AU737584B2 - Compositions and methods to prevent toxicity induced by nonsteroidal antiinflammatory drugs - Google Patents

Description

N

S&FRef: 393590D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

*r *r *t 0 Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: Nitromed, Inc.

801 Albany Street Boston Massachusetts 02118 United States of America David S. Garvey, L. Gordon Letts, H. Burt Renfroe and Sang W. Tam Spruson Ferguson St Martins Tower 31 Market Street Sydney NSW 2000 Compositions and Methods to Prevent Toxicity Induced by Nonsteroidal Antiinflammatory Drugs The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c COMPOSITIONS AND METHODS TO PREVENT TOXICITY INDUCED BY NONSTEROIDAL ANTIINFLAMMATORY

DRUGS

This invention relates to the field of "aspirin-like" or nonsteroidal antiinflammatory drug compounds and compositions that prevent, reduce or reverse the gastrointestinal, renal, and other toxicities associated with nonsteroidal antiinflammatory drugs.

Arena et al., W094/12463, discloses the chemistry and pharmacology of nitroxybutylester[(CH2)4-ONO 2 derivatives of several aryl propionic acid nonsteroidal antiinflammatory drugs including ketoprofen, flurbiprofen, suprofen, indobufen and etodolac. Studies on nitroxybutylester derivatives of flurbiprofen and ketoprofen are also reported in Wallace et al., Gastroenterology, 107:173-179 (1994).

See, also, Cuzzolin et al., Pharmacol. Res., 29(1):89-97 (1994); Reuter et al., Life Sci. (USA), 55/1(PL1-PL8) (1994); Reuter et al., Gastroenterology, 106(4):Suppl.

s. A759 (1994); Wallace et al., Eur. J. Pharmacol., 257(3):249-255 (1994); Wallace et al., Gastroenterology, 106(4):Suppl. A208 (1994); and Conforti et al., Agents-Actions, 40(3-4):176-180 (1993). These publications uniformly examine and rely upon the use of indirectly linked nitrogen dioxide substitutions.

-l- 2 The present invention is based on the discovery by the inventors that it is possible to link a nitrogen monoxide group, nitric oxide to a non-steroidal antiinflammatory agent and that the resulting compounds not only possess potent analgesic/antiinflammatory properties but has a much reduced potential for producing gastrointestinal lesions (ulcers).

The present invention is further based on the discovery by the inventors that it is possible to coadminister a nonsteroidal antiinflammatory drug (NSAID) and a compound that directly donates, releases or transfers nitrogen monoxide (preferably as a charged species, particularly nitrosonium) to prevent, reduce, or reverse the gastrointestinal, renal, and other toxicities induced by the NSAID.

NSAIDs are antiinflammatory, analgesic and antipyretic compounds that act as cyclooxygenase, the enzyme responsible for the biosyntheses of the prostaglandins and certain autocoids, inhibitors, including inhibitors of the various isozymes of cyclooxygenase (including but not limited to cyclooxygenase-1 and and as inhibitors of both cyclooxygenase and lipoxygenase. Particularly preferred cyclooxygenase-2 inhibitors are selected from the group comprising CELEBREX (Celecoxib or 4-(5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide), VIOXX (Rofecoxib 1is or 4-(4'-methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone), valdecoxib (4-(5-methyl-3-phenyl-4isoxazolyl)benzenesulfonamide), parecoxib (4-(5-methyl-3-phenyl-4isoxazolyl)phenyl)sulfonyl)propanamide), L-745337 (5-methanesulphonamido-6-(2,4difluorothiophenyl)-1-indanone), L-761066 (1-((4-bromophenyl)methyl)-5-methoxy-p-2-dimethyl-1H- S9 indole-3-butanoic acid), JTE-522 (4-(4-cyclohexyl-2-methyl-5-oxazolyl)-2-fluoro-benzenesulfonamide), 20 and GR-253035. A nitric oxide donor is a compound that contains a nitric oxide moiety and which directly releases or directly chemically transfers nitrogen monoxide (nitric oxide), preferably in its positively charged nitrosonium form, to another molecule. Nitric oxide donors include but are not limited to S-nitrosothiols, nitrites, N-oxo-N-nitrosamines, and substrates of various forms of nitric oxide synthase.

Herein disclosed is a compound comprising a non-steroidal antiinflammatory agent to which is directly or indirectly linked at least one NO group. The non-steroidal antiinflammatory agent can, for example, be an aryl propionic acid or an enolic anilide. The invention also provides compositions comprising such compounds in a pharmaceutically acceptable carrier.

Also herein disclosed is a composition comprising a mixture of a therapeutically effective amount of a nonsteroidal antiinflammatory agent and an NSAID toxicity reducing amount of a compound that donates, transfers or releases nitric oxide.

Further disclosed is a composition comprising a non-steroidal antiinflammatory agent to which is directly or indirectly linked at least one NO group and a compound that donates, transfers or releases nitric oxide. The non-steroidal antiinflammatory agent can, for example, be an aryl propionic cSidor an enolic anilide. Also disclosed are compositions comprising such compounds in a Spharmaceutically acceptable carrier.

Z 6 1l.

ZZ06296 Also herein disclosed is a method for treating inflammation, pain and/or fever in an individual in need thereof which comprises administering to the individual a nonsteroidal antiinflammatory agent, which may optionally be substituted with at least one NO group, and a compound that donates, transfers or releases nitric oxide. The NSAID or NSAID directly or indirectly linked to at least one NO group, and nitric oxide donor can be administered separately or as components of the same composition.

Also herein disclosed is a method of treating inflammation, pain and/or fever in an individual in need thereof which comprises administering to the individual a composition comprising a therapeutically effective amount of an NSAID, which may optionally be substituted with at least one NO group, and an NSAID toxicity reducing amount of a nitric oxide donor in a pharmaceutically acceptable carrier. Such compositions are discussed in more detail below.

Also herein disclosed is a method to decrease or reverse the gastrointestinal toxicity of nonsteroidal antiinflammatory drugs administered to an animal, particularly a human, by coadministerating to said animal a nitric oxide donor. The NSAID and nitric oxide donor can be administered separately or as components of the same composition.

Also herein disclosed is a method to decrease or reverse the renal toxicity of nonsteroidal antiinflammatory drugs administered to an animal, particularly a human, by co-administering to said animal a nitric oxide donor. The NSAID and nitric oxide donor can be administed separately or as OlOOO S components of the same composition.

°*oo 20 Also herein disclosed is a method to accelerate gastrointestinal tissue repair in an animal, particularly a human, by administering to said animal a nitric oxide donor. The NSAID and nitric oxide **donor can be administered separately or as components of the same composition.

According to a first embodiment of the invention, there is provided a method for reducing druginduced gastrointestinal or renal toxicity comprising administering a therapeutically effective amount of a composition comprising a compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase, and (ii) a pharmaceutically acceptable carrier.

According to a second embodiment of the invention, there is provided a method for treating gastrointestinal lesions comprising administering a therapeutically effective amount of a composition comprising a compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate of nitric oxide synthase, and (ii) a pharmaceutically acceptable carrier.

According to a third embodiment of the invention, there is provided a composition comprising (i) a compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthases, and (ii) a pharmaceutically acceptable carrier, _wh!enused for reducing drug-induced gastrointestinal or renal toxicity.

ZZ06296 According to a fourth embodiment.of the invention, there is provided a composition comprising a compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase, and (ii) a pharmaceutically acceptable carrier, when used for treating gastrointestinal lesions.

According to a fifth embodiment of the invention, there is provided use of a composition comprising a compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthases, and (ii) a pharmaceutically acceptable carrier for the manufacture of a medicament for reducing drug-induced gastrointestinal or renal toxicity.

According to a sixth embodiment of the invention, there is provided use of a composition comprising a compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase, and (ii) a pharmaceutically acceptable carrier for the manufacture of a medicament for treating gastrointestinal lesions.

According to a seventh embodiment of the invention, there is provided a composition :15 comprising a selective cyclooxygenase-2 inhibitor and a nitric oxide donor.

According to an eighth embodiment of the invention, there is provided a composition comprising a therapeutically effective amount of a nonsteroidal antiinflammatory drug and (ii) an NSAID toxicity reducing amount of a compound that donates nitric oxide, transfers nitric oxide, releases nitric oxide, or elevates endogenous synthesis levels of nitric oxide, substantially as hereinbefore described with S 20 reference to any one of the examples.

According to a ninth embodiment of the invention, there is provided a composition comprising a non-steroidal antiinflammatory agent to which is directly or indirectly linked at least one NO group and .a compound that donates nitric oxide, transfers nitric oxide, releases nitric oxide, or elevates endogenous synthesis levels of nitric oxide, substantially as hereinbefore described with reference to 25 any one of the examples.

The compounds and compositions disclosed herein are novel and can be utilized to treat numerous inflammatory disease states and disorders. For example, reperfusion injury to an ischemic organ, reperfusion injury to the ischemic myocardium, myocardial infarction, inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, hypertension, psoriasis, organ transplant rejections, organ preservation, impotence, radiation-induced injury, asthma, atherosclerosis, thrombosis, platelet aggregation, metastasis, influenza, stroke, bums, trauma, acute pancreatitis, pyelonephritis, hepatitis, autoimmune diseases, insulin-dependent diabetes mellitus, disseminated intravascular coagulation, fatty embolism, Alzheimer's disease, adult and infantile respiratory diseases, carcinogenesis and hemorrhages in neonates.

The NSAID can be nitrosylated through sites such as oxygen (hydroxyl condensation), sulfur (sulfhydryl condensation), carbon and nitrogen.

The term "lower alkyl" herein refers to branched or straight chain alkyl groups comprising one to n mcarbon atoms, including methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, neopentyl and the like.

h ZI ZZ06296 The term "alkoxy" herein refers to RO-wherein R is lower alkyl as defined above. Representative examples of alkoxy groups include methoxy. ethoxy, t-butoxv and the like.

The term "alkoxyalkyl" herein refers to an alkoxy group as previously defined appended to an alkyl group as previously defined. Examples of alkoxyalkyl include, but are not limited to, methoxymethyl, methoxyethyl, isopropoxymethyl and the like.

The term "alkenyl" herein refers to a branched or straight chain C2-Co hydrocarbon which also comprises one or more carbon-carbon double bonds.

The term "nitrite" herein refers to -O-NO.

The term "amino" herein refers to -NH,.

The term "nitrosothiol" herein refers to -S-NO.

The term "cyano" herein refers to -CN.

The term "hydroxy" herein refers to -OH.

The term "thionitrate" herein refers to -S-NO,.

r The term "alkylsulfinyl" herein refers to R 50

-S(O)

2 wherein R 5 o is a branched or unbranched lower alkyl of up to four carbons.

The term "carboxamido" herein refers to -C(O)NH 2 The term "carbamoyl" herein refers to -O-C(O)NH 2 The term "carboxyl" herein refers to -CO,H.

The term "alkylamino" herein refers to RsNH-wherein Rs, is a lower alkyl group, for example, methylamino, ethylamino, butylamino, and the like.

The term "dialkylamino" herein refers to R,, 2

R

3 N- wherein Rs, and R 53 are independently selected from lower alkyl, for example dimethylamino, diethylamino, methyl propylamino, and the like.

The term "N-alkylcarbamoyl" herein refers to wherein R 5 is as previously defined.

The term "NN-dialkylcarbamoyl" herein refers to -O-C(O)N(R52)(R,3) wherein

R

5 and R.

3 are as previously defined.

The term "nitroso" herein refers to the group -NO and "nitrosylated" refers to compounds that have been substituted therewith.

The term "nitro" herein refers to the group -NO 2 and "nitrosated" refers to compounds that have been substituted therewith.

The term "aryl" herein refers to a mono- or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. Aryl groups (including bicyclic aryl groups) can be unsubstituted or substituted with one, two or three substituents independently selected from loweralkyl, haloalkyl, alkoxy, amino, alkylamino, dialkylamino, hydroxy, halo, and nitro. In addition, substituted aryl groups include tetrafluorophenyl and pentafluorophenyl.

-6o The term "arylalkyl" herein refers to a lower alkyl radical to which is appended an aryl group. Representative arylalkyl groups include benzyl,-phenylethyl.

hydroxybenzyl, fluorobenzyl, fluorophenylethyl and the like.

The term "arylthio" herein refers to R, 5 S- wherein is an aryl group.

The term "cycloalkyl" herein refers to an alicyclic group comprising from 3 to 7 carbon atoms including, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

The term "bridged cycloalkyl" herein refers to two or more cycloalkyl radicals fused via adjacent or non-adjacent carbon atoms, including but not limited to t adamantyl and decahydronapthyl.

The terms halogen" or "halo" herein refer to I, Br, Cl or F. The term "haloalkyl" herein refers to a lower alkyl radical, as defined above, bearing at least one halogen substituent, for example, chloromethyl, fluoroethyl or trifluoromethyl and the like.

The term "heteroaryl" herein refers to a mono- or bi-cyclic ring system containing one or two aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring. Heteroaryl groups (including bicyclic heteroaryl groups) can be unsubstituted or substituted with one, two, or three substituents independently selected from lower alkyl, haloalkyl, alkoxy, amino, alkylamino, 20 dialkylamino, hydroxy, halo and nitro. Examples of heteroaryl groups include but are not limited to pyridine, pyrazine, pyrimidine, pyridazine, pyrazole, triazole, thiazole, isothiazole, benzothiazole, benzoxazole, thiadiazole, oxazole, pyrrole, imidazole, and isoxazole.

-7- The term "heterocyclic ring" herein refers to any 6r 7-membered nonaromatic ring containing at least one nitrogen atom which is bonded to an atom which is not part of the heterocyclic ring. In addition, the heterocyclic ring may also contain a one additional heteroatom which may be nitrogen, oxygen, or sulfur.

The term "heterocyclic compounds" herein refers to mono and polycyclic compounds containing at least one heteroaryl or heterocyclic ring.

Compounds of the invention which have one or more asymmetric carbon atoms may exist as the optically pure enantiomers, pure diastereomers, mixtures of enantiomers, mixtures of diastereomers, racemic mixtures of enantiomers, diastereomeric racemates or mixtures of diastereomeric racemates. It is to be understood that the present invention anticipates and includes within its scope all such isomers and mixtures thereof.

The NSAID used in the compositions of the invention can be any of those known to the art, including those exemplified below.

First, despite the introduction of many new drugs, aspirin (acetylsalicylic acid) is still the most widely prescribed antiinflammatory, analgesic and antipyretic agent and is a standard for the comparison and evaluation of all other NSAIDs. Salicylic acid itself is so irritating that it can only be used externally. However, derivatives, particularly salicylate esters and salts, have been prepared which provide ingestible 20 forms of the salicylates which have the desired antiinflammatory and other properties.

In addition to aspirin which is the acetate ester of salicylic acid, are the diflurophenyl derivative (diflunisal) and salicylsalicylic acid (salsalate). Also available are the salts of salicylic acid, principally sodium salicylate. Sodium salicylate and aspirin are the two most commonly used preparations for systemic treatment. Other salicylates o- include salicylamide, sodium thiosalicylate, choline salicylate and magnesium salicylate. Also available are combinations of choline and magnesium salicylates.

-8go *g* S Also contemplated are 5-aminosalicylic acid (mesalamine). salicylazosulfapyridine (sulfasalazine) and methylsalicylate.

Another group of NSAID drugs included are the pyrazolon derivatives.

Included in this group are, for example, phenylbutazone, oxyphenbutazone. antipyrine.

aminopyrine, dipyrone and apazone (azapropazone).

Another group of such NSAIDs are the para-aminophenol derivatives. These are the so-called "coal tar" analgesics and include phenacetin and its active metabolite acetaminophen.

Another group of compounds contemplated include indomethacin, a methylated to indole derivative, and the structurally related compound, sulindac.

Also contemplated is a group of compounds referred to as the fenamates which are derivatives of N-phenylanthranilic acid. The most well known of these compounds are mefenamic, meclofenamic, flufenamic, tolfenamic and etofenamic acids. They are used either as the acid or as pharmaceutically acceptable salts.

Another contemplated NSAID is tolmetin which, like the other NSAIDs discussed herein, causes gastric erosion and prolonged bleeding time.

Another group of NSAID compounds are the propionic acid derivatives.

Principal members of this group are ibuprofen, naproxen, flurbiprofen, fenoprofen and ketoprofen. Other members of this group, in use or study in countries outside the 2 include fenbufen, pirprofen, oxaprozin. indoprofen and tiaprofenic acid.

Also contemplated are piroxicam and amperoxicam, oxicam derivatives which are a class of antiinflammatory enolic acids. The other related compounds tenoxicam and tenidap are also contemplated. Another compound that is particularly .9 -9g*o *o and tenidap are also contemplated. Another compound that is particularly contemplated is diclophenac, one of the series of phenylacetic acid derivatives that have been developed as antiinflammatory agents. Other NSAIDs which are contemplated as suitable in the compositions of the invention include etodolac and f nabumentone.

Each of the above contemplated NSAIDs is described more fully in the literature, such as in Goodman and Gilman, The Pharmacological Basis of Therapeutics (8th Edition), McGraw-Hill, 1993, Pgs. 638-381.

The compositions of the invention can also include NSAIDs which have been t nitrosylated through sites such as oxygen (hydroxyl condensation), sulfur (sulfhydryl condensation), carbon and nitrogen, including those specifically discussed below and in the working examples that follow.

One embodiment of this aspect includes nitroso substituted compounds of the formula: O-D-O 0 A2\ N H

A

3 S R (o)2 wherein D is selected from a covalent bond; (ii) in which R. is lower alkyl, cycloalkyl, aryl or heteroaryl, Y is oxygen, sulfur, or NR, in which R i is hydrogen or lower alkyl, Rb and R, are independently selected from, hydrogen, lower alkyl, cycloalkyl, aryl, heteroaryl, aminoarylalkyl, alkylamino, dialkylamino or taken together are cycloalkyl or bridged cycloalkyl, p is an integer from 1 to 6 and T is a covalent bond, oxygen, sulfur, or nitrogen and Q is -NO or

NO

2 with the proviso that -T-Q is not -O-NO 2 or T 2 wherein T, and T, are independently selected from T, and wherein RI, p and T are as defined above and with the provision that -T-Q does not equal -O-NO 2 Z is an aryl or heteroaryl; and

A

1

A

2 and A 3 comprise the other subunits of a 5- or 6-membered monocyclic aromatic ring and each is independently selected from C-R, wherein R, at each occurrence is independently selected from hydrogen, lower alkyl, lower haloalkyl.

alkoxyalkyl, halogen or nitro; N-Rd wherein Rd at each occurrence is independently selected from a covalent bond to an adjacent ring atom in order to render the ring aromatic, hydrogen, lower alkyl, cycloalkyl, arylalkyl, aryl, v O heteroaryl; sulfur; oxygen; and Ba=Bb wherein Ba and Bb are each independently selected from nitrogen or C-R, wherein at each occurrence R, is as defined above.

Another embodiment of this aspect is nitroso substituted compounds of the formula: O-D-Q 0 Z N N-Re A, A 3 Rb

A

2 wherein Rb, R, D, Q, Z, A 2 and A 3 are defined as above.

Another embodiment is compounds of the formula: .R R-C-C-X T -11- *o *oo wherein R, is hydrogen or lower ailkyl; Rf is selected from (1) (2) (3) (4) CH 3

S\

CH

3 (7) (8)

F

C Hs

H

N

C2HS 0

H

3 1 -12- (14) (9) (11)

H

N

CI

0

-N

(16) (12)

CH

3 0 (17) 0 0

CH

(13) -13in which n is 0 or 1; and X is 2 wherein G is a covilent bond: (ii) (iii) (iv) wherein Rm is heteroaryl or heterocyclic ring; p, and P2 are independently selected from p and in which Y. Rb, R, p and T are as defined above with the proviso that -T-Q is not -O-NO (2)

O-N

0-NN in which W is a heterocyclic ring or NRhR, wherein Rh and R, are independently selected from lower alkyl, aryl or alkenyl; 2 [C(Rb)(R,]pI-T-Q]p2 wherein and Y 2 are independently selected from Y, S is an integer from 0 to 3, and Rb, RI, Z, T, and Q are as defined above with the proviso o that -T-Q is not is -O-NO 2 Another embodiment of this aspect is compounds of the formula: 0 wherein R, is selected from

*FOH

c

F

H

-14- .0 cI.Is

S

-14- 0 OH M and X is defined as above.

