WO2007098089A2 - Treatment of hyperproliferative diseases with methotrexate n-oxide and analogs - Google Patents

Treatment of hyperproliferative diseases with methotrexate n-oxide and analogs Download PDF

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Publication number
WO2007098089A2
WO2007098089A2 PCT/US2007/004250 US2007004250W WO2007098089A2 WO 2007098089 A2 WO2007098089 A2 WO 2007098089A2 US 2007004250 W US2007004250 W US 2007004250W WO 2007098089 A2 WO2007098089 A2 WO 2007098089A2
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methyl
pteridinyl
diamino
methylamino
carbonyl
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PCT/US2007/004250
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French (fr)
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WO2007098089A3 (en
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John G. Curd
John F.W. Keana
Alshad S. Lalani
Paul B. Westberg
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Novacea, Inc.
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Publication of WO2007098089A3 publication Critical patent/WO2007098089A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D475/00—Heterocyclic compounds containing pteridine ring systems
    • C07D475/02—Heterocyclic compounds containing pteridine ring systems with an oxygen atom directly attached in position 4
    • C07D475/04—Heterocyclic compounds containing pteridine ring systems with an oxygen atom directly attached in position 4 with a nitrogen atom directly attached in position 2

Definitions

  • the present invention relates to novel compounds having activity for treating hyperproliferative disorders, including neoplastic and non-neoplastic disorders, as well as certain inflammatory conditions.
  • the invention also relates to pharmaceutical compositions and formulations comprising the novel compounds. Further, the invention relates to methods of using the novel compounds, alone or in combination with one or more other active agents or treatments, to treat hyperproliferative disorders, including various cancers.
  • Methotrexate is an antifolate and is used for the treatment of acute leukemia, malignant lymphoma and other diseases. It is also known as an immunosuppressive drug and is primarily used for preventing acute graft- versus-host reactions in bone marrow transplantation. Furthermore, administration of low doses of methotrexate is known to be effective for the treatment of rheumatoid arthritis.
  • U.S. Patent No. 6,559,149 describes methotrexate derivatives that are represented by the following general fo ⁇ nula (II):
  • R is a lower alkyl group having 1 -4 carbon atoms
  • R 2 is a lower alkyl group having 1-4 carbon atoms or a trifluoromethyl group
  • R 3 is a hydrogen atom, a lower alkyl group having 1-4 carbon atoms or a trifluoromethyl group
  • R 4 is a hydrogen atom or a lower alkyl group having 1-4 carbon atoms
  • R 5 is a group represented by the general formula COOR ⁇ (where Rs is a hydrogen atom or a lower alkyl group having 1 -4 carbon atoms) or a group represented by the formula SO 3 H; and n is an integer of 1-4, or where W is a group represented by the general formula:
  • R 7 is a lower alkyl group having 1-4 carbon atoms
  • Rs is a hydrogen atom or a lower alkyl group having 1-4 carbon atoms
  • R 9 is a group represented by the general formula COOR 10 (where Rio is a hydrogen atom or a lower alkyl group having 1-4 carbon atoms) or a group represented by the formula SO 3 H; and m is an integer of 1-4], or the general formula:
  • R) i is a hydrogen atom or a lower alkyl group having 1-4 carbon atoms
  • Ri 2 is a hydrogen atom or a lower alkyl group having 1-4 carbon atoms
  • R13 is a group represented by the general formula COORi 4 (where Ri 4 is a hydrogen atom or a lower alkyl group having 1-4 carbon atoms) or a group represented by the formula SO 3 H; and 1 is an integer of 1-4, or W is a group represented by the general formula:
  • Rj 5 is a hydrogen atom or a lower alkyl group having 1-4 carbon atoms
  • R] 6 is a hydrogen atom or a lower alkyl group having 1-4 carbon atoms
  • k is an integer of 2 or 3.
  • 6,559,149 are the following compounds: dimethyl N- ⁇ 4-[ ⁇ -(2,4-diamino-6-pteridinyl)methyl- ⁇ -methylamino]- 3-methyl ⁇ benzoyl-L-2-aminoadipate; iV- ⁇ 4-[iV-(2,4-diamino-6-pteridinyl)methyl-N'-methylamino]-3- methyl ⁇ benzoyl-L-2-aminoadipic acid; iy- ⁇ 4-[7V-(2,4-diamino-6-pteridinyl)methyl-N-methylamino]-3- trifluoromethyl ⁇ benzoyl-L-2-aminoadipic acid; dimethyl N- ⁇ 1 -[2,4-diammo-6-pteridinyl)methyl]-7-methylindoline-5- carbonyl ⁇ -L-2-aminoadipate;
  • N- ⁇ 4-[7V-(2,4-diamino-6-pteridinyI)methylamino]-3-methyl)benzoyl-L- 2-aminoadipic acid diethyl iV-4-[N'-(2,4-diamino-6-pteridinyl)methyl-N 1 -methylamino]-3- methylbenzoyl-L-glutamate; dimethyl N- ⁇ 4-[jV-(2,4-diamino-6-pteridinyl)methyl-N I -methylamino]- 3-methyl ⁇ benzoyl-L-2-aminoadipate;
  • Methotrexate is metabolized upon uptake by mammalian cells such that one or more glutamyl moieties are added to methotrexate to yield a mixture of methotrexate polyglutamates. See U.S. Patent 6,921,667. The number of glutamyl moieties that can be added to methotrexate generally varies from two to seven. [0006] U.S. Patent No. 4,584,375 discloses methotrexate derivatives and the synthesis thereof, wherein the methotrexate derivative has the general formula:
  • m is from 0 to 5.
  • the present invention is related to compounds, compositions and methods for treating hyperproliferative disorders, such as cancer and inflammation.
  • One aspect of the invention is drawn to compounds having Formula I:
  • R) is a lower alkyl group having 1-4 carbon atoms
  • R 2 is hydrogen, halo, hydroxy, cyano, a lower alkyl group having 1-4 carbon atoms, NRi 2 RnRi 4 , a lower alkoxy having 1-4 carbon atoms or a trifluoromethyl group, or R] and R 2 together form a 5 or 6 membered saturated or unsaturated heterocycle having one or two hetero atoms where the second heteroatom, when present, is S or O 3 wherein S is optionally substituted with one or two oxygen atoms;
  • R 3 is hydrogen, halo, cyano, alkoxy, cycloalkyloxy, benzyloxy, alkyl, cycloalkyl, aryl, heteroaryl, aryloxy, aralkyl, NR I 2 R I 3 R M or haloalkyl, or Ri and R 3 together form a 5 or 6 membered saturated or unsaturated heterocycle;
  • R 4 is hydrogen, hydroxy, halo, alkyl, a lower alkoxy or NR I 2 R I 3 R I4 ;
  • R 7 is COOR 17 , SO 3 H, PO 3 H 2 , NHCOOR 18 , NHCOR 18 , or CONR 17 R 18 wherein R 17 is hydrogen, alkyl, optionally substituted phenyl, carboxyalkyl or alkylsulfonyl and R 18 is alkyl, aryl, aralkyl or cycloalkyl; each of R 1 2 and R] 3 is hydrogen or alkyl; each of R 8 , Rn and Ri 4 is O or is absent provided that at least one of m is 0-6; and each of n and p is independently 1, 2 or 3; wherein one or more -CH 2 - groups of the terminal amino acid is optionally substituted with one or more halogen atoms.
  • the compounds having formula I are N 10 -oxide of methotrexate and analogs, selected from the N 10 -oxides of the group consisting of:
  • Methotrexate dimethyl N- ⁇ 4-[N'-(2,4-diamino-6-pteridinyl)methyl-N-methylamino]- 3-methyl ⁇ benzoyl-L-2-aminoadipate;
  • glutamic acid N- ⁇ l-[(2,4-diamino-6-pteridinyl)methyl-7-methylindole-5'-carbonyl ⁇ - L-2-arninoadipic acid; diethyl N- ⁇ 4-[/V'-(2,4-diamino-6-pteridinyl)rnethylarnino]-3- methyl ⁇ benzoyl-L-glutamate; dimethyl TV- ⁇ 4-[/V-(2,4-diamino-6-pteridinyl)methylamino]-3- methyl ⁇ benzoyl-L-2-aminoadipate; and
  • the hyperproliferative disorder is cancer.
  • the cancer is a solid tumor.
  • the cancer is selected from the group consisting of colon cancer, brain cancer, glioma, multiple myeloma, head and neck cancer, hepatocellular cancer, melanoma, ovarian cancer, cervical cancer, renal cancer, and non-small cell lung cancer.
  • the cancer is acute and chronic lymphocytic leukemia, acute granulocytic leukemia, adrenal cortex carcinoma, bladder carcinoma, breast carcinoma, cervical carcinoma, cervical hyperplasia, choriocarcinoma, chronic granulocytic leukemia, chronic lymphocytic leukemia, colon carcinoma, endometrial carcinoma, esophageal carcinoma, essential thrombocytosis, genitourinary carcinoma, hairy cell leukemia, head and neck carcinoma, Hodgkin's disease, Kaposi's sarcoma, lung carcinoma, lymphoma, malignant carcinoid carcinoma, malignant hypercalcemia, malignant melanoma, malignant pancreatic insulinoma, medullary thyroid carcinoma, melanoma, multiple myeloma, mycosis fungoides, myeloid and lymphocytic leukemia, neuroblastoma, non-Hodgkin's lymphoma, osteogenic sarcoma, ova
  • the hyperproliferative disorder is any one of age-related macular degeneration, Crohn's disease, cirrhosis, chronic inflammatory-related disorders, proliferative diabetic retinopathy, proliferative vitreoretinopathy, retinopathy of prematurity, granulomatosis, immune hyperproliferation associated with organ or tissue transplantation, an immunoproliferative disease or disorder, e.g., inflammatory bowel disease, psoriasis, rheumatoid arthritis, systemic lupus erythematosus (SLE), vascular hyperproliferation secondary to retinal hypoxia, or vasculitis.
  • an immunoproliferative disease or disorder e.g., inflammatory bowel disease, psoriasis, rheumatoid arthritis, systemic lupus erythematosus (SLE), vascular hyperproliferation secondary to retinal hypoxia, or vasculitis.
  • the invention is drawn to methods of treating, ameliorating, or preventing hyperproliferative disease in a subject comprising administering to said subject a therapeutically effective amount of an N-oxide of methotrexate or analog thereof.
  • An additional aspect of the present invention is a method for treating, ameliorating, or preventing hyperproliferative disorders in an animal comprising administering to the animal a therapeutically effective amount of a compound having Formula I in combination with one or more active agents or treatments.
  • the one or more active agent or treatment is a chemotherapeutic agent, a radiotherapeutic agent/treatment, an anti-angiogenesis agent, a vascular targeting agent, a hypoxia-inducible factor 1 (HIFl) inhibitor, an Hsp90 inhibitor, a tyrosine kinase inhibitor, a serine/threonine kinase inhibitor, a proteasome inhibitor, an HDAC inibitor, a caspase inducer, a CDK inhibitor, and a proapoptotic molecule.
  • the one or more active agent or treatment is used, has been used, or is known to be useful for the treatment of the hyperproliferative disorder.
  • the method of treating, ameliorating, or preventing hyperproliferative disorder in an animal comprises administering to the animal a therapeutically effective amount of methotrexate N 10 -oxide or analog thereof.
  • the methotrexate analog N I0 -oxide, or a pharmaceutically acceptable salt thereof is provided in combination with one or more active agents or treatments, for example, chemotherapeutic agents or radiotherapeutic agents/treatments.
  • the one or more chemotherapeutic agents can be any chemotherapeutic agent which is used, has been used, or is known to be useful for the treatment of hyperproliferative disorders.
  • the one or more radiotherapeutic agents or treatments can be external-beam radiation therapy, brachytherapy, thermotherapy, radiosurgery, charged-particle radiotherapy, neutron radiotherapy, photodynamic therapy, or radionuclide therapy.
  • the compound having Formula I can be administered prior to, during, and/or beyond administration of the one or more chemotherapeutic agents or radiotherapeutic agents or treatments.
  • the method of administering a compound having Formula I in combination with one or more chemotherapeutic agents or radiotherapeutic agents or treatments is repeated more than once.
  • the combination of a compound having Formula I and one or more chemotherapeutic agents or radiotherapeutic agents or treatments of the present invention will have additive potency or an additive therapeutic effect.
  • the invention also encompasses synergistic combinations where the therapeutic efficacy is greater than additive. Preferably, such combinations will reduce or avoid unwanted or adverse effects.
  • the combination therapies encompassed by the invention will provide an improved overall therapy relative to administration of a compound having Formula I or any chemotherapeutic agent or radiotherapeutic agent or treatment alone.
  • doses of existing or experimental chemotherapeutic agents or radiotherapeutic agents or treatments will be reduced or administered less frequently which will increase patient compliance, thereby improving therapy and reducing unwanted or adverse effects.
  • the methods of the invention will be useful not only with previously untreated patients but also will be useful in the treatment of patients partially or completely refractory to current standard and/or experimental cancer therapies, including but not limited to radiotherapies, chemotherapies, and/or surgery.
  • the invention will provide therapeutic methods for the treatment or amelioration of hyperproliferative disorders that have been shown to be or may be refractory or non-responsive to other therapies.
  • N-oxide compounds of the invention will function as prodrugs with greatly diminished cytotoxicity. It is believed that these N-oxide compounds will be activated under hypoxic conditions within the target tissues ⁇ i.e., reduced at the nitrogen atom), followed by inhibition of dihydrofolate reductase and/or thymidylate synthase, diminishing cells' ability to replicate. Other N-oxide compounds of the invention may have intrinsic cytotoxic activity. Since a number of pathological tissues have significant hypoxic components which promote hyperproliferation, it is believed that this portion of tissue will be preferentially targeted.
  • Ri-R 8 , m, n and p are defined above.
  • Ri is a lower alkyl group having 1-4 carbon atoms
  • each of R 2 and R 3 is independently hydrogen, halo, hydroxy, cyano, a lower alkyl group having 1-4 carbon atoms, a lower alkoxy having 1-4 carbon atoms, a trifluoromethyl group or -NRI 2 R H R H ;
  • R 8 -R 14 , X 1 , Y, Z, m, n and p are as defined above.
  • R 6 -R 8 , m, n and p are as defined above.
  • R & -R 8 , ni, n and p are as defined above.
  • a therapeutically effective amount of a compound having Formula I, or a pharmaceutically acceptable salt thereof, and at least one other active agent is provided in the form of a pharmaceutical composition having at least one pharmaceutically acceptable carrier.
  • the at least one other active agent is a chemotherapeutic agent (including an active vitamin D compound).
  • Compounds having Formula I may be formulated in a single formulation with the other active agent(s), or formulated independently.
  • a therapeutically effective amount of a compound having Formula 1, or a pharmaceutically acceptable salt thereof is administered to an animal in need thereof.
  • the hyperproliferative disorder is cancer.
  • a further aspect of the invention relates to methods for treating, ameliorating, or preventing a hyperproliferative disorder comprising administering a therapeutically effective amount of a compound having Formula I 3 or a pharmaceutically acceptable salt thereof, in combination with at least one other active agent or treatment to a patient in need thereof.
  • combinations of a compound having Formula I with a chemotherapeutic agent are administered.
  • the chemotherapeutic agent is selected from gemcitabine and irinotecan.
  • Hyperproliferative disorders which can be treated with the compounds having Formula I include any hypoxia-aggravated hyperproliferative disease and/or disorder, such as any number of cancers.
  • cancers include, without limitation, cancers of the bladder, brain, breast, cervix, colon, endometrium, esophagus, head and neck, kidney, larynx, liver, lung, oral cavity, ovaries, pancreas, prostate, skin, stomach, and testis.
  • Certain of these cancers may be more specifically referred to as acute and chronic lymphocytic leukemia, acute granulocytic leukemia, adrenal cortex carcinoma, bladder carcinoma, breast carcinoma, cervical carcinoma, cervical hyperplasia, choriocarcinoma, chronic granulocytic leukemia, chronic lymphocytic leukemia, colon carcinoma, endometrial carcinoma, esophageal carcinoma, essential thrombocytosis, genitourinary carcinoma, hairy cell leukemia, head and neck carcinoma, Hodgkin's disease, Kaposi's sarcoma, lung carcinoma, lymphoma, malignant carcinoid carcinoma, malignant hypercalcemia, malignant melanoma, malignant pancreatic insulinoma, medullary thyroid carcinoma, melanoma, multiple myeloma, mycosis fungoides, myeloid and lymphocytic leukemia, neuroblastoma, non-Hodgkin's lymphoma, osteogenic sar
  • the cancer is a solid tumor.
  • the cancer is selected from the group consisting of colon cancer, brain cancer, glioma, multiple myeloma, head and neck cancer hepatocellular cancer, melanoma, ovarian cancer, cervical cancer, renal cancer, and non-small cell lung cancer.
  • alkyl refers to an unsaturated acyclic hydrocarbon radical.
  • lower alkyl refers to acyclic hydrocarbon radicals containing from about 2 to about 10 carbon atoms, preferably from about 2 to about 8 carbon atoms and more preferably 1 to about 6 carbon atoms.
  • alkyl radicals examples include methyl, ethyl, propyl, butyl, isobutyl, pentyl, 2-methylbutyl, 3-methylbutyl, hexyl, heptyl, and octyl, and the like.
  • cycloalkyl refers to saturated hydrocarbon ring structures of from 3 to 12 carbon atoms, and preferably from 3 to 6 carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 2- methylcyclopropyl, cyclopropylmethyl, cyclopentylmethyl, norbornyl, adamantyl, pinanyl, myrtanyl and the like. "Lower cycloalkyl” refers to cycloalkyl of 3 to 6 carbons.
  • alkoxy means a straight, branched or cyclic hydrocarbon configuration and combinations thereof, including from 1 to 20 carbon atoms, preferably from 1 to 8 carbon atoms, more preferably from 1 to about 4 carbon atoms, and an oxygen atom at the point of attachment.
  • Suitable alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec- butoxy, tert-butoxy, cyclopropyloxy, cyclohexyloxy, and the like.
  • “Lower alkoxy” refers to alkoxy groups having from 1 to 4 carbon atoms.
  • aryl refers to an aromatic hydrocarbon radical with 4 to about 16 carbon atoms, preferably of 6 to about 12 carbon atoms, and more preferably of 6 to about 10 carbon atoms.
  • the rings may optionally be substituted with 1-3 substituents selected from alkyl, halogen, hydroxy, alkoxy, aryloxy, haloalkyl, phenyl and heteroaryl. Examples of aryl groups are phenyl, biphenyl and naphthyl.
  • heterocycle refers to a cyclic hydrocarbon structure of from 1 to 6, preferably 5 to 6, atoms, and containing from 1 to 3 heteroatoms selected from O, N and S; or a bicyclic 9- to 10-membered heterocyclic system containing from 1 to 4 heteroatoms chosen from O, N and S.
  • Useful heteroaryl groups include thienyl, benzo[b]thienyl, naphtho[2,3- b]thienyl, thianthrenyl, furyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxanthenyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrirnidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalzinyl, naphthyridinyl, quinozalinyl, cinnolinyl, pteridinyl, carbazolyl, ⁇ -car
  • heteroaryl group contains a nitrogen atom in a ring
  • nitrogen atom may be in the form of an N-oxide, e.g., a pyridyl N- oxide, pyrazinyl N-oxide, pyrimidinyl N-oxide, and the like.
  • prodrug refers to a derivative of a parent
  • N-oxide molecule that requires biotransformation, either solvolytic or enzymatic, within the organism to release the active drug.
  • the N-oxides may be considered as prodrugs of the corresponding non-N-oxide parent compound, the term "prodrug” as used herein does not refer to the N-oxides. Rather, prodrugs of the present inventions are limited to variations or derivatives of the compounds of this invention which have groups cleavable under metabolic conditions to form the compounds of the present invention.
  • prodrugs of the present invention become the compounds of the invention when they undergo solvolysis under physiological conditions or undergo enzymatic degradation (e.g., hydrolysis), oxidation (e.g., hepatic hydroxylation) or reduction, excluding the bioreductive process that transforms the N-oxides of the present invention to the corresponding non-N-oxides of the parent compounds.
  • enzymatic degradation e.g., hydrolysis
  • oxidation e.g., hepatic hydroxylation
  • reduction excluding the bioreductive process that transforms the N-oxides of the present invention to the corresponding non-N-oxides of the parent compounds.
  • Typical enzymatically or solvolytically cleavable groups suitable for preparing the prodrugs of the present invention include esters, imines, carbamates, acetals and ketals.
  • an ester of a carboxylic acid containing compound of the present invention may be prepared by condensation with an alcohol, preferably a lower alkyl alcohol, more preferably a C) -4 alkyl alcohol.
  • an ester of a hydroxy containing compound of the present invention may be prepared by condensation with a carboxylic or a dioic acid, preferably an alkyl carboxylic or dioic acid, more preferably a C 1 4 carboxylic acid or a C 3 6 dioic acid or anhydride thereof.
  • an imine of an amino containing compound of the present invention may be obtained by condensation of the amino group with a carbonyl group of an aldehyde or a ketone.
