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WO1999011644A1 - Composes di-n-heterocycliques, et procedes et compositions permettant d'inhiber l'activite de la parp - Google Patents

Composes di-n-heterocycliques, et procedes et compositions permettant d'inhiber l'activite de la parp Download PDF

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Publication number
WO1999011644A1
WO1999011644A1 PCT/US1998/018188 US9818188W WO9911644A1 WO 1999011644 A1 WO1999011644 A1 WO 1999011644A1 US 9818188 W US9818188 W US 9818188W WO 9911644 A1 WO9911644 A1 WO 9911644A1
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Prior art keywords
compound
cancer
group
formula
aromatic
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PCT/US1998/018188
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English (en)
Inventor
Paul F. Jackson
Keith M. Maclin
Jie Zhang
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Guilford Pharmaceuticals Inc.
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Priority claimed from US09/145,185 external-priority patent/US6121278A/en
Application filed by Guilford Pharmaceuticals Inc. filed Critical Guilford Pharmaceuticals Inc.
Priority to AU92981/98A priority Critical patent/AU9298198A/en
Publication of WO1999011644A1 publication Critical patent/WO1999011644A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/06—Peri-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings

Definitions

  • the present invention relates to mnibitors of the nucleic enzyme poly (adenosme 5 ' -diphospho-ribose) polymerase ["poly (ADP-ribose) polymerase” or “PARP”, which is also sometimes called “PARS” for poly (ADP-ribose) synthetase] .
  • the invention relates to the use of PARP inhibitors to prevent and/or treat tissue damage resulting from cell damage or death due to necrosis or apoptosis; neural tissue damage resulting from ischemia and reperfusion injury; neurological ⁇ isorders and neurodegenerative diseases; to prevent or treat vascular stroke; to treat or prevent cardiovascular disorders; to treat other conditions and/or disorders such as age-related macular degeneration, AIDS and other immune senescence diseases, arthritis, atherosclerosis, cachexia, cancer, degenerative diseases of skeletal muscle involving replicative senescence, diabetes, head trauma, immune senescence, inflammatory bowel disorders (such as colitis and Crohn's disease), muscular dystrophy, osteoarthritis, osteoporosis, chronic ana acute pain (such as ne ropatmc pain), renal failure, retinal ischemia, septic shock (such as endotoxic shock) , and skin aging; to extend the lifespan and proliferative capacity of cells; to alter gene expression of senescent cells; to
  • PARP tne Prior Art Poly (ADP-ribose) polymerase
  • ADP-ribose polymerase is an enzyme located in the nuclei of cells of various organs, including muscle, heart and brain cells. PARP plays a physiological role in the repair of strand breaks in DNA. Once activated by damaged DNA fragments, PARP catalyzes the attachment of up to 100 ADP- ribose units to a variety of nuclear proteins, including histones and PARP itself. While the exact range of functions of PARP has not Deen fully established, th s enzyme s tnought ATTORNEY DOCKE' 0. 23356- 8 to play a role in enhancing DNA repair.
  • NAD the source of ADP-ribose
  • NMDA N-methyl-D- aspartate
  • AMPA AMPA
  • Kamate MGR receptors
  • 35 glutamate binds to these receptors, ion channels in the receptors open, permitting flows of ions across their cell ATTORNEY DOCKE 0. 23356-M8 membranes, e.g., Ca 2+ and Na + into the cells and K * out of the cells.
  • These flows of ions especially the influx of Ca 2+ , cause overstimulation of the neurons.
  • the over-stimulated neurons secrete more glutamate, creating a feedback loop or domino effect which ultimately results in cell damage or death via the production of proteases, lipases and free radicals.
  • NMDA receptors activate neuronal nitric oxide synthase (NNOS) , which causes the formation of nitric oxide (NO) , which more directly mediates neurotoxicity. Protection against NMDA neurotoxicity has occurred following treatment with NOS inhibitors. See Dawson et al., "Nitric Oxide Mediates Glutamate Neurotoxicity in Primary Cortical Cultures", Proc. Na tl . Acad. Sci . USA, 88 : 6368 -11 (1991); and Dawson et al., “Mecnamsms of Nitric Oxide-mediated Neurotoxicity in Primary Brain Cultures", J. Neurcsci . , 23:6, 2651-61 (1993).
  • NNOS neuronal nitric oxide synthase
  • NO nitric oxide
  • PARP inhibitors have been reported to be effective in radiosensitizmg hypoxic tumor cells and effective in preventing tumor cells from recovering from potentially letnal damage of DNA after radiation therapy, presumably by their ability to prevent DNA repair. See U.S. Patent Nos. 5,032,617; 5,215,738; and 5,041,653.
  • PARP inhibitors have been shown to be useful for treating endotoxic shock or septic shock.
  • Zingarelli et al. "Protective Effects of Nicotinamide Against Nitric Oxide-Mediated Delayed Vascular Failure in Endotoxic
  • Zingarelli et al. also found that the actions of nicotinamide ATTORNEY DOCKE". .0. 23356-M8 may be related to inhibition of the NO-mediated activation of the .energy-consuming DNA repair cycle, triggered by poly (ADP ribose) synthetase. See also, Cuzzocrea, "Role of Peroxynitrite and Activation of Poly (ADP-Ribose) Synthetase in the Vascular Failure Induced by Zymosan-activated Plasma," Brit. J. Pharm . , 222:493-503 (1997).
  • PARP inhibitors are used for peripheral nerve injuries, and the resultant pathological pain syndrome known as neuropathic pain, such as that induced by chronic constriction injury (CCI) of the common sciatic nerve and in which transsynaptic alteration of spinal cord dorsal horn characterized by hyperchromatosis of cytoplasm and nucleoplasm (so-called "dark” neurons) occurs.
  • CCI chronic constriction injury
  • nucleoplasm cytoplasm and nucleoplasm
  • PARP inhibitors have also been used to extend the lifespan and proliferative capacity of cells including treatment of diseases such as skin aging, Alzheimer's disease, atherosclerosis, osteoarthritis, osteoporosis, muscular dystrophy, degenerative diseases of skeletal muscle involving replicative senescence, age-related macular degeneration, immune senescence, AIDS, and other immune senescence diseases; and to alter gene expression of senescent cells. See WO ATTORNEY DOCKE .10. 23356-M8 98/27975.
  • Nitrogen-containing tricyclic compounds other than the compounds of the invention are known:
  • R 1 is amino, lower alkylamino, di (lower) alkylamino or di (lower) alkylamino (lower) alkylamino.
  • the compounds are disclosed as having circulatory and central nervous system activities that elicit antidepressant and antihypertensive effects.
  • R x and each are hydrogen, lower alkyl, di (lower) alkylamino (lower) alkyl.
  • the compounds are disclosed as having circulatory and central nervous system activities that elicit antidepressant and antihypertensive effects.
  • Rath discloses three-ring pyracridones having the formula:
  • Xs can be amino or nitro groups.
  • the Petrow compounds are purported to possess “valuable therapeutic properties”.
  • X is a member selected from the group consisting of carboxy and lower-carbalkoxy
  • R is a member selected from the group consisting of lower alkyl and lower alkenyl.
  • R, . and R are selected from the group consisting of hydrogen, hydroxyl and lower alkoxy
  • R 3 is selected from the group consisting of hydrogen, lower alkyl, phenyl and phenyl (lower alkyl)
  • R 5 is selected from the group consisting of hydrogen and lower alkyl
  • R shield and R are selected from the group consisting of hydrogen, lower alkyl and other substituents as listed in the patent.
  • the compounds are said to have antibacterial activity.
  • R is hydroxy, phenoxy, chloro, amino, (lower) - alkylamino and di (lower) alkylamino, wherein R x is hydrogen or acetyl, R 2 and R 3 are hydrogen or methoxy and R 4 is hydrogen, methoxy or dimethylamino.
  • the compounds are said to be antispasmodic agents that abolish spastic contractions and lower hypertonicity of the ileum and colon.
  • R,_ and R 2 are hydrogen, lower alkyl, lower alkoxy or come together to form a methylenedioxy group, wherein R 3 is hydrogen or lower alkyl, and wherein R 4 is hydrogen or a cycloalkyl-lower alkyl substituent.
  • the compounds are said to be bronchodilators
  • Non-azo N-substituted-1, 8-naphthalimide derivatives are shown in Lewis et al., U.S. Patent No. 5,420,136, issued May 30, 1995.
  • Lewis et al. discloses naphthalimide dyes having the following formula or related formulas: ATTORNEY DOCKE 10. 23356-M8
  • X is halogen, sulfonate ester or a nitrogen leaving group
  • R and R' are alkyl or particular groups capable of complexing with a metal ion (as defined in the patent) .
  • the compounds are said to be useful for treating tumors and bacterial infections.
  • the Beverung, Jr. et -al. compounds are useful in the control of hypertension, as anti-clotting agents and as bronchodilators .
  • the Kennewell et al. compounds are said to have antiallergic properties.
  • the Chu et al. compounds are said to have antibacterial and antineoplastic activities.
  • Descarboxylsergic acids and ergolinones such as 6-methyl- 9-ergoline-8-ones are shown in Bach et al., U.S. Patent No. 4,031,097, issued June 21, 1977.
  • Bach et al. discloses compounds having the formula: ATTORNEY DOCKE'. 0 . 23356-M8
  • the Bach et al. compounds are said to have oxytocic, serotonin antagonist, prolactin inhibition and muscle contracting activities . It is not believed that the above disclosed compounds have been shown to inhibit PARP activity per se.
  • Y represents the atoms necessary to form a fused 5- to 6- membered, aromatic or non-aromatic, heterocyclic ring containing at least one nitrogen heteroatom in a 1,3- relationship with the nitrogen atom depicted in formula I, wherein Y may be unsubstituted or substituted with at least one alkyl, alkenyl, cycloalkyl, cycloalkenyl, aralkyl, aryl, double bonded oxygen, or -C00R 5 group, or a moiety selected from the group consisting of: ATTORNEY DOCKE JO. 23356- 8
  • R 5 and R 7 are independently hydrogen or alkyl, alkenyl, cycloalkyl, cycloalkenyl, aralkyl, or aryl; with the proviso that when Y forms a 5-membered unsaturated ring, Y must be substituted with at least one non-hydrogen, non-phenyl substituent.
  • the pharmaceutical composition of the invention comprises a pharmaceutically acceptable carrier and a compound of formula I:
  • Y represents the atoms necessary to form a fused 5- to 6- membered, aromatic or non-aromatic, heterocyclic ring containing at least one nitrogen heteroatom in a 1,3- relationship with the nitrogen atom depicted in formula I, wherein Y may be unsubstituted or substituted with at least one alkyl, alkenyl, cycloalkyl, cycloalkenyl, aralkyl, aryl, double bonded oxygen, or -COOR 5 group, or a moiety selected from the group consisting of: ATTORNEY DOCKET MO . 23356-t>
  • R D and R 7 are independently hydrogen or alkyl, alkenyl, cycloalkyl, cycloalkenyl, aralkyl, or aryl; with the proviso that when Y forms a 5-membered unsaturated ring, Y must be substituted with at least one non-hydrogen, non-phenyl substituent.
