Substituted ideno[1,2-c]isoquinoline derivatives and methods of use thereof

Abstract

The invention provides a novel class of substituted indeno[1,2-c]isoquinoline derivatives. Pharmaceutical compositions and methods of making and using the compounds, are also described.

Description

FIELD OF THE INVENTION

[0001] The invention relates generally to substituted tetracyclic benzamide derivatives and more particularly to indeno[1,2-c]isoquinoline.

BACKGROUND OF THE INVENTION

[0002] Inflammation disorders, such as arthritis, colitis, and autoimmune diabetes, typically manifest themselves as disorders distinct form those associated with reperfusion injury, e.g., stroke and heart attack, and can clinically manifest themselves as different entities. However, there can be common underlying mechanisms between these two types of disorders. In particular, inflammation and reperfusion injury can induce proinflammatory cytokine and chemokine synthesis. Induction of pro-inflammatory cytokines can, in turn, result in production of cytotoxic free radicals such as nitric oxide and superoxide. NO and superoxide can react to form peroxynitrite (ONOO.sup.-) (Szab{acute over (o )} et al., Shock 6:79-88, 1996).

[0003] The peroxynitrite-induced cell necrosis observed in inflammation and reperfusion injury involves, in significant part, the activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS). Activation of PARS is thought to be an important step in the cell-mediated death observed in inflammation and reperfusion injury (Szab et al., Trends Pharmacol. Sci. 19: 287-98, 1998).

[0004] A number of PARS inhibitors have been described in the art. See, e.g., Banasik et al., J. Biol. Chem., 267:1569-75, 1992, and Banasik et al., Mol. Cell. Biochem., 138:185-97, 1994. Additionally, some potent PARS inhibitors are reported in, for example, WO 00/39104, WO 00/39070, WO 99/59975, WO 99/59973, WO 99/11649, WO 99/11645, WO 99/11644, WO 99/11628, WO 99/11623, WO 99/11311, WO 00/42040; Zhang et al., Biochem. Biophys. Res. Commun., 278:590-98, 2000, White et al., J. Med. Chem., 43:4084-4097, 2000; Griffin et al., J. Med. Chem., 41:5247-5256, 1998; Shinkwin et al., Bioorg. Med. Chem., 7:297-308, 1999, Soriano et al., Nature Medicine, 7:108-113, 2001. Furthermore, side effects of some of the best known-PARP inhibitors have been discussed in Milan et al, Science, 223:589-591, 1984.

[0005] Certain indeno[1,2-c]isoquinoline derivatives are known in the art. For example, cytotoxic non-camptothecin topoisomerase I inhibitors are reported in Cushman et al., J. Med. Chem., 43:3688-3698, 2000; Cushman et al., J. Med. Chem. 42:446-57, 1999; indeno[1,2-c]isoquinoline as antineoplastic agents are reported in Cushman et al., WO 99/23900; neoplasm inhibitors are disclosed in Hrbata et al., WO/9305023.

[0006] Syntheses of substituted indeno[1,2-c]isoquinoline, other than the compounds of the invention, are reported in, for example, Wawzonek et al., Org. Prep. Proc. Int. 14:163-8, 1982; Wawzonek et al., Can. J. Chem. 59:2833, 1981; Andoi et al., Bull. Chem. Soc. Japan, 47:1014-17, 1974; Dusemund et al., Arch. Pharm (Weinheim, Ger.), 317:381-2, 1984; and Lal et al, Indian J. Chem., Sect. B, 38B:33-39, 1999.

SUMMARY OF THE INVENTION

[0007] The invention is based in part on the discovery of novel substituted tetracyclic benzamide derivatives and their unexpected effects in inhibiting inflammation, cell death and in treating shock and reperfusion injuries.

[0008] Accordingly, in one aspect the invention includes an indeno[1,2-c]isoquinoline derivative according to Formula I and Formula II, as set forth in the Detailed Description of the Invention, below.

[0009] Also provided by the invention is a method of treating inflammatory and reperfusion conditions in mammals by administering to a mammal in need of such treatment an effective amount of a compound according to Formula I or Formula II.

[0010] In a further aspect, the invention also includes a method for the production of a compound according to Formula I or Formula II. 1

[0011] The substituted indeno[1,2-c]isoquinoline compounds described in the current invention are potent compounds that can be used to treat a variety of conditions and diseases, typically those known to involve inflammatory mediator production and cell death.

[0012] The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications cited in this specification are incorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention provides a novel class of substituted indeno[1,2-c]isoquinoline derivatives according to Formula I and Formula II, as set forth below: 2

[0014] Specifically, the present invention relates to a compound of Formula I, wherein:

[0015] R.sub.5 is O, N or S:

[0016] R.sub.6 is H or straight chain alkyl:

[0017] X is CO, CH.sub.2, CH-Halo, CH(CH.sub.2).sub.nOH, aryl-C--OH, O, NH, S or CH--NR.sub.11R.sub.12;

[0018] wherein n is zero or a positive integer;

[0019] R.sub.11 and R.sub.12 are, independently, H, C.sub.1-C.sub.9 alkyl, or, taken together, N, R.sub.11, and R.sub.12 form an optionally-substituted heterocycle including, but not limited to, piperidine, piperazine, and morpholine;

[0020] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.7, R.sub.8, R.sub.9, and R.sub.10 are, independently, hydrogen, halo, alkylhalo, hydroxy, alkoxy, C.sub.1-C.sub.10 straight or branched chain alkyl, C.sub.2-C.sub.10 straight or branched chain alkenyl group, C.sub.3-C.sub.9 carbocyclic, aryl, alkylamino, amino, carboxy, ester, arylalkyl, nitro or A-B;

[0021] wherein A is --SO.sub.2--, --SO.sub.2NH--, --NHCO--, --NHCONH--, O, CO, OCO, CONH, NH, CH.sub.2, S or CS;

[0022] B is C.sub.1-C.sub.10 straight or branched chain alkyl, C.sub.2-C.sub.10 straight or branched chain alkenyl group, heterocycle, C.sub.3-C.sub.8 carbocycle, aryl, amino, aminoalkyl, aminodialkyl, heterocyclic amine, alkylheterocycle, arylamido, carboxy, ester, or an arylalkyl group, any of which may be optionally substituted with one or more of alkoxy, halogen, alkylhalo, alkylhydroxy, alkylamino, hydroxy, nitro, amino, aminoalkyl, aminodialkyl, heterocyclic amine, C.sub.1-C.sub.10 straight or branched chain alkyl, C.sub.2-C.sub.10 straight or branched chain alkenyl, C.sub.2-C.sub.10 straight or branched chain alkynyl, aryl, benzyl, alkylamido, alkylcarboxy, alkylester, arylalkyl, or a heterocycle or C.sub.3-C.sub.8 carbocycle optionally further substituted with alkyl, alkoxy, halogen, alkylhalo, alkylhydroxy, alkylamino, hydroxy, nitro, or amino.

[0023] In some embodiments, when A is SO.sub.2, B is NZ.sub.1Z.sub.2, where Z.sub.1 and Z.sub.2 are, independently, H, or C1-5 alkyl, optionally-substituted with halo, OH or NZ.sub.3Z.sub.4, where Z.sub.3 and Z.sub.4 are independently, H or C1-C5 alkyl optionally-substituted with halo, OH or amino.

[0024] In embodiments where Z.sub.1 and Z.sub.2 are connected, NZ.sub.1Z.sub.2 forms a heterocyclic amine. Similarly, if Z.sub.3 and Z.sub.4 are joined, NZ.sub.3Z.sub.4 forms an optionally-substituted heterocyclic amine. Such heterocyclic amines include, but are not limited to, piperidine, piperazine, morpholine, N-alkylated or alkylcarbonylated piperazines, pyrole, imidazole, benzimidazole, tetrazoles, indole, isoquinoline, quinoline, pyrrolidine and purine.

[0025] These heterocyclic amines formed by either NZ.sub.1Z.sub.2 or NZ.sub.3Z.sub.4 may be further substituted with one or more of alkoxy, halogen, alkylhalo, alkylhydroxy, alkylamino, hydroxy, nitro, amino, aminoalkyl, aminodialkyl, heterocyclic amine, C.sub.1-C.sub.10 straight or branched chain alkyl, C.sub.2-C.sub.10 straight or branched chain alkenyl, C.sub.2-C.sub.10 straight or branched chain alkynyl, aryl, benzyl, alkylamido, alkylcarboxy, alkylester, arylalkyl, or a heterocycle or C.sub.3-C.sub.8 carbocycle optionally further substituted with alkyl, alkoxy, halogen, alkylhalo, alkylhydroxy, alkylamino, hydroxy, nitro or amino functionalities.

