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Title:
NITRATE ESTERS OF CARBONIC ANHYDRASE INHIBITORS
Document Type and Number:
WIPO Patent Application WO/2008/071421
Kind Code:
A1
Abstract:
Nitroderivatives of carbonic anhydrase inhibitors having improved pharmacological activity and enhanced tolerability are described. They can be employed for the treatment of glaucoma, ocular hypertension, age-related macular degeneration, diabetic macular edema, diabetic retinopathy, hypertensive retinopathy and retinal vasculopathies, cancer, epilepsy, high-altitude disorders and neuromuscular diseases.

Inventors:
SUPURAN, Claudiu (Via dè Magalotti 7, Firenze, I-50122, IT)
BENEDINI, Francesca (Via Spilamberto 16, San Donato Milanese, I-20097, IT)
BIONDI, Stefano (Via Modigliani 2, Milano, I-20144, IT)
ONGINI, Ennio (Via Fratelli Cervi, Residenza Campo, Segrate, I-20090, IT)
Application Number:
EP2007/010915
Publication Date:
June 19, 2008
Filing Date:
December 07, 2007
Export Citation:
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Assignee:
NICOX S.A. (Taissounières HB4, 1681 route des DolinesBP 313, Sophia Antipolis, F-06560, FR)
SUPURAN, Claudiu (Via dè Magalotti 7, Firenze, I-50122, IT)
BENEDINI, Francesca (Via Spilamberto 16, San Donato Milanese, I-20097, IT)
BIONDI, Stefano (Via Modigliani 2, Milano, I-20144, IT)
ONGINI, Ennio (Via Fratelli Cervi, Residenza Campo, Segrate, I-20090, IT)
International Classes:
C07D211/22; A61K31/18; A61K31/21; A61K31/433; A61P27/06; A61P35/00; C07C203/04; C07C311/16; C07C311/17; C07D285/135; C07D295/088
Domestic Patent References:
WO2004002409A2
WO2005021004A1
WO2006091716A2
WO2006052899A2
WO2005068421A1
Other References:
DHAWAN VIJAY ET AL: "Selective nitros(yl)ation induced in vivo by a nitric oxide-donating cyclooxygenase-2 inhibitor: a NObonomic analysis." FREE RADICAL BIOLOGY & MEDICINE 1 NOV 2005, vol. 39, no. 9, 1 November 2005 (2005-11-01), pages 1191-1207, XP002473906 ISSN: 0891-5849
BOLLA MANLIO ET AL: "Therapeutic potential of nitrate esters of commonly used drugs." CURRENT TOPICS IN MEDICINAL CHEMISTRY 2005, vol. 5, no. 7, 2005, pages 707-720, XP002473907 ISSN: 1568-0266
BURGAUD J L ET AL: "NITRIC-OXIDE RELEASING MOLECULES: A NEW CLASS OF DRUGS WITH SEVERAL MAJOR INDICATIONS" CURRENT PHARMACEUTICAL DESIGN, BENTHAM SCIENCE PUBLISHERS, SCHIPHOL, NL, vol. 8, no. 3, 2002, pages 201-213, XP001122072 ISSN: 1381-6128
VULLO D ET AL: "Carbonic anhydrase inhibitors. Inhibition of the transmembrane isozyme XII with sulfonamides-a new target for the design of antitumor and antiglaucoma drugs?" BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, OXFORD, GB, vol. 15, no. 4, 15 February 2005 (2005-02-15), pages 963-969, XP004730373 ISSN: 0960-894X cited in the application
MASEREEL B ET AL: "Carbonic anhydrase inhibitors: Anticonvulsant sulfonamides incorporating valproyl and other lipophilic moieties" JOURNAL OF MEDICINAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY. WASHINGTON, US, vol. 45, 2002, pages 312-320, XP002274910 ISSN: 0022-2623 cited in the application
CASINI ANGELA ET AL: "Carbonic anhydrase inhibitors: topically acting antiglaucoma sulfonamides incorporating esters and amides of 3- and 4-carboxybenzolamide." BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 1 SEP 2003, vol. 13, no. 17, 1 September 2003 (2003-09-01), pages 2867-2873, XP002473908 ISSN: 0960-894X cited in the application
Attorney, Agent or Firm:
BARCHIELLI, Giovanna (Nicox Research Institute SRL, Via Ariosto 21, Bresso, I-20091, IT)
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Claims:
CLAIMS

1. Use of a carbonic anhydrase inhibitor able to release nitric oxide for the preparation of a medicament for treating eye disorders and cancer,

2. Use according to claim 1, wherein the eye disorder is selected from glaucoma, ocular hypertension, age-related macular degeneration, diabetic macular edema, diabetic retinopathy, hypertensive retinopathy or retinal vasculopathies .

3. Use according to claim 1 wherein carbonic anhydrase inhibitor able to release nitric oxide is a compound having an inhibition constant (K i ) against at least one of the isoenzymes CAII, CAIV, CAIX and CAXII in the range of 0.01- 200 nM.

4. Use according to claim 1 wherein the carbonic anhydrase inhibitor able to release nitric oxide is a compound having an EC 50 value in the range of 1-50μM.

5. A compound of general formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof: R-(X-Y-ONO 2 ) s

(I) wherein s is an integer equal to 1 or 2 ; R is selected from the group consisting of :

wherein n is an integer from 0 to 4; m is an integer equal to 0 or 1;

R 1 , R 2 and R 3 are the same or different and are H, Cl, Br, I,

F, CF 3 , SO 2 NH 2 ;

X 1 is -O- or -NH-; N 1 is -OH, -COOH, -O-, -C (O) -, -NHR 1 or -NR 1 - wherein R 1 is H or C 1 -C 6 al kyl ;

R' is H or a group - (X-Y-ONO 2 ) ;

X is -C(O)-, -O-, -NR 1 -, -S-, -C(O)O- or -C(O)S-, wherein R 1 is H or C 1 -C 6 alkyl; with the proviso that i) when the group -(X-Y-ONO 2 ) is bound to N 1 then:

- N 1 is -O- or -NR 1 - and X is -C(O)-, -C(O)O- or -C(O)S-, wherein R 1 is H or C 1 -C 6 alkyl;

- N 1 is -C(O)- and X is -O-, -NR 1 -, -S-, -C(O)O- or -C(O)S-, wherein R 1 is H or C 1 -C 6 alkyl; ii) when R' is -(X-Y-ONO 2 ) then X is -C(O)-, -C(O)O- or -

C(O)S-; iii) when s is 2, then -X and -Y- of the two groups

-(X-Y-ONO 2 ) can be the same or different; Y is a bivalent radical having the following meaning: a) straight or branched C 1 -C 20 alkylene; straight or branched C 1 -C 20 alkylene substituted with one or more of the substituents selected from the group consisting of halogen atoms, hydroxy, -ONO 2 or T,

wherein T is -OC(O)(C 1 -C 10 alkyl) -ONO 2 or -O(C 1 -C 10 alkyl) -ONO 2 ; - cycloalkylene with 5 to 7 carbon atoms into cycloalkylene ring, the ring being optionally substituted with side chains T 1 , wherein T 1 is straight or branched C 1 -C 10 alkyl; b)

C)

wherein n 0 is an integer from 0 to 20, n 1 is an integer from 1 to 20, d)

wherein

X 1 = -OC(O)- or -C(O)O-; n 2 is an integer from 0 to 2;

R 2 is H or CH 3 ;

Z is -(CH 2 ) n 1 - or the bivalent radical defined above under b) , wherein n 1 is an integer from 1 to 20; e)

wherein:

Y 1 is -CH 2 -CH 2 - (CH 2 ) n 2a - or -CH=CH- (CH 2 ) n 2a - wherein n 2a is from 0 to 2,

X 1 . is -OC(O)- or -C(O)O-, n 2 is an integer from 0 to 2, R 2 is H or CH 3 ,

Z is -(CH 2 ) n 1 - or the bivalent radical defined above under b) , wherein n 1 is an integer from 1 to 20; when Y is selected from the bivalent radicals mentioned under b)-e), then the terminal -ONO 2 group is bound to - (CH 2 ) n 1 ; f)

wherein: n 1 is an integer from 1 to 20,

R 3 is H,

R 0 is H or -COCH 3 ; with the proviso that when Y is selected from the bivalent radicals mentioned under f) , then -ONO 2 group is bound to - (CH 2 ) n 1 ;

g)

wherein X 2 is -O- or -S-, n 3 is an integer from 1 to 6, R 2 is H or -CH 3 ; h)

wherein: n 4 is an integer from 0 to 10; n 5 is an integer from 1 to 10;

R 4 , R 5 , R 6 , R 7 are the same or different, and are H or straight or branched C 1 -C 4 alkyl; wherein the -ONO 2 group is linked to

wherein n 5 is as defined above;

Y 2 is an heterocyclic saturated, unsaturated or aromatic 5 or 6 members ring, containing one or more heteroatoms selected from nitrogen, oxygen, sulfur, and is selected from

6. A compound of general formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof according to claim 5, wherein s is 1; R is

wherein in formula ( IIA)

Ri, R 2 and R 3 are H; m is 0 and the group -SO 2 NHR' is in position 4 of the phenyl ring, R' is H; n is 0 ,

N 1 is -C (O) - and N 1 binds the group - (X-Y-ONO 2 ) ;

X is -O-, . -S- or -NR 1 - wherein R 1 is H or C 1 -C 6 alkyl; or

R 1 , R 2 and R 3 are H; R 1 is 0 and the group -SO 2 NHR' is in position 4 of the phenyl ring, R' is H; n is 0 or 1,

N 1 is -O- and N 1 binds the group -(X-Y-ONO 2 );