  • Aldehydes and ketones suitable for condensation with amino containing compounds of the present invention include alkyl and aryl ketones and aldehydes, more preferably alky ketones and aldehydes, more preferably lower alkyl ketones and aldehydes, most preferably Ci -4 alkyl aldehydes and ketones.
  • a carbamate of an amino containing compound of the present invention may be prepared by condensation of the amino group with, for example, benzyloxycarbonyl chloride.
  • an acetal or ketal of an alcohol containing compound of the present invention may be obtained by condensation of the hydroxy group with chloromethyl methyl ether or chloromethyl ethyl ether.
  • non-N-oxide refers to an amine compound that is not oxidized at the nitrogen atom.
  • methotrexate is the non-N-oxide form of methotrexate N-oxide.
  • metalhotrexate N-oxides and analogs and similar terms as used herein are intended to refer to compounds in which a tertiary or an aromatic amine present in the compounds is oxidized. Any tertiary or aromatic amine suitable to afford a hypoxia-activatable N-oxide of the active compound may be oxidized to prepare the N-oxides of the present invention. Particularly preferred N-oxides of the present invention are those in which the N 10 nitrogen of methotrexate, or the corresponding nitrogen of the analog, is oxidized to N 10 -oxide. As used herein, N 10 refers to the nitrogen atom of the compound of formula I to which Ri and R 8 are attached.
  • compositions are to be understood as defining compositions of which the individual components or ingredients are themselves pharmaceutically acceptable, e.g., where oral administration is foreseen, acceptable for oral use; where topical administration is foreseen, topically acceptable; and where intravenous administration is foreseen, intravenously acceptable.
  • a therapeutically effective amount refers to that amount of the therapeutic agent sufficient to result in amelioration of one or more symptoms of a disorder, or prevent advancement of a disorder, or cause regression of the disorder.
  • a therapeutically effective amount preferably refers to the amount of a therapeutic agent that decreases the rate of tumor growth, decreases tumor mass, decreases the number of metastases, increases time to tumor progression, or increases survival time by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.
  • prevention refers to a decrease in the occurrence of pathological cells (e.g., hyperproHferative or neoplastic cells) in an animal.
  • the prevention may be complete, e.g., the total absence of pathological cells in a subject.
  • the prevention may also be partial, such that the occurrence of pathological cells in a subject is less than that which would have occurred without the present invention.
  • compositions having Formula I can be provided as pharmaceutically acceptable salts.
  • pharmaceutically acceptable salts include inorganic and organic acid addition salts such as hydrochloride, hydrobromide, phosphate, sulphate, citrate, lactate, tartrate, maleate, fumarate, mandelate, benzoate and oxalate; and inorganic and organic base addition salts with bases such as sodium hydroxide, Tris(hydroxymethyl)arninornethane (TRIS, tromethane) and /V-mcthyl-glucamine.
  • inorganic and organic acid addition salts such as hydrochloride, hydrobromide, phosphate, sulphate, citrate, lactate, tartrate, maleate, fumarate, mandelate, benzoate and oxalate
  • bases such as sodium hydroxide, Tris(hydroxymethyl)arninornethane (TRIS, tromethane) and /V-mcthyl-glucamine.
  • salts typically have similar physiological properties compared to the free base, certain acid addition salts may demonstrate preferred physicochemical properties, e.g., enhanced solubility, improved stability.
  • One particular pharmaceutically acceptable salt is derived from maleic acid, the salt being either a hydrogen maleate or a dimaleate salt.
  • Certain of the compounds of the present invention may exist as stereoisomers including optical isomers.
  • the invention includes all stereoisomers and both the racemic mixtures of such stereoisomers as well as the individual enantiomers that may be separated according to methods that are well known to those of ordinary skill in the art.
  • Certain of the compounds of the present invention may also exist as diasteroisomers wherein one or more substituents on the methotrexate analog contain one or more chiral centers.
  • compounds having Formula I are administered in combination with one or more other active agents (e.g., chemotherapeutic agents) or treatments.
  • a patient may be treated for a hyperproliferative disorder, such as cancer, by the administration of a therapeutically effective amount of a compound having Formula 1 in combination with radiotherapy agent/treatment or the administration of a chemotherapeutic agent.
  • compounds of the invention are administered in combination with agents, such as anti-angiogenic agents, that block inhibit or modulate tumor neovascularization.
  • agents such as anti-angiogenic agents, that block inhibit or modulate tumor neovascularization.
  • anti- angiogenesis agents can be any anti-angiogenesis agent which is used, has been used, or is known to be useful for the treatment of hyperproliferative disorders.
  • anti-angiogenesis agents examples include bevacizumab (AvastinTM), VEGF-TRAP, anti-VEGF-receptor antibodies, angiostatin, endostatin, batimastat, captopril, cartilage derived inhibitor, genistein, interleukin 12, lavendustin, medroxyprogesterone acetate, recombinant human platelet factor 4, tecogalan, thrombospondin, TNP-470, VEGF antagonists, anti-VEGF monoclonal antibody, soluble VEGF-receptor chimaeric protein, antisense oligonucleotides, antisense oligodexoynucleotides, siRNAs, anti- VEGF aptamers, pigment epithelium derived factor, a tyrosine kinase inhibitor, an inhibitor of epidermal-derived growth factor, an inhibitor of .
  • vastinTM bevacizumab
  • VEGF-TRAP anti
  • fibrob last-derived growth factor an inhibitor of platelet derived growth factor, an MMP (matrix metalloprotease) inhibitor, an integrin blocker, interferon- ⁇ , pentosan polysulfate, a cyclooxygenase inhibitor, carboxyamidotriazole, combretastatin A-4, squalamine, ⁇ -O-chloroacetyl-carbonyO-fumagillol, thalidomide, troponin-1, indolinethiones, pyridopyrimidines, quinoazolines, phenyl-pyrrolo-pyrimidines, trastuz ⁇ mab, calcium influx inhibitor (CAl), neomycin, squalamine, marimastat, prinomastat (AG-3340), metastat (COL-3) and cinnoline derivatives.
  • MMP matrix metalloprotease
  • integrin blocker interferon- ⁇
  • pentosan polysulfate
  • the compounds of the present invention are administered in combination with a vascular targeting agent (also known as vascular damaging agents).
  • vascular targeting agent also known as vascular damaging agents.
  • the vascular targeting agent is for the treatment of malignant or non-malignant vascular proliferative disorders.
  • vascular targeting agents can be any vascular targeting agent which is used, has been used, or is known to be useful for the treatment of hyperproliferative disorders.
  • vascular targeting agents examples include DMXAA 5,6-dimethylxanthenone-4-acetic acid, ZD6126, (5S)-5-(acetylamino)-9,10,l l-trimethoxy-6,7-dihydro-5H- dibenzo[a,c]cyclohepten-3-yl dihydrogen phosphate, also known as iV-acetylcolchinol-O-phosphate (see, for example, U.S. Patent No. 6,906,048); functionalized stilbene derivatives such as combretastatin A4 and its prodrugs (see, e.g., U.S. Patent Nos.
  • dioleoyltrimethyl- ammonium propane DOTAP
  • N-[l-(2,3-dioleoyloxy)-propyl]-N,N,N- trimethylammonium chloride DOTMA
  • DDAB dimethyldioctadecylammonium bromide
  • DMRIE dimethyldioctadecylammonium bromide
  • DODAP dioleoyl-3-dimethylammonium propane
  • DODAC N,N-dioleyl-N,N- dimethylammoni ⁇ m chloride
  • DOSPA N-(l-(2,3-dioleyloxy)propyl)-N- (2-(spe ⁇ iinecarboxamido)ethyl)-N,N-dimethyl ammonium trifluoroacetate
  • DOSPA dioleoyltrimethyl- ammonium propane
  • DOSPA dioleoleyloxypropyl
  • DOSPA N-(l-(
  • the compounds of the present invention are administered in combination with a hypoxia-inducible factor 1 (HIFl) inhibitor.
  • HIFl hypoxia-inducible factor 1
  • the HIFl inhibitor is for the treatment of malignant or non-malignant vascular proliferative disorders.
  • HIFl inhibitors can be any HIFl inhibitor which is used, has been used, or is known to be useful for the treatment of hyperproliferative disorders.
  • HIFl inhibitors suitable for use in combination with compounds of the present invention include topotecan, P13 kinase inhibitors; LY294002; rapamycin; histone deacetylase inhibitors such as [(E)- (lS,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia- 5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene-3,6,9,19,22-pentanone (FR901228, depsipeptide); heat shock protein 90 (Hsp90) inhibitors such as geldanamycin, 17-allylamino-geldanamycin (17-AAG), and other geldanamycin analogs, and radicicol and radicicol derivatives such as KF58333; genistein; indanone; staurosporin; protein kinase-1 (
  • the compounds of the present invention are administered in combination with an Hsp90 inhibitor.
  • the Hsp90 inhibitor is for the treatment of malignant or non-malignant vascular proliferative disorders.
  • Hsp90 inhibitors can be any Hsp90 inhibitor which is used, has been used, or is known to be useful for the treatment of hyperproliferative disorders.
  • Hsp90 inhibitors that may be combined with the compounds of the present invention include geldanamycin, 17-allylamino-l 7-demethoxygeldanamycin, geldanamycin derivatives such as those described in U.S. patent No.
  • the compounds of the present invention are administered in combination with an inhibitor of tyrosine and/or serine/threonine kinases and tyrosine kinase receptors involved in cellular signaling.
  • Examples of tyrosine kinase and serine/threonine kinase inhibitors include (but not limited to): AMG706, ZA6474, BAY 43-9006, Dasatinib, CEP-701, XL647, XL999, Lapatinb, MLN518/CT53518, PKC412, ST1571, AMN107, AEE 788, OSI-930, OSI- 817, SUl 1248, AG-03736, GW-786034m , CEP-7055.
  • the compounds of the present invention are administered in combination with HDAC inhibitors. Examples include (but not limited to) SAHA, MS-275, MGCD0103, LBH589, PXDlOl, FK228.
  • the compounds of the present invention are administered in combination with proteasome inhbitors such as Velcade.
  • the compounds of the present invention are administered in combination with pro-apoptotic agents such as TRAIL, anti- DR4/DR5 (TRA8) antibodies, IAP, Survivin or small molecules that stimulate caspase activation.
  • pro-apoptotic agents such as TRAIL, anti- DR4/DR5 (TRA8) antibodies, IAP, Survivin or small molecules that stimulate caspase activation.
  • the compounds of the present invention are administered in combination with inhibitors of cell cycle regulators such as CDK inhibitors.
  • “In combination” refers to the use of more than one treatment.
  • the use of the term “in combination” does not restrict the order in which treatments are administered to a subject being treated for a hyperproliferative disorder.
  • a first treatment can be administered prior to, concurrently with, after, or within any cycling regimen involving the administration of a second treatment to a subject with a hyperproliferative disorder.
  • the first treatment can be administered 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before a treatment; or the first treatment can be administered 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after a second treatment.
  • Such treatments include, for example, the administration of compounds having Formula I in combination with one or more chemotherapeutic agents or radiotherapeutic agents/treatments.
  • chemotherapeutic agent is intended to refer to any chemotherapeutic agent known to those of skill in the art to be effective for the treatment, prevention or amelioration of hyperproliferative disorders s ⁇ ch as cancer.
  • Chemotherapeutic agents include, but are not limited to, small molecules, synthetic drugs, peptides, polypeptides, proteins, nucleic acids (e.g., DNA and RNA polynucleotides including, but not limited to, antisense nucleotide sequences, triple helices and nucleotide sequences encoding biologically active proteins, polypeptides or peptides), antibodies, synthetic or natural inorganic molecules, mimetic agents, and synthetic or natural organic molecules.
  • nucleic acids e.g., DNA and RNA polynucleotides including, but not limited to, antisense nucleotide sequences, triple helices and nucleotide sequences encoding biologically active proteins, polypeptides or peptides
  • antibodies synthetic or natural inorgan
  • Any agent which is known to be useful, or which has been used or is currently being used for the treatment or amelioration of a hyperproliferative disorder can be used in combination with a compound having Formula I. See, e.g., Hardman et ah, eds., 2002, Goodman & Gilman's The Pharmacological Basis Of Therapeutics 10th Ed, Mc-Graw-Hill, New York, NY for information regarding therapeutic agents which have been or are currently being used for the treatment or amelioration of a hyperproliferative disorder.
  • chemotherapeutic agents useful in the methods and compositions of the invention include alkylating agents, antimetabolites, antimitotic agents, epipodophyllotoxins, antibiotics, hormones and hormone antagonists, enzymes, platinum coordination complexes, anthracenediones, substituted ureas, methylhydrazine derivatives, imidazotetrazine derivatives, cytoprotective agents, DNA topoisomerase inhibitors, biological response modifiers, retinoids, therapeutic antibodies, differentiating agents, immunomodulatory agents, angiogenesis inhibitors and anti-angiogenic agents.
  • chemotherapeutic agents include, but are not limited to, abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, asparaginase, BCG live, bevaceizumab, bexarotene, bleomycin, bortezomib, busulfan, calusterone, camptothecin, capecitabine, carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cinacalcet, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, darbepoetin alfa, daunorubicin, denileukin diftitox, dexrazoxane, docetaxel, doxorubic
  • Chemotherapeutic agents may be administered at doses that are recognized by those of skill in the art to be effective for the treatment of the hyperproliferative disorder. In certain embodiments, chemotherapeutic agents may be administered at doses lower than those used in the art due to the additive or synergistic effect of the compounds having Formula I.
  • Therapeutic agents useful in the methods and compositions of the invention include active vitamin D compound or mimics thereof, antineoplastic agents ⁇ e.g., actinomycin D, irinotecan, vincristine, vinorelbine, SN-38, azacitidine (5-azacytidine, 5AzaC), thalidomide vinblastine, methotrexate, azathioprine, fluorouracil, doxorubicin, mitomycin, docetaxel, paclitaxel), angiogenic inhibitors (e.g., VEGF-TRAP, angiostatin, endostatin, aptamer antogonist of VEGF, batimastat, captopril, cartilage derived inhibitor, genistein, interleukin 12, lavendustin, medroxypregesterone acetate, recombinant human platelet factor 4, tecogalan, thrombospondin and TNP-470), serine/threonine
  • patients are subjected to a hypoxia imaging technique prior to administration of the compositions comprising the compounds of the present invention.
  • imaging techniques suitable for the determination of the presence of hypoxic tumor cells include computed tomography (CT), magnetic resonance imaging (MRI), single photon emission computer tomography (SPECT), and positron emission tomography (PET).
  • CT computed tomography
  • MRI magnetic resonance imaging
  • SPECT single photon emission computer tomography
  • PET positron emission tomography
  • the invention is directed to a method of treating, preventing or ameliorating a hyperproliferative disease in an animal in need thereof, comprising determining whether said hyperproliferative disease is characterized by hypoxic tissue, and treating said animal with an effective amount of a compound of the invention.
  • radiotherapeutic agent is intended to refer to any radiotherapeutic agent known to one of skill in the art to be effective to treat or ameliorate cancer, without limitation.
  • the radiotherapeutic agent can be an agent such as those administered in brachytherapy or radionuclide therapy.
  • Such methods can optionally further comprise the administration of one or more additional cancer therapies, such as, but not limited to, chemotherapies, surgery, and/or another radiotherapy.
  • the present invention relates to a method for treating cancer comprising the administration of methotrexate or analog N-oxide having Formula I, in combination with a treatment comprising a therapeutically effective dose of brachytherapy.
  • the brachytherapy can be administered according to any schedule, dose, or method known to one of skill in the art to be effective in the treatment or amelioration of cancer, without limitation.
  • brachytherapy comprises insertion of radioactive sources into the body of a subject to be treated for cancer, preferably inside the tumor itself, such that the tumor is maximally exposed to the radioactive source, while preferably minimizing the exposure of healthy tissue.
  • the brachytherapy can be intracavitary brachytherapy.
  • the brachytherapy can be interstitial brachytherapy.
  • the brachytherapy can be administered at a high dose rate, a continuous low dose rate, or a pulsed dose rate.
  • a high dose rate brachytherapy regimen can be a dose of 60 Gy administered in ten fractions over six days
  • a continuous low dose rate brachytherapy regimen can be a total dose of about 65 Gy, administered continuously at about 40 to 50 cGy per hour.
  • Other examples of high, continuous low, and pulsed dose rate brachytherapy are well known in the art. See, e.g., Mazeron et al, Sent. Rad. One. 12:95-108 (2002).
  • Radioisotopes that can be administered in any of the above-described brachytherapies include, but are not limited to, phosphorus 32, cobalt 60, palladium 103, ruthenium 106, iodine 125, cesium 137, indium 192, xenon 133, radium 226, californium 252, or gold 198.
  • Other radioisotopes may be selected for administration in brachytherapy according to the desirable physical properties of such a radioisotope.
  • Radioisotope's suitability for use in brachytherapy including, but not limited to, the radioisotope's half-life, the degree to which emitted radiation penetrates surrounding tissue, the energy of emitted radiation, the ease or difficulty of adequately shielding the radioisotope, the availability of the radioisotope, and the ease or difficulty of altering the shape of the radioisotope prior to administration.
  • the present invention relates to a method for treating cancer comprising the administration of methotrexate or analog N-oxide having Formula I, in combination with a treatment comprising a therapeutically effective dose of a radionuclide.
  • the radionuclide therapy can be administered according to any schedule, dose, or method known to one of skill in the art to be effective in the treatment or amelioration of cancer, without limitation.
  • radionuclide therapy comprises systemic administration of a radioisotope that preferentially accumulates in or binds to the surface of cancerous cells.
  • the preferential accumulation of the radionuclide can be mediated by a number of mechanisms, including, but not limited to, incorporation of the radionuclide into rapidly proliferating cells, specific accumulation of the radionuclide by the cancerous tissue without special targeting (e.g., iodine 131 accumulation in thyroid cancer), or conjugation of the radionuclide to a biomolecule specific for a neoplasm.
  • mechanisms including, but not limited to, incorporation of the radionuclide into rapidly proliferating cells, specific accumulation of the radionuclide by the cancerous tissue without special targeting (e.g., iodine 131 accumulation in thyroid cancer), or conjugation of the radionuclide to a biomolecule specific for a neoplasm.
  • a specific biomolecule for use in targeting a particular neoplasm for radionuclide therapy based upon the cell-surface molecules present on that neoplasm.
  • hepatomas may be specifically targeted by an antibody specific for ferritin, which is frequently over-expressed in such tumors.
  • antibody-targeted radioisotopes for the treatment of cancer include ZEVALIN (ibritumomab tiuxetan) and BEXXAR (tositumomab), both of which comprise an antibody specific for the B cell antigen CD20 and are used for the treatment of non-Hodgkin lymphoma.
  • the present invention relates to a method for treating cancer comprising the administration of methotrexate or analog N-oxide having Formula I, in combination with a treatment comprising a therapeutically effective dose of external-beam radiation therapy.
  • the external-beam radiation therapy can be administered according to any schedule, dose, or method known to one of skill in the art to be effective in the treatment or amelioration of cancer, without limitation.
  • external-beam radiation therapy comprises irradiating a defined volume within a subject with a high energy beam, thereby causing cell death within that volume.
  • the irradiated volume preferably contains the entire cancer to be treated, and preferably contains as little healthy tissue as possible.
  • the external-beam radiation therapy can be three-dimensional conformal radiotherapy.
  • the external-beam radiation therapy can be continuous hyperfractionated radiotherapy.
  • the external-beam radiation therapy can be intensity-modulated radiotherapy.
  • the external-beam radiation therapy can be helical tomotherapy.
  • the external-beam radiation therapy can be three-dimensional conformal radiotherapy with dose escalation.
  • the external-beam radiation therapy can be stereotactic radiotherapy, including, but not limited to, single fraction stereotactic radiotherapy, fractionated stereotactic radiotherapy, and fractionated stereotactically guided conformal radiotherapy.
  • the external-beam radiation therapy can be generated or manipulated by any means known to one of skill in the art.
  • the photon beam used in external-beam radiation therapy can be shaped by a multileaf collimator.
  • suitable devices for generating a photon beam for use in external-beam radiation therapy include a gamma knife and a linac- based stereotactic apparatus.
  • administration of the external-beam radiation therapy is controlled by a computer according to a three-dimensional model of the patient in the treatment position.
  • Such a model can be generated, for example, by computed tomography (CT), magnetic resonance imaging (MRI), single photon emission computer tomography (SPECT), and positron emission tomography (PET).
  • CT computed tomography
  • MRI magnetic resonance imaging
  • SPECT single photon emission computer tomography
  • PET positron emission tomography
  • healthy tissues can optionally be protected from the effects of the external-beam radiation therapy by placing blocking devices such as, e.g., lead shields, in locations where such protection is needed.
  • blocking devices such as, e.g., lead shields
  • metal reflecting shields can optionally be located to reflect the photon beam in order to concentrate the radiation on the cancerous tissue to be treated and protect healthy tissue. Placement of either shield is well within the knowledge of one of skill in the art.
  • Methods of administering and apparatuses and compositions useful for external-beam radiation therapy can be found in U.S. Patent Nos.