[0026] The invention also relates to a compound of Formula II 3

[0027] wherein:

[0028] R.sub.1, R.sub.4, R.sub.7, and R.sub.10 are hydrogen;

[0029] R.sub.2 and R.sub.3 are hydrogen, halo, alkylhalo, hydroxy, alkoxy, C1-C3 straight or branched chain alkyl, nitro, amino, amido, carboxy, or ester;

[0030] R.sub.8 and R.sub.9 are either hydrogen or A-B; and A is --SO.sub.2--, --SO.sub.2NH--, or --NHCO, B is C.sub.1-C.sub.3 straight or branched chain alkyl, heterocycle, amino, aminoalkyl, aminodialkyl, or heterocyclic amine, optionally substituted with one or more of alkylhydroxy, alkylamino, aminoalkyl, aminodialkyl, heterocyclic amine, or a heterocycle optionally further substituted with alkyl, or alkyl hydroxy.

[0031] In preferred embodiments, when A=SO.sub.2, B is NZ.sub.1Z.sub.2, where Z.sub.1 and Z.sub.2 are, independently, H, or C1-3 alkyl optionally substituted with OH or NZ.sub.3Z.sub.4, where Z.sub.3 and Z.sub.4 are independently, H or C1-C3 alkyl optionally substituted with OH or amino. When Z.sub.1 and Z.sub.2 are connected, NZ.sub.1Z.sub.2 forms a heterocyclic amine. Similarly, if Z.sub.3 and Z.sub.4 are joined, NZ.sub.3Z.sub.4 forms an optionally substituted heterocyclic amine. Such a heterocyclic amine includes, but is not limited to, piperidine, piperazine, morpholine, N-alkylated or alkylcarbonylated piperazines, pyrrolidine, and imidazole.

[0032] When either NZ.sub.1Z.sub.2 or NZ.sub.3Z.sub.4 is a heterocycle, said heterocycle may be further substituted with alkyl, alkylhydroxy, or alkylamino.

[0033] The invention also includes a pharmaceutical composition that includes a compound according to Formula I or Formula II and a pharmaceutically acceptable carrier. The invention includes a compound according to Formula I or Formula II when provided as a pharmaceutically acceptable prodrug, hydrated salt, such as a pharmaceutically acceptable salt, or mixtures thereof.

[0034] Salts encompassed within the term "pharmaceutically acceptable salts" refer to non-toxic salts of the compounds of the invention which are generally prepared by reacting the free base with a suitable organic or inorganic acid to produce "pharmaceutically-acceptable acid addition salts" of the compounds described herein. These compounds retain the biological effectiveness and properties of the free bases. Representative salts include, e.g., water-soluble and water-insoluble salts, such as the acetate, amsonate (4,4-diaminostilbene-2,2'-disulfonate), benzenesulfonate, benzonate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate (1,1-methylene-bis-2-hydroxy-3-naphthoate, embonate), pantothenate, phosphate/diphosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosaliculate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate salts.

[0035] Methods of Using Substituted indeno[1,2-c]isoquinoline Derivatives

[0036] The invention also includes a method of inhibiting poly(ADP-ribose) synthase activity (PARS) in a cell. This enzyme, which is also known as poly(ADP-ribose)synthetase and PARP (poly(ADP-ribose) polymerase, EC 2.4.99), and ADP-ribosyltransferase (ADPRT, EC 2.4.2.30), is a nuclear enzyme that catalyzes a transfer of the ADP ribose moiety of NAD+ to an acceptor protein.

[0037] The method includes contacting the cell with a compound of Formula I in an amount sufficient to inhibit poly (ADP)-ribose-synthase in the cell. In general, any cell having, or capable of having, PARS activity can be used. The cell can be provided in any form as long as it is accessible to the compound. For example, the cell can be provided in vitro, ex vivo, or in vivo. PARS activity can be measured using any method known in the art, e.g., methods as described in Banasik et al., J. Biol. Chem. 267:1569-75 (1991).

[0038] Also provided in the invention is a method of inhibiting, preventing, or treating inflammation in a subject. The inflammation can be associated, e.g., with an inflammatory disease. Inflammatory diseases refer to diseases or conditions where there is an inflammation of the body tissue. These include local inflammatory responses and systemic inflammation. Examples of such diseases and conditions include: transplant rejection; chronic inflammatory disorders of the joints, including arthritis, rheumatoid arthritis, osteoarthritis and bone diseases associated with increased bone resorption; inflammatory bowel diseases such as ileitis, ulcerative colitis, Barrett's syndrome, and Crohn's disease; inflammatory lung disorders such as asthma, adult respiratory distress syndrome, and chronic obstructive airway disease; inflammatory disorders of the eye including corneal dystrophy, trachoma, onchocerciasis, uveitis, sympathetic ophthalmitis and endophthalmitis; chronic inflammatory disorders of the gum, including gingivitis and periodontitis; tuberculosis; leprosy; inflammatory diseases of the kidney including uremic complications, glomerulonephritis and nephrosis; inflammatory disorders of the skin including sclerodermatitis, psoriasis and eczema; inflammatory diseases of the central nervous system, including chronic demyelinating diseases of the nervous system, multiple sclerosis, AIDS-related neurodegeneration and Alzheimer's disease, infectious meningitis, encephalomyelitis, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and viral or autoimmune encephalitis; autoimmune diseases including diabetes mellitus, immune-complex vasculitis, systemic lupus erythematosus (SLE); inflammatory diseases of the heart such as cardiomyopathy, ischemic heart disease hypercholesterolemia, and atherosclerosis; as well as various other diseases with significant inflammatory components, including preeclampsia; chronic liver failure, brain and spinal cord trauma, cancer. There may also be a systemic inflammation of the body, exemplified by gram-positive or gram negative shock, hemorrhagic or anaphylactic shock, or shock induced by cancer chemotherapy in response to pro-inflammatory cytokines, e.g., shock associated with pro-inflammatory cytokines. Such shock can be induced, e.g., by a chemotherapeutic agent used cancer chemotherapy.

[0039] The invention also includes a method of treating, preventing, or otherwise inhibiting reperfusion injury in a subject in need of treatment, prevention, or inhibition thereof. The method includes administering a compound of Formula I in an amount sufficient to inhibit reperfusion injury in the subject. Reperfusion refers to the process whereby blood flow in the blood vessels is resumed after blood flow has been interrupted, such as occurs following constriction or obstruction of the vessel. Reperfusion is typically associated with ischemia and may result following a naturally occurring episode, such as a myocardial infarction or stroke, or during a surgical procedure where blood flow in vessels is purposely or unintentionally blocked off.

[0040] The subject treated by the compounds of the invention can be, e.g., a mammal, e.g., a human, mouse, rat, dog, cat, horse, cow, pig, or non-human primate. Administration can be systemic or topical, and can be prophylactic or therapeutic.

[0041] In some embodiments, the subject is treated with a pharmacologically effective amount of a compound of the invention. The term "pharmacologically effective amount" means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by a researcher or clinician.

[0042] The invention also includes pharmaceutical compositions suitable for inhibiting or preventing inflammation or reperfusion injury, PARS activity, or more than one of these activities. The compositions are preferably suitable for internal use and include an effective amount of a pharmacologically active compound of the invention, alone or in combination, with one or more pharmaceutically acceptable carriers. The compounds are especially useful in that they have very low, if any toxicity.

[0043] In practice, the compounds or their pharmaceutically acceptable salts, are administered in amounts which will be sufficient to inhibit ischemic or inflammatory conditions or diseases and/or prevent the development of inflammation or inflammatory disease in animals or mammals, and are used in the pharmaceutical form most suitable for such purposes.

[0044] Administration of the active compounds and salts described herein can be via any of the accepted modes of administration for therapeutic agents. These methods include systemic or local administration such as oral, nasal, parenteral, transdermal, subcutaneous, or topical administration modes.