The' present invention also relates to processes for preparing the compounds of formula (III) or (IV) and to the intermediates useful in such processes.

Compounds of the present invention may be synthesized as shown in reaction Schemes I through XI presented below, in which Rb, R, R, R, R, A 2

A

3 p, and Z are as defined above or as depicted in the reaction schemes for formulas I, II, H or IV; P 1 is an oxygen protecting group and P 2 is a sulfur protecting group. The reactions are performed in solvents appropriate to the reagents and materials employed are suitable for the transformations being effected. It is 1O understood by those skilled in the art of organic synthesis that the functionality present in the molecule must be consistent with the chemical transformation proposed. This will, on occasion, necessitate judgment by the routineer as to the order of synthetic steps, protecting groups required, and deprotection conditions.

Substituents on the starting materials may be incompatible with some of the reaction conditions required in some of the methods described, but alternative methods and substituents compatible with the reaction conditions will be readily apparent to skilled S* practitioners in the art. The use of sulfur and oxygen protecting groups is well known in the art for protecting thiol, alcohol, and amino groups against undesirable reactions during a synthetic procedure and many such protecting groups are known, 2 o 0 T.H. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, John Wiley Sons, New York (1991).

Nitroso compounds of formula wherein A 2

A

3 Ra, and Z are defined as above and an O-nitrosyated enol is represenatative of the D group as defined 0 above may be prepared according to reaction Scheme I. The enolic form of the 3keto amide of the formula 1 is reacted with a suitable nitrosylating agent such as thionyl chloride nitrite, thionyl dinitrite Hakimelahi et al., Helvetica Chimica Acta, 67, 907 (1984)], or nitrosium tetrafluoroborate in a suitable anhydrous solvent such as methylene chloride, tetrahydrofuran (THF), dimethylforamide (DMF). or acetonitrile with or without am amine base such as pyridine or triethylamine to afford the O-nitrite IA.

Scheme I OH 0 ONO 0 A N A

N

N H I I

A

3 S _R A 3

R

(0)2 (0) 1

IA

Nitroso compounds of formula wherein p, A 2

A

3

R

2 Rb, R, and Z are defined as above and an O-nitrosylated ester is representative of the D group as defined above may be prepared according to Scheme II. The enolic form of the 3keto amide of the formula 1 is converted to the ester of the formula 2 wherein p, Rb and R, are defined as above by reaction with an appropriate protected alcohol containing activated acylating agent wherein P' is as defined above. Preferred 1 methods for the formation of enol ester are reacting the enol with the preformed acid chloride or symmetrical anhydride of the protected alcohol containing acid. Preferred protecting groups for the alcohol moiety are silyl ethers such as a trimethylsilyl or a tert-butyldimethylsilyl ether. Deprotection of the hydroxyl moiety (fluoride ion is the preferred method for removing silyl ether protecting groups) followed by reaction 10 with a suitable nitrosylating agent such as thionyl chloride nitrite, thionyl dinitrite, or nitrosium tetrafluoroborate in a suitable anhydrous solvent such as dichloromethane, -16oo o THF, DMF, or acetonitrile with or without an amine base such as pyridine or triethylamine affords the compound of the formula IB.

Scheme II OH 0 Rb 0 Rb 0 A P'-0-(CP-C ON-O-(C C A N 0 0 0 0

A

3 S Ra A NZ A (0)A2 H A 2

H

1 A S R* A 3 S" R (oh (0)2 2

IB

Nitroso compounds of formula wherein p, A 2

A

3 Rb, R, and Z Sare defined as above and an S-nitrosyated enol ester is representative of the D group as defined above may be prepared according to reaction Scheme III. The enolic form of the 0-keto amide of the formula 1 is convened to the ester of the formula 3 wherein p, Rb, and R, are defined as above by reaction with an appropriate protected thiol containing activated acylating agent wherein P 2 is as defined above. Preferred 0 methods for the formation of enol ester are reacting the enol with the preformed acid chloride or symmetrical anhydride of the protected thiol containing acid. Preferred protecting groups for the thiol moiety are as a thioester such as a thioacetate or thiobenzoate, as a disulfide, as a thiocarbamate such as N-methoxymethyl thiocarbamate, or as a thioether such as a paramethoxybenzyl thioether, a tetrahydropyranyl thioether or a S-triphenylmethyl thioether. Deprotection of the thiol moiety (zinc in dilute aqueous acid, triphenylphosphine in water and sodium borohydride are preferred methods for reducing disulfide groups while aqueous base 1 -17is typically utilized to hydrolyze thioesters and N-methoxymethyl thiocarbamates and mercuric trifluoroacetate, silver nitrate, or strong acids such, as trifluoFoacetic or hydrochloric acid and heat are used to remove a paramethoxybenzyl thioether, a tetrahydropyranyl thioether or a S-triphenylmethyl thioether group) followed by reaction with a suitable nitrosylating agent such as thionyl chloride nitrite, thionyl dinitrite, a lower alkyl nitrite such as tert-butyl nitrite, or nitrosium tetrafluoroborate in a suitable anhydrous solvent such as methyene chloride, THF, DMF, or acetonitrile with or without an amine base such as pyridine or triethylamine affords the compound of the formula IC. Alternatively, reacting this intermediate with a \0 stoichiometric quantity of sodium nitrite in aqueous acid affords the compound of the formula IC.

Scheme III Rb 0 S0 P9-( C 0 0 Z RC A

NZ

A2, AO H A 3 s R g A ISR. A S R S R (0)2 (0 (0)2 Io 1 ON-S-(C p-C 0 *A N Z

I

(0)2

IC

-18- -18- Nitroso compounds of formula (II) wherein p, A 2

A

3 Rb and R, and Z are defined as above and an O-nitrosylated ester is representative of thli D group as defined above may be prepared according to Scheme IV. The enolic form of the 3keto amide of the formula 4 is converted to the ester of the formula 5 wherein p, Rb and R, are defined as above by reaction with an appropriate protected alcohol containing activated acylating agent wherein P 1 is as defined above. Preferred methods for the formation of enol ester are reacting the enol with the preformed acid chloride or symmetrical anhydride of the protected alcohol containing acid. Preferred protecting groups for the alcohol moiety are silyl ethers such as a trimethylsilyl or a to tert-butyldimethylsilyl ether. Deprotection of the hydroxyl moiety (fluoride ion is the preferred method for removing silyl ether protecting groups) followed by reaction a suitable nitrosylating agent such as thionyl chloride nitrite, thionyl dinitrite, or nitrosium tetrafluoroborate in a suitable anhydrous solvent such as dichloromethane, THF, DMF, or acetonitrile with or without an amine base such as pyridine or triethylamine affords the compound of the formula IA.

-19- -19- Scheme IV 0 Z N4 Rb A2 R 0

I,

I A Nitroso compounds of formula (II) wherein p, A 2

A

3 Rb, Re, and Z are defined as above and an S-nitrosyated enol ester is represenatative of the D group as defined above may be prepared according to reaction Scheme V. The enolic form of the (-keto amide of the formula 4 is converted to the ester of the formula 6 wherein p, Rb and R, are defined as above by reaction with an appropriate protected thiol containing activated acylating agent wherein P 2 is as defined above. Preferred methods for the formation of enol ester are reacting the enol with the preformed acid chloride or symmetrical anhydride of the protected thiol containing acid. Preferred protecting groups for the thiol moiety are as a thioester such as a thioacetate or 10 thiobenzoate, as a disulfide, as a thiocarbamate such as N-methoxymethyl thiocarbamate, or as a thioether such as a paramethoxybenzyl thioether, a tetrahydropyranyl thioether, or a S-triphenylmethyl thioether. Deprotection of the thiol moiety (zinc in dilute aqueous acid, triphenylphosphine in water and sodium borohydride are preferred methods for reducing disulfide groups while aqueous base is typically utilized to hydrolyze thiolesters and N-methoxymethyl thiocarbamates and mercuric trifluoroacetate, silver nitrate, or strong acids such as trifluoroacetic or hydrochloric acid and heat are used to remove a paramethoxybenzyl thioether, a tetrahydropyranyl thioether or a S-triphenylmethyl thioether group) followed by S reaction a suitable nitrosylating agent such as thionyl chloride nitrite, thionvl dinitrite.

a lower alkyl nitrite such as tert-butyl nitrite, or nitrosium tetrafluoroborate in a suitable anhydrous solvent such as methyene chloride, THF, DMF, or acetonitrile with or without an amine base such as pyridine or triethylamine acid affords the compound of the formula IIB. Alternatively, reacting this intermediate with a stiochiometric \O quantity of sodium nitrite in aqueous acid affords the compound of the formula HB.

Scheme V on-s-(c R, 0 Nitroso compounds of formula (III) wherein p, Rb, R, and R are defined 4 OW-S--(C R 0 Nitroso compounds of formula (III) wherein p, Rb, R, R, and Rf are defined as above and an 0-nitrosylated ester is representative of the X group as defined above may be prepared according to Scheme VI. An acid of the formula 7 is converted into the ester of the formula 8 wherein p, Rb and R, are defined as above by reaction with an appropriate monoprotected diol. Preferred methods for the preparation of esters are initially forming the mixed anhydride via reaction of 7 with a chloroformate -21oooo i 1 n aprpit oortetddo.Peere ehd o tepeaaino esesaeintal°o°igoOmxdOnyrd varato o ihachooomt S such as isobutylchloroformate in the presence of a non nucleophilic base such as triethylamine in an anhydrous inert solvent such as dichloromethane, diethylether. or THF. The mixed anhydride is then reacted with the monoprotected alcohol preferably in the presence of a condensation catalyst such as 4-dimethylamine pyridine.

1 Alternatively, the acid 7 may be first converted to the acid chloride by treatment with oxalyl chloride in the presence of a catalytic amount of DMF. The acid chloride is then reacted with the monoprotected alcohol preferably in the presence of a condensation catalyst such as 4-dimethylamine pyridine and a tertiary amine base such as triethyl amine to afford the ester 8. Alternatively, the acid 7 and monoprotected "O diol may be coupled to afford 8 by treatment with a dehydration agent such as 1.3dicyclohexylcarbodiimide (DCC). Alternatively, compound 7 may be first converted into an alkali metal salt such as the sodium, potassium, or lithium salt, and reacted with an alkyl halide which also contains a protected hydroxyl group in an polar solvent such as DMF to afford 8. Preferred protecting groups for the alcohol moiety i\ are silyl ethers such as a trimethylsilyl or a tert-butyldimethylsilyl ether. Deprotection of.the hydroxyl moiety (fluoride ion is the preferred method for removing silyl ether protecting groups) followed by reaction with a suitable nitrosylating agent such as thionyl chloride nitrite, thionyl dinitrite, or nitrosium tetrafluoroborate in a suitable anhydrous solvent such as dichloromethane, THF, DMF, or acetonitrile with or Lr0 without an amine base such as pyridine or triethylamine affords the compound of the formula IIIA.

-22- *°ao Scheme VI R. 0 R. O Rb I II I II I R,--C-C-OH Rf--C-C-0-(Ctp-0- P H H

R

7 a R. 0 Rb p-0-NO I Z I Nitroso compounds of formula (III) wherein p, Rb, Re, Re, and R, are defined as above and a S-nitrosylated ester is representative of the X group as defined above may be prepared according to Scheme VII. An acid of the formula 7 is converted into the ester of the formula 9 by reaction with an appropriate protected thiol containing alcohol. Preferred methods for the preparation of esters are initially forming the mixed anhydride via reaction of 7 with a chloroformate such as isobutylchloroformate in the presence of a non nucleophilic base such as triethylamine in an anhydrous inert solvent such as diethylether or THF. The mixed anhydride is then reacted with the thiol containing alcohol preferably in the presence of a condensation catalyst such as 4-dimethylamine pyridine. Alternatively, the acid 7 may be first converted to the acid chloride by treatment with oxalyl chloride in the :presence of a catalytic amount of DMF. The acid chloride is then reacted with the monoprotected thiol preferably in the presence of a condensation catalyst such as 4- 6 dimethylamine pyridine and a tertiary amine base such as triethyl amine to afford the ester 9. Alternatively, the acid and thiol containing alcohol may be coupled to afford 9 by treatment with a dehydration agent such as DCC. Alternatively, compound 7 may be first converted into an alkali metal salt such as the sodium, potassium, or -23lithium salt, and reacted with an alkyl halide which also contains a protected thiol group in an polar solvent such as DMF to afford 9. Preferred protecting groups for the thipl moiety are as a thioester such as a thioacetate or thiobenzoate, as a disulfide.

as a thiocarbamate such as N-methoxymethyl thiocarbamate, or as a thioether such as f a paramethoxybenzyl thioether, a tetrahydropyranyl thioether, or a S-triphenylmethyl thioether. Deprotection of the thiol moiety (zinc in dilute aqueous acid, triphenylphosphine in water and sodium borohydride are preferred methods for reducing disulfide groups while aqueous base is typically utilized to hydrolyze thiolesters and N-methoxymethyl thiocarbamates and mercuric trifluoroacetate, silver tO nitrate, or strong acids such as trifluoroacetic or hydrochloric acid and heat-are used to remove a paramethoxybenzyl thioether, a tetrahydropyranyl thioether or a Striphenylmethyl thioether group) followed by reaction with a suitable nitrosylating agent such as thionyl chloride nitrite, thionyl dinitrite, a lower alkyl nitrite such as tert-butyl nitrite, or nitrosium tetrafluoroborate in a suitable anhydrous solvent such as )i methylene chloride, THF, DMF, or acetonitrile with or without an amine base such as pyridine or triethylamine affords the compound of the formula IIIB. Alternatively, this intermediate may be reacted with a stoichiometric quantity of sodium nitrite in aqueous acid affords the compound of the formula IIIB.

4° 4 4 -24- Scheme VII I i I II R--C-C-OH p-S- H 7 H 9 R, R, O Rb p-s-NO H R.

zII Nitroso compounds of formula (III) wherein W, R, and R, are defined as above and a 6-W-substituted sydnonimine wherein W is as defined above is representative of the X group as defined above may be prepared according to Scheme VIII. An acid of the formula 7 is converted into the carboximide of the formula IIIC by. reaction with a 6-W-substituted sydnonimine. Preferred methods for the preparation of carboximides are initially forming the mixed anhydride via reaction of 7 with a chloroformate such as isobutylchloroformate in the presence of a non nucleophilic base such as triethylamine in an anhydrous inert solvent such as VO diethylether or THF. The mixed anhydride is then reacted with the 6-W-substituted sydnonimine to afford IIIC. Alternatively, the acid 7 may be coupled to the 6-Wsubstituted sydnonimine to afford IIIC by treatment with a dehydration agent such as DCC. Alternatively, the acid 7 may be converted into an active ester by reaction with a suitably substituted phenol utilizing any of the conditions for ester formation YS described for Scheme VI, followed by reaction with a 6-W-substituted sydnonimine.

Preferred 6-W-substituted sydnonimines are 1,2,6,4-oxatriazolium, 6-amino-6morpholine and 1,2,6,4-oxatriazolium, 6-amino-6-(6-chloro-2-methyl -benzene) and preferred active esters are para-nitrophenyl, 2,4,5-trichlorophenyl, and pentafluorophenyl.

2i Scheme VIII

O-N

R, Re 0 I RII I I R-C C -OH -N W H H 7 IIIC Nitroso compounds of formula (III) wherein p, Rb, R, R, and Rf are defined as above and a S-nitrosated ester is representative of the X group as defined above may be prepared according to Scheme IX. The protected thiol containing ester of the formula 9 is deprotected. Zinc in dilute aqueous acid, triphenylphosphine in water and sodium borohydride are preferred methods for reducing disulfide groups while aqueous base is typically utilized to hydrolyze thiolesters and N-methoxymethyl thiocarbamates and mercuric trifluoroacetate, silver nitrate, or strong acids such as trifluoroacetic or hydrochloric acid and heat are used to remove a paramethoxybenzyl o thioether, a tetrahydropyranyl thioether or a S-triphenylmethyl thioether group.

Reaction of the thiol group(s) excess dinitrogen tetroxide in a solvent such as methylene chloride, THF, DMF, or acetonitrile affords the compound of the formula

IIID.

-26o Scheme IX Re 0 Rb Re 0 Rb I II I I II Rt-C-C-O-(C)p-S-P 2 R-C-C-O-(C p-S-N0 2 H 9 Rc H Re

IIID

Nitroso compounds of formula (IV) wherein p, Rb, Re, and Rg are defined as above and an O-nitrosylated ester is representative of the X group as defined above may be prepared according to Scheme IX. An acid of the formula 10 is converted into the ester of the formula 11 wherein p, Rb, and R, are defined as above, by reaction with an appropriate monoprotected diol. Preferred methods for the preparation of esters are initially forming the mixed anhydride via reaction of 10 with a chloroformate such as isobutylchloroformate in the presence of a non nucleophilic base such as triethylamine in an anhydrous inert solvent such as dichloromethane, \0diethylether or THF. The mixed anhydride is then reacted with the monoprotected alcohol preferably in the presence of a condensation catalyst such as 4 -dimethylamine pyridine. Alternatively, the acid 10 may be first converted to the acid chloride by treatment with oxalyl chloride in the presence of a catalytic amount of DMF. The *acid chloride is then reacted with the monoprotected alcohol preferably in the presence of a condensation catalyst such as 4-dimethylamine pyridine and a tertiary amine base such as triethylamine to afford the ester 11. Alternatively, the acid and monoprotected diol may be coupled to afford 11 by treatment with a dehydration agent such as DCC. Alternatively, compound 10 may be first converted into an alkali metal salt such as the sodium, potassium, or lithium salt, which is then reacted with 2o an alkyl halide which also contains a protected hydroxyl group in an polar solvent -27such as DMF to afford 11. Preferred protecting groups for the alcohol moiety are silyl ethers such as a trimethylsilyl or a tert-butyldimethylsilyl ether. Defprotection of the hydroxyl moiety (fluoride ion is the preferred method for removing silyl ether protecting groups) followed by reaction with a suitable nitrosylating agent such as S thionyl chloride nitrite, thionyl dinitrite, or nitrosium tetrafluoroborate in a suitable anhydrous solvent such as methylene chloride, THF, DMF, or acetonitrile with or without an amine base such as pyridine or triethyl amine affords the compound of the formula IVA.

Scheme X 0 0 Rb 1 Re 0 Rb R p---NO v

R

0 o Nitroso compounds of formula (IV) wherein R, is defined as above and a Snitrosylated ester is representative of the X group as defined above may be prepared according to Scheme X. An acid of the formula 10 is converted into the ester of the formula 12 by reaction with an appropriate protected thiol containing alcohol.

Preferred methods for the preparation of esters are initially forming the mixed anhydride via reaction of 10 with a chloroformate such as isobutylchloroformate in *:.the presence of a non nucleophilic base such as triethylamine in an anhydrous inert solvent such as diethylether or THF. The mixed anhydride is then reacted with the •-28protected thiol containing alcohol preferably in the presence of a condensation catalyst such as 4-dimethylaminopyridine. Alternatively, the acid 10 may be first converted to the acid chloride by treatment with oxalyl chloride in the presence of a catalytic amount of DMF. The acid chloride is then reacted with the protected thiol containing alcohol preferably in the presence of a condensation catalyst such as 4dimethylamineo pyridine and a tertiary amine base such as triethyl amine to afford the ester 12. Alternatively, the acid and protected thiol containing alcohol may be coupled to afford 12 by treatment with a dehydration agent such as DCC.

Alternatively, compound 10 may be first converted into an alkali metal salt such as VO the sodium, potassium, or lithium salt, which is then reacted with an alkyl halide which also contains a protected thiol group in an polar solvent such as DMF to afford 12 Preferred protecting groups for the thiol moiety are as a thioester such as a thioacetate or thiobenzoate, as a disulfide, as a thiocarbamate such as Nmethoxymethyl thiocarbamate, or as a thioether such as a paramethoxybenzyl 4 thioether, a tetrahydropyranyl thioether, or a S-triphenylmethyl thioether.