  • the pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier and a compound of formula I:
  • ATTORNEY DOCKET ..0. 23356-M8 stroke to treat or prevent cardiovascular disorders; to treat other conditions and/or disorders such as age-related macular degeneration, AIDS and other immune senescence diseases, arthritis, atherosclerosis, cachexia, cancer, degenerative diseases of skeletal muscle involving replicative senescence, diabetes, head trauma, immune senescence, inflammatory bowel disorders (such as colitis and Crohn's disease), muscular dystrophy, osteoarthritis, osteoporosis, chronic and/or acute pain (such as neuropathic pain) , renal failure, retinal ischemia, septic shock (such as endotoxic shock) , and skin aging; to extend the lifespan and proliferative capacity of cells; to alter gene expression of senescent cells; or to radiosensitize hypoxic tumor cells, and wherein Y is defined as above for formula I.
  • a method of inhibiting PARP activity comprises administering a compound of formula I, as described above for the pharmaceutical compositions of the invention.
  • the amount of the compound administered in the methods of the invention is sufficient for treating tissue damage resulting from cell damage or death due to necrosis or apoptosis, neural tissue damage resulting from ischemia and reperfusion injury, or neurological disorders and neurodegenerative diseases; to prevent or treat vascular stroke; to treat or prevent cardiovascular disorders; to treat other conditions and/or disorders such as age-related macular degeneration, AIDS and other immune senescence diseases, arthritis, atherosclerosis, cachexia, cancer, degenerative diseases of skeletal muscle involving replicative senescence, diabetes, head trauma, immune senescence, inflammatory bowel disorders (such as colitis and Crohn's disease), muscular dystrophy, osteoarthritis, osteoporosis, chronic and/or acute pain (such as neuropathic pain), renal failure, retinal ischemia, septic shock (such as endotoxic shock
  • Figure 1 shows the distribution of the cross-sectional infarct area at representative levels along the rostrocaudal axis, as measured from the interaural line in non-treated animals and in animals treated with 10 mg/kg of 3, 4-dihydro-5-
  • Figure 2 shows the effect of intraperitoneal administration of 3, 4-dihydro-5- [4- (1-piperidinyl) -butoxy] - 1 (2H) -isoquinolinone on the infarct volume.
  • the compounds of the present invention inhibit PARP activity.
  • they may treat or prevent neural tissue damage resulting from cell damage or death due to necrosis or apoptosis, cerebral ischemia and reperfusion injury or neurodegenerative diseases in an animal; they may extend the lifespan and proliferative capacity of cells and thus be used to treat or prevent diseases associated therewith; they may alter gene expression of senescent cells; and they may radiosensitize hypoxic tumor cells.
  • the compounds of the invention treat or prevent tissue damage resulting from cell damage or death due to necrosis or apoptosis, and/or effect neuronal activity, either mediated or not mediated by NMDA toxicity. These compounds are thought to interfere with more than the glutamate neurotoxicity and NO-mediated biological pathways. Further, the compounds of the invention can treat or prevent other tissue damage related to PARP activation.
  • the compounds of the -invention can treat or prevent cardiovascular tissue damage resulting from cardiac ischemia or reperfusion injury.
  • Reperfusion injury for instance, occurs at the termination of cardiac bypass procedures or during cardiac arrest when the heart, once prevented from receiving blood, begins to reperfuse.
  • the compounds of the present invention can also be used to extend or increase the lifespan or proliferation of cells and ATTORNEY DOCKE. iMO .
  • 23356-M8 thus to treat or prevent diseases associated therewith and induced or exacerbated by cellular senescence including skin aging, atherosclerosis, osteoarthritis, osteoporosis, muscular dystrophy, degenerative diseases of skeletal muscle involving replicative senescence, age-related macular degeneration, immune senescence, AIDS and other immune senescence diseases, and other diseases associated with cellular senescence and aging, as well as to alter the gene expression of senescent cells.
  • These compounds can also be used to treat cancer and to radiosensitize hypoxic tumor cells to render the tumor cells more susceptible to radiation therapy and to prevent the turner cells from recovering from potentially lethal damage of DNA after radiation therapy, presumably by their ability to prevent DNA repair.
  • the compounds of the present invention can be used to prevent or treat vascular stroke; to treat or prevent cardiovascular disorders; to treat other conditions and/or disorders such as age-related macular degeneration, AIDS and other immune senescence diseases, arthritis, atherosclerosis, cachexia, cancer, degenerative diseases of skeletal muscle involving replicative senescence, diabetes, head trauma, immune senescence, inflammatory bowel disorders (such as colitis and Crohn's disease), muscular dystrophy, osteoarthritis, osteoporosis, chronic and/or acute pain (such as neuropathic pain) , renal failure, retinal ischemia, septic shock (such as endotoxic shock) , and skin aging.
  • diseases and/or disorders such as age-related macular degeneration, AIDS and other immune senescence diseases, arthritis, atherosclerosis, cachexia, cancer, degenerative diseases of skeletal muscle involving replicative senescence, diabetes, head trauma, immune senescence, inflammatory bowel disorders (such as colitis and Crohn's disease),
  • the compounds of the invention act as PARP inhibitors to treat or prevent tissue damage resulting from cell death or damage due to necrosis or apoptosis; to treat or prevent neural tissue damage resulting from cerebral ischemia and reperfusion injury or neurodegenerative diseases in an animal; to extend and increase the lifespan and proliferative capacity of cells; to alter gene expression of senescent cells; and to radiosensitize tumor cells.
  • These compounds are thought to interfere with more than the NMDA-neurotoxicity and N0- mediated biological pathways.
  • the compounds of the invention exhibit an IC 50 for inhibiting PARP in vitro of about ATTORNEY DOCKET .0. 23356-M8
  • the inventors have now discovered that select tricyclic nitrogen-containing compounds that inhibit PARP activity can ameliorate tissue damage resulting from cell damage or death due to necrosis or apoptosi and/or neural tissue damage, including that following focal ischemia and reperfusion injury.
  • inhibition of PARP activity spares the cell from energy loss, preventing irreversible depolarization of the neurons and, thus, provides neuroprotection.
  • PARP activation may play a common role in still other excitotoxic mechanisms, perhaps as yet undiscovered, in addition to the production of free radicals and NO.
  • the compounds of the invention act as PARP inhibitors to treat or prevent neural tissue damage resulting from cerebral ischemia and reperfusion injury or neuro-degenerative diseases in a mammal, to extend and increase the lifespan and proliferative capacity of cells; to alter gene expression of senescent cells; and to radiosensitize tumor cells These compounds are thought to interfere with more than the NMDA- neurotoxicity and NO-mediated biological pathways.
  • the compounds of the invention exhibit an IC 50 for inhibiting PARP in vitro of about 100 ⁇ M or lower, more preferably, about 25 ⁇ M or lower.
  • the compound of the invention has the formula:
  • Y represents the atoms necessary to form a fused 5- to 6- membered, aromatic or non-aromatic, heterocyclic ring containing at least one nitrogen heteroatom in a 1,3- ATTORNEY DOCKE' 0.
  • Y may be unsubstituted or substituted with at least one alkyl, alkenyl, cycloalkyl, cycloalkenyl, aralkyl, aryl, double bonded oxygen, or -C00R 5 group, or a moiety selected from the group consisting of:
  • R 5 and R 7 are independently hydrogen or alkyl, alkenyl, cycloalkyl, cycloalkenyl, aralkyl, or aryl; with the proviso that when Y forms a 5-membered unsaturated ring, Y must be substituted with at least one non-hydrogen, non-phenyl substituent.
  • [N] itrogen heteroatom in a 1,3-relationship with the nitrogen atom depicted in formula I means that there are only two direct bonds between the nitrogen atom in ring Y and the nitrogen atom depicted in formula I.
  • Y forms a 5-membered heterocyclic ring
  • examples include such rings as fused pyrrole, isopyrrole, imidazole, isoimidazole, pyrazole, triazole, pyrrolidine, pyrroline, imidazolidine, i idazoline, pyrazolidine, pyrazoline, thiazole, isothiazole, oxazole, isoxazole, oxadiazole, oxathiazole, dioxazole and the like rings.
  • Y forms a 6-membered heterocyclic ring
  • examples include such rings as fused pyridine, pyrazine, pyrimidine, pyridazine, piperidine, piperazine, triazine, oxazine, oxathiazine, isoxazine, oxadiazine, morpholine and the like rings.
  • Y forms a ring that has at least one site of unsaturation.
  • Y may be aromatic, such as a fused pyrrole or pyridine; or non-aromatic such as a fused piperidyl or piperazinyl.
  • Y may be unsubstituted or substituted with one or more non-interfering substituents.
  • Y may be substituted with an alkyl group such as methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, tert-butyl, n- pentyl, 2-methylpentyl, 2-methylhexyl, dodecyl, octadecyl and the like; with an alkenyl group such as ethenyl, propenyl, butenyl, pentenyl, 2-methylpentenyl, vinyl, isopropenyl, 2,2- dimethyl-1-propenyl, decenyl, hexadecenyl and the like; with a cycloalkyl group such as cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cycloo
  • Aryl is defined as an unsaturated carbocyclic or heterocyclic moiety that may be either unsubstituted or substituted w th one or more non-interfering substituent (s) .
  • Examples include, without limitation, phenyl, benzyl, naphthyl, denyl, azulenyl, fluorenyl, anthracenyl, dolyl, isoindolyl, dolmyl, benzofuranyl, benzo-thiophenyl, mdazolyl, benzi idazolyl, benzithiazolyl, tetrahydrof urnayl, tetrahydropyranyl, pyridyl, pyyrolyl, pyrrolid yl, py ⁇ dinyl, py ⁇ midinyl, pu ⁇ nyl, quinolmyl, isoquinolinyl, tetrahydroqumolmyl, qumol
  • Possible substituents on an aryl group can be any non- interfering substituent.
  • preferred substituents include, without limitation, alkyl, alkenyl, alkoxy, phenoxy, benzyloxy, cycloalkyl, cycloalkenyl, nydroxy, carboxy, carbonyi, am o, amido, cyano, isocyano, nitro, nitroso, nitrilo, isonitrilo, imino, azo, diazo, sulfonyl, sulfoxy, thio, thiocarbonyl, sulfhydryl, halo, haloalkyl, t ⁇ f luoromethyl and aryl.
  • aralkyl groups include benzyl, 3- (1) -naphthyl-1-propyl, p-halobenzyl, p-ethylbenzyl, 1-phenyl-l-propyl, 3-py ⁇ d ⁇ nyl-l-propyl, l-phenyl-2-sec-butyl, 4-phenyl-4-methyl-l-pentyl and the like.
  • Y may also be substituted by double bonded oxygen, carboxylic acid (-COOH) , carboxylic acid analogues (-COOR 5 ) or carboxylic acid mimics.
  • carboxylic acid mimics include: ATTORNEY DOCKE lC . 23356-M8
  • R 7 is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, aralkyl, or aryl, such as described above for Y substituents.
  • the acid mimic moiety itself, R 5 , and R 7 may also be unsubstituted or substituted with one or more non- interfering substituents such as the alkyl, alkenyl, cycloalkyl and cycloalkenyl groups, as described above.
  • the above carboxylic acid mimics are shown in R. Silverman, The Organic Chemistry of Drug Design and Drug Action, Academic Press (1992) .
