EP0542867A1 - Methods of preventing or decreasing tissue damage by novel antioxidants and free radical scavengers - Google Patents

Abstract

The methods described make it possible to treat various bodily conditions resulting in tissue damage, the mediators of which are free radicals derived from oxygen and oxidants, by administering to patients riboside AICA or prodrugs and analogs thereof as antioxidants and as free radical scavengers. The invention also relates to the use of these compounds for inhibiting platelet aggregation during thrombolytic therapy and for inhibiting the situations of membrane fusion which are a prerequisite for the entry of viruses, this inhibitory capacity being due to the power of these compounds to neutralize the destruction by oxidation of cell membranes.

Description

DESCRIPTION

Methods of Preventing or Decreasing Tissue Damage by Novel Antioxidants and Free Radical Scavengers

Cross Reference to Related Applications

This application is a continuation-in-part of United States Serial No. 566,197 filed August 10, 1990, the disclosure of which is incorporated herein by reference. This application is related to the commonly-assigned and concurrently filed United States patent application of Bullough et al "AICA Riboside Analogs" which is a continuation-in-part of United States Serial No. 566,196, filed August 10, 1991; the disclosures of both applications are incorporated herein by reference.

Background of the Invention

Ischemia-induced pathological processes are a major cause of cell death and irreversible tissue destruction and make a prime contribution to the morbidity and mortality of heart disease, e.g. acute myocardial infarction and angina, as well as cerebral ischemia, e.g. stroke and neurological dysfunction. Prolonged ischemia alone is sufficient to cause cell death but recent evidence suggests that substantial cell injury may occur in settings of reversible ischemia at the time of reperfusion. [Can J. Physiol. Pharmacol. 60:1346-1352 (1982)] Reperfusion injury or post-ischemic injury has been thought to have limited the success of clinical interventions which allow reperfusion of hypoxic but still viable tissues, most notably in the context of myocardial ischemia, e.g., thrombolysis, percutaneous transluminal coronary angioplasty and coronary artery bypass surgery. Procedures such as organ transplant, reconstructive tissue transplants and dialysis may also result in cell damage due to reversible ischemia. In addition, prolonged or chronic ischemia during angina can lead to myocardial injury termed stunned or hibernating myocardium. These injuries can cause mechanical dysfunction and congestive heart failure or dyspnea.

The biochemical basis for cell damage which occurs at the time of oxygenation at reperfusion has been the subject of a number of studies and has been postulated to be due to the production of a large burst of free radical species, notably superoxide anion (O₂ ) and secondarily derived cytotoxic species i.e. hydroxyl radical and hydrogen peroxide [J. Mol. Cell. Cardiol. 12:797-808 (1980); Basic Res. Cardiol. 77:465-485 (1982); Circulation 72 (Suppl. 3) III-350 (1985)]. A major cause of cell damage mediated by these species has been thought to result from peroxidation of fatty acids in lipid membranes resulting in loss of fluidity and breakdown of the membrane secretory functions and transmembrane ionic gradients. Base hydroxylation, nicking, crosslinking and scission of DNA may also result in mutation and/or inhibition of protein, nucleotide, and fatty acid synthesis. [Ann R. Coll. Surg. Engl. 62:188-194 (1980)]. In addition, production of hypochlorous acid (HOCl) by the action of myeloperoxidase during the respiratory burst of neutrophils may be stimulated during reperfusion. Hypochlorous acid has been reported to cause mammalian cell injury largely due to oxidation of sulfhydryl-containing membrane proteins and enzymes [J. Clin. Invest. 85:554-562 (1990)]. These oxidants can also combine with and inactivate endothelium-derived relaxing factor (nitric oxide) and result in increased vasospasm, reduced blood flow and greater organ injury and, at times, more pain. (See Vane, et al., "Mechanisms of Disease", New England Journal of Medicine 323(1) :27-36 (July 5, 1990)).

Another potential source of the free radical, superoxide anion, produced upon reperfusion has been thought to result from the oxidation of hypoxanthine to uric acid catalyzed by the enzyme xanthine oxidase, (XO), as follows:

During ischemia, the enzyme xanthine dehydrogenase is reported to be converted by a calcium-activated protease to xanthine oxidase and adenosine triphosphate is reported to be catabolized to provide substrate for the enzyme, i.e. xanthine and hypoxanthine. Upon reperfusion, the availability of oxygen would then allow the above reaction to proceed resulting in a large burst of superoxide anions [New Engl. J. Med., 312 (1985)]. The activity of the enzyme, and hence its contribution to reperfusion injury, has been reported to be low in certain tissues. In some tissues, ischemia alone may be sufficient to cause free radical damage.

Free radicals which cause direct cell damage have been reported to be produced under non-ischemic conditions, for example, during the course of prostaglandin metabolism [J. Biol. Chem. 257:4764-4768 (1982)], by activated neutrophils during the course of pathogenesis associated with inflammatory diseases or destruction of invading microorganisms [Am. J. Pathol. 107:397-418 (1982); Arthritis Rheum 23:455-463 (1980)] and by activated neutrophils in lung tissue following aspiration, membrane oxygenators and dialysis membrane usage, sepsis, burns, microembolism, pulmonary emphysema, chronic obstructive pulmonary disease and hyperoxia [Mayo Clin. Proc. 63:390 (1988)].

Free radical-induced platelet aggregation may also result in thrombosis and pulmonary and systemic embolism, as well as contributing to the problem of reocclusion following thrombolysis. Thrombolytic therapy represents a major advance in the treatment of cardiovascular disease; however, its success has also been limited by a number of factors which include the resistance of some thrombi to lysis, delays in reperfusion, and reocclusion following successful thrombolysis. Thus, inhibition of platelet aggregation may comprise an adjunctive thrombolytic therapy.

A recent study has shown that both xanthine dehydrogenase to xanthine oxidase conversion and enhanced adenosine catabolism via adenosine deaminase elevation occurs in the lung tissue and bronchoalveolar lavage fluid of influenza virus-infected mice [J. Clin. Invest. 85:739-745 (1990)]. This combination results in a hyperimmune reaction against virus replication in the lung caused by the generation of superoxide anion and other toxic radical and oxidant species. This hyperimmune response is thought to be one of the major pathogenic mechanisms of infection by influenza virus which may extend to the pathogenicity of other viral species [Proc. Natl. Acad. Sci. (USA) 87:2506-2510 (1990)].

Prevention of cell damage by free radicals and oxidants formed as a result of ischemia-reperfusion or inflammation would be of clinical importance in the treatment of myocardial infarction, angina, congestive heart failure, cardiopulmonary arrest, stroke, atherosclerosis, arthritis, inflammation, viral infection, hemorrhagic shock, inflammatory bowel disease and adult respiratory distress syndrome (ARDS). In addition, mitigating the effects of reperfusion injury that occurs during thrombolytic therapy, angioplasty, coronary artery bypass grafting, cardioplegia and organ transplantation would allow improvements in these advanced clinical practices.

A number of studies have shown that the enzyme superoxide dismutase (SOD) which catalyses the dismutation of superoxide anion yielding molecular oxygen and hydrogen peroxide might be effective in treating various inflammatory lesions in humans including rheumatoid arthritis and chronic cystitis [Lancet 1:1015-1017 (1981)].

SOD alone or in combination with catalase has been reported to have a beneficial effect in experimental models of regional and global ischemia/reperfusion and organ transplantation [Circ. Res. 56:895-898 (1985); Circ. Res. 54:277-285 (1984); Ann. Thorac. Surg. 42:390-393 (1986)]. However, the limited efficacy, short half-life and high cost have limited the development of SOD as a therapeutic agent.

A number of chemical free radical scavengers, such as dimethylsulfoxide [Am. J. Path. 109:270-276 (1982], mannitol [J . Thorac. Cardiovasc . Surg. 86 : 262-272 (1983 ) ] , glucose [J. Cardiovasc. Pharmacol. 5:35-43 (1983)] and allopurinol [Am. Heart J. 82:362-370 (1971)] have been reported to show limited beneficial effects in some animal models of ischemic reperfusion injury. Allopurinol or its metabolite, oxypurinol, has been said to act to limit free radical production indirectly by inhibition of xanthine oxidase and/or directly by scavenging free radicals [Oxygen Radicals in Biology and Medicine, M.G. Simic, K.A. Taylor, J.F. Ward and C. von Sonntag, editors, pp. 951-955 (1988)]. Allopurinol has been reported to improve the survival rate of influenza virus infected mice; it was hypothesized that allopurinol inhibited superoxide generation by xanthine oxidase [Akaike et al., J. Clin. Invest. 85:739-745 (1990)].

N-acetylcysteine, presumably acting as a free radical scavenger, has been reported to counteract leukocyte and platelet aggregation in the lung reducing pathophysiological changes in an endotoxin model of ARDS in pigs [Acta Chirurgica Scandinavica 154:169-177 (1988)]. The natural antioxidants, alpha-tocopherol and vitamin C, have also been reported to inhibit platelet aggregation with associated potential therapeutic benefits [Naunyn-Schmiedebergs, Archives of Pharmacology 338:74-81 (1988); Medical Hypothesis 19:345-357 (1986)].

Cardioplegia refers to the process of cooling and arresting the heart to protect it during the ischemia encountered in a number of cardiac surgical procedures. It may be achieved by perfusing the coronary arteries after cross clamping the aorta with a blood solution containing a high concentration of potassium. This results in myocardial cell membrane depolarization and immediate cessation of electrical and mechanical activity. Although established as a method of choice for myocardial protection during open heart surgery, its success is limited by the duration of ischemia and there is hence a need to develop better protection when the ischemic period exceeds three hours.

A number of studies in models of hypothermic ischemia with cardioplegic arrest have shown that myocardial injury can be reduced by addition of free radical scavengers and antioxidants to the cardioplegic solution [J. Thorac Cardiovas. Surg. 86:262-272 (1983); Ann. Thorac. Surg. 44:291-272 (1987); Surgery 102:334-340 (1987); Cardiovasc. Res. 23:351-358 (1989)].

Other compounds such as (+)-3',4',5,7-tetrahydroxyflavan-3-ol; 2,2,4-trimethyl-1,2-dihydroguinoline; 6,6-methylene-bis(2,2-dimethyl-4-methansulfonic acid sodium-1,2-dihydroquinoline; and 4-(5)-aminoimidazole-5-(4)-carboxamide phosphate salt (AICA-phosphate) were reported to inhibit lipid peroxidation and have membrane stabilizing effects [Feher, et al. Drugs Exptl. Clin. Res. 10:549-562 (1984)]. These drugs were said to demonstrate efficacy in treatment of chronic liver diseases including chemically-induced liver alterations and hepatitis of autoimmune or viral origin [Acta Md. Hungarica 45:265-276; Acta Medica Academae Scien. Hungarica 37:99-103 (1980)]. Some data suggested that these agents may increase expression of SOD in erythrocytes and lymphocytes, accounting for antioxidant activity [Acta Md. Hungarica 45:265-276]. However, no data was reported in support of these compounds, including AICA phosphate, as free radical scavengers or antioxidants.

Recently, 1, 3-dihydro-4-methyl-5-(4-methylthiobenzyl)-2H-imidazole-2-thione [U.S. Patent 4,868,197] and imidazoline amide derivatives [EP 312960] have been reported to prevent or reduce reperfusion injury as measured by reduced myocardial stunning. Some compounds of the 2-imidazolone and 2-imidazolthione class have been reported to be potentially useful as antioxidants in vivo. [Biochemical Pharmacology 36:1457-1460 (1987)].

According to the present invention we have discovered therapeutically useful free radical scavengers and antioxidants, some of which may provide the beneficial effect of increased adenosine production during times of net ATP breakdown as occurs during ischemia.

U.S. Patent 4,912,092 is said to describe a method for increasing extracellular concentrations of adenosine by therapeutic intervention with the purine precursor 5-amino-1-beta-D-ribofuranosylimidazole-4-carboxamide (AICA riboside) and the advantages of such intervention in the management of the treatment of diseases associated with ischemia and inflammation.

U.S. Patent 4,575,498 is said to demonstrate enhanced nucleotide synthesis and concomitant repletion of ATP pools with AICA riboside to enable the amelioration of tissue damage in ischemic canine hearts.

U.S. Patent No. 4,115,641 to Fischer et al. is directed to certain ribofuranosyl derivatives which are said to have cardiac and circulatory-dynamic properties. In particular, Fischer et al. are directed to certain compounds which are said to have intrinsic adenosine-like modes of action as determined by measuring decreased heart rate and blood pressure.

Adenine has been utilized to increase the shelf life of packed red blood cells (RBC's) presumably by increasing ATP pools; however, AICA riboside does not appear to be metabolized to adenine in human red blood cells.

Summary of the Invention

The present invention is directed to methods of decreasing tissue damage in a mammal following a period of diminished or interrupted blood flow to that tissue, including that caused by conditions such as ischemia, surgery, cardioplegia or the like by administering to the mammal or to cells, tissues or organs of the mammal an antioxidant effective amount or a free-radical scavenging effective amount of AICA riboside (1-β-D-ribofuranosyl-5-amino-imidazole-4-carboxamide or 5-amino-4-imidazole carboxamide riboside) or a substituted-imidazole analog of AICA riboside.

In one aspect, the present invention is directed to certain new substituted imidazole analogs of AICA riboside which exhibit surprisingly advantageous activity in decreasing post-ischemic and reperfusion tissue damage, and in increasing post-ischemic cardiac function.

These compounds can be used to treat diseases which arise from, or are aggravated by free radical or oxidant damage caused by insufficient blood flow through a particular organ or portion thereof or other biological sources of free radicals and oxidants. Certain of these compounds have also demonstrated antiplatelet and antiviral properties.

According to one aspect of the present invention, post-ischemic damage to cardiac tissue resulting from hypoperfusion or interrupted perfusion is prevented or decreased by the administration of AICA riboside or substituted-imidazole analogs of AICA riboside. The cardioprotective effects include decreased tissue damage mediated by oxygen-related free radicals and oxidants and improved post-ischemic cardiac function.

According to another aspect of the present invention, we have surprisingly found that AICA riboside and these substituted-imidazole analogs of AICA riboside, when added to collected whole blood or packed red blood cells, act to maintain cellular viability and function of red blood cells, platelets or white blood cells during storage such as for blood banking, blood collection and prolonged storage. AICA riboside does not appear to be metabolized to adenine in red blood cells.

Definitions

As used herein, the following terms have the following meanings, unless expressly stated to the contrary.

The term "hydrocarbyl" refers to an organic radical comprised of primarily carbon and hydrogen and includes alkyl, alkenyl and alkynyl groups as well as aromatic groups such as aryl and aralkyl groups and groups which have a mixture of saturated and unsaturated bonds, alicyclic (carbocyclic or cycloalkyl) groups or such groups substituted with aryl (aromatic) groups or combinations thereof and may refer to straight-chain, branched-chain or cyclic structures or to radicals having a combination thereof.

The term "alkyl" refers to saturated aliphatic groups, including straight, branched and carbocyclic groups. The term "lower alkyl" refers to both straight- and branched-chain alkyl groups having a total of from 1 to 6 carbon atoms and includes primary, secondary and tertiary alkyl groups. Typical lower alkyls include, for example, methyl, ethyl, n-propyl,, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, n-hexyl, and the like.

The term "aryl" refers tc aromatic groups having form about 6 to 14 carbon atoms and includes cyclic aromatic systems such as phenyl and naphthyl.

The term "aralkyl" refers to an alkyl group of about 1 to 4 carbon atoms substituted with an aryl group of form 6 to 10 carbon atoms and includes, for example, benzyl, p-chlorobenzyl, p-methylbenzyl and 2-phenylethyl.

The term "alkenyl" refers to unsaturated alkyl groups having at least one double bond [e.g. CH₃CH=CH(CH₂)₂-] and includes both straight and branched-chain alkenyl groups. The term "alkynyl" refers to unsaturated groups having at least one triple bond [e.g. CH₃C≡C(CH₂)₂-] and includes both straight chain and branched-chain groups.

The term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

The term "acyl" refers to the group wherein R' is hydrocarbyl.

The term "acyloxy" refers to the group wherein R' is hydrocarbyl.

The term "alkylene" refers to straight and branched-chain alkylene groups which are biradicals, and includes, for example, groups such as ethylene, propylene, 2-methylpropylene 3-methylpentylene

and the like .

The term "amide" or "amido" refers to the group wherein each R" is independently hydrogen or hydrocarbyl, or to compounds having at least one such group.

The term "carboxamide" refers to the group wherein each R" is independently hydrogen or hydrocarbyl. The term "unsubstituted carboxamide" refers to the group

- The term "acylamino" refers to the group wherein R' is hydrocarbyl. The term "lower acylamino" refers to acylamino groups wherein R' is alkyl of 1 to 6 carbon atoms.

The term "carbonate ester" refers to the group wherein R' is hydrocarbyl or to compounds having at least one such group.

The term "acyl ester" refers to the group wherein R' is hydrocarbyl or to compounds having at least one such group.

The term "phosphate ester" refers to the group

wherein R" is independently hydrogen or hydrocarbyl and/or to compounds having at least one such group, and includes salts thereof.

The term "mixed ester" refers to compounds having at least one carbonate ester group and at least one acyl ester group or to compounds having combinations of different acyl ester or carbonate ester groups.

The term "carboxylic acid ester" refers to the group wherein R' is hydrocarbyl or to compounds having at least one such group.

The term "carboxyl" refers to the group The term "carbocyclic AICA riboside" refers to an analog of AICA riboside wherein the oxygen atom of the ribosyl ring has been replaced by a methylene (-CH₂-) group.

The term "hydrocarbyloxy" refers to the group R'O- wherein R' is hydrocarbyl.

The term "alkoxy" refers to the group R'O- wherein R1 is alkyl.

The term "hydrocarbylthio" refers to the group having the formula R'S- wherein R' is hydrocarbyl.

The term "hydrocarbylamino" refers to the groups -NHR' or -NR'₂ where R' is an independently selected hydrocarbyl group.

The term, "hydrocarbylimidate" refers to the group wherein R' is hydrocarbyl.

The term "carboxamideoxime" refers to the group

The term "hydrocarbyloxyamidine" refers to the group wherein R' is hydrocarbyl.

The term "hydrocarbyloxycarbonyl" refers to the group

R wherein R' is hydrocarbyl.

The term "hydrocarbyloxycarboxy" refers to the group wherein R' is hydrocarbyl.

The term "thioester" refers to the group

wherein R' is hydrocarbyl. The term "substituted imidazole analog of AICA riboside" includes the compounds set forth in formulas I, II and III described herein in the Detailed Description of the Invention as "Preferred Substituted Imidazole Analogs of AICA Riboside" and "Preferred Novel Substituted Imidazole Analogs of AICA Riboside."

Brief Description of the Drawings

FIG. 1 depicts the effects of AICA riboside administered at reperfusion alone on cardiac function. Detailed Description of the Invention

Preferred Substituted Imidazole Analogs of AICA Riboside:

According to the present invention, preferred substituted-imidazole analogs of AICA riboside include compounds of the formula (I) are useful free radical scavengers and antioxidants:

or a pharmaceutically acceptable salt thereof wherein:

(a) if R₁ is hydrogen or hydrocarbyl of about 1 to about 18 carbon atoms, optionally substituted with from 1 to about 4 substituents independently selected from hydroxy, sulfhydryl, hydrocarbyloxy, hydrocarbylthio, halogen, amino, hydrocarbylamino, aryl; or carboxylic acid or an ester, thioester, amide and salt thereof; then R₂ is amino, R₃ is hydrogen, cyano, or carboxylic acid or an amide, ester, thioester, or salt thereof; and R₄ is hydrogen, hydrocarbyl, halogen, hydroxy, (including tantomeric imidazolones) hydrocarbyloxy, sulfhydryl (including tautomeric imidazolthiones), hydrocarbylthio, amino, or hydrocarbylamino; or

(b) if R₁ is

wherein X is -O- or -CH₂-, R₅ and R₆ are independently hydrogen, hydrocarbyl, acyl or hydrocarbyloxycarbonyl; R₇ is hydrogen, halogen, hydroxy, hydrocarbyloxy, sulfhydryl, hydrocarbylthio, sulfamyloxy, amino, hydrocarbylamino, azido, hydrocarbyl, acyloxy, hydrocarbyloxycarboxy or phosphate ester group or salts thereof; then R₂ is hydrogen, amino, hydrocarbylamino, acylamino, amido or dihydrocarbylaminoalkyleneimino; R₃ is hydrogen, cyano, h y d r o c a r b y l i m i d a t e , c a r b o x a m i d o x i m e , hydrocarbyloxyamidine, carboxamide or carboxylic acid or an ester, thioester, amide or salt thereof; and R₄ is hydrogen, halogen, hydrocarbyl, amino, hydrocarbylamino, hydroxy (including tantomeric imidazolone), hydrocarbyloxy, sulfhydryl (including tautomeric imidazolthione), or hydrocarbylthio and pharmaceutically acceptable salts thereof. Since compounds of the above formula wherein R₄ is hydroxy or sulfhydryl may exist in their isomeric (tautomeric) imidazole-2-one and imidazole-2-thione forms, these isomers are intended to be included in the ambit of Formula I.

Alternatively R₃ may be a group of formula:

which R₂, R₄, R₅, R₆ and R₇ are as previously defined in connection with formulas I and II and alk is an alkylene group of from 2 to 8 carbon atoms. Suitable alk groups include n-hexylene and 1,4-cyclohexylene.

Preferred compounds of Formula I according to subparagraph (a) include those wherein R₁ is hydrogen, R₂ is amino, R₃ is carboxamide and R₄ is hydrogen and pharmaceutically acceptable salts thereof.

Preferred compounds of formula I according to subparagraph (b) include those wherein R₂ is amino, R₃ is carboxamide wherein one of the amide hydrogens is optionally replaced by an optionally substituted hydrocarbyl, more preferably an aralkyl group, R₄ is hydrogen, R₅ is hydrogen, R₆ is hydrogen and R₇ is hydroxy or amino.