Deprotection of the thiol moiety (zinc in dilute aqueous acid, triphenylphosphine in water and sodium borohydride are preferred methods for reducing disulfide groups while aqueous base is typically utilized to hydrolyze thiolesters and N-methoxymethyl thiocarbamates and mercuric trifluoroacetate, silver nitrate, or strong acids such as ?Y trifluoroacetic or hydrochloric acid and heat are used to remove a paramethoxybenzyl thioether, a tetrahydropyranyl thioether or a S-triphenylmethyl thioether group) followed by reaction with a suitable nitrosylating agent such as thionyl chloride nitrite, thionyl dinitrite, a lower alkyl nitrite such as tert-butyl nitrite, or nitrosium tetrafluoroborate in a suitable anhydrous solvent such as methylene chloride, THF, 'DMF, or acetonitrile affords the compound of the formula IVB. Alternatively, this intermediate may be reacted with a stoichiometric quantity of sodium nitrite in aqueous acid affords the compound of the formula IVB S* -29- Scheme XI 0 0 R-C-OH Rg,-C-O-(C P 12 Rc 0 Rb

R,-C-O-(C--S-NO

in Nitroso compounds of formula (IV) wherein Rg is defined as above and a 6substituted sydnonimine is representative of the X group as defined above may be prepared according to Scheme XI. An acid of the formula 10 is converted into the carboximide of the formula IVC by reaction with a 6-W-substituted sydnonimine wherein W is as defined above. Preferred methods for the preparation of carboximides are initially forming the mixed anhydride via reaction of 10 with a chloroformate such as isobutylchloroformate in the presence of a non nucleophilic base such as triethylamine in an anhydrous inert solvent such as diethylether or THF.

10 The mixed anhydride is then reacted with the 6-W-substituted sydnonimine to afford IVC. Alternatively, the acid 10 may be coupled to the 6-W-substituted sydnonimine afford IVC by treatment with a dehydration agent such as DCC. Alternatively, the acid 10 may be converted into an active ester by reaction with a suitably substituted phenol utilizing any of the conditions for ester formation described above, followed f by reaction with a 6-W-substituted sydnonimine. Preferred 6-W-substituted sydnonimines are 1,2,6,4-oxatriazolium, 6-amino-6-morpholine and 1,2,6,4oxatriazolium, 6-amino-6-(6-chloro-2-methyl -benzene) and preferred active esters are para-nitrophenyl, 2,4,5-trichlorophenyl. and pentafluorophenyl. Scheme XII

O-N

o o 4 0 0 0-N II II N- Rg-C-OH R,-C-N W

VC

Nitroso compounds of formula (IV) wherein p, Rb, and are defined as S above and a S-nitrosated ester is representative of the X group as defined above may be prepared according to Scheme XIII. The protected thiol containing ester of the formula 12 is deprotected. Zinc in dilute aqueous acid, triphenylphosphine in water and sodium borohydride are preferred methods for reducing disulfide groups while aqueous base is typically utilized to hydrolyze thiolesters and N-methoxymethyl 1O thiocarbamates and mercuric trifluoroacetate, silver nitrate, or strong acids such as trifluoroacetic or hydrochloric acid and heat are used to remove a paramethoxybenzyl thioether, a tetrahydropyranyl thioether or a S-triphenylmethyl thioether group.

Reaction of the thiol group(s) with excess dinitrogen tetroxide in a solvent such as methylene chloride, THF, DMF, or acetonitrile affords the compound of the formula 31- Scheme XIII 0 0 R 0 Rb 0

R

b p-S-P 2 Rg-C-O-(C p-S-NO, 12 Re Rc The' compounds that donate, transfer or release nitric oxide can be any of those known to the art, including those mentioned and/or exemplified below.

Nitrogen monoxide can exist in three forms: NO- (nitroxyl), NO* (nitric C oxide) and NO' (nitrosonium). NO* is a highly reactive short-lived species that is potentially toxic to cells. This is critical, because the pharmacological efficacy of NO depends upon the form in which it is delivered. In contrast to nitric oxide radical, nitrosonium and nitroxyl do not react with 02 or 0;2 species, and are also resistant to decomposition in the presence of redox metals. Consequently, administration of NO ko equivalents does not result in the generation of toxic by-products or the elimination of the active NO moiety.

Compounds contemplated for use in the invention are nitric oxide and compounds that release nitric oxide or otherwise directly or indirectly deliver or transfer nitric oxide to a site of its activity, such as on a cell membrane, in vivo. As I used here, the term "nitric oxide" encompasses uncharged nitric oxide and charged nitric oxide species, particularly including nitrosonium ion and nitroxyl S.ion The reactive form of nitric oxide can be provided by gaseous nitric oxide.

The nitric oxide releasing, delivering or transferring compounds, having the structure F-NO, wherein F is a nitric oxide releasing, delivering or transferring moiety and v is O an integer of 1 or 2, include any and all such compounds which provide nitric oxide a e** Sto its intended site of action in a form active for their intended purpose. As used here, the term "NO adducts" encompasses any of such nitric oxide releasing.

delivering or transferring compounds, including, for example, S-nitrosothiols. Snitroso amino acids, S-nitroso-polypeptides, organic nitrites and organic thionitrates.

S It. is contemplated that any or all of these "NO adducts" can be mono- or polynittosylated and/or nitrosated at a variety of naturally susceptible or artificially provided binding sites for nitric oxide.

One group of such NO adducts is the S-nitrosothiols, which are compounds that include at least one -S-NO group. Such compounds include S-nitroso- |0 polypeptides (the term "polypeptide" includes proteins and also polyamino acids that do not possess an ascertained biological function, and derivatives thereof); Snitrosylated amino acids (including natural and synthetic amino acids and their stereoisomers and racemic mixtures and derivatives thereof); S-nitrosylated sugars, Snitrosylated-modified and unmodified oligonucleotides (preferably of at least 5, and 1 more particularly 5-200, nucleotides); and an S-nitrosylated hydrocarbon where the hydrocarbon can be a branched or unbranched, and saturated or unsaturated aliphatic hydrocarbon, or an aromatic hydrocarbon; S-nitroso hydrocarbons having one or more substituent groups in addition to the S-nitroso group; and heterocyclic compounds. Snitrosothiols and the methods for preparing them are described in U.S. Patent Application No. 07/943,834, filed September 14, 1992, Oae ei al., Org. Prep. Proc.

Int., 15(3):165-198 (1983); Loscalzo et al., J. Pharmacol. Exp. Ther., 249(3):726-729 (1989) and Kowaluk et al., J. Pharmacol. Exp. Ther., 256:1256-1264 (1990), all of which are incorporated in their entirety by reference.

One particularly preferred embodiment of this aspect relates to S-nitroso amino 9 acids where the nitroso group is linked to a sulfur group of a sulfur-containing amino acid or derivative thereof. For example, such compounds include the following: Snitroso-N-acetylcysteine, S-nitroso-N-acetylpenicillamine, S-nitroso-homocysteine, Snitroso-cysteine and S-nitroso-glutathione.

-33- *o **l Suitable S-nitrosylated proteins include thiol-containing proteins (where the NO group is attached to one or more sulfur group on an amino acid oramino acid derivative thereof) from various functional classes including enzymes, such as tissuetype plasminogen activator(TPA) and cathepsin B; transport proteins, such as lipoproteins, heme proteins such as hemoglobin and serum albumin; and biologically protective proteins, such as the immunoglobulins and the cytokines. Such nitrosylated proteins are described in PCT Publ. Applic. No. WO 93/09806, published May 27, 1993. Examples include polynitrosylated albumin where multiple thiol or other nucleophilic centers in the protein are modified.

1o Further examples of suitable S-nitrosothiols include those having the structures: CH3 [C(Rb)(R)]xSNO wherein x equals 2 to 20 and Rb and R, are as defined above; (ii) HS[C(Rb)(R)]xSNO lS wherein x equals 2 to 20; and (iii) ONS[C(IR)(R)]x

V

wherein x equals 2 to 20 and V is selected from the group consisting of fluoro, alkoxy, cyano, carboxamido, cycloalkyl, arylkoxy, alkylsulfinyl, arylthio, alkylamino, dialkylamino, hydroxy, carbamoyl, N-alkylcarbamoyl, N,N-dialkylcarbamoyl, amino, 23 hydroxyl, carboxyl, hydrogen, nitro and aryl; and x, Rb and R, are as defined above.

Nitrosothiols can be prepared by various methods of synthesis. In general, the thiol precursor is prepared first, then converted to the S-nitrosothiol derivative by nitrosation of the thiol group with NaNO, under acidic conditions (pH is about which yields the S-nitroso derivative. Acids which may be used for this purpose include aqueous sulfuric, acetic and hydrochloric acids. Alternatively, they may be :-34- 0* e S nitrosated by reaction with an organic nitrite such as tert-butyl nitrite, or an nitrosonium salt such as nitrosonium tetraflurorborate in an inert solvent.

Another group of such NO adducts are those wherein the compounds donate, transfer or release nitric oxide and are selected from the group consisting of compounds that include at least one ON-O-, ON-N- or ON-C- group. The compound that includes at least one ON-O-, ON-N- or ON-C- group is preferably selected from the group consisting of ON-O-,ON-N- or ON-C-polypeptides (the term "polypeptide" includes proteins and also polyamino acids that do not possess an ascertained biological function, and derivatives thereof); ON-O-, ON-N- or ON-C- %O amino acids (including natural and synthetic amino acids and their stereoisomers and racemic mixtures); ON-O-, ON-N- or ON-C-sugars; ON-O-, ON-N- or ON-Cmodified and unmodified oligonucleotides (preferably of at least 5, and more particularly 5-200, nucleotides), ON-O-, ON-N- or ON-C-hydrocarbons which can be branched or unbranched, saturated or unsaturated aliphatic hydrocarbons or aromatic S' hydrocarbons; ON-O-, ON-N- or ON-C- hydrocarbons having one or more substituent groups in addition to the ON-O-, ON-N- or ON-C- group; and ON-O-, ON-N- or ON-C-heterocyclic compounds.

Another group of such adducts are 2-hydroxy-2-nitrosohydrazines which donate, transfer or release nitric oxide and have a R 1 0R 20 0 -N(OM)-NO group ao wherein Ro and R 200 include polypeptides, amino acids, sugars, modified and unmodified oligonucleotides, hydrocarbons where the hydrocarbon can be a branched :or unbranched, and saturated or unsaturated aliphatic hydrocarbon or an aromatic hydrocarbon, hydrocarbons having one or more substituent groups and heterocyclic compounds. M' is a metal cation, such as, for example, a Group I metal cation.

Another group of such adducts are thionitrates which donate, transfer or o* C release nitric oxide and have the structure R,0o-(S)-NO, wherein Ro 00 is as described S-35-

C

above for.the N-oxo-N-nitrosoamines. Particularly preferred are those compounds where Ro 00 is a polypeptide or hydrocarbon.

Agents which stimulate endogenous NO synthesis such as L-arginine. the substrate for nitric oxide synthase, are also suitable for use in accordance with the invention.

When administered in vivo, the compositions may be administered in combination with pharmaceutical carriers and in dosages described herein.

The compositions of the present invention may be administered orally, parenterally, by inhalation spray, rectally, or topically in dosage unit formulations 0 i containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants. and vehicles as desired. 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.

Solid dosage forms for oral administration may include capsules, tablets, pills, powders, granules and gels. In such solid dosage forms, the active compounds 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, 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.

Dosage forms for topical administration of the composition can include creams, sprays, lotions, gels, ointments and the like. In such dosage forms the compositions of the invention can be mixed to form white, smooth, homogeneous, Sopaque lotions with, for example, benzyl alcohol 1% (wt/wt) as preservative, -36emulsifying wax, glycerin, isopropyl palmitate. lactic acid, purified water. sorbitol solution and polyethylene glycol 400. They can be mixed to form a white, smooth.

homogeneous, opaque creams with, for example, benzyl alcohol 2% (wt/wt) as preservative, emulsifying wax, glycerin, isopropyl palmitate, lactic acid, purified water, and sorbitol solution. They can be mixed to form ointments with, for example, benzyl alcohol 2% (wt/wt) as preservative, white petrolatum, emulsifying wax, and tenox II (butylated hydroxyanisole, propyl gallate, citric acid, propylene glycol). Woven pads or rolls of bandaging material, e.g. gauge, can be impregnated with the compositions in solution, lotion, cream, ointment or other such form can also O be used for topical application. The compositions can also be applied topically using a transdermal system, such as one of an acrylic-based polymer, adhesive with a resinous crosslinking agent impregnated with the composition and laminated to an impermeable backing.

Suppositories for rectal administration of the drug composition, such as for treating pediatric fever etc., can be prepared by mixing the drug with a suitable nonirritating excipient such as cocoa butter and polyethylene glycols which are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the 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 nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1, 3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed an a solvent or suspending medium.

-37- While the compositions of the invention can be administered as a mixture of an NSAID and a nitric oxide donor, they can also be used in combination -with one or more additional compounds which are known to be effective against the specific disease state that one is targeting for treatment.

The compositions of this invention can further include conventional excipients, i. pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral application which do not deleteriously react with the active compounds.

Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, '0 magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc. The pharmaceutical preparations can be sterilized and if desired, mixed with auxiliary agents, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds. For parenteral application, particularly suitable vehicles consist of solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants. Aqueous suspensions may contain substances which increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbitol JO and/or dextran. Optionally, the suspension may also contain stabilizers.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.

The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.

-38- Various delivery systems are known and can be used to administer a therapeutic compound or composition of the invention, encapsulation in liposomes, microparticles, microcapsules and the like.

The therapeutics of the invention can be formulated as neutral or salt forms.

s Pharmaceutically acceptable salts include, but are not limited to, those formed with free amino groups such as those derived from hydrochloric, phosphoric, sulfuric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The term "therapeutically effective amount," for the purposes of the invention, refers to the amount of the nitric oxide adduct which is effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges for effective amounts of each nitric oxide adduct is within the skill of the art. Generally, the dosage required to provide an effective amount of the composition, and which can be adjusted by one of ordinary skill in the art will vary, depending on the age, health, physical condition, sex, weight, extent of disease of the recipient, frequency of treatment and the nature and scope of the disorder.

The amount of a given NSAID which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. Reference is again made to Goodman and Gilman, supra; The Physician's Desk Reference, Medical Economics Company, Inc., Oradell, 1995; and to Drug Facts and Comparisons, Facts and Comparisons, Inc., St. Louis, MO, 1993. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.

-39- The amount of nitric oxide donor in a pharmaceutical composition may be in amounts of 0.1-10 times the molar equivalent of the NSAID. The usual daily doses of NSAIDs are 3-40 mg/kg body weight and the doses of nitric oxide donors in the pharmaceutical composition may be in amounts of 1-500 mg/kg body weight daily and more usually about 1-50 mg/kg. Effective doses may be extrapolated from doseresponse curves derived from in vitro or animal model test systems and are in the same ranges or less than as described for the commercially available compounds in the Physician's Desk Reference, supra.

The invention also provides a pharmaceutical pack or kit comprising -one or o more-containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

The following non-limitative examples further describe and enable one of ordinary skill in the art to make and use the invention. Flash chromatography was performed on 40 micron silica gel (Baker).

Example 1 "Cholest-5-en-38-O-nitroso alcohol SCholesterol 0.242 g, 0.62 mmol) was dissolved in anhydrous methylene chloride (3 mL) and pyridine (0.103 g, 3.45 mmol) was added, followed by nitrosonium tetrafluoroborate (0.036 g, 0.31 mmol). After stirring for 1 hour at room temperature, an additional nitrosonium tetrafluoroborate (0.099 g, 0.85 mmol) was added. The reaction mixture was stirred at room temperature for 16 hours. The solvent was evaporated and the residue was purified by flash chromatography on silica gel, deactivated with *•o Striethylamine, eluted methylene chloride to give 0.165 g (64 yield) of the title compound as a white solid. 'H NMR (CDCl3, 300 MHz), 6 0.86 6 0.92 3 H).

1.05-1.75 21 1.80-2.01 6 2.25-2.47 2 5.23 1 5 44 (m, 1 H).

Example 2 N-(N-L-y-glutamvl- S-Nitroso-L-cvsteinvI)ylvcine N-(N-L-y-glutamyl-L-cysteinyl)glycine (100 g, 0.325 mol) was dissolved in deoxygenated water (200 ml) and 2N HCI (162 ml) at room temperature and then the reaction mixture was cooled to 0 OC. With rapid stirring, a solution of sodium nitrite 'j (24.4 g, 0.35 mol) in water (40 ml) was added and stirring with cooling of the reaction mixture was continued for approximately 1 hour after which time the pink precipitate which formed was collected by vacuum filtration. The filter cake was resuspended in chilled 40% acetone-water (600 ml) and collected by vacumm filtration. The filter cake was washed with acetone (2 X 200 ml) and ether (100 ml) and then dried under high vacuum at room temperature in the dark to afford the title compound as a pink powder.

'H NMR 6:1.98 2 2.32 (t,2 3.67 1 3.82 (s 2 3.86 (dd, 1 3.98 (dd, 1 4.53 1H).

Example 3 S-Nitroso-triphenvlmethanethiol 20 Triphenylmethyl mercaptan (0.050g, 0.18 mmol) was dissolved in anhydrous methylene chloride and cooled to 0°C. Tert-butyl nitrite (0.186 g, 1.80 mmol) was added and the resulting mixture was stirred at 0°C for 30 minutes. The reaction mixture was allowed to warm to room temperature and stirred at room temperature for 1 hour. The solvent and excess of tert-butyl nitrite were evaporated to give the title compound as a green solid (0.054 g, 98 'H NMR (CDCl1) 6: 7.13-7.18 4 7.25-7.39 (m, 11 H).

S-41- -41- Example 4 4-O-Nitroso-l-(3-benzovl-a-methvlbenzeneacetic acid) butvt ester 4a. 4-Hydroxv- -(3-benzovl-a-methylbenzeneacetic acid) butyl ester 3-Benzoyl-o-methylbenzeneacetic acid (4 g, 16 mmol) and 100 AL DMF were dissolved in benzene (25 mL). Oxalyl chloride (1.6 mL, 18 mmol) was added dropwise.

Stirring was continued for 2 hour before concentration to a syrup. Butanediol (9 mL.

100 mmol) and pyridine (1.67 mL, 21 mmol) were dissolved in methylene chloride (100 mL) and dioxane (15 mL) and cooled to 0°C. A solution of the acid chloride was added in methylene chloride (20 mL). The reaction mixture was stirred cold for 20 minutes 3 then warmed to room temperature with stirring for 2 hour. The solution was washed 1 X 30 H 2 0, 1 N HC1, satd NaHC0 3 and brine; dried over Na:SO,; and the volatiles were evaporated. The residue was filtered through a pad of silica gel eluting with 2:1 Hex:EtOAc to yield 4.8 g (91 of hydroxy ester. 'H NMR (CDCI 3 d 7.41-7.81 (mult, 9 4.08-4.15 (mult, 2 3.79 J 7.2 Hz, 1 3.59 J 6.3 Hz. 2 1.53-1.69 (mult, 4 1.53 J 7.2 Hz, 3H).

4b. 4-O-Nitroso-l-(3-benzovl-a-methvlbenzeneacetic acid) butvl ester The product of Example 4a (1 g, 3.6 mmol) and pyridine (1.4 mL, 18 mmol) were dissolved in dichloromethane (15 mL) and cooled to -78 0 C. Nitrosonium tetrafluoroborate (840 mg, 7.2 mmol) was added and the solution was kept cold for

O

2 minutes. The reaction was warmed to room temperature with continued stirring for 1 hour. The mixture was diluted with dichloromethane and washed with IN HCI, then brine. The solution was dried over sodium sulfate and evaporated. Chromatography on silica gel eluting with 9:1 Hexane:EtOAc gave 840 mg of the title compound. 'H NMR (CDC13): 5 7.41-7.80 9 4.65 1 4.11 J 6.0 Hz, 2 3.79 25(q, J 7.2 Hz, 1 1.65-1.72 4 1.53 J 7.2 Hz, 3H). Anal Calcd for

C

20

H

2 ,NOs: C, 67.59; H, 5.96; N, 3.94. Found: C, 66.72; H, 5.95; N, 2.93 -42- Example 4-O-Nitroso-4-methyl- -(3-benzovl-oa-methvlbenzeneacetic acid) pentvl ester 4-Hvdroxv-4-methvl-l-(3-benzovl-a-methvlbenzeneacetic acid) pentvl ester 3-Benzoyl-a-methylbenzeneacetic acid (1.99 g, 7.7 mmol) in methylene chloride (20 mL) under nitrogen and cooled over ice was treated successively with oxalyl chloride (1.36 mL, 15.7 mmol) and dimethylformamide (5 drops). A vigorous gas evolution was noted and the reaction mixture was stirred with slow warming and then overnight at ambient temperature. The volatile materials were removed in vacuo and the residue dissolved in methylene chloride (10 mL) and added dropwise to a precooled mixture of 1 0 2-methyl-2,5-pentanediol (3.7 g, 31 mmol) and pyridine (0.69 mL, 8.6 mmol) also in methylene chloride (10 mL) under a nitrogen atmosphere. The reaction mixture was stirred under nitrogen with slow warming and then overnight at ambient temperature.

