ITMI20111818A1 - ASPIRIN DERIVATIVES DONORS OF NITRIC OXIDE - Google Patents

Description

Aspirin derivatives donor nitric oxide

The present invention relates to nitric oxide donor aspirin derivatives, to a process for their preparation and pharmaceutical compositions containing them.

Aspirin (acetylsalicylic acid) is a well known drug belonging to the class of non-steroidal anti-inflammatory drugs (NSAIDs) which exhibit a variety of actions including anti-inflammatory, analgesic, antipyretic and antithrombotic activity. The major drawback that limits its use is a significant gastrotoxicity which is responsible for gastric ulceration, exacerbation of peptic ulcer symptoms, gastrointestinal bleeding and erosive gastritis (Goodman & Gilman's The Pharmacological Basis of Therapeutics; 10th Ed .; McGraw-Hill, Chapter 27).

Many attempts have been made to overcome these side effects. A recent strategy to decrease the gastrotoxicity of aspirin has been the combination of aspirin with nitric oxide releasing groups. Aspirin with nitric oxide releasing groups (derivatives of aspirin releasing NO) are known in the art and are reported to have an improved gastrointestinal and cardiovascular safety profile compared to aspirin (WO 92/01668, WO 95 / 30641, WO 97/16405 and US 5,859,053).

A limitation of the aspirins that release NO of the prior art is the high enzymatic lability of the O-acetyloxy group which becomes labile in plasma when the carboxylic acid is converted to ester. Consequently these substances are rapidly metabolised to salicylic acid in human serum without the formation of significant quantities of aspirin.

Further disadvantages of many aspirin derivatives that release NO are due to the high hydrophobility and poor water solubility of the compounds. Due to these disadvantages, their systemic absorption is low.

WO 2009/049961, WO 2010/118968 and Gasco, J. Med. Chem. 2011; 54 (15): 5478â € “84, describe derivatives of aspirin in which the nitric oxide donor group has been linked through a labile bond of labile carbonyloxy methyl ester to the carboxylic group of aspirin. These compounds are capable of releasing aspirin when incubated in human serum, but show poor solubility in water and consequently low bioavailability or have a high molecular weight which results in the use of high doses of drug to obtain acceptable levels of acetylsalicylic acid. in the body.

There is therefore a need for drugs that maintain the therapeutic effects of aspirin with decreased unwanted side effects and good bioavailability. Therefore, the subject of the present invention are compounds of general formula (I) and their pharmaceutically acceptable salts or their stereoisomers

O O <R> 1 R2

OO O <(CH> 2 <)> m <(CH)> n <(NH)> p (CH) q ONO2O

OR

(THE)

where is it:

m is an integer from 0 to 3;

n is 0 or 1;

p is 0 or 1;

q is an integer from 1 to 3;

each of R1 and R2 independently is H, C1-C3alkyl, (CH2) r-NH2 where r is an integer from 0 to 4 or (CH2) s-O-CO- (CH2) t-COOH where s is an integer from 0 to 4 and t is an integer from 1 to 4; provided that:

when p is 1 each of R1 and R2 independently is H or C1-C3alkyl;

when p is 0 one of R1e R2Ã ̈ (CH2) r-NH2o (CH2) s-O-CO- (CH2) t-COOH where r, s, and t are as defined above and the other is H or C1- C3alkyl.

When the compounds of the invention contain a salifiable carboxyl group, examples of pharmaceutically acceptable salts are both those with inorganic bases, such as sodium, potassium, calcium and aluminum hydroxides, or with organic bases such as, lysine, arginine, triethylamine, dibenzylamine, piperidine or other pharmaceutically acceptable organic amines or bases such as those reported for example in Wermuth C.G. and Stahl, P.H. Pharmaceutical Salts: Properties, Selection, and Use - A Handbook Verlag Helvetica Chimica Acta, 2002.

The compounds according to the present invention, when they contain a salifiable nitrogen atom in the molecule, can be transformed into the corresponding salts by reaction, in an organic solvent such as acetonitrile, tetrahydofuran, with the corresponding organic or inorganic acids.

Examples of organic acids are oxalic, tartaric, maleic, succinic, citric and trifluoroacetic acids. Examples of inorganic acids are nitric, hydrochloric, sulfuric, phosphoric and other pharmaceutically acceptable acids such as those reported for example in Wermuth C.G. and Stahl, P.H. Pharmaceutical Salts: Properties, Selection, and Use - A Handbook Verlag Helvetica Chimica Acta, 2002.

The compounds of the invention which have one or more asymmetric atoms can exist as optically pure enantiomers, pure diastereomers, mixtures of enantiomers, mixtures of diastereomers, racemic mixtures of enantiomers, racemates or mixtures of racemates.

The scope of the invention also includes all possible isomers, stereisomers and their mixtures of the compounds of formula (I), including mixtures enriched in a particular isomer.

A preferred embodiment of the invention are the compounds of formula (I) where p is 1, m is 2 or 3, n is 0, q is an integer from 1 to 3, R1 and R2 are H.

A preferred embodiment of the invention are the compounds of formula (I), where p is 0, m is 1 or 2, n is 0 or 1, q is an integer from 1 to 3, one of R1 and R2Ã ̈ (CH2) r-NH2 where r is an integer from 0 to 3 and the other is H.

A preferred embodiment of the invention are the compounds of formula (I) where p is 0, m is an integer from 0 to 2, n is 0 or 1, q is an integer from 1 to 3, one of R1e R2Ã ̈ (CH2) s-0 â € “CO- (CH2) t-COOH where s is 0 or 1 and t is an integer from 1 to 3, preferably 2 and the other is H.

Particularly preferred compounds of formula (I) are:

O O OO O <NH> 2 <.HCl>

ONO OAc2

(1)

OOClH

H.

No.