X is -C(O)-, -C(O)O- or -C(O)S-; or

R 1 is Cl and R 1 is in position 4 of the phenyl ring,

R 2 and R 3 are H; m is 0 and the group -SO 2 NHR' is in position 3 of the phenyl ring, R' is H; n is 0, N 1 is -C(O)- and N 1 binds the group -(X-Y-ONO 2 );

X is -O-, -S- or -NR 1 - wherein R 1 is H or C 1 -C 6 alkyl; or

R 1 R 2 and R 3 are H; m is 0 and the group -SO 2 NHR' is in position 3 of the phenyl ring, R' is H; n is 3 , N 1 is ~C {0) - and N 1 binds the group - (X-Y-ONO 2 ) ;

X is -O-, -S- or -NR 1 - wherein R 1 is H or C 1 -C 6 alkyl ; α compound of general formula ( I ) or a pharmaceutically acceptable salt or stereoisomer thereof according to claim 5 wherein wherein s is 1 ;

R is

(II B) wherein in formula (IIB) N 1 is -C(O)- and N 1 binds the group -(X-Y-ONO 2 ); R' is H; X is -O-, -NR 1 -, -S-; or

N 1 is -COOH; R' is -(X-Y-ONO 2 ) ; X is -C(O)-, -C(O)O- or -C(O)S-; 8. A compound of general formula (I)- or a pharmaceutically acceptable salt or stereoisomer thereof according to claims 5 to 7, wherein Y has the following meaning: ' a) straight or branched C 1 -C 10 alkylene, - straight or branched C 1 -C 10 alkylene substituted with one or more of the substituents selected from -ONO 2 or -OC(O) (C 1 -C 10 alkyl)-ONO 2 or -O(C 1 -C 10 alkyl)-ONO 2 ; b)

c)

wherein n 0 is an integer from 0 to 5, and n 1 is an integer from 1 to 5; d)

wherein :

X 1 = -OC(O)- or -C(O)O-;

Z is -(CH 2 ) n 1 - or the bivalent radical defined above under b) wherein n 1 is an integer from 1 to 10 and n 2 is 1, and R 2 is H or CH 3 ; e)

wherein:

Y 1 is -CH 2 -CH 2 - (CH 2 ) n 2a - or -CH=CH- (CH 2 ) n 2a - wherein n 2a is 0 or

1;

X 1 is -OC(O)- or -C(O)O-, n 2 is 1, R 2 is CH 3 ; Z is -(CH 2 ) n 1 - or the bivalent radical defined above under b) , wherein n 1 is an integer from 1 to 10; with the proviso that: when Y is selected from the bivalent radicals mentioned under b)-e), then the terminal -ONO 2 group is bound to - (CH 2 ) n 1 ; f)

(f") wherein: n 1 is an integer from 1 to 10; R 3 is H, R 0 is -COCH 3 ; with the proviso that when Y is selected from the bivalent radicals mentioned under f) , then -ONO 2 group is bound to - (CH 2 ) n 1 ; g)

wherein X 2 is -O- or -S-, n 3 is 1 and R 2 is H; h)

wherein : n 4 is an integer from 0 to 5; n 5 is an integer from 1 to 5; R 4 , R 5 , R 6 , R 7 are H; wherein the -ONO 2 group is linked to

wherein n 5 is as defined above; Y 2 is selected from

9. A compound according to claim 6, selected from the group consisting of compounds of formula

10. A compound according to claim 7, selected from the group consisting of compounds of formula

11. α compound of general formula (I) according to claims 5 to 10 for use as a medicament.

12. Use of a compound according to claims 5 to 10 for preparing a drug for the treatment or prophylaxis of glaucoma, ocular hypertension, age-related macular degeneration, diabetic macular edema, diabetic retinopathy, hypertensive retinopathy and retinal vasculopathies, cancer, epilepsy, high-altitude disorders and neuromuscular diseases.

13. Use of a compound according to claims 5 to 10 as a drug for the treatment or prophylaxis of glaucoma, ocular hypertension, age-related macular degeneration, diabetic macular edema, diabetic retinopathy, hypertensive retinopathy and retinal vasculopathies, cancer, epilepsy, high-altitude disorders and neuromuscular diseases.

14. A pharmaceutical composition comprising a compound according to claims 5 to 10 and a pharmaceutically acceptable carrier.

15. A pharmaceutical composition according to claim 14 in a suitable form for the oral, parenteral, rectal, topic and transdermic administration, by inhalation spray or aerosol or iontophoresis devices.

16. A pharmaceutical composition according to claim 14 wherein the compound of general formula (I) is administered as a solution, suspension or emulsion in an ophthalmically acceptable vehicle.

17. A pharmaceutical composition comprising a mixture of a compound of general formula (I) according to claims 5 to 10 and (i) a beta-adrenergic antagonists or (ii) a prostaglandin analog or (iii) an α-adrenergic agonist or a nitrooxy derivative thereof.

18. A pharmaceutical composition according to claim 17 wherein the beta-adrenergic antagonists is timolol and the nitrooxy derivative thereof is a nitrooxy derivative of timolol.

19. A pharmaceutical composition according to claim 17 wherein the prostaglandin analog is latanoprost and the nitrooxy derivative thereof is a nitrooxy derivative of latanoprost analog.

20. A pharmaceutical composition comprising a compound of general formula (I) according to claims 5 to 10 and at least a compound used to treat cancer.

21. A pharmaceutical kit for simultaneous, successively or previously administration of a composition according to claim 14 and (i) a beta-adrenergic antagonists or (ii) a prostaglandin analog or (iii) an α-adrenergic agonist or a nitrooxy derivative thereof.

22. α pharmaceutical kit comprising a compound of general formula (I) according to claim 5, a compound used to treat cancer as combined preparation for simultaneous, separated, sequential use for the treatment of cancer.

23. Use of a carbonic anhydrase inhibitor able to release nitric oxide as a drug for treating eye disorders and cancer.

24. Use according to claim 23, wherein the eye disorder is selected from glaucoma, ocular hypertension, age-related macular degeneration, diabetic macular edema, diabetic retinopathy, hypertensive retinopathy and retinal vasculopathies .

25. Use according to claim 23 wherein carbonic anhydrase inhibitor able to release nitric oxide is a compound having an inhibition constant (K i ) against at least one of

the isoenzymes CAII, CAIV, CAIX and CAXII in the range, of 0.01-200 nM.

26. Use according to claim 23 wherein the carbonic anhydrase inhibitor able to release nitric oxide is a compound having an EC50 value in the range of 1-50μM.

Description:

NITRATE ESTERS OF CARBONIC ANHYDRASE INHIBITORS

The present invention relates to new carbonic anhydrase inhibitors (CAIs) derivatives. More particularly, the present invention relates to nitroderivatives of CAIs, pharmaceutical compositions containing them and their use as drugs for treating glaucoma, ocular hypertension, age- related macular degeneration, diabetic macular edema, diabetic retinopathy, hypertensive retinopathy and retinal vasculopathies, cancer, epilepsy, high-altitude disorders and neuromuscular diseases.

In higher vertebrates, including humans, 14 different Carbonic anhydrase (CA) isoenzymes or CA-related proteins (CARP) were described, with very different subcellular localisation and tissue distribution. There are several cytosolic forms (CA I-III, CA VII), four membrane-bound isoenzymes (CA IV, CA IX, CA XII and CAXIV) , one mitochondrial form (CA V) and one secreted CA isozyme (CA

VI) {Supuran CT. ; Scozzafava A. Expert Opin. Ther. Patents

(2002) 12, 217-242) . Carbonic anhydrase is found in many tissues that secrete acidic or alkaline fluids, including the ocular ciliary epithelium.

The physiological function of the CA I is still unknown. The expression of CA XII has been linked to tumour invasiveness, similarly to that of CA IX.

CA II and CA IV are involved in the formation of agueous humor. The human isozyme hCA IV is abundant in a multitude of tissues, such as nasal mucosa, oesophageal

epithelium, kidneys, pancreas, salivary glands, heart muscle (endothelial and muscle cells) , eyes, lungs, brain capillaries, and colon, playing important physiological functions related to the nasal chemiosensitivity to CO 2 , the antireflux defense, bicarbonate reabsorption, NH 4 + output, pH regulation, production of ocular fluids, gas exchange.

CA Va and CA Vb are the targets for new antiobesity agents, whereas CA VII for the development of antiepileptic drugs . S-Glutathiolation of CA III was shown to occur rapidly in hepatocytes under oxidative stress.

With CAIs a class of compounds is intended, comprising as main components acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide and brinzolamide.

Many CAIs were clinically developed as antiglaucoma drugs but other applications were also found such as epilepsy, neuromuscular diseases and high-altitude disorders

(Martindale, The complete drug reference, 33 rd edition, 826- 827) .

Several side effects are associated with oral carbonic anhydrase inhibitors include fatigue, anorexia, depression, paresthesias and serum electrolyte abnormalities (The Merck

Manual of Diagnosis and Therapy, Seventeenth Edition, M. H. Beers and R. Berkow Editors, Sec. 8, Ch. 100) .

WO 2006/052899 discloses novel nitrosated and/or nitrosylated compounds or pharmaceutically acceptable salts thereof, and novel compositions, for treating ophthalmic disorders comprising at least one nitrosated and/or nitrosylated compound, and, optionally, at least one nitric oxide donor and/or at least one therapeutic agent selected from the group consisting of an α-adrenergic receptor agonist, an ACE inhibitor, an antimicrobial, a β-adrenergic

antagonist, a carbonic anhydrase inhibitor, a non-steroidal anti-inflammatory drug, a prostaglandin, a COX-2 inhibitor and a steroid.