  • Examples of particularly useful Y ring structures include:
  • tricyclic nuclei of the compounds of the present invention examples include:
  • the most preferred compound is 2, 3-dihydro-lH- benzo [de] [1, 8] naphthydrin-2-one, which has the following structure:
  • the compounds of the invention may be useful in a free base form, in the form of pharmaceutically acceptable salts, pharmaceutically acceptable hydrates, pharmaceutically acceptable esters, pharmaceutically acceptable solvates, pharmaceutically acceptable prodrugs, pharmaceutically acceptable metabolites, and in the form of pharmaceutically acceptable stereoisomers . These forms are all within the scope of the invention. In practice, the use of these forms amounts to use of the neutral compound.
  • “Pharmaceutically acceptable salt”, “hydrate”, “ester” or “solvate” refers to a salt, hydrate, ester, or solvate of the inventive compounds which possesses the desired pharmacological activity and which is neither biologically nor otherwise undesirable.
  • Organic acids can be used to produce salts, hydrates, esters, or solvates such as acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, p- toluenesulfonate, bisulfate, sulfamate, sulfate, naphthylate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentane- propionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate heptanoate, hexanoate, 2-hydroxy
  • Suitable base salts, hydrates, esters, or solvates include hydroxides, carbonates, and bicarbonates of ammonia, alkali metal salts such as sodium, lithium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, aluminum salts, and zinc salts.
  • Salts, hydrates, esters, or solvates may also be formed with organic bases.
  • Organic bases suitable for the formation of pharmaceutically acceptable base addition salts, hydrates, esters, or solvates of the compounds of the present invention include those that are non-toxic and strong enough to form such salts, hydrates, esters, or solvates.
  • the class of such organic bases may include mono- , di-, and trialkylamines, such as methylamine, dimethylamine, triethylamine and dicyclohexylamine; mono-, di- or trihydroxyalkylamines, such as mono-, di-, and triethanolamine ; amino acids, such as arginine and lysine; guanidine; N-methyl- glucosamine; N-methyl-glucamine; L-glutamine; N-methyl- piperazine; morpholine; ethylenediamine; N-benzyl- phenethylamine; (trihydroxy-methyl) aminoethane; and the like. See, for example, "Pharmaceutical Salts," J. Pharm . Sci . , ATTORNEY DOCKE 0. 23356-M8
  • basic nitrogen-containing grou p s can be quaternized with agents including: lower alkyl hali ⁇ es such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; and aralkyl halides such as benzyl and phenethyl bromides .
  • lower alkyl hali ⁇ es such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
  • dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates
  • long chain halides such as decyl, la
  • the acid addition salts, hydrates, esters, or solvates of the basic compounds may be prepared either by dissolving the free base of a PARP inhibitor in an aqueous or an aqueous alcohol solution or other suitable solvent containing the appropriate acid or base, and isolating the salt by evaporating the solution.
  • the free base of the PARP inhibitor may be reacted with an acid, as well as reacting the PARP inhibitor having an acid group thereon with a base, such that the reactions are in an organic solvent, in which case the salt separates directly or can be obtained by concentrating the solution.
  • “Pharmaceutically acceptable prodrug” refers to a derivative of the inventive compounds which undergoes biotransformation prior to exhibiting its pharmacological effect (s).
  • the prodrug is formulated with the objective (s) of improved chemical stability, improved patient acceptance and compliance, improved bioavailability, prolonged duration of action, improved organ selectivity, improved formulation (e.g., increased hydrosolubility) , and/or decreased side effects (e.g., toxicity) .
  • the prodrug can be readily prepared from the inventive compounds using methods known in the art, such as those described by Burger ' s Medicinal Chemistry and Drug Chemistry, Fifth Ed., Vol. 1, pp. 172-178, 949-982 (1995).
  • the inventive compounds can be transformed into prodrugs by converting one or more of the hydroxy or carboxy groups into esters.
  • "Pharmaceutically acceptable metabolite” refers to drugs that have undergone a metabolic transformation.
  • a feature characteristic of many of these transformations is that the metabolic products are more polar than the parent drugs, although a polar drug does sometimes yield a less polar product.
  • Substances with high lipid/water partition coefficients which pass easily across membranes, also diffuse back readily from tubular urine through the renal tubular cells into the plasma. Thus, such substances tend to have a low renal clearance and a long persistence in the body. If a drug is metabolized to a more polar compound, one with a lower partition coefficient, its tubular reabsorption will be greatly reduced.
  • the specific secretory mechanisms for anions and cations in the proximal renal tubules and in the parenchymal liver cells operate upon highly polar substances.
  • phenacetin acetophenetidin
  • acetanilide is both mild analgesic and antipyretic agents, but are each transformed within the body to a more polar and more effective metabolite, p-hydroxyacetanilid (acetaminophen) , which is widely used today.
  • p-hydroxyacetanilid acetaminophen
  • the successive metabolites peak and decay in the plasma sequentially.
  • acetanilid is the principal plasma component.
  • the metabolite acetaminophen concentration reaches a peak.
  • the ATTORNEY DOCKE the ATTORNEY DOCKE :o.
  • Phase I or functionalization reactions generally consist of (1) oxidative and reductive reactions that alter and create new functional groups and (2) hydrolytic reactions that cleave esters and amides to release masked functional groups. These changes are usually in the direction of increased polarity.
  • Phase II reactions are conjugation reactions in which the drug, or often a metabolite of the drug, is coupled to an endogenous substrate, such as glucuronic acid, acetic acid, or sulfuric acid.
  • an endogenous substrate such as glucuronic acid, acetic acid, or sulfuric acid.
  • the compounds of the present invention possess one or more asymmetric center (s) and thus can be produced as mixtures (racemic and non-racemic) of stereoisomers, or as individual R- and S-stereoisomers.
  • the individual stereoisomers may be obtained by using an optically active starting material, by resolving a racemic or non-racemic mixture of an intermediate at some appropriate stage of synthesis, or by resolving a compound of formula I.
  • isomers refers to compounds having the same number and kind of atoms, and hence, the same molecular weight, but differing in respect to the arrangement or configuration of the atoms.
  • Stepoisomers are isomers that differ only in the arrangement of atoms in space.
  • Enantiomers are a pair of stereoisomers that are non-superimposable mirror images of each other.
  • Diastereoisomers are stereoisomers which are not mirror images of each other.
  • Racemic mixture means a mixture containing equal, or roughly equal, parts of individual enantiomers.
  • a “non-racemic mixture” is a mixture containing unequal, or substantially unequal, parts of individual enantiomers or stereoisomers.
  • PARP inhibitors can be synthesized by known methods from starting materials that are known or are themselves commercially available. They may also be prepared by methods used to prepare corresponding compounds in the literature.
  • the compounds of the present invention can also be readily prepared by standard techniques of organic chemistry, using the general synthetic pathways depicted below.
  • Precursor compounds can be prepared by methods known in the art. See, for example, L. Paquette, Principles of Modern Heterocyclic Chemistry (1968) .
  • An especially preferred process for making the compounds of formula I wherein Y forms a six-membered ring comprises the following steps:
  • the second step of the above process typically takes place in the presence of a solvent such as diethyl ether or ethanol, depending upon the choice of reducing agent.
  • a solvent such as diethyl ether or ethanol, depending upon the choice of reducing agent.
  • LAH LiAlH4
  • a suitable solvent is diethyl ether.
  • NaBH4 NaBH4
  • ethanol may be a more suitable solvent.
  • a preferred process for making compounds of formula I wherein Y forms a 5-membered ring comprises the following steps: ATTORNEY DOCKE: 0. 23356-M8
  • the product a compound of formula I
  • the product is isolated from the reaction mixture by conventional techniques, such as by precipitating out, extraction with an immiscible solvent under appropriate pH conditions, evaporation, filtration, crystallization, or by column chromatography on silica gel and the like. Typically, however, the product is removed by either crystallization or column chromatography on silica gel.
  • conventional techniques such as by precipitating out, extraction with an immiscible solvent under appropriate pH conditions, evaporation, filtration, crystallization, or by column chromatography on silica gel and the like.
  • the product is removed by either crystallization or column chromatography on silica gel.
  • Other variations and modifications of this invention using the synthetic pathways described above will be obvious to those skilled in the art.
  • the compounds of formula I used in the composition of the invention will have an IC 50 for inhibiting poly (ADP-ribose) polymerase in vitro of 100 ⁇ M or lower, preferably 25 ⁇ M or lower, more preferably 12 ⁇ M or lower and, even more preferably, 12 mM or lower.
  • compositions A further aspect of the present invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable carrier or a diluent and a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt, hydrate, ester, solvate, prodrug, metabolite, stereoisomer, or mixtures (hereafter, "a compound of formula I").
  • a pharmaceutically acceptable carrier or a diluent and a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt, hydrate, ester, solvate, prodrug, metabolite, stereoisomer, or mixtures (hereafter, "a compound of formula I").
  • formulations of the present invention suitable for oral administration may be in the form of discrete units such as capsules, cachets, tablets, troche or lozenges, and the like, each containing a predetermined amount of the active ingredient; in the form of a powder or granules; in the form of a solution or a suspension in an aqueous liquid or nonaqueous liquid; or in the form of an oil-in-water emulsion or a water-in-oil emulsion.
  • the active ingredient may also be in the form of a bolus, electuary, or paste.
  • compositions will usually be formulated into a unit dosage form, such as a tablet, capsule, aqueous suspension or solution.
  • a unit dosage form such as a tablet, capsule, aqueous suspension or solution.
  • Such formulations typically include a solid, semisolid, or liquid carrier.
  • Exemplary carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calci-um phosphate, mineral oil, cocoa butter, oil of theobroma, alginates, tragacanth, gelatin, syrup, methyl cellulose, polyoxyethylene sorbitan monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, corn starch and the like.
  • Particularly preferred formulations include tablets and gelatin capsules comprising the active ingredient together with (a) diluents, such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, dried corn starch, and glycine, and the like; and/or (b) lubricants, such as silica, talcum, stearic acid, its magnesium or calcium salt, and polyethylene glycol, and the like.
  • diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, dried corn starch, and glycine, and the like
  • lubricants such as silica, talcum, stearic acid, its magnesium or calcium salt, and polyethylene glycol, and the like.
  • Tablets may also contain binders, such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidone; disintegrants, such as starches, agar, alginic acid or its sodium salt, and effervescent mixtures; and/or absorbents, colorants, flavors, and sweeteners.
  • binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidone
  • disintegrants such as starches, agar, alginic acid or its sodium salt, and effervescent mixtures
  • absorbents colorants, flavors, and sweeteners.
  • the ATTORNEY DOCKE r ⁇ . 23356-M8 compositions of the invention may be sterilized and/or contain adjuvants, such as preserving, stabilizing, swelling or emulsifying agents, solution promoters, salts for
  • Aqueous suspensions may contain emulsifying and suspending agents combined with the active ingredient. All oral dosage forms may further contain sweetening and/or flavoring and/or coloring agents. These compositions are prepared according to conventional mixing, granulating, or coating methods, respectively, and contain about 0.1 to 75% of the active ingredient, preferably about 1 to 50% of the same.
  • a tablet may be made by compressing or molding the active ingredient optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active, or dispersing agent. Molded tablets may be made by molding, in a suitable machine, a mixture of the powdered active ingredient and a suitable carrier moistened with an inert liquid diluent.