[0045] Depending on the intended mode of administration, the compositions may be in solid, semi-solid or liquid dosage form, such as, for example, injectables, tablets, suppositories, pills, time-release capsules, elixirs, tinctures, emulsions, syrups, powders, liquids, suspensions, or the like, preferably in unit dosages and consistent with conventional pharmaceutical practices. Likewise, they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.

[0046] Preferred pharmaceutical compositions are tablets and gelatin capsules comprising the active ingredient, or the pharmaceutically acceptable salt thereof, together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, sodium, saccharin, glucose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and/or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, magnesium carbonate, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, waxes and/or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, methyl cellulose, bentonite, xanthan gum, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners.

[0047] Liquid, particularly injectable compositions can, for example, be prepared by dissolving, dispersing, etc. The active compound is dissolved in or mixed with a pharmaceutically pure solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form the injectable isotonic solution or suspension.

[0048] The active compound defined above, may be also formulated as suppositories which are advantageously prepared from fatty emulsions or suspensions; using for example, polyalkylene glycols, for example, propylene glycol, as the carrier.

[0049] The compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, containing cholesterol, stearylamine or phosphatidylcholines. In some embodiments, a film of lipid components is hydrated with an aqueous solution of drug to a form lipid layer encapsulating the drug, as described in U.S. Pat. No. 5,262,564.

[0050] Active compounds may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-ph- enol, polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.

[0051] Parental injectable administration is generally used for subcutaneous, intramuscular or intravenous injections and infusions. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions or solid forms suitable for dissolving in liquid prior to injection.

[0052] One approach for parenteral administration employs the implantation of a slow-release or sustained-released systems, which assures that a constant level of dosage is maintained, according to U.S. Pat. No. 3,710,795, incorporated herein by reference.

[0053] The compositions may be sterilized and/or contain minor amounts of non-toxic adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure pH buffering agents, and other substances such as for example, sodium acetate, triethanolamine oleate, etc. In addition, they may also contain other therapeutically valuable substances.

[0054] Compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and of the above pharmaceutical compositions may contain 0.1 to 99%, preferably 1 to 70% of the active compounds, especially compounds of the Formula I as active ingredients.

[0055] The dosage regimen utilizing the compounds is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.

[0056] Oral dosages of the present invention, when used for the indicated effects, will range between about 0.05 to 1000 mg/day orally. The compositions are preferably provided in the form of scored tablets containing 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100.0, 250.0, 500.0 and 1000.0 mg of active ingredient. Effective plasma levels of the compounds of the present invention range from 0.002 mg to 50 mg per kg of body weight per day.

[0057] Compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily. Furthermore, preferred compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen. Other preferred topical preparations include creams, ointments, lotions, aerosol sprays and gels, wherein the concentration of active ingredient would range from 0.1% to 15%, w/w or w/v.

[0058] Methods of Making Substituted indeno[1,2-b]isoquinoline Derivatives

[0059] Examples of synthetic pathways for making compounds according to the invention are set forth in the Examples, below and outlined in Schemes 1 and 2. For example, 5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]is- oquinoline was prepared by reacting compound 1 (Aldrich Chemical) with ammonia in methanol.

[0060] (.+-.) 11-hydroxy-5,6-dihydro-5-oxo-11H-indeno[1,2-c]isoquinoline (3a) was prepared by reacting 2 with NaBH.sub.4 in ethanol.

[0061] (.+-.) 11-hydroxy-11-methyl-5,6-dihydro-5-oxo-11H-indeno[1,2-c]isoq- uinoline (3b) was prepared by reacting 2 with MeMgI.

[0062] (.+-.) 11-hydroxy-11-(m-methoxyphenyl)-5,6-dihydro-5-oxo-11H-indeno- [1,2-c]isoquinoline (3b) was prepared from 2 using m-MeO--C.sub.6H.sub.4Mg- I.

[0063] (.+-.) 11-N,N-dimethyl-5,6-dihydro-5-oxo-11H-indeno[1,2-c]isoquinol- ine (5a) was prepared from 3a using chloroacetylchloride followed by reacting with dimethylamine. Similarly prepared are: (.+-.) 11-N,N-diethyl-5,6-dihydro-5-oxo-11H-indeno[1,2-c]isoquinoline (5b), (.+-.) 11-N-piperidino-5,6-dihydro-5-oxo-11H-indeno[1,2-c]isoquinoline (5c), (.+-.) 11-N-(4-methylpiperazino)-5,6-dihydro-5-oxo-11H-indeno[1,2-c- ]isoquinoline (5d), (.+-.) 11-N-morpholino-5,6-dihydro-5-oxo-11H-indeno[1,- 2-c]isoquinoline (5e), (.+-.) 11-N-morpholino-5,6-dihydro-5-oxo-11H-indeno- [1,2-c]isoquinoline (5e) was also prepared from (.+-.) 1-bromo-5,6-dihydro-5-oxo-1H-indeno[1,2-c]isoquinoline (4b).

[0064] 5,6-Dihydro-5-oxo-11H-indeno-[1,2-c]isoquinoline (6)is prepared by reduction of 5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoline (2) or (1)11-hydroxy-5,6-dihydro-5-oxo-11H-indeno[1,2-c]isoquinoline (3a) using CF.sub.3COOH/triethylsilane. 9-Chlorosulphonyl-5,6-dihydro-5-oxo-11H-inde- no-[1,2-c]isoquinoline (7) was prepared by chlorosulfonation of 5,6-dihydro-5-oxo-11H-indeno-[1,2-c]isoquinoline (6). 9-[N-(4-methylpiperazine)sulphonyl]-5,6-dihydro-5-oxo-11H-indeno-[1,2-c]i- soquinoline

[0065] (8a) was prepared from 9-chlorosulphonyl-5,6-dihydro-5-oxo-11H-inde- no-[1,2-c]isoquinoline (7), and N-methylpiperazine. Similarly prepared are: 9-[N-(4-carbomethoxymethylenepiperazine)sulphonyl]-5,6-dihydro-5-oxo- -11H-indeno-[1,2-c]isoquinoline (8b), 9-[N-4-(2-hydroxyethylpiperazine)sul- phonyl]-5,6-dihydro-5-oxo-11H-indeno-[1,2-c]isoquinoline (8c), 9-[N-(1-imidazole)sulphonyl]-5,6-dihydro-5-oxo-11H-indeno-[1,2-c]isoquino- line (8d), 9-[N-(2-hydroxyproline)sulphonyl]-5,6-dihydro-5-oxo-11H-indeno-- [1,2-c]isoquinoline (8e), 9-[N-morpholinesulphonyl]-5,6-dihydro-5-oxo-11H-- indeno-[1,2-c]isoquinoline (8f), 9-[N-(2-N,N-dimethylethyl)-sulphonyl]-5,6- -dihydro-5-oxo-11H-indeno-[1,2-c]isoquinoline (8g), 9-[N-(2-piperidinoethyl)-sulphonyl]-5,6-dihydro-5-oxo-1H-indeno-[1,2-c]is- oquinoline (8h), 9-[N-2-(2-pyridinoethyl)-sulphonyl]-5,6-dihydro-5-oxo-11H- -indeno-[1,2-c]isoquinoline (8i), 9-[N-2-(morpholinoethyl)-sulphonyl]-5,6-- dihydro-5-oxo-11H-indeno-[1,2-c]isoquinoline (8j), 9-[N-2-(N-methyltetrahy- dropyrrolidino)-sulphonyl]-5,6-dihydro-5-oxo-11H-indeno-[1,2-c]isoquinolin- e (8k), 9-[N-(3-morpholinopropyl)-sulphonyl]-5,6-dihydro-5-oxo-11H-indeno-- [1,2-c]isoquinoline (8l), 9-[N-(3-tetrahydropyrrolodinopropyl)-sulphonyl]-- 5,6-dihydro-5-oxo-11H-indeno-[1,2-c]isoquinoline (8m), 9-[N-3-(1-imidazolepropyl)-sulphonyl]-5,6-dihydro-5-oxo-11H-indeno-[1,2-c- ]isoquinoline (8n), 9-[N-3(4-methylpiperazinopropyl)-sulphonyl]-5,6-dihydr- o-5-oxo-11H-indeno-[1,2-c]isoquinoline (8o),9-[N-di-(N,N-diethylethyl)-sul- phonyl]-5,6-dihydro-5-oxo-11H-indeno-[1,2-c]isoquinoline (8p), 9-[N-di-(N,N-dimethylethyl)-sulphonyl]-5,6-dihydro-5-oxo-11H-indeno-[1,2-- c]isoquinoline (8q), and 9-[N-di-(N,N-dihydroxyethyl)-sulphonyl]-5,6-dihyd- ro-5-oxo-11H-indeno-[1,2-c]isoquinoline (8r).