In particular, in view of their demonstration of promising activity as antioxidants and in decreasing free radical levels in certain models, preferred compounds include compound Nos. 21 (1-227), 23 (1-343), 25 (1-360), 27 (1-395), 29 (1-349), 32 (1-262, 43 (1-432), 47 (1-450), 52 (1-467), 53 (1-468), 66 (1-531) and 79 (1-607) of Tables VIII and IX.

One preferred group of compounds of formula I include certain novel substituted-imidazole analogs of AICA riboside of which will be more fully described hereinafter.

Preferred Novel Substituted Imidazole Analogs of AICA Riboside

Preferred novel substituted imidazole analogs of the present invention include those of formula I wherein R₁ is

wherein X is -O- or -CH₂-, R₅ and R₆ are independently hydrogen, alkyl (of 1 to about 18 carbon atoms), acyl or hydrocarbyloxy-carbonyl; and R₇ is hydrogen, hydrocarbyl, hydroxy, hydrocarbyloxy, sulfhydryl, hydrocarbylthio, sulfamyloxy, amino, hydrocarbylamino, azido, acyloxy, hydrocarbyloxycarboxy or phosphate ester or salt thereof; R₂ is amino, hydrocarbylamino, acylamino or dihydrocarbylaminoalkyleneimino; R₃ is carboxamide wherein one of the amide hydrogens (attached to the nitrogen atom) is optionally replaced by alkyl, cycloalkyl, aryl or aralkyl, optionally substituted with 1 to 3 substituents independently selected from halogen, alkyl, aryl, nitro, amino, hydrocarbylamino, sulfhydryl, hydrocarbylthio, hydroxy, hydrocarbyloxy, trifluoromethyl or sulfonamide, R₃ is carboxamide wherein both amide hydrogens are replaced by alkyl or together by an alkylene or aralkylene group to form a ring; R₃ is -C(O)SR₈ wherein R₈ is alkyl, cycloakyl, aryl or aralkyl optionally substituted with 1 to 3 substituents independently selected from halogen, alkyl, aryl, nitro, amino, hydrocarbylamino, hydrocarbylthio, hydroxy, hydrocarbyloxy, trifluoromethyl or sulfonamide; or, further, R₃ is a group of formula III wherein R₂, R₄, R₅, R₆ and R₇ are as defined with formulas I and II and alk is alkylene of 2 to 8 carbon atoms; R₄ is hydrogen, hydrocarbyl, halogen, hydroxy, hydrocarbyloxy, amino, hydrocarbylamino, sulfhydryl, or hydrocarbylthio; provided that when X is -O- or -CH₂-, R₂ is amino, R₃ is unsubstituted carboxamide, R₄ is hydrogen, R₅ and R₆ are independently hydrogen, acyl or hydrocarbyloxycarbonyl, then R₇ is not hydrogen, hydroxy, acyloxy or hydrocarbyloxycarboxy or when both R₅ and R₆ are hydrogen, R₇ is not a phosphate ester; and provided that when X is oxygen, R₂ is amino, R₃ is unsubstituted carboxamide, R₄ is sulfhydryl, and R₅ and R₆ are both hydrogen, then R₇ is not acetoxy; when X is oxygen, R₂ is amino, R₃ is unsubstituted carboxamide, and R₄ is chloro, bromo, amino or methoxy then R₅ and R₆ are not both hydrogen and R₇ is not hydroxy or R₅ and R₆ are not both acetyl and R₇ is not acetoxy; and provided further that when X is oxygen, R₂ is amino, R₃ is benzylcarboxamide or p-iodophenylcarboxamide and R₄ is hydrogen, and R₅ and R₆ are both hydrogen; then R₇ is not hydroxy; or when R₃ is p-iodophenylcarboxamide, then R₅ and R₆ are not both acetyl and R₇ is not acetoxy.

Preferred compounds of formula I include those wherein R₂ is amino, R₃ is carboxamide substituted with an aralkyl group, more preferably a benzyl group, having from 1 to 3 ring substitutions as described above, or cycloalkyl. Preferred dihydrocarbylaminoalkyleneimino groups include dimethylaminomethyleneimino. In view of their promising antioxidant and free radical level decreasing activity, as demonstrated by certain models, preferred compounds include compound Nos. 21 (1-27), 23 (1-343), 25 (1-360), 27 (1-395), 29 (1-349), 32 (1-262), 43 (1-432), 47 (1-450), 52 (1-467), 53 (1-468), 66 (1-531), and 79 (1-607) of Tables VIII and IV.

One example of an especially preferred compound is a compound where X is oxygen, R₁ is amino, R₂ is p-chlorobenzylcarboxamide, R₃, R₄ and R₅ are hydrogen and R₆ is amino and salts, thereof. One particularly preferred salt is the hydrochloride salt.

Preparation of Preferred Novel Substituted Imidazole Analogs of Aica Riboside

The novel substituted imidazole analogs of the present invention can be synthesized by well known chemical reactions as demonstrated in the examples which follow. In general, compounds of formula I where R₁ is hydrogen, hydrocarbyl, substituted hydrocarbyl or the fragment described by formula II can be prepared from 4-methyl-5-nitro-1H-imidazole by the route described by Baker et al. (Baker D., J. Org. Chem. 47: 3457 (1982)) to prepare 1-benzyl-5-nitro-1H-imidazole-4-carboxylic acid followed by the additional step of reduction of the nitro group to give the desired amino group at R₂. Alternatively, the elegant synthesis of AICA riboside reported by Ferris et al. (Ferris, J.P., J. Org. Chem. 50: 747 (1985)) allows a versatile route to 1,4-di-substituted 5-aminoimidazoles starting with the appropriately protected R₁ hydrocarbyl or riboside and diaminomaleonitrile. This route also allows for the introduction of the desired R₄ alkyl, hydrocarbyl and aryl groups by selection of the appropriate ortho ester in the cyclization reaction of the maleonitrile to the imidazole. Other desired R₄ substituent can be introduced by the methods described by Miyoshi et al. (Miyoshi T., Chem. Pharm. Bull. 24 (9): 2089 (1976)) for the preparation of 2-bromo and 5-amino-2-thio-1-(2,3-O-isopropylidene-β-D-ribofuranosyl)-4-imidazolecarboxamide or the method of Ivanovics et al. (Ivanovics, G.A. et al., J. Org. Chem. 25: 3631 (1974)) for the preparation of 2-alkoxy and 2-hydroxy (as tautomeric imidazolones) substituted 5-amino imidazole-4-carboxamides. Compounds where the desired R₂ substituent is acylamino can be prepared by acylation of the corresponding appropriately protected R₂ amino compound with the desired acyl anhydride followed by de-O-acylation with ammonia or sodium methoxide. Compounds where R₂ is hydrocarbylamino can be prepared by reductive alkylation of the corresponding appropriately protected R₂-amino compound with the desired hydrocarbylamine as described by Sato et al. (Chem. Pharm. Bull., 37: 1604 (1989)).

Compounds according to formula I where R₁ is described according to formula II can be prepared by well known reactions as demonstrated in the examples which follow. Preparation of compounds according to formula II where R₇ is acyloxy or hydrocarbyloxycarboxy can be prepared selectively by reaction of the appropriate hydrocarbyl acid anhydride or hydrocarbyl chloro carbonate with the 2,3-O-isopropylidene protected riboside followed by removal of the isopropylidene group with dilute aqueous acid as described by Miyoshi et al. (vide supra). Compounds according to formula II where R₇ is hydrocarbyloxy can be prepared from the protected 5-substituted pentoses (Snyder J.R., Carbonhydr. Res.. 163 : 169 (1987)) using the method of Ferris et al. (vide supra). Compounds according to formula II where R₇ is sulfhydryl, hydrocarbylthio or hydrocarbylamino can be prepared from the 5'-deoxy-5'-iodo-2',3'-isopropylideneimidazole riboside (Srivastava P.C., J. Med. Chem., 18: 1237 (1975)) by nucleophilic displacement of the halogen with the desired amine or mercaptan. Compounds according to formula II where R₇ is acylamino can be prepared from the corresponding 5-amino-5'-deoxy-imidazole riboside by acylation with the desired hydrocarbyl acid anhydride followed by de-O-acylation with ammonia or sodium methoxide. Compounds according to formula II where R₇ is hydrocarbyl can be prepared from the 1-(2,3-O-isopropylidene-β-D-ribo-pento-1,5-dialdo-14,-furanosyl)imidazoles by the Wittig reaction modification of nucleosides described by Montgomery et al. (J. Het. Chem., 11: 211 (1974)). Compounds according to formula II where R₇ is phosphate or a phosphate ester can be prepared by the general method of Khwaja et al. (Tetrahedron, 27: 6189 (1971)) for nucleoside phosphates.

Utility

We have found that 5-amino-1-beta-D-ribofuranosylimidazole-4-carboxamide and analogs and prodrugs thereof can increase post-ischemic function in isolated buffer perfused rat and guinea pig hearts (Langendorff model) subject to hypoperfusion or interruption of perfusion. In addition, we have found that related compounds provide protection from free radical and oxidant damage in a buffer perfused guinea pig heart.

The ability of the compounds of this invention to reduce reperfusion injury presumed to be mediated by free radical and/or oxidant injury, can be demonstrated in a model of ischemia induced by low flow perfusion followed by restoration of normal perfusion. Accordingly, another aspect of the present invention comprises the inclusion of AICA riboside or a substituted-imidazole analog of AICA riboside in the cardioplegia solution to afford better protection from tissue damage due to the prolonged ischemia during cardioplegia.

According to another aspect of the present invention, AICA riboside or a substituted-imidazole analog of AICA riboside is administered to decrease platelet aggregation and preserve platelet function. Preservation of platelet function during hypercoaguable states resulting from diseases such as cancer, thrombocytopenia purpura, anemia, shock and hemorrhagic fever virus infection may serve to mitigate the shock of these diseases. Moreover, the inhibition of platelet aggregation and preservation of platelet function may be used as an adjunct to thrombolytic therapy.

We have found that AICA riboside acts to maintain cellular viability and function in stored whole blood. In particular, the addition of AICA riboside (0.1 μ M to 1000 μM final concentration) to human blood with added citrate as an anticoagulant prolongs red cell viability, suppresses hemoglobin release and maintains viability/function of blood elements when whole blood is stored for prolonged periods of time. Addition of AICA riboside may increase red blood cell shelf life to about 45 days or more. We believe that this mechanism of blood preservation may be related, in part, to the free radical scavenging and antioxidant properties of AICA riboside and its analogs, as well as its ability to become incorporated in the nucleotide pool to attenuate loss of high energy phoshates during storage and in part, by the augmented release of adenosine, a "protective" autacoid. Thus, AICA riboside and these substituted imidazole analogs of AICA riboside may be used in blood-banking to prolong the shelf-life of stored blood and packed red blood cells, platelets or white blood cells for transfusion, and also cross-match samples. AICA riboside or its analogs is added to the whole blood or packed red blood cells, platelets, white blood cells or cross-match sample soon after the blood is drawn, prior to storage.

Another aspect of the present invention is directed to the use of these substituted imidazole analogs of AICA riboside as antiviral agents. These agents may be administered either prophylactically (i.e. before viral infection) or post-infection. These analogs are useful in treating retroviral infections including human immunodeficiency virus (HIV) infections. The antioxidant activity of these analogs may result in prevention of membrane fusion events and viral entry due to neutralization of oxidants.

Particularly preferred substituted imidazole analogs of AICA riboside include those compounds which cannot be phosphorylated.

Compounds of the invention are administered to the affected tissue at the rate of from 0.01 to 3.0 μmole/min/kg, preferably from 0.1 to 1.0 μmole/min/kg. Such rates are easily maintained when these compounds are intravenously administered as discussed below. When other methods are used (e.g., oral administration), use of time-release preparations to control the rate of release of the active ingredient may be preferred. These compounds are administered in a dose of about 0.01 mg/kg/day to about 200 mg/kg/day, preferably from about 0.5 mg/kg/day to about 100 mg/kg/day.

For the purposes of this invention, the compounds of the invention may be administered by a variety of means including orally, parenterally, by inhalation spray, topically, or rectally in formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, and intraarterial injections with a variety of infusion techniques. Intraarterial and intravenous injection as used herein includes administration through catheters. Preferred for certain indications are methods of administration which allow rapid access to the tissue or organ being treated, such as intravenous injections for the treatment of myocardial infarction. When an organ outside a body is being treated, perfusion is preferred.

Pharmaceutical compositions containing the active ingredient may be in any form suitable for the intended method of administration. When used for oral use for example, tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents including those from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipient which are suitable for manufacture of tablets are acceptable. These excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as maize starch, or alginic acid; binding agents, such as starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.

Formulations for oral use may be also presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions of the invention contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as sodium c a r b o x y m e t h y l c e l l u l o s e , m e t h y l c e l l u l o s e , hydroxypropylmethylcelluose, sodium alginate, polyvmylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadeaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension may also contain one or more preservative such as ethyl of n-propyl p-hydroxybenzoate, one or more coloring agent, one or more flavoring agent and one or more sweetening agent, such as sucrose or saccharin.

Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oral suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

Dispersible powders and granules of the invention suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, a mineral oil, such as liquid paraffin, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsion may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, such as glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a flavoring or a coloring agent.

The pharmaceutical compositions of the invention may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, such as a solution in 1,3-butanediol or prepared as a lyophylized powder. 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 may conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injeσtables.

The amount of active ingredient that may be combined with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a time-release formulation intended for oral administration to humans may contain 20 to 200 μmoles of active material compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95% of the total compositions. It is preferred that pharmaceutical composition be prepared which provides easily measurable amounts for administration. For example, an aqueous solution intended for intravenous infusion should contain from about 20 to about 50 μmoles of the active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 ml/hr can occur.

It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the individual being treated; the time and route of administration; the rate of excretion; other drugs which have previously been administered; and the severity of the particular disease undergoing therapy, as is well understood by those skilled in the art.

Examples of use of the method of the invention include the following. It will be understood that these examples are exemplary and that the method of the invention is not limited solely to these examples.

The method may be used following thrombolysis for coronary occlusion. The compound would be given as a sterile injectable preparation with water or isotonic sodium chloride as the solvent. The solution can be administered intravenously or directly into the coronary artery at the time of left heart catheterization or into a carotid artery. The rate of administration could vary from 0.2 to 1 μmole/min/kg with, for example, an infusion volume of 30 ml/hr. Duration of therapy would typically be about 96 hours.

Angina and early myocardial infarcts can be treated by intravenous administration using a sterile injectable preparation using the rates discussed above.

Compounds of the invention can also be administered to patients intravenously during cardiac bypass surgery or to other surgical patients at risk for a myocardial infarct. The compound can be added directly to the solution administered by the membrane oxygenation, or to the cardiac preservation solution, at the rates discussed above.

Organs can be preserved using the method of the invention by perfusing the organ with a solution containing a compound of the invention. The dosage administered would vary with the rate of perfusion of the organ, as is well understood to those skilled in the art.

This method is particularly applicable to organs and tissues used in organ transplantation.

To assist in understanding the present invention, the following examples are included which describe the results of a series of experiments. These experiments demonstrate the antioxidant and free radical scavenging properties of compounds defined according to formulas I, II, and III. The following examples also describe synthetic procedures for preparing some of these substituted imidazole analogs of AICA riboside. The following examples relating to this invention should not, of course, be construed as specifically limiting the invention and such variations of the invention, now known or later developed, which would be within the purview of one skilled in the art are considered to fall within the scope of the present invention as hereinafter claimed. Examples

Example 1

Antioxidant Activity

Solutions of the test compound were prepared in water to a final concentration of 10 mM. 100 μl of this solution was added to 1 ml of approximately 1 mM sodium hypochlorite aqueous solution. The resulting solutions were mixed for approximately two seconds and immediately thereafter tested for oxidizing strength with starch-iodide paper. Compounds which showed no detectable oxidation of starch-iodide paper, i.e., detectable purple color resulting from the formation of a starch-iodine complex, are scored as positive antioxidants. The results are given in Table I. The compounds imidazole, ribavirin, tiazofurin, 5-amino-1-beta-D-ribofuranosylpyrazole-4-carboxamide and adenosine which do not act as antioxidants in this test are included to demonstrate the surprising selective antioxidant properties of AICA riboside and these substituted imidazole analogs of AICA riboside (see Formula I).

TABLE I

COMPOUND

COMPOUND NO.¹ RESULT water (control) neg 5-amino-1-beta-D-ribofuranosylimidazole-4- 1(1-110) poscarboxamide

5-amino-(2,3,5-tri-O-acetyl-beta-D-ribo- 2(1-111) posfuranosyl) imidazole-4-carboxamide

¹ "Compound No." refers to the Compound No. as set forth in Tables VIII and IX. TABLE I (Continued)

COMPOUND COMPOUND NO. RESULT

5-amino-1-beta-D-ribofuranosylimidazole-4- 3(1-115) poscarbonitrile

5-amino-1-beta-D-ribofuranosylimidazole-4- 4(1-122) poscarboxamidoxime

5-amino-1-beta-D-ribofuranosylpyrazole-4- (1-124) negcarboxamide 5-amino-1-[trans-2-trans-3-dihydroxy-cis- 5(1-145) pos4-(hydroxymethyl)cyclopentyl]imidazole-4-carboxamide ethyl 5-amino-1-beta-D-ribofuranosyl- 6(1-155) pos4-imidazolecarboximidate N⁵-dimethylaminomethyleneamino-beta-D- 7(1-164) posribofuranosylimidazole-4-carboxamide

5-aminoimidazole-4-carboxamide-1-beta-D- 8(1-172) posribofuranosyl 5'monophosphate monohydrate

5-acetamido-1-(2,3,5-tri-O-acetyl9(1-177) pos beta-D-ribofuranosyl)imidazole-4-carboxamide

5-amino-1-beta-D-ribofuranosylimidazole-4- 10(1-186) posN-(cyclopentyl)carboxamide

5-amino-1-beta-D-ribofuranosylimidazole- 11(1-226) pos4-N-(benzyl)carboxamide TABLE I (Continued)

COMPOUND

COMPOUND NO. RESULT

5-amino-1-beta-D-ribofuranosylimidazole-4- 12(1-232) posN-(cyclopropyl)carboxamide

5-amino-2-bromo-1-beta-D-ribofuranosyl13(1-240) pos imidazole-4-carboxamide tiazofurin - - - - - neg ribavirin [ (2-beta-D-ribofuranosyl ) -1 , 2 , 4- - - - - - negtriazole-3-carboxamide]

5-amino-a-beta-D-ribofuranosyl-4-imidazole 14(1-260) pos carboxylic acid methyl ester

5-amino-5'-sulfamoyl-1-beta-D-ribofurano15(1-261) pos sylimidazole-4-carboxamide 5-amino-1-beta-D-ribofuranosylimidazole 16(1-273) pos

5-(N-acetamido)-1-beta-D-ribofuranosyl17(1-295) pos imidazole

5-amino-1-beta-D-ribofuranosylimidazole-4- 18(1-335) posmethoxamidine 5-amino-1-(5'-deoxy-beta-D-ribofuranosyl) 19(1-154) posimidazole-4-carboxamide

5-amino-1-(2'-O-methyl-beta-D-ribofuran20(1-188) pososyl)-imidazole-4-carboxamide TABLE I (Continued)

COMPOUND

COMPOUND NO. RESULT

5-amino-1-(3'-O-methyl-beta-D-ribofuran22(1-243) pos osyl)-imidazole-4-carboxamide

5-amino-1-beta-D-ribofuranosylimidazole-4- 23(1-343) posN-[(4-nitropheny1)methyl]carboxamide

5-amino-1-beta-D-ribofuranosylimidazole-4- 24(1-354) posN-[(2-chlorophenyl)methyl]carboxamide 5-amino-1-beta-D-ribofuranosylimidazole-4- 25(1-360) posN-[(2,4-dichlorophenyl)methyl]carboxamide

5-amino-1-(5'-chloro-5'-deoxy-beta-D-ribo26(1-332) pos furanosyl)imidazole-4-carboxamide

5-amino-2-thio-1-beta-D-ribofuranosyl27(1-395) pos imidazole-4-carboxamide

5-amino-1-(5-hydroxylpentyl)imidazole-4- 28(1-304) poscarboxamide

5-amino-1-(5-amino-5-deoxy-B-D-ribo53(1-468) pos furanosyl)-imidazole-4-N-[(4-chlorophenyl)

methyl] carboxamide, hydrochloride

4(5)-aminoimidazole-5(4)-carboxamide pos adenosine neg imidazole neg Example 2

Prevention of Oxidant/Free-Radical Mediated

Rreperfusion Injury

The ability of AICA riboside and these substituted- imidazole AICA riboside analogs to reduce reperfusion injury presumed to be directly mediated by free radical or oxidant injury was demonstrated in a model of electrolysis-induced myocardial dysfunction [See, J. Pharmacol. Meth. 15:305-320 (1986)].