The solution was washed successively with 2N hydrochloric acid and 2N sodium hydroxide, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residual oil was subjected to column chromatography using ethyl acetate/hexane The product was isolated as an oil in 76% yield (2.1 'H NMR (CDCl 3 8: 7.77-7.81 3 7.64-7.43 6 4.18-4.03 2 3.80 J=7.2 Hz, 1 1.62-1.71 2 1.54 J=7.2 Hz, 3 1.42-1.35 2 1.16 6H). Anal calcd for C22H 26 0 4 C, 74.55; H, 7.39. Found: C, 74.26; H, 7.43.

5b. 4-O-Nitroso-4-methvl-1-(3-benzovl-ao-methvlbenzeneacetic acid) pentl ester A solution of the product of example 5a (0.4 g, 1.13 mmol) and pyridine (456 mL, 5.6 mmol) in methylene chloride (4 mL) was cooled to -78 0 C and nitrosonium tetrafluoroborate (262 mg,2.26 mmol) added. The reaction mixture was stirred at -78°C for 3 hours, washed with water and dried over sodium sulfate. After filtration and evaporation of the solvent the residual oil was subjected to column chromatography using ethyl acetate/hexane/triethylamine (18:80:2). The title compound was isolated as an oil -43- *o in 58% yield (0.25 'H NMR (CDCl 3 6: 7.41-7.80 9 4.02-4.17 2 H).

3.79 J=7.2 Hz, 1 1.73-1.79 2 1.62-1.69 2 1.52-1.55 9H).

Example 6 3-S-Nitroso-3-methvl-l-( 3 -benzovl--methylbenzeneacetic acid) butvl ester S6a. 3-Meicapto-3-methvl-l-( 3 -benzovl-x-methvlbenzeneacetic acid) butvl ester To 3 -Benzoyl-a-methylbenzeneacetic acid (529 mg, 2 mmol) in benzene (5 mL) containing 5 ml of DMF was added oxalyl chloride (200 ml 2.2 mmol) dropwise. The reaction mixture was stirred 1.5 hour and then concentrated in vacuo to a syrup. The crude acid chloride was dissolved in dichloromethane (10 mL) and 3-mercapto-3-methyl to butanol (Sweetman et al. J. Med Chem., 14:868 (1971) (350 mg, 2.2 mmol) was added followed by pyridine (180 ml, 2.2 mmol). The reaction was stirred at room temperature for 1 hour and then it was diluted with dichloromethane and wash with IN HCI, followed by saturated sodium bicarbonate, and then brine. The organic phase was dried over sodium sulfate, concentrated in vacuo, and the residue was chromatographed on I silica gel eluting eith 9:1 hexane:ethyl acetate to afford 640 mg (90 of the product.

'H NMR (CDC1 3 6: 7.41-7.81 9 4.28 J 7.1 Hz, 2 3.78 J 7.2 Hz, 1 1.88 J 7.0 Hz, 2 1.69 1 1.54 J 7.3 Hz, 3 1.35 3 1.34 3H).

6b. 3-S-Nitroso-3-methvl-l-(3-benzovl-a-methvlbenzeneacetic acid) butyl ester o To a solution of the product of Example 6a (105 mg, 0.3 mmol) in dichloromethane mL) was added tert-butyl nitrite (70 mg, 0.6 mmol) in a dropwise fashion. The mixture was stirred at room temperature for 30 minutes. The solvent and excess reagent were evaporated to give 113 mg (quantitative) of the title compound. 'H NMR (CDCl,) 7.44-7.81 9 4.29 J 6.9 Hz, 2 3.77 j 7.2 Hz, 1 2.51 j 2 6.9 Hz, 2 1.841 3 1.836 3 1.53 J 7.2 3H).

-44- Example 7 4-0-Nitroso-l-((S)-6-methoxv-oY-methvl-2-naphthaleneacetic ai bdbutvl ester 7a. (S)-6-methoxv-cx-methyl-2-naphthaleneacetic acetyl chloride Under a nitrogen atmosphere, oxalyl chloride (4.13 g, 30 mmol) was combined with methylene chloride (30 mL) and the resulting mixture was cooled to 0°C.

Dimethylformamide (10 drops) was added and after 5 minutes of stirring, a suspension of (S)-6-methoxy-a-methyl-2-naphthaleneacetic acid (3.00 g, 13 mmol) in methylene chloride (30 mL) was added dropwise over a 30 minute period. The reaction mixture was allowed to warm to room temperature and stirred overnight. The solvent was evaporated tO in vacuo to give the product in a quantitative yield. 'H NMR (CDCl 3 8: 1.5 3 H), 3:91 1 4.21 1 7.09-7.14 1 7.15 1 7.42 (dd, 1 7.68 (s, 2 7.71 1H).

7b. 4-Hvdroxy-1-((S)-6-methoxv-a-methyl-2-naphthaleneacetic acid) butyl ester Under a nitrogen atmosphere, 1,4-butanediol (5.30 mL, 60 mmol) and pyridine (0.95g, 12 mmol) were combined in methylene chloride (20 mL). The resulting solution was stirred for 5 minutes and then cooled to 0°C. A solution of the product of Example 7a (3.0 g, 12 mmol) in methylene chloride (15 ml) was added dropwise over 30 minute period. After stirring for 20 hours at room temperature, the reaction mixture was diluted with ethyl acetate and washed with IN hydrochloric acid. The organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography on silica gel using hexane/ethyl acetate (1:1 to 1:3) to afford 3.09 g (79% yield) of the product as a colorless oil. 'H NMR (CDCl 3 6: 1.47-1.68 4H, overlapping with a doublet at 1.57, 3 3.55 2 3.84 1 3.91 3 H), :4.11 2 7.11 2 7.15 1 7.42 (dd, 1 7.67 1 7.70 (d, 2 H).

7c. 4-O-Nitroso- 6 -methoxv-a-methvl-2-naphthaleneacetic acid) butvl ester The product of Example 7b (0.209 g. 0.69 mmol) was dissolved in anhydrous methylene chloride (4 mL) and pyridine (0.273 g, 3.45 mmol) was added. The resulting solution was cooled to -78 0 C and nitrosonium tetrafluoroborate (0.161 g. 1.38 mmol) was added in one portion. The reaction mixture was stirred for 1 hour at -78 0 C. The solvent was evaporated in vacuo and the residue was purified by flash chromatography on silica gel, deactivated with triethylamine, eluted with ethyl acetate/hexane to give 0.180 g (79% yield) of the title compound as an oil. 'H NMR (CDCI 3 6: 1.58 3 1.64- 1.69 4 3.85 1 3.92 3 4.11 2 4.60 2 7.10-7.13 1 7.15 1 7.39 (dd, 1 7.66 1 7.70 2 H).

Example 8 4-O-Nitroso-l-(1-)4-chlorobenzovl)-5-methoxv-2-methvl-lH-indole-3-acetic acid) butvl ester 8a. 4-Hvdroxy-1-(1 -(4-chlorobenzovl)-5-methoxv-2-methvl- 1H-indole-3-acetic acid) I- butyl ester A stirred suspension of 1 (4-chlorobenzoyl)5-methoxy-2-methylindoyl)-3-acetic acid (3.7 g, 10.5 mmol) in methylene chloride (20 mL) under nitrogen and cooled over ice was treated successively with oxalyl chloride (1.8 mL, 20.6 mmol) and dimethylformamide (10 drops). A vigorous gas evolution was noted and the reaction 20 mixture was stirred with gradual warming to room temperature and then at ambient for a total of 5 hours. The volatile materials were evaporated and the residue dissolved in Sdichloromethane (10 mL) and added dropwise to a precooled mixture 1,4-butanediol (4.7 g, 51.7 mmol) and pyridine (0.92 mL, 11.4 mmol) also in methylene chloride (10 mL).

The reaction mixture was stirred with slow warming and then for 5 hours at ambient 2" temperature under a nitrogen atmosphere. The solution was washed with 2N hydrochloric acid, saturated sodium bicarbonate, dried over anhydrous sodium sulfate, -46filtered and concentrated in vacuo. The residual oil was subjected to column chromatography using ethyl acetate/hexane The product was isoldied. as an oil in yield (3.3 g which solidified on standing 'H NMR (CDCl 3 8: 7.67 J=8.4 Hz.

2, 7.47 J=8.5 Hz. 2 6.97 J=2.5 Hz, 1 6.87 J=9 Hz. 1 6.67 (dd, J=2.5 Hz, 9Hz, 1 4.13 J=6.4 Hz, 2 3.83 3 3.66 2 3.59 J=6.4 Hz, 2 2.38 3 1.5 1-1.75 (in, 4H). Anal caled for C, 3

H,

4 1 :C 64.26; H, 5.63; N, 3.26. Found: C, 64.08; H, 5.60; N, 3.18.

8b. 4-0-Nitroso-l1-(1 -(4-chlorobenzoyl)-5-methoxy-2-methvl- 1 H-indole-3-acetic acid) butyl ester ko A stirred solution of the product of Example 8a (1 g, 2.3 nimol). and pyridine (0.90 mnL, 11.6 mmol) in methylene chloride (15 ml) at -78 fiC under a nitrogen atmosphere was treated with nitrosoniumn tetrafluoroborate (0.54 g, 4.6 mnmol). The reaction mixture was stirred at -78 CiC for 3.5 hours, washed with water, dried with anhydrous sodium sulfate and the solvent removed in vacuo. The residual oil was subjected to column Ichromatography using ethyl acetate/hexane The product was isolated as a yellow oil in 69 yield (0.73 'H NMR (CDCl 3 8: 7.66 J=8.5 Hz, 2Hz), 7.47 Hz. 2 6.95 1=2.5 Hz, 1 6.85 1=5 Hz, I 6.66,(dd, J=2.5 Hz, 6.5 Hz, 1 4.66 (hr s, 2 4.16 1=6.6 Hz, 2 3.83 3 3.66 2 2.39 (s, 1.65-1.80 (in, 4H). Anal calcd for C,- 3

H

2 3 C1N 2 0 6 C, 60.2; H, 5.05; N. 6. 1.

dOFound: C. 59.93; H, 4.87; N, 5.85.

Example 9 3-0-Nitroso- 14-1 -(4-chlorobenzoyl)-5-methoxv-2-m ethl- I H-indole-3-acetic acid) butyl ester 9a. 3 -Hvdroxy- 1 -0 -(4-chlorobenzoyl)-5-methoxy-2-methy -I1 H-indole-3 -acetic acid) 4 butyl ester -47- A stirred suspension ofl -(4-chlorobenzoyl)-5-methoxy-2-methyl 1H-indole-3-acetic acid (5 g, 13.9 mmol) in methylene chloride (25 mL) under nitrogen and Eooled over ice was treated successively with oxalyl chloride (2.44 mL. 28 mmol) and dimethylformamide (10 drops). A vigorous gas evolution was noted and the reaction mixture was stirred with gradual warming for a total of 5 hours. The volatile materials were removed in vacuo and the residue dissolved in methylene chloride (15 mL) and added dropwise to a precooled mixture (+/-)-1,3-butanediol (8.83 g, 98 mmol) and pyridine (1.24 mL, 15.4 mmol) also in dichloromethane (10 mL). The reaction mixture was stirred with slow warming and then over the weekend at ambient temperature under o a nitrogen atmosphere. The solution was washed with 2N hydrochloric acid, saturated sodium bicarbonate, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residual oil was subjected to column chromatography using ethyl acetate/hexane The product was isolated as an oil which solidified on standing in 75% yield (4.5 'H NMR indicated that the desired product was contaminated with San isomer and so it was recrystalised three times from diethyl ether/hexanes to give the desired product as a solid in 15 yield (0.9 'H NMR (CDC1 3 6: 7.66 J=8.5 Hz, 2 7.43 J=8.5 Hz, 2 6.95 J=2.4 Hz, 1 6.86 J=9 Hz, 1 6.67 (dd, J=9 Hz, 2.5 Hz), 4.30-4.39 1 4.15-4.4 1 3.83 3 3.75-3.85 1 3.67 2 2.38 3 1.95 1 1.65-2.8 2 1.16 (d, SJ=6.3 Hz, 3H). Anal calcd for C, 3

H

2 4 CINO,: C, 64.26; H, 5.63; N, 3.26. Found: C, 64.29; H, 5.53; N, 3.18.

9b. 3-O-Nitroso-l-(1-(4-chlorobenzovl)-5-methoxv-2-methvl-lH-indole-3-acetic acid) butyl ester stirred solution of the product of Example 9a (0.15 g, 0.34 mmol). and pyridine (0.14 mL, 1.7 mmol) in dichloromethane (2 mL) at -78 0 C under a nitrogen atmosphere was treated with nitrosonium tetrafluoroborate (0.08 g, 0.7 mmol). The reaction mixture was stirred at -78 0 C for 3.5 hours, washed with water, dried with anhydrous sodium sulfate and the solvent removed in vacuo. The residual oil was subjected to column -48chromatography using ethyl acetate/hexane The title compound was isolated as a yellow oilin 79 yield (0.125 'H NMR (CDCl 3 6:.7.66 J38.5 Hz. 2 H).

7.47 J=8.5 Hz), 6.95 J=2.3 Hz, 1 6.86 J=9 Hz, 1 6.67 (dd, J=9 Hz.

Hz), 5.52 (sextet, J=6.5 Hz, 1 4.06-4.24 2 3.83 3 3.65 2 H).

2.38 3 2.05 J=4 Hz, 2 1.37 J=6.5 Hz).

Example 4-O-Nitroso-4 methvl-l-( -(4-chlorobenzovl)-5-methoxv-2-methvl-1H-indole-3acetic. acid) pentvl ester 4-Hydroxv-4 methyl- -(1-(4-chlorobenzovl)-5-methoxv-2-methvl- H-indole-3-acetic Io acid) pentvl ester A stirred suspension of 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3acetic acid (2.8 g, 7.7 mmol) in methylene chloride (25 mL) under nitrogen and cooled over, ice was treated successively with oxalyl chloride (1.36 mL, 15.7 mmol) and dimethylformamide (5 drops). A vigorous gas evolution was noted and the reaction t mixture was stirred over ice for 30 minutes and then at room temperature for 3 hours.

The volatile materials were removed in vacuo and the residue dissolved in methylene chloride (15 mL) and added dropwise to a precooled mixture of 2-methyl-2,5-pentanediol (3.7 g, 31 mmol) and pyridine (0.69 mL, 8.6 mmol) also in methylene chloride (10 mL).

The reaction mixture was stirred under nitrogen with slow warming and then overnight 2 at ambient temperature under a nitrogen atmosphere. The solution was washed with 2N hydrochloric acid, dried over anhydrous sodium sulfate, and filtered to give an oil which was concentrated in vacuo. The residual oil was subjected to column chromatography using ethyl acetate/hexane The product was isolated as an oil which solidified on standing in 100% yield (3.6 'H NMR (CDCl 3 6: 7.69 J=8.9 Hz, 2 7.47 (d, J=8.9 Hz, 2 6.98 J=2.5 Hz, 1 6.87 J=9 Hz, 1 6.67 (dd, J=9 Hz, Hz), 4.09-4.14 2 3.83 3 3.66 3 2.39 3 1.62-1.73 2 -49- 0 SH), 1.37-1.43 2 1.14 6H). Anal calcd for C,,H,gCINO,: C. 65.57: H. 6.16: N, 3.06. Found: C, 65.35; H, 6.25; N, 3.10.

4-O-Nitroso-4-methvl-1-(1-(4-chlorobenzovl)-5-methoxv-2-methvl- H-indole-3acetic acid) pentyl ester A solution the product of Example 10a (0.2 g, 0.44 mmol) and pyridine (176 mL.

2.2 mmol) in methylene chloride (2 mL) was cooled over dry ice and nitrosonium tetrafluoroborate (101 mg, 0.87 mmol) added. The reaction mixture was stirred at -78 iC for 3 hours, allowed to stand at the same temperature overnight, washed with water and dried over sodium sulfate. After filtration and evaporation of the solvent the residual oil was subjected to column chromatography (twice) using ethyl acetate/hexanes/triethylamine (25:73:2). The product was isolated as an oil in 42% yield (0.09 'H NMR (CDC13) 5: 7.66 J=7.5 Hz, 2 7.47 J=7.5 Hz, 2 6.96 J=2.5 Hz, 1 6.86 J=9 Hz, 1 6.66 (dd, J=7.5 Hz, 2.5 Hz), 4.11 J=6Hz, 2 3.83 3 3.66 2 2.39 3 1.75-1.81 2 1.64-1.72 (m, S2 1.51 6H).

Example 11 3-S-Nitroso-3-methvl- -(c-methvl-4-(2-methylpropvl)benzeneacetic acid) butvl ester 11a. 3-Mercapto-3-methvl-1 -(a-methvl-4-(2-methylpropvl)benzeneacetic acid) butvl ester A solution of a-methyl-4-(2-methylpropyl)benzeneacetic acid (1.52 g, 7.4 mmol) in methylene chloride (15 mL) cooled over ice and under nitrogen, was treated successively with oxalyl chloride (1.29 mL, 1.88 g, 14.8 mmol) and dimethylformamide drops). The resultant solution was stirred over ice for 30 minutes and then at Sambient temperature for 2 hours. The excess volatile materials were removed in vacuo Sand the residue, dissolved in methylene chloride (5 mL). added to a.precooled solution of pyridine (0.54 mL, 6.7 mmol) and 3-mercapto-3-methylbutanol (0.8 g: 6.7 mmol) in methylene chloride (15 mL). The reaction mixture was stirred over ice for 30 minutes and then at ambient temperature for 3 hours. The solution was then diluted with Sadditional methylene chloride and washed with 2N hydrochloric acid, saturated sodium bicarbonate and brine and the organic phase dried with sodium sulfate, filtered and the solvent removed in vacuo. The residual oil was subjected to column chromatography using ethyl acetate/hexane The product was isolated as an oil in 68 yield (1.4 'H NMR (CDCl 3 5: 7.18 J=7.5 Hz, 2 7.09 J=7.5 Hz, 2 4.25 ko Hz, 2 3.67 J=7 Hz, 1 2.44 J=7.8 Hz, 2 1.77-1.9 3 1.48 (d.

J=7 Hz, 3 1.32 6 0.89 J=6.6 Hz, 6H).

11 b. 3-S-Nitroso-3-methvl-1 -(a-methvl-4-(2-methvlpropyl)benzeneacetic acid) butvl ester A solution of the product of Example 1 la (0.4 g, 1.2 mmol) in methylene chloride I (8 mL) under nitrogen was treated with tert butyl nitrite (0.62 mL, 0.53 g, 5 mmol).

After stirring for 1 hour at ambient temperature the volatile materials were evaporated.

The residual green oil was subjected to column chromatography using ethyl acetate/hexanes The product was isolated as a green oil in 65 yield (0.25 g).

'H NMR (CDC13) 5: 7.0 J=7.5 Hz, 2 7.10 J=7.5 Hz, 2 4.27 J=6.9 Hz, 3.66 J=7.2 Hz, 1 2.49 J=6.6 Hz, 2 2.44 J=7.2 Hz, 2 1.8- 1.9 1 1.81 3 1.80 3 1.48 J=7.2 Hz, 3 0.89 J=6.6 Hz, 6H).

Example 12 4-O-Nitroso- -(c-methvl-4-(2-methvlDropvl)benzeneacetic acid) butvl ester 12a. 4-Hvroxy-l-(oa-methvl-4-(2-methylpropvl)benzeneacetic acid) butvl ester -51oooo* -5 1- *-o1 o Oo a-Methyl-4-(2-methylpropyl)benzeneacetic acid (4 g, 19 mmol) and: 10 AL DMF were dissolved in benzene (30 mL). Oxalyl chloride was added dropwise. Stirring was continued for 2 hour before concentration to a syrup. Butanediol (9 mL, 100 mmol) and pyridine (1.67 mL, 21 mmol) were dissolved in dichloromethane (100 mL) and dioxane (15 mL) and cooled to 0°C. A solution of the acid chloride was added in dichloromethane (20 mL). The reaction mixture was stirred cold for 20 minutes then warmed to room temperature with stirring for 2 hour. The solution was washed H,O, 1 N HCI, satd sodium bicarbonate and finally brine; dried over sodium sulfate: and evaporated. The residue was filtered through silica gel eluting with 2:1 hexane:EtOAc 0 to yield 4.8 g (91 ofthe product. 'H NMR (CDCl 3 6: 7.19 J 6.2 Hz. 2 H).

