 10 mins at room temperature and then transferred to a cold room at 4°C. After 15 mins, hex-5-enyl 4- nitrobenzoate (10 g, 40.1 mmole) was added and the reaction stirred overnight at 4°C. Then ethyl acetate (250 mL) was added and followed by careful addition of sodium metabisulfite (16 g) . The reaction was left for 30 min at 4°C and then treated with water (250 mL) . The organic layer was extracted and the aqueous extracted twice with ethyl acetate (2 x 100 mL) . The combined organic phases were washed with water and brine, dried over sodium sulfate, evaporated. The residue was dissolved in diethylether (180 mL) and left under stirring overnight to give compound (5R) -5, 6-dihydroxyhexyl 4- nitrobenzoate as white solid (8.4 g, 74%).

¹ H NMR (300 MHz, CDCl ₃ ) δ 8.31 (m, 2H), 8.22 (m, 2H), 4.41 (t, J = 6.6 Hz, 2H), 3.73 (m, 2H), 3.48 (dd, J = 10.9, 7.4 Hz, IH) , 1.69 (m, 9H) .

Step 2

(5R) -5,6-bis (nitrooxy) hexyl 4-nitrobenzoate

At 0°C, to a stirred solution of acetic anhydride (20 mL) was slowly added fuming nitric acid (4 mL, 84.8 mmol, 8 eq) . After 5 min of stirring, (5R) -5, 6-dihydroxyhexyl 4-nitrobenzoate (3.0 g, 10.6 mmol) was added as a solid. The reaction was stirred at this temperature for 30 min and then poured onto ice. After melting, the oil was separated from the aqueous layer and diluted with ethyl acetate (50 mL) . The organic layer was washed with water, saturated NaHCO ₃ , water and brine, dried on Na2SO ₄ , filtered and evaporated to give the desired product as an oil (3.4 g, 86%) .

¹ H NMR (300 MHz, CDCl ₃ ) δ 8.35 - 8.29 (m, 2H), 8.25 - 8.19 (m, 2H), 5.33 (qd, J = 6.3, 3.2 Hz, IH), 4.77 (dd, J = 12.9, 3.1 Hz, IH), 4.52 (dd, J = 12.9, 6.4 Hz, IH), 4.42 (t, J = 6.4 Hz, 2H), 1.95 - 1.81 (m, 4H), 1.72 - 1.55 (m, 2H).

Step 3

(2R) -6-hydroxy-2- (nitrooxy) hexyl nitrate

To a stirred solution of (5R) -5, 6-bis (nitrooxy) hexyl 4- nitrobenzoate (1.6 g, 4.28 mmol) in a 1/1 mixture of ethanol /THF (20 mL of each) at 0 ⁰ C was added a 2M sodium hydroxide solution (2.15 mL, 4.3 mmol, 1 eq) and the reaction was stirred for 2 hrs . The reaction was diluted with ethyl acetate (100 mL) and water (100 mL) and extracted. The aqueous layer was extracted once again with ethyl acetate (50 mL) . The combined organic layers were successively washed with water and brine, dried over sodium sulfate and evaporated. The oily residue was purified by column chromatography (Biotage System, SNAP Cartridge silica 100 g, eluent: n-hexane/ethyl acetate 50/50 isocratic during 12 CV) to give (2R) -6-hydroxy-2- (nitrooxy) hexyl nitrate as colorless oil (0.86 g, 90%).

1H NMR (300 MHz, CDCl ₃ ) δ 5.32 (qd, J = 6.7, 3.0 Hz, IH), 4.77 (dd, J = 12.9, 3.0 Hz, IH), 4.49 (dd, J = 12.9, 6.6 Hz, IH), 3.68 (d, J = 5.5 Hz, 2H), 1.89 - 1.71 (m, 2H), 1.70 - 1.48 (m, 5H) , 1.46 (s, IH) .

Step 4

(S) - (5R) -5,6-bis (nitrooxy) hexyl) 2- (6-methoxynaphthalen-2-yl) propanoate (Compound (II) )

To a stirred solution of (2R) -6-hydroxy-2- (nitrooxy) hexyl nitrate (6) (0.86 g, 3.84 mmol) , (S) -2- ( 6-methoxy-2-naphthyl) - propionic acid (0.88 g, 3.84 mmol, 1 eq) and N, N- dimethylaminopyridine (10 mg, 0.07 mmol, 0.02 eq) at 0 ⁰ C, N-

(3-dimethylaminopropyl) -N' -ethyl-carbodiimide hydrochloride (EDAC, 0.81 g, 4.2 mmol, 1.1 eq) was added and the reaction was stirred for 6 h at RT. The solution was washed with water, HCl IM, water, saturated NaHCO ₃ , water and brine. The residue was purified by column chromatography in silica (Biotage System, SNAP Cartridge silica 100 g, eluent: n-hexane/ethyl acetate 80/20 isocratic during 12 CV) to give a colorless oil (1.46 g, 83%) .

¹ H NMR (300 MHz, CDCl ₃ ) δ 7.72 (d, J = 8.6 Hz, 2H), 7.68 (s, IH), 7.41 (dd, J = 8.6, 1.7 Hz, IH), 7.17 (dd, J = 8.9, 2.4 Hz, IH), 7.13 (d, J = 2.4 Hz, IH), 5.01 (qd, J = 6.9, 2.9 Hz, IH), 4.41 (dd, J = 13.0, 2.9 Hz, IH), 4.21 - 4.11 (m, 2H), 4.11 - 4.01 (m, IH), 3.93 (s, 3H), 3.86 (q, J = 7.1 Hz, IH), 1.66 - 1.46 (m, 7H), 1.37 - 1.17 (m, 2H). ¹³ C NMR (75 MHz, DMSO) δ 174.37, 157.67, 136.18, 133.76, 129.56, 128.85, 127.41, 126.67, 126.05, 119.23, 106.14, 80.58, 72.24, 64.23, 55.61, 44.93, 28.21, 28.04, 21.19, 18.72.

