WO2007051119A1 - Methods and compositions of parp inhibitors as potentiators in cancer therapy - Google Patents

Abstract

The present disclosure relates generally to a series of compounds which are derivatives of tricyclic compounds which inhibit poly (ADP-ribose) polymerase (PARP) and their use in the potentiation of cancer therapies. The disclosure relates more particularly to uses of azaphenanthridone compounds to chemosensitize cancers to the cytotoxic effects of chemopharmaceutical agents.

Description

COMPOSITIONS OF PARP INHIBITORS AND THEIR USE AS POTENTIATORS IN CANCER THERAPY

FIELD OF THE INVENTION

[0001] The present invention relates generally to a series of tricyclic compounds which inhibit poly (ADP-ribose) polymerase (PARP) and their use in the potentiation of cancer therapies. More particularly, the present disclosure relates to the use of azaphenanthridone compounds to chemosensitize tumors and cancers to the cytotoxic effects of chemotherapeutic agents.

BACKGROUND

[0002] PARP (EC 2.4.2.30), also known as PARS (for poly(ADP-ribose) synthetase), or ADPRT (for NADφrotein (ADP-ribosyl) transferase (polymerising)) is a major nuclear protein of 116 kDa. It is mainly present in almost all eukaryotes. The enzyme synthesizes poly( ADP-ribose), a branched polymer that can consist of over 200 ADP- ribose units from NAD. The protein acceptors of poly( ADP-ribose) are directly or indirectly involved in maintaining DNA integrity. They include histones, topoisomerases, DNA and RNA polymerases, DNA ligases, and Ca²⁺- and Mg⁺- dependent endonucleases. PARP protein is expressed at a high level in many tissues, most notably in the immune system, heart, brain and germ-line cells. Under normal physiological conditions, there is minimal PARP activity. However, DNA damage causes an immediate activation of PARP by up to 500-fold. Among the many functions attributed to PARP is its major role in facilitating DNA repair by ADP-ribosylation and therefore coordinating a number of DNA repair proteins. As a result of PARP activation, NAD levels significantly decline. While many endogenous and exogenous agents have been shown to damage DNA and activate PARP, peroxynitrite, formed from a combination of nitric oxide (NO) and superoxide, appears to be a main perpetrator responsible for various reported disease conditions in vivo, e.g., during shock, stroke and inflammation.

[0003] It is also known that PARP inhibitors, such as 3-amino benzamide, affect DNA repair generally in response, for example, to hydrogen peroxide or gamma- radiation. Cristovao et al., "Effect of a Poly(ADP-Ribose) Polymerase Inhibitor on DNA Breakage and Cytotoxicity Induced by Hydrogen Peroxide and γ-Radiation," Terato., Carcino., andMuta., 16, 219-27 (1996). Specifically, Cristovao et al. observed a PARP- dependent recovery of DNA strand breaks in leukocytes treated with hydrogen peroxide.

[0004] PAPvP inhibitors have been reported to be effective, as synergists or potentiators, in radiosensitizing hypoxic tumor cells. PARP inhibitors have also been reported to be effective as synergists in preventing tumor cells from recovering from potentially lethal damage of DNA after radiation therapy, presumably by their ability to prevent DNA repair. U.S. Patent Nos. 5,032,617; 5,215,738; and 5,041,653.

[0005] Temozolomide, a DNA methylating agent used to treat melanoma, induces DNA damage, which is repaired by O⁶-alkylguanine alkyltransferase (ATase) and poly(ADP-ribose) polymerase- 1 (PARP-l)-dependent base excision repair. Temozolomide is an orally available monofunctional DNA alkylating agent used to treat gliomas and malignant melanoma. Temozolomide is rapidly absorbed and undergoes spontaneous breakdown to form the active monomethyl triazene, 5-(3-methyl-l- triazeno)imidazole-4-carboxamide. Monomethyl triazene forms several DNA methylation products, the predominate species being N⁷-methylguanine (70%), N³- methyladenine (9%), and O⁶-methylguanine (5%). Unless repaired by O⁶-alkyIguanine alkyltransferase, O⁶-methylguanine is a cytotoxic lesion due to mispairing with thymine during DNA replication. This mispairing is recognized on the daughter strand by mismatch repair proteins and the thymine excised. However, unless the original O - methylguanine lesion is repaired by ATase-mediated removal of the methyl adduct, thymine can be reinserted. Repetitive futile rounds of thymine excision and incorporation opposite an unrepaired O⁶-methylguanine lesion causes a state of persistent strand breakage and the MutS branch of mismatch repair system signals G2-M cell cycle arrest and the initiation of apoptosis. The quantitatively more important N⁷- methylguanine and N³-methyladenine lesions formed by temozolomide are rapidly repaired by base excision repair. Plummer, et al., CHn. Cancer Res., 11(9), 3402 (2005).

[0006] There is considerable interest in the development of PARP inhibitors as both chemopotentiators and radiopotentiators for use in cancer therapy and to limit cellular damage after ischemia or endotoxic stress. In particular, potentiation of temozolomide cytotoxicity observed in preclinical studies with potent PARP-I inhibitors reflects inhibition of base excision repair and subsequent cytotoxicity due to incomplete processing of N⁷-methylguanine and N³-methyladenine. There is now a large body of preclinical data demonstrating that the cytotoxicity of temozolomide is potentiated by coadministration of a PARP inhibitor either in vitro or in vivo. Plummer, et al, CHn. Cancer Res., 11(9), 3402 (2005).

[0007] Chemosensitization by PARP inhibitors is not limited to temozolomide. Cytotoxic drugs, generally, or radiation can induce activation of PARP-I, and it has been demonstrated that inhibitors of PARP-I can potentiate the DNA damaging and cytotoxic effects of chemotherapy and irradiation. Canon K., et al, J. Med. Chem., 45, 4961 (2002). PARP-I mediated DNA repair in response to DNA damaging agents represents a mechanism of tumor resistance, and inhibition of this enzyme has been shown to enhance the activity of ionizing radiation and several cytotoxic antitumor agents, including temozolomide and topotecan. Suto et al. in U.S. Pat. No. 5,177,075 discuss several isoquinolines used for enhancing the lethal effects of ionizing radiation or chemotherapeutic agents on tumor cells. Weltin et al., "Effect of 6(5H)- Phenanthridinone, an Inhibitor of Poly(ADP-ribose) Polymerase, on Cultured Tumor Cells", Oncol. Res., 6, 399-403 (1994), discuss the inhibition of PARP activity, reduced proliferation of tumor cells, and a marked synergistic effect when tumor cells are co- treated with an alkylating drug. PARP-I is thus a potentially important therapeutic target for enhancing DNA-damaging cancer therapies.

[0008] The ability of PARP inhibitors to potentiate the lethality of cytotoxic agents, whether by radiosensitizing tumor cells to ionizing radiation, or by chemosensitizing tumor cells to the cytotoxic effects of chemotherapeutic agents has been reported in, inter alia., US 2002/0028815; US 2003/0134843; US 2004/0067949; White A., et al, 14 Bioorg. & Med. Chem Letts., 2433 (2004); Canon K., et al, J. Med. Chem. 45, 4961 (2002); Skalitsky D., et al, J. MeJ. Chem., 46, 210 (2003); Farmer H, et al, Nature, 434, 917 (April 2005); Plummer E., et al, Clin. Cancer Res. 11, 3402 (2005); Tikhe J., et al, J. Med. Chem. 47, 5467 (2004); International Publication No. WO 98/33802; and International Publication No. WO 2005/012305. [0009] There continues to be a need for effective and potent PARP inhibitors which enhance the lethal effects of ionizing radiation and/or chemotherapeutic agents on tumor cells while producing minimal side-effects.

SUMMARY OF INVENTION

[0010] The present invention provides azaphenanthridone compounds described herein, derivatives thereof and their uses to inhibit poly(ADP-ribose) polymerase ("PARP"), compositions containing these compounds and methods for making and using these PARP inhibitors to treat, prevent and/or ameliorate the effects of cancers by potentiating the cytotoxic effects of ionizing radiation and/or chemotherapeutic agents on tumor cells.

[0011] The present invention provides a chemosensitization method for treating cancers and or tumors comprising contacting the tumor or cancer cells with a cytotoxicity-potentiating azaphenanthridone compound and further contacting the tumor or cancer cells with an anticancer agent.

[0012] An aspect of the present invention provides a chemosensitization method to treat tumors and/or cancer in an animal, comprising administering to said animal an azaphenanthridone compound of formula I

wherein

R₁, R₂ and R₃ are independently selected from H, halogen, amino, -OH, optionally substituted alkyl, alkenyl, alkynyl, alkoxy, -Obenzyl, cycloalkyl, aryl, heterocyclyl, -NR5R0, and -NR₅COR₇, wherein R₅ and R₆ are each independently selected from hydrogen, optionally substituted alkyl, cycloalkyl, aryl, and heterocyclyl, and

R₇ is selected from an optionally substituted alkyl, cycloalkyl, aryl, and heterocyclyl, and

R₄ is independently selected from hydrogen, halogen, alkoxy, and alkyl.

[0013] In one aspect of the invention, R₄ is halogen. In another aspect, only one R₄ is present on the ring.

[0014] In another aspect of the invention, R₂, R₃, and R₄ are each hydrogen.

[0015] In another aspect of the invention, R₂, R₃, and R₄ are each hydrogen and Ri is an optionally substituted heterocyclyl or -NR₅R₆, wherein R₅ and R₆ are independently hydrogen or an optionally substituted alkyl.

[0016] hi another aspect, the present invention provides a chemosensitization method for treating cancers in a mammal, particularly a human, comprising administering to the mammal an azaphenanthridone compound selected from:

[0017] According to an aspect of the invention, the azaphenanthridone compound used in the chemosensitization method is:

[0018] According to an aspect of the invention, the azaphenanthridone compound used in the chemosensitization method is:

[0019] An aspect of the present invention provides a chemosensitization method wherein a first dose of at least one azaphenanthridone compound of Formula I is administered singly or repeatedly to a patient in need thereof, and wherein subsequently a second dose of at least one chemotherapeutic agent is administered singly or repeatedly to said patient after a time period to provide an effective amount of chemosensitizatioa

[0020] An aspect of the present invention provides a pharmaceutical formulation comprising a chemosensitizing azaphenanthridone derivative of Formula I in a form selected from the group consisting of pharmaceutically acceptable free bases, salts, hydrates, esters, solvates, prodrugs, metabolites, stereoisomers, and mixtures thereof. According to a further aspect, the pharmaceutical formulation further comprises a pharmaceutically acceptable carrier and, optionally, a chemotherapeutic agent. Non- limiting examples of such chemotherapeutic agents are recited below.