O O O ONO2

OCOCH3

ONO2O OR O

OR

O O O OH

OR

OR

OR

(3)

O O OO O <NH> 2 <.HCl> OAc ONO2

(4)

OO <ONO> 2

O O O NH2.HCl OAc

(5)

OR NOT

Or O2

O O O NH2.HCl OAc

(6)

O O ONO2

NH Cl

O O O2.H

OAc

(7)

OR NOT

Or O2

NH

O O O2.HCl

OAc

(8)

NH

O O O2.HCl ONO OAc2

(9)

O O

NH

O O O2.HCl

OAc ONO2 (10)

O O OO O <ONO> 2

<NH2.HCl>

OAc

(11)

OONH2.HCl

O O O ONO2OAc

(12)

NH

O O2.HCl

O O O ONO2OAc

(13)

O O NH2.HCl

OR NOT

O O O2

OAc

(14)

O O

OR NOT

O O O2

NH OAc2.HCl

(15)

O <O> ClH

O O O <N> ONO

H2

OCOCH3

(16)

O O ClH

OR NOT

O O O <N> 2

H.

OCOCH3

(17)

O O ClH

O O O <N> ONO

H2

OCOCH3

(18)

O O H ClH

NINTH

O O O2

OCOCH3

(19)

O O O O O O O OH

OR

OR ONO2

(20)

O O O O O O O OH

OR

OR ONO2

(21)

OR NOT

O O O2

OH

O O O O

OR

OR

OR

(22)

The compounds of the invention can be used for

prevention and treatment of cardiovascular events

thrombotics caused by platelet aggregation,

thrombosis, and following ischemic clinical events, including

thrombotic and thromboembolic stroke, myocardial ischemia,

myocardial infarction, angina pectoris, ischemic attack

transient, reversible ischemic neurological deficits, e

any such thrombotic event in any matrix

vascular (splanchnic, renal, aortic, peripheral, etc.).

The compounds of the invention are useful for the treatment of

pain, fever and inflammation of a variety of

conditions including rheumatic fever, associated symptoms

with flu and other viral infections, colds

pain, back and neck pain, dysmenorrhea, ache

head, toothache, sprains and strains, myositis,

neuralgia, synovitis, arthritis, including rheumatoid arthritis,

degenerative joint disease (osteoarthritis),

gout and ankylosing spodylitis, bursitis, burns, wounds, resulting from surgical and dental operations.

The above compounds of formula (I) can be

used to treat cancer, for example tumors, tumors

malignant, neoplasms and other dysproliferative diseases, which

they can be treated according to the invention, including

leukemias such as myeloid and lymphocytic leukemia, lymphomas,

myeloproliferative diseases, and solid tumors such as, but

without being limited to them, sarcomas and carcinomas, such as

fibrosarcoma, chondrosarcoma, osteogenic sarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelium-sarcoma, synovoma, mesothelioma, colon cancer, gastric cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma basal, adenocarcinoma, carcinoma of the tongue, adeno papillary carcinoma, cystadenocarcinoma, medulla carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choreocarcinoma, cervical cancer, testicular cancer, lung cancer, lung cancer small and non-small cells, spleen carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, hemangioblastoma, oligodendroglioma, melanoma, neuroblastoma and retinoblastoma.

In particular, the above compounds of formula (I) can be used to treat cancers such as colon carcinoma, gastric cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, medulla carcinoma, renal cell carcinoma. , spleen carcinoma.

The compounds of formula (I) can be used in combination with both cell therapy, radiotherapy and any type of chemotherapy such as alkylating agents (nitrogenous mustards, cisplatin, etc.) antimetabolites (methotrexate, 5-fluoro uracil, etc.), natural products ( vincristine, doxorubicin, topotecan, etoposide, etc) hormones and antagonists (tamoxifen, prednisone, flutamide, leuprolide etc), biological response modifiers (IFN-Alpha, IL-2, antibodies, etc) and any other modifier of specific signals (HDAC inhibitors, carbonic anhydrase inhibitors, etc).

The present invention also provides pharmaceutical kits comprising a compound of formula (I) and at least one chemotherapeutic agent.

The present invention also provides pharmaceutical kits comprising one or more containers filled with one or more compounds and / or compositions of the present invention and one or more chemotherapeutic agents listed above as combined preparations for simultaneous, separate, or sequential use. The compounds of the invention can be used alone or in combination with NSAIDs, such as those described in Goodman and Gilman's, The Pharmacological Basis of Therapeutics, Tenth edition, p. 687-716.

Another object of the present invention relates to pharmaceutical compositions containing at least one compound of formula (I) together with non-toxic adjuvants and / or carriers usually used in the pharmaceutical field.

The daily dose of the active ingredient to be administered can be a single dose or it can be an effective amount divided into several smaller doses that need to be administered throughout the day.

Usually, a total daily dose can be preferably in the amount of 50 to 1000 mg.

The dosage regimen and frequency of administration for treating the aforementioned diseases with the compound of the invention and / or the pharmaceutical compositions of the present invention will be chosen according to a variety of factors, including for example age, body weight, sex and medical condition of the patient as well as the severity of the disease, the route of administration, drug considerations and possible concomitant therapy with other drugs.

In some cases, dosage levels above or below the range mentioned above and / or more frequent, may be adequate and this will logically depend on the judgment of the physician and the state of the disease.

The compounds of the invention can be administered orally, parenterally, rectally or topically, by inhalation or aerosol, in formulations optionally containing pharmaceutically acceptable conventional non-toxic carriers, adjuvants and vehicles as desired.

Topical administration may also involve the use of stransdermal administration such as transdermal patches or iontophoresis devices. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intrasternal injection, or infusion techniques.

The injectable preparations, for example sterile injectable aqueous or oily suspensions, can be formulated according to the known art using suitable dispersing or wetting agents or suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a parenterally acceptable non-toxic diluent or solvent.

Acceptable carriers and solvents include water, Ringer's solution and isotonic sodium chloride.

In addition, sterile fixed oils are conventionally used as a solvent or suspension medium. Any fixed oil can be used including synthetic mono or diglycerides, also fatty acids such as oleic acid are used in injectable preparations.

Suppositories for rectal administration of the drug can be prepared by mixing the active ingredient with a suitable non-irritating excipient, such as cocoa butter and polyethylene glycols.

Solid dosage forms for oral administration may include capsules, tablets, pills, powders, granules and gels. In such dosage forms, the active compound can be mixed with at least one inert diluent such as sucrose, lactose and starch. Such dosage forms may also comprise, as in usual practice, additional substances other than inert diluents, for example lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills can also be prepared with enteric coatings. Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art such as water. Such compositions can also comprise adjuvants, such as wetting agents, emulsifying and suspending agents and sweetening, flavoring and the like.