  • the present invention relates to a method for treating cancer comprising the administration of methotrexate or analog N-oxide having Formula I, in combination with a treatment comprising a therapeutically effective dose of thermotherapy.
  • the thermotherapy can be administered according to any schedule, dose, or method known to one of skill in the art to be effective in the treatment or amelioration of cancer, without limitation.
  • the thermotherapy can be cryoablation therapy.
  • the thermotherapy can be hyperthermic therapy.
  • the thermotherapy can be a therapy that elevates the temperature of the tumor higher than in hyperthermic therapy.
  • Cryoablation therapy involves freezing of a neoplastic mass, leading to deposition of intra- and extra-cellular ice crystals; disruption of cellular membranes, proteins, and organelles; and induction of a hyperosmotic environment, thereby causing cell death.
  • Cryoablation can be performed in one, two, or more freeze-thaw cycles, and further the periods of freezing and thawing can be adjusted for maximum tumor cell death by one of skill in the art.
  • One exemplary device that can be used in cryoablation is a cryoprobe incorporating vacuum-insulated liquid nitrogen. See, e.g., Murphy et al., Sem. Urol. Oncol 19: 133-140 (2001).
  • any device that can achieve a local temperature of about -18O 0 C to about -195 0 C can be used in cryoablation therapy.
  • Methods for and apparatuses useful in cryoablation therapy are described in U.S. Patent Nos. 6,383,181, 6,383,180, 5,993,444, 5,654,279, 5,437,673, and 5,147,355, each of which is incorporated herein by reference in its entirety.
  • Hyperthermic therapy typically involves elevating the temperature of a neoplastic mass to a range from about 42°C to about 44 0 C. The temperature of the cancer may be further elevated above this range; however, such temperatures can increase injury to surrounding healthy tissue while not causing increased cell death within the tumor to be treated.
  • the tumor may be heated in hyperthermic therapy by any means known to one of skill in the art without limitation.
  • the tumor may be heated by microwaves, high intensity focused ultrasound, ferromagnetic thermoseeds, localized current fields, infrared radiation, wet or dry radiofrequency ablation, laser photocoagulation, laser interstitial thermic therapy, and electrocautery.
  • Microwaves and radiowaves can be generated by waveguide applicators, horn, spiral, current sheet, and compact applicators.
  • the present invention relates to a method for treating cancer comprising the administration of methotrexate or analog N-oxide having Formula I, in combination with a treatment comprising a therapeutically effective dose of radiosurgery.
  • the radiosurgery can be administered according to any schedule, dose, or method known to one of skill in the art to be effective in the treatment or amelioration of cancer, without limitation.
  • radiosurgery comprises exposing a defined volume within a subject to a manually directed radioactive source, thereby causing cell death within that volume.
  • the irradiated volume preferably contains the entire cancer to be treated, and preferably contains as little healthy tissue as possible.
  • the tissue to be treated is first exposed using conventional surgical techniques, then the radioactive source is manually directed to that area by a surgeon.
  • the radioactive source can be placed near the tissue to be irradiated using, for example, a laparoscope.
  • Methods and apparatuses useful for radiosurgery are further described in Valentini et al, Eur. J. Surg. Oncol. 28:180-185 (2002) and in U.S. Patent Nos. 6,421 ,416, 6,248,056, and 5,547,454, each of which is incorporated herein by reference in its entirety.
  • the present invention relates to a method for treating cancer comprising the administration of methotrexate or analog N-oxide having Formula I, in combination with a treatment comprising a therapeutically effective dose of charged-particle radiotherapy.
  • the charged-particle radiotherapy can be administered according to any schedule, dose, or method known to one of skill in the art to be effective in the treatment or amelioration of cancer, without limitation.
  • the charged-particle radiotherapy can be proton beam radiotherapy.
  • the charged-particle radiotherapy can be helium ion radiotherapy.
  • charged-particle radiotherapy comprises irradiating a defined volume within a subject with a charged-particle beam, thereby causing cellular death within that volume.
  • the irradiated volume preferably contains the entire cancer to be treated, and preferably contains as little healthy tissue as possible.
  • a method for administering charged-particle radiotherapy is described in U.S. Patent No. 5,668,371, which is incorporated herein by reference in its entirety.
  • the present invention relates to a method for treating cancer comprising the administration of methotrexate or analog N-oxide having Formula J, in combination with a treatment comprising a therapeutically effective dose of neutron radiotherapy.
  • the neutron radiotherapy can be administered according to any schedule, dose, or method known to one of skill in the art to be effective in the treatment or amelioration of cancer, without limitation.
  • the neutron radiotherapy can be a neutron capture therapy.
  • a compound that emits radiation when bombarded with neutrons and preferentially accumulates in a neoplastic mass is administered to a subject.
  • the tumor is irradiated with a low energy neutron beam, activating the compound and causing it to emit decay products that kill the cancerous cells.
  • Such compounds are typically boron containing compounds, but any compound that has a significantly larger neutron capture cross-section than common body constituents can be used.
  • the neutrons administered in such therapies are typically relatively low energy neutrons having energies at or below about 0.5 eV.
  • the compound to be activated can be caused to preferentially accumulate in the target tissue according to any of the methods useful for targeting of radionuclides, as described below, or in the methods described in Laramore, Semin. Oncol. 24:612-685 (1997) and in U.S. Patents Nos. 6,400,796, 5,877,165, 5,872,107, and 5,653,957, each of which is incorporated herein by reference in its entirety.
  • the neutron radiotherapy can be a fast neutron radiotherapy.
  • fast neutron radiotherapy comprises irradiating a defined volume within a subject with a neutron beam, thereby causing cellular death within that volume.
  • the irradiated volume preferably contains the entire cancer to be treated, and preferably contains as little healthy tissue as possible.
  • high energy neutrons are administered in such therapies, with energies in the range of about 10 to about 100 million eV.
  • fast neutron radiotherapy can be combined with charged-particle radiotherapy in the administration of mixed proton-neutron radiotherapy.
  • the present invention relates to a method for treating cancer comprising the administration of methotrexate or analog N-oxide having Formula I, in combination with a treatment comprising a therapeutically effective dose of photodynamic therapy.
  • the photodynamic therapy can be administered according to any schedule, dose, or method known to one of skill in the art to be effective in the treatment or amelioration of cancer, without limitation.
  • photodynamic therapy comprises administering a photosensitizing agent that preferentially accumulates in a neoplastic mass and sensitizes the neoplasm to light, then exposing the tumor to light of an appropriate wavelength. " Upon such exposure, the photosensitizing agent catalyzes the production of a cytotoxic agent, such as, e.g., singlet oxygen, which kills the cancerous cells.
  • a cytotoxic agent such as, e.g., singlet oxygen
  • Representative photosensitizing agents that may be used in photodynamic therapy include, but are not limited to, porphyrins such as porfimer sodium, 5-aminolaevulanic acid and verteporfin; chlorins such as temoporfin; texaphyrins such as lutetium texephyrin; purpurins such as tin etiopurpurin; phthalocyanines; and titanium dioxide.
  • the wavelength of light used to activate the photosensitizing agent can be selected according to several factors, including the depth of the tumor beneath the skin and the absorption spectrum of the photosensitizing agent administered. The period of light exposure may also vary according to the efficiency of the absorption of light by the photosensitizing agent and the efficiency of the transfer of energy to the cytotoxic agent. Such determinations are well within the ordinary skill of one in the art.
  • both the particular radiation dose to be utilized in treating a hyperproliferative disorder and the method of administration will depend on a variety of factors.
  • the dosages of radiation that can be used according to the methods of the present invention are determined by the particular requirements of each situation.
  • the dosage will depend on such factors as the size of the tumor, the location of the tumor, the age and sex of the patient, the frequency of the dosage, the presence of other tumors, possible metastases and the like.
  • Those skilled in the art of radiotherapy can readily ascertain the dosage and the method of administration for any particular tumor by reference to Hall, E. J., Radiobiology for the Radiologist, 5th edition, Lippincott Williams & Wilkins Publishers, Philadelphia, PA, 2000; Gunderson, L. L.
  • radiotherapeutic agents and treatments may be administered at doses lower than those known in the art due to the additive or synergistic effect of the compound having Formula T.
  • compositions in accordance with the present invention may be employed for administration in any appropriate manner, e.g., oral or buccal administration, e.g., in unit dosage form, for example in the form of a tablet, in a solution, in hard or soft encapsulated form including gelatin encapsulated form, sachet, or lozenge.
  • Compositions may also be administered parenterally or topically, e.g., for application to the skin, for example in the form of a cream, paste, lotion, gel, ointment, poultice, cataplasm, plaster, dermal patch or the like, or for ophthalmic application, for example in the form of an eyedrop, -lotion or -gel formulation.
  • Readily flowable forms for example solutions, emulsions and suspensions, may also be employed e.g., for . intralesional injection, or may be administered rectally, e.g., as an enema or suppository, or intranasal administration, e.g., as a nasal spray or aerosol.
  • Microcrystalline powders may be formulated for inhalation, e.g., delivery to the nose, sinus, throat or lungs.
  • Transdermal compositions/devices and pessaries may also be employed for delivery of the compounds of the invention.
  • compositions may additionally contain agents that enhance the delivery of the compounds having Formula I (or other active agents), e.g., liposomes, polymers or co-polymers (e.g., branched chain polymers).
  • agents that enhance the delivery of the compounds having Formula I e.g., liposomes, polymers or co-polymers (e.g., branched chain polymers).
  • Preferred dosage forms of the present invention include oral dosage forms and intravenous dosage forms.
  • Intravenous forms include, but are not limited to, bolus and drip injections.
  • the intravenous dosage forms are sterile or capable of being sterilized prior to administration to a subject since they typically bypass the subject's natural defenses against contaminants.
  • intravenous dosage forms include, but are not limited to, Water for Injection USP; aqueous vehicles including, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-rniscible vehicles including, but not limited to, ethyl alcohol, polyethylene glycol and polypropylene glycol; and non-aqueous vehicles including, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate and benzyl benzoate.
  • aqueous vehicles including, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection
  • water-rniscible vehicles including, but not limited to, ethyl alcohol, polyethylene glycol and
  • compositions of the present invention may further comprise one or more additives.
  • additives that are well known in the art include, e.g., detackifiers, anti-foaming agents, buffering agents, antioxidants (e.g., ascorbic acid, ascorbyl palmitate, sodium ascorbate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate, malic acid, fumaric acid, potassium metabisulfite, sodium bisulfite, sodium metabisulfite, and tocopherols, e.g., ⁇ -tocopherol (vitamin E)), preservatives, chelating agents, viscomodulators, tonicif ⁇ ers, flavorants, colorants, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and mixtures thereof.
  • the amounts of such additives can be readily determined by one skilled in the art, according to the particular properties
  • the additive may also comprise a thickening agent.
  • suitable thickening agents may be of those known and employed in the art, including, e.g., pharmaceutically acceptable polymeric materials and inorganic thickening ' agents.
  • Exemplary thickening agents for use in the present pharmaceutical compositions include polyacrylate and polyacrylate co-polymer resins, for example poly-acrylic acid and poly-acrylic acid/methacrylic acid resins; celluloses and cellulose derivatives including: alkyl celluloses, e.g., methyl-, ethyl- and propyl-celluloses; hydroxyalkyl -celluloses, e.g., hydroxypropyl- celluloses and hydroxypropylalkyl-celluloses such as hydroxypropyl-methyl- celluloses; acylated celluloses, e.g., cellulose-acetates, cellulose- acetatephthallates, celliilose-acetatesuccinates and hydroxypropylmethyl- cellulose
  • polyvinylpyrrolidones including for example poly-N- vinylpyrrolidones and vinylpyrrolidone co-polymers such as vinylpyrrolidone- vinylacetate co-polymers
  • polyvinyl resins e.g., including polyvinylacetates and alcohols, as well as other polymeric materials including gum traganth, gum arabicum, alginates, e.g., alginic acid, and salts thereof, e.g., sodium alginates
  • inorganic thickening agents such as atapulgite, bentonite and silicates including hydrophilic silicon dioxide products, e.g., alkylated (for example methylated) silica gels, in particular colloidal silicon dioxide products.
  • Such thickening agents as described above may be included, e.g., to provide a sustained release effect. However, where oral administration is intended, the use of thickening agents may not be required. Use of thickening agents is, on the other hand, indicated, e.g., where topical application is foreseen-.
  • Compounds of Formula I may be formulated similar to methotrexate or polyglutamated methotrexate as may be appropriate.
  • the dosage of the compound having Formula I will vary according to the activity and/or toxicity of the particular compound, the condition being treated, and the physical form of the pharmaceutical composition being employed for administration, it may be stated by way of guidance that a dosage selected in the range from 0.1 to 20 mg/kg of body weight per day will often be suitable, although higher dosages, such as 0.1 to 50 mg/kg of body weight per day may be useful.
  • a dosage selected in the range from 0.1 to 20 mg/kg of body weight per day will often be suitable, although higher dosages, such as 0.1 to 50 mg/kg of body weight per day may be useful.
  • the dosage of the compounds having Formula T will be lower, e.g., when used in combination with at least a second hyperproliferative disorder treatment, and may vary according to the activity and/or toxicity of the particular compound, the condition being treated, and the physical form of the pharmaceutical composition being employed for administration.
  • the compound having Formula I will preferably be present in an amount of between 0.01 and 2000 mg per unit dose. More preferably, the amount of compound having Formula I per unit dose will be about 0.01, 0.05, 0.1 , 0.5, 1 , 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, ' 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, or 2000 mg or any amount therein.
  • the total quantity of ingredients present in the capsule is preferably about 10-1000 ⁇ L. More preferably, the total quantity of ingredients present in the capsule is about 100-300 ⁇ L. In another embodiment, the total quantity of ingredients present in the capsule is preferably about 10-1500 mg, preferably about 100- 1000 mg.
  • the relative proportion of ingredients in the compositions of the invention will, of course, vary considerably depending on the particular type of composition concerned.
  • the relative proportions will also vary depending on the particular function of ingredients in the composition.
  • the relative proportions will also vary depending on the particular ingredients employed and the desired physical characteristics of the product composition, e.g., in the case of a composition for topical use, whether this is to be a free flowing liquid or a paste. Determination of workable proportions in any particular instance will generally be within the capability of a person of ordinary skill in the art. All indicated proportions and relative weight ranges described below are accordingly to be understood as being indicative individually inventive teachings only and not as limiting the invention in its broadest aspect.
  • compositions of the invention will of course vary, e.g., depending on the intended route of administration and to what extent other components are present. In general, however, the compound having Formula I will suitably be present in an amount of from about 0.005% to 20% by weight based upon the total weight of the composition. In certain embodiments, the compound having Formula I is present in an amount of from about 0.01% to 15% by weight based upon the total weight of the composition.
  • the present invention also provides a process for the production of a pharmaceutical composition as hereinbefore defined, which process comprises bringing the individual components thereof into intimate admixture and, when required, compounding the obtained composition in unit dosage form, for example filling said composition into tablets, gelatin, e.g., soft or hard gelatin, capsules, or non-gelatin capsules.
  • Scheme 1 potassium iodide
  • the reaction may, however, start with the alkylation of the amine on 2,4,5,6-tetraaminopyrimidine by the bromoaldehyde. Air oxidation of the dihydro ring in the initial condensation product completes the synthesis of methotrexate analog.
  • Methotrexate analogs may be prepared, for example, by substituting the glutamyl group with an analog, for example 2-aminoadipate group.
  • the p-aminobenzoic acid may be replaced with analogs having, for example, one, two, three or four substituents on the benzene ring.
  • the amino nitrogen of the 4-aminobenzoic acid moiety and the carbon atom to which it is attached may be part of a heterocyclic ring fused with the benzene " ring.
  • Another variation is to introduce polyglutamate or analogs on methotrexate analog.
  • Methotrexate polyglutamates are known metabolites of methotrexate, which are pharmacologically important. See Chabner, B. A. et al. J. Clin. Invest., 76:907-912 (19S5). N-oxides of methotrexate polyglutamate and its analogs are contemplated in this invention.
  • methotrexate polyglutamate or analogs may be synthesized by starting with /V-substituted polyglutamyl-p-aminobenzamide, which in turn may be prepared by successively adding glutamic acid residues to N-protected 4-aminobenzoic acid chloride using methods known in the art.
  • the ⁇ -carboxylic acid of commercially available glutamic acid ⁇ -benzyl ester may first be protected with, for example, 2-(trimethylsilyl)ethoxymethyl protecting group (SEM) to afford ⁇ -SEM- ⁇ -benzyl glutamate. See Jarowicki, K. and Kocienski, P., J. Chem.
  • the ./V-substituted polyglutamyl-p-aminobenzamide may be coupled with 2,4,5, 6-tetraaminopyrimidine and 2,3-dibromopriopionaldehyde in the presence of potassium iodide (Scheme 1) to yield a product that can be air- oxidized to afford polyglutamated analog of methotrexate or its analog.
  • the one or more ⁇ -SEM protecting groups can be removed under conditions that do not affect esters and amides. See Jarowicki, K. and Kocienski, P., J. Chem.
  • any of the pyridine- or pyrimidine- or pyi-azine-type nitrogen atoms or any tertiary amino groups in methotrexate and analogs can be selectively oxidized to the corresponding N-oxide using known oxidizing agents.
  • the tertiary amino group in methotrexate i.e. N 10
  • Primary and secondary amino groups that may be present in methotrexate and analogs may be first selectively alkylated to give the corresponding tertiary amine, which then can be oxidized to the corresponding N-oxide.
  • Certain oxidizing agents that are known in the art from preparing the N-oxides of pyridine- or pyrimidine- or pyrazine-type nitrogen atoms or tertiary amines include, without limitation, potassium monopersulfate, monoperoxyphthalic acid, magnesium monoperoxyphthalate (MMPP), hydrogen peroxide, peracetic acid, trifluoroperacetic acid, perbenzoic acid, 3-chloroperbenzoic acid (CPBA), and 2-phenylsulfony1-3- phenyloxaziridine (Davis reagent).
  • the oxidation reaction can be carried out in a solvent such as chloroform, methylene chloride, 1 ,2-dichloroethane, or acetic acid, optionally in the presence of an alkali or alkaline-earth metal carbonate or bicarbonate.
  • a solvent such as chloroform, methylene chloride, 1 ,2-dichloroethane, or acetic acid
  • the reaction can be run from about 1 to 48 hours at a temperature of 0 0 C to reflux temperature, and checked periodically for the presence of the desired N-oxide.
  • reaction times may need Io be adjusted accordingly to obtain appropriate quantities of the desired bis-N-oxide product.
  • the present invention contemplates the preparation of N-oxide analogs in which one or more of the nitrogen atoms that are suitable for N-oxide formation are present as the N-oxide without regard to the susceptibility of a particular nitrogen atom to N-oxide formation or the susceptibility of a particular N-oxide group to reduction. It is envisaged to employ a combination of suitable protecting groups (see: Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, second edition, Wiley Interscience, 1991) to protect those nitrogen atoms not undergoing oxidation.
  • primary and secondary amines that may be present in a methotrexate analog may be protected using, for example, tert- butyl sulfonyl (BUS) group.
  • BUS tert- butyl sulfonyl
  • the BUS protecting group is introduced by reaction of the amine with tert-butylsulfmyl chloride followed by oxidation of the sulfinyl amide with, for example, dimethyldioxirane, m-chloroperbenzoic acid or RuCl 3 catalyzed NaIO 4 .
  • the oxidation step in the preparation of the BUS-protected primary or secondary amines may also oxidize any tertiary amine and heteroaromatic nitrogen present in the compounds.
  • this protecting group may be introduced into primary and secondary amines while simultaneously oxidizing tertiary and/or heteroaromatic nitrogen atoms.
  • the BUS protecting group is stable towards strong reagents such as alkyllithium, Grignard reagents, 0.1 M HCl in MeOH (20° C, 1 hr), 0.1 M TFA in dichloromethane (20° C, 1 hr) and pyrolysis at 180° C.
  • the BUS-protected secondary amines can be cleaved with 0.1 M triflic acid in dichloromethane containing anisole as a cation scavenger at 0° C for 15-.30 minutes while primary amines are released more slowly at room temperature.
  • both BUS-protected primary and secondary amines may be deprotected with 0.1 M triflic acid in dichloromethane containing anisole as a cation scavenger at 25° C for 2.5 hours.
  • the BUS protecting group may allow protecting primary and secondary amines simultaneously while also oxidizing tertiary amines and heteroaromatic nitrogen atoms to the N-oxides.
  • the BUS protecting group may allow protecting primary and secondary amines simultaneously, oxidizing tertiary amines and heteroaromatic nitrogen atoms to N-oxides, deprotect the secondary amine selectively, alkylate the secondary amine to a tertiaiy amine, oxidize the resulting tertiary amine and deprotect the primary amine.
  • a primary and a secondary amine may be protected with BUS protecting group, the secondary amine maybe deprotected selectively, the secondary amine may be protected with, for example, Boc protecting group, and then the primary amine may be deprotected selectively followed by alkylation and oxidation.
  • a BUS protecting group may be used to transform one of amines to an N-oxide without affecting the other.