It is an object of the present invention to provide new derivatives of CAIs able not only to eliminate or at least reduce the side effects associated with the conventionally carbonic anhydrase inhibitors, but also to possess an improved pharmacological activity. It has been surprisingly found that the compounds of the invention have a significantly improved overall profile as compared to carbonic anhydrase inhibitors both in terms of wider pharmacological activity, enhanced tolerability and long- acting ocular hypotensive activity. In particular, it has been recognized that the carbonic anhydrase inhibitors nitroderivatives can be employed for treating eye disorders in particular, glaucoma, ocular hypertension, age-related macular degeneration, diabetic macular edema, diabetic retinopathy, hypertensive retinopathy and retinal vasculopathies; cancer, epilepsy, high-altitude disorders and neuromuscular diseases.

An object of the present invention is a method for treating eye disorders and cancer in a patient in need thereof comprising administering a therapeutically effective amount of a carbonic anhydrase inhibitor able to release nitric oxide.

A carbonic anhydrase inhibitor is a compound having an inhibition constant (Ki) against at least one of the isoenzymes CAII, CAIV, CAIX and CAXII in the range of 0.01- 200 nM. The carbonic anhydrase activity is measured according to the test on carbonic anhydrase inhibition as reported below.

A carbonic anhydrase inhibitor able to release nitric oxide is a compound having an EC 50 value in the range of 1-

50μM, in a vasorelaxation assay. The vasorelaxation is measured according to the test on vascular tone as reported below.

More particularly, object of the present invention is nitroderivatives of carbonic anhydrase inhibitors of general formula (I) and pharmaceutically acceptable salts or stereoisomers thereof

R- (X-Y-ONO 2 ) s

(I) wherein s is an integer equal to 1 or 2, preferably s is 1; R is selected from the group consisting of:

wherein n is an integer from 0 to 4 ; m is an integer equal to 0 or 1;

R 1 , R 2 and R 3 are the same or different and are H, Cl, Br, I,

F, CF 3 , SO 2 NH 2 ;

X 1 is -O- or -NH-; N 1 is -OH, -COOH, -O-, -C(O)-, -NHR 1 or -NR 1 - wherein R 1 is H or C 1 -C 6 alkyl;

R' is H or a group -(X-Y-ONO 2 ) ;

X is -C(O)-, -O-, -NR 1 -, -S-, -C(O)O- or -C(O)S-; with the proviso that: i) when the group -(X-Y-ONO 2 ) is bound to N 1 , then: - N 1 is -O- or -NR 1 - and X is -C(O)-, -C(O)O- or -C(O)S-, wherein R 1 is H or C 1 -C 6 alkyl, preferably R 1 is H, preferably X is -C(O)- or -C(O)O-;

- N 1 is -C(O)- and X is -O- , -NR 1 -, -S-, -C(O)O- or -C(O)S-, wherein R 1 is H or C 1 -C 6 alkyl, preferably R 1 is H, preferably X is -O- or -NR 1 -; ii) when R' is a group -(X-Y-ONO 2 ) then X is -C(O)-, -C(O)O- or -C(O)S-, preferably X is -C(O)- or -C(O)O-; iii) when s is 2, then -X and -Y- of the two groups

-(X-Y-ONO 2 ) can be the same or different; preferably R is (IIA) or (IIB);

Y is a bivalent radical having the following meaning: a) straight or branched C 1 -C 20 alkylene, preferably straight or branched C 1 -C 10 alkylene; straight or branched C 1 -C 20 alkylene substituted with one or more of the substituents selected from the group consisting of halogen atoms, hydroxy, -ONO 2 or T, wherein T is -OC(O) (C 1 -C 10 alkyl) -ONO 2 or -O(C 1 -C 10 alkyl) -ONO 2 , preferably straight or branched C 1 -C 10 alkylene substituted with one or more of the substituents selected from -ONO 2 or -OC(O)(C 1 -C 10 alkyl)-ONO 2 or -O (C 1 -C 10 alkyl) -ONO 2 ; cycloalkylene with 5 to 7 carbon atoms into cycloalkylene ring, the ring being optionally substituted with side chains T 1 , wherein T 1 is straight or branched C 1 -C 10 alkyl; b)

c)

wherein n 0 is an integer from 0 to 20, preferably n 0 is an integer from 0 to 5, n 1 is an integer from 1 to 20, preferably n 1 is an integer from 1 to 10 or from 1 to 5; d)

wherein

Xi = -OC(O)- or -C(O)O-, preferably Xi is -C(O)O-; n 2 is an integer from 0 to 2, preferably n 2 is 1;

R 2 is H or CH 3 , preferably R 3 is -CH 3 ;

Z is -(CH 2 ) n 1 - or the bivalent radical defined above under b) , wherein n 1 is an integer from 1 to 20, preferably n 1 is an integer from 1 to 10; e)

wherein:

Y 1 is -CH 2 -CH 2 -(CH 2 )n 2a - or -CH=CH- (CH 2 ) n 2a - wherein n 2a is from 0 to 2, preferably n 2a is 0 or 1; Xi is -OC(O)- or -C(O)O-, n 2 is an integer from 0 to 2, preferably n 2 is 1 R 2 is H or CH 3 , preferably R 2 is CH 3 ;

Z is -(CH 2 ) n 1 - or the bivalent radical defined above under b) , wherein n 1 is an integer from 1 to 20;

when Y is selected from the bivalent radicals mentioned under b)-e), then the -ONO 2 group is bound to -(CH 2 ) n 1 ; f)

wherein: n 1 is an integer from 1 to 20, preferably n 1 is an integer from 1 to 10;

R 3 is H or CH 3 , preferably R 3 is H,

R 0 is H or -COCH 3 , preferably R 0 is -COCH 3 ; with the proviso that when Y is selected from the bivalent radicals mentioned under f) , then -ONO2 group is bound to - (CH 2 ) n 1 ; g)

wherein X 2 is -O- or -S-, n 3 is an integer from 1 to 6, preferably n 3 is 1,

R 2 is H or CH 3 , preferably R 2 is H; h)

wherein: n 4 is an integer from 0 to 10, preferably n 4 is an integer from 0 to 5; n 5 is an integer from 1 to 10, preferably n 5 is an integer from 1 to 5;

R 4 , R 5 , R 6 , R 7 are the same or different, and are H or straight or branched C 1 -C 4 alkyl, preferably R 4 , R 5 , R 6 , R 7 are H; wherein the -ONO2 group is linked to

wherein n 5 is as defined above;

Y 2 is an heterocyclic saturated, unsaturated or aromatic 5 or 6 members ring, containing one or more heteroatoms selected from nitrogen, oxygen, sulfur, and is selected from

(Y (Y13) preferably Y 2 is (Y1), (Y2), (Y4), (Y5) , (Y6) or (Y13). Preferred compounds of formula (I) are those wherein Y has the following meaning: a) straight or branched C 1 -C 10 alkylene, straight or branched C 1 -C 10 alkylene substituted with one or more substituents selected from -ONO 2 -OC(O) (C 1 - C 10 alkyl) -ONO 2 or -O (C 1 -C 10 alkyl) -ONO 2 ; b)

c)

wherein n 0 is an integer from 0 to 5, and n 1 is an integer from 1 to 5; d)

wherein:

Xi = -OC(O)- or -C(O)O-;

Z is -(CH 2 ) n 1 - or the bivalent radical defined above under b) wherein n 1 is an integer from 1 to 10 and n 2 is 1, and R 2 is H or CH 3 ; e)

wherein:

Y 1 is -CH 2 -CH 2 - (CH 2 ) n 2a - or -CH=CH- (CH 2 ) n 2a - wherein n 2a is 0 or 1;

Xi is -OC(O)- or -C(O)O-, n 2 is 1, R 2 is CH 3 ;

Z is -(CH 2 ) n 1 - or the bivalent radical defined above under b) , wherein n 1 is an integer from 1 to 10; with the proviso that: when Y is selected from the bivalent radicals mentioned under b) -e) , then the terminal -ONO 2 group is bound to - (CH 2 ) n 1 ; f)

wherein: n 1 is an integer from 1 to 10; R 3 is H,

R 0 is H or -COCH 3 , preferably R 0 is -OCOCH 3 ; ; with the proviso that when Y is selected from the bivalent radicals mentioned under f) , then -ONO 2 group is bound to - (CH 2 ) n 1 ; g)

wherein X 2 is -O- or -S-, n 3 is 1 and R 2 is H; h)

wherein : n 4 is an integer from 0 to 5 ; n 5 is an integer from 1 to 5 ; R 4 , R 5 , R 6 , R 7 are H; wherein the -ONO 2 group is linked to

wherein n 5 is as defined above; Y 2 is selected from

An object of the present invention refers to nitroderivatives of carbonic anhydrase inhibitors of general formula (I) and pharmaceutically acceptable salts or stereoisomers thereof

R-(X-Y-ONO 2 )s

(I) wherein s is 1 ; R is

wherein in formula (IIA)

R 1 , R 2 and R 3 are H;

m is 0 and the group -SO 2 NHR' is in position 4 of the phenyl ring, R' is H; n is 0, N 1 is -C(O)-, and N 1 binds the group -(X-Y-ONO 2 );

X is -O-, -S- or -NR 1 - wherein R 1 is H or C 1 -C 6 alkyl; or

R 1 , R 2 and R 3 are H; m is 0 and the group -SO 2 NHR' is in position 4 of the phenyl ring, R' is H; n is 0 or 1, N 1 is -O- and N 1 binds the group -(X-Y-ONO 2 );

X is -C(O)-, -C(O)O- or -C(O)S-; or

R 1 is Cl and R 1 is in position 4 of the phenyl ring, R 2 and

R 3 are H; m is 0 and the group -SO 2 NHR' is in position 3 of the phenyl ring, R' is H; n is 0, N 1 is -C(O)- and N 1 binds the group -(X-Y-ONO 2 );

X is -O-, -S- or -NR 1 - wherein R 1 is H or C 1 -C 6 alkyl; or

R 1 R 2 and R 3 are H; m is 0 and the group -SO 2 NHR' is in position 3 of the phenyl ring, R' is H; n is 3, N 1 is -C(O)-, and N 1 binds the group -(X-Y-ONO 2 );