Synthesis procedure

The compounds of general formula (I), where R1, R2, m, n, p and q are as defined above, can be obtained through a process comprising the steps:

a) reaction of the silver salt of aspirin of formula (II):

OR

<O +> Ag-

OR

OR

with a compound of formula (III):

Al-CH2O-CO-O-X

(III)

Where Al à ̈ Cl, Br, I and X is selected from the group consisting of para nitrophenyl, pentafluorophenyl, in an organic solvent such as aromatic hydrocarbons, ethers, at a temperature between -40 ° C and the reflux temperature for a time between a few minutes and 72 hours, to obtain a compound of formula (IV):

O OOO O X O

OR

(IV)

where X is as defined above, and

b) reaction of the compound of formula (IV) obtained in step a) with an alcohol of formula (V):

<R '> 1P1R2'

H O <(CH> 2 <)> m <(CH)> n (N) p (CH) qQ

(V)

where m, n, p, and q are as defined above:

each of R1â € ™ and R2â € ™ independently is H, C1-C3alkyl, (CH2) r-NH-P1, where R is as defined above, P1Ã is a protective amino group such as Boc, Fmoc or those described in T. W. Greene â € œProtective groups in organic suynthesisâ €, Haward University Press, 1980, or (CH2) s-O-CO- (CH2) t-COOP2 where P2Ã ̈ a carboxyl protective group such as an allyl ester such as those described in T. W. Greene â € œProtective groups in organic suynthesisâ €, Haward University Press, 1980, s and t are as defined above, Q is ONO2o a chlorine atom, a bromine atom, an iodine atom, a mesyl group, a tosyl group, to give a compound of formula (VI):

O <O>

<R '> 1 P1 R'2

OO O <(CH> 2 <)> m <(CH)> n (N) p (CH) qQ

OR

OR

(YOU)

The reaction can be carried out in a suitable organic solvent such as hydrogenated hydrocarbons, ethers, aromatic hydrocarbons, dipolar aprotic solvents preferably DMF, in the presence of a suitable base such as tertiary amines, in particular triethylamine or cesium carbonate and

c) when Q is a chlorine atom, a bromine atom, an iodine atom, a mesyl group, a tosyl group, conversion of the compound of formula (VI) obtained in step b) into the nitro derived by reaction with a source of nitrate such as silver nitrate, lithium nitrate, sodium nitrate, potassium nitrate, magnesium nitrate, calcium nitrate, iron nitrate, zinc nitrate, or tetralkylammonium nitrate (where alkyl is ̈ C1-C10alkyl) in a suitable organic solvent such as acetonitrile, tetrahydrofuran, methylethyl ketone, ethyl acetate, DMF, the reaction is carried out, in the dark, at a temperature between room temperature and the boiling temperature of the solvent. The preferred source of nitrates is silver nitrate;

And

d) when p is 1 or one of R1â € ™, R2â € ™ independent is (CH2) r-NH-P1o (CH2) s-O-CO- (CH2) t-COOP2, deprotection of the compound obtained in step b) or c) as described in T. W. Greene â € œProtective groups in organic suynthesisâ €, Haward University Press, 1980, 2nd edition. Trifluoroacetic acid or an anhydrous inorganic acid are the preferred methods for removing the Boc protecting group, an organic base such as piperidine is the preferred method for removing the Fmoc protecting group. Dimedone followed by triphenylphosphine and palladium tetrakis is the preferred method for removing the protective allyl ester group.

Compounds (II), (III), and (V) can be obtained by methods known in the art.

Compound (II) can be obtained by reaction of acetylsalicylic acid with a water-soluble salt of Ag such as silver oxide in a mixture of a suitable organic solvent such as acetonitrile, tetrahydrofuran and water. The reaction is carried out, in the dark, at a temperature between room temperature and 40 ° C for a time between a few minutes and 12 hours.

The compounds of formula (III) where Al and X are as defined above can be obtained by reaction of phenols X-OH with a chloroformate of formula Al-CH2O-CO-Cl in an anhydrous inert organic solvent such as N, Nâ € ™ - dimethylformamide, tetrahydrofuran, benzene, toluene, dioxane, a polyhalogenated aliphatic hydrocarbon at a temperature between -20 ° C and 50 ° C. The reaction is completed in a time interval between 30 minutes and 36 hours.

The compounds of formula (V) where m, n, p, q, R1â € ™ and R2â € ™ are as defined above, and Q Ã ̈ ONO2 can be obtained by reaction of compounds of formula (VII)

R'1P1R2 <'>

H O <(CH> 2 <)> m (CH) n (N) p <(CH)> qOP3

(VII)

Where P1Ã ̈ as described above and P3Ã ̈ H or a protective group of hydroxyl such as TBDPS or those described in T. W. Greene â € œProtective groups in organic suynthesisâ €, Haward University Press, 1980, with a nitrate salt selected from tetraethylammonium nitrate , or tetrabutylammonium nitrate in the presence of tri-African anhydride and in the presence of an organic base chosen from: 2,6-di-tert-butyl-4-methylpyridine, sym-collidine, 2,6-lutidine, pyridine or tritylamine. The reaction is carried out in an organic solvent selected from dichloromethane, acetonitrile, tetrahydrofuran, at a temperature between -70 ° C and room temperature. The reaction is completed in a time interval between 30 minutes and 36 hours; and deprotection of the compound obtained as written in T. W. Greene â € œProtective groups in organic suynthesisâ €, Haward University Press, 1980 2nd edition.

Tetrabutylammonium fluoride in tetrahydrofuran in the presence of acetic acid is the preferred method for removing the TBDPS protective group.

The compounds of formula (VII) are commercially available and can be obtained according to the methods known in the literature.

The compounds of formula (V) where m, n, p, q, R1â € ™ and R2â € ™ are as defined above and Q is a chlorine atom, a bromine atom, an iodine atom, a mesyl group, a tosyl group, are commercially available or can be obtained according to methods known in the literature.