7.08 d, J 8.2 Hz, 2H),4.07-4.12 2 3.68 J 7.1 Hz, 1 3.58 J 6.3 Hz, 1 2.44 J 7.2 Hz, 2 1.84 (sept, J 6.8 Hz, 1 1.50-1.69 4 H), 1.48 d, J 7.2 Hz, 3 0.88 J 6.6 Hz, 6H). Anal Calcd for C, 7

H

26 0 3

C,

73.34; H, 9.41. Found: C, 73.17; H, 9.67 12b. 4-O-Nitroso-l-(a-methyl-4-(2-methvlpropvl)benzeneacetic acid) butyl ester The product of Example 12a (1 g, 3.6 mmol) and pyridine (1.4 mL, 18 mmol) were dissolved in dichloromethane (15 mL) and cooled to -78C. Nitrosonium tetrafluoroborate(840 mg, 7.2 mmol) was added and the solution was kept cold for minutes. The reaction was warmed to room temperature with continued stirring for 1 hour. The mixture was diluted with dichloromethane and washed successively with IN HC1, H,O, and brine. The solution was dried over sodium sulfate and evaporated.

Chromatography on silica gel eluting with 9:1 hexane:EtOAc gave 840 mg (76 of the title compound. 'H NMR (CDCl 3 6: 7.18 J 8.1 Hz, 2 7.08 d, J 8.1 Hz, 2 4.62 2 4.07-4.12 2 3.68 J 7.1 Hz, 1 2.44 J 7.2 Hz, 2 1.84 (sept, J 6.7 Hz, 1 1.64-1.68 4 1.48 J 7.2 Hz, 3 H), 0.88 J 6.6 Hz, 6H).

*-52- Example 13 4-O-Nitroso-l-(2-Fluoro-e-methvl-biphenvlacetic acid) butvl- ester 13a. 2-Fluoro-a-methvl-biphenvlacetic acid chloride Under a fitrogen atmosphere, oxalyl chloride (3.8 g, 30 mmol) was combined with methylene chloride (30 mL). The resulting mixture was cooled to 0°C and dimethylformamide (10 drops) was added. After 5 minutes of stirring a solution of 2fluoro-a-methyl-biphenylacetic acid (3.0 g, 12 mmol) in methylene chloride (30 mL) was added dropwise over a 30 minute period. The reaction mixture was allowed to warm to room temperature and stirred overnight. The solvent was evaporated to give the product "0 in'a quantitative yield as a yellow solid. 'H NMR (CDCl 3 5:1.58 3 4.20 1 H), 7.11 2 7.33-7.47 (m,E4 7.54 2 H).

13b. 4-Hvdroxy-l-(2-Fluoro-a-methvl-biphenvlacetic acid) butvl ester Under a nitrogen atmosphere, 1,4-butanediol (5.30 mL, 60 mmol) and pyridine (0.95Eg,E12 mmol) were combined in methylene chloride (20 mL). The resulting solution was stirred for 5 minutes and then cooled to 0*C. A solution of the product of Example 13a (3.0 g, 12 mmol) in methylene chloride (15 ml) was added dropwise over minute period. After stirring for 20 hours at room temperature, the reaction mixture was diluted with ethyl acetate and washed with IN hydrochloric acid. The organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was 3 0 purified by flash chromatography on silica-gel eluting with methylene chloride/hexane to give 1.66 g (44 of the product as a colorless oil. 'H NMR (CDCl 3 6:1.56 3 1.61-1.77 4 3.63 2 3.75 (q,l 4.14 2 7.14 2 H), 7.27-7.45 4 7.53 2 H).

*53- -53g* 13c. 4-O-Nitroso-l-(2-Fluoro-a-methyl-biphenvlacetic acid) burvl ester The product of Example 13b (0.190 g, 0.60 mmol) was dissolved in anhydrous methylene chloride (4 mL) and pyridine (0.237 g, 3.00 mmol) was added. The resulting solution was cooled to -78 iC and nitrosonium tetrafluoroborate (0.084 g. 0.72 mmol) was added. The reaction mixture was stirred for 1 hour at -78 iC and an additional nitrosonium tetrafluoroborate (0.047 g, 0.40 mmol) was added. After 30 minutes of stirring at -78 iC, the solvent was evaporated in vacuo and the residue was purified by flash chromatography on silica gel deactivated with triethylamine, eluted with methylene chloride/hexane to give 0.117 g (57 yield) of the title compound. 'H 1 o NMR (CDCl1) 6: 1.54 3 1.68-1.83 4 3.75 1 4.14 2 2_H),_7.14 2 7.34-7.48 4 7.54 2 H).

Example 14 4-O-Nitroso-l-(2-Fluoro-c-methyl-biphenvlacetic acid) thiobutvl ester 14a. 1-tert-Butvydimethvlsilvloxy-4-chloro-butanol 1I- 4-Chloro-l-butanol (5.43 g, 50 mmol) was dissolved in dimethylformamide and tert-butyldimethylsilylchloride (7.54 g, 50 mmol) was added, followed by imidazole (3.4 g, 50 mmol). After 24 hours of stirring at room temperature, the reaction mixture was diluted with hexane, washed with water and brine and dried over anhydrous sodium sulfate. The solvent was evaporated to give colorless liquid which was purified O by chromatography on silica gel eluting with hexane/ethyl acetate (30:1) to give the product (7.26 g, 56 'H NMR (CDC13) 6: 0.05 6 0.89 9 1.64-1.68 2 1.82-1.86 2 3.57 2 3.64 2 H).

*-54a -54- 14b. 4-tert-Butvldimethvlsilvloxv- 1-acetvl-thio-butvl ester Under a nitrogen atmosphere, potassium thioacetate (0.53 g, 4.7 mmol) was dissolved in dimethylformamide (12 mL) and cooled to 0 C. A solution of the product of Example 14a (1.01 g, 3.91 mmol) in dimethylformamide (14 mL) was added. After 24 hours of stirring at room temperature, the solvent was evaporated and the residue was partioned between hexane and water The organic layer was concentrated in vacuo to give the product (0.820 g, 71 as a yellow liquid. 'H NMR (CDCl 3 6: 0.04 6 0.88 9 1.57-1.64 4 2.32 3 2.89 2 3.61 (t, .2 H).

0 14c. 4-tert-Butyldimethvlsilvlox-1 -butane thiol The product of Example 14b (5.7 g, 19.2 mmol) was dissolved in methanol mL) and degassed with nitrogen gas for 30 minutes. Potassium carbonate (2.92 g, 21.1 mmol) was added in one portion at room temperature. After 1 hour of stirring at room temperature, the solvent was evaporated and the residue was partioned between hexane Sand water. The organic layer was dried over anhydrous sodium sulfate and concentrated in vacuo to give the product (3.2 g, 66 'H NMR (CDCI 3 6: 0.05 6 0.89 9 1 34 1 1.61-1.68 4 2.51-2.57 2 3.62 2 H).

14d. 4-tert-Butvldimethylsilvloxy-1 -(2-Fluoro-ca-methyl-biphenvlacetic acid) thiobutyl- ester The product of Example 14c (1.37g, 5.4 mmol) was combined with pyridine 142Eg, 1.8 mmol) in methylene chloride (5 mL) and the resulting solution was cooled to 0 iC. A solution of the product of Example 13a (0.500 g, 1.8 mmol) in methylene chloride (4 mL) was added dropwise. After 22 hours of stirring at room temperature, the reaction mixture was diluted with ethyl acetate and washed with 1N hydrochloric acid. The organic layer was dried over anhydrous sodium sulfate and concentrated in •:co o 0oo* vacuo to give the product (0.526 g, 59 'H NMR (CDC 3 6: 0.04 0.89 9EH),1.56 3 1.57-1.62 4 1.88-2.29 2 3.61-t, 2 7.15 (t,E2 7.37-7.44 4 7.54 2 H).

14e. 4-Hydroxy-l-(2-Fluoro-a-methyl-biDhenylacetic acid) thio-butyl ester The product of Example 14d (0.320 g, 0.64 mmol) was dissolved in the mixture of glacial acetic acid (0.5 mL), water (1 mL), and tetrahydrofuran (5 mL). The resulting solution was stirred for 24 hours at room temperature. The solvent was evaporated and the residue was partioned between methylene chloride and water. The organic layer was washed with saturated sodium bicarbonate solution and brine, and dried over anhydrous so sodium sulfate. The solvent was evaporated to give the product (0.235 g, 100 'H NMR (CDC 3 6: 1.57 3 1.58-1.69 4 2.87 -2.93 2 3.63 2 3.84-3.92 1 7.14 2 7.37-7.44 4 7.54 2 H).

14f. 4-O-Nitroso-l-(2-Fluoro-a-methyl-biphenvlacetic acid) thio-butvl ester The product of Example 14e (0.235 g, 0.61 mmol) was dissolved in anhydrous methylene chloride (3 mL) and pyridine (0.097 g, 1.23 mmol) was added. The resulting solution was cooled to -78*C and nitrosonium tetrafluoroborate (0.144 g, 1.23 mmol) was added in one portion. The reaction mixture was stirred for 1 hour at -78 0 C, the solvent was evaporated, and the residue was purified by chromatography on silica gel eluted with hexane/ethyl acetate (10:1) to give the title compound (0.110 g, 44 'H 20 NMR (CDCl 3 6: 157 3 1.58-1.80 4 3.85-3.93 1 4.69 2 7.14 2 7.37-7.44 4 7 55 2 H).

*56- -56- *o *o Example Nitroso-2-methyl-N-2-Dvridinvl-2-H-1,2benzothiazine-2-carboxamide-1. 1-dioxide 4-Hydroxy-2-methyl-N-2-pyridinyl-2-H- 1,2-benzothiazine-2-carboxamide- 1,1- S dioxide (10.0 g, 30 mmol) was dissolved in anhydrous methylene chloride and cooled to 0 iC. Nitrosonium tetrafluoroborate (4.407 g, 38 mmol) was added in one portion, followed by pyridine (2.98 g, 38 mmol). The reaction mixture was stirred at room temperature for 7 days and then additional nitrosonium tetrafluoroborate (0.571 g, 1.72 mmol) was added. After stirring for 14 days at room temperature, the reaction mixture 1O was poured into saturated sodium bicarbonate solution and extracted with methylene chloride. The solvent was evaporated, the residue was treated with ethyl acetate and filtered. The precipitate was dissolved in the mixture of methylene chloride/ ethyl acetate and the solution was treated with decolorizing charcoal, filtered and concentrated in vacuo to give the title compound as a solid (1.56 g, 14 'HENMR S(CDC13, 300 MHz), 6_2.96 3 6.84 1 7.17 1 7.60-7.86 5 H), 8.22 1 H).

Example 16 4-O-Nitroso-hydroxvmethylene-(1-(3-benzovl-a-methvlbenzeneacetic acid)) benzvl ester a 16a. 3-benzovl-a-methvlbenzeneacetic acid chloride 3-Benzoyl-a-methylbenzeneacetic acid (3.2 g, 12.6 mmol) was treated in the same manner as set forth in Example 13a. Evaporation of the solvent, affored the the product as a yellow oil in a quantitative yield. 'H NMR (CDCI 3 61.64 3 4.21 1 H), 7.45-7.51 4 7.62 1 7 72-7.82 4 H).

-57- -57- *o O 16b. 4-Hvdroxvmethvlene-(1-(3-benzovl-t-methylbenzeneacetic acid)) benzvl ester Under a nitrogen atmosphere, 1,4-benzenedimethanol (0.507 g, 3.67 mmol) and pyridine (0.145 g, 1.83 mmol) were combined in methylene chloride (5 mL). The resulting solution was stirred for 5 minutes and then cooled to 0°C. A solution of the product of Example 16a (0.500 g, 1.83 mmol) in methylene chloride (5 mL) was added dropwise over 15 minutes. The reaction mixture was allowed to warm to room temperature and was then stirred over 2 days period. The solvent was evaporated and the residue was dissolved in ethyl acetate, washed with IN hydrochloric acid and saturated sodium bicarbonate solution. The organic phase was dried over anhydrous D sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography on silica-gel eluting with hexane/ethyl acetate (5:1 to 2:1) to give 0.092 g (42 of the product. 'H NMR (CDC 3 6:1.60 3 2.19 1 3.90 1 4.71 2 5.17 2 7.32 (dd, 4 7 45-7.82 7 7 84 2 H).

16c. 4-O-Nitroso-hvdroxvmethylene-( 1 -(3-benzovl-a-methylbenzeneacetic acid)) S benzyl ester The product of Example 16b (0.090 g, 0.24 mmol) was treated in the same manner as set. forth in Example 7c. Purification of the crude product was accomplished using flash chromatography on silica gel eluted with hexane/ethyl acetate to give 0.069 g (71 of the title compound as a yellow oil. 'H NMR (CDCl 3 300tMHz). 61.55 (d, X 3 3.85 1 5.11 2 5.67 2 7.27-7.80 9 H).

benvl ester 17a. 3-Hydroxvmethylene-(1-(3-benzoyl-a-methvlbenzeneacetic acid)) benzl ester o-* "0 -58- Under, a nitrogen atmosphere, 1,3-benzenedimethanol (0.500 g, 3.62 mmol) and pyridine (0.193 g, 2.44 mmol) were combined in methylene chloride (7 mL). The resulting solution was stirred for 5 minutes and then cooled to 0°C. A solution of the the product of Example 16a (0.665 g, 2.44 mmol) in methylene chloride (5 mL) was added dropwise over 15 minutes. The reaction mixture was stirred 2 hour 30 minutes at 0°C, concentrated in vacuo, diluted with ethyl acetate, washed with IN hydrochloric acid and saturated sodium bicarbonate solution. The organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography on silica gel eluting with hexane/ethyl acetate to give 0.530 g (58 of the product. 'H NMR (CDC13) 6:1.55 3 3.85 1 4.64 2 H), 5.12 2 7.13-7.18 1 7.22 1 7.26-7.30 2 7.40-7.67 (m, 6 7 73-7.78 3 H).

17b. 3-O-Nitroso-hvdroxvmethylene-(1-(3-benzovl-a-methylbenzeneacetic acid)) benzvl ester S The product of Example 17a (0.74 g, 0.198 mmol) was treated in the same manner as set forth in Example 7c. Purification of the crude product was accomplished using flash chromatography on silica gel eluted with hexane/ethyl acetate to give 0.046 g (71 of the title compound. 'HtNMR (CDCl 3 6:1.55 3 3.85 1 5.12 2 5.65 2 7.18-7.31 4 7.40-7.75 6 7.76-7.79 3 H).

Example 18 3-O-Nitroso-hvdroxvmethvlene-l-(1-(3-benzoyl-a-methvlbenzeneacetic acid))hvdroxvmethvladamantyl ester 18a. 1,3-Dicarboxvmethyl adamantane 1,3-adamantanedicarboxylic acid (1.5 g, 5.95 mmol) was dissolved in methanol 3 (30mL) and concentrated sulfuric acid (0.5 mL, 8.90 mmol) was added. The reaction •-59- Smixture was stirred at room temperature 20 hours. After concentration in vacuo, the residue was dissolved in methylene chloride, washed with water/brine and dried over anhydrous sodium sulfate. The solvent was evaporated to give the product as a white solid in a quantitative yield. 'H NMR (CDCl 3 6:1.65-1.71 2 1.76-1.82 (mi, 8 1.98-2.03 2EH), 2.07-2.18 2 3.66 6 H).

18b. 1,3-Dihydroxymethyl adamantane Under a nitrogen atmosphere, the product of Example 18a (1.33 g, 5.95 mmol) was dissolved in tetrahydrofuran (20 mL) and lithium aluminum hydride (0.316 g, 8.33 mmol) was added in one portion. The reaction mixture was allowed to reflux for O minutes, and was then quenched with water (0.316 mL, 8.33 mmol), 15 sodium hydroxide solution (0.316 mL), and water (0.95 mL). After 15 hours of stirring at room temperature, the reaction mixture was filtered through PTFE and filtrate was partitioned between ethyl acetate and brine. The organic phase was dried over anhydrous sodium sulfate, filtered through PTFE and concentrated in vacuo to give the product (0.370 g, 28- as a white solid. 'H NMR (CDCl 3 6:1.24-1.29 2 1.42-1.52 8 H), 1.61-1.68 2 2.07-2.16 2 3.25 4 H).

18c. 3-Hydroxymethylene- 1-(1-(3-benzovlvx-methvlbenzeneacetic acid))hydroxymethyladamantvl ester The product of Example 18b (0.199 g,0.54 mmol) was dissolved in tetrahydrofuran do mL) and pyridine (0.047 g, 0.59 mmol) was added. A solution of the product of Example 16a (0.161 g, 0.59 mmol) in chloroform (3 mL) was added dropwise. The reaction mixture was stirred at room temperature for 40 hours. The solvent was evaporated, the residue was dissolved in methylene chloride, washed with 1N hydrochloric acid, saturated sodium bicarbonate solution and brine, and dried over anhydrous sodium sulfate. The solvent was evaporated and the residue was purified by flash chromatography on silica gel eluted with hexane/ethyl acetate to give the *0 *product 102 g, 28 as a colorless oil. IH NMR (CDC 3 6:1.13 -1.17 (in. 2 H).

1. 18-1.55 (in, 10 1.98-2.02 (in, 2 3.18 2 3.66 1 H),-3.77 1 H), 3.83 1 7.43-7.68 (in, 6 7.76-7.81 (in, 3 H).

1 8d. .3-0-Nitroso-hydroxymethylene-l1-(1 -(3-benzovl-cy-methylbenzeneacetic acid))hydroxymethyladamantyl ester The product of Example 18c (0.056 g, 0.083 mmol) was dissolved in anhydrous methylene chloride (2 rnL) and pyridine (2 drops) was added. The resulting solution was cooled to -78'C and nitrosonium tetrafluoroborate was added in one portion.

The reaction mixture was stirred for 3 hours at -78'C, washed with water, brine, %0 dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by chromatography on silica gel eluted with hexane/ethyl acetate (15: 1) to give the title compound as a colorless oil. 'H NMR (CDCl 3 6:1.15-1.19 (in, 2 H), 1.29-1.61 (in, 10 1.98-2.03 (in, 2 3.65 1 3.77 1 3.82 1 4.33 2 7.43-7.68 (in, 6 7.76-7.81 (in, 3 H).

Example 19 3-(2-S-Nitroso-2-metthvl DFoDIinc acid Dropvi anmide)-2-amino-l-(ct-methvI-4-(2methylpropvlbbenzeneacetic acid) DFoDVI ester hydrochloride 1 9a. 2-Mercanto-2-methvl-l1-(2-tert-butvloxvcarbainovl-3-hvdroxv-Dropionic acid) vroyl amide S210 2-tert-Butyloxycarbamoyl-3-hydroxy-propionic acid (5 g, 24 mmol), 1-amino-2inethyl-2-propanethiol-HCl (3.5 g, 25 inmol), triethylamine (3.4 inL, 25 mniol), and 4-diinethylaminopyridine (300 mg, 2.4 mmol) were dissolved in inethylene chloride (120 mL). DCC (5.1 g, 24 mxnol) was added and the reaction mixture was stirred at room temperature overnight. The precipitate which formed was removed by )26 filtration and washed with Et 2 O. The mixed solvents were allowed to stand and more solid precipitated. This was removed by filtration and the mother liquor was 9 -61evaporated to leave 7.3 g of syrup. 'H-NMR (DMSO-d 6 6 7.78 J 5.6 Hz. 1 6.69 J 7.8 Hz, 1 4.82 (br s, 1 3.96 (mult. 1 -3.54 (mult. 2 3.32 (mult, 1H, obscured by H20), 2.73 (dd, J 5.6 and 13.3 Hz, 1 1.37 9 1.22 3 1.20 3H). Anal calcd for C12H 2 4

N

2 0 4 S: C, 49.29; H, 8.27; N, 9.58; S, 10.96. Found: C, 49.39; H, 8.01; N, 9.44; S, 10.96.