[M+H] : 437.89

Example 3

Synthesis of (2R) -2 ,3-bis (nitroxy) propyl ester of (S) -naproxen (Compound (III) )

(III)

Step 1

(4S) -2 ,2-dimethyl-l ,3-dioxolan-4-yl] methyl 4-nitrobenzoate

To a stirred solution of (R) - (+) -1, 2-isopropylideneglycerol

(1.62 g, 13.7 mmol) and 4-nitrobenzoyl chloride (2.54 g, 13.7 mmol, 1 eq) in dichloromethane (30 mL) was added dropwise triethylamine (1.9 mL, 18.9 mmol, 1 eq) . After 5 h, the organic layer was washed with HCl IM, water and brine, dried on sodium sulfate, filtered and evaporated. The residue was crystallized from ethyl acetate/n-hexane to give the desired product as a pale yellow solid (3.5 g, 98%) .

1H NMR (300 MHz, CDCl ₃ ) δ 8.34 - 8.29 (m, 2H), 8.29 - 8.23 (m, 2H), 4.55 - 4.36 (m, 3H), 4.19 (dd, J = 8.5, 6.2 Hz, IH), 3.90 (dd, J = 8.5, 5.5 Hz, IH), 1.46 (s, 3H), 1.41 (s, 3H).

Step 2

(2S) -2 ,3-dihydroxypropyl 4-nitrobenzoate

A solution of (4S) -2, 2-dimethyl-1, 3-dioxolan-4-yl] methyl 4- nitrobenzoate (3.5 g, 16.71 mmol) in acetic acid (35 mL) and water (15 mL) was heated at 60 ⁰ C for 4 hours. After cooling to room temperature, the solution was partly evaporated and the residue was dissolved in ethyl acetate, washed successively with saturated NaHCO3, water and brine, dried on sodium sulfate, filtered and evaporated. The residue was crystallized from ethyl acetate/diethyl ether/n-hexane to give the desired compound as a white solid (2.06 g, 65%) .

¹ H NMR (300 MHz, CDCl ₃ ) δ 8.38 - 8.29 (m, 2H), 8.29 - 8.18 (m, 2H), 4.56 - 4.43 (m, 2H), 4.14 (dt, J = 6.2, 4.8 Hz, IH), 3.84 (dd, J = 11.3, 3.9 Hz, IH), 3.73 (dd, J = 11.3, 5.8 Hz, IH). Step 3

(2R) -2,3-bis (nitrooxy) propyl 4-nitrobenzoate

At 0°C, to a stirred solution of acetic anhydride (10 mL) was added drop wisely fuming nitric acid (1.88 mL, 44.6 mmol, 6 eq) . After 5 min of stirring, (2S) -2, 3-dihydroxypropyl 4- nitrobenzoate (1.8 g, 7.5 mmol) was slowly added as a solid. After 30 min of stirring at 0 ⁰ C, the reaction was poured on ice and the solid formed was isolated by filtration, washed with water (2 x 20 mL) . The solid was dissolved in ethyl acetate and washed successively with water, saturated NaHCO ₃ , water and brine, dried on sodium sulfate, filtered and evaporated. The residue precipitated from n-hexane / diethyl ether to give the desired product as a white solid (2.15 g, 87%) .

1H NMR (300 MHz, CDCl ₃ ) δ 8.35 (d, J = 8.8 Hz, 2H), 8.22 (d, J = 8.8 Hz, 2H), 5.71 - 5.62 (m, IH), 4.91 (dd, J = 12.9, 3.8 Hz, IH), 4.79 (dd, J = 12.7, 3.8 Hz, IH), 4.75 (dd, J = 12.2, 5.7 Hz, IH), 4.61 (dd, J = 12.6, 5.7 Hz, IH). Step 4

(IR) -2-hydroxy-1- [ (nitrooxy) methyl] ethyl nitrate

At 0°C, to a stirred solution of (2R) -2, 3-bis (nitrooxy) propyl

4-nitrobenzoate (2.15 g, 6.49 mmol) in tetrahydrofuran/ethanol

(1/1, v/v, 20 mL each) was added 1 M solution of sodium hydroxide (7 mL, 7 mmol, 1.08 eq) . The reaction was left for 2 hour and then diluted with ethyl acetate and water (80 mL of each) . The organic later was extracted, washed successively with water and brine, dried on sodium sulfate, filtered and evaporated. The residue was purified by column chromatography (Biotage System, SNAP Cartridge silica 50 g, eluent: n- hexane/ethyl acetate 80/20 to n-hexane/ethyl acetate 60/40 during 12 CV) to give a colorless oil (0.62 g, 50%) .

1H NMR (300 MHz, CDCl ₃ ) δ 5.42 - 5.33 (m, IH), 4.87 (dd, J = 12.9, 3.5 HZ, IH), 4.70 (dd, J = 12.9, 6.5 Hz, IH), 4.04 - 3.89 (m, 2H), 1.91 (t, J = 6.0 Hz, IH, OH).

Step 5

(2R) -2,3-bis (nitrooxy) propyl (2S) -2- (6-methoxy-2-naphthyl) propanoate (Compound (III) )

To a stirred solution of ( (IR) -2-hydroxy-l- [ (nitrooxy) methyl]ethyl nitrate (0.78 g, 3.40 mmol) , (S) -2- ( 6-methoxy-2- naphthyl) -propionic acid (0.68 g, 3.40 mmol, 1 eq) and N, N- dimethylaminopyridine (7 mg, 0.05 mmol, 0.02 eq) at 0 ⁰ C, N- (3- dimethylaminopropyl) -N' -ethyl-carbodiimide hydrochloride

(EDAC, 0.68 g, 3.57 mmol, 1.05 eq) was added and the reaction was stirred for 6 h at RT. The solution was washed with water,

HCl IM, water, saturated NaHCO ₃ , water and brine. The residue was purified by column chromatography in silica (Biotage System, SNAP Cartridge silica 100 g, eluent: n-hexane/ethyl acetate 80/20 isocratic during 12 CV) to give a colorless oil (0.74 g, 68%) .