[0021] According to additional aspects of the invention, the chemosensitizing azaphenanthridone compound and the chemotherapeutic agent are administered essentially simultaneously.

[0022] According to an aspect of the invention, the chemotherapeutic agent is selected from the group consisting of temozolomide, adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, docetaxel, doxorubicin, interferon (alpha, beta, gamma), interleukin 2, irinotecan, paclitaxel, a taxoid, dactinomycin, danorubicin, 4'- deoxydoxorubicin, bleomycin, pilcamycin, mitomycin, neomycin and gentamycin, etoposide, 4-OH cyclophosphamide, a platinum coordination complex, topotecan, therapeutically effective analogs and derivatives of the same, and mixtures thereof. According to a preferred aspect, the chemotherapeutic agent is temozolomide. [0023] An aspect of the present invention provides a method of using an azaphenanthridone compound of Formula I in the manufacture of a medicament for the treatment of cancer.

[0024] An aspect of the present invention provides a medical device comprising a drag delivering or eluting member; and an azaphenanthridone compound of Formula I disposed on or within said member. According to a further aspect of the invention, the drug delivering or eluting member is selected from the group consisting of a shunt, a colostomy bag attachment device, an ear drainage tube, a lead for a pace maker, a lead for an implantable defibrillator, a suture, a staple, an anastomosis device, a vertebral disk, a bone pin, a suture anchor, a hemostatic barrier, a clamp, a screw, a plate, a clip, a vascular implant, a tissue adhesive, a tissue sealant, a tissue scaffold, a bone substitute, an intraluminal device, a stent, or a vascular support.

[0025] According to an aspect, the present invention provides a pharmaceutical composition comprising a potentiation-effective amount of at least one azaphenanthridone compound of Formula I. According to a further aspect, the potentiation action of the azaphenanthridone compound radiosensitizes tumor cells to the cytotoxic effects of ionizing radiation. According to a further aspect, the potentiation action of the azaphenanthridone compound chemosensitizes tumor cells to the cytotoxic effects of chemotherapeutic agents.

[0026] According to an aspect of the present invention, the pharmaceutical composition comprising a cytotoxicity-potentiation agent of Formula I further comprises a chemotherapeutically effective amount of at least one chemotherapeutic agent. According to a further aspect, the chemotherapeutic agent is selected from the group consisting of temozolomide, adriamycin, camptothecin, carboplatm, cisplatin, daunorubicin, docetaxel, doxorubicin, interferon (alpha, beta, gamma), interleukin 2, irinotecan, paclitaxel, topotecan, therapeutically effective analogs and derivatives of the same, and mixtures thereof. According to a preferred aspect, the chemotherapeutic agent is temozolomide.

[0027] Still other aspects and advantages of the present invention will become readily apparent to those skilled in the art from the following detailed description, wherein are shown and described preferred embodiments of the invention, simply by way of illustration of the best mode contemplated for carrying out the invention. As will be realized the invention is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, without departing from the invention. Accordingly, the description is to be regarded as illustrative in nature and not as restrictive.

DETAILED DESCRIPTION OF THE INVENTION.

[0028] The present invention provides azaphenanthridone compounds described herein, derivatives thereof and their uses to inhibit poly(ADP-ribose) polymerase ("PARP"), compositions containing these compounds and methods for using these compounds to treat, prevent and/or ameliorate the effects of cancers by potentiating the cytotoxic effects of ionizing radiation and/or chemotherapeutic agents on tumor cells.

[0029] The term "alkyl", as used herein, unless otherwise indicated, includes saturated monovalent hydrocarbon radicals having straight, branched, or cyclic moieties (including fused and bridged bicyclic and spirocyclic moieties), or a combination of the foregoing moieties. For an alkyl group to have cyclic moieties, the group must have at least three carbon atoms.

[0030] The term "alkenyl", as used herein, unless otherwise indicated, includes alkyl moieties having at least one carbon-carbon double bond wherein alkyl is as defined above and including E and Z isomers of said alkenyl moiety.

[0031] The term "alkynyl", as used herein, unless otherwise indicated, includes alkyl moieties having at least one carbon-carbon triple bond wherein alkyl is as defined above. [0032] The term "alkoxy", as used herein, unless otherwise indicated, includes O-alkyl groups wherein alkyl is as defined above.

[0033] The term "Me" means methyl, "Et" means ethyl, and "Ac" means acetyl. [0034] The term "cycloalkyl", as used herein, unless otherwise indicated refers to a non-aromatic, saturated or partially saturated, monocyclic or fused, spiro or unfused bicyclic or tricyclic hydrocarbon referred to herein containing a total of from 3 to 10 carbon atoms, preferably 5-8 ring carbon atoms. Exemplary cycloalkyls include monocyclic rings having from 3-7, preferably 3-6, carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like. Illustrative examples of cycloalkyl are derived from, but not limited to, the following:

[0035] The term "aryl", as used herein, unless otherwise indicated, includes an organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, such as phenyl or naphthyl.

[0036] The term "heterocyclic" or "heterocyclyl" as used herein, unless otherwise indicated, includes aromatic and non-aromatic heterocyclic groups containing one to four heteroatoms each selected from O, S and N, wherein each heterocyclic group has from 4-10 atoms in its ring system, and with the proviso that the ring of said group does not contain two adjacent O or S atoms. Non-aromatic heterocyclic groups include groups having only 4 atoms in their ring system, but aromatic heterocyclic groups must have at least 5 atoms in their ring system. The heterocyclic groups include benzo-fused ring systems. An example of a 4 membered heterocyclic group is azetidinyl (derived from azetidine). An example of a 5 membered heterocyclic group is thiazolyl and an example of a 10 membered heterocyclic group is quinolinyl. Examples of non-aromatic heterocyclic groups are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepmyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydroρyridinyl, 2-ρyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-indolyl and quinolizinyl. Examples of aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. The foregoing groups, as derived from the groups listed above, may be C-attached or N-attached where such is possible. For instance, a group derived from pyrrole maybe pyrrol-1-yl (N-attached) or pyrrol-3-yl (C- attached). Further, a group derived from imidazole may be imidazol-1-yl (N-attached) or imidazol-3-yl (C-attached). The 4-10 membered heterocyclic may be optionally substituted on any ring carbon, sulfur, or nitrogen atom(s) by one to two oxo, per ring. An example of a heterocyclic group wherein 2 ring carbon atoms are substituted with oxo moieties is 1,1-dioxo-thiomorpholinyl. Other illustrative examples of 4-10 membered heterocyclic are derived from, but not limited to, the following:

[0037] The term "preventing" refers to the ability of a compound or composition of the invention to prevent a disease identified herein in patients diagnosed as having the disease or who are at risk of developing such disease. The term also encompasses preventing further progression of the disease in patients who are already suffering from or have symptoms of such disease.

[0038] The term "patient" or "subject" means an animal (e.g., cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, guinea pig, etc.) or a mammal, including chimeric and transgenic animals and mammals.

[0039] The term "in combination" refers to the use of more than one prophylactic and/or therapeutic agents simultaneously or sequentially and in a manner that their respective effects are additive or synergistic. [0040] The term "treating" refers to:

(i) preventing a disease, disorder, or condition from occurring in an animal that may be predisposed to the disease, disorder and/or condition, but has not yet been diagnosed as having it;

(ii) inhibiting the disease, disorder, or condition, i.e., arresting its development; and

(iii) relieving the disease, disorder, or condition, i.e., causing regression of the disease, disorder, and/or condition.

[0041] Another aspect of the present invention provides chemosensitization methods to treat cancerous and/or tumor cells in an animal, comprising administering to said animal a pharmaceutical formulation which comprises the azaphenanthridone compound of formula I.

[0042] Optional substituents as described herein include halogen, amino, hydroxyl, nitro, alkylamino, alkoxy, aryloxy, arylalkyl (e.g., benzyl), haloalkyl, alkyl, cycloalkyl, aryl, and heterocyclyl, where each of these in turn are optionally substituted by halogen, haloalkyl, alkyl, and heterocyclyl.

[0043] In an embodiment, the chemosensitizing compounds of the present invention maybe administered to treat cancers including, but not limited to: 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, Ewing's sarcoma, gallbladder cancer, hairy cell leukemia, head and neck cancer, Hodgkm's lymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer (small and/or non-small cell), malignant peritoneal effusion, malignant pleural effusion, melanoma, mesothelioma, multiple myeloma, neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovarian cancer, ovary (germ cell) cancer, prostrate 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.

[0044] In an embodiment, at least one chemotherapeutic agent is administered to treat the cancer. The chemotherapeutic agent may be administered essentially simultaneously with the compound of formula I. Preferably, the chemotherapeutic agent is administered after administering the compound of formula I and after a sufficient time to permit an optimum chemosensitization. The chemotherapeutic agent may include, but is not limited to: temozolomide, adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, docetaxel, doxorubicin, interferon (alpha, beta, gamma), interleukin 2, irinotecan, paclitaxel, topotecan, therapeutically effective analogs and derivatives of the same, and mixtures thereof.

[0045] A preferred chemotherapeutic agent is temozolomide. [0046] The invention further provides compositions, preferably pharmaceutical compositions, which contains a pharmaceutically acceptable carrier or diluent and at least one compound disclosed herein.

[0047] Preferably, the compounds of the invention exhibit an IC_5O for inhibiting PARP in vitro, as measured by the methods described herein, of about 100 niicromolar (μM), or less, preferably less than about 50 micromolar, more preferably less than about 10 micromolar, or less than 1 micromolar, most preferably less than about 0.1 micromolar.

[0048] A convenient method to determine IC₅₀ of a PARP inhibitor compound is a PARP assay using purified recombinant human PARP from Trevigan (Gaithersburg, Md.), as follows: The PARP enzyme assay is set up on ice in a volume of 100 microliters consisting of 100 mM Tris-HCl (pH 8.0), 1 mM MgCl₂, 28 mM KCl, 28 mM NaCl, 3.0 μg/ml of DNase I-activated herring sperm DNA (Sigma, Mo.), 30 micromolar [³H]nicotinamide adenine dinucleotide (62.5 mci/mmole), 15 micrograms/ml PARP enzyme, and various concentrations of the compounds to be tested. The reaction is initiated by adding enzyme and incubating the mixture at 25⁰C. After 2 minutes of incubation, the reaction is terminated by adding 500 microliters of ice cold 30% (w/v) trichloroacetic acid. The precipitate formed is transferred onto a glass fiber filter (Packard Unifilter-GF/C) and washed three times with 70% ethanol. After the filter is dried, the radioactivity is determined by scintillation counting. The compounds of this invention were found to have potent enzymatic activity in the range of a few nanomolar to 20 micromolar in IC₅₀ in this inhibition assay.