Isolated guinea pig hearts were cannulated via the ascending aorta and attached to a perfusion apparatus according to the method of Langendorff. The hearts were perfused at a constant pressure of 60 cm water using a modified Krebs-Henseleit buffer (pH 7.4) at 37 degrees C. As an index of cardiac function, left ventricular developed pressure (LVDP) was continuously monitored. Electrolysis was performed by inserting platinum electrodes directly into the inflowing perfusion buffer above the heart. Following equilibration of the hearts for a period of 30 minutes, the perfusion buffer was subjected to electrolysis for a period of 1 minute at 1 mA. This resulted in a decrease in LVDP to approximately 25% of control values at 10 minutes post-electrolysis. As shown in Table II, addition of 5 or 20 μM 5-amino-1-beta-D-ribofuranosylimidazole-4-carboxamide (AICA riboside) to the perfusion buffer maintained LVDP at 90 to 100% of preelectrolysis values. Addition of 1μM AICA riboside maintained LVDP at 60% and 0.5 μM AICA riboside was no different from control values (see Table II). Evaluation of a number of compounds according to formula (I) is summarized in Table II and shows that the preferred analogs are at least equal to AICA riboside (Compound Nos. 5(1-145) and 11 (1-226)), or in many examples better than AICA riboside (Compound Nos. 21 (1-227), 53 (1-468), and 66(1-531). Imidazole, which is not protective, is included to demonstrate the specificity of the response to compounds according to formula (I). TABLE II

Functional Recovery

(TAB)

Compound Cone. (μM) (%Baseline LVDP) Control (Post Electrolysis)) - - - - - 25 ± 3

1 (1-110) 20 99 ± 1

5 92 ± 4

1 61 ± 6

0.5 22 ± 3 5 (1-145) 20 100 11 (1-226) 20 98 ± 2 21 (1-227) 20 98 ± 2

1 98 ± 1

53 (1-468) 1 99 ± 1

0.5 99 ± 1

0.2 69 ± 10

0.05 31 ± 13

66 (1-531) 89 ± 7

79 (1-607) 54 ± 19 AICA base 20 104 ± 2

5 93 ± 5

Imidazole 20 19 ± 2

Example 3

Reduction of Post-Ischemic Reperfusion Injury

Isolated rat hearts were prepared as described in Example 2. Developed pressures (LVDP) were continuously monitored and coronary flows also measured gravimetrically. After equilibrating the hearts at a constant perfusion pressure of 100 cm water for 30 minutes, the hearts were subjected to low flow ischemia by reducing the perfusion pressure to 10 cm water for a period of 30 minutes and then reperfused by restoring the pressure to its original level (100 cm water) for a further 30 minutes. Addition of 10 μM, 20 μM or 100 μM 5-amino-1-β-D-ribofuranosylimidazole-4-carboxamide to the perfusion buffer at reperfusion alone significantly improved the recovery of LVDP at 30 minutes reperfusion. (See Figure 1) Selected compounds according to formula (I) were evaluated for their ability to reduce post-ischemic injury. The results are summarized in Table III.

TABLE III

Series Compound No. Conc. (μM) Function Recoverv P value

% Baseline LVDP

(# of Hearts)

Perfusion Buffer - - - - - 64 .9+0.7 ( 125) - - - - - Control (Post

Ischemia)

1 (1-110) 20 79.4 ±1.3(34) .0001

5 64.2±1.5(6) NS¹

I 10 (1-186) 20 84.5±3.5(2) .0024

5 83.7±0.7(6) .0001 ¹ NS = not significant

TABLE III (Continued)

Series Compound No. Conc. (μM) Function Recovery P value

% Baseline LVDP

(# of Hearts)

11 (1-226) 20 85.7±6.2(3) .0002

5 77.2±5.8 (7) NS¹

16 (1-273) ² 5 83.1±3.2 (5) .0001

23 (1-343) 179.0±2.3 (6) .0002

25 (1-360) 5 86.8±2.3 (6) .0001

72.4±1.6 (6) .0289

37 (1-270) 5 71.9±3.0 (5j .0500

29 (1-349) 1 76.7±2.9 (7) .0028

40 (1-392)² 20 78.5±3.7 (8) <.005

47 (1-450) 1 74.0±2.8 (6) .0045

² Known compound

TABLE III (Continued)

Series Compound No. Conc. (μM) Function Recovery P value

% Baseline LVDP

(# of Hearts)

52 (1-467) 5 86.0±2.5 (5) .0001

53 (1-468) 5 85.6±1.8 (10) .0001 59 (1-506) 1 75.8 ±2.2 (7) .0001

68 (1-538) 5 75.3±2.2 (4) .0033

69 (1-549) 5 77.0±2.8 (6) .0002 74 (1-572) 5 73.3±3.3 (6) .0012

II 27 (1-395) 5 74.6±3.7 (7) .0060

67 (1-535) 5 77.4±5.7 (3) .0045

III 19 (1-154) 20 85.5±1.7 (5) .0001

21 (1-227) 5 81.0±3.2 (8) .0001

1 77.0±4.4 (10) .0007

TABLE III (Continued)

Series Compound No . Conc. (μM) Function Recobery P value

% Baseline LVDP

(# of Hearts)

26 (1-332) 5 70.7±4.1 (8) .0466

62 (1-510) 5

75.5±2.3 (4) .0049

63 (1-517) 5

79.7±4.8 (4) .0001

65 (1-522) 5

72.3±5.6 (4) .0410

66 (1-531) 5

88.5±1.8 (5) .0001 76 (1-578) 5

79.2±2.0 (3) .0011

Example 4

Effect of AICA Riboside Incorporation in Cardioplegia

Solution

Male rats (n=8/group) were anesthesized with pentobarbital sodium (60 mg/Kg, i.p.) and subjected to a bolus of AICA riboside (100 mg/Kg, i.v.). Control animals received the same amount of saline. After 15 minutes, heparin was administered (1000 IU/Kg, i.v.) and the chest opened; hearts were excised and immediately arrested with a 2 min infusion of the St. Thomas' Hospital Cardioplegic Solution Number 2 (containing in mM: NaCl 110, KCl 16, MgCl₂1.2, NaHCO₃10, pH 7.8) at 20°C with or without added AICA riboside (20 μmol/l). The hearts were then stored for 150 minutes at 20 °C immersed in the same cardioplegic solution. Following the termination of ischaemia, all hearts were reperfused normothermically for 15 minutes in the Langendorff mode with or without AICA riboside (20 μmol/1) added to the perfusion fluid. Hearts were then converted to the working mode for a further 20 minutes. At the end of this period heart rate (HR), coronary flow

(CF), aortic flow (AF), cardiac output (CO), stroke volume (SV), and stroke work (SW) were measured. Cardiac output was calculated as the sum of coronary flow and aortic flow, stroke volume as cardiac output divided by heart rate, and stroke work as the stroke volume multiplied by the peak systolic pressure. Hearts not subjected to ischaemia were perfused for the same period (15 minutes Langendorff plus 20 minutes working heart) to serve as time-matched aerobic controls for comparative purposes.

The values obtained in eight individual hearts in the non-ischaemic aerobic control group (AICA riboside-free) are given in Table IV. Table V shows the individual values for post-ischaemic recovery in the AICA riboside-free control group, and the values from AICA riboside-treated hearts are shown in Table VI. [INSERT R FROM PAGE 82 - PHOTOCOPIED PAGE WITH TABLES IV, V & VI]

Example 5

Effect of AICA Riboside Analoσs on Inhibition of Platelet

Aggregation in Human Whole Blood

The ability of preferred AICA riboside analogs to inhibit platelet aggregation was examined in human whole blood. Whole blood was drawn from healthy donors and collected in 0.1 volume of sodium citrate to prevent coagulation. Platelet aggregation was measured by the impedance technique using a Whole Blood Aggregometer. The test compounds were incubated in whole blood for 10 minutes at 37ºC and 10 μM adenosine was added 5 minutes before eliciting aggregation. Aggregation was induced by addition of ADP (6-25 μM) at the minimum concentration inducing full aggregation in untreated controls.

The results are shown in Table VII.

TABLE VII

Series Compound No. IC₅₀(μM)

1 (1-110) 2700

4 (1-122) 200

23 (1-343) 38

28 (1-348) 180

29 (1-349) 90

51 (1-466) 193

52 (1-467) 480

53 (1-468) 150

56 (1-487) 75

59 (1-506) 70

61 (1-509) 171

71 (1-562) 40

72 (1-563) 300

II 27 (1-395) 950

43 (1-432) 620

IV 32 (1-262) 350 Example A

Preparation of 5-Amino-(2,3,5-tri-O-acetyl-beta

D-ribofuranosyl)imidazole-4-carboxamide (Compound No. 2³

(1-111))

50 g of AICA riboside was dissolved in pyridine (450 ml) and then cooled in an ice bath. Acetic anhydride (80 ml) was added and the ice bath removed. The reaction mixture was stirred for 3 hrs. TLC on silica gel, eluting with 9:1 methylene chloride:methanol, showed the reaction to be complete. Methanol (5 ml) was added to neutralize unreacted acetic anhydride. The solvents were removed by evaporation under high vacuum (bath temperature less than 40 °C). The residue was coevaporated with dimethylformamide (3 × 150 ml). The residue was crystallized from ethanol using seed crystals. The yield of the triacetate 62 g of white solid; melting point 128-129°C.

NMR (DMSO-d₆) δ ppm 2.05-2.15 (2s, 9H, -CH₃), 4.3 (broad s, 3H, 4'-CH, 5'-CH₂), 5.3 (m, 1H, 3'-CH) 5.55 (t, 1H, 2'-CH), 5.87 (d, 1H, 1'-CH), 5.9 (broad s, 2H, 5-NH₂), 6.7-6.9 (broad d, 2H, 4-NH₂), 7.4 (s, 1H, 2-CH)

The preparation of this compound was also described in U.S. Patent No. 3,450,693 to K. Suzuki & I. Kumoshiro (1969); See also Chem. Abs. 71:816982 (1969). Example B

Preparation of N -Dimethylaminomethyleneamino-beta-D-ribofuranosylimidazole-4-carboxamide (Compound No. 7 (1-164))

Dissolved 2',3',5'-tri-O-acetyl AICA riboside (10 g) in dimethylformamide (30 ml) and dimethylformamide dimethyl acetal (20 ml). The reaction mixture was allowed to stir overnight. TLC on silica gel, eluting with 9:1 methylene chloride: methanol, showed that the reaction was ³ As used herein "Compound No. refers to the compounds of Tables VIII and IX. complete by absence of starting material. The solvent was removed by evaporation under high vacuum (bath temperature less than 40°C). The residue was dissolved in cyclohexylamine and stirred overnight. The solvent was removed by evaporation under reduced pressure and the residue was crystallized from ethanol. Yield was 4.6 g of white solid, melting point 173-175°C.

NMR (MeOH-d₄), δ ppm 3.0-3.05 (2s, 6H, N(CH₃)₂), 3.75 (m, 2H, 5'-CH₂), 4.0 (g, 1H, 4'-CH), 4.2 (t, 1H, 3'-CH), 4.35 (t, 1H, 2'-CH), 5.8 (d, 1H, 1'-CH), 7.7 (s, 1H, 2-CH) , 8.25 (s, 1H, 5-N=CH-N)

Example C

Preparation of 5-Amino-1-beta-D-ribofuranosylimidazole- 4-N-(cyclopentyl)carboxamide (Compound No. 10 (1-186)) The literature procedure of P.C. Srivastava, R.W. Mancuso, R.J. Rosseau and R.K. Robins, J. Med. Chem. 17(11): 1207 (1977) was followed to synthesize N-succinimidyl-5-amino-1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-imidazole-4-carboxylate ("intermediate No. 4.") Intermediate No. 4 (3.9 g) was dissolved in methylene chloride (60 ml). Cyclopentylamine (0.8 ml) was added and the solution was stirred overnight. TLC on silica, eluting with 9:1 methylene chloride:methanol, showed the reaction was complete by absence of starting material. The solvent mixture was extracted with 5% hydrochloric acid solution (100 ml), saturated sodium bicarbonate solution (100 ml) and water (200 ml). The organic layer was dried over sodium sulfate and evaporated under reduced pressure to give 3.1 g of yellow foam. The acetyl groups were removed by dissolving the 3.1 g of foam in methanol (70 ml) and cooling in an ice bath. Ammonium hydroxide (60 ml) was added and the ice bath was removed. After 2 ½ hours stirring, TLC on silica gel, eluting with 9:1 methylene chloride:methanol, showed all starting material was gone. The solvent was evaporated under reduced pressure to give a residue which was purified on a silica column, eluting with 9:1 and 6:1 methylene chloride:methanol. Fractions which were alike by TLC were pooled and evaporated under reduced pressure to yield 1.1 g of white foam crystallized from methanol-ethyl acetate, melting point 158-160 °C.

NMR (DMSO-d₆) , δ ppm 1.4-1.9 (m, 8H, -CH₂-CH₂-), 3.6

(m, 2H, 5 ' -CH₂) , 3.9 (d, 1H, NH-CH ), 4.0-4.35 (m, 3H,

2',3',4'-CH), 5.15-5.4 (m, 3H, 2',3',5'-OH), 5.45 (d, 1H,

1'-CH), 5.9 (broad s, 2H, -NH₂), 7.1 (d, 1H, -NH-), 7.3 (s, 1H, 2-CH).

Example D

Preparation of 5-Amino-1-beta-D-ribofuranosylimidazole- 4-N-(cyclopropyl)carboxamide (Compound No. 12 (1-232))

This compound was prepared following the procedure described in Example C except cyclopropylamine (0.5 ml) was substituted for cyclopentylamine (0.8 ml). The yield starting with 6.2 g of intermediate No. 4 (the succinate ester) was 2.3 g.

NMR (DMSO-d₆) δ ppm 0.5 (m, 4H, CH₂-CH₂) 2.7 (m, 1H, N-CH ), 3.6 (m, 2H, 5'-CH₂), 3.8-4.3 (m, 3H, 2',3',4'-CH), 5.15-5.4 (m, 3H, 2^,,3',5'-OH) 5.45 (d, 1H, 1'-CH), 5.9 (s, 2H, NH₂), 7.2 (s, 1H, 2-CH) 7.4 (d, 1H, 4-NH).

Example E

Preparation of 5-Amino-1-beta-D-ribofuranosylimidazole-4-N-(benzyl)carboxamide (Compound No. 11 (1-226))

Inosine (10 g) was suspended in dimethylformamide (100 ml) and dimethylformamidedibenzylacetal (25 ml). The resulting mixture was stirred at 70°C overnight. TLC on silica, eluting with 6:1 methylene chloride:methanol, showed completion of reaction. Solvent was removed by evaporation at reduced pressure. The remainder was dissolved in ammonium hydroxide (130 ml). The mixture was stirred overnight, then evaporated under reduced pressure. Ethanol (80 ml) was added to the residue and the resulting mixture was warmed. The solid was collected by filtration. Yield of 1-benzylinosine was 10.5 g which was characterized by NMR.

The intermediate, 1-benzylinosine (10.5 g), was dissolved in ethanol (1.0 L) and 3 M sodium hydroxide solution (140 ml). This solution was refluxed for 3 hours. TLC on silica showed the reaction was complete. The solvent was removed by evaporation under reduced pressure. The residue was chromatographed on a silica gel column, eluting with 6:1 methylene chloride:methanol. Fractions were collected which were similar by TLC and concentrated until crystals appeared. Yield was 7.4 g of the above-identified compound as a white solid, melting point 178-179°C.

NMR (DMSO-d₆) δ ppm 3.6 (m, 2H, 5'-CH₂) 3.85-4.35 (m, 3H, 2',3',4'-CH), 4.4 (d , 2H , N-CH₂) , 5. 15-5 . 4 (m, 3H , 2 ' , 3 ' , 5 ' -OH) , 5. 5 (d, 1H, 1'-CH), 5.9 (broad s, 2H, 5-NH₂), 7.2-7.4 (m, 6H, 2-CH, C₆H₅) 7.95 (t, 1H, NH).

See also E. Shaw, J.A.C.S. 80:3899 (1958).

Example F

Preparation of 5-Amino-1-β-D-ribofuranosylimidazole- 4-carboxylic acid methyl ester (Compound No. 14 (1-260))

5-amino-1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)-imidazole-4-carboxylic acid (3.85 g, 10 mmol) was dissolved in 40 ml tetrahydrofuran and cooled to 0°C. An excess of diazomethane in ether was added and the mixture warmed to room temperature. Acetic acid was added to destroy excess diazomethane and the mixture was evaporated to dryness. The residue was purified by chromatography on silica gel, eluting with 7:3 ethyl acetate:hexane. The major product fractions, judged by silica thin layer chromatography (TLC) using the above system, were combined and evaporated to yield 1.2 g of a white foam. This was dissolved in 40 ml of methanol containing 20 mg of sodium methoxide and stirred for 30 minutes. Silica TLC, eluting with 6:1 methylene chloride:methanol, showed no remaining starting material and a new slower-moving product spot. The reaction was neutralized with Dowex 50 (H⁺) resin and evaporated to yield 0.64 g of the desired product as a white foam. IR (KBr):1725 cm^-1 (-CO-OCH₃).

NMR (DMSO-d₆) : δ ppm, 3.65 (s, 3H, CH₃), 3.8 (m, 3H, 4'-CH and 5'-CH₂), 4.1 (m, 1H, 3'-CH), 4.2 (m, 1H, 2'-CH), 5.5 (d, 1H, 1'-CH), 8.0 (s, 1H, 2-CH).

Example G

Preparation of 5-Amino-5'-sulfamoyl-1-β-D-ribofuranosylimidazole-4-carboxamide (Compound No. 15 (1-261))

A. Preparation of 5- Amino-2',3'-isopropylidene-1-β-ribofuranosyl-5-sulfamoylimidazole-4-carboxamide

To a solution of 2',3'-isopropylidene-AICA-riboside (2.98 g, 10 mmol) in dry N,N-dimethylformamide (25 ml), sodium hydride (300 mg, 80% dispersion in oil) was added over a period of 10 min. After the evolution of hydrogen gas had ceased, the flask was immersed in an ice bath and the mixture was stirred for 30 min. A solution of sulfamoyl chloride (1.3 g, 11 mmol) in dry tetrahydrofuran (20 ml) was added slowly. TLC of the reaction mixture (silica gel, solvent 9:1 methylene chloride:methanol) indicated presence of some starting material. An additional 200 mg of sulfamoyl chloride in tetrahydrofuran (10 ml) was added and the resulting mixture stirred for one hour. Methanol (1 ml) was added and solvent was evaporated under high vacuum. The residue chromatographed over silica gel, eluting with a mixture of methylene chloride:methanol (9:1). Several fractions were collected. Fractions showing identical TLC patterns were pooled and evaporated to a glassy product. Yield was 1.5 g.

¹H-NMR (DMSO-d₆) δ ppm, 1.25 and 1.55 (2s, 6H, C(CH₃)₂) , 4.1 (d, 2H, 5'-CH₂) , 4.25-4.35 (m, 1H, 4'-CH) , 4.8-4.9 and 5.1-5.2 (2m, 2H, 2'-CH and 3'-CH), 5.8 (d, 1H, 1'-CH), 5.9 (s, 2H, 5-NH₂), 6.65-6.95 (br. d, 2H, CONH₂), 7.35 (s, 1H, 2-CH) , 7.7 (s, 2H, SO₂NH) . The NMR data conformed to the structure of 5-amino-2,3'- isopropylidene-1-B-ribofuranosyl-5'-sulfamoylimidazole- 4-carboxamide. This intermediate product was used in the deblocking step without further purification or isolation.

B. Preparation of 5-Amino-5'-Sulfamoyl-1-β-D- ribofuranosylimidazole-4-carboxamide (Compound No.

15 (1-261))

The compound from the preceeding preparation was dissolved in 60% formic acid (20 ml) and the resulting solution was stirred at room temperature for 48 hours. The solvent was removed by evaporation under high vacuum. The residue was coevaporated with water. The product was crystallized from aqueous ethanol. Yield was 1.0 g of the above-identified product, melting point 174-175°C. ¹H-nmr (DMSO-d₆) δ ppm 3.9-4.3 (m, 5H, 2'-CH, 3'-CH, 4' -CH and 5'-CH₂), 5.4 and 5.5 (2d, 2H, 2'-OH and 3'-OH), 5.5 (d, 1H, 1'-CH), 5.8 (br.s, 2H, 5-NH₂), 6.6-6.9 (br.d, 2H, CONH₂), 7.3 (s, 1H, 2-CH) and 7.6 (s, 2H, SO₂NH₂).

Example H

Preparation of 5'-Amino-5-deoxy-AICA-riboside

(Compound No. 21 (1-227))

A. Preparation of 5'-Azido-5'-deoχy-AICA-riboside

A mixture 5'-deoxy-5'-iodo-2',3'-isopropylidene-AICA riboside (8.0 g) (Ref: P.C. Srivastava, A.R. Newman, T.R. Mathews, and R.K. Robins, J. Med. Chem., 18, 1237 (1975)), lithium azide (4.0 g), and N,N-dimethylformamide was heated at 80-90°C for 5 hours. The mixture was evaporated to dryness and the residue was chromatographed over silica gel column eluting with methylene chloride. The fast moving product-containing fractions were pooled and evaporated to obtain 7.2 g of a product which was subjected to deblocking with 60% formic acid (100 ml) at room temperature for 48 hours. Excess formic, acid was removed by evaporation under high vacuum. The residue was coevaporated with water (3 × 25 ml) to obtain a semi-solid product. This product was crystallized from aqueous ethanol. IR (KBr) cm^-1: 3400-3000 (br. NH₂, CONH₂, OH, etc.), 2150 (S, N₃) 1640 (CONH₂) .

Yield was 5.0 g, of the above-identified product, melting point 138-139ºC.

1H-NMR (DMSO-d₆) δ ppm 3.55 (d, 2H, 5'-CH₂), 3.95 (br. s, 2H, 3'-CH and 4'-CH), 4.2-4.4 (m, 1H, 2'-CH), 5.35 and 5.50 (2d, 2H, 2 '-OH and 3'-OH), 5.55 (d, 1H, 1'-CH), 5.75-5.9 (br. s, 2H, 5-NH₂), 6.6-6.9 (br. d, 2H, CONH₂) and 7.35 (s, 1H, 2-CH). B. Preparation of 5'-Amino-5'-deoxy-AICA-riboside

A solution of 5'-azido-5'-deoxy-AICA-riboside (800 mg) (the product of step (A) in methanol (40 ml) was hydrogenated in a Parr apparatus with palladium on carbon (5%) (100 mg) as the hydrogenation catalyst at 40 psi for 60 min. The catalyst was removed by filtration of the reaction mixture through a celite pad. The clear filtrate was evaporated to dryness. The product was crystallized from boiling ethanol. Yield was 650 mg of the above- identified product, melting point 188-189 °C.