¹ H NMR (300 MHz, CDCl ₃ ) δ 7.74 (d, J = 1.6 Hz, IH), 7.72 (d, J = 2.2 Hz, IH), 7.67 (s, IH), 7.37 (dd, J = 8.5, 1.7 Hz, IH), 7.18 (dd, J = 8.9, 2.5 Hz, IH), 7.14 (d, J = 2.2 Hz, IH), 5.46 - 5.35 (m, IH), 4.56 (dd, J = 12.9, 3.6 Hz, IH), 4.46 - 4.36 (m, 2H), 4.32 (dd, J = 12.5, 5.4 Hz, IH), 3.98 - 3.85 (m, 4H), 1.62 (d, J = 7.2 Hz, 3H) .

Example 4

Synthesis of (2S) -2 ,3-bis (nitrooxy) propyl ester of (S)- naproxen (Compound (IV) )

(IV)

Step 1

(4R) -2 ,2-dimethyl-l ,3-dioxolan-4-yl] methyl 4-nitrobenzoate

At 0 ⁰ C, to a stirred solution of (S) - (+) -1, 2- isopropylideneglycerol (2.50 g, 18.9 mmol) and 4-nitrobenzoyl chloride (3.51 g, 18.9 mmol, 1 eq) was added dropwise triethylamine (2.6 mL, 18.9 mmol, 1 eq) . The reaction was stirred for 5 h and the reaction was transferred in a separating funnel. The organic layer was washed successively with water, HCl IM, water and brine, filtered on sodium sulfate and evaporated. The residue was crystallized from diethyl ether / n-hexane to give the desired product as a pale yellow solid (4.73 g, 89 %) .

1H NMR (300 MHz, CDCl ₃ ) δ 8.34 - 8.30 (m, 2H), 8.28 - 8.23 (m, 2H), 4.55 - 4.37 (m, 3H), 4.19 (dd, J = 8.5, 6.2, IH), 3.90 (dd, J = 8.5, 5.5, IH), 1.48 (s, 3H), 1.41 (s, 3H).

Step 2

(2R) -2 ,3-dihydroxypropyl 4-nitrobenzoate

A solution of (4R) -2, 2-dimethyl-l, 3-dioxolan-4-yl] methyl 4- nitrobenzoate (4.7 g, 16.71 mmol) in acetic acid (80 mL) , THF

(10 mL) and water (10 mL) was heated at 60 ⁰ C for 6 hours.

After cooling to room temperature, the solution was partly evaporated and the residue was crystallized from ethyl acetate / n-hexane to give the desired compound as a white solid (2.12 g, 53%) .

¹ H NMR (300 MHz, CDCl ₃ ) δ 8.38 - 8.29 (m, 2H), 8.29 - 8.18 (m, 2H), 4.56 - 4.43 (m, 2H), 4.14 (qu, J = 4.8 Hz, IH), 3.84 (dd, J = 11.3, 3.9 Hz, IH), 3.73 (dd, J = 11.3, 5.8 Hz, IH). S tep 3

(2S) -2,3-bis (nitrooxy) propyl 4-nitrobenzoate

At 0°C, to a stirred solution of acetic anhydride (10 mL) was added drop wisely fuming nitric acid (2.4 mL, 52.7 mmol, 6 eq) . After 5 min of stirring, (2R) -2, 3-dihydroxypropyl 4- nitrobenzoate (2.1 g, 8.7 mmol) was slowly added as a solid. After 30 min of stirring at 0 ⁰ C, the reaction was poured on ice and the solid formed was isolated by filtration, washed with water (2 x 20 mL) . The solid was dissolved in ethyl acetate and washed successively with water, saturated NaHCO3, water and brine, dried on sodium sulfate, filtered and evaporated. The residue precipitated from n-hexane / diethyl ether to give the desired product as a white solid (2.07 g, 72%) .

1H NMR (300 MHz, CDCl ₃ ) δ 8.35 (d, J = 8.8 Hz, 2H), 8.22 (d, J = 8.8 Hz, 2H), 5.71 - 5.62 (m, IH), 4.91 (dd, J = 12.9, 3.8 Hz, IH), 4.79 (dd, J = 12.7, 3.8 Hz, IH), 4.75 (dd, J = 12.2, 5.7 Hz, IH), 4.61 (dd, J = 12.6, 5.7 Hz, IH).

Step 4

(IS) -2-hydroxy-l- [ (nitrooxy) methyl] ethyl nitrate

At 0°C, to a stirred solution of (2S) -2, 3-bis (nitrooxy) propyl 4-nitrobenzoate (1.89 g, 5.70 mmol) in tetrahydrofuran / ethanol (1/1, v/v, 17 mL each) was added 1 M solution of sodium hydroxide (6 mL, 6 mmol, 1.05 eq) . The reaction was left for 1 hour and then diluted with ethyl acetate and water (60 mL of each) . The organic later was extracted, washed successively with water and brine, dried on sodium sulfate, filtered and evaporated. The residue was purified by column chromatography (Biotage System, SNAP Cartridge silica 100 g, eluent: n-hexane/ethyl acetate 60/40 to n-hexane/ethyl acetate 60/40 during 12 CV) to give a colorless oil (0.89 g, 86%). ¹ H NMR (300 MHz, CDCl ₃ ) δ 5.42 - 5.34 (m, IH), 4.87 (dd, J = 12.9, 3.6 Hz, IH), 4.70 (dd, J = 12.9, 6.5 Hz, IH), 4.05 - 3.88 (m, 2H), 1.91 (t, J = 6.0 Hz, IH, OH).