[0066] The amide derivatives 5,6-dihydro-5-oxo-[11H-indeno-[1,2-c]isoquino- line 3-yl]-N,-morpholinoacetamide (10a), 5,6-dihydro-5-oxo-[11H-indeno-[1,- 2-c]isoquinoline 3-yl]-N,N-dimethylacetamide (10b) and 5,6-dihydro-5-oxo-[11H-indeno-[1,2-c]isoquinoline 2-yl]-N,N-dimethylaceta- mide (14) were prepared from 5,6-dihydro-5-oxo-[11H-indeno-[1,2-c]isoquino- line (6) and 5-chloro-[11H-indeno-[1,2-c]isoquinoline (11) using nitration, then reduction, and followed by amination of chloroacetamide. 5,6-Dihydro-5-oxo-11H-indeno-[1,2-c]isoquinoline (15) was prepared by bromination of chloroamidate 11.

[0067] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. The following examples illustrate the synthesis of novel substituted indeno[1,2-c]isoquinoline derivatives of the invention, and of the use of these compounds to inhibit inflammation and reperfusion. 4 5

EXAMPLES

Example 1

Synthesis of Substituted indeno[1,2-c]isoquinoline

[0068] a) General Methods

[0069] Proton nuclear magnetic resonance (NMR) spectra were obtained from Varian 300 MHz spectrophotometer and chemical shift is reported in parts per million, 6. Thin layer chromatography, TLC, was carried out on precoated TLC plates with silica gel 60 F-254 and preparative TLC on precoated Whatman 60A TLC plates. All intermediates and final compounds were characterized on the basis of .sup.1H NMR and Mass spectral (MS) data.

[0070] b) Synthesis of 5,6-dihydro-5,11-diketo-11H-indeno[1,2-c]isoquinoli- ne (2): 6

[0071] A stirred suspension of 1 (55 g, 0.22 mol) in NH.sub.3/MeOH (7.0 N, 700 mL) was refluxed for 24 h. The reaction mixture was then allowed to come to room temperature where it was filtered and washed thoroughly with MeOH to give 46 g of the orange colored product in 84% yield. .sup.1H NMR (DMSO-D.sub.6): 7.48-7.61 (m, 4H), 7.80-7.88 (m, 1H), 7.86 (d, J=8.7 Hz, 1H), 8.22 (d, J=8.4 Hz, 1H), 8.44 (d, J=7.5 Hz, 1H), 13.05 (s, 11H); .sup.13C NMR (DMSO-D.sub.6): 106.33, 121.63, 122.94, 123.27, 124.80, 128.45, 132.17, 133.60, 134.03, 134.68, 134.68, 134.81, 137.09, 156.41, 163.76, 190.57; MS (ES.sup.-): m/z 246.2 (M-1); Anal. Calcd for C.sub.16H.sub.9NO.sub.2: C, 77.72; H, 3.67; N, 5.67; Found: C, 77.54; H, 3.69, N, 5.69.

[0072] c) Synthesis of (.+-.) 11-hydroxy-5,6-dihydro-5-oxo-11H-indenol1,2-- c]isoquinoline (3a): 7

[0073] To a stirred suspension of 2 (2.5 g, 0.01 mol) in EtOH (25 mL) was added NaBH.sub.4 (3.75 g, 0.1 mol) at room temperature in small portions over 30 min. The reaction mixture was stirred for an additional 2 h and then cooled down to 0.degree. C. where it was triturated with dilute (dil.) HCl (10% soln.). The resulting solid precipitated was filtered, washed with water and MeOH to give 3a (2.326 g, 92%). .sup.1H NMR (DMSO-D.sub.6): 5.58 (d, J=8.1 Hz, 1H), 5.78 (d, J=8.7 Hz, 1H), 7.33-7.89 (m, 6H), 7.95 (d, J=7.8 Hz, 1H), 8.22 (d, J=7.8 Hz, 1H), 12.29 (s, 1H); .sup.13C NMR (DMSO-D.sub.6): 77.44, 118.81, 120.15, 124.28, 125.04, 125.67, 126.34, 128.46, 128.64, 128.95, 133.27, 135.62, 136.12, 139.93, 148.55, 163.69.; MS (ES.sup.+): m/z 250.1 (M+1); Anal. Calcd for C.sub.16H.sub.11NO.sub.2: C, 77.10; H, 4.45; N, 5.62. Found: C, 77.01; H, 4.57, N, 5.59. Similarly by reacting 2 with MeMgI and m-MeO--C.sub.6H.sub.4MgBr compounds (.+-.) 11-hydroxy-11methyl-5,6-dihydr- o-5-oxo-11H-indeno[1,2-c]isoquinoline (3b) and (1) 11-hydroxy-11-(m-methox- yphenyl)-5,6-dihydro-5-oxo-1 1H-indeno[1,2-c]isoquinoline (3c) are prepared.

[0074] d) Synthesis of 5,6-dihydro-5-oxo-11H-indeno[1,2-c]isoquinolines (5a-e): 8

[0075] To a stirred suspension of the 3 (0.5 g, 0.002 mol) in pyridine (10 mL) was added chloroacetyl chloride (0.81 g, 0.006 mol) at 0.degree. C. The reaction mixture was allowed to come to room temperature where it was stirred for 24 h. The reaction mixture was then poured on ice and extracted with EtOAc. The organic layer was separated, dried, concentrated to give crude compound 4a, which was treated further with the amines and stirred at room temperature for 24 h. The reaction mixture was poured on ice, the resulting solid was filtered and transferred in a round bottom flask where it was dissolved in 10% HCl. If a solid had not precipitated after pouring on ice, then the reaction mixture was basified with sat. aq. (saturated aqueous) NaHCO.sub.3 and the resulting solid was filtered to give the desired product. 5a: .sup.1H NMR (DMSO-D.sub.6): 2.31 (s, 6H), 5.00 (s, 1H), 7.28-7.45 (m, 3H), 7.68-7.73 (m, 2H), 7.95 (d, J=6.9 Hz, 1H), 8.10 (d, J=7.8 Hz, 1H), 8.21 (d, J=8.1 Hz, 1H), 12.26 (s, 1H); .sup.13C NMR (DMSO-D.sub.6): 68.09, 116.28, 120.52, 124.58, 125.74, 126.27, 126.34, 127.68, 128.64, 133.02, 136.27, 144.45, 163.80; MS (ES+): m/z 277.2 (M+1).

[0076] The following compounds were also prepared by reacting 4a with the appropriate amines:

[0077] (.+-.) 1-diethylamino-5,6-dihydro-5-oxo-1 1H-indeno[1,2-c]isoquinol- ine (5b)

[0078] (.+-.) 11-piperidino-5,6-dihydro-5-oxo-11H-indeno[1,2-c]isoquinolin- e (5c)

[0079] (.+-.) 11-(m-methylpiperazino)-5,6-dihydro-5-oxo-11H-indeno[1,2-c]i- soquinoline (5d)

[0080] (.+-.) 11-morpholino-5,6-dihydro-5-oxo-11H-indeno[1,2-c]isoquinolin- e (5e).

[0081] e) Synthesis of (.+-.) 11-morpholino-5,6-dihydro-5-oxo-11H-indeno[1- ,2-c]isoquinolines (5e) from 4b 9

[0082] To a stirred suspension of 3 (0.6 g, 2.4 mmol) in trifluoroacetic acid (5 mL) was added phosphorus tribromide (1.0 M soln. in CH.sub.2Cl.sub.2, 3 mL) at room temperature and the reaction mixture was stirred for 8 h. The reaction mixture was poured on ice and the resulting solid was filtered to give bromo compound 4b (0.61 gm, 76%). .sup.1H NMR (DMSO-D.sub.6): 7.35-7.50 (m, 3H), 7.61 (d, J=6.6 Hz, 1H), 7.73-7.82 (m, 2H), 7.94 (d, J=6.6 Hz, 1H), 8.23 (d, J=7.8 Hz, 1H), 12.41 (s, 1H); .sup.13C NMR (DMSO-D.sub.6): 52.06, 79.35, 114.43, 120.56, 123.58, 125.27, 125.50, 126.68, 128.55, 128.86, 129.66, 133.73, 135.91, 136.61, 141.39, 143.95, 163.74.