Example 1)

((3-amino-2- (nitroxy) propoxy) carbonyloxy) methyl-2-acetoxybenzoate hydrochloride

O O

OO O <NH> 2 <.HCl>

ONO OAc2

A) N-Fmoc (2,2-dimethyl-1,3-dioxolan-4-yl) methanamine

O O

H.

No.

Fmoc

An aqueous solution of sodium carbonate 10% ( 35ml).

The mixture was stirred at 0 ° C and FMOC chloride (2.95g; 11.43 mmol) in dioxane (40 ml) was added dropwise. The reaction was kept under stirring at 0 ° C for 4 hours then at room temperature overnight. The solution was diluted with H2O (200 ml) and extracted with dichloromethane (3x100 ml). The combined organic phases were washed with a saturated saline solution (2 x 100 ml), dried over Na2SO4 and concentrated under reduced pressure to give the title compound (4.19 g).

B) N-Fmoc-3-aminopropane-1,2-diol

HO OH

H.

No.

Fmoc

To a solution of compound A) (4.19 g, 11.43 mmoles) in tetrahydrofuran (50 ml), a 3N solution of HCl (7.62 ml) was added. The suspension was stirred at room temperature for 5 hours. The reaction was diluted with H2O (300 ml) and extracted with dichloromethane (3x100 ml). The combined organic phases were washed with saturated brine (2 x 100 ml), dried over concentrated Na2SO4 and under reduced pressure. The residue was purified by flash chromatography (Biotage System, SNAP cartridge in silica 100 g, eluent: 1/1 dichloromethane / acetone gradient to 100% acetone during 10 cv, 100% acetone (7 cv). The product suspended in N-hexane / ACOEt 1/1 (70 ml) was stirred at room temperature for 1 hour The mixture was filtered to give the title compound (2.18 g).

C) N-Fmoc-1-amino-3- (tert-butyldiphenylsilyloxy) propan-2-ol

I have OTBDPS

H.

No.

Fmoc

To a solution of compound B) (0.8 g, 2.55 mmol) and imidazole (0.344 g; 5.1 mmol) in DMF (5ml), tert-butyl (chlorine) diphenyl silane (0.662 ml; 2, 55 mmol).

The solution was stirred at room temperature overnight. The reaction was diluted with H2O (300 ml) and extracted with dichloromethane (3 x 100 ml).

The combined organic phases were washed with saturated saline solution (2x50 ml) dried on NA2SO4e concentrated under reduced pressure. The residue was purified by flash chromatography (Biotage system, SNAP cartridge of silica 50g, eluent: gradient n-hexane 100% to n-hexane / ACOEt, 1/1 during 8 cv) to give the title compound (0.739 g).

D) n-Fmoc-1-amino-3- (tert-butyldiphenylsilyloxy) propan-2-yl-nitrate

<O> 2 <NO> OTBDPS

H.

No.

Fmoc

Tetraethylammonium nitrate (0.35g 1.82 mmol) and 2,6-di-tert-butyl-4-methylpyridine ( 0.468 g): 2.28 mmoles). The solution was cooled to 70 ° C and tri-African anhydride (0.3 ml, 1.82 mmol) added dropwise.

The mixture was kept under stirring at -70 ° C for 2 hours and then was allowed to cool to room temperature. The solution was diluted with an aqueous solution of NaH2PO45% (100 ml) and extracted with dichloromethane (3 x 100 ml).

The combined organic layers were washed with saturated saline solution (2 x 50 ml). Dried over concentrated Na2SO4e under reduced pressure. The residue was purified by flash chromatography (Biotage system, SNAP cartridge of silica 50 g, eluent: gradient N-hexane 100% to N-hexane / AcOEt 1/1 during 8 cv) to give the title compound (0.5g) .

E) 1-amino-3- (tert-butyldiphenylsilyloxy) propan-2-ylnitrate

<O> 2 <NO> OTBDPS

H2N

To a solution of compound D) (0.5g, 0.837 mmol) in acetonitrile, (40 ml), piperidine (0.413 ml; 4.189 mmol) was added.

The mixture was stirred at room temperature for 5 hours. The reaction was concentrated under reduced pressure. The residue was used in the next step without further purification.

F) N-Boc-1-amino-3- (tert-butyldiphenylsilyloxy) propan-2-yl-nitrate

<O> 2 <NO> OTBDPS

H.

No.

Boc

To a solution of compound E) (0.313 g, 0.837 mmol) and triethylamine (0.098 ml; 1.674 mmol) in dichloromethane (36 ml), di-tertzbuthyldicarbonate (0.274 g; 1.85 mmol) in dichloromethane ( 7 ml).

The mixture was stirred at room temperature overnight. The solvent was evaporated in vacuo. The residue was purified by flash chromatography (Biotage system, SNAP cartridge of silica 50 g, eluent: gradient N-hexane 100% to N-hexane / AcOEt 1/1 during 12 cv) to give the title compound (0.385 g).

G) N-Boc-1-amino-3-hydroxypropan-2-yl-nitrate

<O> 2 <NO> OH

H.

No.

<Boc>

To a solution of compound F) (0.385g, 0.5 mmol) in anhydrous tetrahydrofuran (6 ml) cooled to -70 ° C, a solution of 1N tetrabutylammoniofluoride in tetrahydrofuane (1.5 ml; 1.5 mmol) was added and acetic acid (0.092 ml; 1.5 mmoles).

The mixture was kept under stirring at room temperature for 5 hours. The reaction was diluted with an aqueous solution of NaH2PO45% (100 ml) and extracted with dichloromethane (3 x 100 ml). The combined organic phases were washed with a saturated saline solution (2x50ml), dried on Na2SO4 and concentrated under reduced pressure. The residue was purified by flash chromatography (Biotage system, SNAP cartridge of silica 50 g, eluent: gradient N-hexane-ACOEt 8/2 to N-hexane-ACOEt 2/8 during 8 cv) to give the title compound (0.144 g).