Step 5

(2S) -2,3-bis (nitrooxy) propyl (2S) -2- (6-methoxy-2-naphthyl) propanoate (Compound (IV) )

To a stirred solution of ( (IS) -2-hydroxy-l- [ (nitrooxy) methyl]ethyl nitrate (0.50 g, 2.75 mmol) , (S) -2- ( 6-methoxy-2- naphthyl) -propionic acid (0.63 g, 2.75 mmol, 1 eq) and N, N- dimethylaminopyridine (7 mg, 0.05 mmol, 0.02 eq) at 0 ⁰ C, N- (3- dimethylaminopropyl) -N' -ethyl-carbodiimide hydrochloride

(EDAC, 0.55 g, 2.89 mmol, 1.05 eq) was added and the reaction was stirred for 6 h at RT. The solution was washed with water,

HCl IM, water, saturated NaHCO ₃ , water and brine. The residue was purified by column chromatography (Biotage System, SNAP Cartridge silica 100 g, eluent: n-hexane/ethyl acetate 80/20 isocratic during 12 CV) to give a colorless oil (0.74 g, 68%) . ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.73 (d, J = 1.7 Hz, IH), 7.72 (d, J = 2.2 Hz, IH), 7.67 (s, IH), 7.38 (dd, J = 8.9, 1.7 Hz, IH), 7.18 (dd, J = 8.9, 2.5 Hz, IH), 7.14 (d, J = 2.2 Hz, IH), 5.38 (ddd, J = 10.0, 6.0, 4.1 Hz, IH), 4.60 (dd, J = 12.9, 3.6 Hz, IH), 4.51 (dd, J = 12.5, 4.3 Hz, IH), 4.37 (dd, J = 12.9, 6.6 Hz, IH), 4.26 (dd, J = 12.5, 5.5 Hz, IH), 3.97 - 3.87 (m, 4H), 1.62 (d, J = 7.1 Hz, 3H) .

Example 5

Synthesis of 6- (nitrooxy) hexanoate ester of (S) -naproxen (Compound (V) )

(V) Step 1

6-Nitrooxy-hexan-l-ol

To a solution of 6-bromohexan-l-ol (2.2 mL, 16.6 mmol) in CH ₃ CN

(100 mL) was added 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 ₂ O, 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, 86%).

1H NMR (300 MHz, CDCl 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, IH) .

Step 2

6- (nitrooxy) hexyl (2S) -2- (6-methoxy-2-naphthyl)propanoate (Compound (V) )

To a stirred solution of 6-nitrooxy-hexan-l-ol (2.34 g, 15.32 mmol), (S) -2- ( 6-methoxy-2-naphthyl) -propionic acid (3.53 g, 15.32 mmol, 1 eq) and N, N-dimethylaminopyridine (41 mg, 0.31 mmol, 0.02 eq) at 0 ⁰ C, N- (3-dimethylaminopropyl) -N' -ethyl- carbodiimide hydrochloride (EDAC, 3.08 g, 16.09 mmol, 1.05 eq) was added and the reaction was stirred for 6 h at RT. The solution was washed with water, HCl IM, water, saturated NaHCO ₃ , water and brine. The residue was purified by column chromatography in silica (Biotage System, SNAP Cartridge silica 100 g, eluent: n-hexane/ethyl acetate 82/18 isocratic during 12 CV) to give the desired product as a white solid (4.14 g, 72%) . ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.73 (s, IH), 7.70 (s, IH), 7.68 (s, IH), 7.42 (dd, J = 8.5, 1.7, IH), 7.19 - 7.12 (m, 2H), 4.30 (t, J = 6.7, 2H), 4.18 - 4.01 (m, 2H), 3.93 (s, 3H), 3.86 (q, J = 7.1, IH), 1.64 - 1.50 (m, 9H), 1.35 - 1.16 (m, 5H) .

Pharmacological examples

The ability to produce gastric mucosa lesions and also antiinflammatory activity of (55) -5, 6-dinitroxy hexanoate ester of (S) -naproxen (compound (I)) were evaluated in comparison to the following compounds:

- naproxen (parent drug) ;

(5R) -5, 6-bis (nitrooxy) hexanoate ester of (S) -naproxen

(compound (II)); compound (II) is the diastereoisomer of the compound of the invention in that the chiral carbon atom of the dinitrate ester chain bound to the carboxylic function of naproxen and has configuration (R) ;

(2R) -2, 3-bis (nitrooxy) propyl-2- ( 6-methoxy (2-naphthyl) propanoate (Compound (III)); compound (III) was disclosed in WO 2004/004648 (NMI 1182);

(2S) -2, 3-bis (nitrooxy) propyl-2- ( 6-methoxy (2-naphthyl) propanoate (compound (IV)); compound (IV) is the diastereoisomer of compound (III);

- 6- (nitrooxy) hexanoate ester of (S) -naproxen (compound (V)); compound (V) is the correspondent mononitrate derivative of the compound of the invention.

Example Fl

Evaluation of gastric mucosa lesions following single oral administration

Male adult Wistar rats (200-220 body weight) were used (Harlan Italy, Milan) . The compounds under study were orally administered (10 ml/kg) at 30 mg/kg to conscious rats, previously fasted (48 hrs before the experiment) . Under light ether anaesthesia, rats were sacrificed 4 hrs after drug administration. The stomach was removed, opened along the lesser curvature, gently rinsed and examined under a stereomicroscope by two observers unaware of the treatment, to determine the extent of macroscopically visible damage. Each individual hemorrhagic lesion was measured along its greatest length (1 mm = rating of 1; 1-2 mm = rating of 2; >2 mm = rating according to their greatest length) . The overall total is designated as "lesion index". Data are reported in Table 1 as % of naproxen-induced gastric lesion.