1H-NMR (D₂O) δ ppm, 2.7 (d, 2H, 5'-CH₂), 3.8-4.4 (3m, 3H, 2'-CH, 3'-CH and 4'-CH), 5.4 (d, 1H, 1'-CH) and 7.3 (s, 1H, 2-CH). IR (KBr) cm^-1: 3500-3000 (br. OH, NH₂, CONH₂, etc.), 1640-1645 (br.s. C0NH₂) .

Example I

Preparation of 5-Amino-1-(2-0-methyl-β-D-ribσfuranosyl)-imidazole-4-carboxamide (Compound No. 20 (1-188))

and 5-Amino-1-(3-O-methyl-β-D-ribofuranosyl)imidazole-4-carboxamide (Compound No. 22 (1-243))

5-Amino-1-β-D-ribofuranosylimidazole-4-carboxamide (5.2 g, 20 mmol) was dissolved in 40 ml hot dimethylformamide and diluted with 70 ml methanol containing 35 mg tin (II) chloride dihydrate. A solution of 0.1 mol of diazomethane in 200 ml of ether was added in portions over 45 min. After each addition, 20 mg of tin (II) chloride dihydrate was added. The resulting mixture was filtered and evaporated to give a syrup. The syrup was dissolved in 25 ml of methanol and upon cooling yielded crystalline 5-amino-1-(2-0-methyl-,9-D-ribofuranosyl)imidazole-4-carboxamide which was collected by filtration and dried. Yield was 1.2 g, melting point

114-117ºC.

NMR (DMSO-d₆) (for Compound No. 20): δ ppm, 3.3 (s,

3H, CH₃), 3.6 (m, 2H, 5'-CH₂), 3.9 (m, 1H, 4'-CH), 4.1 (m, 1H, 2'-CH), 4.2 (m, 1H, 3'-CH), 5.2 (d, 1H, 3'-OH), 5.3 (t, 1H, 5'-OH), 5.6 (d, 1H, 1'-CH), 6.0 (br. s, 2H, 5-NH₂),

6.7 (br. d, 2H, 4-CONH₂), 7.3 (s, 1H, 2-CH).

The supernatant from the above crystallization was concentrated and applied to a 200 ml column of silica gel.

The column was eluted with 10:1 methylene chloride:methanol (1 L), 8:1 methylene chloride:methanol

(500 ml) and 5:1 methylene chloride:methanol (500 ml).

The 5:1 eluate contained a major product and was evaporated and residue dissolved in 10 ml of methanol.

Upon cooling this yielded crystals which were collected and dried. Yield was 1.4 grams. By NMR decoupling and exchange experiments the product was shown to be 5-amino- 1-(3-0-methyl-β-D-ribofuranosyl)-imidazole-4-carboxamide.

NMR (DMSO-d₆) (for Compound No. 22) δ ppm: 3.3 (s,

3H, CH₃), 3.6 (m, 2H, 5'-CH₂), 3.7 (m, 1H, 4'-CH), 4.0 (m, 1H, 3'-CH), 4.4 (m, 1H, 2'-CH), 5.3 (t, 1H, 5'-OH), 5.4

(2d, 2H, 2'-CHand 1'-CH), 5.9 (br. s, 2H, 5-NH₂), 6.7 (br. d, 2H, CO-NH₂), 7.7 (s, 1H, 2-CH).

Example J

Preparation of 5-Amino-1-β-D-ribofuranosylimidazole-4-N- [ (4-nitrophenyl) methyl ] carboxamide (Compound No. 23 (1-343) )

N-Succinimidyl-5-amino-1- ( 2 , 3 , 5-tri-O-acetyl- β-D-ribofuranosyl ) imidazole-4-carboxylate⁴ ( 0. 50 g) ,

4-nitrobenzylamine hydrochloride (210 mg) and ⁴ Srivastava, P. C . , J . Med . Chem. 17 : 1207 ( 1974) triethylamine (0.16 ml) were stirred in chloroform (30 ml) at room temperature overnight. The solution was washed with saturated sodium bicarbonate solution and water, then evaporated under reduced pressure. The resulting yellow tar was chromatographed on silica gel, eluting with 9:1 methylene chloride:methanol. The collected fractions were monitored by TLC. The like fractions were combined and concentrated under reduced pressure to afford a yellow foam (0.38 g). The foam was dissolved in methanol (20 ml) and methanolic sodium methoxide solution was added (0.3 ml of 0.25 M solution). The solution was stirred under an argon atmosphere for 15 min. TLC indicated the reaction was complete. The solution was neutralized to pH 6 with ion exchange resin. The resin was filtered and the solution concentrated under high vacuum to yield a yellow foam (0.23 g).

NMR (DMSO-d₆) δ ppm, 3.6 (m, 2H, 5'-CH₂) 3.9-4.3 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 4.5 (d, 2H, -CH₂-C₆H₄-NO₂), 5.2- 5.4 (br., 3H, 2 '-OH, 3'-OH, 5'-OH), 5.5 (d, 1H, 1'-CH), 6.0 (br. s, 2H, 5-NH₂), 7.3 (s, 1H, 7-CH), 7.4-8.2 (AB_q, 4H, -C₆H₄-NO₂), 8.3 (t, 1H, 4-CONH).

Example K

Preparation of 5-amino-1-β-D-ribofuranosylimidazole- 4-N-[(3-chlorophenyl)methyl]carboxamide (Compound No. 24 (1-354))

This compound was prepared according to the procedures described in Example J for the 4-p-nitrobenzyl derivative, substituting 2-chlorobenzylamine for 4-nitrobenzylamine hydrochloride.

NMR (DMSO-d₆) δ ppm, 3.6 (m, 2H, 5'-CH₂), 3.9-4.3 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 4.4 (d, 2H, -CH₂-O-Cl), 5.1-5.4 (br., 3H, 2' -OH, 3'-OH, 5'-OH) , 5.5 (d, 1H, 1'-CH), 6.0 (br.s., 2H, 5-NH₂), 7.2-7.4 (m, 4H, -C₆H₄-Cl), 8.0 (t, 1H, 4-CONH). Example L

Preparation of 5-Amino-1-β-D-ribofuranosylimidazole- 4-N-[(2,4-dichlorophenyl)methyl]carboxamide (Compound No. 25 (1-360))

This compound was prepared according to the procedures described in Example J for the 4-p-nitrobenzyl derivative, substituting 2,4-dichlorobenzylamine for 4-nitrobenzyl hydrochloride.

NMR (DMSO-d₆) , δ ppm, 3.6 (m, 2H, 5'-CH₂), 3.9-4.3 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 4.4 (d, 2H, -CH₂-C₆H₄-Cl₂), 5.2-

5.4 (m, 3H, 2'-OH, 3'-OH, 5'-OH), 5.5 (d, 1H, 1'-CH), 6.0

(br. s, 2H, 5-NH2), 7.2-7.6 (m, 3H, -C₆H₃-C1₂) , 8.1 (t, 1H,

4-CONH-).

Example M

Preparation of 5-Amino-2-thio-1-β-D-ribofuranosyl

imidazole-4-carboxamide (Compound No. 27 (1-395))

To 10 ml of 80% formic acid was added 400 mg of 5-amino-2-thio-1-(2,3-O-isopropylidene-β-D-ribofuranosyl)- imidazole-4-carboxamide.⁵ The resulting mixture was stirred for 1 hour at room temperature. Silica TLC, eluting with 4:1 methylene chloride:methanol, showed conversion of staring material to one major product. The mixture was evaporated to dryness, dissolved in 5 ml of methanol and applied to a 50 ml column of silica gel. The column was eluted with methylene chloride:methanol (5:1). The major product, as determined by TLC, was collected and evaporated to dryness. The residue was dissolved in 3 ml of hot methanol and crystallized upon cooling. Yield was 150 mg of the above-identified product, melting point 205- 208°C.

NMR (DMSO-d₆), δ ppm 3.6 (m, 2H, 5'-CH₂), 3.8 (m, 1H, 4'-CH), 4.1 (m, 1H, 3'-CH), 4.5 (m, 1H, 2'-CH), 5.1 (d, 1H, 2' or 3'-OH), 5.2 (d, 1H, 2' or 3'-OH), 5.7 (t, 1H, ⁵ Preparation described in T. Miyoshi, S. Suzaki, A. Yamazaki, Chem. Pharm. Bull, 24 (9) 2089-2093 (1976). 5' -OH), 6.3 (d, 1H, 1'-CH), 6.4 (br. s, 2H, 5-NH₂), 6.9 (br. s, 2H, 4-CONH₂), 11.1 (br. s, 1H, 5'-SH).

Example N

Preparation of 5-Amino-1-(5-chloro-5-deoχy-β-D-ribofuranosyl) imidazole-4-carboxamide (Compound No. 26 (1-332))

AICA riboside (1.00 g) , triphenylphosphine (3.05 g) and carbon tetrachloride (1.15 ml) were stirred in dimethyl formamide (38 ml) at room temperature for 3 hours. The solution was diluted with methanol (15 ml), then concentrated under reduced pressure. The resulting yellow tar was chromatographed on silica gel, eluting with 4:1 methylene chloride:methanol. The like fractions were combined and concentrated under reduced pressure to afford a purple foam. The presence of triphenylphosphine oxide, as determined by ¹H NMR, necessitated a second chromotographic step as above. Yield was 0.43 g of a white foam.

¹H-NMR (DMSO-d₆), δ ppm 3.7-3.9 (m, 2H, 5'-CH₂), 4.0-4.4 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 5.4-5.5 (m, 2H, 2'-OH, 3'-OH), 5.6 (d, 1H, 1'-CH), 5.9 (br. S, 2H, 5-NH₂), 6.7-6.9 (br. d, 2H, 4-CONH₂), 7.3 (s, 1H, 2-CH).

Example 0

Preparation of 5-Amino-1-(2-O-ethyl-β-D-ribofuranosyl)-4-imidazole carboxamide (Compound No. 34 (1-250) ) and 5-Amino-1-(3-O-ethyl-β-D-ribofuranosyl)-4-imidazole carboxamide (Compound No. 31 (1-251))

A solution of approximately 30 mmol diazoethane in 40 ml of ether was prepared by slow addition of 7 g (44 mmol) of 1-ethyl-3-nitro-1-nitrosoguanidine to a mixture of 8 g of potassium hydroxide, 9 ml water and 60 ml of ether followed by distillation. This was slowly added to a solution of 3.2 g (12 mmol) of 5-amino-1-β-D-ribofuranosylimidazole-4-carboxamide (AICA riboside) in 35 ml dimethylformamide containing 50 mg of tin(II) chloride dihydrate. During the addition approximately 20 ml of methanol was added to maintain solubility. The reaction was filtered to remove a trace precipitate and evaporated to a yellow syrup. Thin layer chromatography on silica gel using methylene chloride/methanol (3:1) showed a major product spot moving faster than AICA riboside. The syrup was chromatographed on silica gel using methylene chloride/methanol (8:1) collecting the major product as determined by TLC. The appropriate fractions were evaporated to a white foam. This was dissolved in 7 ml of methanol. Upon cooling to 4°C the mixture crystallized to yield 160 mg of 5-amino-1-(2-O-ethyl-β-ribofuranosyl)imidazole-4-carboxamide (Compound No. 34 (1-250)) confirmed by NMR decoupling and exchange experiments.

1H NMR (DMSO-d₆) (for Compound No. 34): 5 ppm, 1.05 (t, 3H, CH₃), 3.3-3.6 (m, 4H, 2'-OCH₂-, 5'-CH₂), 3.9 (m, 1H, 4'-CH), 4.1-4.3 (m, 2H, 2'-CH, 3'-CH), 5.15 (d, 1H, 3 '-OH), 5.25 (t, 1H, 5' -OH), 5.55 (d, 1H, 1'-CH), 6.0 (br.s, 2H, 5-NH₂) , 6.6-6.9 (br.d, 2H, 4-CONH₂), 7.3 (S, 1H, 7-CH).

The supernatant from the above crystallization was cooled overnight at -12°C yielding a second crop of crystals, 0.58 g, which by NMR decoupling and exchange experiments was shown to be mostly 5-amino-1-(3-O-ethyl-β-D-ribofuranosyl) imidazole-4-carboxamide (Compound No. 31 (1-251)).

¹H NMR (DMSO -d₆) (for Compound No. 31): δ ppm, 1.1 (t, 3H, CH₃) , 3.4-3.7 (m, 4H 3'-OCH₂-, 5'-CH₂), 3.85 (m, 1H, 4'-CH), 4.0 (m, 1H, 3'-CH) 4.4 (q, 1H, 2-CH), 5.25 (t, 1H, 5' -OH), 5.35 (d, 1H, 2'-OH), 5.45 (d, 1H, 1'-CH), 5.9 (br.s, 2H, 5-NH₂), 6.6-6.9 (br.d, 2H, 4-CONH₂), 7.3 (s, 1H, 1-CH). The major impurity was identified as the 2'-O-ethyl isomer. Example P

Preparation of 5-Amino-1-(2-O-n-butyl-β-D-ribofuranosyl) imidazole-4-carboxamide and 5-Amino-1-(3-O-n-butyl-β-D-ribofuranosyl) imidazole-4-carboxamide

(Compound Nos. 32 (1-262) and 33 (1-263))

5-Amino-1-β-D-ribofuranosylimidazole-4-carboxamide (2.50 g, 10.0 mmol) and tin(II) chloride hydrate (35 mg) were dissolved in dimethylformamide (40 ml) and methanol (30 ml). A solution of 0.1 ml of diazobutane⁶ in 150 ml of ether was added in portions. Halfway through the addition, more tin (II) chloride hydrate was added (35 mg). Methanol was added, as needed, to ensure the starting material stayed in solution. The mixture was stirred for 1 hr, then concentrated under reduced pressure to give an oil. Analysis of the oil by ¹H NMR showed mostly N-butylethylcarbamate. The oil was stirred with hexane and decanted to remove the N-butylethylcarbamate. The resulting tar was chromatographed on silica gel using 6:1 methylene chloride:methanol as eluting solvent. The appropriate fractions were combined and concentrated under reduced pressure to give a pink foam. ¹H NMR analysis showed a mixture of 2' and 3' butyl ethers. HPLC analysis showed a 56:28 mixture. The solid was dissolved in isopropanol (2 ml) and cooled. The resulting solid was filtered and dried to give 63 mg. HPLC analysis showed a 77/18 mixture. ¹H NMR decoupling and exchange experiments showed the major product to be the 2'-0-n-butyl ether.

¹H-NMR (DMSO-D₆) (for Compound No. 32): δ ppm, 0.8-1.5 (m, 7H, -CH₂CH₂CH₃), 3.3-4.2 (m, 7H, 2'-OCH₂-, 2'-CH, 3'-CH, 4'-CH, 5'-CH₂), 5.1 (d, 1H, 3'-OH), 5.3 (t, 1H, 5'-OH), 5.6 (d, 1H, 1'-CH), 6.0 (br.s, 2H, 5-NH₂), 7.6- 7.8 (br.d, 2N, 4-CONH₂), 7.3 (s, 1H, 2-CH). ⁶ Diazobutane was prepared by treatment of 16.5 g of N-nitroso-N-n-butylmethane [Wilds, A.L. and Meeder, A.L., SOC 13 (1948)] in ethyl ether (100ml) with potassium hydroxide (55 g) in water (60 ml). The ethereal diazobutane was used without distillation. The supernatant from the above crystallization was concentrated under reduced pressure to give 125 mg of a pink foam. HPL analysis showed a 14/71 mixture. ¹H NMR decoupling and exchange experiments showed the major product to be the 3'-O-n-butyl ether.

¹H NMR (DMSO-D₆) (for Compound No. 33): δ ppm, 0.8- 1.6 (m, 7H,-CH₂CH₂CH₃), 3.4-4.4 (m, 7H, 3'-OCH₂-, 2'-CH,

3'-CH, 4'-CH, 5'-CH₂), 5.2 (t, 1H, 5'-OH), 5.3 (d, 1H,

2'-OH), 5.4 (d, 1H, 1'-CH), 5.9 (br.s, 2H, 5-NH₂), 6.6-6.8 (br.d., 2H, 4-CONH₂), 7.3 (s, 1N, 7-CH).

Example O

Preparation of 5-Amino-1-β-D-ribofurano-sylimidazole-4- N-[(3-nitrophenyl)methyl] carboxamide (Compound No. 28

(1-348))

This compound was prepared according to the procedures described in example J for the 4-p-nitrobenzyl derivative, substituting 3-nitrobenzylamine hydrochloride for 4-nitrobenzylamine hydrochloride.

NMR (DMSO-D₆) δ ppm 3.6 (m, 2H, 5'-CH₂), 3.9-4.3 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 4.4 (d, 2H, -CH₂- NO₂), 5.2-5.4

(br., 3H, 2'-OH, 3'-OH, 5'-O), 5.5 (d, 1H, 1'-CH), 6.0

(br.s., 2H, 5-NH₂), 7.4 (s, 1H, 7-CH) , 7.6-8.2 (m, 4H,

-C₆H₄Cl), 8.3 (t, 1H, 4-CONH).

Example R

Preparation of 5-Amino-1-β-D-ribofurano-sylimidazole-4- N-[(4-chlorophenyl)methyl] carboxamide (Compound No. 29

(1-349))

This compound was prepared according to the procedures described in Example J for the 4-p-nitrobenzyl derivative, substituting 4-chlorobenzene amide for

4-nitrobenzylamine hydrochloride.

NMR (DMSO-D₆) δ ppm 3.6 (m, 2H, 5'-CH₂), 3.9-4.3 (m,

3H, 2'-CH, 3'-CH, 4'-CH), 4.4 (d, 2H, -CH₂-C₆H₄-Cl), 5.2-5.4

(br., 3H, 2'-OH, 3' -OH, 5'-OH), 515 (d, 1H, 1'-CH) 5.9 (br.s., 2H, 5-NH₂) , 7.3-7.4 (m, 5N, -C₆H₄Cl) , 7-CH) , 8.1 (t, 1H, 4-CONH).

Example S

Preparation of 5-Amino-1-β-D-ribofurano-sylimidazole-4-N-[(4-methylphenyl)methyl] carboxamide (Compound No. 30 (1-388))

This compound was prepared according to the procedures described in Example J for the 4-P-Nitrobenzyl derivative, substituting 4-methylbenzylamine for 4-nitrobenzylamine hydrochloride.

NMR (DMSO-D₆) δ ppm 2.2 (s, 3H, -C₆H₄-CH₃), 3.6 (m, 2H,

5'-CH₂), 3.9-4.3 (m, 5H, 2'-CH, 3'-CH, 4'-CH, -CH₂-C₆H₄-Me),

5.2-5.4 (br., 3H, 2' -OH, 3' -OH, 5'-OH), 5.5 (d, 1H, 1'-CH), 5.9 (br.s., 2H, 5-NH₂, 7.1-7.2 (M, 4H, -C₆H₄-Me), 7.3 (S, 1H, 7-CH), 7.9 (t, 1H, 4-CONH).

Example T

Preparation of 5-Amino-1-β-D-ribofuranosyl-imidazole-4- Nf(3-chlorophenyl)methyl]carboxamide (Compound No. 35

(1-355))

This compound was prepared according to the procedures described in Example J for the 4-B-nitrobenzyl derivative, substituting 3-chlorobenzylamine for

4-nitrobenzylamine hydrochloride.

NMR (DMSO,-d₆) δ ppm, 3.6 (m, 2H, 5'-CH₂), 3.9-4.3 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 4.3 (d, 2H, -CH₂ Cl), 5.1-5.4

(br., 3H, 2'-OH, 3'-OH, 5'-OH), 5.5 (d, 1H, 1'-CH), 6.0

(br.s., 2H, 5-NH₂), 7.2-7.4 (m, 4H, -C₆H₄-Cl), 7.4 (s, 1H,

7-CH), 8.1 (t, 1H, 4-CONH).

Example U

Preparation of 5-Amino-4-(1-piperidinocarbamoyl)-1-β-D-ribofuranosylimidazole (Compound No. 36 (1-207))

This compound in Example J for the 4-p-nitrobenzyl derivative, substituting piperidine for 4-nitrobenzylamine hydrochloride. The product was crystallized from ethanol. m.p. 190-192ºC. NMR (DMSO-d₆) δ ppm 1.4-1.7 (M, GH, 3, 4, 5-CH₂ groups of piperidine ring), 3.55 (m, 2H, 5'-CH₂), 3.8-3.95 (m, 5H, 2- and 6-CH₂ groups of piperidine ring, and 4'-CH), 4.0-4.1 (m, 1H, 3'-CH), 4.25-4.35 (m, 7H, 2-CH) 5.15 (d, 1H, 2 ' or 3'-OH), 5.2 (t, 1H, 5' -OH).

Example V

Preparation of 5-Amino-1-β-D-ribofuranosyl-imidazole-4-N-[p-methoxybenzyl)carboxamide (Compound No. 39 (1-390))

A mixture of the activated succinate ester (0.5 g) (prepared according to Example J), 4-methoxybenzylamine (0.15 ml) and methylene chloride (20 ml) was sirred overnight. TLC indicated completion of the reaction. The solvent was evaporated and the residue was chromatographed over a short silica gel column using a mixture of methylene chloride:methanol (9:1). The fractions containing the product were pooled and evaporated. The residue thus obtained was dissolved in methanol (20 ml) and the pH was adjusted to about 10 by adding a sodium methoxide solution. After stirring the reaction mixture for 45 minutes at room temperature, the solution was neutralized with Dowex 50 H+-resin (pH about 6.0). The resin was filtered off, washed with methanol (2 x 2 ml) . The combined filtrate and the washings was evaporated and the residue was crystallized from ethanol. Yield was 100 mg, with a mp of 187-188 °C.