  • composition When administered parenterally, the composition will normally be in a unit dosage, sterile injectable form (aqueous isotonic solution, suspension or emulsion) with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier preferably non-toxic, parenterally-acceptable and contain non- therapeutic diluents or solvents.
  • aqueous solutions such as saline (isotonic sodium chloride solution), Ringer's solution, dextrose solution, and Hanks' solution
  • nonaqueous carriers such as 1,3-butanediol, fixed oils (e.g., corn, cottonseed, peanut, sesame oil, and synthetic mono- or di-glyceride) , ethyl oleate, and isopropyl myristate.
  • Oleaginous suspensions can be formulated according to techniques known in the art using suitable dispersing or ATTORNEY DOCKET O . 23356-M8 wetting agents and suspending agents.
  • the acceptable solvents or suspending mediums are sterile fixed oils.
  • any bland fixed oil may be used.
  • Fatty acids such as oleic acid and its glyceride derivatives, including olive oil and castor oil, especially in their polyoxyethylated forms, are also useful in the preparation of injectables.
  • These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants.
  • Sterile saline is a preferred carrier, and the compounds are often sufficiently water soluble to be made up as a solution for all foreseeable needs.
  • the carrier may contain minor amounts of additives, such as substances that enhance solubility, isotonicity, and chemical stability, e.g., anti- oxidants, buffers and preservatives.
  • the composition When administered rectally, the composition will usually be formulated into a unit dosage form such as a suppository or cachet. These compositions can be prepared by mixing the compound with suitable non-irritating excipients that are solid at room temperature, but liquid at rectal temperature, such that they will melt in the rectum to release the compound.
  • Common excipients include cocoa butter, beeswax and polyethylene glycols or other fatty emulsions or suspensions.
  • the compounds may be administered topically, especially when the conditions addressed for treatment involve areas or organs readily accessible by topical application, including neurological disorders of the eye, the skin or the lower intestinal tract.
  • the compounds can be formulated as micronized suspensions in isotonic, pH-adjusted sterile saline or, preferably, as a solution in isotonic, pH-adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride.
  • the compounds may be formulated into ointments, such as petrolatum.
  • suitable ointments containing the compounds ATTORNEY DOCKE O are examples of suitable ointments containing the compounds ATTORNEY DOCKE O .
  • the compounds can be formulated into suitable lotions or creams containing the active compound suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • Topical application to the lower intestinal tract can be effected in rectal suppository formulations (see above) or in suitable enema formulations.
  • Formulations suitable for nasal or buccal administration may comprise about 0.1% to about 5% w/w of the active ingredient or, for example, about 1% w/w of the same.
  • some formulations can be compounded into a sublingual troche or lozenge.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.
  • the carrier is a solid biodegradable polymer or mixture of biodegradable polymers with appropriate time release characteristics and release kinetics.
  • the composition of the invention may then be molded into a solid implant suitable for providing efficacious concentrations of the compounds of the invention over a prolonged period of time without the need for frequent redosing.
  • the composition ATTORNEY DOCKET .40. 23356-M8 of the present invention can be incorporated into the biodegradable polymer or polymer mixture in any suitable manner known to one of ordinary skill in the art and may form a homogeneous matrix with the biodegradable polymer, or may be encapsulated in some way within the polymer, or may be molded into a solid implant.
  • the biodegradable polymer or polymer mixture is used to form a soft "depot" containing the pharmaceutical composition of the present invention that can be administered as a flowable liquid, for example, by injection, but which remains sufficiently viscous to maintain the pharmaceutical composition within the localized area around the injection site.
  • the degradation time of the depot so formed can be varied from several days to a few years, depending upon the polymer selected and its molecular wight.
  • the pharmaceutical composition of the present invention is used in amounts that are therapeutically effective and the amounts used may depend upon the desired release profile, the concentration of the pharmaceutical composition required for the sensitizing effect, and the length of time that the pharmaceutical composition has to be released for treatment.
  • composition of the invention is preferably administered as a capsule or tablet containing a single or divided dose of the compound, or as a sterile solution, suspension, or emulsion, for parenteral administration in a single or divided dose.
  • the compounds of the invention can be prepared in lyophilized form.
  • 1 to 100 mg of a- PARP inhibitor may be lyophilized in individual vials, together with a carrier and a buffer, such as mannitol and sodium phosphate.
  • the composition may then be ATTORNEY DOCK! NO. 23356-M8 reconstituted in the vials with bacteriostatic water before administration .
  • the compounds of the invention are used in the composition in amounts that are therapeutically effective. While the effective amount of the PARP inhibitor will depend upon the particular compound being used, amounts of these compounds varying from about 1% to about 65% have been easily incorporated into liquid or solid carrier delivery systems.
  • an effective therapeutic amount of the compounds and compositions described above are administered to animals to effect a neuronal activity, preferably one that is not mediated by NMDA neurotoxicity.
  • a neuronal activity may consist of stimulation of damaged neurons, promotion of neuronal regeneration, prevention of neurodegeneration and treatment of a neurological disorder.
  • the present invention further relates to a method of effecting a neuronal activity in an animal, comprising administering an effective amount of the compound of formula I to said animal.
  • the compounds of the invention inhibit PARP activity and, thus, are believed to be useful for treating neural tissue damage, particularly damage resulting from cerebral ischemia and reperfusion injury or neurodegenerative diseases in mammals.
  • neural tissue refers to the various components that make up the nervous system including, without limitation, neurons, neural support cells, glia, Schwann cells, vasculature contained within and supplying these structures, the central nervous system, the brain, the brain stem, the spinal cord, the junction of the central nervous system with the peripheral nervous system, the peripheral nervous system, and allied structures.
  • ischemia refers to localized tissue anemia due to obstruction of the inflow of arterial blood.
  • Global ischemia occurs when blood flow to the entire brain ceases for ATTORNEY DOCKE' 0. 23356-M8 a period of time.
  • Global ischemia may result from cardiac arrest.
  • Focal ischemia occurs when a portion of the brain is deprived of its normal blood supply.
  • Focal ischemia may result from thromboembolytic occlusion of a cerebral vessel, traumatic head injury, edema or brain tumor. Even if transient, both global and focal ischemia can cause widespread neuronal damage.
  • nerve tissue damage occurs over hours or even days following the onset of ischemia, some permanent nerve tissue damage may develop in the initial minutes following the cessation of blood flow to the brain. Much of this damage has been attributed to glutamate toxicity and to the secondary consequences of tissue reperfusion, such as the release of vasoactive products by damaged endothelium and the release of cytotoxic products, such as free radicals and leukotrines, by the damaged tissue. Ischemia can also occur in the heart in myocardial infarction and other cardiovascular disorders in which the coronary arteries have been obstructed as a result of atherosclerosis, thrombi, or spasm.
  • neural tissue damage resulting from ischemia and reperfusion injury and neurodegenerative diseases includes neurotoxicity, such as seen in vascular stroke and global and focal ischemia.
  • neural insult refers to any damage to nervous tissue and any disability or death resulting therefrom.
  • the cause of nervous insult may be metabolic, toxic, neurotoxic, iatrogenic, thermal or chemical, and includes without limitation, ischemia, hypoxia, cerebrovascular accident, trauma, surgery, pressure, mass effect, hemmorrhage, radiation, vasospasm, neurodegenerative disease, infection, Parkinson's disease, amyotrophic lateral sclerosis (ALS ) , myelination/dernyelination process, epilepsy, cognitive disorder, glutamate abnormality and secondary effects thereof.
  • ALS amyotrophic lateral sclerosis
  • neuroprotective refers to the effect of reducing, arresting or ameliorating nervous insult, and ATTORNEY DOCKE JO. 23356- 8 protecting, resuscitating, or reviving nervous tissue that has suffered nervous insult.
  • preventing neurodegeneration includes the ability to prevent neurodegeneration in patients diagnosed as having a neurodegenerative disease or who are at risk of developing a neurodegenerative disease. The term also encompasses preventing further neurodegeneration in patients who are already suffering from or have symptoms of a neurodegenerative disease.
  • treating refers to:
  • neurological disorders that are treatable by the method of using the present invention include, without limitation, trigeminal neuralgia; glossopharyngeal neuralgia;
  • Bell's Palsy myasthenia gravis; muscular dystrophy; amyotrophic lateral sclerosis; progressive muscular atrophy; progressive bulbar inherited muscular atrophy; herniated, ruptured or prolapsed invertebrate disk syndromes; cervical spondylosis; plexus disorders; thoracic outlet destruction syndromes; peripheral neuropathies such as those caused by lead, dapsone, ticks, porphyria, or Guillain-Barre syndrome;
  • Alzheimer's disease Huntington's Disease and Parkinson's disease.
  • the method of the present invention is particularly useful for treating a neurological disorder selected from the group consisting of: peripheral neuropathy caused by physical injury or disease state; head trauma, such as traumatic brain injury; physical damage to the spinal cord; stroke associated with brain damage, such as vascular stroke associated with hypoxia ATTORNEY DOCKET 0. 23356-M8 and brain damage, focal cerebral ischemia, global cerebral ischemia, and cerebral reperfusion injury; demyelinating diseases, such as multiple sclerosis; and neurological disorders related to neurodegeneration, such as Alzheimer's Disease, Parkinson's Disease, Huntington's Disease and amyotrophic lateral sclerosis (ALS) .
  • a neurological disorder selected from the group consisting of: peripheral neuropathy caused by physical injury or disease state; head trauma, such as traumatic brain injury; physical damage to the spinal cord; stroke associated with brain damage, such as vascular stroke associated with hypoxia ATTORNEY DOCKET 0. 23356-M8 and brain damage, focal cerebral ischemia, global cerebral ischemia, and cerebral reperfusion injury; dem
  • the compounds, compositions and methods of the present invention are particularly useful for treating or preventing tissue damage resulting from cell death or damage due to necrosis or apoptosis.
  • the compounds, compositions and methods of the invention can also be used to treat a cardiovascular disorder in an animal, by administering an effective amount of the compound of formula to the animal.
  • cardiovascular disorders refers to those disorders that can either cause ischemia or are caused by reperfusion of the heart. Examples include, but are not limited to, coronary artery disease, angina pectoris, myocardial infarction, cardiovascular tissue damage caused by cardiac arrest, cardiovascular tissue damage caused by cardiac bypass, cardiogenic shock, and related conditions that would be known by those of ordinary skill in the art or which involve dysfunction of or tissue damage to the heart or vasculature, especially, but not limited to, tissue damage related to PARP activation.
  • the methods of the invention are believed to be useful for treating cardiac tissue damage, particularly damage resulting from cardiac ischemia or caused by reperfusion injury in animals.
  • the methods of the invention are particularly useful for treating cardiovascular disorders selected from the group consisting of: coronary artery disease, such as atherosclerosis; angina pectoris; myocardial infarction; myocardial ischemia and cardiac arrest; cardiac bypass; and cardiogenic shock.
  • coronary artery disease such as atherosclerosis
  • angina pectoris myocardial infarction
  • myocardial ischemia and cardiac arrest cardiac bypass
  • cardiogenic shock e.g., ATTORNEY DOCKI .vie 23356-M8 are particularly helpful in treating the acute forms of the above cardiovascular disorders.