  • Boc group to protect amines allows introduction and removal of the group under mild conditions.
  • a methotrexate analog amine group may be protected with Boc group by simply mixing the analog and Boc-ON (2-(Boc-oxyimino)-2-phenylacetonitrile, available from Aldrich Co.) in benzene at 25° C for 20 minutes (or 6 hours if the amine is an electron deficient aniline) in the presence of powdered zinc.
  • Boc-ON 2-(Boc-oxyimino)-2-phenylacetonitrile
  • Bsmoc base-sensitive amino protecting group l,l-dioxobenzo[6]thiophene-methoxycarbonyl
  • Bsmoc is introduced via its chloroformate or ./V-hydroxy-succinimide derivative.
  • the Bsmoc group is stable towards tertiary amines for 24 hours but is removed within 3-5 minutes using piperidine.
  • primary and secondary amines present in methotrexate analogs may conveniently be protected with Bsmoc protecting group followed by oxidation of the tertiary amines and removal of the protecting group under mild conditions.
  • heteroaromatic nitrogen atoms can be oxidized selectively in the presence of certain aromatic primary amines and certain secondary amines adjacent to a double bond.
  • oxidation of cytosine with m-chloroperbenzoic acid results in cytosine 3-N-oxide despite the presence of aromatic primary amine and a secondary amine.
  • heteroaromatic nitrogen atoms and tertiary amines may be oxidized in the presence of certain aromatic primary amines and secondary amines.
  • a variety of substituted commercially available 4-aminobenzoic acids may be used as a starting point for the synthesis of methotrexate analog N-oxides in which the benzoic acid is substituted with one, two, three or four substituents.
  • 4-aminobenzoic acid substituted with one or more of, for example, alkyl, halo, cyan ⁇ , hydroxy, alkoxy, fluorinated alkoxy, benzyloxy and cyclohexyloxy, which are commercially available may be used as starting material for the synthesis of methotrexate analogs.
  • the list of the compounds is intended to provide working examples but not intended to limit the invention.
  • a precursor of the 4-amino group can be a 4-nitro group, which upon catalytic reduction with H 2 generates the amino group.
  • a precursor of the 4-amino group can be a 4-nitro group, which upon catalytic reduction with H 2 generates the amino group.
  • the nitro group itself can be introduced into an aromatic precursor by way of a nitration reaction.
  • the carboxyl group may be introduced at the correct position by forming a Grignard reagent with a suitable aromatic halide and then quenching the reagent with anhydrous carbon dioxide.
  • position selective metallation of the carboxyl group followed by reaction with a suitable electrophile introduces the electrophile into a position either ortho or meta to the carboxyl group, depending on the reagent used (see: Nguyen, T.-H. Chou, N. T. T.; Castanet, A.-S.; Nguyen, K. P. P.; Mortier, J. Org. Lett., 7:2445 (2005) and references cited therein).
  • fluorinated alkyl groups may be introduced to the benzoic acid moiety by choosing appropriately substituted starting material.
  • substituted 4-aminobenzoic acid in which R 2 or R 3 is a trifluoromethyl group may be prepared by starting with, for example, 4-cyano-2- (trifluoromethyl)acetanilide. Simultaneously hydrolyzing the amide and the cyano groups of 4-cyano-2-(trifluoromethyl)acetanilide will yield 3-trifluromethyl-4-aminobenzoic acid.
  • substituted 4-aminobenzoic acid in which R 4 or R 5 is a trifluoromethyl group may be prepared by starting with 4-amino-2-trifluorornethylbenzonitrile, which, upon hydrolysis of the cyano group, affords 2-trifluormethyl ⁇ 4-aminobenzoic acid.
  • the carboxylic acid group of substituted 4-aminobenzoic acid is protected by transforming it into, for example, an ester (Scheme 2).
  • the substituted 4-aminobenzoic acid is dissolved in anhydrous alcohol followed by catalytic amount of a strong acid.
  • the equilibrium between the ester and the acid can be shifted in favor of the ester if an excess of the alcohol is used, if the ester or water is removed from the reaction mixture by, for example, distillation or if water is removed by a dehydrating agent, silica gel or molecular sieves.
  • Scheme 2 See, for example, Smith, M. B. and March, J., "March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure," 5 th ed., Chapter 10, John Wiley & Sons, Inc., New York, NY (2001).
  • the amine nitrogen of substituted 4-aminobenzoic acid ester may also be protected with, for example, a p-toluenesulfonyl group, a benzyloxycarbonyl group or an acetyl group.
  • a substituted 4-aminobenzoic acid ester may be dissolved in ether in which sodium hydrogen carbonate is suspended.
  • An ether solution of an acid chloride, for example benzyloxycarbonyl chloride is thereafter added while cooling the reaction mixture with ice. Following stirring for about 2.5 h at room temperature, additional amount of the ether solution of the acid chloride may be added to the reaction mixture while keeping the mixture in ice bath.
  • the stoichiometry of the reaction is such that an excess amount of the acid chloride is used.
  • the reaction mixture will be washed with water and dried with anhydrous sodium sulfate crystals. Evaporation of the ether will yield a residue that can be purified by recrystallization or column chromatography to afford substituted /V-benzyloxycarbonyl-4-aminobenzoic acid ester. See, e.g., 6,559,149, example 12.
  • N-benzyloxycarbonyl-4-amino-benzoic acid ester will then be N-alkylated following methods well known in the art. For example, a solution of the ./V-benzyloxycarbonyl-4-amino-benzoic acid ester in dimethylformamide (DMF) will be added slowly to a suspension of sodium hydride in DMF while maintaining the reaction temperature at or near 0° C. The mixture will be allowed to warm up to room temperature. After stirring the reaction mixture for 1 h at room temperature, an alkyl halide (for example, methyl iodide) will be added slowly and the mixture will be stirred at room temperature for 2 h.
  • DMF dimethylformamide
  • reaction solution will be poured into a saturated aqueous solution of sodium hydrogen carbonate and the mixture will be extracted with toluene.
  • organic layer will be dried with sodium sulfate and the solvent will be distilled off under vacuum.
  • the resulting residue will be purified, for example, by column chromatography. See, e.g., 6,559,149, example 13.
  • the ester group of the substituted /V-methyl-/V-benzyloxycarbonyl-4- aminobenzoic acid ester will be hydrolyzed with a base.
  • a base For example, treatment ⁇ f-methyl- ⁇ f-benzyloxycarbonyl-4-aminobenzoic acid ester with 2N NaOH solution aqueous/ethanol solution at reflux temperature for two hours will yield N-methyl-7V r -benzyloxycarbonyl-4-aminobenzoic acid sodium salt.
  • the acid can be isolated by acidifying the mixture with, for example, 4N HCl, a pH below 4, followed by extraction with, for example, chloroform. See, e.g., 6,559,149, example 14.
  • substituted /V-methyl-/V-benzyloxycarbonyl-4-aminobenzoic acid will be coupled with a glutamic acid or analog according to methods known in the art.
  • substituted 7V-methyl-/V-benzyloxycarbonyl-4- aminobenzoic acid will be suspended in a chlorinated hydrocarbon solvent, for example dichloromethane.
  • a chlorinated hydrocarbon solvent for example dichloromethane.
  • a large excess of thionyl chloride will be added to the solution while keeping the mixture in an ice path. After stirring the reaction mixture for 2 hours at room temperature, the solution will be concentrated to dryness under vacuum.
  • reaction mixture After redissolving the reaction mixture in dichloromethane and suspending potassium carbonate in the reaction mixture, an equivalent amount of diethyl glutamate (or its hydrochloride salt) or its analog will then be added.
  • the volume of the reaction mixture is approximately doubled with water followed by stirring at about 0° C to about 50° C, more preferably between 15 and 40° C, more preferably at or near room temperature for between about 2 hours to 3 days, more preferably between 4 hours and 24 hours, more preferable 8 to 15 hours.
  • the reaction mixture will be poured into water and extracted with chloroform.
  • the organic layer will be washed with IN HCl and dried with sodium sulfate.
  • ⁇ r -(N'-benzyloxycarbonyl- ⁇ r '-methyl-4-aminobenzoyl)-L-glutamic acid diester or analog will be stirred in a solution of 30% hydrogen bromide/acetic acid (about 10 ml) in the presence of about 1 gram of anisole for about 1 to 10 hours at about 0° C to about 50° C. See, e.g., U.S. Patent No. 6,559,149, example 16.
  • the reaction product will them be extracted and purified according to methods known in the art.
  • Compounds of formula TIT may be obtained by starting with the commercially available (Apollo Scientific Ltd., U.K.) l,2,3,4 ⁇ tetrahydro-6- quinolinecarboxylic acid (THQA) and using methods described for compounds of Formula I.
  • the carboxylic acid group of THQA will be esterified followed by protection of N-amine with, for example, benzyloxycarbonyl protecting group.
  • N-benzyloxycarbonyl-l.ZjB ⁇ -tetrahydro- ⁇ -quinolinecarboxylic acid ester will be hydrolyzed followed by coupling the resulting 7V-benzyloxycarbonyl ⁇ l,2,3,4-tetrahydro-6-quinolinecarboxylic acid with glutamic acid diester or analog.
  • Compounds of formula IV may be obtained by coupling iV-benzyloxycarbonyl-5-carboxyindole with glutamic acid or analog followed by reaction with 2,4,5,6-tetraaminopyrimidine and
  • the TV-benzyloxycarbonyl-5-carboxyindole (obtained by reaction between benzyloxycarbonyl chloride and methyl 5-indolecarboxylate followed by hydrolysis of the ester) will be coupled with glutamic acid or analog to afford iV ⁇ N'-benzyloxycarbonyl-S-carboxyindoly ⁇ -L-glutamic acid diester or its analog.
  • N-(N'-benzyloxycarbonyI-5-carboxyindolyl)-L-glutamic acid or its analog will be reacted with 2,4,5, 6-tetraaminopyrimidine and 2,3-dibromopropionaldehyde in the presence of KI to afford the dihydro compound of methotrexate analog that can be air oxidized to afford non-N-oxide of compounds of IV (i.e., methotrexate analogs).
  • methotrexate analogs i.e., methotrexate analogs.
  • Scheme 1 TV-oxidation of the tertiary amine group of the methotrexate analogs with MCPBA will yield compounds of Formula IV.
  • Primary, secondary or tertiary amines may also be introduced into methotrexate analog by using a modified procedure.
  • mono- or di-fluorinated e.g., 2-fluoro-4-nitrobenzoic acid, 3-fluoro-4-nitrobenzoic acid, or 2,5-difluoro-4-nirobenzoic acid, which are commercially available
  • a dialkyl amine e.g., dimethylamine
  • reaction between 2-fluoro-4-nitrobenzoic acid and dimethylamine will yield N 1 N- dimethyl-4-nitro-2-aminobenzoic acid.
  • N,N-dimethyl-4-nitro-3-aminobenzoic acid may similarly be obtained.
  • dialkylamine for example, JV,iV-dimethyl amine, TV.TV-diethyl amine, 7V,N-dipropyl amine, etc.
  • dialkylamine for example, JV,iV-dimethyl amine, TV.TV-diethyl amine, 7V,N-dipropyl amine, etc.
  • 4-nitrobenzoic acid will introduce amino or alkyl amino substituent on the 4-nitrobenzoic acid.
  • Suitable protection scheme may be used to protect the amino groups.
  • the nitro group of the substituted benzoic acid may selectively be reduced to amine to afford 4-aminobenzoic acid substituted with one or two amino groups.
  • the nitro group is susceptible to reduction by lithium triethylborohydride in tetrahydrofuran (THF) at 0° C while the carboxylic acid salts are not. See Table 2, Brown, H. C. and Krishnamurthy, S. Tetrahedron, 35:567-601, 591, 604 (1978).
  • ⁇ yV-dimethyl-4-nitro-3- aminobenzoic acid tetraethyl ammonium salt in THF will be added into a suspension of lithium triethylborohydride in THF while keeping the pot temperature at between -20° C and 10° C, more preferably at -10° C to 5° C, most preferably at about 0° C.
  • the mixture will be stirred at about 0° C while following the reaction progression using a suitable analytical procedure (e.g., TLC or gas chromatography).
  • the reaction will be quenched by, for example, slowly adding a moist alcohol, which decomposes the excess lithium triethylborohydride, followed by isolation of the reaction product.
  • the product, 3-(iV,N-dimethylamino)-4-aminobenzoic acid may then be used to synthesize a methotrexate analog according to methods described above.
  • MCPBA m-chloroperbenzoic acid
  • the resulting solution was filtered to remove any traces of solid unreacted methotrexate and then it was extracted with methylene chloride (3 x 5 mL) and ether (1 x 5 mL) to remove m-chiorobenzoic acid.
  • the aqueous phase was lyophilized to give 0.020 g (97%) of methotrexate N-oxide as a yellow-orange powder.
  • the crude N-oxide product is reasonably clean and typically contains about 10% of impurities.
  • the crude product was loaded onto a silica gel column as a suspension in water/acetonitrile (1 :1, 1 mL) and then it was chromatographed using an acetonitrile/water/acetic acid (5:2:2) mixture as eluent.
  • the desired fractions were collected and extracted with methylene chloride (1OmL x 3) and ether (1OmL x 1) to remove some of the acetonitrile and acetic acid. Note that extraction is preferable to evaporation due to sensitivity of the product to light. Lyophilization of the aqueous phase gave 0.01 1 g (52% yield) of methotrexate N-oxide as a greenish-yellow powder with an estimated purity of >90% based on the 1 H NMR spectrum.
  • the aqueous phase was lyophilized to give 0.011 g (97%) of methotrexate sodium salt N-oxide as a yellow-orange powder.
  • the crude product was loaded on a silica gel column as a suspension in water/acetonitrile mixture (1 :1, ImL) and then chromatographed using acetonitrile/water/acetic acid (5:2:2) mixture as eluent.
  • the desired fractions were collected and extracted with methylene chloride (10 mL x 3) and ether (1OmL x 1) to remove some of the acetonitrile and acetic acid.
  • Liophilization of the aqueous phase gave 0.006 g (54% yield) of the sodium salt of methotrexate N-oxide as a greenish-yellow powder with an estimated purity of >90% based on the 1 H NMR spectrum.
  • Procedure 3 A suspension containing 0.010 g (0.022 mmol) of methotrexate, 0.5 mL water and 0.5 mL methanol was stirred for 20 minutes. To this suspension magnesium bis(monoperoxyphthalate) hexahydrate (0.02 g, 0.044 mmol) was added. The methotrexate dissolved over 1 hour. The mixture was stirred additional hour and solvents were evaporated until about 0.5 mL of water remains. This solution was transferred on silica gel column chromatography and eluted with acetonitrile/water/acetic acid mixture. Magnesium phthalate eluted with acetonitrile.
  • methotrexate and analog N-oxides thereof on different lymphoma, leukemia, and multiple myeloma cell lines will be tested in vitro under normoxic as well as 1% O2 hypoxic conditions. Standard cytotoxicity assays using MTS dye will be run to determine the IC50 for each compound. Cells will be exposed to the compounds for 24 hours and cells will be stained 24-72 hours post-drug exposure. Positive controls will use chemotherapeutic agents at doses shown in the art to be effective. The results should indicate that methotrexate analogs are cytotoxic to many of the cell lines, with IC50 values in the nanomolar to sub-nanomolar range.
  • Methotrexate analog N-oxides are expected to be less active or inactive compared to non-N-oxide under normoxic conditions. However, under 1% O2 hypoxic conditions, methotrexate analog N-oxides are expected to be converted to the corresponding parent non-N-oxide, which are expected to be cytotoxic with IC50 values in the millimolar to sub-nanomolar range.
  • methotrexate and analog N-oxides thereof are expected to be less active or inactive compared to the corresponding methotrexate analogs.
  • the anti-proliferative activity of methotrexate and analog N-oxides thereof on established and primary tumor cell lines will be tested in vitro under normoxic and 1% O2 hypoxic conditions at concentrations ranging from 1 nM to 10 mM.
  • the anti-proliferative effect will be measured using the 5-bromo- 2'-deoxyuridine ("BrDU") incorporation technique.
  • the cells will be exposed to the compounds in the presence of BrDU for 24 hours.
  • BrDU is incorporated into the replicating cellular DNA. After cell fixation and washing, the incorporated BrDU is determined in a specific ELlSA using an antibody specific to BrDU coupled to peroxidase.
  • N-oxides are expected not to have significant anti-proliferative activity in cancer cells at concentrations of up to 10 mM under normoxia. However, the N-oxides are expected to exhibit significant anti-proliferative effect on the cancer cell lines under 1% O2 hypoxia.
  • methotrexate and analog ⁇ -oxides will be evaluated using xenograft murine models.
  • male 5 to 6 week old nude mice will be inoculated subcutaneously in the mammary fat pad on each side with an injection of a human cancer cell line, for example about 1x10 6 MDA-MB-231 (2LMP) in 0.3 ml serum free medium.
  • a human cancer cell line for example about 1x10 6 MDA-MB-231 (2LMP) in 0.3 ml serum free medium.
  • the best xenograft recipients will be used.
  • Treatments with methotrexate and analog ⁇ -oxides will begin when tumors averaged about 5-7 mm in diameter and will be continued for 4 weeks with a 2 month follow up period.
  • Test animals will be divided into cohort groups of 5-8 animals into the following treatment groups: Control (Group 1); Vehicle Control (Group 2), daily administration of the vehicle only; Methotrexate and analog ⁇ -oxide (Group 3), 0.001 mg/kg administered orally per day for 4 weeks; Methotrexate and analog ⁇ -oxide (Group 4), 0.01 mg/kg administered orally per day for 4 weeks; Methotrexate and analog ⁇ -oxide (Group 5), 0.05 mg/kg administered orally per day for 4 weeks; Methotrexate and analog ⁇ -oxide (Group 6), 0.10 mg/kg administered orally per day for 4 weeks; Methotrexate and analog ⁇ - oxide (Group 7), 0.15 mg/kg administered orally per day for 4 weeks; Methotrexate and analog ⁇ -oxide (Group 8), 1.0 mg/kg administered orally per day for 4 weeks.
  • the test doses may further be increased or decreased upon recognition that such a modification is warranted for a particular methotrexate analog ⁇ -oxide.
  • a particular methotrexate analog ⁇ -oxide may exert its own, for example, antiproliferative effect, making it more effective than it would have been had methotrexate analog ⁇ -oxide been biologically inactive. In such a circumstance, lower doses of the methotrexate analog ⁇ -oxide may prove effective.
  • More cohort groups may be added to test various doses of methotrexate and analog ⁇ -oxides.
  • tumor sizes and animal weights will be measured periodically, for example 1-3 times per week for each animal. Following treatment, tumor sizes will be measured periodically, for example, 1-3 times per week, and animal weights once per week. Tumor sizes and weight observations will be made without knowledge of the animal's treatment group.
  • the data will be modeled using, for example, the natural logarithm of tumor volume versus time. Comparisons of the growth rates of animals administered with varying dosages will be performed. For example, Tumor Growth Delay and Tumor Growth Inhibition for varying dosages will be compared to the control.
  • N-oxide in combination with chemotherapeutic agents and/or radiotherapy will be evaluated using a xenograft model in nude mice.

Abstract

The present invention relates to methotrexate and analog N-oxides having activity for treating hyperproliferative disorders. Further, the invention relates to pharmaceutical compositions and methods of using methotrexate and analog N-oxides, alone or in combination with one or more other active agents or treatments, to treat hyperproliferative disorders.

Description

TREATMENT OF HYPERPROLIFERATIVE DISEASES WITH METHOTREXATE N-OXIDE AND ANALOGS
BACKGROUND OF THE INVENTION
Field of the Invention
[0001] The present invention relates to novel compounds having activity for treating hyperproliferative disorders, including neoplastic and non-neoplastic disorders, as well as certain inflammatory conditions. The invention also relates to pharmaceutical compositions and formulations comprising the novel compounds. Further, the invention relates to methods of using the novel compounds, alone or in combination with one or more other active agents or treatments, to treat hyperproliferative disorders, including various cancers.
Related Art
[0002] Methotrexate is an antifolate and is used for the treatment of acute leukemia, malignant lymphoma and other diseases. It is also known as an immunosuppressive drug and is primarily used for preventing acute graft- versus-host reactions in bone marrow transplantation. Furthermore, administration of low doses of methotrexate is known to be effective for the treatment of rheumatoid arthritis. U.S. patent 5,958,928.