[0083] Compound 4b (0.5 g) was suspended in MeOH (10 mL) and treated with excess amount of morpholine (.about.10 eq.) at room temperature and stirred at 60.degree. C. for 3 h. The reaction mixture was poured on ice, and diluted with ethyl acetate (40 mL). Organic layer was separated and extracted in dil. HCl (10% soln.), aqueous layer was then basified with sat. aq. NaHCO.sub.3 and the solid precipitated was filtered, and dried to give 5e (0.46 g, 90% ). .sup.1H NMR (DMSO-D.sub.6): 2.56 (m, 4H), 3.49 (m, 4H), 5.04 (s, 1H), 7.31-7.45 (m, 3H), 7.65-7.76 (m, 2H), 7.96 (d, J=7.2 Hz, 1H), 8.20-8.24 (m, 2H) 12.29 (s, 1H); .sup.13C NMR (DMSO-D.sub.6): 49.36, 67.62, 68.11, 115.20, 120.60, 124.47, 125.84, 126.34, 126.41, 127.76, 128.30, 128.72, 133.09, 136.30, 136.96, 140.35, 144.44, 163.67.

[0084] f) Synthesis of 5,6-dihydro-5-oxo-11H-indeno[1,2-c]isoquinolines (6): 10

[0085] Method I: To a stirred solution of the alcohol 3a (0.35 g, 1.4 mmol) in trifluoroacetic acid (10 mL), was added at room temperature triethylsilane (0.812 g, 7 mmol) and the reaction mixture was stirred for 24 h. Trifluoroacetic acid was evaporated under vacuo and EtOAc was added to the crude product. The resulting solid was filtered and washed with H.sub.2O and EtOAc to give the desired product 6 (0.285 g, 87%). .sup.1H NMR (DMSO-D.sub.6): 3.89 (s, 2H), 7.30-7.47 (m, 3H), 7.59 (d, J=6.9 Hz, 1H), 7.72-7.74 (m, 2H), 7.98 (d, J=7.8 Hz, 1H), 8.23 (d, J=8.4 Hz, 1H), 12.31 (s, 1H); .sup.13CNMR DMSO-D.sub.6): 33.51, 116.50, 120.19, 124.01, 125.51, 125.55, 126.42, 127.50, 127.68, 128.56, 133.45, 136.39, 137.53, 140.18, 143.80, 163.46; MS (ES.sup.-): m/Z 232.1 (M-1); Anal. Calcd for C.sub.16H.sub.11NO: C, 82.38; H, 4.75; N, 6.00. Found: C, 81.79; H, 4.45, N, 5.99.

[0086] Method II: To a stirred suspension of 2 (40 g, 0.16 mol) in trifluoroacetic acid (2.5 L) was added triethylsilane (94 g, 0.8 mol) in small portions at room temperature and the reaction mixture was stirred for 96 h until the disappearance of the starting material (monitored by TLC, 5% MeOH/CH.sub.2Cl.sub.2). The reaction mixture was slowly poured on ice and filtered and washed with copious amounts of H.sub.2O and MeOH and dried under vacuo to give the desired product 6 (33.1 g, 88%). The product gave identical spectral data with 6 obtained from Method I.

[0087] g) Synthesis of 9-chlorosulfonyl-5,6-dihydro-5-oxo-1H-indeno[1,2-c] isoquinolines (7): 11

[0088] Compound 6 (40 g, 0.17 mol) was added in small portions into chlorosulfonyl chloride (112 mL, 1.71 mol) at 0.degree. C. and the reaction mixture was allowed to come to room temperature where it was stirred for 2 h. The reaction mixture was slowly poured on ice and the resulting yellow solid was filtered and washed thoroughly with water and EtOAc and dried under vacuo to give the desired product 7 (52 g, 92%). .sup.1H NMR (DMSO-D.sub.6): 3.91 (s, 2H), 7.43-7.48 (m, 1H), 7.60 (d, J=7.2 Hz, 1H), 7.74-7.76 (m, 2H), 7.79 (s, 1H), 7.90 (d, J=7.5 Hz, 1H), 8.23 (d, J=7.8 Hz, 1H), Anal. Calcd for C.sub.16H.sub.12ClNO.sub.4S: C, 54.94; H, 3.46; N, 4.00. Found: C, 55.28; H, 3.43, N, 3.68, KF, 2.95.

[0089] h) Synthesis of 9-sulphonamido Derivatives of 5,6-dihydro-5-oxo-11H-indeno[1,2-c]isoquinolines (8a-r) from 7: 12

[0090] Method I: To a stirred suspension of 3-(4-morpholino)-1-propylamine (17.28, 0.12 mol) in EtOAc was added sat. aq. NaHCO.sub.3 (300 mL) and stirred for 15 min, where 7 (4.0 gm, 0.012 mol) was introduced in small portions at room temperature. The reaction mixture was stirred for 24h, filtered and washed with H.sub.2O, EtOAc and MeOH. Then it was boiled in MeOH for 30 min. and was filtered while still warm and washed with MeOH to give the desired product 8l as a free base (2.330 gm, 44%). .sup.1H NMR (DMSO-D.sub.6): 1.47-1.52 (m, 2H), 2.16-2.21 (m, 4H), 2.47-2.48 (m, 2H), 3.44-3.48 (m, 2H), 3.23 (m, 4H), 4.02 (s, 2H), 7.49-7.58 (m, 1H), 7.78-7.82 (m, 3H), 7.97 (s, 1H), 8.14 (d, J=7.8 Hz, 1H), 8.26 (d, J=7.8 Hz, 1H), 9.59 (s, 1H), 12.42 (s, 1H).

[0091] The free bases of 8d, 8g, 8h, 8j, 8l, 8m-8r were also prepared by Method I.

[0092] 9-(N-sulphonylimidazole)-5,6-dihydro-5-oxo-11H-indeno[1,2-c]isoquin- olines (8d).

[0093] Method II: To a stirred suspension of 3-(4-morpholino)-1-propylamin- e (4.250 g) in CH.sub.2Cl.sub.2 (100 mL) was added 7 (1.950 gm, 5.89 mmol) and the resulting mixture was stirred for 5 minutes. Subsequently, triethylamine (3 mL) was added and the reaction mixture was stirred for 24 hr at room temperature. After this time the precipitate was collected and washed with MeOH (2.times.100 mL) and the crude solid product transferred to a round bottom flask. This material was boiled in MeOH (200 mL) for 30 min. and the resulting solid was collected while the solution was still warm. The filter cake was washed with MeOH (200 mL) to give the desired product as the free base of 8l (1.460 gm, 56%).

[0094] Using Method II, the free bases of compounds 8a-r were prepared.

[0095] i) General Procedure for the Preparation of Mesylate Salts of 8a-r:

[0096] Free base 8l was added to methanesulphonic acid at 0.degree. C. and the resulting mixture was allowed to come to room temperature, after which it was stirred for 2 h. The reaction mixture was then poured into cold MeOH (between -10.degree. C. and 0.degree. C.) and the precipitated product was filtered, washed with MeOH (100 mL) and dried in vacuo. The dried solid was then dissolved in water (.about.200 mL) and lyophilized to give the desired methanesulphonate monohydrate salt 8l. (1.020 gm, 84%). .sup.1H NMR (DMSO-D.sub.6): 1.75-1.85 (m, 2H), 2.35 (s, 3H), 2.78-2.84 (m, 2H), 2.96-3.12 (m, 4H), 3.36 (d, J=12.3 Hz, 2H), 3.61 (t, J=11.4 Hz, 2H), 3.94 (d, J=12.9 Hz, 2H), 4.03 (s, 2H), 7.49-7.55 (m, 1H), 7.76-7.84 (m, 3H), 7.99 (d, J=0.9 Hz, 1H), 8.15 (d, J=8.4 Hz, 1H), 8.25 (d, J=8.4 Hz, 1H), 9.59 (s, 1H), 12.42 (s, 1H); .sup.13C NMR (DMSO-D.sub.6): 24.27, 33.86, 51.89, 54.51, 64.02, 119.70, 120.39, 123.53, 126.09, 126.45, 128.63, 133.66, 135.80, 138.71, 141.21, 144.57, 163.29; Anal. Calcd for C.sub.24H.sub.31N.sub.3O.sub.8S.sub.2: C, 52.06; H, 5.46; N, 7.59, KF, 3.36. Found: C, 51.85; H, 5.35, N, 7.30, KF, 4.32.