¹H NMR (DMSO-d₆): δ ppm, 3.55 (m, 2H, 5'-CH₂), 37 (s, 3H, -OCH₃), 3.7-4.1 (m, 3H, 2'-CH, 3'-CH, and 4'-CH), 4.35-4.2 (dd, 2H, -CH₂-N-), 5.1-5.4 (3,m, 3H, 2'-OH, 3'-OH, and 5'-OH), 5.45(d, 1H, 1-CH), 5.9 (br. 2H, NH₂), 6.8-7.2 (m, 4H, aromatic-phenyl), 7.3(s, 17H, C₂-H) , and 7.85 (t, 1H, C-NH). Example W

Preparation of 5-Amino-1-β-D-ribofuranosylimidazole-4-N(4-dimethylaminobenzyl)-carboxamide hydrochloride (Compound No. 41 (1-396-3))

To a suspension of 4-dimethylaminobenzylamine hydrochloride (245 mg, 2 mmol) in methylene chloride (25 ml), triethylamine (222 mg, 2 mmol) was added and the resulting mixture stirred 45 minutes to it was added the activated succinate ester prepared according to example J (500 mg); the resulting mixture was stirred at room temperature overnight. TLC indicated completion of the reaction. The reaction mixture was evaporated and the residue was chromatographed through a short silica gel column using a mixture of methylene chloride-methanol (9:1). Fractions showing the major product were pooled and evaporated to dryness. The residue was dissolved in methanol (15 ml) and the pH was adjusted to about 10 using a sodium methoxide solution. After stirring at room temperature for 45 mintues, the solution was neutralized with Dowex 50-resin. The resin was filtered off and washed with methanol (2 × 5 ml). The combined filtrate and the washings were evaporated to dryness. The residue which was in the form of a foam was dissolved in absolute ethanol (10 ml). The pH of the solution was adjusted to about 5 with an ethanolic-HCl solution. Solvent was evaporated to dryness and the residue was treated with anhydrous ether. The amorphous solid that separated was collected by filtration and washed with ether (2 × 10 ml), and dried under high vacuum to yield 250 mg. The compound obtained was highly hygroscopic; no melting point could be obtained.

¹H NMR (D₂O) δ ppm, 3.05 (s, 6H, N(CH₃)₂), 3.6 (m, 2H, 5'-CH₂), 3.8-4.3 (3m, 3H, 2'-CH, 3'-CH, and 4'-CH), 4.4 (s, 2H, CH₂-N-) , 5.5 (d, 1H, 1'-CH), 7.3-7.4 (m, 4H, phenyl), and 7.9 (s, 1H, 2-CH). Example X

Preparation of (R)-5-Amino-1-β-D-ribofuranosylimidazole-4-N-[2-hydroxy-2-(3,4-dihvdroxyphenγl) ethyl]carboxamide (Compound 42 (1-431))

This compound was prepared according to the procedure described in Example J substituting (R)-norepinephrine for 4-nitrobenzylamine hydrochloride and dimethylformamide in place of chloroform as the reaction solvent.

¹H NMR (DMSO-d₆) : δ ppm, 3.1 - 3.3 (m, 2H,-CH₂-N), 3.5-3.6 (m, 2H, 5'-CH₂), 3.8-3.9 (m, 1H, 4'-CH) 4.0-4.1 (m, 1H, 3'-CH) 4.2-4.3 (m, 1H, 2'-CH), 4.4-4.5 (m, 1H, phenyl- CH-OH), 5.2-5.2 (m, 1H, 2' or 3' -OH), 5.2-5.3 (t, 1H, 5'-OH) 5.3-5.4 (m, 1H, 2' or 3'-OH), 5.4-5.5 (d, 1H, 1'-CH), 5.9 (br. s, 2H, 5-NH₂), 6.5-6.8 (m, 3H, aryl of catechol), 7.1 (t, 1H, 4-CONH), 7.3 (s, 1H, 2-CH), 7.2-7.8 (br. s, 2H, catechol-OH).

Example Y

Preparation of 5-Amino-2-thiophenyl-1-β-D-ribofuranosylmidazole-4-carboxamide (Compound No. 43 (1-432))

5-Amino-2-bromo-1-(2,3-0-isopropylidene-β-D-ribofuranosyl)imidazole-4-carboxamide¹ (1.1 g), thiophenol (1.3 g) and triethylamine (0.61 g) were refluxed in a mixture of 25 ml methanol and 3 ml of 1 N sodium hydroxide for 18 hours. The reaction mixture was concentrated and the residue mixed with 40 ml of methylene chloride. The methylene chloride mixture was washed with water and saturated sodium bicarbonate and dried over magnesium sulfate. The methylene chloride was evaporated and the residue purified by chromatography on 200 ml of silica gel using a mixture of methylene chloride and methanol (95:5), yielding 0.5 g of 5-amino-2-thiophenyl-1-(2,3-0-isopropylidene-β-D-ribofuranosyl)imidazole-4-carboxamide. Treatment of that compound with 80% formic acid for 3 hours at room termperature to remove the isopropylidene ¹ Miyosi T., Chem. Pharm. Bull. 24:2089 (1976) group followed by evaporation and purification by silica chromatography using methylene chloride:methanol (9:1) yielded 250 mg of the title compound as a white foam.

¹H NMR (DMSO-d₆) δ ppm, 3.3-3.5 (m, 2H, 5'-CH₂), 3.8-3.9 (m, 1H, 4'-CH) 4.0-4.1(m, 1H, 3'-CH), 4.5 (q, 1H, 2'-CH) 5.1 (d, 1H, 2'- or 3' -OH), 5.3 (d, 1H, 2' -or 3' -OH), 5.7 (t, 1H, 5'-OH), 5.9 (d, 1H, 1'-CH) 7.5 (br. s, 2H, 4-NH₂), 6.7 and 7.1 (br s, 2H, CONH₂) 7.1-7.5 (m, 5H, phenyl). Example Z

Preparation of 5-Amino-1-β-D-ribofuranosylimidazole-4-N-(2-endo-norbornyl)carboxamide) (Compound No. 45 (1-438))

A mixture of (±) endo-2-aminonorbornane hydrochloride (240 mg), triethylamine (160 mg) and methylene chloride was stirred at room temperature for 45 minutes under argon. To it was added activated succinate ester (See Example J) (750 mg) and stirred overnight. TLC indicated completion of the reaction. Solvent was evaporated and the residue chromatographed over silica gel column using a mixture of methylene chloride.methanol (9:1). Fractions containing the product were pooled and evaporated. The residue was dissolved in methanol (25 ml) and the pH was adjusted to about 10 with a sodium methoxide solution. After stirring for 45 minutes at room temperature the solution was neutralized with H+ resin (pH approximately 6) . The resin was filtered off and washed with methanol. The combined washings and the filtrate was evaporated and the residue kept under high vacuum to obtain a solid glossy product. Yield was 280 mg.

1H NMR (DMSO-d₆) δ ppm, 1.1-2.4 (m, 10H, norbonyl), 3.6 (br.M, 2H, 5'-CH₂), 3.9 (m, 1H, -N-CH), 4-4.4 (2 m, 3H, 2'-CH, 3'-CH and 4'-CH), 5.05, and 5.35 (2-d, 2H, 2'-OH and 3' -OH), 5.25 (t, 1H, 5'-OH), 5.5 (d, 1H, 1'-CH), 5.9 (br. 2H, NH₂) 6.8 (d, 1H,-NH-CO), 7.25 (S, 1H, 2-CH). Example AA

Preparation of 5-Amino-1-β-D-ribofuranosyl- imidazole-4-N-[(3-iodophenγl)methyl]carboxamide

(Compound No. 44 (1-434))

This compound was prepared according to the procedures described in Example J for the 4-p-nitrobenzyl derivative, substituting 3-iodobenzylamine hydrochloride for 4-nitrobenzylamine hydrochloride.

¹H NMR (DMSO-d₆) δ ppm, 3.6 (m, 2H, 5'-CH₂), 3.9-4.3 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 4.3 (d, 2H, -CH₂-C₆H₄-I), 5.2-5.4 (m, 3H, 2'-OH, 3'-OH, 5'-OH), 5.5 (d, 1H, 1'-CH), 5.9 (br.s., 2H, 5-NH₂), 7.1-7.7 (m, 4H, -C₆H₄), 7.3 (s, 1H, 2-CH), 8.1 (t, 1H, 4-CONH-)

Example AB

Preparation of 5-Amino-1-(5-iodo-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-[(4-nitrophenyl)

methyl]carboxamide (Compound No. 46(1-445))

The compound used in this procedure, 5-amino-1- (5-iodo-5-deoxy-2,3-isopropylidene-β-D-ribofuranosyl)imidazole-4-N-[(4-nitrophenyl)methylcarboxamide, was prepared by the same reaction sequence (stopping at step B) described in Example AH for compound 53 (1-468), substituting the 4-N-p-nitrobenzylamide (compound 23 (1-343)) for the 4-N-p-chlorobenzylamide (compound 29 (1-349)).

5-Amino-1-(5-iodo-5-deoxy-2,3-O-isopropylidene-β-D-r i b o f u r a n o s y l ) i m i d a z o l e - 4 - N -[(4-nitrophenyl)methylcarboxamide (200 mg) was dissolved in 10 ml of 80% formic acid. The solution was stirred at 45°C for 2 hours. The solvents were evaporated under reduced pressure and the resulting residue co-evaporated twice with water and twice with methanol. The residue was chromatographed on silica gel, using 6/1 methylene chloride/methanol as eluting solvent. The appropriate fractions were combined and concentrated under reduced pressure to yield 60 mg of the above-identified compound as a yellow foam.

¹H NMR (DMSO-d₆) δ ppm, 3.3-3.6 (m, 2H, 5'-CH₂),

3.8-4.4 (m, 3H, 2'-CH, 3'-CH4'-CH), 4.5 (d, 2H, CH₂-C₆H₄NO₂) , 5.4-5.5 (m, 2H, 2 ' -OH, 3 ' -OH) , 5. 6 (d, 2H,

1 ' -CH) , 5 . 9 (br. s . , 2H, 5-NH₂) , 7 .4 (S , 1H, 2-CH) , 7 . 5- 8 . 2 (m, 4H , C₆H₄-NO₂, 8 . 3 (4 , 1H, 4-CONH-) .

Example AC

Preparation of 5-Amino-1-β-D-ribofurano- sylimidazole-4-carboxylic Acid, p-Nitrobenzylthio Ester (Compound No. 47 (1-450))

5-Amino-1(2,3,5-tri-O-acetyl-β-D-ribofuranosyl) imidazole-4-carboxylic acid¹ (1.0 g) was dissolved in 8 ml of thionyl chloride under argon with stirring for 10 minutes. The mixture was evaporated under vacuum and the residue was dissolved in 15 ml of tetrahydrofuran containing 2.0 g of p-nitrobenzyl mercaptan. Triethylamine (1.5 ml) was added and the mixture stirred under argon for 20 minutes. The reaction is evaporated to a gum and the residue mixed with 50 ml of methylene chloride and washed with 2 × 25 ml of water. The methylene chloride phase was dried over magnesium sulfate and evaporated to a syrup which was purified by chromatography on silica gel using a mixture of ethyl acetate and methylene chloride (1:1) yielding 500 mg of 5-amino-1-(2,3,5-tri-O-acetyl-,9-D-ribofuranosyl) imidazole-4-carboxylic acid, p-nitrobenzylthio ester. Treatment with sodium methoxide in 30 ml of dry methanol such that a slightly basic pH was maintained until deacetylation was complete (as determined by thin layer chromatography), followed by neutralization with Dowex 50 (H+) and evaporation yielded the desired compound contaminated with a product presumed to be the methyl ester. Purification by chromatography on silica using a mixture of methylene ¹ Srivastava, P.C., J. Med. Chem. 17:1207 (1974) chloride and methanol (9:1) gave 38 mg of the desired compound as a yellow foam.

¹H NMR (DMSO-d₆) c5 ppm, 3.5-3.7 (m, 2H, 5'-CH₂),

3.9-4.0 (m, 1H, 4'-CH), 4.2-4.4 (m, 2H, 2' -and 3'-CH), 5.2 (d, 1H, 2'-or 3'-OH), 5.3-5.5 (m, 2H, 5' and 2 ' -or 3'-OH),

5.6 (d, 1H, 1'-CH), 6.9 (br. s, 2H, 5-NH₂) , 7.4 (s, lh,

2-CH) , 7.6 and 8.2 (d, 2H, phenyl).

Example AD

Preparation of 5-Amino-1-β-D-ribofuranosyl-imidazole-4-N-indolinylcarboxamide (Compound No. 48

( 1-452))

This compound was prepared according to the procedures described in Example J for the 4-p-nitrobenzyl derivative, substituting indoline for 4-nitrobenzylamine hydrochloride.

¹H NMR (DMSO-d₆) δ ppm, 3.1 (t, 2H, indolinyl-CH₂), 3.6

(m, 2H, 5'-CH₂-), 5.2-5.4 (m, 3H, 2'-OH, 3'-OH, 5'-OH), 5.5

(d, 1H, 1'-CH), 6.4 (br.s., 2H, 5-NH₂), 6.9-8.1 (m, 4H, indolinyl aromatics), 7.4 (S, 1H, 2-CH). Example AE

Preparation of (R)-5-Amino-1-β-D-ribofuranosylimidazole 4-N-[1-4-nitrophenyl)ethyl]

carboaxamide (Compound No. 49(1-453))

This compound was prepared accoridng to the procedures described in Example J for the 4-p-nitrobenzyl derivative, substituting (R)-4-nitro-α-methylbenzylamine hydrochloride for 4-nitrobenzylamine hydrochloride.

¹H NMR (DMSO-d₆) δ ppm, 1.5 (d, 3H, α-methyl on

N4-benzyl carboxamide), 3.6 (m, 2H, 5'-CH₂), 3.9-4.3 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 5.1 (m, 1H, methine proton on

N4-benzylcarboxamide), 5.1-5.4(m, 3H, 2'-OH3'-OH, 5'-OH),

5.5 (d, 1H, 1'-CH), 7.3 (s, 1H, 2-CH) , 7.6-8.2 (m, 4H,

C₆H₄-NO₂) , 8.0 (d, 1H, 4-C0NH-) . Example AF

Preparation of (S)-5-Amino-1-β-D-ribofuranosylimidazole-4-N-[1-(4-nitrophenyl)ethyl]

carboxamide (Compound No. 50(1-459))

This compound was prepared according to the procedures described in Example J for the 4-p-nitrobenzyl derivative, substituting (S)-4-nitro-α-methylbenzylamine hydrochloride for 4-nitrobenzylamine hydrochloride.

'H NMR (DMSO-d₆) δ ppm, 1.5 (d, 3H, α-methyl on N4-benzyl carboxamide), 3.6 (m, 2H, 5-CH₂), 3.9-4.3 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 5.1 (m, 1H, methine proton on N4-benzylcarboximide), 5.1-5.4 (m, 3H, 2'-OH, 3' -OH, 5'-OH), 5.5 (d, 1H, 1'-CH' 5.9 (br.s., 2H, 5-NH₂), 7.4 (s, 1H, 2-CH), 7.6-8.2 (m, 4H, C₆H₄NO₂) 8.0 (d, 1H, 4-CONH-). Example AG

Preparation of 5-Amino-1-(5-chloro-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-[4-nitrophenyl)

methyl]carboxamide (Compound No. 51(1-466))

5-amino-1-β-D-ribofuranosylimidazole-N-[(4-nitrophenyl)methyl]carboxamide, Compound 23 (1-343) (0.5g), triphenylphosphine (1.00 g), carbon tetrachloride (0.37 ml), and THF (25 ml) were combined and stirred at ambient temperature, under argon, overnight. A white precipitate formed. Dimethylformamide (8 ml) was added and the solution was stirred at ambient temperature, under argon, overnight. The solvent was evaporated under reduced pressure and the resulting oil co-evaporated with methanol (3 × 20 ml). The resulting viscous oil was chromatographed on silica gel, using 7:1 methylene chloride:methanol as eluting solvent. The appropriate fractions were combined and concentrated in vacuo to give a yellow foam (0.28 g). The foam was crystallized from cold methanol to give yellow crystals (200 mg), mp = 174-176°C.

'H NMR (DMSO-d₆) δ ppm 3.7-3.9 (m, 2H, 5'-CH₂), 4.0-4.4 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 4.5 (d, 2H , -CH₂-C₆H₄NO₂) , 5 . 4-5 . 6 (m, 2H , 2 ' -OH, 3 ' -OH) , 5 . 6 (d, 1H, 1 ' -CH) , 5. 9 (br. s . , 2H, 5-NH₂) , 7 . 4 (s , 1H, 2-CH) , 7. 5 - 8 . 2 (m, 4H , -C₆H₄NO₂) , 8 . 3 (t , 1H, 4-CONH-) .

Example AH

Preparation of 5-Amino-1-(5-azido-5-deoxy-β-D- ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]- carboxamide (compound 52 (1-467)) and 5-Amino-1- (5-amino-5-deoxy-β-D-ribofuranosyl)imidazole-4-N- [(4-chlorophenyl)methyl]carboxamide Hydrochloride

(Compound No. 53 (1-468))

A. Preparation of 5-Amino-1-(2,3-O-isopropylidene-β-D-ribofuranosyl)imidazole-4-N- [(4-chlorophenyl)methylcarboxamide

Compound 29 (1-349), (6.8 g, 17.8 mmole), was dissolved in a mixture of 100 ml DMF, 15 ml acetone and 15 ml 2,2-dimethoxypropane. Hydrogen chloride gas

(approximately 1.0 g) was added and the mixture stirred under argon for 4 hours. The mixture was poured into 50 ml of saturated sodium bicarbonate and evaporated under vacuum at 45°C. The residue dissolved in a mixture of 100 ml ethyl acetate and 25 ml water. The ethyl acetate phase was separated and washed with 25 ml of water, dried over magnesium sulfate and concentrated to a foam. TLC (silica gel, 9:1 methylene chloride:methanol) showed a significant faster moving impurity in the product which was identified as the 5'-(2-methoxypropane) mixed ketal of the above-identified compound. This was converted to the above-identified compound by dissolving the foam in 100 ml of methanol and adjusting the pH to 2.5 with ethanolic hydrogen chloride. After 30 minutes the mixture was neutralized with saturated sodium bicarbonate and concentrated to a slurry. This was dissolved in 100 ml of methylene chloride, washed with 25 ml of water. The methylene chloride phase was dried over magnesium sulfate and concentrated to a foam. Drying under vacuum at 40°C for 18 hours yielded 7.2 g (96%) of the above-identified compound.

B. Preparation of 5-Amino-1-(5-iodo-5-deoxy-2,3-isopropylidene-β-D-ribofuranosyl)imidazole-4-N- [(4-chlorophenyl)methyl]carboxamide

A mixture of the product of Step A (25 g, 59 mmole) and methyltriphenoxyphosphonium iodide (76 g, 166 mmole) in 500 ml of methylene chloride was stirred for 30 minutes at room temperature under argon. The resulting solution was extracted with 150 ml of water, 150 ml of 5% sodium thiosulfate, 150 ml of 1 N sodium hydroxide, 100 ml of water and dried over magnesium sulfate. The solvent was removed under vacuum and the resulting oil applied to a 1.31 column of flash grade silica gel prepared in 2:1 hexane: ethyl actetate. The column was eluted with the same solvent to remove impurities then 1:1 hexane: ethyl acetate was used to elute the desired product. Appropriate fractions were combined and evaporated to yield 24.4 g of the above-identified compound as a gummy solid. Impure fractions were again subjected to chromatography to yield an additional 2.3 g of the above- identified product. Total yield was 26.7 g (85%) .

C . Preparation of 5-amino-1-(5-azido-5-deoxy- 2,3-O-isopropylidene-β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]carboxamide

A mixture of the product of Step B (26.7 g, 50 mmole) , lithium azide (14 g, 285 mmole) and 100 mg of 18-crown-6 in 350 ml of DMF was stirred for 8 hours at room temperature under argon. The slurry was concentrated to remove solvent and the residue dissolved in a mixture of 500 ml of ethyl acetate and 100 ml of water. The ethyl acetate phase was separated, washed with water and saturated sodium chloride, and then dried over magnesium sulfate. Evaporation of the solvent yielded 25 g of the above-identified compound as a yellow gum which still contained solvent. This was used in the next step without further purification.

D. Preparation of 5-Amino-1-(5-azido-5-deoxγ- β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]carboxamide, (Compound No. 52 (1-467))

The product of Step C, as obtained, was dissolved in 150 ml of 80% trifluoracetic acid and warmed to 50°C for 30 minutes. The solution was evaporated to a syrup at 40°C under vacuum and the residue evaporated twice from 25 ml of water. The syrupy residue was dissolved in 100 ml of ethyl acetate and gently stirred over 100 ml of saturated sodium bicarbonate. Crystallization began in the ethyl acetate phase and after 1 hour crystals were collected by filtration. These crystals were combined with two additional crops or crystals obtained by concentration of the ethyl acetate phase to yield 15.7 g (77% yield based on the product of Step B). Melting point of an analytical sample was 182-183°C.