19b. 3-(2-Mercapto-2-methvl propionic acid propvl amide)-2-tert-butvloxvcarbamovl- 1-(a-methvl-4-(2-methvlpropvl)benzeneacetic acid) propyl ester 1-a-methyl-4-(2-methylpropyl)benzeneacetic acid (1.4 g, 6.8 mmol) and 10 pL of DMF were slurried in benzene (10 mL). Oxalyl chloride (630 L, 7.2 mmol) was \O added dropwise. The reaction mixture was allowed to stir at room temperature for 1 hour. The volatiles were removed on a rotary evaporator and the residue was reconcentrated from 5 mL of benzene. The reisdue was taken up in methylene chloride (10 mL) and cooled to 0*C. To this solution was added the product of Example 19a (2 g, 6.8 mmol) and pyridine (570 uL, 6.8 mmol) in methylene 6 chloride (14 mL). The reaction was kept cold for 15 minutes then allowed to warm to room temperature. After 1 hour the mixture was diluted with methylene chloride and ,washed (1 X 10 ml) with 0.3 N HC1 and satd NaHCO 3 The solvent was dried over Na 2

SO

4 and evoporated in vacuo to leave 3.04 g of product as a mixture of inseparable diastereomers. 'H-NMR (CDCl 3 6: 7.15-7.18 (mult, 2 7.07-7.14 (mult, 2 6.57 (mult, 1 5.24 and 5.04 (br s, 1 4.48 (dd, J 4.3 and 10.6 Hz, 1 4.44 (dd, J 5.0 and 10.6 Hz, 1 4.28 (dd, J 5.0 and 11.5 Hz, 1 4.25 (mult, 1 3.71 J 7.2 Hz, 1 3.69 J 7.2 Hz, 1 3.32 (dd, J 6.7 and 13.6 Hz, 1 3.21-3.24 (mult, 1 3.17 (dd, J 5.4 and 12.7 Hz, 1 2.43 and 2.41 J 7.1 Hz, 2 1.84 (sept, J 6.7 Hz, 1 1.46 and 1.47 J 7.1 Hz, 3 1.43 9 1.30 3 1.28 3 0.88 J 6.6 Hz, 6H). Anal calcd for C 2 5 H4oN20S: C, 62.47; H, 8.39; N, 5.83; S, 6.67. Found: C, 62.78; H, 8.30; N, 5.69; S, 6.31.

*4 G e -62- 19c. 3-(2-S-Nitroso-2-methyl propionic acid propyl amide)-2-amino- I -(ce-methvi-4- (2-methylpropvyl)benzeneacetic acid) propyl ester hydrochloride The product of Example 19b (630 mg, 1.3 mmol) and tert-butyl nitrite (190 uL, 1.6 tumol) were dissolved in methylene chloride (8 mL) and stirred at room Stemperature for 1.5 hour. The solvent was evaporated and the residue was filtered thrbugh silica gel to give 430 mg of nitrosothiol. The amine protecting group was removed by stirring in 3N HC1 in EtOAc (6 mL) for 1 hour. The solvent was removed to give 360 mg (62 overall) of nitrosothiol hydrochloride (mixture of diastereomers) as a green solid. IH-NMR (CDCl 3 6 8.94-9.00 (mult, 1 8.49 0O (br s, 3 7.04-7.18 (mult, 4 4.40-4.47 (mult, 1 4.13 (mult, 2 3,944.01 (mult, 1 3.71-3.77 (mult, 2 2.39/2.37 J 6.0 Hz, 2 H), 1.83/1.80/1.78/1.73 6 1.36 J 6.0 Hz, 3 0.83 J 6.4 Hz, 6H).

Example 3-(2-S-Nitroso-2-methyl propionic acid propyl amide)-2-amino-l-(3-benzol-ymethylbenzeneacetic acid) DroDVI ester hydrochloride 20a..3-(2-Mercgpto-2-methYl propionic acid propyl amide)-2-tert-butvloxycarbamoyl- 1 -(3-benzovl-ca-methylbenzeneacetic acid) propyl ester 3-Benzoyl-ca-methylbenzeneacetic acid (1.75 g, 6.8 mmol) and 10 uL of DMF were slurried in benzene (10 mL). Oxalyl chloride (630 uL, 7.2 mmol) was added dropwise. The reaction mixture was allowed to stir at room temperature for I hour.

The. volatiles were removed on a rotary evaporator and the residue was reconcentrated from 5 mL of benzene. The reisdue was taken up in methylene ~:chloride (10 mL) and cooled to 0 To this solution was added the product of 25 Example 19a (2 g, 6.8 minol) and pyridine (570 uL, 6.8 mmol) in methylene chloride (14 mL). The reaction was kept cold for 15 minutes then allowed to warm to room temperature. After 1 hour the mixture was diluted with methylene chloride and washed (I X 10) with 0.3 N HCl and satd NaHCO 3 The solvent was dried over Na.,S0 4 and removed on a rotary evaporator to leave 3.4 g of product.

-63- *Chromatography on silica gel eluting with 2:1 Hex:EtOAc gave 1.89 g of an inseparable mixture of diastereomers. 'H-NMR (CDCl 3 6:7.77-7.82 (mnult, 3 H), 7 .55-7 .67 (mult, 2 7.41-7.52 (mult, 4 6.72-6.77 (mult, 1 5.24 and 5.01 .(br s, 1 4.26-4.55 (mult, 3 3.83 J 7.2 Hz, 1 3.14-3.78 (mult, 2 1.42 and 1.41 9 1.32, 1.30, and 1.28 6H). Anal calcd for

C

28

H

36

N

2 0 6 S: C, 63.61; H, 6.86; N, 5.30; S, 6.06. Found: C, 63.80; H, 6.76; N, 5.10; S, 5.88.

3-(2-S-Nitroso-2-methyl propionic acid propyl amide)-2-amino-l-(3-benzol-ymethylbenzeneacetic acid) propyl ester hydrochloride '11 The product of Example 20a (520 mg, 1.0 mmol) and tert-butyl nitrite (140 AL, 1:'2 mmol) were dissolved in methylene chloride (8 mL) and stirred at rooom.

temperature for 1.5 hour. The solvent was evaporated and the residue was filtered through a plug of silica gel to give 350 mg of nitrosothiol. The amine protecting group was removed by stirring in 3N HCl in EtOAc (6 mL) for 1 hour. The solvent 11was evaporated to give 290 mg (58 overall) of the title compound (mixture of diastereomers) as a green solid. 'H-NMR (CDC1 3 6:8.94-9.01 (mult, 1 8.47 (3 7.48-7.73 (mult, 9 4.39-4.47 (mult, 1 4.16 (mult, 2 3.89-4.06 (mult, 2 3.69-3.77 (mult, 1 1.89/1.81/1.79/1/70 6 1.43/1.42 J =7.1 Hz, 3H).

Example 21 3-2-S-Nitroso-2-fliethyl Dropioflic acid propyl amidde)-2-amino-l-((S)-6-methoxvoa-methyl-2-naphthaleneacetic acid) Dropyl ester hydrochloride 21 a. 3-(2-Mercapto-2-methyl propionic acid propyl amide)-2-tert-butyloxycarbamoyl-I1 ((S)-6-methoxy-ca-methyl-2-naphthaleneacetic acid) pronl ester (S)-6-methoxy-ca-methyl-2-naphthaleneacetic acid (1.75 g, 7.6 mmol) and 10 AsL of DMF were slurried in benzene (10 tuL). Oxalyl chloride (760 A~L, 7.6 mmol) was *.:added dropwise. The reaction mixture was allowed to stir at room temperature for 1 hour. The volatiles were evaporated on a rotary evaporator and the -residue was reconcentrated from 5 mL of benzene. The reisdue was taken up in methylene chloride mL) and cooled to 0 0 C. To this solution was added the the product of Example 19a (2.2 g, 7.6 mmol) and pyridine (630 pL, 7.6 mmol) in methylene chloride (14 mL).

The reaction was kept cold for 15 minutes then allowed to warm to room temperature.

After 1 hour the mixture was diluted with methylene chloride and washed (1 X 10) with 0.3 N HC1 and satd NaHCO 3 The solvent was dried over Na 2

SO

4 and evaporated on a rotary evaporator. 'H-NMR (CDC13) 6: 7.72 J 8.5 Hz, 1 7.70 J 8.6 Hz, 1 7.64 1 7.37 (dd, J 1.8 and 8.5 Hz, 1 7.15 (dd, J 2.5 and 0 8.9 Hz, 1 7.11 j 2.5 Hz, 1 6.35 J 1 5.05 J 1 H), 4.47 (dd, J 4.6 and 11 Hz, 1 4.10-4.35 (mult, 2H),3.91 3 3.88 J 7.2 Hz, 1 3.04 (dd, J 6.3 ands 13.6 Hz, 1 2.88-2.95 (mult, 1 1.56 J 7.2 Hz, 3 1.39 9 1.19 3 1.17 3H). Anal calcd for

C

26

H

36 N,20S: C, 61.88; H, 7.19; N, 5.55; S, 6.35. Found: C, 62.14; H, 7.07; N, i1 5.20; S, 6.02.

21b. 3-(2-S-Nitroso-2-methvl propionic acid propvl amide)-2-amino-l-((S)-6-methoxvc-methvl-2-naphthaleneacetic acid) propyl ester hvdrochloride The product of Example 21a (500 mg, 1.0 mmol) and tert-butyl nitrite (150 tiL, 1.2 mmol) were dissolved in methylene chloride (8 mL) and stirred at room temperature for 1.5 hour. The solvent was evaporated and the residue was filtered through a plug of-silica gel to give 470 mg of nitrosothiol. The amine protecting group was removed by stirring in 3N HCI in EtOAc (6 mL) for 1 hour. The solvent was evaporated to give 330 mg (69 overall) of the title compound as a green solid. 'H-NMR (CDCl 3 6: 9.00 J 6.0 Hz, 1 8.5 (br s, 3 7.78 J 8.9 Hz, 1 7.76 j 8.5 Hz, 1 7.20 1 7.39 (dd, J 1.8 and 8.5 Hz, 1 7.14 (dd, J and 8.9 Hz, 1 4.49 (pent. J 6.5 Hz, 1 4.14-4.22 (mult, 2 3.87-3.97 (mult 2 3.85 3 3.72 (dd, J 5.6 and 13.9 Hz, 1 H), 1.78/1.80/1.89/1.97 6 1.45 J 7.2 Hz, 3H).

o *e Example 22 4-((2-S-Nitroso-2-methl)-Drovl amide)-1-((S)-6-methoxv-o!-nethvl-2naphthaleneacetic acid) butyl ester 22a. 3-Carboxv-propionic acid-(2-mercapto-2-methvl)-propvl amide §To a solution of -succinic anhydride (15 g, 0. 15 mol), pyridine (54 g, 0. 69 mol), isopropyl alcohol (50 ml), and methylene chloride (150 ml) was added l-ainino-2methyl-2 propanethiol hydrochloride (23.3 g, 0.16 mol) and the reaction was stirred at room temperature for 4 hours. The reaction was concentrated in vacuo and the residue partioned between ethyl acetate and 1N HCL The organic phase was dried over sodium 'kO sulfate and the volatiles evaporated. The residual oil was recrystalized from ethyl acetate/hexane to afford the product as colorless prisms (22.3g, 73 yield). IH-NMR (CDCl 3 :6 6.20 (br s, 1 3.35 J 6.2 Hz, 2 2.74 (in, 2 2.58 (in, 2 1.35 6H).

22b. 4-Hyroxy-bulyric acid-(2-mercapto-2-methyl)-propyl amide To a solution of the product of Example 22a (1.20 g 5.8 mmol) in anhydrous tetrahydrofuran (10 ml) was added borane diznethylsulfide complex (656 Al, 6.8 mmol) and the reaction mixture was allowed to stand at room temperature for 6 hours. The reaction mixture was concentrated in vacuo and the residue partioned between ethyl acetate and 1N HCL. The organic phase was dried over sodium sulfate to afford the .0 cnide product which was used without further purification. 1 H-NMR (CDC1 3 6: 6.16 o (br s, 1 3.71 J 5.7 Hz, 2 3.33 J 6.2 Hz, 2H) 2.41 J 6.8 Hz, 2 1.63 (in, 2H) 1.30 6H).

.1% 22c. 4-((2-Mercalpto-2-methvl)-propvl amide)- 1-((S)-6-methoxv-ca-methvl-2- :nap~hthaleneacetic acid) butyl ester The-product of Example 7a (0.204 g, 0.82 mmol) was dissolved in anhydrous o methylene chloride (2 ml-) and pyridine (66 AL, 0.82 mniol) was added. The reaction -66mixture was cooled to -78 0 C and a solution of the product of Example 22b (0.187 g.

0.98 mmol) in anhydrous methylene chloride (3 mL) was added. The reaction mixture was allowed to warm to room temperature and stirred overnight. The solvent was evaporated and the residue was purified by flash chromatography on silica gel eluting with hexane/ethyl acetate to give 0.190g (59 yield) of the product as a white solid. 'H NMR (CDC13) 6 1.25 6 1.53-1.58 3 1.86-1.95 2 1.98- 2.08 2 3.15-3.21 (dd, 2 3.80-3.87 1 3.88 3 4.02-4.18 (m, 5.74 1 7.07-7.10 1 7.10-7.15 (dd, 1 7.38-7.43 (dd, 1 7.65 1 7.65-7.69 1 7.69-7.72 1 H).

\O 22d. 4-((2-S-Nitroso-2-methvl)-propyl amide)-l-((S)-6-methoxy-cy-methvl-2naphthaleneacetic acid) butyl ester The product of Example 22c (0.102 g, 0.26 mmol) was dissolved in anhydrous methylene chloride (2 mL) and tert-butyl nitrite (46 AL, 0.39 mmol) was added. The reaction mixture was stirred for 15 minutes at room temperature and the solvent was evaporated in vacuo to give 0.105 g (93 yield) of the title compound as a green oil.

'H NMR (CDCl 3 6 1.53-1.59 3 1.78 6 1.81-1.99 4 3.79-3.86 1 3.86-3.90 (dd, 2 3.91 3 3.97-4.18 2 5.41 1 7.07- 7;10 1 7.10-7.15 (dd, 1 7.36-7.40 (dd, 1 7.65-7.70 3 H).

Example 23 2-((2-S-Nitroso-2-methvl) propyl amide)-l-((S)-6-methoxv-ar-methvl-2naphthaleneacetic acid) ethyl ester 23a. Chloroacetic acid (2-tetrahydropvranvl thioether-2-methvl-propyl)-amide To a stirred solution of pyridine (2.37 g, 30 mmol), l-amino-2-methyl-2 propanethiol hydrochloride (2 g, 14 mmol in methylene chloride (30 ml) at 0 oC was added dropwise chloroacetyl chloride (1.7 g, 15 mmol). After the addition was complete and the reaction mixture was stirred overnight with slow warming to room temperature. The reaction was washed with 4N HCI and the organic phase was dried -67- Sover sodium sulfate and then concentrated in vacuo. A portion of the residue (0.370g.

2.04 mmol) was combined with dihydropyran (326 gL, 2.24 mmol) and cooled to 0°C.

A 4M solution of hydrochloric acid in ethyl ether (14 AL) was added and the reaction mixture was stirred for 3 hours at room temperature. The solvent was evaporated in i vacuo to give 0.530 g (98 yield) of the product as a colorless oil. 'H NMR (CDC1 3 6: 1.23-1.42 6 1.51-1.73 4 1.74-1.91 2 3.23-3.35 (dd, 1 H), 3.42-3.58 2 4.05 2 4.05-4.11 (dd, 1 4.81-4.89 (dd, 1 7.54 (s, 1 H).

23b. 2-((2-tetrahvdropvranvl thioether 2-methyl) propvl amide)-l-((S)-6-methoxy-a- \O methvl-2-naphthaleneacetic acid) ethyl ester Under a nitrogen atmosphere (S)-6-methoxy-ca-methyl-2-naphthaleneacetic acid sodium salt (0.514 g, 2.04 mmol) was suspended in anhydrous dimethylformamide mL) and a solution of the product of Example 23a (0.519 g, 2.04 mmol) in anhydrous dimethylformamide (5 mL) was added. The reaction mixture was stirred for '17 hours at room temperature. The solvent was evaporated, the residue was suspended in methylene chloride, and the precipitate was filtered. The filtrate was concentrated in vacuo and the residue was purified by flash chromatography on silica gel eluting with hexane/ethyl acetate to give 0.263 (28 yield of the the product as an oil. 'H NMR (CDC1) 6: 1.06-1.15 3 1.23-1.26 3 1.43- 1.59 4 1.60-1.66 3 1.51-1.84 2 3.04-3.22 (ddd, 1 3.24- 3.48 2 3.87 3 3.91-4.03 2 4.41-4.64 2 4.70-4.76 1 6.94-7.05 1 7.06-7.10 1 7.11-7.15 (dd, 1 7.37-7.46 (dd, 1 7.63-7.71 3 H).

23c. 2-((2-Mercapto-2-methyl) propyl amide)- 1-((S)-6-methoxy-a-methyl-2- A naphthaleneacetic acid) ethyl ester The product of Example 23b (0.180 g, 0.39 mmol) was dissolved in methanol and a solution of silver nitrate (0.133g, 0.79 mmol) in water (0.5 mL) was added.

The reaction mixture was stirred for 30 minutes at room temperature and the solvent Swas evaporated. The residue was suspended in dichloromethane (50 mL) :and a 4M solution of hydrochloric acid in ethyl ether (1 mL) was added. After 12- hours stirring at room temperature, the precipitate was filtered, the filtrate washed with brine and dried over anhydrous sodium sulfate. The solvent was evaporated and the residue was purified by flash chromatography on silica gel eluting with hexane/ethyl acetate to to give 0.046 g (31 yield) of the title compound (2c) as a yellow oil. 'H NMR (CDCl 3 6: 1.01-1.11 6 1.19 1 1.59-1.64 3 2.94-3.03 (dd, 1 3.15-3.24 (dd, 1 3.90 3 3.91-4.00 1 H), 4.43-4.50 1 4.70-4.77 1 6.13 1 7.07-7.11 1 7.12-7.17 (dd, 1 7.37-7.44 (dd, 1 7.65-7.74 3 H).

23d. 2-((2-S-Nitroso-2-methvl) propvl amide)-l-((S)-6-methoxv-a-methvl-2naphthaleneacetic acid) ethyl ester The product of Example 23c (0.040 g, 0.11 mmol) was dissolved in anhydrous methylene chloride (1 mL) and tert-butyl nitrite (19 AL, 0.16 mmol) was added. The 1 reaction mixture was stirred for 15 minutes at room temperature and the solvent was evaporated in vacuo to give 0.043 g (100 yield) of the title compound as a green oil.

'H NMR (CDC13) 6: 1.54 3 1.56-1.61 6 3.59-3.68 (dd, 1 3.92 (s, 3.82-3.91 2 4.02-4.46 1 4.69-4.75 1 5.90 1 7.09- 7.12 d, 1 7.13-7.18 (dd, 1 7.29-7.34 (dd, 1 7.61-7.71 3 H).

2 0 Example 24 3-S-Nitro-3-methvl-1-(3-benzovl-a-methvlbenzeneacetic acid) butyl ester To a solution of the product of Example 6a (103 mg, 0.29 mmol) in methylene chloride (3 ml) was bubbled in dinitrogen tetroxide till saturation. The reaction mixture was allowed to stand at room temperature for 20 minutes and the the excess dinitrogen tetroxide was blown off by bubbling nitrogen gas through the solution. The volatiles were evaporated and the residue purified by flash silica gel chromatography eluting with ether/hexanes to afford 93.7 mg (80 of the title compound as a colorless oil.

0...6 -69- 'H NMR (CDC13) 6:_1.44 3 1.45 3 1.53 J 7.0 Hz, 3 1.69 (br s, 1 2.26 J 6.4 Hz, 2 3.78 J 7.0 Hz, 3 4.23-(td, J 6.35 Hz, J 2.25.Hz, 2 7.4-7.8 9H).