H) Chloromethyl - nitrophenyl carbonate

ClO <O>

NO2

To a solution of 4-nitrophenol (4 g, 28.75 mmol) in anhydrous dichloromethane (80 mL) and pyridine (2.32 mL, 28.75 mmol) cooled to 0 ° C, chloromethyl chloroformate (2.32 mL, 28.75 mmol) was added. The mixture was kept under stirring at room temperature for 5 hours. The reaction was evaporated in vacuo. The residue was diluted in an aqueous solution with 10% citric acid (200 mL) and extracted with diethyl ether (3 x 150 mL). The combined organic phases were washed with a saturated saline solution (2 x 50 ml), dried over Na2SO4 and concentrated under reduced pressure to give the title compound (6 g).

I) Iodomethyl 4-nitrophenyl carbonate

OR

I <O>

NO2

Sodium iodate (16.53 g; 110 mmol) was added to a solution of compound H) (5.11 g, 22 mmol) in acetonitrile. The mixture was refluxed for 6 hours. The reaction was evaporated in vacuo. The residue was dissolved in diethyl ether (200 ml) and filtered. The organic phases were washed with water (2 x 50 ml), dried on NasSO4 and concentrated under reduced pressure. The residue was purified by flash chromatography (Biotage System, SNAP cartridge of silica 100 g, eluent: gradient n-hexane 100% to n-hexane / AcOEt 6/4 during 8 cv) to give the title compound (5.75 g).

J) ((4-nitrophenoxy) carbonyloxy) methyl 2-acetoxybenzoate

O O

OOO

OAc

<NO2>

Silver oxide (2.78 g, 12 mmol) was added to a solution of acetyl salicylic acid (3.6 g, 20 mmol) in acetonitrile (72 ml) and water (36 ml). The mixture was stirred at room temperature for 6 hours in the dark. The reaction was filtered and the solvent was evaporated in vacuo to give 5.79 g of silver 2-acetoxybenzoate. The compound was added to a solution of compound I) (5.75 g; 17.8 mmol) in toluene (115 ml). The mixture was stirred in the dark overnight. The salts were filtered and the solvent was evaporated under vacuum. The residue was purified by flash chromatography (Biotage System, SNAP cartridge of silica 100 g eluent: gradient n-hexane 100% to n-hexane / AcOEt 1/1 during 10 cv) to give the title compound (5.2 g).

K) ((N-Boc-3-amino-2- (nitroxy) propoxy) carbonyloxy) methyl 2-acetoxybenzoate.

O O

OO ONH-Boc

ONO OAc2

To a solution of compound G) (0.118 g, 0.5 mmoles) and compound J) 0.206 g, 0.55 mmoles) in dichloromethane (15 ml), triethylamine (0.077 ml; 0.55 mmoles) was added. The mixture was evaporated at room temperature for 70 hours. The solvent was evaporated in vacuo. The residue was purified by flash chromatography (Biotage system; SNAP cartridge of silica 50 g, eluent: gradient n-hexane 100% to n-hexane / AcOEt 1/1 during 10 cv) to give the title compound (0.2 g).

<1> H-NMR (CDCl3) Î ́ 8.08 (1H, dd), 7.62 (1H, dt), 7.35 (1H, dt), 7.13 (1H, dd), 5.96 (2H, s) 5.35 (1H, m ), 4.98 (1H, m) 4.52 (1H, m), 4.33 (1H, m), 3.452H, m), 2.38 (3H, s), 1.46 (9H, s).

L) ((3-amino-2- (nitroxy-propoxy) carbonyloxy) methyl 2-acetoxybenzoate hydrochloride

Compound K) (0.08g, 0.169 mmol) was added to a solution of 4N HCl in dioxane (1.7 ml). The reaction was stirred at room temperature for 1 hour. The solvent was evaporated in vacuo to give the title compound (0.077 g).

Example 2

((2- (2-nitroxyethylamino) ethoxy) carbonyloxy) methyl 2-acetoxy benzoate hydrochloride

OOClH

H.

No.

O O O ONO2

OCOCH3

M) -Boc-2- (2-hydroxyethylamino) ethyl nitrate

Boc

No.

H O ONO2

To a solution of N-Boc-diethanolamine (2g, 9.74 mmol) in dichloromethane (100 ml), tetraethylammonium nitrate (1.68 g; 8.76 mmol) and 2,6-di-tert-butyl- were added. 4-methylpyridine (2.4 g; 11.69 mmol). The solution was cooled to â € “70 ° C and tri-African anhydride (1.44 ml; 8.76 mmol) was added dropwise.

The mixture was kept under stirring at -70 ° C for 2 hours and then was allowed to reach room temperature. The reaction was diluted with an aqueous solution of NaH2PO45% (200 ml) and extracted with dichloromethane (3 x 150 ml).

The combined organic phases were washed with a saturated saline solution (2x50 ml), dried on Na2SO4 and concentrated under reduced pressure. The residue was purified by flash chromatography (Biotage system, SNAP cartridge of silica 100 g, eluent: gradient N-hexane 100% (2cv) to N-hexane / ACOET 2/8 during 10 cv to give the title compound (0.275 g ).

N) N-Boc - ((2 - ((2- (nitroxy) ethyl) amino) ethoxy) carbonyloxy) methyl 2-acetoxy benzoate

OR OR OR ONO

N2

OAc <O> O Boc

Triethylamine (0.167 ml, 1.2 mmol) was added to a solution of compound M) (0.275g, 1.1 mmol) and compound J) (0.435g, 1.2 mmol) in dichloromethane (30 ml). The mixture was stirred at room temperature for 48 hours. The solvent was evaporated in vacuo. The residue was purified by flash chromatography (Biotage system, SNAP cartridge in silica 100 g, eluent: gradient N-hexane 100% to N-hexane / AcOEt 6/4 during 12 cv) to give the title compound (0.25g) .

<1> H-NMR (CDCl3) Î ́ 8.18 (1H, dd), 7.63 (1H, dt), 7.36 (1H, dt), 7.14 (1H, dd), 5.96 (2H, s), 4.55 (2H, m), 4.34 (2H, m), 2.38 (3H, s), 1.47 (9H, s).

O) ((2- (2-nitroxyethylamino) ethoxy) carbonyloxy) methyl 2-acetoxy benzoate hydrochloride

Compound N) (0.118 g, 0.24 mmoles) was added to a solution of 4N HCl in dioxane (2.4 ml). The reaction was kept at room temperature for 1 hour. The solvent was evaporated in vacuo to give the title compound (0.094g).