[0049] Specific embodiments of the present invention include the azaphenanthridone compounds shown and neutral and/or salt forms thereof, as well as enantiomer and racemic mixtures thereof, where appropriate.

[0050] Broadly, in addition to being useful in the chemosensitization methods disclosed herein, the compounds and compositions of the present invention can be used to treat or prevent cell damage or death due to necrosis or apoptosis, cerebral ischemia and reperfusion injury or neurodegenerative diseases in an animal, such as a human. The compounds and compositions of the present invention can be used to extend the lifespan and proliferative capacity of cells and thus can be used to treat or prevent diseases associated therewith; they alter gene expression of senescent cells; and they radiosensitize hypoxic tumor cells. Preferably, the compounds and compositions of the invention can be used to 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. The compounds of the present invention are not limited to being useful in treating glutamate mediated neurotoxicity and/or NO-mediated biological pathways. Further, the compounds of the invention can be used to treat or prevent other tissue damage related to PARP activation, as described herein.

[0051] The present invention provides compounds which inhibit the in vitro and/or in vivo polymerase activity of poly(ADP-ribose) polymerase (PAJRP), and compositions containing the disclosed compounds.

[0052] The present invention provides methods to inhibit, limit and/or control the in vitro and/or in vivo polymerase activity of poly(ADP-ribose) polymerase (PARP) in any of solutions, cells, tissues, organs or organ systems. In one embodiment, the present invention provides methods of limiting or inhibiting PARP activity in a mammal, such as a human, either locally or systemically.

[0053] The compounds of the present invention can also be used to extend or increase the lifespan or proliferation of cells and 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 muscular 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 tumor 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 muscular degeneration, AIDS and other immune senescence diseases, inflammation, arthritis, gout, atherosclerosis, cachexia, cancer, degenerative diseases of skeletal muscle involving replicative senescence, diabetes, head trauma, immune senescence, gout, 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.

[0054] Preferably, the compounds of the invention act as PARP inhibitors to treat or prevent cancers by chemopotentiating the cytotoxic or cell cycle arrest-inducing effects of other chemotherapeutic agents.

[0055] The compounds of the present invention may possess one or more asymmetric center(s) and thus can be produced as mixtures (racemic and non-racemic) of stereoisomers, or as individual enantiomers or diastereomers. The individual stereoisomers may be obtained by using an optically active staring material, by resolving a racemic or non-racemic mixture of an intermediate at some appropriate stage of the synthesis, or by resolution of the compound of Formula I. It is understood that the individual stereoisomers as well as mixtures (racemic and non-racemic) of stereoisomers are encompassed by the scope of the present invention.

[0056] The compounds of the invention are 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.

[0057] "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- hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, tosylate and undecanoate. Inorganic acids can be used to produce salts, hydrates, esters, or solvates such as hydrochloride, hydrobromide, hydroiodide, and thiocyanate.

[0058] Examples of 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.

[0059] 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. For purposes of illustration, the class of such organic bases may include mono-, di-, and trialkylamines, such as methylamine, dimethylamine, triethylamine and dicyclohexylarnine; mono-, di- or trihydroxyalkylamines, such as mono-, di-, and triethanolamine; amino acids, such as argmine 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. ScL, 66:1, 1-19 (1977). Accordingly, basic nitrogen-containing groups can be quaternized with agents including: lower alkyl halides 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.

[0060] The acid addition salts, hydrates, esters, or solvates of the basic compounds may be prepared either by dissolving the free base of a compound of the present invention 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. Alternatively, the free base of the compound of the present invention can be reacted with an acid, as well as reacting the compound 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.

[0061] "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 Burgers Medicinal Chemistry and Drug Chemistry, Fifth Ed, Vol. 1, pp. 172-178, 949-982 (1995). For example, the inventive compounds can be transformed into prodrugs by converting one or more of the hydroxy or carboxy groups into esters.

[0062] "Pharmaceutically acceptable metabolite" refers to drugs that have undergone a metabolic transformation. After entry into the body, most drugs are substrates for chemical reactions that may change their physical properties and biologic effects. These metabolic conversions, which usually affect the polarity of the compound, alter the way in which drugs are distributed in and excreted from the body. However, in some cases, metabolism of a drug is required for therapeutic effect. For example, anticancer drugs of the antimetabolite class must be converted to their active forms after they have been transported into a cancer cell. Since most drugs undergo metabolic transformation of some kind, the biochemical reactions that play a role in drug metabolism may be numerous and diverse. The main site of drug metabolism is the liver, although other tissues may also participate.

[0063] Further still, the methods of the invention can be used to treat cancer and to chemosensitize tumor cells. The term "cancer," as used herein, is defined broadly. The compounds of the present invention can potentiate the effects of "anti-cancer agents," which term also encompasses "anti-tumor cell growth agents," "chemotherapeutic agents," "cytostatic agents," "cytotoxic agents," "anti-neoplastic agents," and the like, as will be appreciated by those skilled in the art. "Chemosensitization," as used herein, refers to the ability of the compounds of the invention to potentiate the antitumoral activity of chemotherapeutic agents. Such chemosensitization is useful, for example, to increase the tumor growth-retarding or -arresting effect of a given dose of a chemotherapeutic agent, or to improve the side-effect profile of a chemotherapeutic agent by allowing for reductions in its dose while maintaining its antitumoral efficacy. For example, 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, Ewing'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), 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.

[0064] Pharmaceutical Compositions of the Invention

[0065] The present invention also relates to a pharmaceutical composition comprising (i) a therapeutically effective amount of a compound of Formula I and (ii) a pharmaceutically acceptable carrier.

[0066] The above discussion relating to the preferred embodiments' utility and administration of the compounds of the present invention also applies to the pharmaceutical composition of the present invention.

[0067] The term "pharmaceutically acceptable carrier" as used herein refers to any carrier, diluent, excipient, suspending agent, lubricating agent, adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent, preservative, surfactant, colorant, flavorant, or sweetener. [0068] For these purposes, the composition of the invention may be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, bucally, vaginally, intraventricularly, via an implanted reservoir in dosage formulations containing conventional non-toxic pharmaceutically-acceptable carriers, or by any other convenient dosage form. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal intraventricular, intrasternal, and intracranial injection or infusion techniques.

[0069] When administered parenterally, the composition will normally be in a unit dosage, sterile injectable form (solution, suspension or emulsion) which is preferably isotonic with the blood of the recipient with a pharmaceutically acceptable carrier. Examples of such sterile injectable forms are sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable forms may also be sterile injectable solutions or suspensions in non-toxic parenterally-acceptable diluents or solvents, for example, as solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, saline, Ringer's solution, dextrose solution, isotonic sodium chloride solution, and Hanks' solution. In addition, sterile, fixed oils are conventionally employed as solvents or suspending mediums. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides, corn, cottonseed, peanut, and sesame oil. Fatty acids such as ethyl oleate, isopropyl myristate, and oleic acid and its glyceride derivatives, including olive oil and castor oil, especially in their polyoxyethylated versions, are useful in the preparation of injectables. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants.

[0070] 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.

[0071] Formulations suitable for nasal or buccal administration (such as self- propelling powder dispensing formulations) may comprise about 0.1% to about 5% w/w, for example 1% w/w of active ingredient. The formulations for human medical use of the present invention comprise an active ingredient in association with a pharmaceutically acceptable carrier therefore and optionally other therapeutic ingredient(s).

[0072] When administered orally, the composition will usually be formulated into unit dosage forms such as tablets, cachets, powder, granules, beads, chewable lozenges, capsules, liquids, aqueous suspensions or solutions, or similar dosage forms, using conventional equipment and techniques known in the art. Such formulations typically include a solid, semisolid, or liquid carrier. Exemplary carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, alginates, tragacanth, gelatin, syrup, methyl cellulose, polyoxyethylene sorbitan monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and the like.

[0073] The composition of the invention is preferably administered as a capsule or tablet containing a single or divided dose of the compound of Formula I. Preferably, the composition is administered as a sterile solution, suspension, or emulsion, in a single or divided dose. Tablets may contain carriers such as lactose and corn starch, and/or lubricating agents such as magnesium stearate. Capsules may contain diluents including lactose and dried corn starch.

[0074] 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.

[0075] The compounds of this invention may also be administered rectally in the form of suppositories. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at room temperature, but liquid at rectal temperature, and, therefore, will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycols. [0076] Compositions and methods of the invention also may utilize controlled release technology. Thus, for example, the inventive compounds may be incorporated into a hydrophobic polymer matrix for controlled release over a period of days. The composition of the invention may then be molded into a solid implant, or externally applied patch, suitable for providing efficacious concentrations of the compounds of Formula I over a prolonged period of time without the need for frequent re-dosing. Such controlled release films are well known to the art. Particularly preferred are transdermal delivery systems. Other examples of polymers commonly employed for this purpose that may be used in the present invention include nondegradable ethylene- vinyl acetate copolymer an degradable lactic acid-glycolic acid copolymers which may be used externally or internally. Certain hydrogels such as poly(hydroxyethylmethacrylate) or poly(vinylalcohol) also may be useful, but for shorter release cycles than the other polymer release systems, such as those mentioned above.

[0077] In a preferred embodiment, 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 re-dosing. The composition 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

[0078] In one embodiment, 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 weight. By using a polymer composition in injectable form, even the need to make an incision may be eliminated. In any event, a flexible or flowable delivery "depot" will adjust to the shape of the space it occupies with the body with a minimum of trauma to surrounding tissues. The pharmaceutical composition of the present invention is used in amounts that are therapeutically effective, and 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.

[0079] The compounds of the invention are used in the composition in amounts that are therapeutically effective. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, welling, or emulsifying agents, solution promoters, salts for regulating the osmotic pressure, and/or buffers. In addition, they may also contain other therapeutically valuable substances, such as, without limitation, the specific chemotherapeutic agents recited herein. The compositions are prepared according to conventional mixing, granulating, or coating methods, and contain about 0.1 to 75% by weight, preferably about 1 to 50% by weight, of the compound of the invention.

[0080] To be effective therapeutically as central nervous system targets, the compounds of the present invention should readily penetrate the blood-brain barrier when peripherally administered. Compounds which cannot penetrate the blood-brain barrier can be effectively administered by an intraventricular route or other appropriate delivery system suitable for administration to the brain.