[0097] Similarly HC.sub.1, H.sub.2SO.sub.4, CH.sub.3COOH, and succinic acid salts of 8l were prepared.

Example 2

Effect of Selected Compounds on PARS Activity in Cultured Macrophages, Using a Whole-Cell Based Assay and a Purified Enzyme Assay

[0098] Testing of the compounds for PARP inhibitory potency and prevention of peroxynitrite induced cytotoxicity was performed as described (Virag et al., Br J Pharmacol. 1999 February; 126(3):769-77; Immunology. 1998 July; 94(3):345-55). RAW mouse macrophages were cultured in DMEM medium with high glucose and supplemented with 10% fetal bovine serum. Cells were used at 80% confluence in 12-well plates. Cells were pretreated with various concentrations (100 nM-11M) of a PARP inhibitor compound of the invention for 10 min. Peroxynitrite, a prototypical oxidant which induces DNA single strand breakage, was used to induce PARP activation. Peroxynitrite was diluted in phosphate buffered saline (PBS) (pH 11.0) and added to the cells in a bolus of 50 .mu.l. Cells were then incubated for 20 min. Peroxynitrite (decomposed by incubation for 30 min at pH 7.0) was used as a control, and failed to influence the parameter studied. After the 20 min incubation, the cells were spun, the medium was aspirated and the cells were resuspended in 0.5 ml assay buffer (56 mM HEPES pH 7.5, 28 mM KCl, 28 mM NaCl, 2 mM MgCl.sub.2, 0.01% w/v digitonin and 0.125 .mu.M NAD.sup.+ and 0.5 .mu.Ci/ml.sup.3H-NAD.sup.+). Following an incubation in assay buffer, (10 min at 37.degree. C.), PARP activity was measured as follows: 200 .mu.l ice cold 50% w/v TCA was added and the samples were incubated for 4 hours at 4.degree. C. Samples were then spun (10,000 g, 10 min) and pellets washed twice with ice cold 5% w/v TCA and solubilized overnight in 250 .mu.l 2% w/v SDS/0.1 N NaOH at 37.degree. C. The contents of the tubes were added to 6.5 ml ScintiSafe Plus scintillation liquid (Fisher Scientific) and radioactivity was determined using a liquid scintillation counter (Wallac, Gaithersburg, Md.). The results shown in Table 1 demonstrate that the compounds pf the invention potently and dose-dependently inhibit the activation of PARS in the macrophage assay.

1TABLE 1 Inhibitory effect of various novel substituted indeno[1,2- c]isoquinolines on PARS activation in cultured murine macrophages. % PARS % PARS % PARS Compound inhibition inhibition inhibition No. at 1 uM at 300 nM at 100 nM 2 60 NT 16 3a 67 NT 8 3b 25 0 NT 3c 21 9 NT 4b 88 NT 51 5a 55 NT 10 5b 33 NT 0 5c 24 NT 0 5d 48 NT 0 5e 21 NT 0 6 65 NT 30% 7 50 NT 0 8a NT 47 NT 8b NT NT NT 8c NT 27 NT 8d NT 82 77 8e NT 68 NT 8f NT NT NT 8g NT 55 34 8h NT 76 56 8j NT 76 34 8k NT 38 24 8l NT 84 34 8m NT 50 NT 8n NT 82 74 8o NT 55 48 8p NT 45 27 8q NT 28 20 8r NT 28 20 10a NT 59 55 10b NT 17 17 NT = Not tested

[0099] The potency of inhibition on purified PARP enzyme was subsequently determined for selected compounds, and the potency was compared with that of 3-aminobenzamide, a prototypical benchmark PARP inhibitor. The assay was performed in 96 well ELISA plates according to instructions provided with a commercially available PARP inhibition assay kit (Trevigen, Gaithersburg, Md.). Briefly, wells were coated with 1 mg/ml histone (50 .mu.l/well) at 4.degree. C. overnight. Plates were then washed four times with PBS and then blocked by adding 50 .mu.l Strep-Diluent (supplied with the kit). After incubation (1 h, room temperature), the plates were washed four times with PBS. Appropriate dilutions of PARP inhibitors were combined with 2.times. PARP cocktail (1.95 mM NAD.sup.+, 50 .mu.M biotinylated NAD.sup.+ in 50 mM TRIS pH 8.0, 25 mM MgCl.sub.2) and high specific activity PARP enzyme (both were supplied with the kit) in a volume of 50 .mu.l. The reaction was allowed to proceed for 30 min at room temperature. After 4 washes in PBS, incorporated biotin was detected by peroxidase-conjugated streptavidin (1:500 dilution) and TACS Sapphire substrate. The assay confirmed the results of the macrophage-based PARS assay. For example, the PARP inhibitor 8l, exerted 50% inhibition of PARS activity in this assay at 3 nM, and thus was approximately 50,000 times more potent than the reference compound 3-aminobenzamide.

Example 3

Effects of Substituted indeno[1,2-c]isoquinoline in Various Models of Inflammation and Reperfusion Injury

[0100] a: Effects of Selected PARS Inhibitors on in vitro Cell Injury Models

[0101] In additional in vitro studies in isolated thymocytes, cells were exposed to peroxynitrite or hydrogen peroxide (toxic oxidant species) to induce cytotoxicity. There is now evidence that in this system the toxicity is, at least in part, related to activation of the nuclear enzyme PARS (see Introduction). In this assay (described, in detail, in Virag et al., Immunology 94(3):345-55, 1998), the compounds tested prevented the oxidant-induced suppression of the viability of the cells and did so at the low nanomolar concentration range. An example of this response (Compound 8l) is shown in Table 2. This assay (the oxidant-stimulated thymocyte) represents an in vitro model of a situation where cells are dying because of exposure to pro-oxidant species, as it occurs in during the reperfusion of ischemic organs.

2TABLE 2 Reduction of peroxynitrite induced cytotoxicity by 30 nM-3 .mu.M of the PARS inhibitor compound 81. +81 +81 +81 +81 +81 Control 30 nM 100 nM 300 nM 1 .mu.M 3 .mu.M Cyto-toxicity 98% 74% 39% 2% 0% 0%

[0102] b: Effect of Selected PARS Inhibitors on in vivo Inflammation Models

[0103] In order to substantiate the efficacy of the compounds in inflammatory conditions, the effect of the compounds was tested in a systemic inflammatory model induced by bacterial lipopolysaccharide. Injection of bacterial lipopolysaccharide (LPS) causes the production of the pro-inflammatory cytokine TNF-alpha, which is a mediator of systemic inflammation and shock. In a series of experiments, mice were pretreated with intraperitoneal injection of 0.1 and 1 mg/kg of compounds 8l, 8p and 8j, and LPS at 10 mg/kg was injected i.p., and TNF-alpha was measured in the plasma at 90 minutes. As shown in Table 3, all compounds substantially reduced TNF production, indicative of an anti-inflammatory activity.

3TABLE 3 Reduction of LPS induced TNF production by 0.1-1 mg/kg intraperitoneal injection of the PARS inhibitor compounds 8L, 8P and 8J in mice in vivo 8j (0.1) 8j (1) 8p (0.1) 8p (1) 8l (0.1) 8l (1) Vehicle TNF 3831.6 .+-. 5038.8 .+-. 4470.0 .+-. 5090.8 .+-. 3714.6 .+-. 3509.8 .+-. 6994 .+-. (ng/ml) 385.2 377.1 184.4 203.7 300.9 311.5 904.4

[0104] All compounds markedly suppressed LPS induced TNF production when compared to control.

[0105] At high doses, LPS causes multiple organ dysfunction resembling of septic shock, and ultimately death (in part because of the early release of TNF-alpha). Similarly, in a model induced by cecal ligation and puncture (CLP), the live bacteria that derive from the intestinal flora induce systemic inflammation and shock. Agents that inhibit inflammatory mediator production, PARS activation, and cell death in this model will prevent mortality induced by LPS or CLP. In experiments with Balb/c mice, injection of 100 mg/kg LPS intraperitoneally caused death in 50% of the animals over 24h, whereas treatment of the animals with 3 mg/kg/day of compound 8l reduced the endotoxin-induced mortality to 10% under the same experimental conditions. In response to CLP induced shock, compound 8l (3 mg/kg/day) caused an improvement in the mortality from 100% death to 60% death over 24 hours.