Example 3

1- (2-acetoxyphenyl) -7- (nitroxymethyl) -1,5,10-trioxy-2,4,6,9-tetraoxatridecan-13-oic acid

OR NOT

Or O2O

OR

O O O OH

O O O

P) 4 - ((2,2-dimethyl-1,3-dioxolan-4-yl) methoxy) -4-oxobutanoic acid

OR

OOCOOH

OR

Succinic anhydride (6.1g, 61 mmol) and triethylamine were added to a solution of D, L-isopropylidene glycerol (2.6 g, 20 mmol) in tert-butanol (100 ml).

The mixture was kept at room temperature overnight. The solution was concentrated under vacuum to a small volume, diluted with dichloromethane (150 ml) and washed with an aqueous solution of H3PO45% (200 ml). The organic phase was dried over concentrated Na2SO4e under reduced pressure. The residue was used in the next step without further purification.

Q) Allyl (2,2-dimethyl-1,3-dioxolan-4-yl) methylsuccinate

O O

OO

OR

OR

A solution of Compound P) (4.6 g, 20 mmol), allyl alcohol (2.04 ml, 30 mmol), EDAC (5.75 g, 30 mmol) and DMAP (0.244 g, 2 mmol) in dichloromethane ( 400 ml) was kept at room temperature for 20 hours.

The solution was concentrated in vacuo. The residue was purified by flash chromatography (Biotage system, SNAP cartridge in silica 100 g, eluent: N-hexane gradient (AcOEt 9/1 to n-hexane / AcOEt 1/1 during 8 cv) to give the title compound (4 , 25g).

R) Allyl 2,3-dihydroxypropylsuccinate

OH O

H OOO

OR

To a solution of compound R) (4.25 g, 15.6 mmoles) in tetrahydrofuran (72 ml), 3N HCl (10.4 ml) was added. The reaction was stirred at room temperature for 3 hours. The mixture was diluted with water (200 ml) and extracted with AcOEt (3 x 150 ml). The combined organic phases were washed with saturated saline solution (2x50 ml), dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by flash chromatography (Biotage system, SNAP cartridge in silica 50, eluent: gradient N-hexane / AcOEet 1/1 to n-hexane / AcOEet 2/8 during 10 cv) to give the title compound (1, 62g).

S) Allyl 1-hydroxy-3- (nitroxy) propane -2-succinate

OH O

OR

OR

OR

ONO2

To a solution of compound R), (1.62 g, 7 mmol) in dichloromethane (70 ml), tetraethylammonium nitrate (1.41 g; 7.35 mmol) and 2,6-di-tert-butyl-4-methylpyridine (2g, 9.9 mmol). The solution was cooled to -70 ° C and triflic anhydride (1.21 ml; 7.35 moles) was added dropwise.

The mixture was kept under stirring at -70 ° C for 2 hours and then was allowed to reach room temperature. The reaction was diluted with a 5% NaH2PO4 aqueous solution (200 ml) and extracted with dichloromethane (3 x 150 ml). The combined organic phases were washed with a saturated saline solution (2x50 ml), dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by flash chromatography (Biotage system, SNAP cartridge in silica 50, eluent: gradient N-hexane 100% (2cv), N-hexane to n-hexane / AcOEt 2/8 during 8 cv) to give the compound of title (0.624g).

T) 2 - (((2-acetoxybenzoyloxy) methoxy) carbonyloxy) -3- (nitroxy) propylallilsuccinate

OR

O O O O

OR

OAc <O> O O

ONO2

To a solution of compound S) (0.277g, 1.19 mmol) and compound J) (0.492g, 1.31 mmol) in dichloromethane (30 ml) was added tritylamine (0.182 ml, 1.31 mmol). The mixture was stirred at room temperature for 48 hours. The solvent was evaporated in vacuo. The residue was purified by chromatography (Biotage system, SNAP cartridge in silica 50 g, eluent: gradient N-hexane / AcOEt 9/1 to n-hexane / AcOEt 1/1 during 10 cv) to give the title compound (0.430 g ).

U) 1- (2-aceotoxyphenyl) -7- (nitroxymethyl) -1,5-10-trioxy-2,4,6,9-tetraoxatridecan-13-oic acid

To a solution of compound T) (0.43g, 0.83mmol), dimedone (0.117g, 0.83mmol) in anhydrous CH2CL2 (20ml) was added, followed by triphenylphosphine (0.441g; 0.83mmol) and palladium tetrakis (0.058g; 0.05mmol). The solution was stirred for 2 hours and then concentrated under reduced pressure. The residue was purified by flash chromatography (Biotage system; SNAP cartridge in silica 50 g, eluent: gradient N-hexane / acetone 9/1 to N-hexane / acetone 1/1 during 8cv, N-hexane-acetone 1/1 with 0.1% acetic acid 2cv) to give the title composition (0.165 g).

<1> H-NMR (CDCl3) Î ́ 8.10 (1H, dd), 7.63 (1H, dt), 7.35 (1H, dt), 7.14 (1H, dd), 5.97 (2H, s), 5.21 (1H, m), 4.75 (1H, dd), 4.59 (1H, dd), 4.46 (1H, dd), 4.28 (1H, dd), 2.65 84H, s), 2.38 (3H, s).

Stability in human plasma

990 µl of human plasma at 37 ° C were added with 10 µl of a 1 mM concentrated solution of each compound to be tested in DMSO. At each time (5,10,15,30,60 min), 50 µl of the solution to be tested were treated in duplicate with 150 µl of acetonitrile and 10 µl of DMSO to eliminate the proteins, vortexed and centrifuged 10 min at 4 ° C at 400 rpm. The supernatant was transferred to a clean plate and injected into a UPLC-MS system.

A calibration curve (CC) was prepared for each compound to be tested in human plasma in a range of 0.1 -10 µM.