1H NMR (DMS0-d₆) δ ppm, 3.6 (M, 2H, 5'-CH₂), 4.0-4.3 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 4.3 (d, 2H, -CH₂C₆H₄Cl), 5.4-5.5 (m, 2H, 2'-OH, 3'-OH), 5.5 (d, 1H, 1'-CH), 5.9 (br.s., 2H, 5-NH₂), 7.3-7.4 (m, 4H, C₆H₄Cl), 7.4 (s, 1H, 2-CH), 8.1 (t, 1H, 4-CONH-). IR (KBr) cm^-1, 2110. E. Preparation of 5-Amino-1-(5-amino-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]carboxamide

Compound 52 (1-467) (6.5 g, 159 mmole) was dissolved in 500 ml of boiling ethanol. After cooling to 40°C the solution was saturated with argon and 0.5 g of 10% palladium on carbon added. The mixture was stirred under a hydrogen atmosphere for 8 hours. The mixture was saturated with argon and filtered through Celite 505 and concentrated to a syrup which was used in the next step without further purification. F. Preparation of 5-Amino-1-(5-amino-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]carboxamide Hydrochloride (Compound

No. 53 (1-468))

The product of Step E (theoretically 159 mmole) was dissolved in 100 ml of ethanol and 3.5 ml of 6 N hydrochloric acid added (pH to wet pH paper approximately 3). The solution was evaporated to a hard syrup. This syrup was dissolved in 50 ml of hot ethanol and diluted with 150 ml of ethyl ether. The resulting gummy precipitate was stirred sealed for 12 hours and the resulting white precipitate collected by filtration and washed with ether. Drying under vacuum at 40ºC yielded 6.0 g of the above-identified compound (90% yield based on the compound from Step D).

¹H NMR (DMSO d₆) δ ppm, 3.0-3.2 (m, 2H, 5'-CH₂) , 4.0-4.4 (M, 3H, 2'-CH, 3'-CH, 4'-CH) , 4.4 (d, 2H, -CH₂-C₆H₄Cl) , 5.8-6.2 (br. , 2H, 2' -OH, 3'-OH) , 7.2-7.4 (m, 4H, C₆H₄Cl) , 7.8 (s, 1H, 2-CH) , 8.3 (br., 3H, NH₂.HCl) . Example Al

Preparation of 5-Amino-1-(5-amino-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-(cyclopentyl)carboxamide Hydrochloride

((Compound No. 37) 1-270))

This compound was prepared by the same reaction sequence described in Example AH for compound 53 (1-468), substituting the 4-N-cyclopentylamide, compound 10 (1-186), of Table XII for the 4-N-p-chlorobenzylamide compound 29 (1-349) of Table XII.

1H NMR(DMSO-d₆) δ ppm, 1.4-1.9(m, 9H, cyclopentyl aliphatic protons), 3.0-3.2 (m, 2H, S'-CH₂), 4.0-4.3 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 5.5(d, ¹H, 1'-CH), 5.9(br.s, 2H, 5-NH₂), 7.1(d, 1H, 4-CONH-), 7.4 (s, 1H, 2-CH). Example AJ

Preparation of 5-Amino-1-(5-deoxγ-5-methylthio- β-D-ribofuranosyl)imidazole-4-carboxamide

(Compound No. 54 (1-483))

The intermediate, 5-amino-1-(5-chloro-5-deoxy-β-D- ribofuranosyl)imidazole-4-carboxamide, was prepared according to the procedures described in Example Al for compound 5¹ (1-466), substituting 5-amino-1-β-D-ribofuranosylimidazole-4-carboxamide for 5-amino-1-β-D-r i b o f u r a n o s y l i m i d a z o l e - 4 - N - [ ( 4-nitrophenylmethyl ] carboxamide .

To a 0.1 N sodium methoxide/methanol solution, at 0° under argon, was bubbled methyl mercaptan. To the resulting 0.1 N sodium methylthiolate/methanol solution was added 5-amino-1-(5-chloro-5-deoxy-β-D-ribofuranosyl)imidazole-4-carboxamide (0.40 g). The solution was heated of reflux overnight. The solution was cooled and neutralized with Dowex 50 strongly acidic ion exchange resin. The mixture was filtered and concentrated under reduced pressure. The resulting residue was chromatographed on silica gel, using 4:1 methylene chloride:methanol as the eluting solvent. The appropriate fractions were combined, concentrated under reduced pressure, and vacuum dried to give the above-identified compound as a a white foam (0.28 g) .

1H NMR (DMSO-d₆) δ ppm, 2.1(s, 3H, - S-CH₃), 3.7- 3.9(m, 2H, 5'-CH₂), 3.9-4.4 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 5.3-5.4 (m, 2H, 2 '-OH, 3'-OH), 5.5(d, 1H, 1'-CH), 5.8(br.s., 2H, 5-NH₂) , 6.6-6.9 (br.m, 2H, 4-CONH₂), 7.3(s, 1H, 2-CH).

Example AK

Preparation 5-Amino-1-β-D-ribofuranosylimidazole-4-N-(4-bromophenyl)carboxamide

(Compound No. 55 (1-484))

5-Amino-1-(2,3,5-tri-0-acetyl-β-D-ribofuranosyl)imidazole-4-carboxylic acid (Srivastava, P.C., et al., J. Med. Chem. 17 1207, (1974), (0.75 g) and thionyl chloride (7 ml) were stirred at ambient temperature under a drying tube, for 15 minutes. The excess thionyl chloride was evaporated under reduced pressure and the resulting residue co-evaporated with methylene chloride (3 × 20 ml). The resulting yellow foam was dissolved in methylene chloride (40 ml) and 4-bromoaniline (0.35 g) was added. Triethylamine (approximately 0.75 ml) was added until the solution was basic. The solution was stirred at ambient temperature under a drying tube for 2 hours. The solution was washed with water, dried with magnesium sulfate, and concentrated under reduced pressure to give a yellow foam. The foam was dissolved in methanol (35 ml). A sodium methoxide methanol solution (approximately 0.75 ml of a 0.5 N solution) was added and the resulting solution stirred at ambient temperature under a drying tube, for 30 minutes. The solution was neutralized with methanol-washed Dowex 50 (strongly acidic ion-exchange resin). The mixture was filtered and concentrated under reduced pressure to give a pale yellow residue. The residue was crystallized from methanol (15 ml) /methylene chloride (10 ml) to give tan crystals (0.23 g). The crystals were recrystallized to give off-white crystals (90 mg). Mp: 214-216ºC (decomp).

1H NMR (DMSO-d₆) δ ppm, 3.6(m, 2H, 5'-CH₂), 3.9-4.3 (m, 3H, 1'-CH, 3'-CH, 4'-CH), 5.2-5.4 (m, 3H, 2'-OH, 3'-OH, 5'-OH), 5.5(d, 1H, 1'-CH), 6.2(br.s., 2H, 5-NH₂), 7.4-7.8 (m, 4H, -C₆H₄Br), 7.4 (s, 1H, 2-CH), 9.5(s, 1H, 4-CONH).

Example AL

Preparation of 5-Amino-1-β-D-ribofuranosylimidazole-4-N-[(4-bromophenyl)methyl]carboxamide

(Compound No. 56 (1-487))

This compound was prepared according to the procedures described in Example J for the 4-p-nitrobenzyl derivative, substituting 4-bromobenzylamine hydrochloride for 4-nitrobenzylamine hydrochloride. ¹H NMR(DMSO-d₆) δ ppm, 3.5-3.6(m, 2H, 5'-CH₂), 3.9-

4.3 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 4.3 (d, 2H, CH₂-C₆H₄Br) ,

5.1-5.4 (m, 3H, 2'-OH, 3'-OH, 5'-OH), 5.5 (d, 1H, 1'-CH),

5.9(br.s, 2H, 5-NH₂) , 7.2-7.5(m, 4H, -C₆H₄Br) , 7.3(s, 1H, 2-CH), 8.0(t, 1H, 4-CONH-).

Example AM

Preparation of 5-Amino-1-β-D-ribofuranosyl-imidazole-4-N-(4-iodophenyl)

carboxamide (Compound No. 57 (1-488))

This compound was prepared according to the procedures described in Example AK for the 4-p-bromophenyl derivative, substituting 4-iodoaniline for 4-bromoaniline.

The final product was recrystallized from ethanol. Mp:

227-229°C H NMR (DMSO-d₆) δ ppm, 3.5-3.6(m, 2H, 5'-CH₂), 3.9-4.4(m, 3H, 2'-CH, 3'-CH, 4'-CH), 5.2-5.4 (m, 3H, 2'- OH, 3'-OH, 5'-OH), 5.5(d, 1H, 1'-CH), 6.2(br.s., 2H, 5- NH₂₎, 7.4(s, 1H, 2-CH) , 7.6-7.7(m, 4H, -C₆H₄I), 9.5(s, 1H,

4-CONH) .

Example AN

Preparation of 5-Amino-1-β-D-ribofuranosylimidazole-4-N-(4-nitrophenyl)carboxamide

(Compound No. 58 (1-489))

This compound was prepared according to the procedures described in Example AK for the 4-p-bromophenyl derivative, substituting 4-nitroaniline for 4- bromoaniline. The final product was recrystallized from methanol to give a yellow powder.

¹H NMR (DMSO-d₆) δ ppm, 3.5-3.6(m, 2H, 5'-CH₂), 3.9- 4.4(m, 3H, 2'-CH, 3'-CH, 4'-CH), 5.2-5.4 (m, 3H, 2'-OH, 3'-OH, 5'-OH), 5.6(d, 1H, 1'-CH), 6.4(br.s., 2H, 5-NH₂),

7.5(s, 1H, 2-CH), 8.1-8.3 (m, 4H, C₆H₄NO₂), 10.1(s, 1H, 4- CONH).

SUBSTITUTE SHEET Example AO

Preparation of 5-Amino-l-β-D-ribofuranosylimidazole-4-N-[2-(4-nitrophenyl)ethyl

carboxamide (Compound No. 59 (1-506))

This compound was prepared according to the procedures described in Example J for the 4-p-nitrobenzyl derivative, substituting 4-nitrophenethylamine hydrochloride for 4-nitrobenzylamine hydrochloride.

¹H NMR (DMSO-d₆) δ ppm, 2.9-3.0(t, 2H, -CH₂-C₂H₄-NO₂), 3.4-3.6 (m, 2H, 5'-CH₂), 3.9-4.3 (m, 3H, 2'-CH, 3'-CH, 4'- CH), 4.8-5.4(br., 3H, 2'-OH, 3'-OH, 5'-OH), 5.5(d, 1H, 1'-CH), 5.9-6.2(br., 2H, 5-NH₂), 7.5-8.2 (m, 4H, -C₆H₄NO₂), 7.6(s, 1H, 2-CH), 7.7 (t, 1H, 4-CONH).

Example AP

Preparation of 5-Amino-4-[1-[4-(4-nitrophenyl)]piperazinocarbamoyl]-1-β-D-ribofuranosylimidazole (Compound No. 60 (1-508))

This compound was prepared according to the procedures described in Example J for the 4-nitrobenzyl derivative, but substituting 1-(4-nitrophenyl)piperazine for 4-nitrobenzylamine hydrochloride. The product as recrystallized from cold methanol and had a mp of 199- 200ºC.

'H NMR (DMSO-d₆) δ ppm, 3.4-3.6 (m, 10H, 3'-CH₂, piperazonyl methylenes), 3.9-4.3 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 5.2-5.4(m, 3H, 2'-OH, 3'-OH, 5'-OH), 5.5(d, 1H, 1'-CH), 6.3 (br.s., 2H, 5-NH₂) , 7.0-8.1(m, 4H, -C₆H₄NO₂) ,7.3(s, 1H, 2-CH).

Example AO

Preparation of 5-Amino-1-(5-deoxy-β-D-ribofuranosyl)imidazole-4 N-[(4-chlorophenyl)

methyl]carboxamide (Compound No. 61 (1-509))

5-Amino-1-(5-iodo-5-deoxy-2,3-isopropylidene-β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]carboxamide (see procedures described in Example AH for preparation of Compound 53 (1-468), step B) (0.64 g) was stirred in 30 ml of 50% formic acid overnight. The excess solvent was evaporated under reduced pressure. The resulting residue was co-evaporated with water (25 ml) and methanol (25 ml). The resulting yellow foam was chromatographed on silica gel, using 9:1 methylene chloride:methanol as eluting solvent. The appropriate fractions were combined and concentrated under reduced pressure to give 0.47 g of 5-amino-1-(5-iodo-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]carboxamide.

5-Amino-1-(5-iodo-5-deoxy-β-O-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl] carboxamide (0.04 g), palladium on carbon 10% (20 mg), and ethanol (20 ml) were charged to a Parr bottle. The bottle and contents were charged with 45 p.ε.i. hydrogen. The reaction progress was monitored by HPLC (Waters C18, 55% methanol/45% 0.1 N acetic acid, 260 nm, 1.0 ml/min). After 24 hour, there was 34% starting material. Fresh catalyst was added (20 mg) and the mixture re-charged with hydrogen (45 p.s.i.). The mixture was shaken for an additional 48 hours. The reaction mixture contained 30% starting material. The mixture was filtered through Celite, and concentrated under reduced pressure. The resulting residue was chromatographed on silica gel, using ethyl acetate (400 ml) and 5% methanol in ethyl acetate

(200 ml) as the eluting solvent. The appropriate fractions were combined and concentrated under reduced pressure to yield 70 mg of a white foam. HPLC indicated 9% starting material. The material was rechromatographed on silica gel, using ethyl acetate as eluting solvent. All fractions containing less than 3% starting material were combined and concentrated under reduced pressure to yield 36 mg of the above-identified compound as a pink foam.

¹H NMR (DMSO-d₆) δ ppm, 1. 2-1. 3 (d , 3H , 5 ' -CH₃) , 3 . 7 -4 . 3 (m , 3H , 2 ' -CH, 3 ' -CH₂ 4 ' -CH) , 4 . 3 (d , 2H , CH₂-C₆H₄Cl ) , 5.1-5.4 (m, 3H, 2'-OH, 3'-OH, 1'-CH), 5.8(br.s., 2H, 5-NH₂), 7.2-7.4 (m, 5H, C₆H₄Cl, 2-CH), 8.1(t, 1H, 4-CONH).

Example AR

Preparation of 5-Amino-1-(5-deoxy-5-methylsulfinyl-β-D-ribofuranosyl)-imidazole-4-carboxyamide (Compound No. 62 (1-510))

5-Amino-1-(5-deoxy-5-methylthio-β-D-ribofuranosyl ) imidazole-4-carboxamide (compound 54 (1-483)) of Example AK (0.40 g) was dissolved in water (20 ml). Hydrogen peroxide, 30 weight percent, (0.42 ml), was added and the solution stirred for 30 minutes. TLC (6/1, methylene chloride/methanol) indicated some starting material present. An additional 1.0 ml of hydrogen peroxide was added and the solution stirred for 15 minutes. TLC indicated no starting material. The solvent was evaporated under reduced pressure to give a yellow foam. The foam was chromatographed on silica gel, using 3/1, methylene chloride/methanol, as eluting solvent. The appropriate fractions were combined and concentrated in vacuo to give 75 mg of the above-identified compound as a yellow foam.

HPLC (Waters C18, 100% 0.1 N acetic acid, 1.0 ml/minutes, 260 nm) indicated 2 equimolar products. This is consistent with oxidation of the product to a diaster omeric mixture of sulfoxides.

1H NMR (DMSO-d₆) δ ppm, 2.6(s, 3H, CH₃S(O)-), 3.0-3.2 (m, 2H, 5'-CH₂), 4.0-4.4 (m, 3H, 2'-CH, 3'-CH, 4'-CH) 5.4-5.6(m, 3H, 2'-OH, 3'-OH, 1'-CH), 5.9(br.s., 2H, 5-NH₂), 6.6-6.9 (br., 2H, 4-CONH₆), 7.3(s, 1H, 2-CH). Example AS

Preparation of 5-Amino-1-β-D-(5-deoxy-5-methylaminoribofuranosyl)imidazole-4-carboxamide

(Compound No. 63 (1-517))

5'-Deoxy-5'-iodo-2',3'-O-isopropylidene-AICA riboside (1.00 g) (ref: P.C. Srivastava, A.R. Newman, T.R. Mathews, and T.R. Mathews, and R.K. Robins, J. Med. Chem., 18, 1237 (1975)), methylamine 40% weight in water (3 ml), and methanol (30 ml) were combined and heated at reflux for 18 hours. The reaction gave a mixture of products. The solution was cooled and the solvents evaporated under reduced pressure. The resulting residue was chromatographed on silica gel, using 6/1 methylene chloride/methanol (400 ml) and 3/1 methylene chloride/methanol (300 ml) as the eluting solvent. The fractions containing the slow-eluting component which was desired product were combined and evaporated under reduced pressure to give 0.13 g of 5'-deoxy-5'-methylamino-2',3'-isopropylidene-AICA riboside.

5'-deoxy-5'-methylamine-2',3'-isopropylidene AICA riboside (0.13 g) was heated at 60 °C in 75% formic acid (20 ml) for 1.5 hour. The solution was cooled and the solvent evaporated under reduced pressure to yield a white foam. The foam was dissolved in water (5 ml) and applied to a short column of Dowex 50 strongly acidic ion-exchange resin. The column was washed with water then eluted with 1 M NH₄OH in 20% methanol/water. The solvent was evaporated under reduced pressure and the resulting residue co-evaporated with methanol (3 × 20 ml) to yield 75 mg of the above-identified product as an off-white foam.

¹H NMR (D₆-DMSO-d₆) δ ppm, 2.3(s, 3H, CH₃N) , 2.5- 2.7 (m, 2H, 5'-CH₂) , 3.3-3.4 (br., 1H, MENH) , 3.9-4.3 (m, 3H, 2'-CH, 3'-CH, 4'-CH) , 5.1-5.4 (m, 2H, 2'-OH, 3'-OH) , 5.4 (d, 1H, 1'-CH) , 6.2(br.s., 2H, 5- NH₂) , 6.6-6.8 (br., 2H, 4- CONH), 7.2 (s, 1H, 2-CH).

Example AT

Preparation of 5-Amino-1-β-D-ribofuranosylimidazole-4-N-(2-chlorophenyl)carboxamide

(Compound No. 64 (1-519))

This compound was prepared according to the procedures decribed in Examples AK for compound 55 (1- 484) for the 4-p-bromophenyl derivative, substituting 2-chloraniline for 4-bromaniline. The final product was recrystallized from methylene chloride (20 ml) /methanol (1 ml) to yield 0.25 g of the above-identified product. Mp = 131-135ºC.

¹H NMR (DMSO-d₆) δ ppm, 3.5-3.6 (m, 2H, 5'-CH₂), 3.9 - 4.3 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 5.2-5.4 (m, 3H, 2'-OH, 3'-OH, 5'-OH), 5.5(d, 1H, 1'-CH), 6.2 (br.s., 2H, 5-NH₂), 7.0-8.4 (m, 5H, C₆H₄Br, 2'-CH), 9.1(s, 1H, 4-CONH). Example AU

Preparation of 5-Amino-1-β-D-(5-benzylamino-5-deoxyribofuranosyl)imidazole-4-carboxamide

(Compound No. 66(1-531))

5'-deoxy-5'-iodo-2',3'-isopropylidene AICA riboside (1.00 g) (ref: P.C. Srivastava, A.R. Newman, T.R. Mathews, and R.K. Robins, J. Med. Chem. 18: 1237 (1975)), benzylamine (2.0 ml), and methanol (40 ml) were combined and heated at reflux for 24 hours. Then, the procedures described in Example AS for Compound 63 (1-517) were followed to give the above-identified compound.

¹H NMR (DMSO-d₆) δ ppm, 2.7 (d 2H, -CH₂-C₆H₅), 3.3-3.4(br., 1H, -NH -CH₂C₆H₅), 3.9-4.3 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 5.1-5.4 (m, 2H, 2'-OH, 3'-OH), 5.4 (d, 1H, 1-CH), 6.1(br.s., 2H, 5-NH₂), 6.6-6.8(br., 2H, 4-CONH₂), 7.2-7.4 (m, 6H, -C₆H₅, 2-CH).

Example AV

Preparation of 5-Amino-2-thio-1-β-D- (5-deoxyribofuranosyl) imidazole-4-carboxamide

(Compound No. 67 (1-535))

A. Preparation of 5'-Deoxγ-2',3'-isopropylidene-2-bromo-AICA Riboside

To a solution of 5'-deoxy-2',3'-isopropylidene-AICA riboside (2.90 g) (ref: P.C. Srivastava, A.R. Newman,

T.R. Mathews, and R.K. Robins, J. Med. Chem., 18: 1237 (1975)) in chloroform (100 ml), was added N- bromosuccinimide in small portions over 20 minutes. The solution was stirred at ambient temperature for 30 minutes. The solution was washed with water, twice with brine, and then dried over magnesium sulfate. The solvent was evaporated in vacuo to yield a dark foam. The foam was passed through a column of silca gel , eluting with 9 : 1 methylene chloride:methanol. The fractions containing product were combined and concentrated under reduced pressure to yield 2.02 g of reddish-brown foam. B. Preparation of 5'-Deoxy-2',3'-O-isopropylidene-2-thio AICA Riboside

Postassium sulfate (3.7 g) was heated at reflux in ethanol (20 ml) for 15 minutes. The mixture was filtered. To the filtrate was added 5'-deoxy-2',3'-isopropylidene-2-bromo AICA riboside (from step A). The mixture was heated at 100°C in a steel bomb for 5.5 hours. The mixture was cooled and filtered. The pH of the filtrate was adjusted to about 5-6 with acetic acid, and the solvent evaporated under reduced pressure. The resulting residue was passed through a column of silica gel, eluting with 7/1, methylene chloride/methanol. The fractions containing the product were combined and concentrated under reduced pressure to give a dark brown foam. The foam was stirred in methylene chloride (50 ml), then filtered to yield a pale purple powder. The powder was stirred in cold methanol, then filtered and vacuum dried to yield 0.52 g of a pale yellow solid. Mp = 211-214 (decomposition).

C. Preparation of 5-Amino-2-thio-1-(deoxy-β-D-ribofuranosyl)imidazole-4-carboxamide

(Compound 67 (1-535))

5 '-deoxy-2',3'-isopropylidene-2-thiol AICA riboside

(0.45 g) (from step B) was stirred in 50% formic acid (30 ml) at 50°C for 1 hour. The solvent was evaporated under reduced pressure. The resulting residue was co-evaporated with methanol (2 × 20 ml). The resulting solid was warmed in methanol (25 ml), then stirred at room temperature overnight. The mixture was filtered and the filtrate concentrated under reduced pressure to yield a greenish foam. The foam was chromatographed on silica gel, using 5/1, methylene chloride/methanol, as the eluting solvent. The appropriate fractions were combined and concentrated under reduced pressure to give a yellow foam. The foam was crystallized from cold methanol to yield 69 mg. of the above-identified compound mp = 201-203 °C, (decomposition).