[0106] The data demonstrating the reduction of TNF production by the PARS inhibitor compounds in animals subjected to an inflammation model, coupled with the fact that TNF production is an important trigger of inflammation in various inflammatory diseases (such as, for example, colitis, arthritis and neuroinflammation and shock) indicate that the compounds of the invention have therapeutic effects in various systemic and local inflammatory conditions, including the rejection of transplanted organs, which entails both an inflammatory- and a reperfusion injury component.

[0107] c: Effect of Selected PARS Inhibitors on in vivo Reperfusion Injury Models

[0108] In order to substantiate the efficacy of the compounds of the invention in ischemia-reperfusion conditions, the effect of a selected compound in a mouse model of ischemic and reperfused gut was tested. The superior mesenteric artery was occluded for 45 min, followed by a reperfusion for 1 h. Following the end of the reperfusion, gut permeability was measured with the FD4 method in evened gut sacks (Liaudet et al; Shock, 2000 August; 14(2):134-41). Ischemia-reperfusion increased the permeability of the gut from 11.+-.4 to 216.+-.27 ml/min/cm.sup.2, indicating of severe damage of the reperfused gut. Treatment with Compound 8l (3 mg/kg i.v. injected 10 min prior to the start of reperfusion), reduced the increase in the permeability of the gut by approximately 73%, indicating a marked maintenance of the gut function. The ischemia-reperfusion studies in the gut were associated with a 80% mortality over 12 hours, whereas only 15% mortality was noted in the animals treated with the PARS inhibitor compound.

[0109] In another set of experiments, the effect of Compound 8l in a rat model of middle cerebral artery occlusion/reperfusion was assayed as described in Abdelkarim et al., Int J Mol Med. 2001 ;7(3):255-60. Occlusion lasted for 2 hours, followed by reperfusion for 24 hours. Infarct size was quantified with the tetrazolium staining. compound 8l was administered at 3 mg/kg/day in 3 divided intraperitoneally injected doses, the first dose being 10 min before the start of reperfusion. There was an approximately 80% reduction in the degree of cortical necrosis and neuronal death in the PARS inhibitor treated animals, when compared to vehicle-treated controls. This protection also translated into functional benefit, such as neurological improvements in the PARS inhibitor treated group.

[0110] These data indicate that the compounds of the invention have therapeutic effects in various systemic and local conditions of ischemia-reperfusion, including the rejection of transplanted organs, which entails both an inflammatory- and a reperfusion injury component.

[0111] d: Effect of Selected PARS Inhibitors in a Diabetes Model

[0112] PARS inhibitors and PARS deficiency is known to reduce the development of diabetes and the incidence of diabetic complications (Mabley et al., Br J Pharmacol. 2001 July; 133(6):909-19; Soriano et al., Nat Med. 2001 January; 7(1):108-13). In order to substantiate the efficacy of the compounds in a diabetes model, a single high-dose streptozotocin model of diabetes was conducted as previously described 0. Briefly, 160 mg/kg streptozotocin was injected to mice treated with vehicle or with selected novel PARS inhibitor compounds described in the current application intraperitoneally (3 mg/kg) and 3 days later blood sugar levels were determined using a blood glucose meter. The data shown in Table 4 demonstrate that the PARS inhibitors attenuate the streptozotocin-induced onset of diabetes, as they reduce the hyperglycemia.

4TABLE 4 Reduction of streptozotocin (STZ) induced hyperglycemia by 3 mg/kg intraperitoneal injection of the PARS inhibitor compounds 8L, 8P and 8J in mice in vivo Basal STZ + Vehicle STZ + 8j STZ + 8p 8l Glucose 153 .+-. 21 320 .+-. 13 253 .+-. 24 264 .+-. 24 244 .+-. 21 (mg/ml)

Claims

What is claimed is:

1. A compound of Formula I 13wherein: R.sub.5 is O, N or S; R6 is H or straight chain alkyl; X is CO, CH.sub.2, CH-Halo, CH(CH.sub.2).sub.nOH, aryl-C--OH, O, NH, S or CH--NR.sub.11R.sub.12, wherein R.sub.11 and R.sub.12 are, independently, H, C.sub.1-C.sub.9 alkyl, or NR.sub.1, R.sub.12, taken together, form an optionally-substituted heterocycle and wherein n is zero or a positive integer; R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.7, R.sub.8, R.sub.9, and R10 are, independently, hydrogen, halo, alkylhalo, hydroxy, alkoxy, C.sub.1-C.sub.10 straight or branched chain alkyl, C.sub.2-C.sub.10 straight or branched chain alkenyl group, C.sub.3-C.sub.8 carbocyclic, aryl, alkylamino, amino, carboxy, ester, arylalkyl, nitro, or A-B; wherein A is --SO.sub.2--, --SO.sub.2NH--, --NHCO--, --NHCONH--, O, CO, OCO, CONH, NH, CH.sub.2, S or CS; and B is C.sub.1-C.sub.10 straight or branched chain alkyl, C.sub.2-C.sub.10 straight or branched chain alkenyl group, heterocycle, C.sub.3-C.sub.8 carbocycle, aryl, amino, aminoalkyl, aminodialkyl, heterocyclic amine, alkylheterocycle, arylamido, carboxy, ester, arylalkyl, or NZ.sub.1Z.sub.2.

2 The compound of claim 1 wherein R.sub.5 is O, and R.sub.6 is H.

3 The compound of claim 2 wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.7, R.sub.8, R.sub.9, R.sub.10 are independently H, methyl, ethyl, halo, nitro, hydroxy, methoxy, ethoxy, amino or substituted amino, or A-B.

4 The compound of claim 3, wherein either R.sub.8 or R.sub.9 is A-B.

5 The compound of claim 4 wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.7, and R.sub.10 are H.

6 The compound of claim 2, wherein R.sub.9 is A-B.

7 The compound of claim 3, wherein R.sub.9 is A-B.

8 The compound of claim 5, wherein R.sub.9 is A-B and R.sub.8 is H.

9 The compound of claim 4, wherein A is --SO.sub.2--, --SO.sub.2NH-- or --NHCO--.

10 The compound of claim 5, wherein A is --SO.sub.2--, SO.sub.2NH-- or --NHCO--.

11 The compound of claim 6, wherein A is --SO.sub.2--, --SO.sub.2NH-- or --NHCO--.

12 The compound of claim 7, wherein A is --SO.sub.2--, --SO.sub.2NH-- or --NHCO--.

13 The compound of claim 8, wherein A is --SO.sub.2--, --SO.sub.2NH-- or --NHCO--.

14 The compound of claim 2, wherein R.sub.9 is AB and B is an optionally substituted C.sub.1-C.sub.4 straight or branched chain alkyl, heterocycle, C.sub.3-C.sub.8 carbocyclic, aryl, alkylamino, alkylhydroxy, or alkylheterocycle.

15 The compound of claim 3, wherein A is SO.sub.2 and B is NZ.sub.1Z.sub.2, and Z.sub.1 and Z.sub.2 are, independently, H, or C1-C5 alkyl, optionally substituted with halo, OH or NZ.sub.3Z.sub.4, wherein Z.sub.3 and Z.sub.4 are, independently, H, C1-C5 alkyl, optionally substituted with halo, OH or amino, or wherein Z.sub.1 and Z.sub.2, taken together, NZ.sub.1Z.sub.2 form a heterocyclic amine.

16 The compound of claim 15, wherein Z1 and Z2 are --(CH.sub.2).sub.nD; wherein n is 1-5, D is H, hydroxy, heterocyclic amine or NZ.sub.3Z.sub.4; wherein Z.sub.3 and Z.sub.4 are, independently, H, methyl, or ethyl.

17 The compound of claim 15, wherein B is a heterocycle, and is optionally substituted with methyl, ethyl, or alkylhydroxy.

18 The compound of claim 15, wherein Z.sub.1 is H and Z.sub.2 is --(CH.sub.2).sub.nNZ.sub.3Z.sub.4; wherein n is 2 or 3, and Z.sub.3 and Z.sub.4 are, independently, methyl or ethyl, or, taken together, NZ.sub.3Z.sub.4 forms a heterocyclic amine.