Example F2

Anti-inflammatory activity evaluated using the carrageenan- induced paw edema model

Male adult Wistar rats (200-220 body weight) were used (Harlan Italy, Milan) . Briefly, 0.1 ml of carrageenan (1% suspension in CMC) was administered by subplantar injection to conscious animals. Paw volume was measured by a plethysmometer (Basile, Varese) before and at several time-points (2, 3 or 4 h) after carrageenan administration. Drugs under study were orally administered (10 ml/kg) at 30 mg/kg, immediately before carrageenan injection. Control rats received equivalent volumes of vehicle (2% DMSO, 5% castor oil, 5% tween 80, 88% methocel 1%) . Each group of rats comprised of 8-10 animals.

Paw edema was determined in each rat by subtracting the initial volume displacement from subsequent post-carrageenan measurements and expressed as percent increase in paw volume over basal.

Table 1 shows both the gastric damage (as percent of the naproxen-induced gastric damage) and the anti-inflammatory activity of compounds under study (expressed as percent inhibition of maximum paw volume increase observed in control groups, arbitrarily considered as 100).

The results of the gastric mucosa lesions test show that the compound of the invention has significantly and unexpectaly improved gastrointestinal safety compared to its diastereoisomer (compound (II)) and to the other compounds.

Oral administration of the compound (I) reduced paw edema formation in the carrageenan rat model, with similar potency and efficacy relative to naproxen.

The results show that (55) -5, 6-dinitroxy hexanoate ester of (S) -naproxen (compound (I)) has a significantly improved gastrointestinal safety compared to its diastereoisomer (compound (II)) while it shows same anti-inflammatory activity of its diastereoisomer

Table 1

Gastrointestinal

Antiinflammatory lesion index

Compound Activity

(% compared with

(% inhibition) naproxen)

Naproxen 100 46

(D 3.6 ± 2.7 58 ± 5.2

(H) 25 ± 11.3 58 ± 6.6

(III) 25 ± 5.4 52 ± 2.6

(IV) 32 ± 15 56 ± 4.0

(V) 17.5 ± 2.1 49 ± 3.9

Example F3

Evaluation of intestinal mucosa lesions following repeated administration of (5S) -5, 6-dinitroxy hexanoate ester of (S)- naproxen (compound (I)), 6- (nitrooxy) hexanoate ester of (S)- naproxen (compound (V)) or naproxen. Male Wistar rats (-200 g) were treated twice daily with naproxen (0.3-30 mg/kg, p.o.), 6- (nitrooxy) hexanoate ester of

(S)-naproxen (compound (V)) (0.5-49 mg/kg, p.o.) or (5S)-5,6- dinitroxy hexanoate ester of (S) -naproxen (compound (I)) (0.5-

57 mg/kg, p.o.) for 4.5 days. Rats were euthanized 4 h after the final dose of the test substance and the intestinal damage was blindly scored. The length of all lesions (mm) was summed up to give the intestinal damage score.

Table 2 shows the intestinal damage at the highest dose used (equimolar doses) as damage score (mm) of the test compounds. The results of the intestinal mucosa lesions show that compound of the invention at the highest dose tested had significantly improved intestinal safety compared to the parent compound naproxen and its correspondent mononitrate derivative, 6- (nitrooxy) hexanoate ester of (S) -naproxen (compound (V) ) .

Table 2

Compound Intestinal lesion

(mm)

naproxen 143 ± 39

(I) 3.2 ± 10.9

(V) 14.5 ± 39

Example F4

Effects on blood pressure in aged spontaneously hypertensive rats (SHR) of (5S) -5, 6-dinitroxy hexanoate ester of (S)- naproxen (compound (I) ) compared to naproxen Male aged (28-week-old) spontaneously hypertensive rats (SHR) (n=4-5/group) were supplied by Charles River (Calco, Lecco) . They were acclimatized to standard laboratory conditions for 1 month before surgery, with free access to food and water.

Surgery

Rats were anesthetized with an intraperitoneal administration of a mixture of ketamine/xylazine/acepromazine and a segment

(about 1 cm below the renal arteries) of the abdominal aorta was isolated. The catheter tip was inserted into the aorta just above the iliac bifurcation, the transmitter was fixed to the muscles, and the abdominal wall was sutured. After recovery from anesthesia, the rats were housed individually in cages placed on the receivers. Rimadyl Veterinario (carprofene 4 mg/kg subcutaneous, once daily) was administered for 2 days after surgery to prevent infection.

Hemodynamic Measurements

Blood pressure (BP) and heart rate (HR) were recorded and analyzed by a Dataquest A. R. T. software (Data Sciences). The system consists of blood pressure sensors (model TA11PAC40), receivers (model RPC-I), Dataquest A. R. T Gold Acquisition version 4.0 and Dataquest A. R. T Gold Analysis.

Hemodynamic recordings were taken every 2 min, starting 1 day before drug administration until the return to the basal value. Each recording lasted 10 sec, and the average of the values calculated for each hour is compared to the basal value

(1 hr before the treatment) of the same day.

Results

(55) -5, 6-dinitroxy hexanoate ester of (S) -naproxen (compound (I)) was orally administered to spontaneously hypertensive rats (SHR) at the equal molar dose of 30 mg/kg naproxen and blood pressure was monitored for the following 24 hours.

Single oral administration to SHR of (55) -5, 6-dinitroxy hexanoate ester of (S) -naproxen caused a reduction of systolic blood pressure of about 20 mmHg over basal (194 mmHg) within the first 6 hours after the treatment. On the contrary, naproxen-treated animals did not show any changes in blood pressure over basal (-4 mm Hg) .