Example 3-(2-S-Nitroso-2-methvl)-diDroDvl amide)-1-((S)-6-methoxv-a-methyl-2naphthaleneacetic acid) isophthalic ester 5-Acetoxvisophthalic acid To a stirred solution of isophthalic acid (2.0 g, 11.0 mmol) in pyridine (10 ml) was added acetic anhydride 1.23 g, 12.1 mmol) and the reaction was allowed to stir at room temperature for 3 hours. The reaction mixture was concentrated in vacuo and the residue partioned between ethyl acetate and 2N HC1. The organic phase was dried over sodium sulfate and the volatiles evaporated to afford 2.17 g (88 of the product as a white solid. 'H NMR (CDCl 3 /DMSO) 6:2.33 3 7.71 2 8.60 2 H).

5-Acetoxy-(1, 3-(2-mercapto-2-methvl)-dipropvl) amide To a solution of the product of Example 25a (506 mg, 2.26 mmol) in anhydrous tetrahydrofuran (6 ml) was added dimethylformamide 1 drop) and oxalyl chloride (631 mg, 5 mmol) and the reaction mixture was stirred at room temperature for minutes. Concentration of the volatiles in vacuo folloed by azeotroping the residue with additional tetrahydrofuran (2 x 5 ml) afforded the crude acid chloride which was 0O used without further purification in the next step. To a solution of 2-amino-2-methyl-2propanethiol hydrochloride (720 mg, 5 mmol), pyridine (2.34 g, 29 mmol) in methylene chloride (10 ml) was added the acid chloride in methylene chloride (5 ml) and the reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was concentrated in vacuo and the residue partioned between methylene chloride and 1N HCl-brine. The organic phase was dried over sodium sufate and the volatiles evaporated to afford 693 mg (82 the crude product as a white solid. 'H NMR (CDC1 3 6:_1.43 o 12 1.71 2 2.36 3 3.55 J 6.2 Hz, 4 6,80 2 7.27 2 7.71 J 1.5 Hz, 1 H).

5-Hydroxv-1, 3-(2-mercaDto-2-methvl)-dipropvl amide To the product of Example 25b (690 mg, 1.8 mmol) in methanol (10 ml) was added lithium hydroxide monohydrate (90 mg, 2.1 mmol) and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated in vatuo and the residue partioned between ethyl acetate and 1N HCl-brine. The organic phase was dried over sodium sulfate and and the volatiles evaporated to afford 540 mg of the crude product as a white solid. 'H NMR (CDC1 3 6:1.42 12 1.71 \0 2 3.53 J 6.1 Hz, 4 6.92 2 7.62 2 7.74 1 H).

3-(2-Mercapto-2-methvl)-dipropyl amide)-l-((S)-6-methoxv-a-methvl-2naphthaleneacetic acid) isophthalic ester To a stirred solution of (S)-6-methoxy-a-methyl-2-naphthaleneacetic acid (69 mg, 0.30 mmol) in tetrhydrofuran (2 ml) at 0°C was added triethylamine (32 mg, 0.32 i> mmol) and isobutyl chloroformate (40 mg, 0.30 mmol) and the reaction mixture was stirred for an additional 10 minutes. The product of Example 25c (100 mg, 0.30 mmol) and pyridine (5 ml) were added and the reaction mixture stirred at room temperature for 18 hours. The reaction mixture was concentrated in vacuo and the residue purified by flash silica gel chromatography to afford 22 mg (13 of the product as a white solid.

'H NMR (CDCI 3 6:1.38 12 1.52-1.74 5 3.48 J 6.1 Hz, 4 H), 3.91 3 4.12 J 7.0 Hz, 1 6.73 2 7.18 2 7.48 J 7.2 Hz, 1 7.50 2 7.58-7.77 3 8.05 1 H).

25e. 3-(2-S-Nitroso-2-methyl)-dipropyl amide)-l-((S)-6-methoxv-a--methvl-2naphthaleneacetic acid) isophthalic ester The product of Example 25d (0.018 g, 0.032 mmol) was dissolved in anhydrous methylene chloride (1 mL) and cooled to 0 C. Tert-butyl nitrite (20 AL, 0.17 mmol) -71-

S

was added and the resulting mixture was stirred for 25 minutes. The solvent was evaporated in vacuo to give 0.018 g (90 yield) of the title compound asa green solid.

'H NMR (CDC 3 6: 1.66-1.73 3 1.89 12 3.93 3 4.04-4.13 (q.

1 4.15-4.19 4 6.54-6.58 2 7.15 1 7.16-7.19 1 7.42- 7.48 3 7.70-7.80 4 H).

Example 26 Comparative In Vivo Analgesic, Antiinflammatorv and Gastric Lesion Activities The phenylbenzoquinone-induced writhing test in mice was used to measure analgesic o activity. The ability of the compounds to inhibit phenylbenzoquinone-induced writhing in mice was measured using the method of Siegmund et al., Proc. Soc. Exp. Biol. Med.

729-731, 1957. Male CD-1 mice (Charles River Laboratories, Wilmington, MA) weighing 20-25 g were fasted overnight. Vehicle or compounds were administered by oral gavage 1 hour prior to i.p. injection of 2 mg/kg of phenylbenzoquinone. In the case of a nitric oxide adduct being given in combination with a NSAID, the nitric oxide adduct was administered immediately before the NSAID. Five minutes after the i.p.

injection of phenylbenzoquinone, the number of writhes in a 5 minute period was counted.

The rat paw edema test was used to measure antiinflammatory activity. The rat paw X edema test was performed according to the method of Winter et al., Proc. Soc. Exp.

Biol. Med. 111: 544-547, 1962. Male Sprague-Dawley rats (250-275 g) were fasted overnight and dosed by oral gavage with vehicle or suspensions of compound one hour prior to the subplantar injection of 50 /l of 1% suspension of carrageenin. Three hours later, the paw volume was measured and compared with the initial volume measured immediately after carrageenin injection.

*-7 -72-

C

SThe rat gastric lesion test (Kitagawa et al., J. Pharmacol. Exp. Ther., 253:1133-1137.

1990; Al-Ghamdi et al., J. Int. Med. Res., 19: 2242, 1991) was used tcr evaluate the potential of compounds to produce gastric lesion. Male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 230-250 g were used for the Sexperiments. The rats were housed with laboratory chow and water ad libitum prior to the study. The rats were fasted for 24-30 hours with free access to water and then dosed by oral gavage with vehicle or with drugs given at a volume of 0.5 mL/100 g.

For the unmodified NSAIDs being given in combination with a nitric oxide adduct (NOadduct), the NO-adduct was administered by oral gavage immediately prior to the 0O administration of NSAID by oral gavage. Food was withheld for 18 hours after the inital dosing. For acute studies, rats were euthanized by CO, eighteen hours after dosing and the stomachs were dissected. For the multiple dosing studies, the results of which are in Table 3, food was given eighteen hours after the first dose and the rats were maintained on food and water ad libitum while receiving a single daily dose for the remainder of the experiment. For the multiple dosing studies, the results of which are in Table 4, the rats were either fasted 24-30 hours before the first dosing and for 4 hours after the first dosing, (4 day study with ketoprofen, Example 4, and Example allowed access to food and water ad libitum before as well as during the experiment, (7 day study with ketoprofen and Example or fasted 24-30 hours prior 0 to the first dosing and for 18 hours after the first dosing, (7 day study with ibuprofen, Example 11, and Example 12). The stomachs were dissected along the greater curvature, washed with a directed stream of 0.9% saline and pinned open on a sylgard based petridish for examination of the hemorrhagic lesion. Gastric lesion score was expressed in mm and calculated by summing the length of each lesion.

.2 Table 1 shows the relative activities of compounds in the analgesic, antiinflammatory and gastric lesion tests, and are expressed, for each novel NSAID compound, as described according to the general formulas (HI) and or NSAID coadministered with an NO-adduct, as the ratio of activitiy relative to the parent

NSAID.

73- -73- Table 1 Relative Activity Analgzesia Antiinflammation Gastric Lesion ComDound Ketoprofen Example 4 Example 6 Example 5 Example 16 Flurbiprofen Example 13 Indomethacin Example 8 Ibuprofen Example 12 Example I11 Piroxicam Piroxicam Example 2 1 0.7

ND

ND

ND

1 1.83 1 0.03

ND

ND

ND

1 0.31 1 0.08

ND

ND

ND

1 <0.03 <0.05 0.08 ND not determined -74- Table 2 shows the results of single dose treatment studies in which various NOadducts were administered in combination with various NSAIDs. The combinations are able to protect against the NSAID induced gastric toxicity.

Table 2 Molar Dose Ratio NSAID NO-Adduct NSAID (mg/kg) Gastric Lesion Protection NO-Adduct Piroxicam 16 Example 2 Piroxicam 8 Example 2 Piroxicam 8 Isoamyl nitrite Piroxicam 8 Isosorbide dinitrate Piroxicam 8 Example 1 Flurbiprofen 16 Example 2 Tenidap 16 Example 2 Indomethaacin 20 Example 2 1:1 1:1 1:3 1:3 1:2 1:1 1:1 1:1 1 1 Tenidap 22.5 Example 1 1 1 70-100% Protection 40-69% Protection 20-39% Protection Table 3 shows the results of multiple dose treatment studies in which various NO-adducts were administered in combination with various NSAIDs. The combinations are able to protect against the NSAID induced gastric toxicity.

Table 3 Treatment Molar Dose Ratio Gastric Lesion NO-Donor NSAID NO-Adduct (Days) NSAID (mg/k) Piroxicam 16 Piroxicam 16 Ibuprofen 40 Ibuprofen 30 Protection Example 2 Example 2 Example 2 Example 2 1:1 1:1 1:1 1:1 1 1 70-100% Protection Protection 40-69% Protection 20-39% Table 4 shows the results of multiple dose treatment studies in which various novel NSAID compounds directly or indirectly linked to various NO-adducts were administered. The modified NSAIDs containing NO-adducts produced significantly less gastric toxcity.

Table 4

S

Compound Ketoprofen Example 4 Example 6 Ketoprofen Example 4 Ibuprofen (mg/kg) 14 10 14 Treatment (Days) Relative Gastric Lesion Activity Example 11 50 7 Example 12 45 7 Vehicle 7 100% of the gastric toxcity induced by the parent NSAID 21-40% of the gastric toxcity induced by the parent NSAID 1-20% of the gastric toxcity induced by the parent NSAID -77-

Claims (82)

1. A method for reducing drug-induced gastrointestinal or renal toxicity comprising administering a therapeutically effective amount of a composition comprising a compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase, and (ii) a pharmaceutically acceptable carrier.

2. A method according to claim 1, wherein the compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase is an S-nitrosothiol.

3. A method according to claim 2, wherein the S-nitrosothiol is S-nitroso-N-acetylcysteine, S-nitroso-N-acetylpenicillamine, S-nitroso-homocysteine, S-nitroso-cysteine, or S-nitroso-glutathione.

4. A method according to claim 2, wherein the S-nitrosothiol is: CH3[C(Rb)(Rc)]xSNO; (ii) HS[C(Rb)(Rc)]xSNO; or (iii) ONS[C(Rb)(Rc)]xV; 15 wherein x equals 2 to 20; V is fluoro, alkoxy, cyano, carboxamido, cycloalkyl, arylalkoxy, Salkylsulfinyl, arylthio, alkylamino, dialkylamino, hydroxy, carbamoyl, N-alkylcarbamoyl, N,N-dialkylcarbamoyl, amino, carboxyl, hydrogen, nitro or aryl; and Rb and Rc are each independently hydrogen, lower alkyl, cycloalkyl, aryl, heteroaryl, aminoarylalkyl, alkylamino, or dialkylamino, or Rb and Rc taken together are cycloalkyl or bridged cycloalkyl. 20 5. A method according to claim 1, wherein the compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase is L-arginine. A method according to claim 1, wherein the compound that donates, transfers or .releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric 25 oxide synthase is: a compound comprising at least one ON-O-, ON-N- or ON-C- group; (ii) a compound of the formula RiooR 2 ooN(O-M+)-NO, wherein Rioo and R20 0 are each independently a polypeptide, an amino acid, a sugar, a modified or unmodified oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic hydrocarbon, or a heterocyclic group; and M+ is a metal cation; or (iii) a thionitrate of the formula Rioo(S)-NO2, wherein Rioo is a polypeptide, an amino acid, a sugar, a modified or unmodified oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic hydrocarbon, or a heterocyclic group.

7. A method according to claim 6, wherein the compound comprising at least one ON-O-, ON-N- or ON-C- group is an ON-O- polypeptide, an ON-O- amino acid, an ON-O- sugar, a modified or unmodified ON-O- oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic ON-O- hydrocarbon, a ON-O- heterocyclic group, an ON-N- polypeptide, an ON-N- amino acid, an ON-N- sugar, a modified or unmodified ON-N- oligonucleotide, f US4branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic -629 ON-N- hydrocarbon, an ON-N- heterocyclic group, an ON-C- polypeptide, an ON-C- amino acid, an ON-C- sugar, a modified or unmodified ON-C- oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic ON-C- hydrocarbon, or an ON-C- heterocyclic group.

8. A method according to claim 6, wherein R 1 oo in the compound of formula Rioo(S)-N0 2 is a polypeptide or a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic hydrocarbon.

9. A method according to any one of claims 1 to 8, wherein said toxicity is associated with NSAID administration.

10. A method according to any one of claims 1 to 9, wherein said composition also comprises a NSAID.

11. A method according to claim 10, wherein said NSAID is directly or indirectly linked to at least one NO group.

12. A method according to claim 10, wherein said NSAID is a selective cyclooxygenase-2 inhibitor.

13. A method according to claim 12, wherein said NSAID, wherein the selective cyclooxygenase-2 inhibitor is selected from the group comprising CELEBREX (Celecoxib or methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide), VIOXX (Rofecoxib or methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone), valdecoxib (4-(5-methyl-3-phenyl-4- 20 isoxazolyl)benzenesulfonamide), parecoxib (4-(5-methyl-3-phenyl-4- l* isoxazolyl)phenyl)sulfonyl)propanamide), L-745337 (5-methanesulphonamido-6-(2,4- difluorothiophenyl)-1-indanone), L-761066 (1-((4-bromophenyl)methyl)-5-methoxy-p-2-dimethyl-1H- indole-3-butanoic acid), JTE-522 (4-(4-cyclohexyl-2-methyl-5-oxazolyl)-2-fluoro-benzenesulfonamide), and GR-253035. 25 14. A method for reducing drug-induced gastrointestinal or renal toxicity comprising administering a therapeutically effective amount of a composition comprising a compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase, and (ii) a pharmaceutically acceptable carrier, substantially as hereinbefore described.

15. A method for treating gastrointestinal lesions comprising administering a therapeutically effective amount of a composition comprising a compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate of nitric oxide synthase, and (ii) a pharmaceutically acceptable carrier.

16. A method according to claim 15, wherein the compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels or nitric oxide or is a substrate for nitric oxide synthase is an S-nitrosothiol.

17. A method according to claim 16, wherein the S-nitrosothiol is S-nitroso-N-acetylcysteine, S-nitroso-N-acetylpenicillamine, S-nitroso-homocysteine, S-nitroso-cysteine, or S-nitroso-glutathione. -K' f

18. A method according to claim 16, wherein the S-nitrosothiol is: ZZ06296 CH3[C(Rb)(Rc)]xSNO; (ii) HS[C(Rb)(Rc)]xSNO; or (iii) ONS[C(Rb)(Rc)]xV; wherein x equals 2 to 20; V is fluoro, alkoxy, cyano, carboxamido, cycloalkyl, arylalkoxy, alkylsulfinyl, arylthio, alkylamino, dialkylamino, hydroxy, carbamoyl, N-alkylcarbamoyl, N,N- dialkylcarbamoyl, amino, carboxyl, hydrogen, nitro or aryl; and Rb and Re are each independently hydrogen, lower alkyl, cycloalkyl, aryl, heteroaryl, aminoarylalkyl, alkylamino, or dialkylamino, or Rb and Re taken together are cycloalkyl or bridged cycloalkyl.

19. A method according to claim 15, wherein the compound that donates, transfers or 1o releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase is L-arginine. A method according to claim 15, wherein the compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric :oxide synthase is: 15 a compound comprising at least one ON-O-, ON-N- or ON-C- group; S. (ii) a compound of the formula RlooR200N(O-M+)-NO, wherein Rioo and R200 are each independently a polypeptide, an amino acid, a sugar, a modified or unmodified oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic hydrocarbon, or a heterocyclic group; and M+ is a metal cation; or 20 (iii) a thionitrate of the formula Rloo(S)-NO2, wherein Rioo is a polypeptide, an amino acid, a sugar, a modified or unmodified oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic hydrocarbon, or a heterocyclic group.

21. A method according to claim 20, wherein the compound comprising at least one ON-O-, ON-N- or ON-C- group is an ON-O- polypeptide, an ON-O- amino acid, an ON-O- sugar, a modified or 25 unmodified ON-O- oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic ON-O- hydrocarbon, a ON-O- heterocyclic group, an ON-N- polypeptide, an ON-N- amino acid, an ON-N- sugar, a modified or unmodified ON-N- oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic ON-N- hydrocarbon, an ON-N- heterocyclic group, an ON-C- polypeptide, an ON-C- amino acid, an ON-C- sugar, a modified or unmodified ON-C- oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic ON-C- hydrocarbon, or an ON-C- heterocyclic group.

22. A method according to claim 20, wherein Rioo in the compound of formula Rioo(S)-N0 2 is a polypeptide or a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic hydrocarbon.

23. A method according to any one of claims 16 to 22, wherein said lesions are associated with NSAID administration.

24. A method according to any one of claims 15 to 23, wherein said composition also S comprises a NSAID. ZZ06296 81 A method according to claim 24, wherein said NSAID is directly or indirectly linked to at least one NO group.

26. A method according to claim 24, wherein said NSAID is a selective cyclooxygenase-2 inhibitor.

27. A method according to claim 26, wherein said NSAID, wherein the selective cyclooxygenase-2 inhibitor is selected from the group comprising CELEBREX (Celecoxib or methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide), VIOXX (Rofecoxib or methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone), valdecoxib (4-(5-methyl-3-phenyl-4- isoxazolyl)benzenesulfonamide), parecoxib (4-(5-methyl-3-phenyl-4- o0 isoxazolyl)phenyl)sulfonyl)propanamide), L-745337 (5-methanesulphonamido-6-(2,4- difluorothiophenyl)-1-indanone), L-761066 (1-((4-bromophenyl)methyl)-5-methoxy-p-2-dimethyl-1H- indole-3-butanoic acid), JTE-522 (4-(4-cyclohexyl-2-methyl-5-oxazolyl)-2-fluoro-benzenesulfonamide), and GR-253035. S28. A method for treating gastrointestinal lesions comprising administering a therapeutically S: 15 effective amount of a composition comprising a compound that donates, transfers or releases nitric S"oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate of nitric oxide synthase, and (ii) a pharmaceutically acceptable carrier, substantially as hereinbefore described.

29. A composition comprising a compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthases, and •20 (ii) a pharmaceutically acceptable carrier, when used for reducing drug-induced gastrointestinal or renal toxicity.

30. A composition when used according to claim 29, composition according to claim 6, or use according to claim 7, wherein the compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase is an S- 25 nitrosothiol.

31. A composition when used according to claim 30, wherein the S-nitrosothiol is S-nitroso- N-acetylcysteine, S-nitroso-N-acetylpenicillamine, S-nitroso-homocysteine, S-nitroso-cysteine, or S- nitroso-glutathione.

32. A composition when used according to claim 30, wherein the S-nitrosothiol is: CH3[C(Rb)(Rc)]xSNO; (ii) HS[C(Rb)(Rc)]xSNO; or (iii) ONS[C(Rb)(Rc)]xV; wherein x equals 2 to 20; V is fluoro, alkoxy, cyano, carboxamido, cycloalkyl, arylalkoxy, alkylsulfinyl, arylthio, alkylamino, dialkylamino, hydroxy, carbamoyl, N-alkylcarbamoyl, N,N- dialkylcarbamoyl, amino, carboxyl, hydrogen, nitro or aryl; and Rb and Rc are each independently hydrogen, lower alkyl, cycloalkyl, aryl, heteroaryl, aminoarylalkyl, alkylamino, or dialkylamino, or Rb and Rc taken together are cycloalkyl or bridged cycloalkyl. ZZ06296 k 82

33. A composition when used according to claim 29, wherein the compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase is L-arginine.