19 The compound of claim 13, wherein X is CH.sub.2, A is SO.sub.2 and B is NZ.sub.1Z.sub.2; wherein Z.sub.1 and Z.sub.2 are, independently, H, C1-C5 alkyl optionally substituted with halo, OH or NZ.sub.3Z.sub.4; wherein Z.sub.3 and Z.sub.4 are, independently, H, C1-C5 alkyl, optionally substituted with halo, OH or amino; or wherein Z.sub.1 and Z.sub.2, taken together, form a heterocyclic amine.

20 The compound of claim 19, wherein Z.sub.1 and Z.sub.2 are --(CH.sub.2).sub.nD, wherein n is 1-5, D is H, hydroxy, a heterocyclic amine or NZ.sub.3Z.sub.4, wherein Z.sub.3 and Z.sub.4 are, independently, H, methyl, or ethyl.

21 The compound of claim 15, wherein B is a heterocycle, optionally substituted with methyl, ethyl, or alkylhydroxy.

22 The compound of claim 15, wherein Z.sub.1 is H and Z.sub.2 is --CH.sub.2).sub.nNZ.sub.3Z.sub.4, wherein n is 2 or 3, and Z.sub.3 and Z.sub.4 are, independently, methyl or ethyl, or, taken together, NZ.sub.3Z.sub.4 form a heterocyclic amine.

23 The compound of claim 2 wherein X is CO, CHOH, CHBr, CH.sub.2 or CH--NR.sub.11R.sub.12 wherein R.sub.11 and R.sub.12 are H, C.sub.1-C.sub.9 alkyl, or NR.sub.11R.sub.12, taken together, form an optionally substituted heterocycle.

24 The compound of claim 4 wherein X is CO, CHOH, CHBr, CH.sub.2 or CH--NR.sub.11R.sub.12 wherein R.sub.11 and R.sub.12 are H, C.sub.1-C.sub.9 alkyl, or NR.sub.11R.sub.12, taken together, form an optionally substituted heterocycle.

25 The compound of claim 6 wherein X is CO, CHOH, CHBr, CH.sub.2 or CH--NR.sub.11R.sub.12 wherein R.sub.11 and R.sub.12 are H, C.sub.1-C.sub.9 alkyl, or NR.sub.11R.sub.12, taken together, form an optionally substituted heterocycle.

26 The compound of claim 7 wherein X is CO, CHOH, CHBr, CH.sub.2 or CH--NR.sub.11R.sub.12 wherein R.sub.11 and R.sub.12 are H, C.sub.1-C.sub.9 alkyl, or NR.sub.11R.sub.12, taken together, form an optionally substituted heterocycle.

27 The compound of claim 8 wherein X is CO, CHOH, CHBr, CH.sub.2 or CH--NR.sub.11R.sub.12 wherein R.sub.11 and R.sub.12 are H, C.sub.1-C.sub.9 alkyl, or NR.sub.11R.sub.12, taken together, form an optionally substituted heterocycle.

28 The compound of claim 4 wherein X is CO, CHOH, CHBr, or CH.sub.2.

29 The compound of claim 4 wherein X is CH.sub.2.

30 The compound of claim 6 wherein X is CO, CHOH, CHBr, or CH.sub.2.

31 The compound of claim 6 wherein X is CH.sub.2.

32 The compound of claim 7 wherein X is CO, CHOH, CHBr, or CH.sub.2.

33 The compound of claim 7 wherein X is CH.sub.2.

34 The compound of claim 8 wherein X is CO, CHOH, CHBr, or CH.sub.2.

35 The compound of claim 8 wherein X is CH.sub.2.

36 The compound of claim 11 wherein X is CO, CHOH, CHBr, or CH.sub.2.

37 The compound of claim 11 wherein X is CH.sub.2.

38 The compound of claim 12 wherein X is CO, CHOH, CHBr, or CH.sub.2.

39 The compound of claim 12 wherein X is CH.sub.2.

40 The compound of claim 15 wherein X is CO, CHOH, CHBr, or CH.sub.2.

41 The compound of claim 15 wherein X is CH.sub.2.

42 The compound of claim 16 wherein X is CO, CHOH, CHBr, or CH.sub.2.

43 The compound of claim 16 wherein X is CH.sub.2.

44 The compound of claim 17 wherein X is CO, CHOH, CHBr, or CH.sub.2.

45 The compound of claim 17 wherein X is CH.sub.2.

46 The compound of claim 18 wherein X is CO, CHOH, CHBr, or CH.sub.2.

47 The compound of claim 18 wherein X is CH.sub.2.

48 A compound of Formula II 14wherein: R.sub.1, R.sub.4, R.sub.7, and R.sub.10 are hydrogen; R.sub.2 and R.sub.3 are hydrogen, halo, alkylhalo, hydroxy, alkoxy, C.sub.1-C.sub.3 straight or branched chain alkyl, nitro, amino, amido, carboxy, or ester; R.sub.8 and R.sub.9 are either hydrogen or A-B; wherein A is --SO.sub.2--, --SO.sub.2NH--, or --NHCO; B is an optionally substituted C.sub.1-C.sub.3 straight or branched chain alkyl, heterocycle, amino, aminoalkyl, aminodialkyl, or heterocyclic amine, or NZ.sub.1Z.sub.2.

49. The compound of claim 48, wherein A is SO.sub.2 and B is NZ.sub.1Z.sub.2.

50. The compound of claim 49, wherein Z.sub.1 and Z.sub.2 are, independently, H, or C1-C3 alkyl, optionally substituted with OH or NZ.sub.3Z.sub.4, or, taken together, NZ.sub.1Z.sub.2 form a heterocyclic amine.

51. The compound of claim 50, wherein Z.sub.3 and Z.sub.4 are, independently, H or C1-C3 alkyl optionally substituted with OH or amino, or, taken together, NZ.sub.3Z.sub.4 form an optionally substituted heterocyclic amine.

52. The compound of claim 51, the heterocyclic amine is selected from the group consisting of piperidine, piperazine, morpholine, N-alkylated or alkylcarbonylated piperazines, pyrrolidine, and imidazole.

53. The compound of claim 52, wherein either NZ.sub.1Z.sub.2 or NZ.sub.3Z.sub.4 is a heterocycle substituted with alkyl, alkylhydroxy or alkylamino.

54. A method of inhibiting poly(ADP)-ribose synthase activity in a cell, the method comprising contacting said cell with the compound of claim 1 or claim 48 in an amount sufficient to inhibit poly (ADP)-ribose-synthase in said cell.

55. A method of treating or preventing local or systemic inflammation in a subject, the method comprising administering the compound of claim 1 or claim 48 in an amount sufficient to inhibit inflammation in said subject.

56. The method of claim 55, wherein said subject is a human subject.

57. The method of claim 55, wherein administering is systemic.

58. The method of claim 55, wherein administering is topical.

59. The method of claim 55, where said local inflammatory condition is caused by an inflammatory disorder of a joint, an inflammatory bowel disease, an inflammatory lung disorder, an inflammatory disease of the central nervous system, or an inflammatory disease of the eye.

60. The method of claim 55, wherein said systemic inflammatory condition is caused by a condition selected from the group consisting of gram-positive shock, gram negative shock, hemorrhagic shock, anaphylactic shock, traumatic shock, and systemic inflammation and chemotherapeutic shock.

61. A method of treating or preventing reperfusion injury in a subject, the method comprising administering the compound of claim 1 or claim 48 in an amount sufficient to inhibit reperfusion injury in said subject.

62. The method of claim 61, wherein said compound is administered prophylactically.

63. The method of claim 61, wherein said compound is administered therapeutically.

64. The method of claim 61, wherein said reperfusion injury is myocardial infarction.

65. The method of claim 61, wherein said reperfusion injury is stroke.

66. The method of claim 61, wherein said subject is a human subject.

67. The method of claim 61, wherein administering is systemic.

68. The method of claim 61, wherein administering is topical.

69. A method of treating or preventing diabetes or diabetic complications in a subject, the method comprising administering the compound of claim 1 or claim 48 in an amount sufficient to inhibit inflammation in said subject.

70. A method of treating or preventing the rejection of transplanted organs in a subject, the method comprising administering the compound of claim 1 or claim 48 in an amount sufficient to inhibit inflammation in said subject.