Example F5

Evaluation of the ability to release nitric oxide of (5S) -5,6- dinitroxy hexanoate ester of (S) -naproxen (Compound (I) ) , (2R) -2,3-bis (nitroxy) propyl ester of (S) -naproxen (Compound (III)) and naproxen. Thoracic aortas from male New Zealand rabbits were used. The aortas were placed immediately in Krebs-HEPES buffer (pH 7.4; composition mM: NaCl 130.0, KCl 3.7, NaHCO3 14.9, KH2PO4 1.2, MgSO4»7H2O 1.2, Glucose 11.0, HEPES 10.0, CaC12»2H2O 1.6). Connective tissue was removed and aortas were cut into ring segments (4-5 mm in length) . Each ring was placed in a 5 ml tissue bath filled with Krebs-HEPES buffer (37°C) aerated with

95% 02 and 5% CO2 and was attached to a force transducer

(Grass FT03) , connected to a BIOPAC MP150 System for measurement of the isometric tension. Preparations were allowed to equilibrate for 1 h at a resting tension of 2 g with changes of the buffer every 15 min. Then the rings were stimulated by exposure to 90 mM KCl (3 times) with intervening washings. After equilibration, they were precontracted submaximally with methoxamine (3 μM) and, when the contraction was stable, acetylcholine (ACh, 3 μM) was added. A relaxant response to ACh indicated the presence of a functional endothelium. After washout, the rings were precontracted submaximally with methoxamine 3 μM. When a steady-state level of contraction was obtained, a cumulative concentration- response curve to test drug (0.01-100 μM) was obtained in the presence of a functional endothelium. The time intervals between doses were based on the time needed to reach a full response. The results are given as mean ± SEM. The responses are expressed as percentage relaxation and plotted vs concentration. The sensitivity of rabbit aorta to different vasodilators is expressed as the concentration that elicited 50% of the maximal responses (EC50) . The responses are quantified in terms of EC50 and Emax (maximal vasodilating effect) values, obtained from the concentration-response curve by nonlinear curve fitting, using GraphPad software.

Results

(55) -5, 6-dinitroxy hexanoate ester of (S) -naproxen evoked concentration-dependent relaxation with an EC ₅ O of 0.8 μM, achieving 96.6% relaxation at the highest concentration tested of 100 μM.

Naproxen did not induce vasorelaxation at any concentration tested (same as vehicle, DMSO 0.01%). Moreover, the reference compound (2R) -2, 3-bis (nitroxy) propyl ester of (S) -naproxen (Compound (III)) relaxed the rabbit aorta with an EC ₅ O of 3.3 μM and E _max of 62.5%. These data show that (55) -5, 6-dinitroxy hexanoate ester of (S) -naproxen is more potent than the reference compound (compound (III)) in inducing relaxation of isolated aortic rings .

Table 3 shows the rabbit aorta rings vasorelaxation parameters of (55) -5, 6-dinitroxy hexanoate ester of (S) -naproxen and (2R)- 2, 3-bis (nitroxy) propyl ester of (S) -naproxen (Compound (III)).

Table 3

Compound EC50 (μM) Emax

naproxen - -

(D 0 .8 96. 6 ± 3 .3

(III) 3 .3 62. 5 ± 2 .5 Example F6

Evaluation of the ability to relax human mammary arteries of (5S) -5, 6-dinitroxy hexanoate ester of (S) -naproxen (compound (I)) and naproxen .

Human internal mammary artery (HIMA) segments from patients (n=6; 5 male and 1 female, aged 69±9 years old) undergoing coronary artery bypass graft (CABG) were obtained from the cardiovascular division at "L. Sacco" Hospital (Milan, Italy). All patients gave their informed consent for excision of remaining tissue according to the declaration of Helsinki and approval to use the discarded arterial segments was given by the Ethical Committee. Only arteries without macroscopic evidence of atherosclerosis were used. The selected IMA segments were collected, placed into cold (4°C) Krebs-HEPES buffer (NaCl 130 mM, KCl 3.7 mM, NaHCO3 14.9 mM, KH2PO4 1.2 mM, MgSO4 • 7 H20 1.2 mM, Glucose 11 mM, HEPES 10 mM, CaC12 • 2 H2O 1.6 mM and ascorbic acid 0.16 mM) and taken to the laboratory immediately. The vessels were dissected out from the surrounding connective tissue and cut into 3-mm long rings . The rings were suspended in organ chambers containing Krebs-HEPES buffer at 37°C and aerated with 95% 02 and 5% CO2, and connected to a force displacement transducer (Grass FT03) for the measurement of isometric force. The rings were stretched to an optimal load (about 1 g) and after 1 h of equilibration were primed by repeated exposure to 9OmM KCl with intervening washings. The endothelium was preserved by cautiously dissecting and mounting the arterial rings. One to four rings were obtained from each vessel sample and each ring was subjected to only one experiment.

To evaluate both the endothelium-dependent and endothelium- independent vasorelaxation, all the arterial rings used in this study were precontracted with KCl (90 mM) and when the contraction reached a stable plateau, acetylcholine (ACh) at 10-5M or sodium nitroprusside (SNP) at 10-6M was added. A relaxant response to ACh indicates the presence of a functional endothelium. Then the vascular rings were re- exposed to KCl (90 mM) and, after obtaining a stable contraction, a cumulative concentration-relaxation curve of test drugs (10-8M - 10-4M)was established. The results are given as mean ± SEM. The vasodilating responses were expressed as percentage relaxation and plotted vs concentration. The responses were quantified in terms of EC50 and Emax (maximal vasodilating effect) values, obtained from the concentration- response curve by nonlinear curve fitting, using GraphPad software . Results

(55) -5, 6-dinitroxy hexanoate ester of (S) -naproxen (compound (I)) induced a significant and sustained relaxation of hIMA rings. In particular, the compound of this invention resulted in 60% maximal relaxation of the hIMA segments contracted with KCl. Moreover, the relaxing effect of (55) -5, 6-dinitroxy hexanoate ester of (S) -naproxen (compound (I)) was concentration-dependent with an EC50 of 20 μM. On the contrary, the artery rings exposed to naproxen presented a weak tendency to contract.