'H NMR (DMSO-d₆) δ ppm 1.3(d, 3H, 5'-CH₃), 3.6-4.5 (m,

3H, 2'-CH, 3'-CH, 4'-CH), 5.0-5.2 (m, 2H, 2'-OH, 3'-OH),

5.6(br.s., 2H, 5-NH₂), 6.0(d, 1H, 1'-CH), 7.0(br., 2H, 4-CONH), 12.0 (br.s., 1H, -SH).

Example AW

Preparation of N,N'-bis-(5-amino-1-β-D-ribofuranosyl imidazole-4-carbonyl)-1,6-diaminohexane (Compound No. 68 (1-538))

N-succinimidyl-5-amino-1- (2,3, 5-tri-O-acetyl-β-D-ribofuranosyl-imidazole-4-carboxylate (2.50 g) (ref: Srivastava, P.C, et al., J. Med. Chem. 12:1207 (1974)), 1,6-hexane diamine (0.300 g) , triethylamine (0.5 ml), and methylene chloride (35 ml) were combined and stirred at room temperature for 18 hours. The title compound was prepared according to the procedures described in Example J. The final product was crystallized from methanol to yield 0.32 g of the above-identified compound. Mp - 181- 185ºC.

1H NMR data reported as for half the symmetrical dimer. ¹H NMR (DMSO-d₆) δ ppm, 1.2-1.5(m, 4H, β and δ methylenes of N-hexyldicarboxamide), 3.0-3.2 (m, 2H, α methylene of N-hexyl dicarboxamide), 3.5-3.6 (m, 2H, 5'-CH₂), 3.8-4.3 (m, 3H, 2'-H, 3'-CH, 4'-CH), 5.1-5.4(m, 3H, 2'-OH, 3'-OH, 5'-OH), 5.5(d, 1H, 1'-CH), 5.9(br.s., 2H, 5-NH₂), 7.3 (s, 1H, 2-Ch), 7.4 (t, 1H, 4-CONH). Example AX

Preparation of N,N'-Bis-(5-Amino-1-β-D-ribofuranosylimidazole-4-carbonyl)-1.4-diaminocγclohexane

(Compound No. 69 (1-549))

This compound was prepared according to the procedures described in Example AW for compound 68 (1- 538), substituting 1,4-diaminocyclohexane for 1,6-hexanediamine.

¹H NMR data are reported as for half the symmetrical dimer. ¹H NMR (DMSO-d₆) <5 ppm 1.3-1.8(m, 4H, cyclohexane methylene protons), 3.5-3.7 (m, 3H, 5'-CH₂, cyclohexane methine), 3.8-4.3 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 5.1-5.4 (m, 3H, 2'-OH, 3'-OH, 5'-OH), 5.5(d, 1H, 1'-CH), 5.9(br.s., 2H, 5-NH₂), 7.1(d, 1H, 4-CONH) 7.3 (s, 1H, 2-CH) . Example AY

Preparation of 5-Amino-2-thio-1-(5-amino-5-deoxy-β-D-ribofuranosyl) imidazole-4-carboxamide

(Compound No. 70(1-551))

A. Preparation of 5-Deoxy-5'-iodo-2-bromo-2',-3'-isopropylidene AICA Riboside

2-Bromo-2'3'-isopropylidene AICA riboside (4.50 g) (ref: T.Miyoshi, S.Suzaki, A. Yamazaki, Chem. Pharm. Bull. 29, 9: 2089, (1976) methyltriphenoxyphosphonium iodide (16.2 g), and methylene chloride (125 ml) were combined and stirred at room temperature for 16 hours. The mixture was washed with water, 0.5 M NAOH (100 ml), 5% NaS₂O₃ (150 ml), and brine, then dried over magnesium sulfate. The solvent was evaporated under reduced pressure to give an orange oil. The oil was triturated in cold diethylether. The resulting mixture was filtered to give 3.53 g of a grey powder. The mother liquor was concentrated under reduced pressure to give an orange oil. The oil was applied to a short column of silica gel. The column was washed with methylene chloride, then the product eluted with 9/1, methylene chloride/methanol (250 ml). The appropriate fractions were combined and concentrated under reduced pressure to give an orange tar. The tar was triturated with cold diethyl ether. The mixture was filtered to yield an additional 0.94 g of a gray powder. The combined powder (4.47 g) was chromatographed on silica gel, using 2/1, ethylacetate/hexane, as eluting solvent. The appropriate fractions were combined and concentrated under reduced pressure to yield a yellow foam (4.02 g). B. Preparation of 5'-Azido-5'

deoxγ-2-bromo-2',3'-isopropylidene AICA Riboside

5'-deoxy-5'-iodo-2-bromo-2',3'-isopropylidene AICA riboside (4.02 g) lithium azide (1.82 g) , and DMF (65 ml) were combined and stirred at ambient temperature for 2 hours. The solvent was evaporated under reduced pressure to give a yellow oil. The oil was dissolved in ethyl acetate (200 ml), washed with water and brine, then dried over magnesium sulfate. The solvent was evaporated under reduced pressure to give a yellow foam (3.01 g). C. Preparation of 5'-Amino-5'-deoxγ-2-bromo-2',3'-isopropylidene AICA Riboside

5'-azido-5'-deoxy-2-bromo-2',3'-isopropylidene AICA riboside (2.00 g), triphenylphosphine (1.83 g), and THF (100 g) were combined and stirred at room temperature for 16 hours. Concentrated NH₄OH (15 ml) was added and the solution heated at reflux for 6 hours. The solution was cooled and the solvent evaporated under reduced pressure. The resulting residue was coevaporated with methanol (2 × 30 ml). The resulting residue was stirred in cold methanol (25 ml) for 30 minutes. The mixture was filtered to give an off-white powder. The solid was recrystallized from methanol to give a white powder (0.73 g). D. Preparation of 5-Amino-2-thio-1-(5- amino-5-deoxy-β-D-ribofuranosyl)imidazole- 4-carboxamide (Compound No. 70 (1-551))

Potassium sulfide (1.0 g) was heated at reflux in ethanol (10 ml) for 15 minutes. The mixture was filtered and to the filtrate was added 5'-amino-5'-deoxy-2-bromo- 2',3'-isopropylidene AICA riboside (0.50 g). The mixture was heated in a steel bomb at 110°C for 5 hours. The mixture was cooled and filtered. The filtrate was again filtered, then concentrated under reduced pressure to give a yellow tar. The tar was chromatographed on silica gel, using 3/1, methylene chloride/methanol, as eluting solvent. The appropriate fractions were combined and concentrated under reduced pressure to give a yellow glass (0.12 g). The glass was dissolved in 80% of trifluoroacetic acid (8 ml) and stirred at room temperature for 1 hour. The solvent was evaporated under reduced pressure to give a yellow solid. The solid was stirred in diethylether/ethanol (10 ml of 95/5), then filtered and dried to yield a yellow solid (55 mg).

1H NMR (DMSO-d₆ + D₂O) δ ppm, 2.6-2.9 (m, 2H,

5'-CH₂-), 3.8-4.5(m, 3H, 2'-CH, 3' CH, 4'-CH), 6.2(d, 1H, 1'-CH).

Example AZ

Preparation of 5-Amino-1-(5-azido-5-deoxy-β-D-ribofuranosyl)imidazole-4-N- [(4-nitrophenyl)-methyl]carboxamide

(Compound No. 71 (1-562))

This compound was prepared according to the procedures described in example AH for compound 52 (1- 467), substituting compound 23 (1-343) (p-nitrobenzyl derivative), for compound 29 (1-349) (p-chlorobenzyl derivative).

1H NMR (DMSO-d₆) δ ppm, 3.5-3.7 (m, 2H, 5'-CH₂), 3.9 - 4.4 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 4.4-4.5(d, 2H, -CH₂-

PhNO₂), 5.4-5.5 (m, 2H, 2' -OH, 3'-OH), 5.5(d, 1H, 1'-CH), 5.9(br.s., 2H, 5-NH₂), 7.4(s, 1H, 2-CH), 6.5-8.2 (m, 4H, - C₆H₄NO₂), 8.3(4, 1H, 4-CONH-).

Example BA

Preparation of 5-Amino-1-(5-amino-5-deoxγ-β-D-ribofuranosyl)imidazole-4-N-[4-nitrophenyl)

methyl]carboxamide (Compound No. 72 (1-563))

This compound was prepared according to the procedures described in Example AH for compared 53 (1- 468), substituting the p-nitrobenzyl amide derivative (compound 23 (1-343)) for the p-chlorobenzyl amide derivative (compound 29 (1-349)).

¹H NMR (DMSO + D₂O) δ ppm 2.6-2.8 (m, 2H, 5'-CH₂-) , 3.8- 4.3 (m, 3H, 2'-CH, 3'-CH, 4'-CH) , 4.4-4.5 (m, 2H, -CH₂- C₆H₄NO₂) , 5.4(d, 1H, 1'-CH), 7.3(s, 1H, 2-CH), 7.5-8.3 (m, 5H, CH₂C₆H₄NO₂, 4-CONH) .

Example BB

Preparation of 5-Amino-1-β-D-ribofuranosylimidazole-4-N-[(4-(trifluoromethylphenyl)

methyl]carboxamide (Compound No. 74 (1-572))

This compound was prepared according to the procedures described in Example J for the p-nitrobenzyl derivative substituting 4-(trifluoromethyl)benzylamine for 4-nitrobenzyl amine hydrochloride. The final product was recrystallized from methylene chloride/methanol. Mp = 137 - 140.

¹H NMR (DMSO-d₆) δ ppm 3.5 - 3.7 (m, 2H, 5'-CH₂), 3.9 -

4.4 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 4.4 - 4.5 (d, 2H, -

CH₂-PhCF₃), 5.2 - 5.5 (m, 3H, 2'-OH, 3' -OH, 5'-OH), 5.5 (d, 1H, 1'-CH), 5.9 (br.s., 2H, 5-NH₂), 7.3 (S, 1H, 2-CH), 7.4 - 7.7 (m, 4H,

-C₆H₄CF₃) , 8.2 (t, 1H, 4-CONH). Example BC

Preparation of 5-Amino-1-β-D-ribofuranosylimidazole- 4-N-[(4-sulfamoylphenyl)methyl]carboxamide

(Compound No. 75 (1-577))

This compound was prepared according to the procedures described in Example J for the p-nitrobenzyl derivative, substituting 4-(aminomethyl)benzene sulfonamide hydrochloride for 4-nitrobenzylamine hydrochloride.

1H NMR (DMSO-d₆) δ ppm, 3.5-3.7 (m, 2H, 5'-CH₂-), 3.9-

4.4 (m, 3H, 2'-CH, 3'-CH, 4'-CH) , 4.4-4.5(d, 2H, -CH₂- C₆H₄SO₂) , 5.2-5.4 (m, 3H, 2'-OH, 3'-OH, 5'-OH), 5.5(d, 1H, 1'-CH) , 6.0 (br.s., 2H, 5-NH₂), 7.3 (br.s., 2H, -SO₂NH₂), 7.4(s, 1H, 2-CH), 7.4-7.8(m, 4H, -C₆H₄-), 8.2 (t, 1H, 4-CONH-).

Example BD

Preparation of 5-Amino-1-(5-(4-chlorobenzylamino)-5-deoxyβ-D-ribofuranosyl)imidazole-4-carboxamide (Compound No. 76 (1-578))

5'-amino-5'-deoxy-AICA-riboside (0.50 g) (compound No. 21 (1-227)) of Table VIII, 4-chlorobenzyl iodide (0.50 g), potassium carbonate (0.26 g), and DMF (15 ml) were combined and stirred at room temperature for 16 hours. The solvent was evaporated under reduced pressure and the resulting residue stirred in warm ethanol (35 ml). The insolubles were removed by filtration and the filtrate concentrated under reduced pressure. The resulting residue was chromatrographed on silica gel, using 3:1, methylene chloride:methanol, as eluting solvent. The fractions containing the slower moving of the two products were combined and concentrated under reduced pressure to yield a tan foam (0.21 g)

1H NMR (DMSO-d₆ + D₂) δ ppm 2.9-3.0 (m, 2H, 5'-CH₂-), 3.9 (s, 2H, -CH₂-C₆H₄), 3.9-4.3 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 5.5(d, 1H, 1'-CH), 7.3(s, 1H, 2-CH), 7.4 (m, 4H, - C₆H₄Cl). Example BE

Preparation of 5-Amino-1-(5-deoxy-β,D-ribofuranosyl)-imidazole; (Compound No. 77 (1-588))

5'-deoxy AICA riboside (1.00 g) (ref: P.C. Srivastava, A.R. Newman, T.R. Mathews, and R.F. Robins, J. Med. CHem. 18:1237 (1975) was heated at reflux in N potassium hydroxide (4.0 ml) for 5 hours. The solvent was evaporated under reduced pressure and the resulting residue co-evaporated with ethanol (4 × 10 ml). The resulting residue was diluted with ethanol (15 ml) and a fine precipitate was filtered. Upon setting for several days, the filtrate gave an additional precipitate. The microscopic solid was collected, and the combined solid material was dissolved in water (20 ml) and neutralized with Dowex 50W strongly acidic ion exchange resin. The solvent was evaporated under reduced pressure to give a dark tar. The tar was dissolved in 80% acetic acid (20 ml) and gently heated (60ºC). The solvent was evaporated under reduced presure to give a dark tar. The tar was co-evaporated with methanol (2 × 15 ml). The resulting residue was chromatographed on silica gel, using 3/1, methylene chloride/methanol, as eluting solvent. The appropriate fractions were combined and concentrated under reduced pressure to yield a dark tar. The tar was co-evaporated with tolune (3 × 20 ml), then vacuum dried to yield a dark brown, hygroscopic foam (110 mg).

1H NMR (D₂) δ ppm, 1.3 (d, 3H, 5'-CH₃), 4.0-4.5 (m, 3H, 2--CH, 3'-CH, 4'-CH), 5.6(d, 1H, 1'-CH), 6.4(s, 1H, 4-CH), 7.7(s, 1H, 2-CH). Example BF

Preparation of 5-Amino-1-(5-deoχy-5-diethylaminoribo-β ,D-furanosyl)imidazole-4-carboxamide

(Compound No.65 (1-522)

5'-deoxy-5'-iodo-2',3'-isopropylidene AICA riboside (1.00 g) (ref.: P.C. Srivastava, A.R. Newman, T.R. Mathews, and R.K. Robins, J. Med. Chem. 18: 1237, (1975)), diethylamine (2.5 ml of 40 wt% in water), and methanol (30 ml) were combined and heated at reflux for 18 hours. The procedures described in Example AS for compound 63 (1- 519) were followed to give the above-identified compound.

¹H NMR (DMSO-d₆) δ ppm 0.9 (t, 6H, methyl groups on

5'-diethylamine), 2.4-2.7 (m, 6H, 5'-CH₂, methylene groups on 5'-diethylamine), 3.3-4.2 (m, 3H, 2'-CH, 3'-CH, 4'- CH), 5.2 (br., 2H, 2'-OH, 3'-OH), 5.4(d, 1H, 1'-CH), 5.9(br.s., 2H, 5-NH₂), 5.7-5.9 (br., 2H, 4-CONH₂), 7.3(s, 1H, 2-CH).

Example BG

Preparation of 5-Amino-1-β-D-ribofuranosylimidazole-4-N-[3-4-nitrophenyl)

propyl]carboxamide (Compound No. 73 (1-566))

This compound was prepared according to the procedures described in Example J for the p-nitrophenyl derivative, substituting 3-(4-nitrophenyl)propylamine (ref: G.W. Hardy, et al., J. Med. Chem. 32: 1108, (1989)) for p-nitrobenzylamine hydrochloride.

¹H NMR (DMSO-d₆) δ ppm 1.7-3.2 (m, 6H, - CH₂ CH₂-), 3.5-3.6 (m, 2H, 5'-CH₂), 3.9-4.3 (m, 3H, 2'-CH, 3'-CH, 4'-CH), 5.2-5.4 (m, 3H, 2'-OH, 3'-OH, 5'-OH), 5.5(d, 2H, 1'-CH), 5.9(br.s., 2H, 5-NH₂), 7.3 (s, 1H, 2-CH), 7.5-8.2 (m, 5H, -CH₆H₄NO₂, 4-CONH-).

101

TABLE IX - Physic:al Characteristics and Preparation

Calculated

Compound No. Physical Elemental Analysis Preparation

(Table XII) State %C %H %N %O Reference or Source

1(1-110) White or light pink powder 41.86 5.46 21.70 30.98 Sigma Chemical Co.

2(1-111) White powder 46.88 5.25 14.58 33.30 Example A

3(1-115) mp 205.00-206.00ºC 45.00 5.04 23.32 26.64 U.S. Patent No. 3,450,693; Suzuki et al.

4(1-122) Foam 39.56 5.53 25.63 29.28 Chem. Pharm. Bull 25:1959 (1977)

5(1-145) mp 209.00-209.00ºC 46.87 6.29 21.86 24.97 European Patent Appn 0278,501

6(1-155) mp 192.00-192.00ºC 46.15 6.34 19.57 27.94 Chem. Pharm. Bull 29(7):1870 (1981)

7(1-164) White solid, mp 173 .5-175ºC 46.00 6.11 22.35 25.53 Example B

%P

8(1-172) White powder 30.35 4.81 15.73 40.42 8.69 Sigma Chemical Co.

9(1-177) White solid 47.89 5.20 13.14 33.77 J. Het. Chem. 9:623 (1972)

10(1-186) Foam 51.53 6.79 17.17 24.51 Example C

11(1-226) White solid, mp 178* -179"C Example E

12(1-232) Off-white solid, mp 206-207* C 48.32 6.08 18.78 26.82 Example D

TABLE IX - Physical Characteristics and Preparation (Continued)

Calculated

Compound No. Physical Elemental Analysis Preparation

(Table XII) State %C %H %N %O Reference or Source

23(1-343) Yellow foam 48.86 4.87 17.80 28.47 Example J

%Cl

24(1-354) White foam 50.20 5.00 14.64 20.90 9.26 Example K

25(1-360) White foam 46.06 4.35 13.43 19.17 16.99 Example L

26(1-332) White foam 39.07 4.74 12.81 23.13 12.81 Example N

%S

27(1-395) Yellow crystals, mp 205-208ºC 33.33 4.97 17.27 24.66 19.77 Example M

8(1-348) Pale yellow foam 48.86 4.87 17.80 28.47 Example Q

%Cl

9(1-349) Pale yellow foam 50.20 5.00 14.64 20.90 9.26 Example R

0(1-388) Off-white foam 56.35 6.12 15.46 22.07 Example S

1(1-251) White crystals 46.15 6.34 19.57 27.94 Example O

2(1-262) Off-white powder 49.67 7.05 17.82 25.45 Example P

3(1-263) Pink foam 49.67 7.05 17.82 25.45 Example P

4(1-250) White crystall 46.15 6.34 19.57 27.94 Example O

%Cl

5(1-355) White foam 50.20 5.00 14.64 20.90 9.26 Example T

TABLE IX - Physical Characteristics and Preparation (Continued)

Calculated

Compound No. Phvsical Elemental Analysis Preparation

(Table XII) State %C %H %N %O Reference or Source

36(1-207) White powder 51.53 6.79 17.17 24.51 Example U

% Cl

37(1-270) pale yellow powder, 46.47 6.69 19.36 17.69 9.80 Example Al

mp 135-155ºC

38(1-351)+ off-white powder, 44.19 5.42 27.75 22.64 - - - - mp 92-95ºC

39 (1-390) white crystals, mp 187-188 ºC 53.96 5.86 14 .81 25.37 - - - - Example V

%S

40 (1-392)+ mp 126ºC 39.41 5.14 20.43 23.33 11.69 R. Muramoto, et al.

Chem. Pharm. Butt. (Japan) 23:759

(1975) .

%Cl

41(1-396-3) light green solid 50.53 6.12 16.37 18.70 8.29 Example W

42 (1-431) pink foam 49.76 5.40 13.65 31.19 - - - - Example X

%S

43(1-432) white powder 49.17 4.95 15.29 21.83 8.75 Example Y

- - - - - - - - - - - - - - - - -+= known compound

TABLE IX - Physi cal characteristics and Preparation (Continued)

Calculated

Compound No. Physical Elemental Analysis Preparation

(Table XII) State %C %H %N %O Reference or Source

55(1-484) off-white crystals, 43.60 4.15 13.56 19.36 %Br 19.34 Example AK

mp 214-216ºC

56(1-487) white foam 44.98 4.48 13.11 18.72 %Br 18.70 Example AL

57(1-488) off-white crystals, 39.15 3.72 12.17 17.38 %I 27.57 Example AM

58(1-489) yellow powder 47.50 4.52 18.46 29.52 - - - - Example AN

mp 227-229ºC

59(1-506) yellow foam 50.12 5.20 17.19 27.49 - - - - Example AO

60(1-508) yellow/orange solid, 50.89 5.39 18.74 24.97 - - - - Example AP

mp 199-200ºC

61(1-509) pink foam 52.39 5.22 15.27 17.45 %Cl 9.67 Example AQ

62(1-510) pale yellow foam 39.47 5.30 18.41 26.29 %S 10.54 Example AR

63 ( 1-517) off-white foam 44.28 6.32 25.82 23.59 - - - - Example AS

64(1-519) brown crystals, 48.86 4.65 15.19 21.69 %Cl 9.61 Example AT

mp 131-135ºC

65(1-522) brown foam 49.83 7.40 22.35 20.42 - - - - Example BF

TABLE IX - Physical Characteristics and Preparation (Continued)

Calculated

Compound No. Physical Elemental Analysis Preparation (Table XII) State %C %H %N %O Reference or Source

77(1-588) brown foam 48.23 6.58 21.09 24.09 - - - - Example BE 78(1-599) white foam 51.56 5.19 15.03 20.60 %Cl 7.61 Example BH 79(1-607) white foam 39.41 5.14 20.43 23.33 %S 11.69

Claims

Claims

1. A method of decreasing or preventing tissue damage in a mammal following a period of diminished or interrupted blood flow to that tissue which comprises administering to said mammal an anti-oxidant or anti-free radical effective amount of compound selected from AICA riboside and a substituted-imidazole analog of AICA riboside.