[0081] For medical use, the amount required of the active ingredient to achieve a therapeutic effect will vary with the particular compound, the route of administration, the mammal under treatment, and the particular disorder or disease being treated. A suitable systemic dose of a compound of the present invention or a pharmacologically acceptable salt thereof for a mammal suffering from, or likely to suffer from, any of condition as described hereinbefore is in the range of about 0.1 mg/kg to about 100 mg/kg of the active ingredient compound, the most preferred dosage being about 1 to about 10 mg/kg.

[0082] It is understood, however, that a specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated and form of administration.

[0083] It is understood that the ordinarily skilled physician or veterinarian will readily determine and prescribe the effective amount of the compound for prophylactic or therapeutic treatment of the condition for which treatment is administered. In so proceeding, the physician or veterinarian can, for example, employ an intravenous bolus followed by an intravenous infusion and repeated administrations, parenterally or orally, as considered appropriate. While it is possible for an active ingredient to be administered alone, it is preferable to present it as a formulation.

[0084] When preparing dosage form incorporating the compositions of the invention, the compounds may also be blended with conventional excipients such as binders, including gelatin, pregelatinized starch, and the like; lubricants, such as hydrogenated vegetable oil, stearic acid, and the like; diluents, such as lactose, mannose, and sucrose; disintegrants, such as carboxymethylcellulose and sodium starch glycolate; suspending agents, such as povidone, polyvinyl alcohol, and the like; absorbants, such as silicon dioxide; preservatives, such as methylparaben, propylparaben, and sodium benzoate; surfactants, such as sodium lauryl sulfate, polysorbate 80, and the like; colorants such as F.D.& C. dyes and lakes; flavorants; and sweeteners.

[0085] The present invention relates to the use of a compound of Formula I in the preparation of a medicament for the treatment of any disease or disorder in an animal described herein. In an embodiment, the compounds of the present invention are used to treat cancer. In a preferred embodiment, the compounds of the present invention are used to potentiate the cytotoxic effects of ionizing radiation. In such an embodiment, the compounds of the present invention act as a radiosensitizer. In an alternative preferred embodiment, the compounds of the present invention are used to potentiate the cytotoxic effects of chemotherapeutic agents. In such an embodiment, the compounds of the present invention act as a chemosensitizer.

[0086] Any pharmacologically-acceptable chemotherapeutic agent that acts by damaging DNA is suitable as the chemotherapeutic agent of the present invention. In particular, the present invention contemplates the use of a chemotherapeutically effective amount of at least one chemotherapeutic agent including, but not limited to: temozolomide, adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, docetaxel, doxorubicin, interferon (alpha, beta, gamma), interleukin 2, irinotecan, paclitaxel, topotecan, therapeutically effective analogs and derivatives of the same, and mixtures thereof. According to a preferred aspect, the chemotherapeutic agent is temozolomide.

[0087] The disclosure contained herein demonstrates the usefulness of the compounds and compositions of the present invention in treating and/or preventing cancer, such as by radiosensitizing and/or chemosensitizing tumor and/or cancer cells to chemotherapeutic agents.

[0088] The compounds of the present invention can be synthesized by methods disclosed in US 6,887,996 and in US 2005/0148575 (which are incorporated herein by reference) from starting materials described therein.

[0089] Compounds of the general formula I may be synthesized, for example, by the following methods.

Scheme 1 : General Scheme for Synthesis of Azaphenanthridones 4a-c. [0090] General procedure for the synthesis of amines 3a-c. The boronic acid 1 (2.0 g, 8.0 mmol) prepared according to Brimble, M. A.; Chan, S. H. Aust. J. Chem. 1998, 51, 235-242 was added to a solution of potassium carbonate (2.2 g in 8 mL H₂O) and 2- chloro-3-amino pyridine (0.94 g, 7.3 mmol) in 100 mL toluene/EtOH (8:1). This mixture was deoxygenated in vacuo and refilled with nitrogen. After stirring the mixture under nitrogen for 30 min, palladium tetrakistriphenylphosphine (250 mg) was added to the mixture. The solution was heated to 80° C. until complete conversion according to TLC (50/50 Hexanes/EtOAc). The reaction was then extracted with water and the toluene layer was dried and concentrated to yield a crude solid which was triturated with diethyl ether (10-20 mL) to yield 1.84 g (85%) of the desired amine 3a. ¹H NMR (CDCl₃) δ 8.05 (d, IH), 7.45 (m, 3H), 7.28 (d, IH), 7.06 (m, IH), 7.00 (d, IH), 4.01 (s, 2H), 3.78 (m, IH), 3.31 (m, IH), 1.48 (d, 3H), 1.13 (d, 3H), 1.01 (d, 3H), 0.84 (d, 3H).

[0091 J R=5-chloro amine 3b. Amide 3b was synthesized from 3-amino-2,5-dichloro- pyridine 2b (X=Cl, R=5-C1) as stated above with the exception of purification by flash chromatography on the minimum amount of silica gel (10% EtOAc/Hexanes→- 50% EtOAc/Hexanes). Dry yield was 1.40 g (70%). ¹H NMR (CDCl₃) δ 7.98 (s, IH), 7.45 (m, 2H), 7.26 (m, 2H), 7.00 (d, IH), 3.76 (m, IH), 3.35 (m, IH), 1.48 (d, 3H), 1.19 (d, 3H), 1.04 (d, 3H), 0.91 (d, 3H).

[0092] R=6-methoxy amine 3c. Amide 3c was synthesized from 3-amino-2-bromo-6- methoxypyridine 2c and boronic acid 1 as stated above. The dry yield was 74%. ¹H NMR (CDCl₃) δ 7 A3 (m, 3H, 7.27 (m, IH), 7.08 (d, IH), 6.61 (d, IH), 3.83 (s, 3H), 3.70 (m, IH), 3.30 (m, IH), 1.49 (d, 3H), 1.15 (d, 3H), 0.99 (d, 3H), 0.75 (d, 3H).

4a.

[0093] Azaphenanthridone 4a. The amide 3a (1.74 g, 5.8 mmol) was dissolved in dry tetrahydrofuran (25 mL) and cooled to -78° C. under nitrogen. Lithium diisopropylamide (2.0 M, 7.6 mL) was added to dropwise to the solution and this mixture was stirred for several hours and warmed to room temperature overnight. The reaction was quenched with water (50 mL) and extracted with 10% MeOH/DCM. The combined organics were dried and concentrated to yield the crude solid which was triturated with boiling diethyl ether to yield the pure material 4a 0.95 g (89%). Mp=300-320° C. (dec); ¹H NMR (d₆- DMSO) δ 11.78 (s, IH), 8.77 (d, IH), 8.55 (d, IH), 8.32 (d, IH), 7.93 (d, IH), 7.74 (m, 2H), 7.54 (m, IH). Anal Calcd. for Ci₂H₂₈N₂O: C, 73.46; H, 4.11; N, 14.28. Found: C, 72.80; H, 4.19; N, 14.06.

4b

[0094] Chloroazaphenanthridone 4b. Chloride 4b was made in an analogous manner to compound 4a. Yield=74%; mp=295-300° C; ¹H NMR (d₆-DMSQ) δ 11.80 (bs, IH), 8.69 (d, IH), 8.56 (s, IH), 8.31 (d, IH), 7.94 (t, IH), 7.78 (m, 2H). Anal Calcd. for C₁₂H₇ClN₂O: C, 62.49; H, 3.06; N, 12.15. Found: C, 61.53; H, 3.21; N, 11.87.

4c

[0095] Methoxyazaphenanthridone 4c. Compound 4c was made from amide 3c in a similar manner to 4a. Yield 99%; mp=290-300° C; ¹H NMR (de-DMSO) δ 11.67 (bs, IH), 8.69 (d, IH), 8.30 (d, IH), 7.93 (t, IH), 7.72 (m, 2H), 7.03 (d, IH)₃ 4.01 (s, 3H). Anal Calcd. for C₁₃Hi₀N₂O₂: C, 69.02; H, 4.46; N, 12.38 Found: C, 67.89; H, 4.49; N, 12.08.

4d

[0096] Hydroxyazaphenanthridone 4d. The methyl ether 4c (500 mg, 2.2 mmol) was dissolved in 10 mL HBr (48% in HOAc) in a sealed tube. The reaction was heated to 100° C. for 1O h. After cooling, the reaction was filtered and washed with acetic acid (3x10 mL) and dried in vacuo. The dry weight of the hydrobromide salt 4d was 421 mg, (90%). 1H NMR (d₆-DMSO) δ 11.61 (bs, IH), 10.50 (bs, IH), 8.62 (d, IH), 8.30 (d, IH), 7.89 (t, IH), 7.71 (t, IH), 7.65 (d, IH), 6.81 (d, IH). Anal Calcd. For C₁₂H₉BrN₂O₂: C, 49.17; H, 3.09; N, 9.56. Found: C, 48.75; H, 3.15; N, 9.36.

4e

[0097] Benzyloxyazaphenanthridone 4e. The hydrobromide salt 4d (100 mg, 0.34 mmol) was dissolved in 3 mL DMF. Potassium carbonate (100 mg) and benzyl bromide (60 μL, 0.50 mmol) were added to the solution and the mixture was heated to 60° C. for 14 h. The solvent was removed in vacuo and the residue was washed with water (5 mL) and boiling MeOH (10 mL) and filtered. The solid 4e (47 mg, 46%) was pure while the filtrate contained a mixture of isomers. Mp=271-276° C. ¹H NMR (d₆-DMSO) δ 11.68 (bs, IH), 8.71 (d, IH), 8.30 (d, IH), 7.93 (t, IH), 7.74 (m, 2H), 7.55 (d, 2H), 7.40 (t, 2H), 7.32 (t, IH), 7.09 (d, IH), 5.54 (s, 2H). Anal Calcd. for C₁₉H₁₄N₂O₂(H₂O): C, 71.24; H, 5.03; N, 8.74. Found: C, 71.28; H, 4.83; N, 8.38.

Scheme 2. Synthesis of Arninoazaphenanthridone 4f.

[0098] Dinitroamide 3f. The coupling of 2-chloro-3 ,5-dmitropyridine 2f with boronic acid 1 was accomplished as outlined in the procedure for 3a-c. ¹H NMR (CDCb) δ 9.55 (s, IH), 9.04 (s, IH), 7.41 (m, 2H), 7.39 (t, IH), 7.28 (d, IH), 3.99 (ra, IH), 3.40 (m, IH), 1.58 (bd, 3H), 1.50 (bd, 3H), 1.33 (bd, 3H), 1.20 (bd, 3H).