  • the methods of the invention can be used to treat tissue damage resulting from cell damage or death due to necrosis or apoptosis, neural tissue damage resulting from ischemia and reperfusion injury, neurological disorders and neurodegenerative diseases; to prevent or treat vascular stroke; to treat or prevent cardiovascular disorders; to treat other conditions and/or disorders such as age-related macular degeneration, AIDS and other immune senescence diseases, arthritis, atherosclerosis, cachexia, cancer, degenerative diseases of skeletal muscle involving replicative senescence, diabetes, head trauma, immune senescence, inflammatory bowel disorders (such as colitis and Crohn's disease), muscular dystrophy, osteoarthritis, osteoporosis, chronic and/or acute pain (such as neuropathic pain), renal failure, retinal ischemia, septic shock (such as endotoxic shock) , and skin aging; to extend the lifespan and proliferative capacity of cells; to alter gene expression of senescent cells; or to radiosensitize tumor cells
  • the methods of the invention can be used to treat cancer and to radiosensitize tumor cells.
  • cancer is interpreted broadly.
  • the compounds of the present invention can be "anti-cancer agents”, which term also encompasses “anti-tumor cell growth agents” and "anti- neoplastic agents”.
  • the methods of the invention are useful for treating cancers and radiosensitizing tumor cells in cancers such as ACTH-producing tumors, acute lymphocytic leukemia, acute nonlymphocytic leukemia, cancer of the adrenal cortex, bladder cancer, brain cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia, chronic myelocytic leukemia, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, esophageal cancer, Swing's sarcoma, gallbladder , cancer, hairy cell leukemia, head & neck cancer, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer (small and/or non-small cell) , ATTORNEY DOCKET ..0.
  • cancers such as ACTH-producing tumors, acute lymphocytic leukemia, acute nonlymphocytic leukemia, cancer of the adrenal cortex, bladder cancer, brain cancer, breast cancer,
  • 23356-M8 malignant peritoneal effusion, malignant pleural effusion, melanoma, mesothelioma, multiple myeloma, neuroblastoma, non- Hodgkin's lymphoma, osteosarcoma, ovarian cancer, ovary (germ cell) cancer, prostate cancer, pancreatic cancer, penile cancer, retinoblastoma, skin cancer, soft-tissue sarcoma, squamous cell carcinomas, stomach cancer, testicular cancer, thyroid cancer, trophoblastic neoplasms, uterine cancer, vaginal cancer, cancer of the vulva and Wilm's tumor.
  • radiosensitizer is defined as a molecule, preferably a low molecular weight molecule, administered to animals in therapeutically effective amounts to increase the sensitivity of the cells to be radiosensitized to electromagnetic radiation and/or to promote the treatment of diseases which are treatable with electromagnetic radiation.
  • Diseases which are treatable with electromagnetic radiation include neoplastic diseases, benign and malignant tumors, and cancerous cells. Electromagnetic radiation treatment of other diseases not listed herein are also contemplated by the present invention.
  • electromagnetic radiation and “radiation” as used herein includes, but is not limited to, radiation having the wavelength of 10 "20 to 10° meters.
  • Preferred embodiments of the present invention employ the electromagnetic radiation of: gamma-radiation (10 ⁇ 20 to 10 "13 m) x-ray radiation (10" u to 10" 9 m) , ultraviolet light (10 nm to 400 nm) , visible light (400 nm to 700 nm) , infrared radiation (700 nm to 1.0 mm), and microwave radiation (1 mm to 30 cm).
  • Radiosensitizers are known to increase the sensitivity of cancerous cells to the toxic effects of electromagnetic radiation.
  • hypoxic cell radiosensitizers e.g., 2- nitroimidazole compounds, and benzotriazine dioxide compounds
  • non-hypoxic cell radiosensitizers e.g., halogenated pyrimidines
  • various other potential mechanisms of action have been hypothesized for radiosensitizers in the treatment of disease.
  • radiosensitizers activated by the electromagnetic radiation of x-rays.
  • x-ray activated radiosensitizers include, but are not limited to, the following: metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, E09, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR) , 5-iododeoxyuridine (IUdR) , bromodeoxycytidine, fluorodeoxyuridine (FudR) , hydroxyurea, cisplatin, and therapeutically effective analogs and derivatives of the same.
  • metronidazole misonidazole
  • desmethylmisonidazole pimonidazole
  • etanidazole nimorazole
  • mitomycin C RSU 1069
  • Photodynamic therapy (PDT) of cancers employs visible light as the radiation activator of the sensitizing agent.
  • photodynamic radiosensitizers include the following, but are not limited to: hematoporphyrin derivatives, Photofrin, benzoporphyrin derivatives, NPe ⁇ , tin etioporphyrin SnET2, pheoborbide-a, bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc phthalocyanine, and therapeutically effective analogs and derivatives of the same.
  • Radiosensitizers may be administered in conjunction with a therapeutically effective amount of one or more other compounds, including but not limited to: compounds which promote the incorporation of radiosensitizers to the target cells; compounds which control the flow of therapeutics, nutrients, and/or oxygen to the target cells; chemotherapeutic agents which act on the tumor with or without additional radiation; or other therapeutically effective compounds for treating cancer or other disease.
  • radiosensitizers examples include, but are not limited to: 5- fluorouracil, leucovorin, 5 ' -amino-5 ' deoxythymidine, oxygen, carbogen, red cell transfusions, perfluorocarbons (e.g., ATTORNEY DOCKET .,0. 23356-M8
  • Fluosol-DA Fluosol-DA
  • 2,3-DPG 2,3-DPG, BW12C, calcium channel blockers
  • pentoxyfylline antiangiogenesis compounds
  • hydralazine and L- BSO.
  • chemotherapeutic agents that may be used in conjunction with radiosensitizers include, but are not limited to: adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, docetaxel, doxorubicin, interferon (alpha, beta, gamma) , interleukin 2, irinotecan, paclitaxel, topotecan, and therapeutically effective analogs and derivatives of the same.
  • the compounds of the present invention may also be used for radiosensitizing tumor cells.
  • treating refers to:
  • the amount required of a compound of formula I to achieve a therapeutic effect will vary according to the particular compound administered, the route of administration, the mammal under treatment, and the particular disorder or disease concerned.
  • a suitable systemic dose of a compound of formula I for a mammal suffering from, or likely to suffer from, any condition as described herein is typically in the range of about 0.1 to about 100 mg of base per kilogram of body weight, preferably from about 1 to about 10 mg/kg of mammal body weight. It is understood that the ordinarily skilled physician or veterinarian will readily be able to determine and prescribe the amount of the compound effective for the desired prophylactic or therapeutic treatment.
  • the physician or veterinarian may employ an intravenous bolus followed by an intravenous infusion and ATTORNEY DOCKET WO. 23356-M8 repeated administrations, as considered appropriate.
  • the compounds may be administered, for example, orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, sublingually, vaginally, intraventricularly, or via an implanted reservoir in dosage formulations containing conventional non-toxic pharmaceutically-acceptable carriers, adjuvants and vehicles.
  • Parenteral includes, but is not limited to, the following examples of administration: intravenous, subcutaneous, intramuscular, intraspinal, intraosseous, intraperitoneal, intrathecal, intraventricular, intrasternal or intracranial injection and infusion techniques, such as by subdural pump. Invasive techniques are preferred, particularly direct administration to damaged neuronal tissue. While it is possible for the compound of formula I to be administered alone, it is preferable to provide it as a part of a pharmaceutical formulation.
  • the compounds used in the methods of the present invention should readily penetrate the blood-brain barrier when peripherally administered. Compounds which cannot penetrate the blood-brain barrier, however, can still be effectively administered by an intraventricular route.
  • the compounds used in the methods of the present invention may be administered by a single dose, multiple discrete doses or continuous infusion. Since the compounds are small, easily diffusible and relatively stable, they are well suited to continuous infusion. Pump means, particularly subcutaneous or subdural pump means, are preferred for continuous infusion. For the methods of the present invention, any effective administration regimen regulating the timing and sequence of doses may be used. Doses of the compounds preferably include pharmaceutical dosage units comprising an efficacious quantity of active compound. ⁇ By an efficacious quantity is meant a quantity sufficient to inhibit PARP activity and/or derive the desired beneficial effects therefrom through administration of ATTORNEY DOCKET 0. 23356-M8 one or more of the pharmaceutical dosage units.
  • the dose is sufficient to prevent or reduce the effects of vascular stroke or other neurodegenerative diseases.
  • An exemplary daily dosage unit for a vertebrate host comprises an amount of from about 0.001 mg/kg to about 50 mg/kg.
  • dosage levels on the order of about 0.1 mg to about 10,000 mg of the active ingredient compound are useful in the treatment of the above conditions, with preferred levels being about 0.1 mg to about 1,000 mg.
  • the specific dose level for any particular patient will vary depending upon a variety of factors, including the activity of the specific compound employed; the age, body weight, general health, sex, and diet of the patient; the time of administration; the rate of excretion; any combination of the compound with other drugs; the severity of the particular disease being treated; and the form and route of administration.
  • in vitro dosage- effect results provide useful guidance on the proper doses for patient administration. Studies in animal models can also be helpful. The considerations for determining the proper dose levels are well-known in the art.
  • the compounds of the invention can be co-administered with one or more other therapeutic agents, preferably agents which can reduce the risk of stroke (such as aspirin) and, more preferably, agents which can reduce the risk of a second ischemic event (such as ticlopidine) .
  • agents which can reduce the risk of stroke such as aspirin
  • agents which can reduce the risk of a second ischemic event such as ticlopidine
  • the compounds and compositions can be co-administered with one or more therapeutic agents either (i) together in a single formulation, or (ii) separately in individual formulations designed for optimal release rates of their respective active agent.
  • Each formulation may contain from about 0.01% to about 99.99% by weight, preferably from about 3.5% to about 60% by weight, of the compound of the invention, as well as one or more pharmaceutical excipients, such as wetting, emulsifying ATTORNEY DOCKE. ,0. 23356-M8 and pH buffering agents.
  • specific dose levels for those agents will depend upon considerations such as those identified above for compositions and methods of the invention in general.
  • Table II below provides known median dosages for selected chemotherapeutic agents that may be administered in combination with the compounds of the invention to such diseases or various cancers.
  • any administration regimen regulating the timing and sequence of delivery of the compound can be used and repeated as necessary to effect treatment.
  • Such regimen may include pretreatment ATTORNEY DOCKET 0. 23356-M8 and/or co-administration with additional therapeutic agents.
  • the compounds of the invention should be administered to the affected cells as soon as possible.
  • the compounds are advantageously administered before the expected nervous insult.
  • Such situations of increased likelihood of nervous insult include surgery, such as carotid endarterectomy, cardiac, vascular, aortic, orthopedic surgery; endovascular procedures, such as arterial catheterization (carotid, vertebral, aortic, cardia, renal, spinal, Adamkiewicz) ; injections of embolic agents; the use of coils or balloons for hemostasis; interruptions of vascularity for treatment of brain lesions; and predisposing medical conditions such as crescendo transient ischemic attacks, emboli and sequential strokes.
  • a particularly advantageous mode of administration with a patient diagnosed with acute multiple vascular strokes is by implantation of a subdural pump to deliver the compound (s) of the invention directly to the infarct area of the brain. Even if comatose, it is expected that the patient would recover more quickly that he or she would without this treatment. Moreover, in any conscious state of the patient, it is expected that any residual neurological symptoms, as well as the re-occurrence of stroke, would be reduced.
  • the compound of the invention should also be administered as soon as possible in a single or divided dose.