34. A composition when used according to claim 29, wherein the compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase is: a compound comprising at least one ON-O-, ON-N- or ON-C- group; (ii) a compound of the formula Ro0oR 2 ooN(O-M+)-NO, wherein Roio and R 2 oo are each independently a polypeptide, an amino acid, a sugar, a modified or unmodified oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic hydrocarbon, or a heterocyclic group; and M+ is a metal cation; or (iii) a thionitrate of the formula Rioo(S)-NO 2 wherein Rico is a polypeptide, an amino acid, a sugar, a modified or unmodified oligonucleotide, a branched or straight, saturated or Sunsaturated, substituted or unsubstituted, aliphatic or aromatic hydrocarbon, or a heterocyclic group. 15 35. A composition when used according to claim 34, wherein the compound comprising at least one ON-O-, ON-N- or ON-C- group is an ON-O- polypeptide, an ON-O- amino acid, an ON-O- sugar, a modified or unmodified ON-O- oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic ON-O- hydrocarbon, a ON-O- heterocyclic group, an ON-N- polypeptide, an ON-N- amino acid, an ON-N- sugar, a modified or 20 unmodified ON-N- oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic ON-N- hydrocarbon, an ON-N- heterocyclic group, an ON-C- polypeptide, an ON-C- amino acid, an ON-C- sugar, a modified or unmodified ON-C- oligonucleotide, -a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic ON-C- hydrocarbon, or an ON-C- heterocyclic group. 25 36. A composition when used according to claim 34, wherein Rioo in the compound of formula Rioo(S)-NO2 is a polypeptide or a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic hydrocarbon.

37. A composition when used according to any one of claims 29 to 36, wherein said toxicity is associated with NSAID administration.

38. A composition when used according to any one of claims 29 to 37, wherein said composition also comprises a NSAID.

39. A composition when used according to claim 38, wherein said NSAID is directly or indirectly linked to at least one NO group. A composition when used according to claim 38, wherein said NSAID is a selective cyclooxygenase-2 inhibitor.

41. A composition when used according to claim 40, wherein said NSAID, wherein the selective cyclooxygenase-2 inhibitor is selected from the group comprising CELEBREX (Celecoxib or ALi/, 4-(5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide), VIOXX (Rofecoxib or S4-(4'-methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone), valdecoxib (4-(5-methyl-3-phenyl-4- ZZ62 ZZ06296 S. A 83 isoxazolyl)benzenesulfonamide), parecoxib (4-(5-methyl-3-phenyl-4- isoxazolyl)phenyl)sulfonyl)propanamide), L-745337 (5-methanesulphonamido-6-(2,4- difluorothiophenyl)-1-indanone), L-761066 (1-((4-bromophenyl)methyl)-5-methoxy-p-2-dimethyl-1H- indole-3-butanoic acid), JTE-522 (4-(4-cyclohexyl-2-methyl-5-oxazolyl)-2-fluoro-benzenesulfonamide), and GR-253035.

42. A composition comprising a compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase, and (ii) a pharmaceutically acceptable carrier, when used for reducing drug-induced gastrointestinal or renal toxicity, substantially as hereinbefore described.

43. A composition comprising a compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase, and (ii) a pharmaceutically acceptable carrier, when used for treating gastrointestinal lesions.

44. A composition when used according to claim 43, wherein the compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels or nitric oxide or is a substrate 15 for nitric oxide synthase is an S-nitrosothiol.

45. A composition when used according to claim 44, wherein the S-nitrosothiol is S-nitroso- S. N-acetylcysteine, S-nitroso-N-acetylpenicillamine, S-nitroso-homocysteine, S-nitroso-cysteine, or S- nitroso-glutathione.

46. A composition when used according to claim 44, wherein the S-nitrosothiol is: 20 CH3[C(Rb)(Rc)]xSNO; (ii) HS[C(Rb)(Rc)]xSNO; or (iii) ONS[C(Rb)(Rc)]xV; wherein x equals 2 to 20; V is fluoro, alkoxy, cyano, carboxamido, cycloalkyl, arylalkoxy, alkylsulfinyl, arylthio, alkylamino, dialkylamino, hydroxy, carbamoyl, N-alkylcarbamoyl, N,N- i: 25 dialkylcarbamoyl, amino, carboxyl, hydrogen, nitro or aryl; and Rb and Rc are each independently hydrogen, lower alkyl, cycloalkyl, aryl, heteroaryl, aminoarylalkyl, alkylamino, or dialkylamino, or Rb and Re taken together are cycloalkyl or bridged cycloalkyl.

47. A composition when used according to claim 43, wherein the compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase is L-arginine.

48. A composition when used according to claim 43, wherein the compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase is: a compound comprising at least one ON-O-, ON-N- or ON-C- group; (ii) a compound of the formula R1ooR200N(O-M+)-NO, wherein Rioo and R200 are each independently a polypeptide, an amino acid, a sugar, a modified or unmodified oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic Shydrocarbon, or a heterocyclic group; and M+ is a metal cation; or SZZ06296 Y P c ,f 84 (iii) a thionitrate of the formula Rio(S)-NO 2 wherein Rioo is a polypeptide, an amino acid, a sugar, a modified or unmodified oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic hydrocarbon, or a heterocyclic group.

49. A composition when used according to claim 48, wherein the compound comprising at least one ON-O-, ON-N- or ON-C- group is an ON-O- polypeptide, an ON-O- amino acid, an ON-O- sugar, a modified or unmodified ON-O- oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic ON-O- hydrocarbon, a ON-O- heterocyclic group, an ON-N- polypeptide, an ON-N- amino acid, an ON-N- sugar, a modified or unmodified ON-N- oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic ON-N- hydrocarbon, an ON-N- heterocyclic group, an ON-C- polypeptide, an ON-C- amino acid, an ON-C- sugar, a modified or unmodified ON-C- oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic ON-C- hydrocarbon, or an ON-C- heterocyclic group.

50. A composition when used according to claim 48, wherein Rioo in the compound of formula Rioo(S)-N0 2 is a polypeptide or a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic hydrocarbon.

51. A composition when used according to any one of claims 43 to 50, wherein said lesions are associated with NSAID administration.

52. A composition when used according to any one of claims 43 to 51, wherein said S 20 composition also comprises a NSAID.

53. A composition when used according to claim 52, wherein said NSAID is directly or indirectly linked to at least one NO group.

54. A composition when used according to claim 52, wherein said NSAID is a selective cyclooxygenase-2 inhibitor. 25 55. A composition when used according to claim 54, wherein said NSAID, wherein the selective cyclooxygenase-2 inhibitor is selected from the group comprising CELEBREX (Celecoxib or 4-(5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide), VIOXX (Rofecoxib or 4 -(4'-methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone), valdecoxib (4-(5-methyl-3-phenyl-4- isoxazolyl)benzenesulfonamide), parecoxib (4-(5-methyl-3-phenyl-4- isoxazolyl)phenyl)sulfonyl)propanamide), L-745337 (5-methanesulphonamido-6-(2,4- difluorothiophenyl)-1-indanone), L-761066 4 -bromophenyl)methyl)-5-methoxy-p-2-dimethyl-1H- indole-3-butanoic acid), JTE-522 4 4 -cyclohexyl- 2 -methyl-5-oxazolyl)-2-fluoro-benzenesulfonamide), and GR-253035.

56. A composition comprising a compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate of nitric oxide synthase, and (ii) a pharmaceutically acceptable carrier, when used for treating gastrointestinal lesions, substantially as hereinbefore described.

57. Use of a composition comprising a compound that donates, transfers or releases nitric oxide elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthases, ZZ06296 Y 'f 19, and (ii) a pharmaceutically acceptable carrier for the manufacture of a medicament for reducing drug- induced gastrointestinal or renal toxicity.

58. A use according to claim 57, wherein the compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase is an S-nitrosothiol.

59. A use according to claim 58, wherein the S-nitrosothiol is S-nitroso-N-acetylcysteine, S- nitroso-N-acetylpenicillamine, S-nitroso-homocysteine, S-nitroso-cysteine, or S-nitroso-glutathione. A use according to claim 58, wherein the S-nitrosothiol is: CH3[C(Rb)(Rc)]xSNO; 1o (ii) HS[C(Rb)(Rc)]xSNO; or (iii) ONS[C(Rb)(Rc)]xV; wherein x equals 2 to 20; V is fluoro, alkoxy, cyano, carboxamido, cycloalkyl, arylalkoxy, alkylsulfinyl, arylthio, alkylamino, dialkylamino, hydroxy, carbamoyl, N-alkylcarbamoyl, N,N- dialkylcarbamoyl, amino, carboxyl, hydrogen, nitro or aryl; and Rb and Re are each independently hydrogen, lower alkyl, cycloalkyl, aryl, heteroaryl, aminoarylalkyl, alkylamino, or dialkylamino, or Rb and Re taken together are cycloalkyl or bridged cycloalkyl.

61. A use according to claim 57, wherein the compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase is L-arginine.

62. A use according to claim 57, wherein the compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase is: a compound comprising at least one ON-O-, ON-N- or ON-C- group; (ii) a compound of the formula RiooR 2 00N(O-M+)-NO, wherein Rloo and R200 are each 25 independently a polypeptide, an amino acid, a sugar, a modified or unmodified oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic hydrocarbon, or a heterocyclic group; and M+ is a metal cation; or (iii) a thionitrate of the formula R 1 oo(S)-NO2, wherein Rioo is a polypeptide, an amino acid, a sugar, a modified or unmodified oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic hydrocarbon, or a heterocyclic group.

63. A use according to claim 62, wherein the compound comprising at least one ON-O-, ON- N- or ON-C- group is an ON-O- polypeptide, an ON-O- amino acid, an ON-O- sugar, a modified or unmodified ON-O- oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic ON-O- hydrocarbon, a ON-O- heterocyclic group, an ON-N- polypeptide, an ON-N- amino acid, an ON-N- sugar, a modified or unmodified ON-N- oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic ON-N- hydrocarbon, an ON-N- heterocyclic group, an ON-C- polypeptide, an ON-C- amino acid, an NiiN-C- sugar, a modified or unmodified ON-C- oligonucleotide, a branched or straight, saturated or ZZ 6296 ZZ06296 V ,V .i 86 unsaturated, substituted or unsubstituted, aliphatic or aromatic ON-C- hydrocarbon, or an ON-C- heterocyclic group.

64. A use according to claim 62, wherein Rioo in the compound of formula Rioo(S)-N0 2 is a polypeptide or a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic hydrocarbon. A use according to any one of claims 57 to 64, wherein said toxicity is associated with NSAID administration.

66. A use according to any one of claims 57 to 65, wherein said composition also comprises a NSAID. 1o 67. A use according to claim 66, wherein said NSAID is directly or indirectly linked to at least one NO group.

68. A use according to claim 66, wherein said NSAID is a selective cyclooxygenase-2 i inhibitor. :69. A use according to claim 68, wherein said NSAID, wherein the selective cyclooxygenase- to 5 is 2 inhibitor is selected from the group comprising CELEBREX (Celecoxib or 4-(5-(4-methylphenyl)-3- (trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide), VIOXX (Rofecoxib or methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone), valdecoxib (4-(5-methyl-3-phenyl-4- isoxazolyl)benzenesulfonamide), parecoxib (4-(5-methyl-3-phenyl-4- isoxazolyl)phenyl)sulfonyl)propanamide), L-745337 (5-methanesulphonamido-6-(2,4- 20 difluorothiophenyl)-1-indanone), L-761066 (1-((4-bromophenyl)methyl)-5-methoxy-p-2-dimethyl-1H- indole-3-butanoic acid), JTE-522 (4-(4-cyclohexyl-2-methyl-5-oxazolyl)-2-fluoro-benzenesulfonamide), Sand GR-253035.

70. Use of a composition comprising a compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase, :25 and (ii) a pharmaceutically acceptable carrier for the manufacture of a medicament for reducing drug- induced gastrointestinal or renal toxicity, substantially as hereinbefore described.

71. Use of a composition comprising a compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase, and (ii) a pharmaceutically acceptable carrier for the manufacture of a medicament for treating gastrointestinal lesions.

72. A use according to claim 71, wherein the compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels or nitric oxide or is a substrate for nitric oxide synthase is an S-nitrosothiol.

73. A use according to claim 72, wherein the S-nitrosothiol is S-nitroso-N-acetylcysteine, S- nitroso-N-acetylpenicillamine, S-nitroso-homocysteine, S-nitroso-cysteine, or S-nitroso-glutathione.

74. A use according to claim 72, wherein the S-nitrosothiol is: CH3[C(Rb)(Rc)]xSNO; (ii) HS[C(Rb)(Rc)]xSNO; or I/ (iii) ONS[C(Rb)(Rc)]xV; ZZ06296 y. i 87 wherein x equals 2 to 20; V is fluoro, alkoxy, cyano, carboxamido, cycloalkyl, arylalkoxy, alkylsulfinyl, arylthio, alkylamino, dialkylamino, hydroxy, carbamoyl, N-alkylcarbamoyl, N,N- dialkylcarbamoyl, amino, carboxyl, hydrogen, nitro or aryl; and Rb and Re are each independently hydrogen, lower alkyl, cycloalkyl, aryl, heteroaryl, aminoarylalkyl, alkylamino, or dialkylamino, or Rb and Re taken together are cycloalkyl or bridged cycloalkyl. A use according to claim 71, wherein the compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase is L-arginine.

76. A use according to claim 71, wherein the compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate for nitric oxide synthase is: a compound comprising at least one ON-O-, ON-N- or ON-C- group; (ii) a compound of the formula RiooR 2 ooN(O-M+-NO, wherein Rioo and R2 00 are each independently a polypeptide, an amino acid, a sugar, a modified or unmodified oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic hydrocarbon, or a heterocyclic group; and M+ is a metal cation; or S: (iii) a thionitrate of the formula Rioo(S)-NO2, wherein R 1 oo is a polypeptide, an amino acid, a sugar, a modified or unmodified oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic hydrocarbon, or a heterocyclic group. 20 77. A use according to claim 76, wherein the compound comprising at least one ON-O-, ON- N- or ON-C- group is an ON-O- polypeptide, an ON-O- amino acid, an ON-O- sugar, a modified or unmodified ON-O- oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic ON-O- hydrocarbon, a ON-O- heterocyclic group, an ON-N- polypeptide, an ON-N- amino acid, an ON-N- sugar, a modified or unmodified ON-N- oligonucleotide, 25 a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic ON-N- hydrocarbon, an ON-N- heterocyclic group, an ON-C- polypeptide, an ON-C- amino acid, an ON-C- sugar, a modified or unmodified ON-C- oligonucleotide, a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic ON-C- hydrocarbon, or an ON-C- heterocyclic group.

78. A use according to claim 76, wherein Rioo in the compound of formula Rloo(S)-N0 2 is a polypeptide or a branched or straight, saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic hydrocarbon.

79. A use according to any one of claims 71 to 78, wherein said lesions are associated with NSAID administration.

80. A use according to any one of claims 71 to 79, wherein said composition also comprises a NSAID.

81. A use according to claim 80, wherein said NSAID is directly or indirectly linked to at least one NO group. 2 1.. La ZZ06296 hl I; 88

82. A use according to claim 80, wherein said NSAID is a selective cyclooxygenase-2 inhibitor.

83. A use according to claim 82, wherein said NSAID, wherein the selective cyclooxygenase- 2 inhibitor is selected from the group comprising CELEBREX (Celecoxib or 4-(5-(4-methylphenyl)-3- (trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide), VIOXX (Rofecoxib or methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone), valdecoxib (4-(5-methyl-3-phenyl-4- isoxazolyl)benzenesulfonamide), parecoxib (4-(5-methyl-3-phenyl-4- isoxazolyl)phenyl)sulfonyl)propanamide), L-745337 (5-methanesulphonamido-6-(2,4- difluorothiophenyl)-1-indanone), L-761066 (1-((4-bromophenyl)methyl)-5-methoxy-p-2-dimethyl-1H- indole-3-butanoic acid), JTE-522 (4-(4-cyclohexyl-2-methyl-5-oxazolyl)-2-fluoro-benzenesulfonamide), and GR-253035.

84. Use of a composition comprising a compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide or is a substrate of nitric oxide synthase, and (ii) a pharmaceutically acceptable carrier for the manufacture of a medicament for treating gastrointestinal lesions, substantially as hereinbefore described. A medicament manufactured by a use according to any one of claims 57 to 84.

86. A composition comprising a selective cyclooxygenase-2 inhibitor and a nitric oxide donor.

87. A composition according to claim 86, wherein the nitric oxide donor is a compound that donates, transfers or releases nitric oxide, elevates endogenous synthesis levels of nitric oxide, 20 stimulates endogenous nitric oxide syntheses, or is a substrate for nitric oxide synthase.

88. A composition according to claim 86, wherein the selective cyclooxygenase-2 inhibitor is selected from the group comprising CELEBREX (Celecoxib or 4-(5-(4-methylphenyl)-3- (trifluoromethyl)-1H-pyrazol-1-yl)benzenesulfonamide), VIOXX (Rofecoxib or methylsulfonylphenyl)-3-phenyl-2-(5H)-furanone), valdecoxib (4-(5-methyl-3-phenyl-4- 25 isoxazolyl)benzenesulfonamide), parecoxib (4-(5-methyl-3-phenyl-4- isoxazolyl)phenyl)sulfonyl)propanamide), L-745337 (5-methanesulphonamido-6-(2,4- difluorothiophenyl)-1-indanone), L-761066 (1-((4-bromophenyl)methyl)-5-methoxy-p-2-dimethyl-1H- indole-3-butanoic acid), JTE-522 (4-(4-cyclohexyl-2-methyl-5-oxazolyl)-2-fluoro-benzenesulfonamide), and GR-253035.

89. A composition according to claim 86, wherein said selective cyclooxygenase-2 inhibitor is directly or indirectly linked to at least one NO group. A composition comprising a therapeutically effective amount of a nonsteroidal antiinflammatory drug and (ii) an NSAID toxicity reducing amount of a compound that donates nitric oxide, transfers nitric oxide, releases nitric oxide, or elevates endogenous synthesis levels of nitric oxide, substantially as hereinbefore described with reference to any one of the examples.

91. A composition comprising a non-steroidal antiinflammatory agent to which is directly or indirectly linked at least one NO group and a compound that donates nitric oxide, transfers nitric oxide, releases nitric oxide, or elevates endogenous synthesis levels of nitric oxide, substantially as ,"AL'L-/,j1hereinbefore described with reference to any one of the examples. L u SZZ06296 89

92. A method of preparing a composition comprising a selective cyclooxygenase-2 inhibitor and a nitric oxide donor, substantially as hereinbefore described.

93. A composition comprising a selective cyclooxygenase-2 inhibitor and a nitric oxide donor, prepared by a method according to claim 92.

94. A composition comprising a selective cyclooxygenase-2 inhibitor and a nitric oxide donor, substantially as hereinbefore described with reference to any one of the examples. A method of preparing a composition comprising an antiinflammatory agent and a compound that donates nitric oxide, transfers nitric oxide, releases nitric oxide, or elevates endogenous synthesis levels of nitric oxide, substantially as hereinbefore described with reference to any one of the examples.

96. A composition comprising an antiinflammatory agent and a compound that donates nitric oxide, transfers nitric oxide, releases nitric oxide, or elevates endogenous synthesis levels of nitric oxide, prepared by a method according to claim

97. A composition comprising an antiinflammatory agent and a compound that donates nitric 1s oxide, transfers nitric oxide, releases nitric oxide, or elevates endogenous synthesis levels of nitric oxide, substantially as hereinbefore described with reference to any one of the examples.

98. A method for treating inflammation, pain, gastrointestinal lesions or fever in an animal in need thereof, said method comprising administering a therapeutically effective amount of a composition according to any one of claims 86 to 91, 93, 94, 96 or 97, or a medicament according to 20 claim

99. A composition according to any one of claims 86 to 91, 93, 94, 96 or 97, or a :0 medicament according to claim 85, when used for treating inflammation, pain, gastrointestinal lesions or fever in an animal in need thereof.

100. Use of a composition according to any one of claims 86 to 91, 93, 94, 96 or 97, for the 25 manufacture of a medicament for treating inflammation, pain, gastrointestinal lesions or fever in an animal in need thereof.

101. A medicament manufactured by a use according to claim 100. Dated 4 July 2001 Nitromed, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON -NT 0ZZ06296