[0003] U.S. Patent No. 6,559,149 describes methotrexate derivatives that are represented by the following general foπnula (II):
Figure imgf000002_0001
where W is a group represented by the general formula:
Figure imgf000003_0001
where R) is a lower alkyl group having 1 -4 carbon atoms; R2 is a lower alkyl group having 1-4 carbon atoms or a trifluoromethyl group; R3 is a hydrogen atom, a lower alkyl group having 1-4 carbon atoms or a trifluoromethyl group; R4 is a hydrogen atom or a lower alkyl group having 1-4 carbon atoms; R5 is a group represented by the general formula COORβ (where Rs is a hydrogen atom or a lower alkyl group having 1 -4 carbon atoms) or a group represented by the formula SO3H; and n is an integer of 1-4, or where W is a group represented by the general formula:
Figure imgf000003_0002
where R7 is a lower alkyl group having 1-4 carbon atoms; Rs is a hydrogen atom or a lower alkyl group having 1-4 carbon atoms; R9 is a group represented by the general formula COOR 10 (where Rio is a hydrogen atom or a lower alkyl group having 1-4 carbon atoms) or a group represented by the formula SO3H; and m is an integer of 1-4], or the general formula:
3
Figure imgf000003_0003
where R) i is a hydrogen atom or a lower alkyl group having 1-4 carbon atoms; Ri2 is a hydrogen atom or a lower alkyl group having 1-4 carbon atoms; R13 is a group represented by the general formula COORi4 (where Ri4 is a hydrogen atom or a lower alkyl group having 1-4 carbon atoms) or a group represented by the formula SO3H; and 1 is an integer of 1-4, or W is a group represented by the general formula:
Figure imgf000004_0001
where Rj 5 is a hydrogen atom or a lower alkyl group having 1-4 carbon atoms; R] 6 is a hydrogen atom or a lower alkyl group having 1-4 carbon atoms; and k is an integer of 2 or 3. Among the compounds disclosed in U.S. Patent No. 6,559,149 are the following compounds: dimethyl N- {4-[ΛΛ-(2,4-diamino-6-pteridinyl)methyl-Λ^-methylamino]- 3-methyl}benzoyl-L-2-aminoadipate; iV-{4-[iV-(2,4-diamino-6-pteridinyl)methyl-N'-methylamino]-3- methyl}benzoyl-L-2-aminoadipic acid; iy-{4-[7V-(2,4-diamino-6-pteridinyl)methyl-N-methylamino]-3- trifluoromethyl}benzoyl-L-2-aminoadipic acid; dimethyl N- { 1 -[2,4-diammo-6-pteridinyl)methyl]-7-methylindoline-5- carbonyl} -L-2-aminoadipate;
//-{l-[2,4-diamino-6-pteridinyl)methyl]-7-methyIindoline-5-carbonyl}- L-2-aminoadipic acid; iV-{l-[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl}-L-2- aminoadipic acid;
N-{l-[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl}-L- homocysteic acid ammonium salt;
N- { 1 -[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl} -L-glutamic acid; 7V-{l-[2,4-diamino-6-pteridinyl)methyl]-7-methylindole-5-carbonyl}- L-2-aminoadipic acid; dimethyl N-{4-[N'-(2,4-diamino-6-pteridinyl)methylamino]-3- methyl)benzoyl-L-2-aminoadipate;
N-{4-[7V-(2,4-diamino-6-pteridinyI)methylamino]-3-methyl)benzoyl-L- 2-aminoadipic acid. diethyl iV-4-[N'-(2,4-diamino-6-pteridinyl)methyl-N1-methylamino]-3- methylbenzoyl-L-glutamate; dimethyl N- {4-[jV-(2,4-diamino-6-pteridinyl)methyl-NI-methylamino]- 3-methyl}benzoyl-L-2-aminoadipate;
7V-{4-[A''-(2,4-diamino-6-pteridinyl)methyl-N1-methylamino]-3- methylbenzoyl}-L-glutamic acid; diethyl N- {4-[N'-(2,4-diamino-6-pteridinyl).methyl-iV-ethylamino]-3- methylbenzoyl} -L-glutamate;
Λ^-{4-[Λ''-(234-diamino-6-ρteridinyl)methyl-Λ''-ethylamino]-3- methylbenzoyl}-L-glutamic acid; diethyl /V-{4-[7V-(2,4-diamino-6-pteridinyl)methyl-//'-ethylamino]-3- methylbeπzoyl} -L-glutamate;
N-{4-[N'-(2,4-diamino-6-pteridinyl)methyl-Λ''-ethylamino]-3- methyl}benzoyI-L-glutamic acid; dimethyl yV-{4-[Λ''-(2,4-diamino-6-pteridinyl)methyl-N'-methylaniino]- 3-melhylbenzoyl}-L-2-aminoadipate;
N-{4-[Λ/'-(2,4-diamino-6-pteridinyl)methyl-iV-methylamino]-3- methyl}benzoyl-L-2-aminoadipic acid;
7V-(4-[Λ?I-(2,4-diamino-6-pteridinyl)methyl-Λ''-methylamino]-3- methylbenzoyl}-L-homocysteic acid ammonium salt; diethyl Λr-{4-[N'-(2,4-diamino-6-ptcridinyl)methyI-N'-methylamino]-3- ethylbenzoyl } -L-glutamate;
N-{4-[7V-(2.4-diamino-6-pteridinyl)methyl--Y-methylamino]-3- ethylbenzoyl}-L-glutamic acid; diethyl N- {4-[iV-(2,4-diamino-6-pteridinyl)methyl-Λ^-methylamino] - 3,5-dimethylbenzoyl}-L-glutamate;
N-{4-[7V-(2,4-diamino-6-pteridinyl)methyl-7V-methylamino]-3,5- dimethylbenzoyl}-L-glutamic acid;
N-{4-[Λ?'-(2,4-diamino-6-pteridinyl)methyl-iV-methylamino]-3- trifluoromethylbenzoyl}-L-2-aminoadipic acid; dimethyl N- {4-[N-(2,4-diamino-6~pteridinyl)methyl-7-methylindoline- 5'-carbonyl}-L-2-aminoadipate;
N- { 1 -[Λ/τ-(2,4-diamino-6-pteridinyl)methyl-7-methylindoline-5l- carbonyl}-L-2-aminoadipic acid;
N- { l-[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl} -L- aminoadipic acid;
N- { 1 -[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl} -L-2- aminoadipic acid;
Λf-{l-[2,4-diammo-6-pteridinyl)methyl]indole-5'-carbonyl}-L- homocysteic acid ammonium salt;
N-{l-[(2,4-diamino-6-pteridinyl)methyl]indole-5'-carbonyl}-L- glutamic acid;
N-{l-[(2,4-diamino-6-pteridinyl)methyl-7-methylindole-5'-carbonyl}- L-2-aminoadipic acid; diethyl N- {4-[Λ^-(2,4-diamino-6-pteridinyl)methylamino]-3- methyl}benzoyl-L-glutamatc; dimethyl N- {4-[Λ/1-(2,4-diamino-6-pteridinyl)methylamino]-3- methyl } benzoyl -L-2-aminoadipate; and
N-{4-[-Y-(2,4-diamino-6-pteridinyl)methylamino]-3-methyl)benzoyl-L- 2-aminoadipic acid; Methotrexate is metabolized upon uptake by mammalian cells such that one or more glutamyl moieties are added to methotrexate to yield a mixture of methotrexate polyglutamates. See U.S. Patent 6,921,667. The number of glutamyl moieties that can be added to methotrexate generally varies from two to seven. [0006] U.S. Patent No. 4,584,375 discloses methotrexate derivatives and the synthesis thereof, wherein the methotrexate derivative has the general formula:
Figure imgf000007_0001
wherein m is from 0 to 5.
BRIEF SUMMARY OF THE INVENTION
[0007] The present invention is related to compounds, compositions and methods for treating hyperproliferative disorders, such as cancer and inflammation. One aspect of the invention is drawn to compounds having Formula I:
Figure imgf000007_0002
or a pharmaceutically acceptable salt or prodrug thereof, wherein:
Xl is hydrogen; deuterium; halo; amino; C(=NH)NH2; C(=0)NH2; C(=S)NH2; NR9R10RiI wherein each of Rg and Ri0 is independently hydrogen, alkyl, haloalkyl, cyanoalkyl, hydroxyalkyl or alkoxyalkyl; carboxyalkyl; phenyl; alkylphenyl; C(=O)R* wherein R' is hydrogen, alkyl, phenyl, or alkylphenyl; or C(=O)OR" wherein R" is hydrogen, alkyl, phenyl or alkylphenyl; each of Y and Z is independently hydrogen or deuterium;
R) is a lower alkyl group having 1-4 carbon atoms, R2 is hydrogen, halo, hydroxy, cyano, a lower alkyl group having 1-4 carbon atoms, NRi2RnRi4, a lower alkoxy having 1-4 carbon atoms or a trifluoromethyl group, or R] and R2 together form a 5 or 6 membered saturated or unsaturated heterocycle having one or two hetero atoms where the second heteroatom, when present, is S or O3 wherein S is optionally substituted with one or two oxygen atoms;
R3 is hydrogen, halo, cyano, alkoxy, cycloalkyloxy, benzyloxy, alkyl, cycloalkyl, aryl, heteroaryl, aryloxy, aralkyl, NRI 2RI 3RM or haloalkyl, or Ri and R3 together form a 5 or 6 membered saturated or unsaturated heterocycle;
R4 is hydrogen, hydroxy, halo, alkyl, a lower alkoxy or NRI 2RI 3RI4;
R5 is hydrogen, hydroxy, halo OrNRj2Ri3Ri4; each R6 is independently COOR15Or CONHR16, wherein each of R15 and R16 is independently hydrogen, alkyl, aryl, aralkyl or cycloalkyl;
R7 is COOR17, SO3H, PO3H2, NHCOOR18, NHCOR18, or CONR17R18 wherein R17 is hydrogen, alkyl, optionally substituted phenyl, carboxyalkyl or alkylsulfonyl and R18 is alkyl, aryl, aralkyl or cycloalkyl; each of R12 and R]3 is hydrogen or alkyl; each of R8, Rn and Ri4 is O or is absent provided that at least one of
Figure imgf000008_0001
m is 0-6; and each of n and p is independently 1, 2 or 3; wherein one or more -CH2- groups of the terminal amino acid is optionally substituted with one or more halogen atoms. In one embodiment, the compounds having formula I are N10-oxide of methotrexate and analogs, selected from the N10-oxides of the group consisting of:
Methotrexate; dimethyl N-{4-[N'-(2,4-diamino-6-pteridinyl)methyl-N-methylamino]- 3-methyl}benzoyl-L-2-aminoadipate;
/V-{4-[Λr-(2,4-diamino-6-pteridinyl)methyl-N-methylamino]-3- methyl}benzoyl-L-2-aminoadipic acid; N-{4-[iV-(2,4-diamino-6-pteridinyl)methyl-7V'-methylamino]-3- trifluoromethyl}benzoyl-L-2-aminoadip'ic acid; dimethyl N~{l-[2,4-diamino-6-pteridinyl)methyl]-7-methylindoline-5- carbonyl} -L-2-aminoadipate;
N-{l-[2,4-diamino-6-pteridinyl)methyl]-7-methylindoline-5-carbonyl}- L-2-aminoadipic acid;
N- { 1 -[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl} -L-2- aminoadipic acid;
N- {I -[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl} -L- homocysteic acid ammonium salt;
N- {l-[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl}-L-glutamic acid;
N-{l-[2,4-diamino-6-pteridinyl)methyl]-7-methylindole-5-carbonyl}- L-2-aminoadipic acid; dimethyl N- {4-[N'-(2,4-diamino-6-pteridinyl)methylamino]-3- methyl)benzoyI-L-2-aminoadipate;
N-{4-[iV-(2,4-diamino-6-pteridinyl)methylamino]-3-methyl)benzoyl-L- 2-aminoadipic acid. diethyl //-4-[Λ^-(2,4-diamino-6-pteridinyl)methyl-/V-methylamino]-3- methylbenzoyl-L-glutamate; dimethyl Λ/-{4-[7V-(2,4-diamino-6-pteridinyl)methyl-Λ/1-methylamino]- 3-methyl}benzoyl-L-2-aminoadipate;
7V-{4-[7V-(2,4-diamino-6-pteridinyl)methyl-7V-methylamino]-3- methylbenzoyl}-L-glutamic acid; diethyl N-{4-[N1-(2,4-diamino-6-pteridinyl)methyl--V-ethylamino]-3- methylbenzoyl} -L-glutamate;
Λr-{4-[Λ"-(2,4-diamino-6-pteridinyl)methyl-7S/'-ethylamino]-3- methylbenzoyl}-L-glutamic acid; diethyl Λ^-{4-[7V-(2,4-diamino-6-pteridinyl)methyl-Λ/ri-ethylamino]-3- methylbenzoyl} -L-glutamate; Λr-{4-[N'-(2,4-diamino-6-|>teridinyl)methyl-/v"-ethylamino]-3- methyl}benzoyl-L-glutamic acid; dimethyl N-{4-[7V-(2,4-diamino-6-pteridinyl)methyl-7VT-methylamino]- 3 -m ethylb en zoyl } -L-2- aminoadipate;
N-{4-[Λ^-(2,4-diamino-6-pteridinyl)methyl-Λ?1-niethylamino]-3- methyl} benzoyl-L-2-aminoadipic acid;
N-(4-[Λ''-(2,4-diamino-6-pteridinyl)methyl-Λ'l-methylamino]-3- methylbenzoyl } -L-homocysteic acid ammonium salt; diethyl N- {4-[yV-(2,4-diamino-6-pteridinyl)methyl-N1-methylamino]-3- ethylbenzoyl } -L-glutamate;
N-{4-[7V-(2,4-diamino-6-pteridinyl)methyl-Λ'1-methylamino]-3- ethylbenzoyl}-L-glutamic acid; diethyl N-{4-[AP-(2,4-diamino-6-pteridinyl)methyI-A^-mel±tylamino]- 3,5-dimethylbenzoyl}-L-glutamate;
N-{4-[7V-(2,4-diamino-6-pteridinyl)methyl-/l/'-methylamino]-3,5- dimethylbenzoyl}-L-glutamic acid;
Λ^-{4-[W-(2:,4-diamino-6-pteridinyl)methyl-Λ''-methylamino]-3- trifluoromethyIbenzoyl}-L-2-aminoadipic acid; dimethyl N-{4-[N-(2;,4-diamino-6-pteridinyl)methyl-7-methylindoline- 5'-carbonyl} -L-2-aminoadipate;
N-{l-[N'-(2,4-diamino-6-pteridinyl)methyl-7-methylindoline-5'- carbonyI}-L-2-aminoadipic acid;
Λ/-{l-[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl}-L- aminoadipic acid;
N-ll-p^-diamino-δ-pteridiny^methylJindole-S-carbony^-L^- aminoadipic acid;
N- { 1 -[2,4-diamino-6-pteridinyl)methyl]indole-5'-carbonyl} -L- homocysteic acid ammonium salt;
N-{l-[(2,4-diamino-6-pteridinyl)methyl]indole-5'-carbonyl}-L- . glutamic acid; N-{l-[(2,4-diamino-6-pteridinyl)methyl-7-methylindole-5'-carbonyl}- L-2-arninoadipic acid; diethyl N-{4-[/V'-(2,4-diamino-6-pteridinyl)rnethylarnino]-3- methyl}benzoyl-L-glutamate; dimethyl TV- {4-[/V-(2,4-diamino-6-pteridinyl)methylamino]-3- methyl}benzoyl-L-2-aminoadipate; and
Λ/-{4-[Λ'r-(2,4-diamino-6-pteridinyl)methylamino]-3-methyl)benzoyl-L- 2-aminoadipic acid; or a pharmaceutically acceptable salt or prodrug thereof. Another aspect of the present invention relates to methods for treating, ameliorating, or preventing hyperproliferative disorders. In certain aspects of the invention, the hyperproliferative disorder is cancer. In one embodiment, the cancer is a solid tumor. In another embodiment, the cancer is selected from the group consisting of colon cancer, brain cancer, glioma, multiple myeloma, head and neck cancer, hepatocellular cancer, melanoma, ovarian cancer, cervical cancer, renal cancer, and non-small cell lung cancer. In a further embodiment, the cancer is acute and chronic lymphocytic leukemia, acute granulocytic leukemia, adrenal cortex carcinoma, bladder carcinoma, breast carcinoma, cervical carcinoma, cervical hyperplasia, choriocarcinoma, chronic granulocytic leukemia, chronic lymphocytic leukemia, colon carcinoma, endometrial carcinoma, esophageal carcinoma, essential thrombocytosis, genitourinary carcinoma, hairy cell leukemia, head and neck carcinoma, Hodgkin's disease, Kaposi's sarcoma, lung carcinoma, lymphoma, malignant carcinoid carcinoma, malignant hypercalcemia, malignant melanoma, malignant pancreatic insulinoma, medullary thyroid carcinoma, melanoma, multiple myeloma, mycosis fungoides, myeloid and lymphocytic leukemia, neuroblastoma, non-Hodgkin's lymphoma, osteogenic sarcoma, ovarian carcinoma, pancreatic carcinoma, polycythemia vera, primary brain carcinoma, primary macroglobulinemia, prostatic carcinoma, renal cell carcinoma, rhabdomyosarcoma, skin cancer, small-cell lung carcinoma, soft- tissue sarcoma, squamous cell carcinoma, stomach carcinoma, testicular carcinoma, thyroid carcinoma, or Wilms' tumor.
[0010] In further aspects of the invention the hyperproliferative disorder is any one of age-related macular degeneration, Crohn's disease, cirrhosis, chronic inflammatory-related disorders, proliferative diabetic retinopathy, proliferative vitreoretinopathy, retinopathy of prematurity, granulomatosis, immune hyperproliferation associated with organ or tissue transplantation, an immunoproliferative disease or disorder, e.g., inflammatory bowel disease, psoriasis, rheumatoid arthritis, systemic lupus erythematosus (SLE), vascular hyperproliferation secondary to retinal hypoxia, or vasculitis.
[0011] In one embodiment the invention is drawn to methods of treating, ameliorating, or preventing hyperproliferative disease in a subject comprising administering to said subject a therapeutically effective amount of an N-oxide of methotrexate or analog thereof.
[0012] An additional aspect of the present invention is a method for treating, ameliorating, or preventing hyperproliferative disorders in an animal comprising administering to the animal a therapeutically effective amount of a compound having Formula I in combination with one or more active agents or treatments. In one embodiment, the one or more active agent or treatment is a chemotherapeutic agent, a radiotherapeutic agent/treatment, an anti-angiogenesis agent, a vascular targeting agent, a hypoxia-inducible factor 1 (HIFl) inhibitor, an Hsp90 inhibitor, a tyrosine kinase inhibitor, a serine/threonine kinase inhibitor, a proteasome inhibitor, an HDAC inibitor, a caspase inducer, a CDK inhibitor, and a proapoptotic molecule. In another embodiment, the one or more active agent or treatment is used, has been used, or is known to be useful for the treatment of the hyperproliferative disorder.
[0013] In one embodiment, the method of treating, ameliorating, or preventing hyperproliferative disorder in an animal comprises administering to the animal a therapeutically effective amount of methotrexate N10-oxide or analog thereof. In particular embodiments, the methotrexate analog NI0-oxide, or a pharmaceutically acceptable salt thereof, is provided in combination with one or more active agents or treatments, for example, chemotherapeutic agents or radiotherapeutic agents/treatments.
[0014] In preferred embodiments of the invention, the one or more chemotherapeutic agents can be any chemotherapeutic agent which is used, has been used, or is known to be useful for the treatment of hyperproliferative disorders.
[0015] In preferred embodiments of the invention, the one or more radiotherapeutic agents or treatments can be external-beam radiation therapy, brachytherapy, thermotherapy, radiosurgery, charged-particle radiotherapy, neutron radiotherapy, photodynamic therapy, or radionuclide therapy.
[0016] In one embodiment of the invention, the compound having Formula I can be administered prior to, during, and/or beyond administration of the one or more chemotherapeutic agents or radiotherapeutic agents or treatments. In another embodiment of the invention, the method of administering a compound having Formula I in combination with one or more chemotherapeutic agents or radiotherapeutic agents or treatments is repeated more than once.
[0017] The combination of a compound having Formula I and one or more chemotherapeutic agents or radiotherapeutic agents or treatments of the present invention will have additive potency or an additive therapeutic effect. The invention also encompasses synergistic combinations where the therapeutic efficacy is greater than additive. Preferably, such combinations will reduce or avoid unwanted or adverse effects. In certain embodiments, the combination therapies encompassed by the invention will provide an improved overall therapy relative to administration of a compound having Formula I or any chemotherapeutic agent or radiotherapeutic agent or treatment alone. In certain embodiments, doses of existing or experimental chemotherapeutic agents or radiotherapeutic agents or treatments will be reduced or administered less frequently which will increase patient compliance, thereby improving therapy and reducing unwanted or adverse effects. 10018] Further, the methods of the invention will be useful not only with previously untreated patients but also will be useful in the treatment of patients partially or completely refractory to current standard and/or experimental cancer therapies, including but not limited to radiotherapies, chemotherapies, and/or surgery. In a preferred embodiment, the invention will provide therapeutic methods for the treatment or amelioration of hyperproliferative disorders that have been shown to be or may be refractory or non-responsive to other therapies.
[0019] While not wishing to be bound by any theory, it is believed that some of the N-oxide compounds of the invention will function as prodrugs with greatly diminished cytotoxicity. It is believed that these N-oxide compounds will be activated under hypoxic conditions within the target tissues {i.e., reduced at the nitrogen atom), followed by inhibition of dihydrofolate reductase and/or thymidylate synthase, diminishing cells' ability to replicate. Other N-oxide compounds of the invention may have intrinsic cytotoxic activity. Since a number of pathological tissues have significant hypoxic components which promote hyperproliferation, it is believed that this portion of tissue will be preferentially targeted.