  • the patient may further receive additional doses of the same or different compounds of the invention, by one of the following routes: parenterally, such as by injection or by intravenous administration; orally, such as by capsule or tablet; by implantation of a biocompatible, biodegradable polymeric matrix delivery system comprising the compound; or by direct administration to the infarct area by insertion of a subdural pump or a central line.
  • parenterally such as by injection or by intravenous administration
  • orally such as by capsule or tablet
  • direct administration to the infarct area by insertion of a subdural pump or a central line.
  • the patient's condition may deteriorate due to the acute disorder and become a chronic disorder by the time that the compounds are available. Even when a patient receives a compound of formula I for the chronic disorder, it is also expected that the patient's condition would stabilize and actually improve as a result of receiving the compound.
  • the IC 50 of with respect to PARP inhibition was determined for several compounds by a PARP assay using purified recombinant human PARP from Trevigen (Gaithersburg, MD) , as ATTORNEY DOCKET 0. 23356-M8 follows:
  • the PARP enzyme assay was set up on ice in a volume of 100 microliters consisting of 10 mM Tris-HCl (pH 8.0), 1 mM MgCl 2 , 28 mM KCl, 28 mM NaCl, 0.1 mg/ml of herring sperm DNA (activated as a 1 mg/ml stock for 10 minutes in a 0.15% hydrogen peroxide solution), 3.0 micromolar [3H] nicotinamide adenine dinucleotide (470 mci/mmole) , 7 micrograms/ml PARP enzyme, and various concentrations of the compounds to be tested.
  • the reaction was initiated by incubating the mixture at 25°C. After 15 minutes' incubation, the reaction was terminated by adding 500 microliters of ice cold 20% (w/v) trichloroacetic acid. The precipitate formed was transferred onto a glass fiber filter (Packard Unifilter-GF/B) and washed three times with ethanol. After the filter was dried, the radioactivity was determined by scintillation counting. Using the PARP assay described above, approximate IC S0 values were obtained for the following compounds:
  • Focal cerebral ischemia was produced by cauterization of the right distal MCA (middle cerebral artery) with bilateral temporary common carotid artery occlusion in male Long-Evans rats for 90 minutes. All procedures performed on the animals were approved by the University Institutional Animal Care and Use Committee of the University of Pennsylvania. A total of 42 rats (weights: 230-340 g) obtained from Charles River were used in this study. The animals fasted overnight with free access to water prior to the surgical procedure.
  • the rats were then anesthetized with halothane (4% for induction and 0.8%-1.2% for the surgical procedure) in a mixture of 70% nitrous oxide and 30% oxygen.
  • the body temperature was monitored by a rectal probe and maintained at 37.5 + 0.5°C with a heating blanket regulated by a homeothermic blanket control unit (Harvard Apparatus Limited, Kent, U.K.).
  • a catheter (PE-50) was placed into the tail artery, and arterial pressure was continuously monitored and recorded on a Grass polygraph recorder (Model 7D, Grass Instruments, Quincy, Massachusetts) .
  • Samples for blood gas analysis (arterial pH, Pa0 2 and PaC0 2 ) were also taken from the tail artery catheter and measured with a blood gas analyzer (ABL 30, Radiometer, Copenhagen, Denmark) .
  • Arterial blood samples were obtained 30 minutes after MCA occlusion.
  • the head of the animal was positioned in a stereotaxic frame, and a right parietal incision between the right lateral canthus and the external auditory meatus was made.
  • a dental drill constantly cooled with saline, a 3 mm burr hole was prepared over the cortex supplied by the right MCA, 4 mm lateral to the sagittal suture and 5 mm caudal to the coronal suture.
  • the dura mater and a thin inner bone layer were kept, care being taken to position the probe over a tissue area devoid of large blood vessels.
  • the flow probe (tip diameter of 1 mm, fiber separation of 0.25 mm) was lowered to the bottom of the cranial burr hole using a micromanipulator .
  • the probe was held stationary by a probe holder secured to the skull with dental cement.
  • the microvascular blood flow in the right parietal cortex was continuously monitored with a laser Doppler flowmeter (FloLab, Moor, Devon, U.K., and Periflux 4001, Perimed, Sweden) .
  • Focal cerebral ischemia was produced by cauterization of the distal portion of the right MCA with bilateral temporary common carotid artery (CCA) occlusion by the procedure of Chen et al., "A Model of Focal Ischemic Stroke in the Rat: Reproducible Extensive Cortical Infarction", Strode 17:738-43 (1986) and/or Liu et al., "Polyethylene Glycol-conjugated Superoxide Dismutase and Catalase Reduce Ischemic Brain Injury", Am. J. Physiol . 256:H589-93 (1989), both of which are hereby incorporated by reference.
  • CCA common carotid artery
  • bilateral CCA's were isolated, and loops made from polyethylene (PE-10) catheter were carefully passed around the CCA's for later remote occlusion.
  • the incision made ATTORNEY DOCKE' 0. 23356-M8 previously for placement of the laser doppler probe was extended to allow observation of the rostral end of the zygomatic arch at the fusion point using a dental drill, and the dura mater overlying the MCA was cut.
  • the MCA distal to its crossing with the inferior cerebral vein was lifted by a fine stainless steel hook attached to a micromanipulator and, following bilateral CCA occlusion, the MCA was cauterized with an electrocoagulator .
  • the burr hole was covered with a small piece of Gelform, and the wound was sutured to maintain the brain temperature within the normal or near-normal range.
  • mice Twenty-four hours after MCA occlusion, the rats were sacrificed with an intraperitoneal injection of pentobarbital sodium (150 mg/kg) .
  • the brain was carefully removed from the skull and cooled in ice-cold artificial CSF for five minutes.
  • the cooled brain was then sectioned in the coronal plane at 2 mm intervals using a rodent brain matrix (RBM-4000C, ASI Instruments, Warren, Michigan) .
  • the brain slices were incubated in phosphate-buffered saline containing 2% 2, 3, 5-triphenyl- tetrazolium chloride (TTC) at 37°C for ten minutes.
  • TTC 5-triphenyl- tetrazolium chloride
  • the total volume of infarction was calculated by summation of the damaged volume of the brain slices.
  • ** Significantly different from the steady state value, p ⁇ 0.01.
  • MABP mean arterial blood pressure
  • blood flow showed no significant changes throughout the entire occlusion period in any group.
  • Focal cerebral ischemia experiments are performed using male Wistar rats weighing 250 - 300 g, which are anesthetized with 4% halothane. Anesthesia is maintained with 1.0-1.5% halothane until the end of surgery. The animals are installed in a warm environment to avoid a decrease in body temperature during surgery. An anterior midline cervical incision is made.
  • the right common carotid artery (CCA) is exposed and isolated from the vagus nerve.
  • a silk suture is placed and tied around the CCA in proximity to the heart.
  • the external carotid artery (ECA) is then exposed and ligated with a silk suture.
  • a puncture is made in the CCA and a small catheter (PE 10, Ulrich & Co., St-Gallen, Switzerland) is gently advanced to the lumen of the internal carotid artery (ICA).
  • ICA internal carotid artery
  • the pterygopalatine artery is not occluded.
  • the catheter is tied in place with a silk suture.
  • a 4-0 nylon suture (Braun Medical, Crissier, Switzerland) is introduced into the catheter lumen and is pushed until the tip blocks the anterior cerebral artery.
  • the length of catheter into the ICA is approximately 19 mm from the origin of the ECA.
  • the suture is maintained in this position by occlusion of the catheter with heat.
  • One cm of catheter and nylon suture are left protruding so that the suture can be withdrawn to allow reperfusion.
  • the skin incision is then closed with wound clips.
  • the animals are maintained in a warm environment during recovery from anesthesia. Two hours later, the animals are re- anesthetized, the clips are discarded, and the wound is re-opened. The catheter is cut, and the suture is pulled out. The catheter is then obturated again by heat, and wound clips are placed on the wound. The animals are allowed to survive for 24 hours with free access to food and water. The rats are then sacrificed with C0 2 and decapitated.
  • the brains are immediately removed, frozen on dry ice and stored at -80°C.
  • the brains are then cut in 0.02 mm-thick sections in a cryocut at -19°C, selecting one of every 20 sections for further examination.
  • the selected sections are ATTORNEY DOCKE AO . 23356-M8 stained with cresyl violet according to the Nissl procedure.
  • Each stained section is examined under a light microscope, and the regional infarct area is determined according to the presence of cells with morphological changes.
  • Various doses of the compounds of the invention are tested in this model.
  • the compounds are administered in either a single dose or a series of multiple doses, i.p. or i.v., at different times, both before or after the onset of ischemia.
  • Compounds of the invention are found to provide protection from ischemia in the range of about 20 to 80%.
  • mice Female Sprague-Dawley rats, each weighing about 300-350 g are anesthetized with intraperitoneal ketamine at a dose of 150 mg/kg.
  • the rats are endotracheally intubated and ventilated with oxygen-enriched room air using a Harvard rodent ventilator.
  • Polyethylene catheters inserted into the carotid artery and the femoral vein are used for artery blood pressure monitoring and fluid administration respectively.
  • Arterial pC0 2 is maintained between 35 and 45mm Hg by adjusting the respirator rate.
  • the rat chests are opened by median sternotomy, the pericardium is incised, and the hearts are cradled with a latex membrane tent.
  • Hemodynamic data are obtained at baseline after at least a 15-minute stabilization period following the end of the surgical operation.
  • the LAD (left anterior descending) coronary artery is ligated for 40 minutes, and then re-perfused for 120 minutes. After 120 minutes' reperfusion, the LAD artery is re-occluded, and a 0.1 ml bolus of monastral blue dye is injected into the left atrium to determine the ischemic risk region.
  • the hearts are then arrested with potassium chloride and cut into five 2-3 mm thick transverse slices. Each slice is weighed and incubated in a 1% solution of trimethyltetrazoliu chloride to visualize the infarcted myocardium located within the risk region. Infarct size is calculated by summing the values for each left ventricular slice and is further expressed ATTORNEY DOCKET . 23356-M8 as a fraction of the risk region of the left ventricle.
  • the compounds of the invention are tested in this model.
  • the compounds are given either in a single dose or a series of multiple doses, i.p. or i.v., at different times, both before or after the onset of ischemia.
  • the compounds of the invention are found to have ischemia/reperfusion injury protection in the range of 10 to 40 percent. Therefore, they protect against ischemia-induced degeneration of rat hippocampal neurons in vitro.
  • a patient just diagnosed with acute retinal ischemia is immediately administered parenterally, either by intermittent or continuous intravenous administration, a compound of formula I, either as a single dose or a series of divided doses of the compound.
  • the patient optionally may receive the same or a different compound of the invention in the form of another parenteral dose. It is expected by the inventors that significant prevention of neural tissue damage would ensue and that the patient's neurological symptoms would considerably lessen due to the administration of the compound, leaving fewer residual neurological effects post- stroke. In addition, it is expected that the re-occurrence of retinal ischemia would be prevented or reduced.
  • a patient has just been diagnosed with acute retinal ischemia.
  • a physician or a nurse parenterally administers a compound of formula I, either as a single dose or as a series of divided doses.