X is -O-, -S- or -NR 1 - wherein R 1 is H or C 1 -C 6 alkyl; Y is selected from: a) straight or branched C 1 -C 10 alkylene, straight or branched C 1 -C 10 alkylene substituted with one or more substituents selected from -ONO 2 -OC(O) (C 1 - C 10 alkyl) -ONO 2 or -O (C 1 -C 10 alkyl) -ONO 2 ; b)

C )

wherein n 0 is an integer from 0 to 5, and n 1 is an integer from 1 to 5; d)

wherein:

Xi = -OC(O)- or -C(O)O-;

Z is -(CH 2 ) n 1 - or the bivalent radical defined above under b) wherein n 1 is an integer from 1 to 10 and n 2 is 1,

R 2 is H or CH 3 , preferably R 2 is CH 3 ; e)

wherein:

Y 1 is -CH 2 -CH 2 - (CH 2 ) n 2a - or -CH=CH- (CH 2 ) n 2a - wherein n 2a is 0 or 1 ;

Xi is -OC(O)- or -C(O)O-, n 2 is 1, R 2 is CH 3 ;

Z is -(CH 2 ) n 1 - or the bivalent radical defined above under b) , wherein n 1 is an integer from 1 to 10; with the proviso that: when Y is selected from the bivalent radicals mentioned under b) -e) , then the terminal -ONO 2 group is bound to - (CH 2 ) n 1 ; f)

wherein: n 1 is an integer from 1 to 10;

R 3 is H,

R 0 is H or -COCH 3 , preferably R 0 is -COCH 3 ; with the proviso that when Y is selected from the bivalent radicals mentioned under f) , then -ONO 2 group is bound to -

(CH 2 ) n 1 ; g)

wherein X 2 is -O- or -S-, n 3 is 1, R 2 is H; h)

wherein: n 4 is an integer from 0 to 5; n 5 is an integer from 1 to 5;

R 4 , R 5 , R 6 , R 7 are H; wherein the -ONO 2 group is linked to

wherein n 5 is as defined above; Y 2 is selected from

Another object of the present invention deals with nitroderivatives of carbonic anhydrase inhibitors of general formula (I) and pharmaceutically acceptable salts or stereoisomers thereof

R-(X-Y-ONO 2 )s (I) wherein s is 1 ; R is

(II B) wherein in formula (IIB) N 1 is -C(O)- and N 1 binds the group -(X-Y-ONO 2 ); R' is H;

X is -O-, -NR 1 -, -S- wherein R 1 is H or a or C 1 -C 6 alkyl; or N 1 is -COOH; R' is the group -(X-Y-ONO 2 );

X is -C(O)-, -C(O)O- or -C(O)S-; Y is a) straight or branched C 1 -C 10 alkylene, - straight or branched C 1 -C 10 alkylene substituted with one or more substituents selected from -ONO 2 or -OC(O) (C 1 -C 10 alkyl) -ONO 2 or -O(C 1 -C 10 alkyl) -ONO 2 ; b)

c)

wherein n 0 is an integer from 0 to 5, and n 1 is an integer from 1 to 5; d)

wherein:

X 1 = -OC(O)- or -C(O)O-;

Z is -(CH 2 ) n 1 - or the bivalent radical defined above under b) wherein n 1 is an integer from 1 to 10 and n 2 is 1,

R 2 is H or CH 3 , preferably R 2 is CH 3 ; e)

wherein:

Y 1 is -CH 2 -CH 2 -(CH 2 ) n 2a - or -CH=CH- (CH 2 ) n 2a - wherein n 2a is 0 or 1;

Xi is -OC(O)- or -C(O)O-, n 2 is 1, R 2 is CH 3 ;

Z is -(CH 2 ) n 1 - or the bivalent radical defined above under b) , wherein n 1 is an integer from 1 to 10; with the proviso that:

when Y is selected from the bivalent radicals mentioned under b) -e) , then the terminal -ONO 2 group is bound to - (CH 2 ) n 1 ; f)

wherein: n 1 is an integer from 1 to 10;

R 3 is H,

R 0 is H or -COCH 3 , preferably R 0 is -COCH 3 ; with the proviso that when Y is selected from the bivalent radicals mentioned under f) , then -ONO 2 group is bound to - (CH 2 ) n 1 ; g )

wherein X 2 is -O- or -S- , n 3 is 1 and R 2 is H; h )

wherein : n 4 is an integer from 0 to 5 ; n 5 is an integer from 1 to 5 ;

R 4 , R 5 , R 6 , R 7 are H; wherein the -ONO2 group is linked to

wherein n 5 is as defined above; Y 2 is selected from

The term "C 1 -C 20 alkylene" as used herein refers to branched or straight chain C 1 -C 20 hydrocarbon, preferably having from 1 to 10 carbon atoms such as methylene, ethylene, propylene, isopropylene, n-butylene, pentylene, n- hexylene and the like.

The term "C 1 -C 10 alkyl" as used herein refers to branched or straight chain alkyl groups comprising one to ten carbon atoms, including methyl, ethyl, n-propyl,

isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, octyl and the like.

The term "cycloalkylene" as used herein refers to ring having from 5 to 7 carbon atoms including, but not limited to, cyclopentylene, cyclohexylene optionally substituted with side chains such as straight or branched (C 1 -C 10 ) - alkyl, preferably CH 3 .

The term "heterocyclic" as used herein refers to saturated, unsaturated or aromatic 5 or 6 members ring, containing one or more heteroatoms selected from nitrogen, oxygen, sulphur, such as for example pyridine, pyrazine, pyrimidine, pyrrolidine, morpholine, imidazole and the like. The following are the most preferred compounds according to the present invention:

As stated above, the invention includes also the pharmaceutically acceptable salts of the compounds of formula (I) and stereoisomers thereof.

Examples of pharmaceutically acceptable salts are either those with inorganic bases, such as sodium, potassium, calcium and aluminium hydroxides, or with organic bases, such as lysine, arginine, triethylamine, dibenzylamine, piperidine and other acceptable organic amines .

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

Examples of organic acids are: oxalic, tartaric, maleic, succinic, citric acids. Examples of inorganic acids are: nitric, hydrochloric, sulphuric, phosphoric acids. Salts with nitric acid are preferred. The compounds of the invention which have one or more asymmetric carbon atoms can exist as optically pure enantiomers, pure diastereomers, enantiomers mixtures, diastereomers mixtures, enantiomer racemic mixtures, racemates or racemate mixtures. Within the scope of the invention are also all the possible isomers, stereoisomers and their mixtures of the compounds of formula (I), including mixtures enriched in a particular isomer.

The invention also relates to the use of the compounds of formula (I) as antiglaucoma agents or as drugs able to reduce the intraocular pressure and to maintain said pressure on a reduced level.

The invention also relates to the use of the compounds of formula (I) as drugs for treating glaucoma, ocular hypertension, age-related macular degeneration, diabetic macular edema, diabetic retinopathy, hypertensive retinopathy and retinal vasculopathies .

As mentioned above, objects of the present invention are also pharmaceutical compositions containing at least a compound of the present invention of formula (I) together with non toxic adjuvants and/or carriers usually employed in the pharmaceutical field.

The daily dose of active ingredient administered to a host can be a single dose or it can be an effective amount

divided into several smaller doses that are to be administered throughout the day. The dosage regimen and administration frequency for treating the mentioned diseases with the compound of the invention and/or with the pharmaceutical compositions of the present invention will be selected in accordance with a variety of factors, including for example age, body weight, sex and medical condition of the patient as well as severity of the disease, route of administration, pharmacological considerations and eventual concomitant therapy with other drugs. In some instances, dosage levels below or above the aforesaid range and/or more frequent may be adequate, and this logically will be within the judgment of the physician and will depend on the disease state . The compounds of the invention may be administered orally, parenterally, rectally or topically, by inhalation spray o aerosol, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles as desired. Topical preparations can be administered as solutions, suspensions or emulsions (dispersions) in an ophthalmically acceptable vehicle. The term "ophthalmically acceptable vehicle" as used herein refers to any substance or combination of substances which are non-reactive with the compounds and suitable for administration to patients. Preferred are aqueous vehicles suitable for topical application to the patient's eyes.

Other ingredients which may be desirable to use in the ophthalmic compositions of the present invention include antimicrobials, preservatives, co-solvents, surfactants and viscosity building agents.

Topical administration may also involve the use of transdermal administration such as transdermal patches or

iontophoresis devices. The term "parenteral" as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques . Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent o solvent. Among the acceptable vehicles and solvents are water, Ringer' s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides, in addition fatty acids such as oleic acid find use in the preparation of injectables .

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 solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as in normal practice, additional substances other than inert diluents, e.g. 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 additionally 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 may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and the like.

It is further contemplated that the compounds of the present invention can be used with other medicaments known to be useful in the treatment of glaucoma or ocular hypertension, either separately or in combination. For example the compounds of the present invention can be combined with (i) beta-blockers, such as timolol, betaxolol, levobunolol and the like described in U.S. Pat. No. 4,952,581; (ii) prostaglandin analogs, such as bimatoprost, latanoprost, travoprost or unoprostone (iii) α-adrenergic agonists including clonidine derivatives, such as apraclonidine or brimonidine described in U.S. Pat. No. 5,811,443. Also contemplated is the combination with nitrooxy derivatives of the above reported compounds, for example nitrooxy derivatives of beta-blockers described in US 6,242,432 or nitrooxy derivatives of prostaglandin analogs described in WO 2005/068421.

It is also contemplated that the compounds of the present invention can be used with other medicaments known to be useful in the treatment of cancer, either separately or in combination.

The compounds of the present invention can be synthesized as follows.