DETAILED DESCRIPTION OF THE INVENTION
[0020] One aspect of the invention is drawn to compounds having Formula I:
Figure imgf000014_0001
or a pharmaceutically acceptable salt thereof, wherein: Ri-R8, m, n and p are defined above.
[0021] In another aspect the invention is drawn to compounds having Formula
II:
Figure imgf000015_0001
or a pharmaceutically acceptable salt thereof, wherein:
Ri is a lower alkyl group having 1-4 carbon atoms, each of R2 and R3 is independently hydrogen, halo, hydroxy, cyano, a lower alkyl group having 1-4 carbon atoms, a lower alkoxy having 1-4 carbon atoms, a trifluoromethyl group or -NRI2RHRH;
R8-R14, X1, Y, Z, m, n and p are as defined above.
[0022] In another aspect the invention is drawn to compounds having Formula
III:
Figure imgf000015_0002
or a pharmaceutically acceptable salt thereof, wherein R6-R8, m, n and p are as defined above.
[0023] In another aspect the invention is drawn to compounds having Formula
IV:
Figure imgf000015_0003
or a pharmaceutically acceptable salt thereof, wherein Re-R8, m. n a°d p are as defined above. [0024] A further aspect the invention is drawn to compounds having Formula
V:
Figure imgf000016_0001
or a pharmaceutically acceptable salt thereof, wherein R&-R8, ni, n and p are as defined above.
[0025] According to another aspect of the invention, a therapeutically effective amount of a compound having Formula I, or a pharmaceutically acceptable salt thereof, and at least one other active agent is provided in the form of a pharmaceutical composition having at least one pharmaceutically acceptable carrier. In certain instances, the at least one other active agent is a chemotherapeutic agent (including an active vitamin D compound). Compounds having Formula I may be formulated in a single formulation with the other active agent(s), or formulated independently.
[0026] According to one aspect of the invention, methods for treating, ameliorating, or preventing hyperproliferative disorders are provided, wherein a therapeutically effective amount of a compound having Formula 1, or a pharmaceutically acceptable salt thereof, is administered to an animal in need thereof. In certain aspects of the invention, the hyperproliferative disorder is cancer.
[0027] A further aspect of the invention relates to methods for treating, ameliorating, or preventing a hyperproliferative disorder comprising administering a therapeutically effective amount of a compound having Formula I3 or a pharmaceutically acceptable salt thereof, in combination with at least one other active agent or treatment to a patient in need thereof. In certain embodiments, combinations of a compound having Formula I with a chemotherapeutic agent are administered. In one embodiment, the chemotherapeutic agent is selected from gemcitabine and irinotecan. [0028] Hyperproliferative disorders which can be treated with the compounds having Formula I include any hypoxia-aggravated hyperproliferative disease and/or disorder, such as any number of cancers. Generally, such cancers include, without limitation, cancers of the bladder, brain, breast, cervix, colon, endometrium, esophagus, head and neck, kidney, larynx, liver, lung, oral cavity, ovaries, pancreas, prostate, skin, stomach, and testis. Certain of these cancers may be more specifically referred to as acute and chronic lymphocytic leukemia, acute granulocytic leukemia, adrenal cortex carcinoma, bladder carcinoma, breast carcinoma, cervical carcinoma, cervical hyperplasia, choriocarcinoma, chronic granulocytic leukemia, chronic lymphocytic leukemia, colon carcinoma, endometrial carcinoma, esophageal carcinoma, essential thrombocytosis, genitourinary carcinoma, hairy cell leukemia, head and neck carcinoma, Hodgkin's disease, Kaposi's sarcoma, lung carcinoma, lymphoma, malignant carcinoid carcinoma, malignant hypercalcemia, malignant melanoma, malignant pancreatic insulinoma, medullary thyroid carcinoma, melanoma, multiple myeloma, mycosis fungoides, myeloid and lymphocytic leukemia, neuroblastoma, non-Hodgkin's lymphoma, osteogenic sarcoma, ovarian carcinoma, pancreatic carcinoma, polycythemia vera, primary brain carcinoma, primary macroglobulinemia, prostatic carcinoma, renal cell carcinoma, rhabdomyosarcoma, skin cancer, small-cell lung carcinoma, sofi-tissue sarcoma, squamous cell carcinoma, stomach carcinoma, testicular carcinoma, thyroid carcinoma, and Wilms1 tumor. In one embodiment, the cancer is a solid tumor. In another embodiment, the cancer is selected from the group consisting of colon cancer, brain cancer, glioma, multiple myeloma, head and neck cancer hepatocellular cancer, melanoma, ovarian cancer, cervical cancer, renal cancer, and non-small cell lung cancer.
[0029] Animals which may be treated according to the present invention include all animals which may benefit from administration of compounds having Formula 1. Such animals include humans, pets such as dogs and cats, and veterinary animals such as cows, pigs, sheep, goats and the like. [0030] The term "alkyl" as used herein refers to an unsaturated acyclic hydrocarbon radical. The term "lower alkyl" refers to acyclic hydrocarbon radicals containing from about 2 to about 10 carbon atoms, preferably from about 2 to about 8 carbon atoms and more preferably 1 to about 6 carbon atoms. Examples of suitable alkyl radicals include methyl, ethyl, propyl, butyl, isobutyl, pentyl, 2-methylbutyl, 3-methylbutyl, hexyl, heptyl, and octyl, and the like.
[0031] The term "cycloalkyl" as used herein refers to saturated hydrocarbon ring structures of from 3 to 12 carbon atoms, and preferably from 3 to 6 carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 2- methylcyclopropyl, cyclopropylmethyl, cyclopentylmethyl, norbornyl, adamantyl, pinanyl, myrtanyl and the like. "Lower cycloalkyl" refers to cycloalkyl of 3 to 6 carbons.
[0032] The term "alkoxy" means a straight, branched or cyclic hydrocarbon configuration and combinations thereof, including from 1 to 20 carbon atoms, preferably from 1 to 8 carbon atoms, more preferably from 1 to about 4 carbon atoms, and an oxygen atom at the point of attachment. Suitable alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec- butoxy, tert-butoxy, cyclopropyloxy, cyclohexyloxy, and the like. "Lower alkoxy" refers to alkoxy groups having from 1 to 4 carbon atoms.
[0033] The term "aryl" as used herein refers to an aromatic hydrocarbon radical with 4 to about 16 carbon atoms, preferably of 6 to about 12 carbon atoms, and more preferably of 6 to about 10 carbon atoms. The rings may optionally be substituted with 1-3 substituents selected from alkyl, halogen, hydroxy, alkoxy, aryloxy, haloalkyl, phenyl and heteroaryl. Examples of aryl groups are phenyl, biphenyl and naphthyl.
[0034] The term "heterocycle" as used herein refers to a cyclic hydrocarbon structure of from 1 to 6, preferably 5 to 6, atoms, and containing from 1 to 3 heteroatoms selected from O, N and S; or a bicyclic 9- to 10-membered heterocyclic system containing from 1 to 4 heteroatoms chosen from O, N and S. [0035] Useful heteroaryl groups include thienyl, benzo[b]thienyl, naphtho[2,3- b]thienyl, thianthrenyl, furyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxanthenyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrirnidinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalzinyl, naphthyridinyl, quinozalinyl, cinnolinyl, pteridinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl, phenoxazinyl, 1,4- dihydroquinoxaIine-2,3-dione, 7-aminoisocoumarin, pyrido[l ,2-a]pyrimidin-4- one, 1 ,2-benzoisoxazol-3-yl, benzimidazolyl, 2-oxindolyl, and 2- oxobenzimidazolyl. Where the heteroaryl group contains a nitrogen atom in a ring, such nitrogen atom may be in the form of an N-oxide, e.g., a pyridyl N- oxide, pyrazinyl N-oxide, pyrimidinyl N-oxide, and the like.
[0036] As used herein, the term "prodrug" refers to a derivative of a parent
N-oxide molecule that requires biotransformation, either solvolytic or enzymatic, within the organism to release the active drug. Although the N-oxides may be considered as prodrugs of the corresponding non-N-oxide parent compound, the term "prodrug" as used herein does not refer to the N-oxides. Rather, prodrugs of the present inventions are limited to variations or derivatives of the compounds of this invention which have groups cleavable under metabolic conditions to form the compounds of the present invention. Thus, prodrugs of the present invention become the compounds of the invention when they undergo solvolysis under physiological conditions or undergo enzymatic degradation (e.g., hydrolysis), oxidation (e.g., hepatic hydroxylation) or reduction, excluding the bioreductive process that transforms the N-oxides of the present invention to the corresponding non-N-oxides of the parent compounds.
[0037] Typical enzymatically or solvolytically cleavable groups suitable for preparing the prodrugs of the present invention include esters, imines, carbamates, acetals and ketals. For example, an ester of a carboxylic acid containing compound of the present invention may be prepared by condensation with an alcohol, preferably a lower alkyl alcohol, more preferably a C)-4 alkyl alcohol. Similarly, an ester of a hydroxy containing compound of the present invention may be prepared by condensation with a carboxylic or a dioic acid, preferably an alkyl carboxylic or dioic acid, more preferably a C1 4 carboxylic acid or a C3 6 dioic acid or anhydride thereof. Moreover, an imine of an amino containing compound of the present invention may be obtained by condensation of the amino group with a carbonyl group of an aldehyde or a ketone. Aldehydes and ketones suitable for condensation with amino containing compounds of the present invention include alkyl and aryl ketones and aldehydes, more preferably alky ketones and aldehydes, more preferably lower alkyl ketones and aldehydes, most preferably Ci-4 alkyl aldehydes and ketones. A carbamate of an amino containing compound of the present invention may be prepared by condensation of the amino group with, for example, benzyloxycarbonyl chloride. In addition, an acetal or ketal of an alcohol containing compound of the present invention may be obtained by condensation of the hydroxy group with chloromethyl methyl ether or chloromethyl ethyl ether.
[0038] The term "non-N-oxide" as used herein refers to an amine compound that is not oxidized at the nitrogen atom. As an example, methotrexate is the non-N-oxide form of methotrexate N-oxide.
[0039] The term "methotrexate N-oxides and analogs" and similar terms as used herein are intended to refer to compounds in which a tertiary or an aromatic amine present in the compounds is oxidized. Any tertiary or aromatic amine suitable to afford a hypoxia-activatable N-oxide of the active compound may be oxidized to prepare the N-oxides of the present invention. Particularly preferred N-oxides of the present invention are those in which the N10 nitrogen of methotrexate, or the corresponding nitrogen of the analog, is oxidized to N10-oxide. As used herein, N10 refers to the nitrogen atom of the compound of formula I to which Ri and R8 are attached.
[0040] The term "pharmaceutical composition" as used herein, is to be understood as defining compositions of which the individual components or ingredients are themselves pharmaceutically acceptable, e.g., where oral administration is foreseen, acceptable for oral use; where topical administration is foreseen, topically acceptable; and where intravenous administration is foreseen, intravenously acceptable.
[0041] As used herein, the term "therapeutically effective amount" refers to that amount of the therapeutic agent sufficient to result in amelioration of one or more symptoms of a disorder, or prevent advancement of a disorder, or cause regression of the disorder. For example, with respect to the treatment of cancer, a therapeutically effective amount preferably refers to the amount of a therapeutic agent that decreases the rate of tumor growth, decreases tumor mass, decreases the number of metastases, increases time to tumor progression, or increases survival time by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.
[0042] The terms "prevent," "preventing," and "prevention," as used herein, refer to a decrease in the occurrence of pathological cells (e.g., hyperproHferative or neoplastic cells) in an animal. The prevention may be complete, e.g., the total absence of pathological cells in a subject. The prevention may also be partial, such that the occurrence of pathological cells in a subject is less than that which would have occurred without the present invention.
[0043] Compounds having Formula I can be provided as pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts (i.e., addition salts) include inorganic and organic acid addition salts such as hydrochloride, hydrobromide, phosphate, sulphate, citrate, lactate, tartrate, maleate, fumarate, mandelate, benzoate and oxalate; and inorganic and organic base addition salts with bases such as sodium hydroxide, Tris(hydroxymethyl)arninornethane (TRIS, tromethane) and /V-mcthyl-glucamine. Although the salts typically have similar physiological properties compared to the free base, certain acid addition salts may demonstrate preferred physicochemical properties, e.g., enhanced solubility, improved stability. One particular pharmaceutically acceptable salt is derived from maleic acid, the salt being either a hydrogen maleate or a dimaleate salt.
[0044] Certain of the compounds of the present invention may exist as stereoisomers including optical isomers. The invention includes all stereoisomers and both the racemic mixtures of such stereoisomers as well as the individual enantiomers that may be separated according to methods that are well known to those of ordinary skill in the art. Certain of the compounds of the present invention may also exist as diasteroisomers wherein one or more substituents on the methotrexate analog contain one or more chiral centers.
[0045] In certain embodiments of the invention, compounds having Formula I are administered in combination with one or more other active agents (e.g., chemotherapeutic agents) or treatments. By way of non-limiting example, a patient may be treated for a hyperproliferative disorder, such as cancer, by the administration of a therapeutically effective amount of a compound having Formula 1 in combination with radiotherapy agent/treatment or the administration of a chemotherapeutic agent.
[0046] In other embodiments, compounds of the invention are administered in combination with agents, such as anti-angiogenic agents, that block inhibit or modulate tumor neovascularization. In preferred embodiments, anti- angiogenesis agents can be any anti-angiogenesis agent which is used, has been used, or is known to be useful for the treatment of hyperproliferative disorders. Examples of anti-angiogenesis agents include bevacizumab (Avastin™), VEGF-TRAP, anti-VEGF-receptor antibodies, angiostatin, endostatin, batimastat, captopril, cartilage derived inhibitor, genistein, interleukin 12, lavendustin, medroxyprogesterone acetate, recombinant human platelet factor 4, tecogalan, thrombospondin, TNP-470, VEGF antagonists, anti-VEGF monoclonal antibody, soluble VEGF-receptor chimaeric protein, antisense oligonucleotides, antisense oligodexoynucleotides, siRNAs, anti- VEGF aptamers, pigment epithelium derived factor, a tyrosine kinase inhibitor, an inhibitor of epidermal-derived growth factor, an inhibitor of . fibrob last-derived growth factor, an inhibitor of platelet derived growth factor, an MMP (matrix metalloprotease) inhibitor, an integrin blocker, interferon-α, pentosan polysulfate, a cyclooxygenase inhibitor, carboxyamidotriazole, combretastatin A-4, squalamine, ό-O-chloroacetyl-carbonyO-fumagillol, thalidomide, troponin-1, indolinethiones, pyridopyrimidines, quinoazolines, phenyl-pyrrolo-pyrimidines, trastuzυmab, calcium influx inhibitor (CAl), neomycin, squalamine, marimastat, prinomastat (AG-3340), metastat (COL-3) and cinnoline derivatives. Additional anti-angiogenic compounds that may be administered in combination with the compounds of the present invention are described in U.S. Patent Nos. 5,192,744, 5,426,100, 5,733,876, 5,840,692, 5,854,205, 5,990,280, 5,994,292, 6,342,219, 6,342,221, 6,346,510, 6,479,512, 6,719,540, 6,797,488, 6,849,599, 6,869,952, 6,887,874, 6,958,340 and 6,979,682.
[0047] In certain embodiments, the compounds of the present invention are administered in combination with a vascular targeting agent (also known as vascular damaging agents). In one embodiment, the vascular targeting agent is for the treatment of malignant or non-malignant vascular proliferative disorders. In other embodiments, vascular targeting agents can be any vascular targeting agent which is used, has been used, or is known to be useful for the treatment of hyperproliferative disorders. Examples of vascular targeting agents that may be administered in combination with the compounds of the present invention include DMXAA 5,6-dimethylxanthenone-4-acetic acid, ZD6126, (5S)-5-(acetylamino)-9,10,l l-trimethoxy-6,7-dihydro-5H- dibenzo[a,c]cyclohepten-3-yl dihydrogen phosphate, also known as iV-acetylcolchinol-O-phosphate (see, for example, U.S. Patent No. 6,906,048); functionalized stilbene derivatives such as combretastatin A4 and its prodrugs (see, e.g., U.S. Patent Nos. 6,919,324 and 6,773,702); dioleoyltrimethyl- ammonium propane (DOTAP), N-[l-(2,3-dioleoyloxy)-propyl]-N,N,N- trimethylammonium chloride (DOTMA), dimethyldioctadecylammonium bromide (DDAB), l ,2-dimyristyloxypropyl-3-dimethylhydroxyethyl (DMRIE), dioleoyl-3-dimethylammonium propane (DODAP), N,N-dioleyl-N,N- dimethylammoniυm chloride (DODAC), or N-(l-(2,3-dioleyloxy)propyl)-N- (2-(speπτiinecarboxamido)ethyl)-N,N-dimethyl ammonium trifluoroacetate (DOSPA), or any other natural or synthetic cationic lipids, including, for example, dioleoylphosphatidyl-choline (DOPC), dipalmitoylphosphatidylcholine (DPPC), disteroylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholϊne (DMPC), or 1,2-sn- dioleoylphosphatidylcholine (DOPE), or any other natural or synthetic electrostatically neutral lipids (see, for example, U.S. patent No. 6,680,068); vascular targeting agents which incorporate benzo[6]thiophene, indole, and benzofuran molecular skeletons such as those described in U.S. Patent No. 6,593,374. In other embodiments, the compounds of the present invention are administered in combination with a hypoxia-inducible factor 1 (HIFl) inhibitor. In one embodiment, the HIFl inhibitor is for the treatment of malignant or non-malignant vascular proliferative disorders. In other embodiments, HIFl inhibitors can be any HIFl inhibitor which is used, has been used, or is known to be useful for the treatment of hyperproliferative disorders. Examples of HIFl inhibitors suitable for use in combination with compounds of the present invention include topotecan, P13 kinase inhibitors; LY294002; rapamycin; histone deacetylase inhibitors such as [(E)- (lS,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia- 5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene-3,6,9,19,22-pentanone (FR901228, depsipeptide); heat shock protein 90 (Hsp90) inhibitors such as geldanamycin, 17-allylamino-geldanamycin (17-AAG), and other geldanamycin analogs, and radicicol and radicicol derivatives such as KF58333; genistein; indanone; staurosporin; protein kinase-1 (MEK-I) inhibitors such as PD98059 (2'-amino-3'-methoxyfiavone); PX-12 (1- methylpropyl 2-imidazolyl disulfide); pleurotin PX478; quinoxaline 1,4- dioxides; sodium butyrate (NaB); sodium nitropurruside (SNP) and other NO donors; microtubule inhibitors such as novobiocin, panzem (2- methoxyestradiol or 2-ME2), vincristines, taxanes, epothilones, discodermolide, and derivatives of any of the foregoing; coumarins; barbituric and thiobarbituric acid analogs; camptothecins; and YC-I . See U.S. Patent No. 6,979,675.
[0049] In certain embodiments, the compounds of the present invention are administered in combination with an Hsp90 inhibitor. In one embodiment, the Hsp90 inhibitor is for the treatment of malignant or non-malignant vascular proliferative disorders. In other embodiments, Hsp90 inhibitors can be any Hsp90 inhibitor which is used, has been used, or is known to be useful for the treatment of hyperproliferative disorders. Examples of Hsp90 inhibitors that may be combined with the compounds of the present invention include geldanamycin, 17-allylamino-l 7-demethoxygeldanamycin, geldanamycin derivatives such as those described in U.S. patent No. 6,890,917, dexamethasone and benzoquinone ansamycins such as those described in U.S. patent No. 6,872,715. Additional Hsp90 inhibitors are disclosed in U.S. Patent Nos. 6,613,780, 6,281,229 and 6,903,1 16.
[00501 In other embodiments, the compounds of the present invention are administered in combination with an inhibitor of tyrosine and/or serine/threonine kinases and tyrosine kinase receptors involved in cellular signaling. These include tyrosine kinase inhibitors of Src, AbI, Platelet Derived Growth Factor Receptors, Vascular Endothelial Growth Factor Receptors, c-Met, Fibroblast Growth Factor receptors, Epidermal Growth Factor Receptors, Insulin Growth Factor Receptors, mTOR, Flt-3, CSF-I Receptor, AKT, Polo kinases, Aurora Kinases, STAT-3, PI-3 Kinase, Ras, Raf and Mitogen Activated Kinases, MEK, ERK. Examples of tyrosine kinase and serine/threonine kinase inhibitors include (but not limited to): AMG706, ZA6474, BAY 43-9006, Dasatinib, CEP-701, XL647, XL999, Lapatinb, MLN518/CT53518, PKC412, ST1571, AMN107, AEE 788, OSI-930, OSI- 817, SUl 1248, AG-03736, GW-786034m , CEP-7055. [0051] In other embodiments, the compounds of the present invention are administered in combination with HDAC inhibitors. Examples include (but not limited to) SAHA, MS-275, MGCD0103, LBH589, PXDlOl, FK228.
[0052 J In other embodiments, the compounds of the present invention are administered in combination with proteasome inhbitors such as Velcade.