  • the patient also receives the same or a different PARP inhibitor by intermittent or continuous administration via implantation of a biocompatible, biodegradable polymeric matrix delivery system comprising a compound of formula I, or via a subdural pump inserted to administer the compound directly to the infarct area of the ATTORNEY DOCKET 0. 23356-M8 brain. It is expected by the inventors that the patient would awaken from the coma more quickly than if the compound of the invention were not administered.
  • the treatment is also expected to reduce the severity of the patient's residual neurological symptoms. In addition, it is expected that reoccurrence of retinal ischemia would be reduced.
  • a patient just diagnosed with acute vascular stroke is immediately administered parenterally, either by intermittent or continuous intravenous administration, a compound of formula I, either as a single dose or a series of divided doses of the compound.
  • the patient optionally may receive the same or a different compound of the invention in the form of another parenteral dose. It is expected by the inventors that significant prevention of neural tissue damage would ensue and that the patient's neurological symptoms would considerably lessen due to the administration of the compound, leaving fewer residual neurological effects post- stroke. In addition, it is expected that the re-occurrence of vascular stroke would be prevented or reduced.
  • Example 8 Treatment of Vascular Stroke A patient has just been diagnosed with acute multiple vascular strokes and is comatose. Immediately, a physician or a nurse parenterally administers a compound of formula I, either as a single dose or as a series of divided doses. Due to the comatose state of the patient, the patient also receives the same or a different PARP inhibitor by intermittent or continuous administration via implantation of a biocompatible, biodegradable polymeric matrix delivery system comprising a compound of formula I, or via a subdural pump inserted to administer the compound directly to the infarct area of the brain.
  • the patient would awaken from the coma more quickly than if the compound of the ATTORNEY DOCKET , . 23356-M8 invention were not administered.
  • the treatment is also expected to reduce the severity of the patient's residual neurological symptoms. In addition, it is expected that reoccurrence of vascular stroke would be reduced.
  • a patient is diagnosed with life-threatening cardio- myopathy and requires a heart transplant. Until a donor heart is found, the patient is maintained on Extra Corporeal Oxygenation Monitoring (ECMO) .
  • ECMO Extra Corporeal Oxygenation Monitoring
  • a donor heart is then located, and the patient undergoes a surgical transplant procedure, during which the patient is placed on a heart-lung pump.
  • the patient receives a compound of the invention intracardiac within a specified period of time prior to re-routing his or her circulation from the heart-lung pump to his or her new heart, thus preventing cardiac reperfusion injury as the new heart begins to beat.
  • Example 10 Septic Shock Assay Groups of 10 C57/BL male mice weighing 18 to 20 g were administered a test compound, 1-carboxynaphthalene-l- carboxamide at the doses of 60, 20, 6 and 2 mg/kg, daily, by intraperitoneal (IP) injection for three consecutive days. Each animal was first challenged with lipopolysaccharide (LPS, from E. Coli, LD 100 of 20 mg/animal IV) plus galactosamine (20 mg/animal IV) . The first dose of test compound in a suitable vehicle was given 30 minutes after challenge, and the second and third doses were given 24 hours later on day 2 and day 3 respectively, with only the surviving animals receiving the second or third dose of the test compound.
  • LPS lipopolysaccharide
  • galactosamine 20 mg/animal IV
  • the human prostate cancer cell line, PC-3s were plated in 6 well dishes and grown at monolayer cultures in RPMI1640 supplemented with 10% FCS. The cells are maintained at 37 °C in 5% C0 2 and 95% air. The cells were exposed to a dose response (0.1 mM to 0.1 ⁇ M) of 3 different PARP inhibitors of Formula I disclosed herein prior to irradiation at one sublethal dose level. For all treatment groups, the six well plates were exposed at room temperature in a Seifert 250kV/15mA irradiator with a 0.5 mm Cu/1 mm. Cell viability was examined by exclusion of 0.4% trypan blue.
  • Dye exclusion was assessed visually by microscopy and viable cell number was calculated by subtracting the number of cells from the viable cell number and dividing by the total number of cells.
  • Cell proliferation rates were calculated by the amount of 3 H-thymidine incorporation post-irradiation.
  • the PARP inhibitors show radiosensitization of the cells.
  • Example 12 In vivo Radiosensitization Before undergoing radiation therapy to treat cancer, a patient is administered an effective amount of a compound or a pharmaceutical composition of the present invention.
  • the compound or pharmaceutical composition acts as a radiosensitizer and making the tumor more susceptible to radiation therapy.
  • Human fibroblast BJ cells at Population Doubling (PDL) 94, are plated in regular growth medium and then changed to low serum medium to reflect physiological conditions described in
  • a medium of DMEM/199 wupplemented with 0.5% bovine calf serum is used.
  • the cells are treated daily for 13 days with the PARP inhibitor of Formula I as disclosed herein.
  • the control cells are treated with and without the solvent used to administer the
  • RNA is prepared from the treated and control cells according to the techniques described in PCT Publication No. 96/13610 and Northern blotting is conducted. Probes specific for senescence-related genes are analyzed, and treated and control cells compared. In analyzing the results, the lowest level of gene expression is arbitrarily set at 1 to provide a basis for comparison. Three genes particularly relevant to age-related changes in the skin are collagen, collagenase and elastin. West, Arch . Derm . 130:87-95 (1994). Elastin expression of the cells treated with the PARP inhibitor of Formula I is significantly increased in comparison with the control cells.
  • Elastin expression is significantly higher in young cells compared to senescent cells, and thus treatment with the PARP inhibitor of Formula I causes elastin expression levels in senescent cells to change to levels similar to those found in much younger cells. Similarly, a beneficial effect is seen in collagenase and collagen expression with treatment with the PARP inhibitors of Formula I.
  • Approximately 105 BJ cells, at PDL 95-100 are plated and grown in 15 cm dishes.
  • the growth medium is DMEM/199 supplemented with 10% bovice calf serum.
  • the cells are treated daily for 24 hours with the PARP inhibitors of Formula I (100 ⁇ g/ 1 mL of medium) .
  • the cells are washed with phosphate buffered solution (PBS), then permeablized with 4% paraformaldehyde for 5 minutes, then washed with PBS, and treated with 100% cold methanol for 10 minutes.
  • the methanol is removed and the cells are washed with PBS, and then treated with 10% serum to block nonspecific antibody binding.
  • Vector is added to the cells and the mixture incubated for 1 hour. The cells are rinsed and washed three times with PBS. A secondary antibody, goat anti-mouse IgG (1 mL) with a biotin tag is added along with 1 mL of a solution containing streptavidin conjugated to alkaline phosphatase and ATTORNEY DOCKET 0. 23356-M8
  • human fibroblast cells lines (either W138 at Population Doubling (PDL) 23 or BJ cells at PDL 71) are thawed and plated on T75 flasks and allowed to grow in normal medium (DMEM/M199 plus 10% bovine calf serum) for about a week, at which time the cells are confluent, and the cultures are therefor ready to be subdivided.
  • normal medium DMEM/M199 plus 10% bovine calf serum
  • the media is aspirated, and the cells rinsed with phosphate buffer saline (PBS) and then trypsinized.
  • PBS phosphate buffer saline
  • Treatment of cells with 10 ⁇ M 3-AB appears to have little or no effect in contrast to treatment with 100 ⁇ M 3-AB which appears lengthen the lifespan of the cells and treatment with 1 mM 3-AB which dramatically increases the lifespan and proliferative capacity of the cells.
  • the cells treated with 1 mM 3-AB will still divide after 60 days m culture.
  • Nerve ligation is performed by exposing one side of the rat's sciatic nerves and dissecting a 5-7 mm-long nerve segment and closing with four loose ligatures at a 1.0- 1.5-mm, followed by implanting of an intrathecal catheter and inserting of a gentamicin sulfate-flushed polyethylene (PE-10) tube into the subarachnoid space through an incision at the cisterna magna.
  • PE-10 gentamicin sulfate-flushed polyethylene
  • the caudal end of the catheter is gently threaded to the lumbar enlargement and the rostral end is secured with dental cement to a screw embedded in the skull and the skin wound is closed with wound clips.
  • Thermal hyperalgesia to radiant heat is assessed by using a paw-withdrawal test.
  • the rat is placed in a plastic cylinder on a 3-mm thick glass plate with a radiant heat source from a projection bulb placed directly under the plantar surface of the rat's hindpaw.
  • the paw-withdrawal latency is defined as the time elapsed from the onset of radiant heat stimulation to withdrawal of the rat's hindpaw.
  • Mechanical hyperalgesia is assessed by placing the rat in a cage with a bottom made of perforated metal sheet with many small square holes. Duration of paw-withdrawal is recorded after pricking the mid-plantar surface of the rat's hindpaw with the tip of a safety pin inserted through the cage bottom.
  • Mechano-allodynia is assessed by placing a rat in a cage similar to the previous test, and applying von Frey filaments in ascending order of bending force ranging from 0.07 to 76 g to the mid-plantar surface of the rat's hindpaw. A von Frey filament is applied perpendicular to the skin and depressed slowly until it bends. A threshold force of response is ATTORNEY DOCKET ..0. 23356-M8 defined as the first filament in .the series to evoke at least one .clear paw-withdrawal out of five applications.

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Abstract

L'invention concerne un composé de formule I: ou un sel, un hydrate, un ester, un produit de solvatation, un promédicament, un métabolite, un stéréoisomère, ou des mélanges pharmaceutiquement acceptables de celui-ci, Y représentant les atomes nécessaires pour former un cycle hétérocyclique condensé, aromatique ou non aromatique, à 5 ou 6 éléments, ce cycle renfermant au moins un hétéroatome d'azote dans une relation 1,3 par rapport à l'atome d'azote décrit dans la formule I, Y pouvant être substitué ou non par au moins un alkyle, un alkényle, un cycloalkyle, un cycloalkényle, un aralkyle, un aryle, ou un oxygène à double liaison, ou un groupe -COOR5, ou un fragment choisi dans le groupe composé par: dans lequel R5 et R7 représentent indépendamment hydrogène ou alkyle, alkényle, cycloalkyle, cycloalkényle, aralkyle, ou aryle.