To do this, working solutions (WS: working solution) of each compound in DMSO were prepared by serial dilution of a 10 mM concentrated solution to the following final concentrations

WS (µM) 50/25/10 / 5.0 / 2.5 / 1.0 / 0.5

2.5 mL of human plasma was treated in duplicate with 7.5 mL of acetonitrile to precipitate proteins; 200 µl of each deproteneized matrix was spiked with 10 µl of each working solution to create a calibration curve for each compound in each matrix at the following final nominal concentrations:

CC (µM) 10/5/2/1 / 0.5 / 0.2 / 0.1

The analytical conditions to detect acetylsalicylic acid (ASA), salicylic acid (SA) and nitro compounds were the following:

Acquity BEH SCHIELD 2.1 x 50 mm (1.7 µ) column at 40 ° C

Mobile phase A. water 0.1% HCOOH

B. acetonitrile 0.1% HCOOH

Flow rate: 0.5 ml / min

Gradient profile

Time

A (%) B (%)

(min)

0.0 90 10

2.0 0 100

2.5 0 100

2.6 90 10

Injection volume 5 µl

MS methods

Detector: Quattro Micro API Mass Spectrometer (Waters)

Mode of ionization: ESI negative (ASA, SA)

ESI positive (compounds (1), (2), (3))

Acquisition mode: Single Ion Reaction

m / z: 179.09 (ASA)

136.96 (SA)

373.10 (compound (1))

387.21 (compound (2))

473.35 (compound (3))

Pause time: 0.08 sec

Table 1. Stability of compounds in human plasma

compounds Compounds t1 / 2ASA Conc.maxASA

(min) (Î1⁄4M) Conc.1h

(Î1⁄4M)

ASA - - 44.59

(1) <5 54.64 27.39

(2) <5 64.06 32.76

Stability in the S9 fraction of the human liver

300 µl of 20 mg / ml human liver S9 were thawed and diluted to 0.5 mg / ml (1:40) with cold 0.1 M PBS at pH 7.4.

Aliquots of 980 µl of diluted S9 were placed in 1.5 ml eppendorf vials in a thermomixer at 37 ° C and allowed to reach the temperature under gentle stirring (300 rpm) for a couple of minutes. Meanwhile, 4 ml of diluted S9 were treated with 12 ml of ACN to precipitate the proteins, vortexed and centrifuged for 10 minutes at 4 ° C - 3200 g: the supernatant was stored at 0 ° C pending preparation of the calibration curves. 10 µl of 50 mM NADPH in water were added to the thermostated S9 for the solutions to be tested w / NADPH; 10 µl of water were added to S9 for the solutions to be tested w / o NADPH. The 50 mM NADPH solution was freshly prepared or stored at -80 ° C immediately after preparation and only thawed only once. 10 µl of a 1 mM solution of each compound to be tested in DMSO was added to the solutions to be tested and the kinetics started. The final concentrations were: compound 10 µM

S9 0.5 mg / ml of protein

NADPH 0.5 mM

At each time (5, 10,15,30,60 min) 50 µl of each solution to be tested w o w / o NADPH were treated in duplicate with 150 µl of acetonitrile and 10 µl of DMSO to precipitate the proteins, vortexed and centrifuged 10 min at 4 ° C at 3200 g.

Working solutions of the nitrocompounds, ASA and SA, were prepared by serial dilution of 10 mM concentrated solutions using DMSO at the following final concentrations:

WS (µM) 50/25/10 / 5.0 / 2.5 / 1.0 / 0.5

200 µl of deproteinized matrix was spiked with 10 µl of each working solution to create a calibration curve for each analyte in each matrix up to the following final nominal concentrations:

CC (µM) 10/5/2/1 / 0.5 / 0.2 / 0.1

The analytical conditions for detecting ASA, SA and NO-compounds were the following:

Acquity column BEH SCHIELD 2.1 x 50 mm (1.7 µ) at 40 ° C

Mobile phase A. water 0.1% HCOOH

B. acetonitrile 0.1% HCOOH

Flow rate 0.5 ml / min Gradient profile

Time

A (%) B (%)

(min)

0.0 90 10

3.0 0 100

4.5 0 100

4.6 90 10

Injection volume 5 µl

MS methods

Detector: Quattro Micro API Mass Spectrometer (Waters)

Mode of ionization: ESI negative (ASA, SA)

ESI positive (compounds (1), (2), (3))

Acquisition mode: Single Ion Reaction

m / z: 179.09 (ASA)

136.96 (SA)

373.10 (compound (1))

387.21 (compound (2))

473.35 (compound (3))

Pause time: 0.08 sec

Table 2. Stability of compounds in human liver S9

<Compounds t1 / 2> ASA Conc. 5 minASA t1 / 2 Compounds

(min) (Î1⁄4M) (min)

ASA 35 10 35

(1) <5 6 15

(2) <5 8 25 Pharmacokinetics in vivo

The formulation was prepared by dissolving an appropriate amount of test compounds in Methocel 1% / DMSO 98/2 on the day of dosing, immediately prior to administration.

Pharmacokinetic evaluations were made in blood samples (approximately 0.3 ml) taken intracardium using syringes containing NA-heparin as anticoagulant, from anesthetized mice (chloral hydrate 380 mg kg <-1>, ip from Sigma Chemicals, St Louis, MO , USA) at 5, 15, 30, 60, 120, 240 min and 24 h after the equimolar oral administration of 10 mg Kg <-1> of the compound (1), of 12.6 mg Kg <-1> of the compound (2) and 4.4 mg kg <-1> of ASA. The animals were then sacrificed. The samples were transferred to pre-cooled tubes (containing 15 µl of dichlorvos (6 mg / ml) to inhibit plasma esterase activity, protected from light.

The blood was centrifuged at 4000 rpm for 10 min at 4 ° C to collect the plasma.

Plasma aliquots of 25 µl were treated in triplicate with 75 µl of methanol to which 5 µl of DMSO were also added to precipitate the proteins, vortexed and centrifuged 10 min at 4 ° C at 4000 rpm.

The supernatant was transferred to a clean plate and injected into an LC-MS system together with the calibration curves.