Synthesis procedure

1. The compound of general formula (I) as above defined wherein:

X is -CO-, s is 1, Y and R are as above defined and N 1 is - O- or -NR 1 - wherein R 1 is as above defined can be obtained by a process comprising:

Ia. reacting a compound of formula B with a compound of formula (Ilia) :

B + HOOC-Y-ONO 2

(IIIa)

wherein Y is as above defined; B is equal to R with N 1 is - OH or -NR 1 H and R' is H or PG wherein PG is an sulfonamido protective group such as dimethylformamidine, in presence of a condensing agent like dicyclohexylcarbodiimide (DCC), N- (3-dimethylaminopropyl) -N f -ethylcarbodimide hydrochloride (EDAC) or N, N' -carbonyldiimidazole (CDI) or other known condensing reagents such as HATU in solvent such as DMF, THF, chloroform at a temperature in the range from -5 °C to 50°C in the presence or not of a base as for example DMAP and optionally deprotecting the compounds by reaction with hydrochloric acid in methanol. The nitric acid ester compounds of formula (Ilia) can be obtained from the corresponding alcohols of formula HOOC-Y- OH (IIIb), that are commercially available, by reaction with nitric acid and acetic anhydride in a temperature range from -50°C to 0°C or reacting the corresponding halogen derivatives of formula HOOC-Y-HaI (IIIc) wherein Hal is an alogen atom preferable Cl, Br, I, that are commercially available, with AgNO 3 as already described in the international application No. WO 2006/008196.

1b. reacting a compound of formula B as above defined with a compound of formula (IIId):

B + ACt-CO-Y-ONO 2 (IIId )

wherein Y is as above defined; Act is an Halogen atom or a carboxylic acid activating group used in peptide chemistry as :

The reaction is generally carried out in presence of a inorganic or organic base in an aprotic polar/non-polar solvent such as DMF, THF or CH 2 Cl 2 at temperatures range between 0°-65°C or in a double phase system H 2 O/Et 2 O at temperatures range between 20°- 40°C; or in the presence of DMAP and a Lewis acid such as Sc(OTf) 3 or Bi(OTf) 3 in solvents such as DMF, CH 2 Cl 2 and optionally deprotecting the compounds by reaction with hydrochloric acid in methanol. The compounds of formula (IIId) can be obtained as described in the international application No. WO 2006/008196.

Ic. reacting a compound of formula R-X-Y-HaI (IVa), wherein R and X are as above defined, with AgNO 3 as already described. Compounds (IVa) can be obtained by reacting compound B with compounds (IIIc), as above defined, with a condensing reagent such as DCC or CDI as above described and optionally deprotecting the compounds by reaction with hydrochloric acid in methanol.

Id. reacting a compound of formula R-X-Y-OH (Va) , wherein R and X are as above defined, with triflic anhydride/tetraalkylammonium nitrate salt in an aprotic polar/non-polar solvent such as DMF, THF or CH 2 Cl 2 at temperatures range between -60° to 65°C as already described. Compounds (Va) can be obtained by reacting compound B with compounds (IIIb), as above defined, with a condensing reagent as above described and optionally deprotecting the compounds by reaction with hydrochloric acid in methanol.

2. The compound of general formula (I) as above defined wherein:

X is -COO-, s is 1, Y and R are as above defined and N 1 is - O- or -NR 1 can be obtained by a process comprising:

2a. reacting a compound of formula B with a compound of formula (VIa) :

B + Act—CO O—Y—ONO 2

(VIa) wherein B is equal to R with N 1 is -OH or -NR 1 H and R' is H or PG which is an sulfonamido protective group such as dimethylformamidine; Act and Y are as above described. The reaction is generally carried out in presence of a inorganic or organic base in an aprotic polar/non-polar solvent such as DMF, THF or CH 2 Cl 2 at temperatures range between 0°-65°C or in a double phase system H 2 O/Et 2 O at temperatures range between 20°- 40°C; or in the presence of DMAP and a Lewis acid such as Sc(OTf) 3 or Bi(OTf) 3 in solvents such as DMF, CH 2 Cl 2 and optionally deprotecting the compounds by reaction with hydrochloric acid in methanol.

The synthesis of compounds (VIa) has already been described in the international application No. WO 2006/008196.

2b. reacting a compound of formula R-X-Y-HaI (VIla) wherein R, X, Y and Hal are as above defined, with AgNO 3 as above described. The compounds of formula (VIla) can be obtained by reacting compound B with compounds Act-CO-O-Y-Hal (VIlla) . The reaction is generally carried out in presence of an inorganic or organic base in an aprotic polar/non-polar solvent such as DMF, THF or CH2Cl 2 at temperatures range between 0°-65°C as above described and optionally deprotecting the compounds by reaction with hydrochloric acid in methanol .

Compound (VIlla) are commercially available or can be synthesized as already described in WO 2005/011646.

3. The compound of general formula (I) as above defined wherein:

X is -O-, s is 1, Y and R are as above defined and N 1 is -

CO-, can be obtained by a process comprising: 3a. reacting a compound of formula B with a compound of formula (VIIIb) :

B + HO-Y-ONO 2

(VIIIb)

wherein Y is as above defined; B is equal to R with N 1 = - COOH and R' = H; in presence of a condensing agent like dicyclohexylcarbodiimide (DCC) or N, N' -carbonyldiimidazole (CDI) or other known condensing reagents such as HATU in solvent such as DMF, THF, chloroform at a temperature in the range from -5°C to 50°C in the presence or not of a base as for example DMAP.

The nitric acid ester compounds of formula (VIIIb) can be obtained from the corresponding alcohols of formula HO-Y-OH (VIIIc) , that are commercially available, by reaction with nitric acid and acetic anhydride in a temperature range from -50°C to 0°C or reacting the corresponding halogen derivatives of formula HO-Y-HaI (VIIId) wherein Hal is an alogen atom preferable Cl, Br, I, that are commercially available, with AgNO 3 as already described in the international application No. WO 2006/008196.

3b. reacting a compound of formula R-X-Y-HaI (IXa), wherein R and X are as above defined, with AgNO 3 as already described. Compounds (IXa) can be obtained by reacting compound B with compounds (VIIId) , as above defined, with a condensing reagent such as DCC or CDI as above described.

3c. reacting a compound of formula R-X-Y-OH (Xa) , wherein R and X are as above defined, with triflic anhydride/tetraalkylammonium nitrate salt in an aprotic polar/non-polar solvent such as DMF, THF or CH 2 Cl 2 at temperatures range between -60° to 65 °C as already described. Compounds (Xa) can be obtained by reacting compound B with compounds (VIIIc) , as above defined, with a condensing reagent as above described.

4. The compound of general formula (I) as above defined wherein:

X is -NR 1 -, s is 1, Y and R are as above defined and N 1 is - CO-, can be obtained by a process comprising: 4a. reacting a compound of formula B with a compound of formula (XIa) :

B + R 1 HN Y-ONO 2

(XIa)

wherein Y is as above defined; B is equal to R with N 1 = - COOH and R' = H; in presence of a condensing agent like dicyclohexylcarbodiimide (DCC) or N, N' -carbonyldiimidazol (CDI) or other known condensing reagents such as HATU in solvent such as DMF, THF, chloroform at a temperature in the range from -5°C to 50°C in the presence or not of a base as for example DMAP.

The nitric acid ester compounds of formula (XIa) can be obtained from the corresponding alcohols of formula R 1 HN-Y- OH (XIb) , that are commercially available, by reaction with nitric acid and acetic anhydride in a temperature range from -50°C to 0°C or reacting the corresponding halogen derivatives of formula R 1 NH-Y-HaI (XIc) wherein Hal is an alogen atom preferable Cl, Br, I, that are commercially available, with AgNO 3 .

4b. reacting a compound of formula R-X-Y-HaI (XIIa) , wherein R and X are as above defined, with AgNO 3 as already described. Compounds (XIIa) can be obtained by reacting compound B with compounds (XIc) , as above defined, with a condensing reagent such as DCC or CDI as above described.

4c. reacting a compound of formula R-X-Y-OH (XIIIa) , wherein R and X are as above defined, with triflic anhydride/tetraalkylammonium nitrate salt in an aprotic polar/non-polar solvent such as DMF, THF or CH 2 Cl 2 at temperatures range between -60° to 65°C as already described. Compounds (XIIIa) can be obtained by reacting compound B with compounds (XIb) , as above defined, with a condensing reagent as above described.

5. The compound of general formula (I) as above defined wherein:

X is -CO-, s is 1, Y and R are as above defined can be obtained by a process comprising:

5a. reacting a compound of formula B with a compound of formula (Ilia) :

B + HOOC-Y-ONO 2

(IIIa)

wherein Y is as above defined; B is equal to R with R' = free valence and N 1 = -O-PG 1 , -CO-PG 2 or -NR 1 -PG 3 wherein PG 1 is an hydroxyl protective group such as TBDMS, PG 2 is an carboxyl protective group such as 2- (p-toluenesulfonyl) ethyl ester, PG 3 is an amino protective group such as BOC; in presence of a condensing agent like dicyclohexylcarbodiimide

(DCC) , N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide hydrochloride (EDAC) or N, N' -carbonyldiimidazole (CDI) or other known condensing reagents such as HATU in solvent such as DMF, THF, chloroform at a temperature in the range from - 5°C to 50°C in the presence or not of a base as for example DMAP and hydrolysing the protective group as known in the literature.

The nitric acid ester compounds of formula (Ilia) can be obtained from the corresponding alcohols of formula HOOC-Y- OH (IIIb), that are commercially available, by reaction with nitric acid and acetic anhydride in a temperature range from -50°C to 0°C or reacting the corresponding halogen derivatives of formula HOOC-Y-HaI (IIIc) wherein Hal is an alogen atom preferable Cl, Br, I, that are commercially available, with AgNO 3 as already described in the international application No. WO 2006/008196.