These results show that the NO release from (55) -5, 6-dinitroxy hexanoate ester of (S) -naproxen (compound (I)) is able to counteract the contracting effects of naproxen due to COX inhibition in arteries from patients with supposed dysfunctional endothelium. These data indicate that (55) -5,6- dinitroxy hexanoate ester of (S) -naproxen have beneficial effects in disorders associated with endothelial alteration. Example F7

Evaluation of COX-independent platelets aggregation of (5S) - 5,6-dinitroxy hexanoate ester of (S) -naproxen (compound (I)), 6- (nitrooxy) hexanoate ester of (S) -naproxen (compound (V)) and naproxen .

Human blood was obtained from healthy volunteers who had not taken drugs for 10 days prior to the study. Prostacyclin- washed platelets were prepared from blood collected into a trisodium citrate/prostacyclin mixture and they were resuspended in Tyrode's solution containing 2 mM Ca ²⁺ . Aggregation was induced by the U46619 (5μg/ml) and was monitored for 6 min in a Payton dual-channel aggregometer by the method of Born and Cross (Born GV and Cross MJ, J Physiol. 1963; 168:178-195). Platelets were treated with test compounds (1-100 μM) 10 min before the induction of aggregation.

Results

(55) -5, 6-dinitroxy hexanoate ester of (S) -naproxen (compound

(I)) inhibited platelet aggregation induced by U46619 in a dose-dependent manner. Likewise, 6- (nitrooxy) hexanoate ester of (S) -naproxen (compound (V)) antagonized U46619-induced platelet aggregation. On the contrary, naproxen did not affect aggregation on its own.

The results show that the (55) -5, 6-dinitroxy hexanoate ester of (S) -naproxen is more potent than its mononitrate derivative

(compound (V)) in inhibiting platelet aggregation (IC ₅ O=

16±1.3and 63±1.2μM, respectively).

These data indicate that (55) -5, 6-dinitroxy hexanoate ester of

(S) -naproxen exerts additional effects in disorders associated with thrombotic events. Example 8

In vivo mouse model of bleomycin-induced lung fibrosis

This test was an evaluation of the effect of compound (I) on lung fibrosis. Compound (I) was compared with its parent drug naproxen.

The results of this study show that compound (I) has therapeutic effects in an in vivo mouse model of bleomycin- induced lung fibrosis [Kaminski et al . , 2000; Moeller et al . , 2006] .

Test compounds and doses:

Compound (I): [ (S) - ( (S) -5, 6-bis (nitrooxy) hexyl) -2- ( 6-methoxy naphthalen-2-yl) propanoate] ; (1.9 or 19 mg/kg)

Naproxen: equimolar to Compound (I) (1 or 10 mg/kg)

Vehicle: 2% DMSO, 5% castor oil, 5% Tween 80, 88% Methocel, 1% in water; mixed under stirring in the above order.

Methods

46 Male C57BL/6 mice, about 2-months old and weighing 25-30 g were anaesthesised with 100 μl zolazepam (Zoletil, 50 μg/g b.wt.) i.p. and operated: some of them were treated with bleomycin (0.05 IU in 100 μl saline) and the remaining ones with saline (non-fibrotic negative controls, n=6) . Bleomycin was delivered by tracheal puncture. All the animals awakened from anaesthesia and survived until the next experimental step.

The bleomycin-treated mice were divided into 5 groups and treated daily orally by an intra-gastric catheter with 100 μl vehicle (fibrotic positive controls, n=6) or compound (I) (1.9 or 19 mg/kg, n=8/group) or naproxen (1 or 10 mg/kg, n=8/group) , dissolved in 100 μl vehicle. The non-fibrotic negative control mice did not undergo further treatment. Oral administration of vehicle or compounds was repeated at h. 09.00 AM until day 14 from surgery (with the exclusion of week-ends) , according to the protocol, for a total of 10 daily doses .

Lung tissue sampling

At day 14 from surgery, the animals were killed and the whole left lungs were excised and fixed by immersion in 4% formaldehyde in PBS for histological analysis. The right lungs were weighed, quickly frozen and stored at -80° C. When needed for the biochemical assays, the latter samples were thawed at 4° C, homogenized on ice in 50 mM Tris-HCl buffer containing 180 mM KCl and 10 mM EDTA, final pH 7.4, and then centrifuged at 10,000 g, 4° C, for 30 min, unless otherwise reported. The supernatants and the pellets were collected and used for separate assays, as detailed below. Determination of myeloperoxidase (MPO) activity. This tissue marker of leukocyte accumulation was determined on aliquots

(100 μl) of the supernatants, using a commercial ELISA kit

(CardioMPO™, Prognostix Inc., Cleveland, OH, USA), according to the manufacturer's instructions. The values are expressed as mU/mg lung tissue (wet wt . ) .

Determination of thiobarbituric acid-reactive substances (TBARS) . TBARS, such as malondialdehyde, are end-products of cell membrane lipid peroxidation by ROS and are considered reliable markers of oxidative tissue injury. They were determined by measurement of the chromogen obtained from the reaction of TBARS with 2-thiobarbituric acid [Aruoma et al . , 1989]. Briefly, 0.5 ml of 2-thiobarbituric acid (1% w/v) in 50 mM NaOH and 0.5 ml of HCl (25% w./v. in water) were added to the lung tissue pellets. The mixture was placed in test tubes, sealed with screw caps, and heated in boiling water for 10 min. After cooling, the chromogen was extracted in 3 ml of 1- butanol, and the organic phase was separated by centrifugation at 2,000 x g for 10 min. The absorbance of the organic phase was read spectrophotometrically at 532 nm wavelength over a Standard curve of 1, 1, 3, 3-tetramethoxypropane . Protein concentration was determined with the Bradford method over an albumin standard curve. The values are expressed as nmol/mg of proteins.