10099] Aminoazaphenanthridone 4f. The dinitroamide 3f (700 mg, 1.88 mmol) was dissolved in 25 mL MeOH and added to a Parr flask under nitrogen with 100 mg of palladium on carbon. This mixture was reduced under an atmosphere of 30 psi of hydrogen for 2 h. The palladium was filtered off through a plug of celite and the filtrate was concentrated in vacuo and the crude diamine (550 mg, 94%) was used in the cyclization without any further purification. The diamine was redissolved in dry tetrahydrofuran and cyclized with LDA (3 eq) in a similar manner to amides 3a-c. Compound 4f was isolated in 56% yield (227 mg). Mρ=>300° C. (dec); ¹H NMR (d₆- DMSO) δ 11.46 (bs, IH), 8.49 (d, IH), 8.17 (d, IH), 7.95 (s, IH), 7.77 (t, IH), 6.79 (s, IH), 5.92 (d, 2H). Anal Calcd. for C₁₂H₉N₃O₂: C, 62.87; H, 4.84; N, 18.33. Found: C, 62.18; H, 4.74; N, 18.17.

Scheme 3. Synthesis of Chloroazaphenanthridone 4g.

[00100] Chloroazaphenanthridone 4g. Amide 5 was prepared from commercial reagents, benzoyl chloride and 3-amino-2,6-dichloropyridine in DCM in high yield. The desired product 4g was prepared by dissolving amide 5 (4.45 g, 16.7 mmol) in DMA (35 mL) and adding sodium carbonate (1.8g, 16.7 mmol) and palladium acetate (400 mg, catalytic amt). The reaction mixture was heated to 125° C. for several hours until the starting material was no longer present by TLC. The reaction was then cooled down to room temperature and concentrated in vacuo and the crude residue was suspended in boiling EtOAc (100 mL) and filtered through a plug of celite. The filtrate was concentrated and the solid that precipitated out was filtered off and determined to be compound 4g (520 mg, 13% yield). Mp=285-295 (dec.) ° C; ¹H NMR (d₆-DMSO) δ 11.92 (bs, IH), 8.62 (d, IH), 8.32 (d, IH), 7.95 (t, IH), 7.77 (m, 2H), 7.62 (d, IH). Anal Calcd. for C₁₂H₇ClN₂O: C, 62.49; H, 3.06; N, 12.15. Found: C, 61.40; H, 3.19; N, 11.77.

4h-x

Scheme 4. General Scheme for Synthesis of Azaphenanthridone Amines 4h-x.

[00101] General procedure for the synthesis of nitro compound 3g. The boronic acid 1 (2.0 g, 8.0 mmol) prepared according to the literature was added to a solution of potassium carbonate (2.2 g in 8 mL H₂O) and 2,5-dichloro-3-nitro pyridine 2g (1.4 g, 7.3 mmol) in 100 mL toluene/EtOH (8:1). This mixture was deoxygenated in vacuo and refilled with nitrogen. After stirring the mixture under nitrogen for 30 min, palladium tetrakistriphenylphosphine (250 mg) was added to the mixture. The solution was heated to 80° C. until complete conversion (no starting material) according to TLC (50/50 Hexanes/EtOAc). The reaction was then extracted with water and the toluene layer was dried and concentrated to yield a crude oil which was columned on silica gel to afford the desired isomer 3g in 45% yield (1.20 g). ¹H NMR (CDCl₃) δ 8.26 (d, IH), 7.45 (m, 3H), 7.34 (m, 2H), 3.99 (m, IH), 3.41 (m, IH), 1.38 (bs, 9H), 1.21 (d, 3H).

[00102] General procedure for the synthesis of amines 6a-x. Chloride 3g (300 mg, 0.83 mmol) was dissolved in THF (5 mL) followed by the addition of diisopropylethylamine (160 μL, 0.91 mmol), and 2-(4-aminoethyl)morpholine (220 μL, 1.66 mmol). The reaction was heated to 65° C. overnight and TLC analysis indicated a low running spot on the baseline (EtOAc). Water (5 mL) was added to the mixture followed by extraction with DCM (3*10 mL). The combined organics were dried and concentrated to yield a crude foam which solidified upon drying in vacuo. The solid was triturated with hexanes and filtered to yield 320 mg (85%) of the desired amine 6a.

[00103] NR₂=aminoethylmorpholine (6a). Yield=85%; ¹H NMR (DMSOd₆) δ 8.21 (m, IH), 7.40 (m, 5H), 6.36 (d, IH), 3.97 (m, IH), 3.70 (m, 6H), 3.47 (m, 2H), 3.31 (m, IH), 2.45 (m, 6H), 1.48 (bs, 3H), 1.24 (bs, 3H), 1.06 (bs, 3H), 0.87 (bs, 3H).

[00104] NR₂=N-methylpiperazine (6b). Yield=72%; MS (ES+)=426.21.

[00105] NR₂=N-boc-[2.2.1]diazabicycloheptane (6c). Yield=72%; MS (ES+)=486.43.

[00106] NR₂=N-boc-piperazine (6d). Yield=72%; MS (ES+)=473.23.

[00107] NR₂=amino (6e). Yield=72%; MS (ES+)=343.31.

[00108] General procedure for the synthesis of anilines 7a-x. Nitro compound 6a (300 mg, 0.66 mmol) was dissolved in MeOH (20 mL) with Pd/C (100 mg) and hydrogenated at 30 psi for 2 h. TLC indicated complete conversion of the intra compound (10% MeOH/EtOAc). The reaction mixture was filtered through a plug of celite and the filtrate was concentrated and dried. The crude foam was used in the cyclization step without further purification. The dry yield of the aniline 7a was 275 mg (99%). ¹H NMR (CDCl₃) δ 7.41 (m, 3H), 6.98 (d, IH), 6.31 (d, IH), 4.75 (bs, 2H), 3.78 (m, IH), 3.70 (m, 4H), 3.28 (m, 3H), 2.56 (m, 2H), 2.47 (m, 4H), 1.50 (d, 3H), 1.19 (d, 3), 1.00 (d, 3H), 0.83 (d, 3H).

4h

[00109] General procedure for the cyclization of anilines 7a-x. The crude aniline 7a (270 mg, 0.64 mmol) was dissolved in THF (20 mL) and cooled to -78° C. A 2.0M solution of LDA (1 mL) was added to the aniline and the reaction was slowly warmed to room temperature overnight. The mixture was quenched with water (10 mL) and extracted several times with EtOAc (3><15 mL). The combined organics were dried and concentrated and the resulting solid was triturated with EtOAc (3 mL) and filtered yielding 125 mg (58%) of the desired amine 4h. ¹H NMR (DMSO dβ) δ 11.46 (s, IH), 8.70 (d, IH), 8.32 (d, IH), 7.92 (t, IH), 7.71 (t, IH), 7.49 (d, IH), 6.82 (d, IH), 6.63 (t, IH), 3.66 (m, 4H), 3.58 (m, 2H), 2.60 (m, 4H). MS ES+=324.9). Mp=250-255° C. Anal Calcd for C₁₈H₂₀N₄O₂(0.5 H₂O) C, 64.85; H, 6.35; N, 16.81. Found: C, 65.44; H, 6.27; N, 16.71.

4i [00110] NR₂=N-methylpiperazine (4i). Yield=46%; mp=285-288° C; ¹H NMR (CDCl₃) δ 11.50 (s, IH), 8.65 (d, IH), 8.27 (d, IH), 7.88 (t, IH), 7.69 (t, IH), 7.56 (d, IH), 7.14 (d, IH), 3.56 (t, 4H), 2.46 (t, 4H), 2.24 (s, 3H). Anal. Calcd for Ci₇Hj₈N₄O: C, 69.37: H, 6.16: N, 19.03; found: C, 69.38: H, 6.15; N, 18.84.

[00111] NR₂=(S,S)-N-Boc-[2.2.1] diazabicycloheptane (4j). Yield=36%; mp=259- 261° C; ¹H NMR (DMSOd₆) δ 11.44 (s, IH), 8.65 (d, IH)₃ 8.26 (d, IH), 7.67 (t, IH), 7.45 (d, IH), 6.85 (t, IH), 4.92 (dd, 2H), 3.38 (m, 2H), 3.30 (s, IH), 3.23 (m, IH), 1.96 (d, 2H), 1.60 (s, 9H). Anal Calcd. for C₂₂H₂₄N₄O₃; C, 67.33; H, 6.16; N, 14.28. Found: C, 67.30; H, 6.19; N, 14.21.

[00112] NR₂=[2.2.1]-diazabicycloheptane (4k). This compound was made by the deprotection of the boc group by 10% TFA/DCM (16h). Yield=98%; mp=160-165° C; ¹H NMR (DMSO-d₆) δ 11.54 (s, IH), 8.68 (d, IH)₅ 8.59 (d, IH), 7.90 (t, IH), 7.72 (t, IH), 7.61 (d, IH), 6.92 (d, IH), 5.04 (s, IH), 4.53 (s, IH), 3.68 (m, 2H), 3.29 (m, 2H), 2.19 (d, IH)₃ 2.00 (d, IH). Anal Calcd. for C₁₇H₁₆N₄O (1.1 C₂HF₃O₂)(0.4 H₂O): C, 54.41; H, 4.00; N, 13.22. Found: C, 54.12; H, 4.26; N, 12.98.

41

[00113] NR₂=Boc-piperazine (41). Yield=48%; mp=231 -235° C; ¹H NMR (DMSO- d₆) δ 11.52 (s, IH), 8.67 (d, IH), 8.27 (d, IH), 7.89 (t, 2H), 7.70 (t, 2H), 7.58 (d, IH), 3.50 (dd, 8H), 1.44 (s, 9H). Anal Calcd. for C₂₁H₂₄N₄O₃ (0.5 H₂O): C, 64.77; H, 6.47; N, 14.39. Found: C, 64.83; H, 6.39; N, 14.23.

4m

[00114] NR₂=piρerazine (4m). This compound was made by the deprotection of the boc group with 10%TFA/DCM (16h). Yield=99%.; mp=250-252° C; ¹H NMR (DMSO- d₆) δ 11.56 (s, IH), 8.93 (bs, IH), 8.65 (d, IH), 8.27 (d, IH), 7.87 (t, IH), 7.70 (t, IH), 7.61 (d, IH), 7.21 (d, IH), 3.61 (t, 4H), 3.26 (bs, 4H), Anal Calcd. for C₁₆H₁₆N₄O (0.5 H₂O) (1.9 TFA): C, 47.18; H, 3.40; N, 11.11. Found: C, 47.39; H, 3.64; N, 11.18.