5b. reacting a compound of formula B as above defined with a compound of formula (IIId) :

B + ACt-CO-Y-ONO 2 ( IIId)

wherein Y is as above defined; Act is an Halogen atom or a carboxylic acid activating group used in peptide chemistry as :

The reaction is generally carried out in presence of a inorganic or organic base in an aprotic polar/non-polar solvent such as DMF, THF or CH 2 Cl 2 at temperatures range between 0°-65°C or in a double phase system H 2 O/Et 2 O at temperatures range between 20°- 40°C; or in the presence of DMAP and a Lewis acid such as Sc(OTf) 3 or Bi(OTf) 3 in solvents such as DMF, CH 2 Cl 2 and hydrolysing the protective group as known in the literature.

The compounds of formula (IIId) can be obtained as described in the international application No. WO 2006/008196.

5c. reacting a compound of formula R-X-Y-HaI (IVa), wherein R, X and Y are as above defined, with AgNO 3 as already described. Compounds (IVa) can be obtained by reacting compound B with compounds (IIIc), as above defined, with a condensing reagent such as DCC or CDI as above described and hydrolysing the protective group as known in the literature.

5d. reacting a compound of formula R-X-Y-OH (Va) , wherein R, X and Y are as above defined, with triflic anhydride/tetraalkylammonium nitrate salt in an aprotic polar/non-polar solvent such as DMF, THF or CH 2 Cl 2 at temperatures range between -60° to 65°C as already described and hydrolysing the protective group as known in the literature. Compounds (Va) can be obtained by reacting compound B with compounds (IIIb), as above defined, with a condensing reagent as above described.

6. The compound of general formula (I) as above defined wherein:

X is -COO-, s is 1, Y and R are as above defined can be obtained by a process comprising: 6a. reacting a compound of formula B with a compound of formula (VIa) :

B + Act—X—Y—ONO 2

(VIa) wherein Act, X and Y are as above described; B is equal to R with R' = free valence and N 1 = -O-PG 1 , -CO-PG 2 or -NR 1 -PG 3 wherein PG 1 is an hydroxyl protective group such as TBDMS, PG2 is an carboxyl protective group such as 2- (p- toluenesulfonyl) ethyl ester, PG 3 is an amino protective group such as BOC. The reaction is generally carried out in presence of a inorganic or organic base in an aprotic polar/non-polar solvent such as DMF, THF or CH 2 Cl 2 at temperatures range between 0°-65°C or in a double phase system H 2 O/Et 2 O at temperatures range between 20°- 40°C; or in the presence of DMAP and a Lewis acid such as Sc(OTf) 3 or Bi(OTf) 3 in solvents such as DMF, CH 2 Cl 2 and hydrolysing the protective group as known in the literature.

The synthesis of compounds (VIa) has already been described in the international application No. WO 2006/008196.

6b. reacting a compound of formula R-X-Y-HaI (VIla) wherein R, X, Y and Hal are as above defined, with AgNO 3 as above described.

The compounds of formula (VIla) can be obtained by reacting compound B with compounds Act-X-Y-Hal (VIlla) . The reaction is generally carried out in presence of an inorganic or organic base in an aprotic polar/non-polar solvent such as DMF, THF or CH 2 Cl 2 at temperatures range between 0°-65°C as above described and hydrolysing the protective group as known in the literature. Compound (VIlla) are commercially available or can be synthesized as already described in WO 2005/011646.

7. The compound of general formula (I) as above defined wherein:

X is -CO-, s is 2, Y and R are as above defined and N 1 is - O- or -NR 1 - wherein R 1 is as above defined can be obtained by a process comprising:

7a. reacting a compound of formula B with a compound of formula (Ilia) :

B + HOOC-Y-ONO 2

(IlIa)

wherein Y is as above defined; B is equal to R with N 1 is - OH or -NR 1 H and R' is H, in presence of a condensing agent like dicyclohexylcarbodiimide (DCC), N-(3- dimethylaminopropyl)-N'-ethylcarbodimide hydrochloride (EDAC) or N, N' -carbonyldiimidazole (CDI) or other known condensing reagents such as HATU in solvent such as DMF,

THF, chloroform at a temperature in the range from -5°C to 50°C in the presence or not of a base as for example DMAP. The nitric acid ester compounds of formula (Ilia) can be obtained as above described.

7b. reacting a compound of formula B as above defined with a compound of formula (IIId) :

B + ACt-CO-Y-ONO 2 (IIId)

wherein Y is as above defined; Act is an Halogen atom or a carboxylic acid activating group used in peptide chemistry as :

The reaction is generally carried out in presence of a inorganic or organic base in an aprotic polar/non-polar solvent such as DMF, THF or CH 2 Cl 2 at temperatures range between 0°-65°C or in a double phase system H 2 O/Et 2 O at temperatures range between 20°- 40°C; or in the presence of DMAP and a Lewis acid such as Sc(OTf) 3 or Bi(OTf) 3 in solvents such as DMF, CH 2 Cl 2 The compounds of formula (IIId) can be obtained as above described

7c. reacting a compound of formula R-(X-Y-HaI) s (VIlla), wherein R, X, Y and Hal are as above defined, s is 2, with

AgNO 3 as already described. Compounds (VIlla) can be obtained by reacting compound B with compounds (IIIc), as above defined, with a condensing reagent such as DCC or CDI as above described.

7d. reacting a compound of formula R-(X-Y-OH) 3 (IXa), wherein R, X and Y are as above defined, s is 2, with triflic anhydride/tetraalkylammonium nitrate salt in an aprotic polar/non-polar solvent such as DMF, THF or CH 2 Cl 2 at temperatures range between -60° to 65°C as already described. Compounds (IXa) can be obtained by reacting compound B with compounds (IIIb), as above defined, with a condensing reagent as above described

8. The compound of general formula (I) as above defined wherein:

X is -COO-, s is 2, Y and R are as above defined and N 1 is - O- or -NR 1 can be obtained by a process comprising: 8a. reacting a compound of formula B with a compound of formula (VIa) :

B + Act—CO O—Y—ONO 2

(VIa) wherein B is equal to R with N 1 is -OH or -NR 1 H and R' is H; Act and Y are as above described. The reaction is generally carried out in presence of a inorganic or organic base in an aprotic polar/non-polar solvent such as DMF, THF or CH 2 Cl 2 at temperatures range between 0°-65°C or in a double phase system H 2 O/Et 2 O at temperatures range between 20°- 40°C; or in the presence of DMAP and a Lewis acid such as Sc(OTf) 3 or Bi(OTf) 3 in solvents such as DMF, CH 2 Cl 2 .

The synthesis of compounds (VIa) has already been described in the international application No. WO 2006/008196.

8b. reacting a compound of formula R-(X-Y-HaI) 3 (Xa) wherein R, X, Y and Hal are as above defined, s is 2, with AgNO 3 as above described.

The compounds of formula (Xa) can be obtained by reacting compound B with compounds Act-CO-O-Y-Hal (VIlla) . The reaction is generally carried out in presence of an inorganic or organic base in an aprotic polar/non-polar solvent such as DMF, THF or CH 2 Cl2 at temperatures range between 0°-65°C as above described.

Compound (VIlla) are commercially available or can be synthesized as already described in WO 2005/011646.

Compounds B of formula (HA) wherein N 1 is -NH 2 , -OH or - COOH; R 1 , R 2 and R 3 are the same or different and are H, Cl, Br, I, F, CF 3 , SO 2 NH 2 ; n is an integer from 0 to 2, m = 0, and R' is H can be prepared as described in D. Vullo et al. Bioorg. Med. Chem. Lett. 15 (2005) 963-969.

Compound B of formula (HB) wherein N 1 is -NH 2 and R' is H can be prepared as described in D. Vullo et al. Bioorg. Med. Chem. Lett. 15 (2005) 963-969. Compound B of formula (HC) wherein N 1 is -NH 2 , n = 1 and R' is H can be prepared as described in D. Vullo et al . Bioorg. Med. Chem. Lett. 15 (2005) 963-969.

Compound B of formula (HD) wherein N 1 is -NH2, m = 1 and R' is H can be prepared as described in B. Masereel et al . J. Med. Chem. (2002) 45, 312-320. Compound B of formula (HD) wherein N 1 is - NH 2 , m = 0 and R' is H can be prepared as described in D. Vullo et al. Bioorg. Med. Chem. Lett. 15 (2005) 963-969.

Compound B of formula (HE) wherein N 1 is -COOH and R' is H can be prepared as described in A. Casini et al. Bioorg. Med. Chem. Lett. 13 (2003) 2867-2873.

Compound B of formula (HF) wherein N 1 is -OH, m = 1, X 1 is -O- and R' is H can be prepared as described in B. Masereel et al. J. Med. Chem. (2002) 45, 312-320.

Compound B of formula (HF) wherein N 1 is -OH, m = 0 and R' is H can be prepared as described in D. Vullo et al. Bioorg. Med. Chem. Lett. 15 (2005) 963-969. Compound B of formula (HG) wherein N 1 is -NH 2 and R' is H can be prepared as described in D. Vullo et al. Bioorg. Med. Chem. Lett. 15 (2005) 963-969.

Example 1

Synthesis of compound of formula (1):

A) 4-Bromobutanol

Tetrahydrofuran (12.5 g , 173 mmol) was charged under nitrogen in a reactor cooled to 5-10°C. Hydrogen bromide

(7.0 g, 86.5 mmol) was then added slowly and the reaction medium was stirred over a period of 4.5 hours at 5-10°C. The mixture was diluted with 22.5 g of cold water and the pH of this solution was adjusted to pH=5-7 by adding 27.65% sodium hydroxide (2.0 g) keeping the temperature at 5-10°C. The solution was then extracted twice with dichloromethane (13.25 g) . The combined organic phases were washed with 25% brine (7.5 g) , adjusted to pH=6-7 with 27.65% sodium hydroxide and dried over magnesium sulfate. Dichloromethane

was distilled off and crude 4-bromobutanol (10.3 g, 66.9 mmol) was obtained in a yield of about 77%.