PCT/US1998/018188 1997-09-03 1998-09-02 Composes di-n-heterocycliques, et procedes et compositions permettant d'inhiber l'activite de la parp WO1999011644A1 (fr)

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Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999059973A1 (fr) * 1998-05-15 1999-11-25 Guilford Pharmaceuticals Inc. Composes de carboxamide, compositions et methodes d'inhibition de l'activite de type poly(adp-ribose) polymerase (parp)
WO2000025787A1 (fr) * 1998-10-30 2000-05-11 Guilford Pharmaceuticals Inc. Compositions pharmaceutiques contenant des inhibiteurs de poly(adp-ribose) glycohydrolase et utilisations de ces compositions
US6235748B1 (en) 1997-09-03 2001-05-22 Guilford Pharmaceuticals Inc. Oxo-substituted compounds, process of making, and compositions and methods for inhibiting parp activity
WO2001023390A3 (fr) * 1999-09-28 2001-12-27 Basf Ag Derives d'azepinoindol, preparation et utilisation desdits derives
US6476048B1 (en) 1999-12-07 2002-11-05 Inotek Pharamaceuticals Corporation Substituted phenanthridinones and methods of use thereof
US6495541B1 (en) 1999-01-11 2002-12-17 Agouron Pharmaceuticals, Inc. Tricyclic inhibitors of poly(ADP-ribose) polymerases
US6531464B1 (en) 1999-12-07 2003-03-11 Inotek Pharmaceutical Corporation Methods for the treatment of neurodegenerative disorders using substituted phenanthridinone derivatives
WO2003020700A2 (fr) 2001-08-31 2003-03-13 Inotek Pharmaceuticals Corporation Derives d'indeno[1,2-c]isoquinoleine substitutee et procedes d'utilisation de ces composes
US6534651B2 (en) 2000-04-06 2003-03-18 Inotek Pharmaceuticals Corp. 7-Substituted isoindolinone inhibitors of inflammation and reperfusion injury and methods of use thereof
US6548494B1 (en) 1999-08-31 2003-04-15 Agouron Pharmaceuticals, Inc. Tricyclic inhibitors of poly(ADP-ribose) polymerases
EP1172360A4 (fr) * 1999-04-19 2004-02-04 Sankyo Co Potentialisateurs de la neurotrophine
US6956035B2 (en) 2001-08-31 2005-10-18 Inotek Pharmaceuticals Corporation Isoquinoline derivatives and methods of use thereof
WO2006078711A2 (fr) 2005-01-19 2006-07-27 Mgi Gp, Inc. Composes de diazabenzo[de]anthracene-3-one et utilisation dans l'inhibition de parp
US7122679B2 (en) 2000-05-09 2006-10-17 Cephalon, Inc. Multicyclic compounds and the use thereof
US7217709B2 (en) 2003-02-28 2007-05-15 Inotek Pharmaceuticals Corporation Tetracyclic benzamide derivatives and methods of use thereof
US7307080B2 (en) 1999-09-01 2007-12-11 Mgi Gp, Inc. Compounds, methods and pharmaceutical compositions for treating cellular damage, such as neural or cardiovascular tissue damage
US7381722B2 (en) 2005-02-25 2008-06-03 Inotek Pharmaceuticals Corporation Tetracyclic amino and carboxamido compounds and methods of use thereof
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US7652028B2 (en) 2005-08-24 2010-01-26 Inotek Pharmaceuticals Corporation Indenoisoquinolinone analogs and methods of use thereof
US8119654B2 (en) 2007-02-28 2012-02-21 Inotek Pharmaceuticals Corporation Indenoisoquinolinone analogs and methods of use thereof
WO2012071684A1 (fr) 2010-12-02 2012-06-07 Shanghai De Novo Pharmatech Co Ltd. Dérivés hétérocycliques, leurs procédés de préparation et leurs utilisations médicales
EP2842956A1 (fr) 2007-10-03 2015-03-04 Eisai Inc. Composés inhibiteurs de la PARP, compositions et procédés d'utilisation
US9150628B2 (en) 2005-11-14 2015-10-06 Centre National De La Recherche Scientifique (Cnrs) PARP inhibitors
WO2016094897A1 (fr) 2014-12-12 2016-06-16 The Jackson Laboratory Compositions et méthodes se rapportant au traitement de cancers, de maladies autoimmunes et de maladies neurodégénératives
WO2016138574A1 (fr) 2015-03-02 2016-09-09 Sinai Health System Facteurs de recombinaison homologue

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995024379A1 (fr) * 1994-03-09 1995-09-14 Newcastle University Ventures Limited Analogues de benzamides utiles en tant qu'inhibiteurs de l'enzyme parp (adp-ribosyltransferase, adprt) de reparation de l'adn
US5589483A (en) * 1994-12-21 1996-12-31 Geron Corporation Isoquinoline poly (ADP-ribose) polymerase inhibitors to treat skin diseases associated with cellular senescence

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995024379A1 (fr) * 1994-03-09 1995-09-14 Newcastle University Ventures Limited Analogues de benzamides utiles en tant qu'inhibiteurs de l'enzyme parp (adp-ribosyltransferase, adprt) de reparation de l'adn
US5589483A (en) * 1994-12-21 1996-12-31 Geron Corporation Isoquinoline poly (ADP-ribose) polymerase inhibitors to treat skin diseases associated with cellular senescence

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BANASIK M ET AL: "SPECIFIC INHIBITORS OF POLY(ADP-RIBOSE) SYNTHETASE AND MONO(ADP-RIBOSYL)TRANSFERASE", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 267, no. 3, 25 January 1992 (1992-01-25), pages 1569 - 1575, XP000574735 *
GRIFFIN R J ET AL: "NOVEL POTENT INHIBITORS OF THE DNA REPAIR ENZYME POLY(ADP-RIBOSE)POLYMERASE (PARP)", ANTI-CANCER DRUG DESIGN, vol. 10, no. 6, September 1995 (1995-09-01), pages 507 - 514, XP002065156 *
M.J.SUTO ET AL: "DIHYDROISOQUINOLINONES:THE DESIGN AND SYNTHESIS OF A NEW SERIES OF POTENT INHIBITORS OF POLY(ADP-RIBOSE)POLYMERASE", ANTI-CANCER DRUG DESIGN, vol. 6, no. 2, May 1991 (1991-05-01), pages 107 - 117, XP002086825 *

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US6235748B1 (en) 1997-09-03 2001-05-22 Guilford Pharmaceuticals Inc. Oxo-substituted compounds, process of making, and compositions and methods for inhibiting parp activity
WO1999059973A1 (fr) * 1998-05-15 1999-11-25 Guilford Pharmaceuticals Inc. Composes de carboxamide, compositions et methodes d'inhibition de l'activite de type poly(adp-ribose) polymerase (parp)
WO2000025787A1 (fr) * 1998-10-30 2000-05-11 Guilford Pharmaceuticals Inc. Compositions pharmaceutiques contenant des inhibiteurs de poly(adp-ribose) glycohydrolase et utilisations de ces compositions
US7429578B2 (en) 1999-01-11 2008-09-30 Agouron Pharmaceuticals, Inc. Tricyclic inhibitors of poly(ADP-ribose) polymerases
US6495541B1 (en) 1999-01-11 2002-12-17 Agouron Pharmaceuticals, Inc. Tricyclic inhibitors of poly(ADP-ribose) polymerases
US6977298B2 (en) 1999-01-11 2005-12-20 Agouron Pharmacetucals, Inc. Tricyclic inhibitors of poly(ADP-ribose) polymerases
EP1172360A4 (fr) * 1999-04-19 2004-02-04 Sankyo Co Potentialisateurs de la neurotrophine
US6548494B1 (en) 1999-08-31 2003-04-15 Agouron Pharmaceuticals, Inc. Tricyclic inhibitors of poly(ADP-ribose) polymerases
US7307080B2 (en) 1999-09-01 2007-12-11 Mgi Gp, Inc. Compounds, methods and pharmaceutical compositions for treating cellular damage, such as neural or cardiovascular tissue damage
WO2001023390A3 (fr) * 1999-09-28 2001-12-27 Basf Ag Derives d'azepinoindol, preparation et utilisation desdits derives
US6476048B1 (en) 1999-12-07 2002-11-05 Inotek Pharamaceuticals Corporation Substituted phenanthridinones and methods of use thereof
US6531464B1 (en) 1999-12-07 2003-03-11 Inotek Pharmaceutical Corporation Methods for the treatment of neurodegenerative disorders using substituted phenanthridinone derivatives
US6534651B2 (en) 2000-04-06 2003-03-18 Inotek Pharmaceuticals Corp. 7-Substituted isoindolinone inhibitors of inflammation and reperfusion injury and methods of use thereof
US6903079B2 (en) 2000-04-06 2005-06-07 Inotek Pharmaceuticals Corporation Nucleoside compounds and compositions thereof
EP2050750A2 (fr) 2000-05-09 2009-04-22 Cephalon, Inc. Composés multi-cycliques et leur utilisation comme inhibiteurs des enzymes PARP, VEGFR2 et MLK3
EP1754707A2 (fr) 2000-05-09 2007-02-21 Cephalon, Inc. Composés multicycliques et leur utilisation comme inhibiteurs des enzymes PARP, VEGFR2 et MLK3
US7122679B2 (en) 2000-05-09 2006-10-17 Cephalon, Inc. Multicyclic compounds and the use thereof
EP2174659A1 (fr) 2001-08-31 2010-04-14 Inotek Pharmaceuticals Corporation Dérivé d'indeno[1,2-c]-isoquinoléine substitutués et utilisation de ces composés
US7268143B2 (en) 2001-08-31 2007-09-11 Inotek Pharmaceuticals Corporation Isoquinoline derivatives and methods of use thereof
US7393955B2 (en) 2001-08-31 2008-07-01 Inotek Pharmaceuticals Corporation Isoquinoline derivatives and methods of use thereof
US6956035B2 (en) 2001-08-31 2005-10-18 Inotek Pharmaceuticals Corporation Isoquinoline derivatives and methods of use thereof
US6828319B2 (en) 2001-08-31 2004-12-07 Inotek Pharmaceuticals Corporation Substituted indeno[1,2-c]isoquinoline derivatives and methods of use thereof
WO2003020700A2 (fr) 2001-08-31 2003-03-13 Inotek Pharmaceuticals Corporation Derives d'indeno[1,2-c]isoquinoleine substitutee et procedes d'utilisation de ces composes
US7217709B2 (en) 2003-02-28 2007-05-15 Inotek Pharmaceuticals Corporation Tetracyclic benzamide derivatives and methods of use thereof
EP2033645A1 (fr) 2004-02-26 2009-03-11 Inotek Pharmaceuticals Corporation Dérivés d'isoquinoléine et leurs procédés d'utilisation
EP2319847A2 (fr) 2005-01-19 2011-05-11 Eisai Inc. Composés de diazabenzo[de]anthracene-3-one et méthodes pour l'inhibition de PARP
WO2006078711A2 (fr) 2005-01-19 2006-07-27 Mgi Gp, Inc. Composes de diazabenzo[de]anthracene-3-one et utilisation dans l'inhibition de parp
US7381722B2 (en) 2005-02-25 2008-06-03 Inotek Pharmaceuticals Corporation Tetracyclic amino and carboxamido compounds and methods of use thereof
US7652028B2 (en) 2005-08-24 2010-01-26 Inotek Pharmaceuticals Corporation Indenoisoquinolinone analogs and methods of use thereof
US9150628B2 (en) 2005-11-14 2015-10-06 Centre National De La Recherche Scientifique (Cnrs) PARP inhibitors
US8119654B2 (en) 2007-02-28 2012-02-21 Inotek Pharmaceuticals Corporation Indenoisoquinolinone analogs and methods of use thereof
EP2842956A1 (fr) 2007-10-03 2015-03-04 Eisai Inc. Composés inhibiteurs de la PARP, compositions et procédés d'utilisation
WO2012071684A1 (fr) 2010-12-02 2012-06-07 Shanghai De Novo Pharmatech Co Ltd. Dérivés hétérocycliques, leurs procédés de préparation et leurs utilisations médicales
US8999985B2 (en) 2010-12-02 2015-04-07 Shanghai De Novo Pharmatech Co Ltd. Substituted phthalazin-1(2H)-ones, preparation processes and medical uses thereof
WO2016094897A1 (fr) 2014-12-12 2016-06-16 The Jackson Laboratory Compositions et méthodes se rapportant au traitement de cancers, de maladies autoimmunes et de maladies neurodégénératives
WO2016138574A1 (fr) 2015-03-02 2016-09-09 Sinai Health System Facteurs de recombinaison homologue

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