[00115] NR₂=amino (4n). Yield=50%; mp=310-315° C; ¹H NMR (DMSO-d₆) δ 11.42 (s, IH), 8.50 (d, IH), 8.26 (d, IH), 7.85 (t, IH), 7.66 (t, IH), 7.44 (d, IH), 6.70 (d, IH), 6.02 (d, 2H), Anal Calcd. for Ci₂H₉N₃O (0.11 EtOAc): C, 67.64; H, 4.51; N, 19.02. Found: C, 67.98; H, 4.57; N, 18.67.

[00116] NR₂=N,N-diethyl aminopropyl (4o). Yield=45%; mp=l 14-116° C; ¹H NMR (DMSO-d₆) δ 300 MHz 0.96(t, 6H, J-7.07, 7.73), 1.72(m, 2H), 2.49(m, 6H), 3.39(m, 2H), 6.70(d, IH, J=8.84), 7.41(d, IH, J=8.84), 7.65(t, IH, J=8.08, 8.09), 7.84(t, IH, J=8.09, 8.34), 8.24(d, IH, J=7.83), 8.83 (d, IH, J=7.83), 11.4 (s, IH).

[00117] NR₂=N-isopropyl piperazine (4p). mp=260-264° C; ¹H NMR (DMSO-(I₆) δ 300 MHz 11.48(s, IH), 8.63(d, J=7.44 Hz, IH), 8.25(d, J=7.25 Hz, IH), 7.86(t, J=8.2, 8.39 Hz, IH), 7.67(t, J=8.20, 6.87 Hz, IH), 7.53(d, J=9.15 Hz, IH), 7.10(d, J=9.16 Hz, IH), 3.55(t, J=4.96, 4.58 Hz, 4H), 2.68(m, IH), 2.56(t, J=4.77, 4.77 Hz, 4H)_; 1.00(d, J=6.48 Hz, 6H). Anal Calcd. for C₁₉H₂₄N₄O: C, 70.8; H, 6.9; N, 17.2. Found: C, 70.9; H, 6.9; N, 17.2.

[00118] NR₂=pyrrolylpiρeridine (4q). mp=l 70-175° C; ¹H NMR (D₂O) δ 300 MHz 7.89(d, J=7.63 Hz, IH), 7.80(d, J=7.82 Hz, IH), 7.54(t, J=7.06, 7.63 Hz, IH), 7.43(t, J=6.48, 7.44 Hz, IH), 6.94(d, J=8.21 Hz, IH), 6.54(d, J=7.25 Hz, IH), 4.08(d, J=12.21 Hz, 2H), 3.63(t, J=9.72, 8.01 Hz, 2H), 3.31(m, IH), 3.13(m, 2H), 2.70(t, J=9.92, 12.02 Hz, 2H), 2.1 l-2.2(m, 4H), 1.94(q, 2H), 1.63(q, 2H), Anal Calcd. for C₂₁H₂₄N₄O (1 H₂O) (1.4 HCl): C, 58.4; H, 6.4; N, 13.0; Cl, 11.5. Found: C, 58.7; H, 6.8; N, 12.8; Cl, 11.1.

4r

[00119] NR₂=N-cyclopentylpiperazine (4r). mp=285-290° Cj ¹H NMR (DMSO-d₆) δ 300 MHz 11.50 (s, IH), 8.65(d, J=7.82 Hz, IH), 8.28(d, J=8.01 Hz, IH), 7.88(t, J=7.06, 6.86 Hz, IH), 7.70(t, J=7.06, 7.06 Hz, IH), 7.56(d, J=8.96 Hz, IH), 7.13(d, J=9.16 Hz, IH), 3.58(t, J=4.96, 4.20 Hz, 4H), 2.57(t, J=4.58, 4.57 Hz, 4H), 1.83(m, 2H), 1.64(m, 2H), 1.57(m, IH), 1.50(m, 2H), 1.37(m, 2H). Anal Calcd. for C₂₁H₂₄N₄O (0.2 H₂O): C, 71.7; H, 7.0; N, 15.9. Found: C, 71.6; H, 7.0; N, 15.7.

4s [00120] NR₂=N-oxo-N-methylpiperidine (4s). The synthesis of this compound and 4t was performed by the oxidation of 4i with excess mCPBA. ¹H NMR (D₂O) δ 300 MHz 7.62 (d, J=6 Hz, IH), 7.52 (d, J=9 Hz, IH), 7.33 (m, 2H), 6.59 (d, J=9 Hz, IH), 6.14 (d, J=9 Hz, IH), 3.73 (m, 4H), 3.62 (t, 2H), 3.55 (s, 3H), 3.08 (t, 2H).

4t

[00121] NR₂=N-oxo-methylpiperidine-N-oxide (4t). mp=230-235° C; ¹H NMR (D₂O) δ 300 MHz 7.97 (m, 2H), 7.51 (d, J=9 Hz, IH), 7.43 (d, J=9 Hz, IH), 7.36 (t, J=9 Hz₃ IH), 7.27 (t, J=9 Hz, IH), 4.88 (bt, 2H), 4.56 (bt, 2H), 3.96 (bd, 2H), 3.69 (s, 3H), 3.64 (bd, 2H). Anal Calcd. for C₁₇H₁₈N₄O₃ (1.25 H₂O): C, 50.1; H, 50.1; N, 13.8. Found: C, 50.7; H, 5.2; N, 13.8.

4u [00122] NR₂=N-methylfuranylpiperazine (4u). mp=270-275° C; ¹U NMR (DMSOd₆) 300 MHz 11.49(s, IH), 6.63(d, J=8.01 Hz, IH), 8.25(d, J=7.82 Hz, IH), 7.86(t, J=8.20, 8.21 Hz, IH), 7.67(t, J=7.06, 7.06 Hz, IH), 7.53(d, J=8.96, IH), 7.1 l(d, J=8.96 Hz, IH), 3.96(m, IH), 3.73(q, IH), 3.59(q, IH), 3.55(t, J=4.96, 3.44 Hz, 4H), 2.61(m, 2H), 2.55(m, 2H), 2.41(m, 2H), 1.86-L98(m, IH), 1.75-1.82 (m, 2H), 1.43-1.47 (m, IH). Anal Calcd. for C₂₁H₂₄N₄O₂(OJ H₂O): C, 68.2; H, 6.7; N, 15.2. Found: C, 68.2; H, 6.7; N, 15.0.

[00123] NR₂=N-cycloproρylmethylpiperazine (4v). ¹H NMR (DMSO-d₆) 300 MHz 11.38 (bs, IH), 8.55 (d, IH), 8.17 (d, IH), 7.76 (t, IH), 7.58 (t, IH), 7.46 (d, IH), 7.05 (d, IH), 3.49 (m, 4H), 2.40 (m, 4H), 2.12 (m, 2H), 0.77 (m, IH), 0.39 (m, 2H), 0.00 (m, 2H).

[00124] NR₂=N-methyl-[2.2.1]-diazabicycloheptane (4w). ¹H NMR (DMSO-d₆) 300 MHz 11.43 (bs, IH), 8.66 (d, IH), 8.27 (d, IH), 7.84 (t, IH)₃ 7.70 (t, IH), 7.53 (d, IH), 6.81 (d, IH), 4.73 (m, IH), 3.52 (m, IH), 3.48 (m, IH), 3.37 (m, IH), 3.34 (m, IH), 2.87 (d, IH), 2.28 (s, 3H), 1.86 (dd, 2H).

4x

[00125] NR₂=N-cycloproρylmethyl-[2.2. l]-diazabicycloheptane (4x). MS (ES+) 347.28.

Scheme 5. Synthesis of Amines 8a-r.

[00126] General procedure for the alkylation of amine 4k. Amine 4k (10 mg), excess K₂CO₃, and corresponding benzyl chloride were placed in test tubes in 1 mL CH₃CN. Those mixtures were heated to 60° C. on heat block overnight. EtOAc and 10% HCl were added. The organic layer was removed and the aqueous layer was basified with 10% NaOH. Extracted with EtOAc and then evaporated under reduced pressure. Compounds 8a-r were made in this manner.

[00127] R=CR₂CN (8a). MS (ES+)=332.41.

{00128] R=CH₂COOEt (8b). MS (ES+)=378.45.

[00129] R=CH₂-(2,5-dimethylphenyl) (8c). MS (ES+)=311.51.

[00130] R=CH₂-(4-fluorophenyl) (8d). MS (ES+)=401.45.

[00131] R=CH₂-(4-methoxyphenyl) (8e). MS (ES+)=413.49.

[00132] R=CH₂-(3,4-dimethylphenyl) (8f). MS (ES+)=411.23.

[00133] R=CH₂-(3,4-dichlorophenyl) (8g). MS (ES+)=451.36.

[00134] R=CH₂-(2-fluorophenyl) (8h). MS (ES+)=401.42.

[00135] R=CH₂-(3-methylphenyl) (8i). MS (ES+)=397.49.

[00136] R=CH₂-(3-chlorophenyl) (8j). MS (ES+)=417.83.

[00137] R=CH₂-(2-methylphenyl) (8k). MS (ES+)=397.41.

[00138] R=CH₂-(2-chlorophenyl) (81). MS (ES+)=417.73.

[00139] R=CH₂-(4-carboxyphenyl) (8m). MS (ES+)=427.43.

[00140] R=CH₂-(3-carboxyphenyl) (8n). MS (ES+)=427.41.

[00141] R=CH₂-(4-methylphenyl) (8o). MS (ES+)=397.42.

[00142] R=CH₂-(4-benzyloxyphenyl) (8p). MS (ES+)=489.44.

[00143] R=CH₂-(3-fluorophenyl) (8q). MS (ES+)=400.42.

[00144] R=CH₂-(3-methylρhenyl) (8r). MS (ES+)=412.43.

9a-c

Scheme 6. Synthesis of Amines 9a-c

[00145] General procedures for parallel synthesis of amines 9a-c. To a mixture of the amine 4m, excess K₂CO₃, and corresponding bromomethylpyridines was added 1 rnL CH₃CN. The reaction mixtures were heated to 90° C. for 4 h. Tris(2-aminoethyl)arnine resin was added and heated to 70° C. for 1 h to remove excess of bromomethylpyridines. The mixtures were filtered and added to water (2 mL). Extracted with EtOAc. The organic layers were dried over Na₂SO₄ and evaporated under reduced pressure.

[00146] R=CH₂-(o-pyridine) (9a). MS (ES+)=372.41. [00147] R=CH₂-(m-pyridine) (9b). MS (ES+)=372.40. [00148] R=CH₂-(o-pyridine) (9c). MS (ES+)=372.41.

Ua-D

Scheme 7. Synthesis of Amines lla-x.