[0053] In other embodiments, the compounds of the present invention are administered in combination with pro-apoptotic agents such as TRAIL, anti- DR4/DR5 (TRA8) antibodies, IAP, Survivin or small molecules that stimulate caspase activation.
[0054] In other embodiments, the compounds of the present invention are administered in combination with inhibitors of cell cycle regulators such as CDK inhibitors.
[0055] "In combination" refers to the use of more than one treatment. The use of the term "in combination" does not restrict the order in which treatments are administered to a subject being treated for a hyperproliferative disorder. A first treatment can be administered prior to, concurrently with, after, or within any cycling regimen involving the administration of a second treatment to a subject with a hyperproliferative disorder. For example, the first treatment can be administered 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before a treatment; or the first treatment can be administered 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after a second treatment. Such treatments include, for example, the administration of compounds having Formula I in combination with one or more chemotherapeutic agents or radiotherapeutic agents/treatments.
[0056] The term "chemotherapeutic agent," as used herein, is intended to refer to any chemotherapeutic agent known to those of skill in the art to be effective for the treatment, prevention or amelioration of hyperproliferative disorders sυch as cancer. Chemotherapeutic agents include, but are not limited to, small molecules, synthetic drugs, peptides, polypeptides, proteins, nucleic acids (e.g., DNA and RNA polynucleotides including, but not limited to, antisense nucleotide sequences, triple helices and nucleotide sequences encoding biologically active proteins, polypeptides or peptides), antibodies, synthetic or natural inorganic molecules, mimetic agents, and synthetic or natural organic molecules. Any agent which is known to be useful, or which has been used or is currently being used for the treatment or amelioration of a hyperproliferative disorder can be used in combination with a compound having Formula I. See, e.g., Hardman et ah, eds., 2002, Goodman & Gilman's The Pharmacological Basis Of Therapeutics 10th Ed, Mc-Graw-Hill, New York, NY for information regarding therapeutic agents which have been or are currently being used for the treatment or amelioration of a hyperproliferative disorder.
[00571 Particular chemotherapeutic agents useful in the methods and compositions of the invention include alkylating agents, antimetabolites, antimitotic agents, epipodophyllotoxins, antibiotics, hormones and hormone antagonists, enzymes, platinum coordination complexes, anthracenediones, substituted ureas, methylhydrazine derivatives, imidazotetrazine derivatives, cytoprotective agents, DNA topoisomerase inhibitors, biological response modifiers, retinoids, therapeutic antibodies, differentiating agents, immunomodulatory agents, angiogenesis inhibitors and anti-angiogenic agents.
[0058] Certain chemotherapeutic agents include, but are not limited to, abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, asparaginase, BCG live, bevaceizumab, bexarotene, bleomycin, bortezomib, busulfan, calusterone, camptothecin, capecitabine, carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cinacalcet, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, darbepoetin alfa, daunorubicin, denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone, Elliott's B solution, epirubicin, epoetin alfa, estramustine, etoposide, exemestane, filgrastim, floxuridine, fludarabine, fluorouracil, fulvestrant, gemcitabine, gemtuzumab ozogamicin, gefitinib, goserelin, hydroxyurea, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib, interferon alfa-2a, interferon alfa-2b, irinotecan, letrozole, leucovorin, levamisole, lomustine, meclorethamine, megestrol, melphalan, mercaptopurine, mesna, methotrexate, methoxsalen, methylprednisolone, mitomycin C, mitotane, mitoxantrone, nandrolone, nofetumomab, oblimersen, oprelvekin, oxaliplatin, paclitaxel, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed, pentostatin, pipobroman, plicamyciπ, polifeprosan, porfimer, procarbazine, quinacrine, rasburicase, rituximab, sargramostim, streptozocin, talc, tamoxifen, tarceva, temozolomide, teniposide, testolactone, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, and zoledronate. In certain embodiments, chemotherapeutic agents are selected from gemcitabine and irinotecan.
[00591 Chemotherapeutic agents may be administered at doses that are recognized by those of skill in the art to be effective for the treatment of the hyperproliferative disorder. In certain embodiments, chemotherapeutic agents may be administered at doses lower than those used in the art due to the additive or synergistic effect of the compounds having Formula I.
[0060] Therapeutic agents useful in the methods and compositions of the invention include active vitamin D compound or mimics thereof, antineoplastic agents {e.g., actinomycin D, irinotecan, vincristine, vinorelbine, SN-38, azacitidine (5-azacytidine, 5AzaC), thalidomide vinblastine, methotrexate, azathioprine, fluorouracil, doxorubicin, mitomycin, docetaxel, paclitaxel), angiogenic inhibitors (e.g., VEGF-TRAP, angiostatin, endostatin, aptamer antogonist of VEGF, batimastat, captopril, cartilage derived inhibitor, genistein, interleukin 12, lavendustin, medroxypregesterone acetate, recombinant human platelet factor 4, tecogalan, thrombospondin and TNP-470), serine/threonine kinase inhibitors, tyrosine kinase inhibitors, HDAC inhibitors, Proteasome inhibitors, CDK inhibitors, HSP inhibitors, vasodilators (e.g., nitrates, calcium channel blockers), anticoagulants (e.g., heparin), anti-platelet agents (e.g., aspirin, blockers of Ilb/IIIa receptors, clopidogrel), anti-thrombins (e.g., hirudin, iloprost), immunosuppressants (e.g., sirolimus, tranilast, dexamethasone, tacrolimus, everolimus, A24), collagen synthetase inhibitors (e.g., halofuginone,' propyl hydroxylase, C- proteinase inhibitor, metalloproteinase inhibitor), anti-inflammatories (e.g., corticosteroids, non-steroidal anti-inflammatory drugs), 17β-estradiol, angiotensin converting enzyme inhibitors, colchicine, fibroblast growth factor antagonists, histamine antagonists, lovastatin, nitroprusside, phosphodiesterase inhibitors, prostaglandin inhibitors, suramin, serotonin blockers, thioprotease inhibitors, platelet-derived growth factor antagonists, nitric oxide, and angiopeptin. In one embodiment, the therapeutic agent is a taxane, e.g., paclitaxel or docetaxel.
[0061] In certain embodiments, patients are subjected to a hypoxia imaging technique prior to administration of the compositions comprising the compounds of the present invention. Examples of imaging techniques suitable for the determination of the presence of hypoxic tumor cells include computed tomography (CT), magnetic resonance imaging (MRI), single photon emission computer tomography (SPECT), and positron emission tomography (PET). Use of such visualization methods can advantageously be used to select a subset of patients that are particularly suitable for treatment with hypoxia activated antiproliferative compositions of the present invention.
[0062] In this embodiment, the invention is directed to a method of treating, preventing or ameliorating a hyperproliferative disease in an animal in need thereof, comprising determining whether said hyperproliferative disease is characterized by hypoxic tissue, and treating said animal with an effective amount of a compound of the invention.
[0063] The term "radiotherapeutic agent," as used herein, is intended to refer to any radiotherapeutic agent known to one of skill in the art to be effective to treat or ameliorate cancer, without limitation. For instance, the radiotherapeutic agent can be an agent such as those administered in brachytherapy or radionuclide therapy. Such methods can optionally further comprise the administration of one or more additional cancer therapies, such as, but not limited to, chemotherapies, surgery, and/or another radiotherapy.
10064] In certain embodiments involving radiotherapeutic agents or treatments, the present invention relates to a method for treating cancer comprising the administration of methotrexate or analog N-oxide having Formula I, in combination with a treatment comprising a therapeutically effective dose of brachytherapy. The brachytherapy can be administered according to any schedule, dose, or method known to one of skill in the art to be effective in the treatment or amelioration of cancer, without limitation. In general, brachytherapy comprises insertion of radioactive sources into the body of a subject to be treated for cancer, preferably inside the tumor itself, such that the tumor is maximally exposed to the radioactive source, while preferably minimizing the exposure of healthy tissue.
[0065] In certain embodiments, the brachytherapy can be intracavitary brachytherapy. In other embodiments, the brachytherapy can be interstitial brachytherapy. Whether the brachytherapy is intracavitary brachytherapy or interstitial brachytherapy, the brachytherapy can be administered at a high dose rate, a continuous low dose rate, or a pulsed dose rate. For example, and not by way of limitation, a high dose rate brachytherapy regimen can be a dose of 60 Gy administered in ten fractions over six days, while a continuous low dose rate brachytherapy regimen can be a total dose of about 65 Gy, administered continuously at about 40 to 50 cGy per hour. Other examples of high, continuous low, and pulsed dose rate brachytherapy are well known in the art. See, e.g., Mazeron et al, Sent. Rad. One. 12:95-108 (2002).
[0066] Representative radioisotopes that can be administered in any of the above-described brachytherapies include, but are not limited to, phosphorus 32, cobalt 60, palladium 103, ruthenium 106, iodine 125, cesium 137, indium 192, xenon 133, radium 226, californium 252, or gold 198. Other radioisotopes may be selected for administration in brachytherapy according to the desirable physical properties of such a radioisotope. One of skill in the art will readily recognize that many properties will affect a radioisotope's suitability for use in brachytherapy, including, but not limited to, the radioisotope's half-life, the degree to which emitted radiation penetrates surrounding tissue, the energy of emitted radiation, the ease or difficulty of adequately shielding the radioisotope, the availability of the radioisotope, and the ease or difficulty of altering the shape of the radioisotope prior to administration.
[0067] Additional methods of administering and apparatuses and compositions useful for brachytherapy are described in U.S. Patent Nos. 6,319,189, 6,179,766, 6,168,777, 6,149,889, and 5,611 ,767, each of which is incorporated herein by reference in its entirety.
[0068] In certain embodiments involving radiotherapeutic agents or treatments, the present invention relates to a method for treating cancer comprising the administration of methotrexate or analog N-oxide having Formula I, in combination with a treatment comprising a therapeutically effective dose of a radionuclide. The radionuclide therapy can be administered according to any schedule, dose, or method known to one of skill in the art to be effective in the treatment or amelioration of cancer, without limitation. In general, radionuclide therapy comprises systemic administration of a radioisotope that preferentially accumulates in or binds to the surface of cancerous cells. The preferential accumulation of the radionuclide can be mediated by a number of mechanisms, including, but not limited to, incorporation of the radionuclide into rapidly proliferating cells, specific accumulation of the radionuclide by the cancerous tissue without special targeting (e.g., iodine 131 accumulation in thyroid cancer), or conjugation of the radionuclide to a biomolecule specific for a neoplasm.
[0069] Representative radioisotopes that can be administered in radionuclide therapy include, but are not limited to, phosphorus 32, yttrium 90, dysprosium 165, indium 111, strontium 89, samarium 153, rhenium 186, iodine 131, iodine 125, lutetium 177, and bismuth 213. While all of these radioisotopes may be linked to a biomolecule providing specificity of targeting, iodine 131, indium 1 1 1, phosphorus 32, samarium 153, and rhenium 186 may be administered systemically without such conjugation. One of skill in the art may select a specific biomolecule for use in targeting a particular neoplasm for radionuclide therapy based upon the cell-surface molecules present on that neoplasm. For example, hepatomas may be specifically targeted by an antibody specific for ferritin, which is frequently over-expressed in such tumors. Examples of antibody-targeted radioisotopes for the treatment of cancer include ZEVALIN (ibritumomab tiuxetan) and BEXXAR (tositumomab), both of which comprise an antibody specific for the B cell antigen CD20 and are used for the treatment of non-Hodgkin lymphoma.
(0070] Other examples of biomolecules providing specificity for particular cell are reviewed in an article by Thomas, Cancer Biother. Radiopharm. J 7:71-82 (2002), which is incorporated herein by reference in its entirety. Furthermore, methods of administering and compositions useful for radionuclide therapy may be found in U.S. Patent Nos. 6,426,400, 6,358,194, 5,766,571, and 5,563,250, each of which is incorporated herein by reference in its entirety.
[0071] In certain embodiments involving radiotherapeutic agents or treatments, the present invention relates to a method for treating cancer comprising the administration of methotrexate or analog N-oxide having Formula I, in combination with a treatment comprising a therapeutically effective dose of external-beam radiation therapy. The external-beam radiation therapy can be administered according to any schedule, dose, or method known to one of skill in the art to be effective in the treatment or amelioration of cancer, without limitation. In general, external-beam radiation therapy comprises irradiating a defined volume within a subject with a high energy beam, thereby causing cell death within that volume. The irradiated volume preferably contains the entire cancer to be treated, and preferably contains as little healthy tissue as possible.
|0072] In certain embodiments, the external-beam radiation therapy can be three-dimensional conformal radiotherapy. In other embodiments, the external-beam radiation therapy can be continuous hyperfractionated radiotherapy. In still other embodiments, the external-beam radiation therapy can be intensity-modulated radiotherapy. In yet other embodiments, the external-beam radiation therapy can be helical tomotherapy. In still other embodiments, the external-beam radiation therapy can be three-dimensional conformal radiotherapy with dose escalation. In yet other embodiments, the external-beam radiation therapy can be stereotactic radiotherapy, including, but not limited to, single fraction stereotactic radiotherapy, fractionated stereotactic radiotherapy, and fractionated stereotactically guided conformal radiotherapy.
[0073] The external-beam radiation therapy can be generated or manipulated by any means known to one of skill in the art. For example, the photon beam used in external-beam radiation therapy can be shaped by a multileaf collimator. Other examples of suitable devices for generating a photon beam for use in external-beam radiation therapy include a gamma knife and a linac- based stereotactic apparatus. In certain embodiments, administration of the external-beam radiation therapy is controlled by a computer according to a three-dimensional model of the patient in the treatment position. Such a model can be generated, for example, by computed tomography (CT), magnetic resonance imaging (MRI), single photon emission computer tomography (SPECT), and positron emission tomography (PET). Use of such visualization methods can advantageously minimize the volume of healthy tissue treated, thereby allowing higher total doses of radiation to be administered to the patient.
[0074] In addition, healthy tissues can optionally be protected from the effects of the external-beam radiation therapy by placing blocking devices such as, e.g., lead shields, in locations where such protection is needed. Alternatively or additionally, metal reflecting shields can optionally be located to reflect the photon beam in order to concentrate the radiation on the cancerous tissue to be treated and protect healthy tissue. Placement of either shield is well within the knowledge of one of skill in the art. [0075] Methods of administering and apparatuses and compositions useful for external-beam radiation therapy can be found in U.S. Patent Nos. 6,449,336, 6,398,710, 6,393,096, 6,335,961 , 6,307,914, 6,256,591, 6,245,005, 6,038,283, 6,001,054, 5,802,136, 5,596,619, and 5,528,652, each of which is incorporated herein by reference in its entirety.
[0076] In certain embodiments involving radiotherapeutic agents or treatments, the present invention relates to a method for treating cancer comprising the administration of methotrexate or analog N-oxide having Formula I, in combination with a treatment comprising a therapeutically effective dose of thermotherapy. The thermotherapy can be administered according to any schedule, dose, or method known to one of skill in the art to be effective in the treatment or amelioration of cancer, without limitation. In certain embodiments, the thermotherapy can be cryoablation therapy. In other embodiments, the thermotherapy can be hyperthermic therapy. In still other embodiments, the thermotherapy can be a therapy that elevates the temperature of the tumor higher than in hyperthermic therapy.
[0077] Cryoablation therapy involves freezing of a neoplastic mass, leading to deposition of intra- and extra-cellular ice crystals; disruption of cellular membranes, proteins, and organelles; and induction of a hyperosmotic environment, thereby causing cell death. Cryoablation can be performed in one, two, or more freeze-thaw cycles, and further the periods of freezing and thawing can be adjusted for maximum tumor cell death by one of skill in the art. One exemplary device that can be used in cryoablation is a cryoprobe incorporating vacuum-insulated liquid nitrogen. See, e.g., Murphy et al., Sem. Urol. Oncol 19: 133-140 (2001). However, any device that can achieve a local temperature of about -18O0C to about -1950C can be used in cryoablation therapy. Methods for and apparatuses useful in cryoablation therapy are described in U.S. Patent Nos. 6,383,181, 6,383,180, 5,993,444, 5,654,279, 5,437,673, and 5,147,355, each of which is incorporated herein by reference in its entirety. [0078| Hyperthermic therapy typically involves elevating the temperature of a neoplastic mass to a range from about 42°C to about 440C. The temperature of the cancer may be further elevated above this range; however, such temperatures can increase injury to surrounding healthy tissue while not causing increased cell death within the tumor to be treated. The tumor may be heated in hyperthermic therapy by any means known to one of skill in the art without limitation. For example, and not by way of limitation, the tumor may be heated by microwaves, high intensity focused ultrasound, ferromagnetic thermoseeds, localized current fields, infrared radiation, wet or dry radiofrequency ablation, laser photocoagulation, laser interstitial thermic therapy, and electrocautery. Microwaves and radiowaves can be generated by waveguide applicators, horn, spiral, current sheet, and compact applicators.
[0079] Other methods of and apparatuses and compositions for raising the temperature of a tumor are reviewed in an article by Wust et al, Lancet Oncol. 3:487-97 (2002), and described in U.S. Patent Nos. 6,470,217, 6,379,347, 6,165,440, 6,163,726, 6,099,554, 6,009,351, 5,776,175, 5,707,401, 5,658,234, 5,620,479, 5,549,639, and 5,523,058, each of which is incorporated herein by reference in its entirety.
[0080] In certain embodiments involving radiotherapeutic agents or treatments, the present invention relates to a method for treating cancer comprising the administration of methotrexate or analog N-oxide having Formula I, in combination with a treatment comprising a therapeutically effective dose of radiosurgery. The radiosurgery can be administered according to any schedule, dose, or method known to one of skill in the art to be effective in the treatment or amelioration of cancer, without limitation. In general, radiosurgery comprises exposing a defined volume within a subject to a manually directed radioactive source, thereby causing cell death within that volume. The irradiated volume preferably contains the entire cancer to be treated, and preferably contains as little healthy tissue as possible. Typically, the tissue to be treated is first exposed using conventional surgical techniques, then the radioactive source is manually directed to that area by a surgeon. Alternatively, the radioactive source can be placed near the tissue to be irradiated using, for example, a laparoscope. Methods and apparatuses useful for radiosurgery are further described in Valentini et al, Eur. J. Surg. Oncol. 28:180-185 (2002) and in U.S. Patent Nos. 6,421 ,416, 6,248,056, and 5,547,454, each of which is incorporated herein by reference in its entirety.
[0081] In certain embodiments involving radiotherapeutic agents or treatments, the present invention relates to a method for treating cancer comprising the administration of methotrexate or analog N-oxide having Formula I, in combination with a treatment comprising a therapeutically effective dose of charged-particle radiotherapy. The charged-particle radiotherapy can be administered according to any schedule, dose, or method known to one of skill in the art to be effective in the treatment or amelioration of cancer, without limitation. In certain embodiments, the charged-particle radiotherapy can be proton beam radiotherapy. In other embodiments, the charged-particle radiotherapy can be helium ion radiotherapy. In general, charged-particle radiotherapy comprises irradiating a defined volume within a subject with a charged-particle beam, thereby causing cellular death within that volume. The irradiated volume preferably contains the entire cancer to be treated, and preferably contains as little healthy tissue as possible. A method for administering charged-particle radiotherapy is described in U.S. Patent No. 5,668,371, which is incorporated herein by reference in its entirety.
[0082J In certain embodiments involving radiotherapeutic agents or treatments, the present invention relates to a method for treating cancer comprising the administration of methotrexate or analog N-oxide having Formula J, in combination with a treatment comprising a therapeutically effective dose of neutron radiotherapy. The neutron radiotherapy can be administered according to any schedule, dose, or method known to one of skill in the art to be effective in the treatment or amelioration of cancer, without limitation.
[0083] In certain embodiments, the neutron radiotherapy can be a neutron capture therapy. In such embodiments, a compound that emits radiation when bombarded with neutrons and preferentially accumulates in a neoplastic mass is administered to a subject. Subsequently, the tumor is irradiated with a low energy neutron beam, activating the compound and causing it to emit decay products that kill the cancerous cells. Such compounds are typically boron containing compounds, but any compound that has a significantly larger neutron capture cross-section than common body constituents can be used. The neutrons administered in such therapies are typically relatively low energy neutrons having energies at or below about 0.5 eV. The compound to be activated can be caused to preferentially accumulate in the target tissue according to any of the methods useful for targeting of radionuclides, as described below, or in the methods described in Laramore, Semin. Oncol. 24:612-685 (1997) and in U.S. Patents Nos. 6,400,796, 5,877,165, 5,872,107, and 5,653,957, each of which is incorporated herein by reference in its entirety.
[0084) In other embodiments, the neutron radiotherapy can be a fast neutron radiotherapy. In general, fast neutron radiotherapy comprises irradiating a defined volume within a subject with a neutron beam, thereby causing cellular death within that volume. The irradiated volume preferably contains the entire cancer to be treated, and preferably contains as little healthy tissue as possible. Generally, high energy neutrons are administered in such therapies, with energies in the range of about 10 to about 100 million eV. Optionally, fast neutron radiotherapy can be combined with charged-particle radiotherapy in the administration of mixed proton-neutron radiotherapy.