2. A method according to claim 1 wherein said compound decreases peroxidation of fatty acids.

3. A method according to claim 1 wherein said compound decreases oxidation of sulfhydryl containing proteins.

4. A method according to claim 1 wherein said compound decreases hypochlorous acid levels in said tissue.

5. A method according to claim 4 whereby oxidation of sulfhydryl groups is decreased.

6. A method according to claim 1 which comprises administering a compound of the formula:

or a pharmaceutically acceptable salt thereof wherein:

(a) if R₁ is hydrogen or hydrocarbyl of about 1 to about 18 carbon atoms, optionally substituted with from 1 to about 4 substituents independently selected from hydroxy, sulfhydryl, hydrocarbyl, hydrocarbylthio, halogen, amino, hydrocarbylamino, aryl; or carboxylic acid or an ester, thioester, amide or salt thereof; then R₂ is amino, R₃ is hydrogen, cyano, or carboxylic acid or amides, esters, thioesters, or salts thereof; and R₄ is hydrogen, hydrocarbyl, halogen, hydroxy, hydrocarbyloxy, sulfhydryl, hydrocarbylthio, amino or hydrocarbylamino or

(b) if R₁ is

wherein X is -O- or -CH₂-, R₅ and R₆ are independently hydrogen, hydrocarbyl, acyl or hydrocarbyloxycarbonyl; R₇ is hydrogen, halogen, hydroxy, hydrocarbyloxy, sulfhydryl, hydrocarbylthio, sulfamyloxy, amino, hydrocarbylamino, azido, hydrocarbyl, acyloxy, hydrocarbyloxycarboxy or phosphate ester group or salts thereof; then R₂ is hydrogen, amino, hydrocarbylamino acylamino, amido, or dihydrocarbylaminoalkyleneimino; R₃ is hydrogen, cyano, h y d r o c a r b y l i m i d a t e , c a r b o x a m i d e o x i m e , hydrocarbyloxyamidine, carboxamide or carboxylic acid or an ester, thioester, amide or salt thereof or R₃ has the formula

wherein alk is alkylene of 2 to 8 carbon atoms; and R₄ is hydrogen, halogen, hydrocarbyl, amino, hydrocarbylamino, hydroxy, hydrocarbyloxy, sulfhydryl, or hydrocarbylthio; and pharmaceutically acceptable salts thereof.

7. A method according to claim 1 wherein said compound is AICA riboside.

8. A method according to claim 1 wherein said compound is 5-amino-1-(5-amino-5-deoxy-β-D-ribofuranosyl)imidazole-4-N-[(4-chlorophenyl)methyl]-carboxamide.

9. A method of preventing or decreasing tissue damage in a mammal following diminished or decreased blood flow to that tissue which comprises administering to said mammal an effective amount of a compound selected from AICA riboside and a substituted-imidazole analog of AICA riboside to decrease free radical or oxidant levels in said tissue.

10. A method according to claim 9 wherein said decreased blood flow results from myocardial infarction, angina, congestive heart failure, angioplasty, coronary artery bypass grafting, ischemic bowel disease, reconstructive tissue transplant surgery, organ transplants, atherosclerosis, stroke, hemorrhagic shock, vasospasm, inflammation, thrombosis or emboli.

11. A method according to claim 9 wherein said compound decreases levels of superoxide anions, hypochlorous acid or other oxygen-derived free radicals or oxidants in said tissue.

12. A method according to claim 9 wherein said substituted-imidazole comprises a compound of the formula:

or a pharmaceutically acceptable salt thereof wherein:

(a) if R₁ is hydrogen or hydrocarbyl of about 1 to about 18 carbon atoms, optionally substituted with from 1 to about 4 substituents independently selected from hydroxy, sulfhydryl, hydrocarbyl, hydrocarbylthio, halogen, amino, hydrocarbylamino, aryl; or carboxylic acid or an ester, thioester, amide or salt thereof; then R₂ is amino, R₃ is hydrogen, cyano, or carboxylic acid or amides, esters, thioesters, or salts thereof; and R₄ is hydrogen, hydrocarbyl, halogen, hydroxy, hydrocarbyloxy, sulfhydryl, hydrocarbylthio, amino or hydrocarbylamino or

(b) if R₁ is wherein X is -O- or -CH₂-, R₅ and R₆ are independently hydrogen, hydrocarbyl, acyl or hydrocarbyloxycarbonyl; R₇ is hydrogen, halogen, hydroxy, hydrocarbyloxy, sulfhydryl, hydrocarbylthio, sulfamyloxy, amino, hydrocarbylamino, azido, hydrocarbyl, acyloxy, hydrocarbyloxycarboxy or phosphate ester group or salts thereof; then R₂ is hydrogen, amino, hydrocarbylamino acylamino, amido, or dihydrocarbylaminoalkyleneimino; R₃ is hydrogen, cyano, h y d r o c a r b y l i m i d a t e , c a r b o x a m i d e o x i m e , hydrocarbyloxyamidine, carboxamide or carboxylic acid or an ester, thioester, amide or salt thereof or R₃ has the formula

wherein alk is alkylene of 2 to 8 carbon atoms; and R₄ is hydrogen, halogen, hydrocarbyl, amino, hydrocarbylamino, hydroxy, hydrocarbyloxy, sulfhydryl, or hydrocarbylthio; and pharmaceutically acceptable salts thereof.

13. A method of decreasing tissue damage in a mammal due to respiratory burst of leukocytes which comprises administering to said mammal a therapeutically effective amount of AICA riboside or a substituted-imidazole AICA riboside analog which decreases free radical or oxidant levels present in said tissue.

14. A method according to claim 13 wherein said respiratory burst follows decreased or interrupted blood flow to said tissue.

15. A method according to claim 14 wherein said respiratory burst results from infectious microorganisms or viruses.

16. A method according to claim 13 wherein said substituted imidazole comprises a compound of the formula:

(a) if R₁ is hydrogen or hydrocarbyl of about 1 to about 18 carbon atoms, optionally substituted with from 1 to about 4 substituents independently selected from hydroxy, sulfhydryl, hydrocarbyl, hydrocarbylthio, halogen, amino, hydrocarbylamino, aryl; or carboxylic acid or an ester, thioester, amide or salt thereof; then R₂ is amino, R₃ is hydrogen, cyano, or carboxylic acid or amides, esters, thioesters, or salts thereof; and R₄ is hydrogen, hydrocarbyl, halogen, hydroxy, hydrocarbyloxy, sulfhydryl, hydrocarbylthio, amino or hydrocarbylamino or

(b) if R₁ is

wherein X is -O- or -CH₂-, R₅- and R₆ are independently hydrogen, hydrocarbyl, acyl or hydrocarbyloxycarbonyl; R₇ is hydrogen, halogen, hydroxy, hydrocarbyloxy, sulfhydryl, hydrocarbylthio, sulfamyloxy, amino, hydrocarbylamino, azido, hydrocarbyl, acyloxy, hydrocarbyloxycarboxy or phosphate ester group or salts thereof; then R₂ is hydrogen, amino, hydrocarbylamino acylamino, amido or dihydrocarbylaminoalkyleneimino; R₃ is hydrogen, cyano, hydrocarbylimidate, carboxamideoxime, hydrocarbyloxyamidme, carboxamide or carboxylic acid or an ester, thioester, amide or salt thereof or R₃ has the formula

wherein alk is alkylene of 2 to 8 carbon atoms; and R₄ is hydrogen, halogen, hydrocarbyl, amino, hydrocarbylamino, hydroxy, hydrocarbyloxy, sulfhydryl, or hydrocarbylthio; and pharmaceutically acceptable salts thereof.

17. A method of preventing or decreasing reperfusion injury in a mammal following diminished or decreased blood flow to that tissue which comprises administering to said mammal an effective amount of a compound selected from AICA riboside or a substituted-imidazole analog of AICA riboside to decrease free radical or oxidant production or increase oxidant or free radical scavenging.

18. A method according to claim 17 wherein said substituted-imidazole comprises a compound of the formula:

( ) or a pharmaceutically acceptable salt thereof wherein:

(a) if R₁ is hydrogen or hydrocarbyl of about 1 to about 18 carbon atoms, optionally substituted with from 1 to about 4 substituents independently selected from hydroxy, sulfhydryl, hydrocarbyl, hydrocarbylthio, halogen, amino, hydrocarbylamino, aryl; or carboxylic acid or an ester, thioester, amide or salt thereof; then R₂ is amino, R₃ is hydrogen, cyano, or carboxylic acid or amides, esters, thioesters, or salts thereof; and R₄ is hydrogen, hydrocarbyl, halogen, hydroxy, hydrocarbyloxy, sulfhydryl, hydrocarbylthio, amino or hydrocarbylamino or

(b) if R₁ is

wherein X is -O- or -CH₂-, R₅ and R₆ are independently hydrogen, hydrocarbyl, acyl or hydrocarbyloxycarbonyl; R₇ is hydrogen, halogen, hydroxy, hydrocarbyloxy, sulfhydryl, hydrocarbylthio, sulfamyloxy, amino, hydrocarbylamino, azido, hydrocarbyl, acyloxy, hydrocarbyloxycarboxy or phosphate ester group or salts thereof; then R₂ is hydrogen, amino, hydrocarbylamino acylamino, amido, or dihydrocarbylaminoalkyleneimino; R₃ is hydrogen, cyano, h y d r o c a r b y l i m i d a t e , c a r b o x a m i d e o x i m e, hydrocarbyloxyamidme, carboxamide or carboxylic acid or an ester, thioester, amide or salt thereof or R₃ has the formula

wherein alk is alkylene of 2 to 8 carbon atoms; and R₄ is hydrogen, halogen, hydrocarbyl, amino, hydrocarbylamino, hydroxy, hydrocarbyloxy, sulfhydryl, or hydrocarbylthio; and pharmaceutically acceptable salts thereof.

19. A compound which comprises a substituted-imidazole analog of AICA riboside of the formula:

wherein R₁ is

wherein X is -O- or -CH₂-, R₅ and R₆ are independently hydrogen, hydrocarbyl (of 1 to about 18 carbon atoms), acyl or hydrocarbyloxy-carbonyl; and R₇ is hydrogen, hydrocarbyl, hydroxy, hydrocarbyloxy, sulfhydryl, hydrocarbylthio, sulfamyloxy, amino, hydrocarbylamino, azido, acyloxy, hydrocarbyloxycarboxy or phosphate ester or salt thereof; R₂ is amino, hydrocarbylamino, or dihydrocarbylaminoalkyleneimino; R₃ is carboxamide wherein one of the amide hydrogens (attached to the nitrogen atom) is optionally replaced by alkyl, cycloalkyl, or aryl or aralkyl optionally substituted with 1 to 3 substituents independently selected from halogen, alkyl, aryl, nitro, amino, hydrocarbylamino, sulfhydryl, hydrocarbylthio, hydroxy, hydrocarbyl, trifluoromethyl or sulfonamide; R₃ is carboxamide wherein both amide hydrogens are replaced by alkyl or together are an alkylene or aralkylene group to form a ring; R₃ is -C(O)SR₈ wherein R₈ is alkyl, cycloalkyl, aryl or aralkyl optionally substituted with 1 to 3 substituents independently selected from halogen, alkyl, aryl, nitro, amino, hydrocarbylamino, sulfhydryl, hydrocarbylthio, hydroxy, hydrocarbyloxy, trifluoromethyl or sulfonamide; or R₃ is a qroup of the formula:

wherein alk is alkylene of 2 to 8 carbon atoms; R₄ is hydrogen, hydrocarbyl, halogen, hydroxy, hydrocarbyloxy, amino, hydrocarbylamino sulfhydryl, or hydrocarbylthio; provided that when X is -O- or -CH₂-, R₂ is amino, R₃ is substituted carboxamide, R₄ is hydrogen, R₅ and R₆ are independently hydrogen, acyl or hydrocarbyloxycar-bonyl, then R₇ is not hydrogen, hydroxy, acyloxy or hydrocarbyloxycarboxy or when both R₅ and R₆ are hydrogen, R₇ is not a phosphate ester; and provided that when X is oxygen, R₂ is amino, R₃ is unsubstituted carboxamide, R₄ is sulfhydryl, and R₅ and R₆ are both hydrogen, then R₇ is not acetoxy; when X is oxygen, R₂ is amino, R₃ is unsubstituted carboxamide, and R₄ is chloro, bromo, amino or methoxy, and R₅ and R₆ are both hydrogen, then R₇ is not hydroxy or R_g and R₆ are not both acetyl and R₇ is not acetoxy; and provided further that when X is oxygen, R₂ is amino, R₃ is benzylcarboxamide or p-iodophenylcarboxamide, R₄ is hydrogen, then R₅ and R₆ are not both hydrogen, and R₇ is not hydroxy; or when R₃ is p-iodophenylcarboxamide, then R₅ and R₆ are not both acetyl and R₇ is not acetoxy.

20. A compound according to claim 19 wherein R₂ is amino and R₃ has an amide hydrogen replaced by cycloalkyl or substituted aralkyl having from 1 to 3 substituents independently selected from alkyl, aryl, halogen, nitro, amino, hydrocarbylamino, sulfhydryl, hydrocarbylthio, hydroxy, hydrocarbyloxy, trifluoromethyl or sulfonamide.

21. A compound according to claim 20 wherein R₂ is amino and R₄, R₅ and R₆ are independently hydrogen.

22. A compound according to claim 21 wherein R₇ is hydroxy.

23. The compound according to claim 22 wherein X is oxygen and R₃ is N-(cyclopropyl)carboxamide.

24. The compound according to claim 22 wherein X is oxygen and R₃ is N-(cyclopentyl)carboxamide.

25. A compound according to claim 19 wherein R₇ is amino or hydrocarbylamino.

26. A compound according to claim 25 wherein R₇ is amino.

27. A compound according to claim 19 wherein R₇ is benzylamino optionally substituted with 1 to 3 substituents independently selected from halogen, alkyl of 1 to 8 carbon atoms, halogen, nitro, alkylamino, alkylthio or alkoxy.

28. A compound according to claim 26 or 27 wherein R₂ is amino, R₃ is unsubstituted carboxamide and R₄, R₅ and

R₆ are hydrogen.

29. A compound according to claim 19 wherein R₂ is amino and R₃ is hydrogen.

30. A compound according to claim 29 wherein R₃ is carboxamide wherein an amide hydrogen is replaced by aralkyl optionally substituted with 1 to 3 substituents independently selected from halogen, alkyl, aryl, nitro, amino, hydrocarbyloxy, trifluoromethyl or sulfonamide.

31. A compound according to claim 30 wherein R₇ is hydroxy, azido or amino.

32. A compound according to claim 31 wherein R₃ has an amide hydrogen replaced by a para-substituted benzyl group.

33. A compound according to claim 32 wherein R₃ is N- (4-chlorobenzyl)carboxamide.

34. A compound according to claim 33 wherein R₇ is amino.

35. A compound according to claim 34 wherein R₅ and R₆ are hydrogen and X is oxygen.

36. A compound according to claim 34 wherein R₅ and R₆ are independently alkyl, acyl or hydrocarbylloxycarbonyl and X is oxygen.

37. A compound according to claim 36 wherein R₅ and R₆ are acetyl and X is oxygen.

38. A compound according to either of claims 35 or 37 which comprises a hydrochloride salt.

39. A compound according to either of claim 35 or 37 which comprises a salt selected from hydrobromide, hydrosulfate, sulfate, hydrophosphate or oxalate.

40. A compound according to claim 33 wherein R₇ is azido.

41. A compound according to claim 32 wherein R₃ is N- (4-nitrobenzyl)carboxamide.

42. A compound according to claim 32 wherein R₃ is N- (1,4-dichlorobenzyl)-carboxamide.

43. A compound according to either of claims 41 or 42 wherein R₅ and R₆ are hydrogen, R₇ is hydroxy and X is oxygen.

44. A compound according to claim 29 wherein R₃ is -C(O)-S-R₈.

45. A method of preventing or decreasing viral infectivity or post-viral injury in a mammal which comprises administering to said mammal an effective amount of a compound selected from AICA riboside or a substituted-imidazole analog of AICA riboside to decrease free radical or oxidant production or increase oxidant or free radical scavenging.

46. A method of preventing or decreasing viral infectivity or post-viral injury in a mammal which comprises administering to said mammal an effective amount of a compound selected from AICA riboside or a substituted imidazole analog of AICA riboside to decrease membrane fusion and viral entry or budding.

47. A method of decreasing platelet aggregation in a mammal which comprises administering to said mammal an effective amount of a compound selected from AICA riboside, or a substituted-imidazole analog of AICA riboside to decrease free radical or oxidant production or increase oxidant or free radical scavenging.

48. A method of preventing or decreasing thrombosis, deep vein thrombosis, or systemic or pulmonary embolism with a mammal which comprises administering to said mammal an effective amount of a compound selected from AICA riboside, or a substituted-imidazole analog of AICA riboside to decrease platelet aggregation.

49. A method according to claim 48 wherein said compounds are used or administered in conjunction with a thrombolytic agent.

50. A method of preventing or decreasing tissue damage in a mammal resulting from free radical or oxidant accumulation which comprises administering to said mammal an effective amount of a compound selected from AICA riboside, or a substituted-imidazole analog of AICA riboside to decrease free radical or oxidant production or increase oxidant or free radical scavenging.

51. A method according to claim 50 wherein such tissue damage results from arthritis, autoimmune disease, sepsis, burns, hyperoxia, inflammatory bowel disease, dialysis, aspiration, adult respiratory distress syndrome, chronic cystitis inflammation, or chronic obstructive pulmonary disease.

52. A method of preventing or reducing tissue damage during the process of cardioplegia which comprises addition of a therapeutically effective amount of AICA riboside or a substituted-imidazole analog of AICA riboside to a cardioplegia solution.

53. A method of preventing or reducing damage to cardiac tissue following interruption of cardiac contraction by means of a chemical agent or hypothermia which comprises administering a therapeutically effective amount of AICA riboside or a substituted-imidazole analog of AICA riboside to said tissue or its environment.

54. A method of preventing or reducing tissue damage in a mammal during cardioplegia or general surgery which comprises administering to said mammal a therapeutically effective amount of AICA riboside or a substituted imidazole analog of AICA riboside.

55. A method of prolonging cellular viability and function in stored whole blood, red blood cells, white blood cells or platelets or in blood samples (for diagnosis or cross-matching purposes) which comprises adding to said blood, cells, platelets or sample, a viability prolonging effective amount of AICA riboside or a substituted imidazole analog of AICA riboside.

56. A method according to claim 55 wherein AICA riboside or the substituted imidazole analog is added to said blood, cells, platelets or sample soon after it is drawn and prior to storage.

57. A method according to claim 56 wherein AICA riboside or the substituted-imidazole analog is added in an amount sufficient to give a final concentration of 0.1 μM to 1000 μM.

58. A method of treating angina pectoris in a mammal which comprises administering to said mammal a therapeutically effective amount of AICA riboside or a substituted imidazole analog of AICA riboside effective to decrease inactivation or destruction of nitric oxide.

59. A method of decreasing destruction or inactivation of endothelium derived relaxing factor which comprises treating a mammal or isolated tissue or cells thereof with an effective amount of AICA riboside or a substituted-imidazole analog of AICA riboside that decreases free radical or oxidant production or increases oxidant or free radical scavenging.

60. A method of decreasing or preventing damage from defects in calcium translocation in a mammal or tissues or cell thereof following a period of diminished or interrupted blood flow or hypoxia in a tissue or cells which comprises administering an amount of AICA riboside or a substituted-imidazole analog of AICA riboside effective to decrease oxidant or free radical levels in said tissue or cells.

61. A method of decreasing or preventing free radical or oxidant-mediated defects in calcium translocation in a mammal or tissue, cells or isolated organ thereof, which comprises adminstering to said mammal, tissue, cells or organ, an effective amount of AICA riboside or a substituted imidazole analog of AICA riboside effective to decrease oxidant or free-radical production or increase free radical or oxidant scavenging.

62. A compound of the formula:

wherein X is -O- or -CH₂-, _R2 is amino, hydrocarbylamino, or dihydrocarbylaminoalkyleneimino; R₃ is piperizinocarbamoyl optionally substituted with hydrocarbyl optionally substituted with 1 to 3 substituents independently selected from halogen, alkyl, aryl, nitro, amino, hydrocarbylamino, sulfhydryl, hydrocarbylthio, hydroxy, hydrocarbyl, trifluoromethyl or sulfonamide; R₄ is hydrogen, hydrocarbyl, halogen, hydroxy, hydrocarbyloxy, amino, hydrocarbylamino sulfhydryl, or hydrocarbylthio; R₅ and R₆ are independently hydrogen, hydrocarbyl (of 1 to about 18 carbon atoms), acyl or hydrocarbyloxycarbonyl; and R₇ is hydrogen, hydrocarbyl, hydroxy, hydrocarbyloxy, sulfhydryl, hydrocarbylthio, sulfamyloxy, amino, hydrocarbylamino, azido, acyloxy, hydrocarbyloxycarboxy or phosphate ester or salt thereof.

63. A compound according to claim 62 wherein R₂ is piperazinylcarbamoyl substituted at N-4 of the piperazine ring.