[00149] Procedure for synthesizing chloracetyl derivative (10). Amine 4n was dissolved in N,N -dimethylacetamide and was cooled to 0° C. in ice bath. Triethylamine (1.1 eq) and Chloroacetylchloride (0.44 ml, 5.5 mmol) were added. The reaction mixture was stirred at room temperature under nitrogen over night. Solvent was evaporated under reduced pressure and the resulting brown residue was added water and 10% NaHCO₃. Solid was collected by filtration to yield 1.03 g (84% yield). Mρ=287-290° C; ¹H NMR (DMSOd₆) δ 300 MHz 11.81(s, IH), 10.94(s, IH), 8.66(d, J=8.39, IH), 8.3 l(d, J=7.63, IH), 8.20(d, J=8.01, IH), 7.96(t, J=7.82, 7.44, IH), 7.77(m 2H), 4.42(s, 2H). Anal Calcd. for Ci₄H₁₀ClN₃O₂: C, 58.5; H, 3.5; N, 14.6; Cl, 14.6. Found: C, 58.5; H₃ 3.6; N, 14.6; Cl, 14.6.

11a

[00150] General procedure for the animation of chloride 10. Animation was carried out in a similar manner to 8a-r. NR₂=dimethylaminoacetyl (Ha). mp=195-198° C; ¹H NMR (DMSO-d₆) δ 300 MHz 7.87(d, J-7.44 Hz, IH), 7.76(d, J=7.82 Hz, IH), 7.56(t, J=7.24, 7.25 Hz, IH), 7.47(m, 2H), 7.06(d, J-8.96 Hz, IH), 4.15(s, 2H), 3.00(s, 6H). Anal Calcd. for C₁₆H₁₆N₄O₂(U H₂O) (1.2 HCl): C, 50.8; H, 5.5; N, 14.8; Cl, 11.3. Found: C, 50.8; H, 5.5; N 14.7; Cl, 11.2.

lib

[00151] NR₂=piperidinylacetyl (lib). mp=175-180° C; ¹H NMR (DMSO-(I₆) δ 400 MHz 8.03(bd, J=8.33 Hz, IH), 7.90(d, J=8.09 Hz, IH), 7.66(t, J=7.33, 7.32 Hz, IH), 7.59(d, J=7.83 Hz, IH), 7.54(t, J=7.58, 7.33 Hz, IH), 7.22(d, J=8.59 Hz, IH), 4.12(s, 2H), 3.63(s, 2H), 3.13(s, 2H), 1.86(m, 6H). Anal Calcd. for C₁₉H₂₀N₄O₂ (2 H₂O) (HCl): C, 54.4; H, 6.3; N, 13.4; Cl, 8.4. Found: C, 54.2; H, 6.0; N, 13.1; Cl, 8.6.

[00152] NR₂=pyiτolydylpiperidinylacetyl (1 Ic). 300 MHz 7.61 (m, 2H), 7.47 (t, J=9 Hz₃ IH), 7.38 (m, 2H), 6.90 (d, J=9 Hz, IH), 3.68 (m, 2H), 3.50 (s, 2H), 3.33 (m, 4H), 2.68 (m, 2H), 2.39 (m, 3H), 1.87-2.12 (m, 6H). Anal Calcd. for C₂₃H₂₇N₅O₂(2 H₂O)(HCl): C, 57.8; H, 6.8 N, 14.7. Found: C, 57.8; H, 6.7; N, 14.6.

[00153] NR₂=2,3 tetrahydropyridine (lid). MS (ES-)=333.

[00154] NR₂=isoindole (lie). MS (ES-)=369.

[00155] NR₂=dipenylamine (Hf). MS (ES-)=407.

[00156] NR₂=N-methylanisole (11 g). MS (ES-)=387.

[00157] NR₂=N-methylbenzylamine (Hh). MS ES-)=371.

[00158] NR₂=N-benzyl— N-phenethylamine (Hi). MS (ES-)=461.

[00159] NR₂-N-hydroxyethylpiperazine (Hj). MS(ES-)=381.

[00160] NR₂=N,N-dipropylamine (Hk). MS (ES-)=351.

[00161] NR₂=4-oxopiperidine (111). MS (ES-)=349.

[00162] NR₂=N,N-dibutylamine (Hm). MS (ES-)=379.

[00163] NR₂=morpholine (Hn). MS (ES-)=337.

[00164] NR₂=imidazole (Ho). MS (ES-)= 318.

13a-q

Scheme 8. Amide Derivatives of Aniline 4f.

[00165] Chloride 12. The chloride 12 was synthesized identically to chloride 10. ¹H NMR (de-DMSO, 300 MHz): 11.81 (bs, IH), 10.94 (s, IH), 8.66 (s, IH), 8.31 (s, IH), 8.20 (d, IH), 7.94 (t, IH), 7.77 (m, 2H), 4.44 (s, 2H).

[00166] General procedure for the animation of chloride 12. The animation was carried out in a manner similar to amination of chloride 10 stated above.

13a [00167] NR₂ dimethylamine hydrochloride (13a). ¹HNMR (D₂O, 300 MHz): 7.82 (d, J=7.44 Hz, IH), 7.76 (d, J=7.63 Hz, IH), 7.70 (d, J=2.10 Hz), 7.63 (t, J=7.05, 7.63 Hz, IH), 7.50 (t, J=7.06, 7.82 Hz, IH), 7.40 (d, J=2.29 Hz, IH), 4.16 (s, 2H), 3.03 (s, 6H).

13b

[00168] NR₂=piperidine hydrochloride (13b). ¹HNMR (D₂O, 300 MHz): 400 MHz 8.03 (bd, J=8.33 Hz, IH), 7.90 (d, J=8.09 Hz, IH), 7.66 (t, J=7.33, 7.32 Hz, IH), 7.59 (d, J=7.83 Hz, IH), 7.54 (t, J=7.58, 7.33 Hz, IH), 7.22 (d, J=8.59 Hz, IH), 4.12 (s, 2H), 3.63 (s, 2H), 3.13 (s, 2H), 1.86 (m, 6H).

13c

[00169] NR₂=pyrollylpiperidine (13c). ¹HNMR (D₂O, 300 MHz) δ 7.48 (d, J=9 Hz, IH), 7.31 (d, J=9 Hz, IH), 7.29 (m, 2H), 7.17 (t, J=9 Hz, IH), 6.99 (s, IH), 3.58 (s, 2H), 3.43-3.31 (m, 5H), 3.22 (m, 2H), 2.91 (m, 2H), 2.74 (m, 2H), 2.48 (s, 6H), 2.14 (m, 2H), 1.89-1.65 (m, 6H). Anal Calcd. for C₂₃H₂₇N₅O₂^ H₂O)(2 MsOH): C, 50.2; H, 5.9 N, 11.7. Found: C, 50.2; H, 6.0 N, 11.6.

[00170] NR₂=N-isopropylpiperidine (13d). ¹HNMR (D₂O, 300 MHz) δ 300 MHz 7.78 (d, J=9 Hz, IH), 7.60 (m, 3H), 7.50 (t, J=9 Hz, IH), 7.23 (s, IH), 3.74 (m, 3H), 3.47 (s, 2H), 3.40 (m, 4H), 2.90 (m, 2H), 1.55 (d, J=6 Hz, 6H). Anal Calcd. for C₂₁H₂₅N₅O₂ (2.25 H₂O) (1 HCl): C, 55.4; H, 6.5; N, 15.4. Found: C, 55.4; H, 6.6; N, 15.4. [00171] NR₂=aminoethylpyrrolidine (13e). MS (ES+)=366.35.

[00172] NR₂=2-aminopropyl— N-methylpyrrolidine (13f). MS (ES+)=394.41.

[00173] NR₂=o-aminoethylpyridine (13g). MS (ES+)=374.30.

[00174] NR₂-m-aminoethylpyridine (13h). MS (ES+)=374.25.

[00175] NR₂=N-benzylpiperazine (13i). MS (ES+)=428.42.

[00176] NR₂=aminoethylmorpholine (13j). MS (ES+)=382.32.

[00177] NR₂=N,N-diethylethylenediamine (13k). MS (ES+)=368.31.

[00178] NR₂=N,N-dimethylethylenediamine (131). MS=(ES+)=340.21.

[00179] NR₂=N,N-diethylpropylenediamine, (13m). MS (ES+)=382.41.

[00180] NR₂-N,N,N-trimethylproρylenediamine (13n). MS (ES+)=368.32.

[00181] NR₂=homopiperazine (13o). MS (ES+)=352.23.

[00182] NR₂=N-methylpiperazine (13p). MS (ES+)=352.32.

[00183] NR₂=piperonylpiperazine (13q). MS (ES+)=472.44.

[00184] NR₂=aminoethylpyrrolidin-2-one (13r). MS (ES+)=394.40.

[00185] NR₂=aminoethylpiperidine (13s). MS (ES+)=380.32.

Scheme 9. Alternative Synthesis of Amines 4a-x.

[00186] General alternate synthesis of amines 4a-x (example 4i). The proposed synthesis below would be more amenable to scale up. The boronic ester 14 can be made on a large scale (20 g) according to the literature reference (Kristensen, J. et al. Org. Lett. 2001, 3(10), 1435-1437). This synthesis cuts off one step, the LDA cyclization since the reduction/cyclization should work in the same step.

[00187] Synthesis of nitro chloride 15. The boronic ester 14 (16.0 g, 61.0 mmol), dinitro chloride 2g (11.7 g, 61 mmol) and potassium carbonate (21 g, 152 mmol) were dissolved in toluene/EtOH (20:1, 300 mL). This mixture was evacuated and refilled several times with nitrogen. Then, tetrakis-palladiumtriphenylphosphine (~2 g) was added followed by heating the mixture to 80° C. overnight. The reaction was then concentrated in vacuo and partitioned between EtOAc (200 mL) and H₂O (200 mL). The organic layer was dried with sodium sulfate and concentrated in vacuo, The crude residue was chromatographed using a gradient system (5% EtOAc/Hexanes→ 20% EtOAc/Hexanes). The final product (Rρ0.3, 10% EtOAc/Hexanes) was isolated as a low melting solid/foam (7.12 g, 38%). Another 1.3 g (7.0%) of a mixture of isomers (other isomer Rr=0.25, 10% EtOAc/Hexanes) was isolated from the column. ¹H NMR (CDCl₃, 300 MHz) δ 8.40 (d, IH), 8.14 (d, IH), 7.65 (t, IH), 7.55 (m, 2H), 7.32 (d, IH), 4.16 (q, 2H), 1.19 (t, 3H).