Determination of 8-hydroxy-2' -deoxyguanosine (8-OHdG). Frozen lung samples were thawed at room temperature and cell DNA isolation was performed according to [Lodovici et al . , 2000], with minor modifications. The samples were homogenized in 1 ml of 10 mM phosphate-buffered saline, pH 7.4, sonicated on ice for 1 min., added with 1 ml of 10 mM Tris-HCl buffer, pH 8, containing 10 mM EDTA, 10 mM NaCl, 0.5 % SDS, and incubated for 1 h at 37° C with 20 μg/ml RNAse (Sigma, Milan, Italy) . Then, the samples were incubated overnight at 37° C in the presence of 100 μg/ml proteinase K (Sigma) . After incubation, the mixture was extracted with chloroform/isoamyl alcohol (10:2 v/v) . DNA was precipitated from the aqueous phase with 0.2 volume of 10 M ammonium acetate, solubilised in 200 μl of 20 mM acetate buffer, pH 5.3, and denaturised at 90° C for 3 min. The extract was then supplemented with 10 IU of Pl nuclease in 10 μl and incubated for 1 h, at 37° C with 5 IU of alkaline phosphatase in 0.4 M phosphate buffer, pH 8.8. All the procedures were performed in the dark. The mixture was filtered by an Amicon Micropure-EZ filter (Amicon, MA, USA) and 100 μl of each sample were used for 8OHdG determination using an ELISA kit (JaICA, Shizuoka, Japan) , following the instructions provided by the manufacturer. The values are expressed as ng/mg of protein (determined by the Bradford method) .

RESULTS

Myeloperoxidase (MPO) activity is an index of leukocyte accumulation into the inflamed lung tissue. Determination of myeloperoxidase (MPO) activity showed (Table 1) that this parameter was markedly increased in the bleomycin-treated mice which received the vehicle alone (0.464 mU/mg lung tissue) as compared with the saline-treated controls (0.124 mU/mg lung tissue) . Administration of compound (I) as well as naproxen caused a statistically significant, dose-dependent reduction of MPO activity. However, compound (I) at both low and high dose was significantly more effective (0.255 and 0.192 mU/mg lung tissue, p<0.05) than equimolar naproxen (0.359 and 0.274 mU/mg lung tissue) .

Table 1: Myeloperoxidase mU/mg lung tissue (wet wt . )

Saline 0.124 ± 0.028

Bleomycin + vehicle 0.464 ± 0.077

Bleomycin + Comp. (I) (1.9 mg/kg) 0.255 ± 0.043

Bleomycin + Comp. (I) (19 mg/kg) 0.192 ± 0.057

Bleomycin + Naproxen (1 mg/kg) 0.359 ± 0.048

Bleomycin + Naproxen (10 mg/kg) 0.274 ± 0.031

TBARS, such as malondialdehyde, are end-products of cell membrane lipid peroxidation by ROS and are considered reliable markers of oxidative tissue injury.

Determination of thiobarbituric acid-reactive substances (TBARS) showed (Table 2) that this parameter was markedly increased in the bleomycin-treated mice which received the vehicle alone (57.77 mmol/mg protein) as compared with the saline-treated controls (16.71 mmol/mg protein). Administration of compound (I) as well as naproxen caused a statistically significant, dose-dependent reduction of TBARS. However, compound (I) at the high dose was significantly more effective (20.7 mmol/mg protein, p<0.001) than equimolar naproxen (32.27 mmol/mg protein). Table 2: TBARS (mmol/mg protein)

Saline 16.71 ± 2.215

Bleomycin + vehicle 57.77 ± 5.028

Bleomycin + Comp. (I) (1.9 mg/kg) 35.49 ± 2.185

Bleomycin + Comp. (I) (19 mg/kg) 20.7 ± 2.096

Bleomycin + Naproxen (1 mg/kg) 39.29 ± 4.27

Bleomycin + Naproxen (10 mg/kg) 32.27 ± 3.901

8-hydroxy-2' -deoxyguanosine (8-OHdG) is an indicator of oxidative DNA damage. Determination of 8-hydroxy-2' - deoxyguanosine (8-OHdG) showed a similar trend as TBARS (Table 3) . In fact, this parameter was markedly increased in the bleomycin-treated mice which received the vehicle alone (65.75 ng/mg protein) as compared with the saline-treated controls

(15.3 ng/mg protein). Administration of compound (I) as well as naproxen caused a statistically significant, dose-dependent reduction of TBARS. Also for this parameter, compound (I) at both low and high dose was significantly more effective (40.74 and 21.73 ng/mg protein, p<0.001) than equimolar naproxen

(51.13 and ng/mg 33.84 protein).

Table 3: 8OH-dG (ng/mg protein)

Saline 15.3 ± 2.417

Bleomycin + vehicle 65.75 ± 7.192

Bleomycin + Comp. (I) (1.9 mg/kg) 40.74 ± 7.67

Bleomycin + Comp. (I) (19 mg/kg) 21.73 ± 2.776

Bleomycin + Naproxen (1 mg/kg) 51.13 ± 5.767

Bleomycin + Naproxen (10 mg/kg) 33.84 ± 2.544

In conclusion, in an animal model of lung fibrosis, Compound

(I) showed improved efficacy over naproxen in reducing different inflammatory parameters, including leukocyte accumulation and markers of oxidative stress.