Working solutions of the nitrocompounds, ASA and SA were prepared for serial dilutions of 10 mM concentrated solutions using DMSO at the following final concentrations

WS (µM) 50/25/10 / 5.0 / 2.5 / 1.0 / 0.5

1.5 ml of control mouse plasma (treated with dichlorvos and centrifuged) were deproteneized using 4.5 ml of methanol vortexed and centrifuged 10 min at 4 ° C at 4000 rpm. The supernatant was spiked (100 µl) with 5 µl of each working solution to create a calibration curve for each analyte at the following final nominal concentrations:

CC (µM) 10/5/2/1 / 0.5 / 0.2 / 0.1

The analytical conditions were the following:

Acquity column BEH SCHIELD 2.1 x 50 mm (1.7 µ) at 40 ° C

Mobile phase A. water 0.1% HCOOH

B. methanol 0.1% HCOOH

Flow rate 0.5 ml / min

Gradient profile

Time

A (%) B (%)

(min)

0.0 90 10

1.0 0 100

1.5 0 100

1.6 90 10

Injection volume 5 µl

MS methods

Detector: Quattro Micro API Mass Spectrometer (Waters)

Mode of ionization: ESI negative (ASA, SA)

ESI positive (compounds (1), (2))

Acquisition mode: Single Ion Reaction

m / z: 179.09 (ASA)

136.96 (SA)

373.10 (compound (1))

387.21 (compound (2))

Pause time: 0.08 sec

The level of AS after administration of ASA (4.4 mg / kg) reached a Cmax of 73 µM at 15 minutes post dose, after which the plasma concentrations of AS declined for up to 24 hours. (Figure 1)

100.0

80.0

60.0

40.0

20.0

0.0

0 1 2 3 4 5

Time (hours)

Figure 1. Concentration of salicylic acid in mouse blood after the assay of acetyl salicylic acid

The level of AS after administration of compound (1) (10 mg kg <-1>) reached Cmax of 41 µM at 15 minutes post dose. AS plasma concentrations declined for up to 4 hours (Figure 2)

50.0

40.0

Salycilic Acid

30.0

20.0

10.0

0.0

0 4 8 12 16 20 24

time (hours)

Figure 2. Salicylic acid concentration in mouse plasma after compound (1) PO assay

The SA level after administration of compound (2) (12.6 mg kg <-1>) reached Cmax of 45 µM at 2 hours post dose. Plasma concentrations of AS declined for up to 4 hours (Figure 3)

50.0

Salycilic Acid

40.0

30.0

20.0

10.0

0.0

0 4 8 12 16 20 24

Time (hours)

Figure 3. Salicylic acid concentration in mouse plasma after compound (2) PO assay

Claims (1)

Claims 1. A compound of general formula (I) and its pharmaceutically acceptable salts or stereoisomers O O <R> 1 R2 OO O <(CH> 2 <)> m <(CH)> n <(NH)> p (CH) q ONO2O OR (THE) where is it: m is an integer between 0 and 3; n is 0 or 1; p is 0 or 1; q is an integer between 1 and 3; each of R1 and R2 independently is H, C1-C3alkyl, (CH2) r-NH2 where r is an integer between 0 and 4, or (CH2) s-O-CO- (CH2) t-COOH where s is an integer between 0 e 4 and t is an integer between 1 and 4; provided that: when p is 1 each of R1 and R2 independently is H or C1-C3alkyl; when p is 0 one of R1e R2Ã ̈ (CH2) r-NH2or (CH2) s-O-CO- (CH2) t-COOH where r, s and t are as defined above and the other is H or C1-C3alkyl. 2. A compound of formula (I) according to claim 1 where p is 1, m is 2 or 3, n is 0, q is an integer between 1 and 3, R1 and R2 are H. 3. A compound of formula (I) according to claim 1 where p is 0, m is 1 or 2, n is 0 or 1, q is an integer between 1 and 3, one of R1, and R2Ã ̈ (CH2) r-NH2 where r is an integer between 0 and 3 and the other is H. 4. A compound of formula (I) where p is 0, m an integer between 0 and 2, n is 0 or 1, q is an integer between 1 and 3, one of R1, and R2Ã ̈ (CH2) s-O-CO- (CH2) t-COOH where s is 0 or 1 and t is an integer between 1 and 3, preferably 2 and the other is H. 5. A compound of formula (I) selected from the group consisting of: O O OO O <NH> 2 <.HCl> ONO OAc2 (1) OOClH H. No. O O O ONO2 OCOCH3 (2) ONO OR O2O OR O O O OH OR OR OR (3) O O OO O <NH> 2 <.HCl> OAc ONO2 (4) OO <ONO> 2 O O O NH2.HCl OAc (5) OR NOT Or O2 O O O NH2.HCl OAc (6) O O ONO2 NH O O O2.HCl OAc (7) OR NOT Or O2 NH.HCl O O O2 OAc (8) O O NH O O O2.HCl ONO OAc2 (9) O O NH O O O2.HCl OAc ONO2 (10) O O OO O <ONO> 2 NH OAc2.HCl (11) OONH2.HCl O O O ONO2 OAc (12) O O2.HCl O O O ONO2 OAc (13) O O NH2.HCl OR NOT O O O2 OAc (14) O O OR NOT O O O2 <NH2.HCl> OAc (15) O <O> ClH O O O <N> ONO H2 OCOCH3 (16) O O ClH OR NOT O O O <N> 2 H. OCOCH3 (17) O O ClH OOO <N> ONO H2 OCOCH3 (18) O O H ClH NINTH O O O2 OCOCH3 (19) O O O O O O O OH OR OR ONO2 OR (20) O O O O O O O OH OR OR ONO O2 (21) OR NOT O O O2 OH O O O O OR OR OR (22) 6. a compound of formula (I) according to claims 1-5 for use as a medicament. 7. A compound of formula (I) according to claims 1-5 for use in the treatment of cardiovascular diseases, thrombosis and platelet aggregation. 8. A compound of formula (I) according to claims 1-5 for use in the treatment of inflammation, pain and fever. 9. A compound of formula (I) according to claims 1-5 for use in the prevention or treatment of tumor diseases. 10. A pharmaceutical composition, a pharmaceutically acceptable non-toxic adjuvant and / or a carrier agent and a pharmaceutically acceptable amount of a compound of general formula (I) and / or a salt or stereoisomer thereof as defined in claims 1-5.