B) 4-Bromobutyl nitrate In reactor cooled to -5 to 5°C, nitric acid fuming (8.5 g, 135 mmol) was slowly added to a solution of 98% sulfuric acid (13.0 g, 130 mmol) in dichloromethane (18.0 g, 212 mmol). 4-bromobutanol (10.2 g, 66.6 mmol) was then added to this mixture and the reaction medium was stirred at -5 to 5°C over a period of 2-5 hours. The mixture was poured into cold water (110 g) keeping the temperature between -5 °C and 3 °C. After decantation, the upper aqueous phase was extracted with dichloromethane and the combined organic phases were washed with water, adjusted to pH=6-7 by addition of 27.65% sodium hydroxide, washed with brine and dried over magnesium sulfate. Dichloromethane was distilled off under vacuum and crude 4-bromobutyl nitrate (12.7 g, 64.1 mmol) was recovered in a yield of about 96%.

C) 5-(4-carboxyl(4-nitrooxybutyl)benzenesulphonamido)-1,3,4- thiadiazole-2-sulphonamide

To a solution of 5-(4-carboxybenzenesulphonamido)-1,3,4- thiadiazole-2-sulphonamide (0.05 g, 0.14 mmol) in DMF (3 ml), 4-bromo-butylnitrate (0.33 g, 25% w/w in methylene chloride, 0.28 mmol) and cesium carbonate (0.068 g, 0.21 mmol) ) were added. The reaction was stirred at room temperature for 12 hours. The solution was treated with HCl 1N and diethyl ether , the organic layer was dried with sodium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography, eluent methylene chloride/methanol 10/2. The product (0.05 g) was obtained.

1 H-NMR (DMSO) δ: 8.02 (2H, d) ; 7.85 (4H, m) ; 4.60 (2H, t); 4.32 (2H, t) ; 1.80 (4H, m) .

Example 2 Synthesis of compound of formula (11) :

D) Ferulic acid 4-(bromo)butyl ester

To a solution of ferulic acid (Ig, 5.15mmol) in tetrahydrofurane (40ml), triphenylphosphine (2.7g, 10.3mmol) and tetrabromomethane (3.41g, 10.3mmol) were added. The mixture was stirred at room temperature for 4 hours. The mixture was filtered and the solvent was evaporated under vacuum. The crude residue was purified by silica gel chromatography, eluent n-hexane/ethyl acetate 7/3. The product (0.77g) was obtained as a yellow solid. (Yield 46%) M.p.=83-88°C

E) Ferulic acid 4-(nitrooxy)butyl ester

A solution of compound A (0.8g, 2.43mmol) and silver nitrate (1.2g, 7.29mmol) in acetonitrile (50ml) was stirred at 40°C, in the dark, for 16 hours. The precipitate (silver salts) was filtered off and the solvent was evaporated under vacuum. The residue was purified by flash chromatography, eluent n-hexane/ethyl acetate 75/25. The product (0.4g) was obtained as white powder (yield 53%) M.p.=63-64°C F)

To a solution of 5- (4-carboxybenzenesulphonamido) -1, 3, 4- thiadiazole-2-sulphonamide (0.05 g, 0.137 mmol) in DMF (3 ml), ferulic acid 4- (nitrooxy) butyl ester (0.043 g, 0.137 mmol), DMAP (cat. amount) and EDAC (0.04 g, 0.205 mmol) were added. The reaction was stirred at room temperature for 24 hours. The solution was treated with water and ethyl acetate, the organic layers were dried with sodium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography, eluent methylene chloride/methanol 9/1. The product (0.03 g) was obtained.

1 H-NMR (DMSO) δ: 8.20 (2H,d); 7.95 (2H,d); 7.80 ((2H,s); 7.18 (1H, d) ; 7.10 (1H, s) ; 7.42 (2H, m) ; 6.75 (1H, d); 4.60 (2H, t); 4.20 (2H, t) ; 3.81 (3H, s) ; 1.75 (4H, m) .

Example 3

Synthesis of compound of formula (35):

G) 4-Sulphonamidobenzoic acid 4- (chloromethyl) benzyl ester

To a solution of 4-sulphonamidobenzoic acid (0.1 g, 0.5 mmol) in DMF (5 ml), alpha, alpha 1 dichloro p-xilene (0.27 g, 1.5 mmol) and cesium carbonate (0.25 g, 0.75 mmol)) were added. The reaction was stirred at room temperature for 20 hours. The solution was concentrated under reduced pressure. The residue was purified by flash chromatography, eluent ethyl acetate/n-hexane 3/7. The product (0,05 g) was obtained.

H) 4-Sulphonamidobenzoic acid 4-(nitrooxymethyl)benzyl ester

A solution of compound G) (0.05 g, 0.15 mmol) and silver nitrate (0.08 g, 0.44 mmol) in acetonitrile (2.5 ml) was stirred at 40°C, in the dark, for 5 hours. The precipitated (silver salts) was filtered off and the solvent was evaporated under vacuum. The residue was purified by flash chromatography, eluent n-hexane/ethyl acetate 7/3. The product (0.04g) was obtained. 1 H-NMR (DMSO) δ: 8.20 (2H,d); 7.98 (2H,d); 7.56 ((6H,m); 5.62 (2H, s) ; 5.39 (2H, s) .

Example 4 Synthesis of compound of formula (79) :

I) 4-bromobutyrric acid 4-sulphonamidobenzyl ester To a solution of 4-(hydroxymethyl)benzensulphonamide

(0.2 g, 1.07 mmol) in DMF (5 ml), 4-bromobutyrric acid (0.18 g, 1.07 mmol), and DCC (0.267 g, 1.29 mmol) were added. The reaction was stirred at room temperature for 24 hours. The solution was treated with water and ethyl acetate, the organic layers were dried with sodium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography, eluent methylene chloride/methanol 94/6. The product (0.035 g) was obtained.

L) 4-(nitrooxy)butyrric acid 4-sulphonamidobenzyl ester A solution of compound I) (0.035 g, 0.09 mmol) and silver nitrate (0.02 g, 0.14 mmol) in acetonitrile (2.5 ml) was stirred at 40°C, in the dark, for 16 hours. The

precipitated (silver salts) was filtered off and the solvent was evaporated under vacuum. The residue was purified by flash chromatography, eluent methylene chloride/methanol 94/6. The product (0.025 g) was obtained. 1 H-NMR (DMSO) δ: 7.80 (2H,d); 7.41 (2H,d); 7.30 ((2H, s); 5.75 (2H, s); 4.63 (2H, t) ; 2.32 (2H,t); 1.94 (4H,m ).

Example 5

Synthesis of compound of formula (27):

To a solution of 4-sulphonamidobenoic acid (0.2 g, 0.99 mmol) in DMA (10 ml)potassium carbonate (0.27 g, 2 mmol) , potassium iodide (0.15 g, 1 mmol) and 4-bromo-butylnitrate

(0.33 g, 20% w/w in methylene chloride, 2 mmol) were added.

The reaction was stirred at room temperature for 24 hours.

The solution was treated with ethyl acetate and water , the organic layer was dried with sodium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography, eluent methylene chloride/methanol 10/1. The product (0.22 g) was obtained.

1 H-NMR (DMSO) δ: 8.15 (2H, m) ; 7.98 (2H, m) ; 7.55 (2H, m) ; 4.60 (2H, t); 4.32 (2H, t) ; 1.82 (4H, m) .

Example 6

Synthesis of compound of formula (37):

To a solution of 4-sulphonamidobenzoic acid (0.1 g, 0.497 mmol) in DMA (10 ml), ferulic acid 4- (nitrooxy) butyl ester, compound B), (0.155 g, 0.497 mmol), DMAP (cat. amount) and DCC (0.114 g, 0.548 mmol) were added. The reaction was stirred at room temperature for 36 hours. The solution was treated with water and ethyl acetate, the organic layers were dried with sodium sulfate and concentrated under reduced pressure. The residue was purified by flash chromatography, eluent methylene chloride/methanol 10/1. The product (0.085 g) was obtained.

1 H-NMR (DMSO) δ: 8.28 (2H, d) ; 8.02 (2H, d) ; 7.61 (4H, m) ; 7.35 (2H, m) ; 6.75 (1H, d) ; 4.59 (2H, t) ; 4.19 (2H, t) ; 3 :82 (3H, S) ; 1.76 (4H, m) .

Example 7

Synthesis of compound of formula (162) :

The compound was synthesized using procedure described in Example 1 staring from 5- (3-carboxybenzenesulphonamido) - 1, 3, 4-thiadiazole-2-sulphonamide and 4-bromo-butylnitrate (compound B) .

1 H-NMR (DMSO) δ: 8.30 (1H, s); 8.02 (2H, dd) ; 7.80 (2H, m) ; 7.12 (1H, t); 4.60 (2H, t) ; 4.32 (2H, t) ; 1.80 (4H, m) .

Example 8

Synthesis of compound of formula (172):

The compound was synthesized using procedure described in Example 2 staring from 5- (3-carboxybenzenesulphonamido) - 1, 3, 4-thiadiazole-2-sulphonamide and ferulic acid 4- (nitrooxy) butyl ester (compound E). 1 H-NMR (DMSO) δ: 8.42 (1H, s) 8.18 (2H,dd); 7.95 (1H, d); 7.75 (2H,m); 7.60 (1H, s); 7.40 (2H, m) ; 6.75 (1H, d); 4.60 (2H, t); 4.20 (2H, t) ; 3.81 (3H, s); 1.80 (4H, m) .