[00188] Synthesis of Diamine 16. The chloride 14 (7.12 g, 23.2 mmol) was dissolved in DCM (250 mL). Diisopropylethylamine (3.3 g, 25.5 mmol) was added to this solution followed by N-methylpiperazine (4.6 g, 46.4 mmol). This mixture was stirred overnight until complete conversion of the chloride was evident by TLC (R_f of diamine=0.1, EtOAc). The reaction was worked up by extraction with water (2x100 mL). The organic layer was dried with sodium sulfate and concentrated in vacuo to yield the crude diamine 16 (6.56 g, 77%). ¹HNMR (CDCl₃, 300 MHz) δ 8.32 (d, IH), 8.07 (d, IH), 7.58 (t, IH), 7.48 (t, IH), 7.26 (d, IH), 6.60 (d, IH), 4,13 (q, 2H), 3.73 (t, 4H), 2.46 (t, 4H), 2.33 (s, 3H), 1.13 (t, 3H).

[00189] Reduction/cyclization to form (4i). The crude diamine 16 was dissolved in MeOH (300 mL). Wet Raney nickel was added (500 mg, catalytic amount) followed by dropwise addition of hydrazine hydrate (4.1 g, 82 mmol). The mixture was heated to reflux and monitored by TLC until completion (approximately 3 h). The product R_f value was 0.1 in 10% MeOH/EtOAc. The Raney nickel was then filtered off and the filtrate was concentrated and suspended in 1 N HCl/EtOAc (150 mL/100 mL) and the solid that resulted was filtered off and triturated with 50 mL of CH₃CN and filtered. The resulting light yellow solid was dried under high vac for 2 h to yield 4.1 g (84% yield) of 4i. ¹H NMR (DMSO-d₆, 300 MHz) δ 11.50 (bs, IH), 8.67 (d, IH), 8.28 (d, IH), 7.88 (t, IH), 7.69 (t, IH), 7.58 (d, IH), 7.15 (d, IH), 3.58 (t, 4H), 2.46 (t, 4H), 2.24 (s, 3H). [00190] Mesylate salt formation (4i^r). A solution of the diamine 4i (2.85 g, 9.7 mmol) in 500 niL dry THF was added methanesulfonic acid (0.65 mL, 10 mmol). The reaction mixture was stirred under N₂ at room temperature overnight. Off white solid was collected by filtration and washed with ether. The solid was vacuum dried to yield 3.2 g (85% yield).

[00191] In Vivo Chemosensitization.

[00192] Example I: Oral administration of Compound 4p + Temozolomide enhances survival of mice bearing malignancies at the CNS site.

[00193] The intracranial transplantation procedure was performed as previously described (Tentori L. et ah, "Effects of single or split exposure of leukemic cells to temozolomide, combined with poly(ADP-ribose) polymerase inhibitors on cell growth, chromosomal aberrations and base excision repair components," Cancer Chemother Pharmacol, 47, 361-69 (2001)). Murine melanoma B16 (10⁴) were injected intracranially (ic) into male B6D2F1 (C57BL/6 x DBA/2) mice. Histological evaluation of tumor growth in the brain was performed 1-5 days after tumor challenge, in order to determine the timing of treatment.

[00194] Compound 4p was dissolved in 70 inM PBS without potassium and administered po 1 h before temozolomide (TMZ). TMZ was dissolved in dimethyl- sulfoxide (40 mg/ml), diluted in saline (5 mg/ml) and administered ip at a dose of 100 mg/Kg. Mice were treated with compound 4p by oral gavages once a day for five days, at doses of 10, 40, or 100 mg/kg/day. Median survival times (MST) were determined and the percentage of increase in lifespan (ILS) was calculated as: {[MST (days) of treated mice/MST (days) of control mice]-l}xlOO. Efficacy of treatments was evaluated by comparing survival curves between treated and control groups.

[00195] In mice bearing B 16 melanoma, the results indicated that the mean survival time of the groups treated with compound 4p + TMZ combination was significantly higher than that observed in animals receiving TMZ as single agent (Figure 1 and Table D- [00196] Figure 1. Compound 4p per os ± TMZ x 5 in B 16 ic.

12 15 18 21 24

Day

Where: open circles represent control, grey-filled triangles represent TMZ x 5, filled diamonds represent 10 mg/kg compound 4p + TMZ (x5); filled squares represent 40 mg/kg compound 4p + TMZ (x5); and (x) represent 100 mg/kg compound 4p + TMZ (x5).

Table 1. Chemopotentiation of TMZ efficacy against intra-cerebral malignant melanoma by oral administration of Compound 4p

Example II: Oral administration of Compound 4h + Temozolomide enhances survival of mice bearing malignancies at the CNS site.

[00197] Following the above procedure for compound 4p, compound 4h was administered at doses of 10 and 40 mg/kg. As indicated by the data in Figure 2, the mean survival times of the groups treated with the combination of temozolomide and compound 4h were significantly higher than that observed in animals receiving TMZ as single agent.

10 15 20

Day

Where S% is percent survival; open circles indicate vehicle control; grey-filled triangles indicate TMZ (100 mg/kg) x 5; open diamonds indicate 40 mg/kg compound 4h + 100 mg/kg TMZ) x 5; and filled diamonds indicate (10 mg/kg compound 4h + 100 mg/kg TMZ) x 5.

[00198] The present invention also provides a medical device for the administration of the compounds of the present invention. A medical device comprising a drug delivering or eluting member, wherein said drug delivering or eluting member has disposed therein or thereon a compound of the formula I. The medical device may be a shunt, a colostomy bag attachment device, an ear drainage tube, a lead for a pace maker, a lead for an implantable defibrillator, a suture, a staple, an anastomosis device, a vertebral disk, a bone pin, a suture anchor, a hemostatic barrier, a clamp, a screw, a plate, a clip, a vascular implant, a tissue adhesive, a tissue sealant, a tissue scaffold, a bone substitute, an intraluminal device, a stent, or a vascular support. In a preferred embodiment, the drug delivering or eluting member is a stent.

[00199] Any pharmacologically-acceptable chemotherapeutic agent that acts by damaging DNA is suitable as the chemotherapeutic agent of the present invention. In particular, the present invention comprehends a chemotherapeutically effective amount of at least one chemotherapeutic agent including, but not limited to, temozolomide, adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, docetaxel, doxorubicin, interferon (alpha, beta, gamma), interleukin 2, irinotecan, paclitaxel, topotecan, therapeutically effective analogs and derivatives of the same, and mixtures thereof. According to a preferred aspect, the chemotherapeutic agent is temozolomide.

[00200] The disclosure contained herein demonstrates the compounds and compositions of the present invention may be useful in treating and/or preventing cancer, such as by radiosensitizing and/or chemosensitizing tumor cells to cytotoxic agents.

[00201] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications are intended to be included within the scope of the following claims.

Claims

1. A chemosensitization method to treat cancers in a mammal, comprising administering to said mammal an azophenanthridone compound of formula I and at least one chemotherapeutic agent, wherein said azaphenanthridone compound of formula I is

wherein

R₁, R₂ and R₃ are independently selected from H, halogen, amino, -OH, optionally substituted alkyl, alkenyl, alkynyl, alkoxy, -Obenzyl, cycloalkyl, aryl, heterocyclyl, -NR₅R₆, and -NR₅COR₇, wherein

R₅ and R₆ are each independently selected from hydrogen, optionally substituted alkyl, cycloalkyl, aryl, and heterocyclyl, and

R₇ is selected from an optionally substituted alkyl, cycloalkyl, aryl, and heterocyclyl, and

R₄ is independently selected from hydrogen, halogen, alkoxy, and alkyl.

2. The chemosensitization method of Claim 1 wherein only one R₄ is present on the ring.

3. The chemosensitization method of claim 1 wherein R₂, R₃, and R₄ are each hydrogen.

4. The chemosensitization method of claim 3 wherein R₁ is an optionally substituted heterocyclyl or -NR₅R₆, wherein R₅ and R₆ are independently hydrogen or an optionally substituted alkyl.

5. The chemosensitization method of Claim 1 wherein said compound of formula I is selected from the group consisting of:

6. The chemosensitization method of Claim 1 wherein the compound is

7. The chemosensitization method of Claim 1 wherein the compound is

8. The chemosensitization method of Claim 1 wherein said cancers are selected from the group consisting of 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, Ewing's sarcoma, gallbladder cancer, hairy cell leukemia, head and neck cancer, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, liver cancer, lung cancer (small and/or non-small cell), malignant peritoneal effusion, malignant pleural effusion, melanoma, mesothelioma, multiple myeloma, neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovarian cancer, ovary (germ cell) cancer, prostrate 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.

9. The chemosensitization method according to claim 8 wherein said cancers are selected from melanoma, mesothelioma, multiple myeloma, skin cancer, and brain cancer.

10. The chemosensitization method according to Claim 1 wherein said chemotherapeutic agent is selected from temozolomide, adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, docetaxel, doxorubicin, interferon (alpha, beta, gamma), interleukin 2, irinotecan, paclitaxel, topotecan, therapeutically effective analogs and derivatives of the same, and mixtures thereof.

11. The chemosensitization method according to Claim 10, wherein said chemotherapeutic agent is temozolomide, ironotecan, or topotecan.

12. A pharmaceutical composition comprising a chemosensitizing effective amount of at least one compound of formula I

wherein

Ri, R₂ and R₃ are independently selected from H, halogen, amino, -OH, optionally substituted alkyl, alkenyl, alkynyl, alkoxy, -Obenzyl, cycloalkyl, aryl, heterocyclyl, -NR₅R₆, and -NR₅COR₇, wherein

R₅ and R₆ are each independently selected from hydrogen, optionally substituted alkyl, cycloalkyl, aryl, and heterocyclyl, and

R₇ is selected from an optionally substituted alkyl, cycloalkyl, aryl, and heterocyclyl, and

R₄ is independently selected from hydrogen, halogen, alkoxy, and alkyl.

13. The chemosensitization method of claim 12 wherein R₂, R₃, and R₄ are each hydrogen.

14. The chemosensitization method of claim 12 wherein R₁ is an optionally substituted heterocyciyl Or-NR₅R₆, wherein R₅ and R₆ are independently hydrogen or an optionally substituted alkyl.

15. The pharmaceutical composition according to Claim 12 wherein said compound of formula I is selected from the group consisting of:

16. The pharmaceutical composition, according to Claim 12, wherein said compound of formula I is

17. The pharmaceutical composition, according to Claim 12, wherein said compound of formula I is

18. The pharmaceutical composition, according to Claim 12, further comprising a chemotherapeutically effective amount of at least one chemotherapeutic agent.