Example 9 Synthesis of compound of formula (205): 4-(nitrooxy)butyrric acid 4-aminosulfonylphenyl ester

M) 4-bromobutyrric acid 4-aminosulfonylphenyl ester

4-Hydroxy-benzene-sulfonamide (500 mg, 2,86 mmol) and 4-bromo-butanoic acid (477 mg, 2,86 mmol) were dissolved in

15 ml of DMA. EDAC was added portion wise (1,645 g, 8,58 mmol) followed by catalytic amount of DMAP and the resulting suspension stirred at room temperature for 48 hours. 300 ml of ethyl acetate was added and the organic solution was extracted with water, 5% aqueous HCl and water. The organic layer was dried with anhydrous sodium sulphate and the

solvent evaporated. The crude residue was purified by flash chromatography, eluent methylene chloride/methanol 20/1. The compound M (785 mg) was obtained (85%) . 4- (nitrooxy)butyrric acid 4-aminosulfonylphenγl ester Compound M (300 mg, 0,93 mmol) was suspended in 10 ml of acetonitrile and AgNO 3 (236 mg, 1,39 mmol) was added. The mixture was stirred and heated at 40°C for 3 days. The solution was filtered and the solvent evaporated. The crude was purified by flash chromatography eluent methylene chloride/methanol 15/1. The product (189 mg) was obtained

(66%) .

1 H NMR: δ 7.90 (2H, d) , 7.45 (2H, s), 7.35 (2H, d) , 4.65 (2H, t), 2.80 (2H, t), 2.05 (2H m)

Example 10

Synthesis of compound of formula (206) : 5-nitrooxypentanoic acid 4-aminosulfonylphenyl ester

N) 5-bromopentanoic acid 4-aminosulfonylphenyl ester

To a solution of 5-bromo-valeric acid (535 mg, 2,86 mmol) in 15 ml of DMA, in a 50ml round bottom flask, was added 4-hydroxy-benzene-sulfonamide (500 mg, 2,86 mmol). EDAC (1,645 g, 8,58 mmol) was then added portion wise followed by a catalytic amount of DMAP and the resulting suspension was stirred at room temperature for 24 hours. 300 ml of ethyl acetate was added and the organic solution was extracted with water, 5% aqueous HCl and water. The organic layer was dried with anhydrous sodium sulphate and the solvent evaporated. The crude residue was purified by flash

chromatography eluent methylene chloride/methanol 20/1. Compound N (640 mg) was obtained (67%).

5-nitrooxypentanoic acid 4-aminosulfonylphenyl ester Compound N (300 mg, 0,89 mmol) was dissolved in 10 ml of acetonitrile and AgNO 3 (227 mg, 1,34 mmol) was added. The mixture was stirred and heated at 40°C for 2 days. The solution was filtered and the solvent evaporated. The crude was purified by flash chromatography eluent methylene chloride/methanol 15/1. The product (160 mg) was obtained

(56%) .

1 H NMR: δ 7.90 (2H, d) , 7.40 (2H, s) , 7.35 (2H, d) , 4.60 (2H, t), 2.70 (2H, t), 1.8 (4H, m)

Example 11

Synthesis of compound of formula (207) : 6-nitrooxyhexanoic acid 4-aminosulfonylphenyl ester

O) 6-bromohexanoic acid 4-aminosulfonylphenyl ester

4-Hydroxy-benzene-sulfonamide (500 mg, 2,86 mmol) and 6-bromo-hexanoic acid (558 mg, 2,86 mmol) were dissolved in 15 ml of DMA. EDAC (1,645 g, 8,58 mmol) was added portion wise followed by a catalytic amount of DMAP and the resulting suspension was stirred at room temperature for 24 hours. 300 ml of ethyl acetate was added and the organic solution was extracted with water, 5% aqueous HCl and water. The organic layer was dried with anhydrous sodium sulphate and the solvent evaporated. The crude residue was purified by flash chromatography eluent methylene chloride/methanol 20/1. Compound O (705 mg) was obtained (70%) .

6-nitrooxyhexanoic acid 4-aminosulfonylphenyl ester

Compound O (300 mg, 0,86 mmol) was dissolved in 15 ml of acetonitrile and AgNO 3 (219mg, 1,29 mmol) was added. The mixture was stirred and heated at 40°C for 2 days. A further equivalent of AgNO 3 (146mg, 0.86 mmol) was added and the solution was heated at 40°C for 24 hours. The solution was filtered and the solvent evaporated. The crude was purified by flash chromatography eluent methylene chloride/methanol 15/1. The product (147 mg) was obtained (52%).

1 H NMR: δ 7.90 (2H, d) , 7.40 (2H, s) , 7.35 (2H, d) , 4.60

Example 12

Synthesis of compound of formula (208): 3-[4-(p- Aminosulfonyldihydrocinnamoil) -3-methoxy] phenyl-2-propenoic acid 4- (nitrooxy) butyl ester

p-Aminosulfonyldihydrocinnamic acid (295 mg, 1,28 mmol) and 3- (4-Hydroxy-3-methoxyphenyl) -2-propenoic acid 4- (nitrooxy) butyl ester (400 mg, 1,28 mmol) were dissolved in 10 ml of DMF. EDAC (370 mg, 1,93 mmol) was added followed by a catalytic amount of DMAP and the suspension was stirred at room temperature for 72 hours. 400 ml of ethyl acetate was added and the organic solution was extracted with water, 5% aqueous HCl and water. The organic layer was dried with anhydrous sodium sulphate and the solvent evaporated. The crude residue was purified by

flash chromatography eluent ethyl acetate/petroleum ether 45/55. The product(396 mg) was obtained (59%). 1 HNMR: δ 7.80 (2H, m) , 7.70 (1H, d) , 7.50 (3H, m) , 7.30 (3H, m) , 7.10 (1H, m) , 6.75 (1H, d) , 4.6 (2H, m) , 4.3 (2H, m) , 3.8 (3H, s), 3.0 (4H, m) , 1.8 (4H, m)

Pharmacological experiments

Evaluation of the carbonic anhydrase inhibition against isoenzymes CA I, II, IX and XII.

An SX.18MV-R Applied Photophysics stopped flow instrument has been used for assaying the CA CO 2 hydration activity. Phenol red (0.2 inM) was used as indicator (pH 6.8-8.4), working at the absorbance maximum of 557 nm. The buffer solution was constituted by 10 mM HEPES, 0.1 M Na 2 SO 4 , and TRIZMA hydrochloride 0.01 M, adjusting the pH solution at 7.5 with NaOH (0,1 M) .

The CA-catalyzed CO 2 hydration reaction was followed for a period of 1-20 s, depending on the isoform used. Satured CO 2 solution in bidistilled water at 20°C was used as substrate.

Stock solutions of inhibitor (1 mM) were prepared with buffer solution with 10-20% (v/v) DMSO, and dilutions up to

0.1 nM. To allow for the formation of the E-I complex, inhibitor and enzyme solutions were preincubated during 15 min at room temperature prior to assay. Enzyme concentrations were 0.1 μM for CA I and II, 10 μM for CA IV and 1 μM for CA IX and CA XII. The human CA I and II are commercialy available, whereas the hCA IV, hCA IX (tumor) and hCA XII (tumor) are cloned isoforms.

Each experiment was done in triplicate.

As reported in Table 1, the compounds of the invention show inhibitory activity in the nanomolar range against at least one of the isoforms.

Table 1

IC 5O is the concentration which inhibits 50% of the response .

Test on vascular -tone

The ability of the carbonic anhydrase inhibitors nitroderivatives to induce vasorelaxation in comparison to native CAI, was tested in vitro in isolated rabbit thoracic aorta preparations (Wanstall J. C. et al., Br. J. Pharmacol., 134:463-472, 2001). Male New Zealand rabbits were anaesthetized with thiopental-Na (50 mg/kg, iv) , sacrificed by exsanguinations and then the thorax was opened and the aorta dissected. Aortic ring preparations (4 mm in length) were set up in physiological salt solution (PSS) at 37°C in small organ chambers (5 ml) . The composition of PSS was (mM) : NaCl 130, NaHCO 3 14.9, KH 2 PO 4 1.2, MgSO 4 1.2, HEPES 10, CaCl 2 , ascorbic acid 170 and glucose 1.1 (95% O 2 /5% CO 2 ; pH 7.4). Each ring was mounted under 2 g passive tension. Isometric tension was recorded with a Grass transducer (Grass FT03) attached to a BIOPAC MP150 System. Preparations

were allowed to equilibrate for 1h, and then contracted submaximally with noradrenaline (NA, 1 μM) and, when the contraction was stable, acetylcholine (ACh, 10 μM) was added. A relaxant response to ACh indicated the presence of a functional endothelium. Vessels that were unable to contract NA or showed no relaxation to Ach were discarded. When a stable precontraction was reached, a cumulative concentration-response curve to either of the vasorelaxant agents was obtained in the presence of a functional endothelium. Each arterial ring was exposed to only one combination of inhibitor and vasorelaxant. Moreover, the effect of the soluble guanylyl cyclase inhibitor ODQ (1-H- (1,2,4)-oxadiazol(4,3-a)quinoxalin-1-one) on vasorelaxation elicited by the compounds was examined preincubating the aortic rings with ODQ (10 μM) for 20 min.

Responses to relaxing agents are expressed as a percentage of residual contraction and plotted against concentration of test compound. EC 50 values (where EC 50 is the concentration producing 50% of the maximum relaxation to the test compound) were interpolated from these plots.

During the experimental period, the plateau obtained with NA was stable without significant spontaneous loss of contraction in the aortic rings. Under these experimental conditions, the carbonic anhydrase inhibitors did not produce relaxation at any of the concentration tested, the curve being not different from that built up in the presence of vehicle alone.

The nitroderivatives of the invention have EC 50 values in the range of 1-50μM. Furthermore, in experiments performed in the presence of ODQ (10 μM) , the vasorelaxant responses to tested compounds were inhibited.