[0085] In certain embodiments involving radiotherapeutic agents or treatments, the present invention relates to a method for treating cancer comprising the administration of methotrexate or analog N-oxide having Formula I, in combination with a treatment comprising a therapeutically effective dose of photodynamic therapy. The photodynamic therapy can be administered according to any schedule, dose, or method known to one of skill in the art to be effective in the treatment or amelioration of cancer, without limitation. In general, photodynamic therapy comprises administering a photosensitizing agent that preferentially accumulates in a neoplastic mass and sensitizes the neoplasm to light, then exposing the tumor to light of an appropriate wavelength. "Upon such exposure, the photosensitizing agent catalyzes the production of a cytotoxic agent, such as, e.g., singlet oxygen, which kills the cancerous cells.
[0086] Representative photosensitizing agents that may be used in photodynamic therapy include, but are not limited to, porphyrins such as porfimer sodium, 5-aminolaevulanic acid and verteporfin; chlorins such as temoporfin; texaphyrins such as lutetium texephyrin; purpurins such as tin etiopurpurin; phthalocyanines; and titanium dioxide. The wavelength of light used to activate the photosensitizing agent can be selected according to several factors, including the depth of the tumor beneath the skin and the absorption spectrum of the photosensitizing agent administered. The period of light exposure may also vary according to the efficiency of the absorption of light by the photosensitizing agent and the efficiency of the transfer of energy to the cytotoxic agent. Such determinations are well within the ordinary skill of one in the art.
[0087] Methods of administering and apparatuses and compositions useful for photodynamic therapy are disclosed in Hopper, Lancet Oncol. 7:212-219 (2000) and U.S. Patent Nos. .6,283,957, 6,071,908, 6,011,563, 5,855,595, 5,716,595, and 5,707,401, each of which is incorporated herein by reference in its entirety.
[0088] It will be appreciated that both the particular radiation dose to be utilized in treating a hyperproliferative disorder and the method of administration will depend on a variety of factors. Thus, the dosages of radiation that can be used according to the methods of the present invention are determined by the particular requirements of each situation. The dosage will depend on such factors as the size of the tumor, the location of the tumor, the age and sex of the patient, the frequency of the dosage, the presence of other tumors, possible metastases and the like. Those skilled in the art of radiotherapy can readily ascertain the dosage and the method of administration for any particular tumor by reference to Hall, E. J., Radiobiology for the Radiologist, 5th edition, Lippincott Williams & Wilkins Publishers, Philadelphia, PA, 2000; Gunderson, L. L. and Tepper J. E., eds., Clinical Radiation Oncology, Churchill Livingstone, London, England, 2000; and Grosch, D. S., Biological Effects of Radiation, 2nd edition, Academic Press, San Francisco, CA, 1980. In certain embodiments, radiotherapeutic agents and treatments may be administered at doses lower than those known in the art due to the additive or synergistic effect of the compound having Formula T.
[0089] Compositions in accordance with the present invention may be employed for administration in any appropriate manner, e.g., oral or buccal administration, e.g., in unit dosage form, for example in the form of a tablet, in a solution, in hard or soft encapsulated form including gelatin encapsulated form, sachet, or lozenge. Compositions may also be administered parenterally or topically, e.g., for application to the skin, for example in the form of a cream, paste, lotion, gel, ointment, poultice, cataplasm, plaster, dermal patch or the like, or for ophthalmic application, for example in the form of an eyedrop, -lotion or -gel formulation. Readily flowable forms, for example solutions, emulsions and suspensions, may also be employed e.g., for . intralesional injection, or may be administered rectally, e.g., as an enema or suppository, or intranasal administration, e.g., as a nasal spray or aerosol. Microcrystalline powders may be formulated for inhalation, e.g., delivery to the nose, sinus, throat or lungs. Transdermal compositions/devices and pessaries may also be employed for delivery of the compounds of the invention. The compositions may additionally contain agents that enhance the delivery of the compounds having Formula I (or other active agents), e.g., liposomes, polymers or co-polymers (e.g., branched chain polymers). Preferred dosage forms of the present invention include oral dosage forms and intravenous dosage forms.
[0090] Intravenous forms include, but are not limited to, bolus and drip injections. In preferred embodiments, the intravenous dosage forms are sterile or capable of being sterilized prior to administration to a subject since they typically bypass the subject's natural defenses against contaminants. Examples of intravenous dosage forms include, but are not limited to, Water for Injection USP; aqueous vehicles including, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-rniscible vehicles including, but not limited to, ethyl alcohol, polyethylene glycol and polypropylene glycol; and non-aqueous vehicles including, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate and benzyl benzoate.
[0091] The pharmaceutical compositions of the present invention may further comprise one or more additives. Additives that are well known in the art include, e.g., detackifiers, anti-foaming agents, buffering agents, antioxidants (e.g., ascorbic acid, ascorbyl palmitate, sodium ascorbate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate, malic acid, fumaric acid, potassium metabisulfite, sodium bisulfite, sodium metabisulfite, and tocopherols, e.g., α-tocopherol (vitamin E)), preservatives, chelating agents, viscomodulators, tonicifϊers, flavorants, colorants, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and mixtures thereof. The amounts of such additives can be readily determined by one skilled in the art, according to the particular properties desired, and can be formulated such that compounds having Formula I are stable, e.g., not reduced by antioxidant additives.
[0092) The additive may also comprise a thickening agent. Suitable thickening agents may be of those known and employed in the art, including, e.g., pharmaceutically acceptable polymeric materials and inorganic thickening ' agents. Exemplary thickening agents for use in the present pharmaceutical compositions include polyacrylate and polyacrylate co-polymer resins, for example poly-acrylic acid and poly-acrylic acid/methacrylic acid resins; celluloses and cellulose derivatives including: alkyl celluloses, e.g., methyl-, ethyl- and propyl-celluloses; hydroxyalkyl -celluloses, e.g., hydroxypropyl- celluloses and hydroxypropylalkyl-celluloses such as hydroxypropyl-methyl- celluloses; acylated celluloses, e.g., cellulose-acetates, cellulose- acetatephthallates, celliilose-acetatesuccinates and hydroxypropylmethyl- cellulose phthallates; and salts thereof such as . sodium-carboxymethyl- celluloses; polyvinylpyrrolidones, including for example poly-N- vinylpyrrolidones and vinylpyrrolidone co-polymers such as vinylpyrrolidone- vinylacetate co-polymers; polyvinyl resins, e.g., including polyvinylacetates and alcohols, as well as other polymeric materials including gum traganth, gum arabicum, alginates, e.g., alginic acid, and salts thereof, e.g., sodium alginates; and inorganic thickening agents such as atapulgite, bentonite and silicates including hydrophilic silicon dioxide products, e.g., alkylated (for example methylated) silica gels, in particular colloidal silicon dioxide products.
[0093] Such thickening agents as described above may be included, e.g., to provide a sustained release effect. However, where oral administration is intended, the use of thickening agents may not be required. Use of thickening agents is, on the other hand, indicated, e.g., where topical application is foreseen-.
[0094] Compounds of Formula I may be formulated similar to methotrexate or polyglutamated methotrexate as may be appropriate.
[0095] Although the dosage of the compound having Formula I will vary according to the activity and/or toxicity of the particular compound, the condition being treated, and the physical form of the pharmaceutical composition being employed for administration, it may be stated by way of guidance that a dosage selected in the range from 0.1 to 20 mg/kg of body weight per day will often be suitable, although higher dosages, such as 0.1 to 50 mg/kg of body weight per day may be useful. Those of ordinary skill in the art are familiar with methods for determining the appropriate dosage. Methods for assessing the toxicity, activity and/or selectivity of the compounds having Formula I may be carried out using any of the methods known in the art, including the antiproliferative activity test. f0096] In certain instances, the dosage of the compounds having Formula T will be lower, e.g., when used in combination with at least a second hyperproliferative disorder treatment, and may vary according to the activity and/or toxicity of the particular compound, the condition being treated, and the physical form of the pharmaceutical composition being employed for administration.
[0097] When the composition of the present invention is formulated in unit dosage form, the compound having Formula I will preferably be present in an amount of between 0.01 and 2000 mg per unit dose. More preferably, the amount of compound having Formula I per unit dose will be about 0.01, 0.05, 0.1 , 0.5, 1 , 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350,' 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, or 2000 mg or any amount therein.
[0098] When the unit dosage form of the composition is a capsule, the total quantity of ingredients present in the capsule is preferably about 10-1000 μL. More preferably, the total quantity of ingredients present in the capsule is about 100-300 μL. In another embodiment, the total quantity of ingredients present in the capsule is preferably about 10-1500 mg, preferably about 100- 1000 mg.
[0099] The relative proportion of ingredients in the compositions of the invention will, of course, vary considerably depending on the particular type of composition concerned. The relative proportions will also vary depending on the particular function of ingredients in the composition. The relative proportions will also vary depending on the particular ingredients employed and the desired physical characteristics of the product composition, e.g., in the case of a composition for topical use, whether this is to be a free flowing liquid or a paste. Determination of workable proportions in any particular instance will generally be within the capability of a person of ordinary skill in the art. All indicated proportions and relative weight ranges described below are accordingly to be understood as being indicative individually inventive teachings only and not as limiting the invention in its broadest aspect.
[00100] The amount of compound having Formula I in compositions of the invention will of course vary, e.g., depending on the intended route of administration and to what extent other components are present. In general, however, the compound having Formula I will suitably be present in an amount of from about 0.005% to 20% by weight based upon the total weight of the composition. In certain embodiments, the compound having Formula I is present in an amount of from about 0.01% to 15% by weight based upon the total weight of the composition.
[00101] In addition to the foregoing, the present invention also provides a process for the production of a pharmaceutical composition as hereinbefore defined, which process comprises bringing the individual components thereof into intimate admixture and, when required, compounding the obtained composition in unit dosage form, for example filling said composition into tablets, gelatin, e.g., soft or hard gelatin, capsules, or non-gelatin capsules.
[00102] The starting materials of the N-oxides of the present invention are known and described, for example, in U.S. Patent Nos. 4,490,529, 4,584,375, 4,956,461, 5,354,753, 5,382,582, 5,698,556, 5,728,692, 5,958,928, 6,559,149 and 6,921,667.
[00103] Compounds having Formula I can be prepared by methods well known in the art and as illustrated by exemplary reactions in the following Schemes. The compounds of this invention may be prepared using methods known to those skilled in the art. Specifically, the compounds of this invention with Formulae I can be prepared as illustrated by the exemplary reaction in Scheme 1. Scheme 1
Figure imgf000044_0001
Synthesis of Methotrexate and Analogs
[00104] Synthesis of methotrexate (Rj is CH3, R2-R5 are H, R6 and R7 are
COOH, m=0 and n=l) typically involves the three component condensation of 2,4,5 ,6-tetraaminopyrimidine, ./V-substituted glutamyl-p-aminobenzamide and 2,3-dibromopriopionaldehydc in the presence of potassium iodide (Scheme 1 ). See Lendicer, D., "Strategies for Organic Drug Synthesis and Design, " Chap. 15, John Wiley and Sons, Inc., New York, NY (1998). The initial step of the condensation reaction can be envisioned as condensation of the aldehyde with the aminopyrimidine to give 2,4-diamino-6-(bromornethyl)pteridine as an intermediate. The reaction may, however, start with the alkylation of the amine on 2,4,5,6-tetraaminopyrimidine by the bromoaldehyde. Air oxidation of the dihydro ring in the initial condensation product completes the synthesis of methotrexate analog.
[00105] Methotrexate analogs may be prepared, for example, by substituting the glutamyl group with an analog, for example 2-aminoadipate group. The p-aminobenzoic acid may be replaced with analogs having, for example, one, two, three or four substituents on the benzene ring. The amino nitrogen of the 4-aminobenzoic acid moiety and the carbon atom to which it is attached may be part of a heterocyclic ring fused with the benzene "ring. [00106] Another variation is to introduce polyglutamate or analogs on methotrexate analog. Methotrexate polyglutamates are known metabolites of methotrexate, which are pharmacologically important. See Chabner, B. A. et al. J. Clin. Invest., 76:907-912 (19S5). N-oxides of methotrexate polyglutamate and its analogs are contemplated in this invention.
[00107] Thus, methotrexate polyglutamate or analogs may be synthesized by starting with /V-substituted polyglutamyl-p-aminobenzamide, which in turn may be prepared by successively adding glutamic acid residues to N-protected 4-aminobenzoic acid chloride using methods known in the art. For example, the α-carboxylic acid of commercially available glutamic acid γ-benzyl ester may first be protected with, for example, 2-(trimethylsilyl)ethoxymethyl protecting group (SEM) to afford α-SEM-γ-benzyl glutamate. See Jarowicki, K. and Kocienski, P., J. Chem. Soc, Perkin Trans 1, 4005-4037, 4017 (1998). Coupling the α,γ-diprotected glutamic acid with substituted Λf-benzyloxycarbonyl-4-aminobenzoic acid will yield N-substituted glutamyl- p-aminobenzamide. Following hydrolysis of the γ-ester of the N-substituted glutamyl-p-aminobenzamide and treatment with thionyl chloride, a second glutamic acid group may be introduced. After the desired number of glutamic acid groups are introduced into the N-substituted glutamyl-p-aminobenzamide, the ./V-substituted polyglutamyl-p-aminobenzamide may be coupled with 2,4,5, 6-tetraaminopyrimidine and 2,3-dibromopriopionaldehyde in the presence of potassium iodide (Scheme 1) to yield a product that can be air- oxidized to afford polyglutamated analog of methotrexate or its analog. The one or more α-SEM protecting groups can be removed under conditions that do not affect esters and amides. See Jarowicki, K. and Kocienski, P., J. Chem. Soc, Perkin Trans 1, 4005-4037, 4017 (1998). N-oxidation of polyglutamated methotrexate analog will yield compounds of Formula I. Polyglutamation of the other analogs to afford compounds of Formulae H-IV will similarly be executed. Thus, Scheme 1 contemplates introducing various substituents and' analogs to the structure of the methotrexate analog. Routes to N-oxidation of Methotrexate and Analogs
[00108] Any of the pyridine- or pyrimidine- or pyi-azine-type nitrogen atoms or any tertiary amino groups in methotrexate and analogs can be selectively oxidized to the corresponding N-oxide using known oxidizing agents. For example, the tertiary amino group in methotrexate, i.e. N10, can be oxidized to the corresponding N-oxide. Primary and secondary amino groups that may be present in methotrexate and analogs may be first selectively alkylated to give the corresponding tertiary amine, which then can be oxidized to the corresponding N-oxide. Certain oxidizing agents that are known in the art from preparing the N-oxides of pyridine- or pyrimidine- or pyrazine-type nitrogen atoms or tertiary amines include, without limitation, potassium monopersulfate, monoperoxyphthalic acid, magnesium monoperoxyphthalate (MMPP), hydrogen peroxide, peracetic acid, trifluoroperacetic acid, perbenzoic acid, 3-chloroperbenzoic acid (CPBA), and 2-phenylsulfony1-3- phenyloxaziridine (Davis reagent). The oxidation reaction can be carried out in a solvent such as chloroform, methylene chloride, 1 ,2-dichloroethane, or acetic acid, optionally in the presence of an alkali or alkaline-earth metal carbonate or bicarbonate. The reaction can be run from about 1 to 48 hours at a temperature of 0 0C to reflux temperature, and checked periodically for the presence of the desired N-oxide. Depending on the groups bound to the amine, reaction times may need Io be adjusted accordingly to obtain appropriate quantities of the desired bis-N-oxide product. See also Lee et ah, "Nitracrine N-oxides: effects of variations in the nature of the side chain N-oxide on hypoxia-selective cytotoxicity" Anticancer Drug Des. 14(6JASl- 497 (1999).
[00109] In other situations, more than one of the nitrogen atoms of the methotrexate analog may be oxidized simultaneously. In certain cases, one or more of the multiple N-oxide groups may be reduced selectively, leaving one or more of the other N-oxide groups in place. Thus, the present invention contemplates the preparation of N-oxide analogs in which one or more of the nitrogen atoms that are suitable for N-oxide formation are present as the N-oxide without regard to the susceptibility of a particular nitrogen atom to N-oxide formation or the susceptibility of a particular N-oxide group to reduction. It is envisaged to employ a combination of suitable protecting groups (see: Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, second edition, Wiley Interscience, 1991) to protect those nitrogen atoms not undergoing oxidation.
[00110] By way of an example, primary and secondary amines that may be present in a methotrexate analog may be protected using, for example, tert- butyl sulfonyl (BUS) group. Jarowicki, K. and Kocienski, P., J. Chem. Soc, Perkin Trans J, 4005-4037, 4029 (1998); Sun, P. and Weinreb, S. M. J. Org. Chem. (52:8604-08 (1997). The BUS protecting group is introduced by reaction of the amine with tert-butylsulfmyl chloride followed by oxidation of the sulfinyl amide with, for example, dimethyldioxirane, m-chloroperbenzoic acid or RuCl3 catalyzed NaIO4. The oxidation step in the preparation of the BUS-protected primary or secondary amines may also oxidize any tertiary amine and heteroaromatic nitrogen present in the compounds. Thus, this protecting group may be introduced into primary and secondary amines while simultaneously oxidizing tertiary and/or heteroaromatic nitrogen atoms.
[00111] The BUS protecting group is stable towards strong reagents such as alkyllithium, Grignard reagents, 0.1 M HCl in MeOH (20° C, 1 hr), 0.1 M TFA in dichloromethane (20° C, 1 hr) and pyrolysis at 180° C. The BUS-protected secondary amines can be cleaved with 0.1 M triflic acid in dichloromethane containing anisole as a cation scavenger at 0° C for 15-.30 minutes while primary amines are released more slowly at room temperature. If desired, both BUS-protected primary and secondary amines may be deprotected with 0.1 M triflic acid in dichloromethane containing anisole as a cation scavenger at 25° C for 2.5 hours. Thus, the BUS protecting group may allow protecting primary and secondary amines simultaneously while also oxidizing tertiary amines and heteroaromatic nitrogen atoms to the N-oxides. Moreover, the BUS protecting group may allow protecting primary and secondary amines simultaneously, oxidizing tertiary amines and heteroaromatic nitrogen atoms to N-oxides, deprotect the secondary amine selectively, alkylate the secondary amine to a tertiaiy amine, oxidize the resulting tertiary amine and deprotect the primary amine. Alternatively, a primary and a secondary amine may be protected with BUS protecting group, the secondary amine maybe deprotected selectively, the secondary amine may be protected with, for example, Boc protecting group, and then the primary amine may be deprotected selectively followed by alkylation and oxidation. Thus, when a primary amine and a secondary amine are present in a methotrexate analog, a BUS protecting group may be used to transform one of amines to an N-oxide without affecting the other.
[00112] Recent development in the use of Boc group to protect amines allows introduction and removal of the group under mild conditions. For example, a methotrexate analog amine group may be protected with Boc group by simply mixing the analog and Boc-ON (2-(Boc-oxyimino)-2-phenylacetonitrile, available from Aldrich Co.) in benzene at 25° C for 20 minutes (or 6 hours if the amine is an electron deficient aniline) in the presence of powdered zinc. See Spivey, A. C. and Maddaford A. Annu. Rep. Prog. Chem., Sect. B, 95:83- 95 (1999). Alkyl esters are tolerated.
[00113] Boc-protected amines are generally deprotected using triflic acid although recent developments generally use mildly acidic conditions that leave acid-labile groups unaffected. For example, heating Boc protected p-anisidines at 180° C in the presence of 4-chlorophenol deprotects the amine group without affecting acid sensitive methoxy enols (-CH=C(OCH3)-). Jarowicki, K. and Kocienski, P., J. Chem. Soc, Perkin Trans 1, 4005-4037, 4025 (1998). Thus, primary and secondary amines in methotrexate may be protected with Boc group followed by oxidation of the tertiary amines and deprotection of the primary and secondary amines.
[00114] It has also recently been reported a new base-sensitive amino protecting group l,l-dioxobenzo[6]thiophene-methoxycarbonyl (Bsmoc). Bsmoc is introduced via its chloroformate or ./V-hydroxy-succinimide derivative. The Bsmoc group is stable towards tertiary amines for 24 hours but is removed within 3-5 minutes using piperidine. Jarowicki, K. and Kocienski, P., J. Chem. Soc, Perkin Trans 1, 4005-4037, 4027 (1998). Thus, primary and secondary amines present in methotrexate analogs may conveniently be protected with Bsmoc protecting group followed by oxidation of the tertiary amines and removal of the protecting group under mild conditions.
[00115] It has also been reported that certain heteroaromatic nitrogen atoms can be oxidized selectively in the presence of certain aromatic primary amines and certain secondary amines adjacent to a double bond. Delia, T. J. et al. J. Org. Chem. 30:2766-68 (1965). For example, oxidation of cytosine with m-chloroperbenzoic acid results in cytosine 3-N-oxide despite the presence of aromatic primary amine and a secondary amine. Thus, heteroaromatic nitrogen atoms and tertiary amines may be oxidized in the presence of certain aromatic primary amines and secondary amines.
Route to Substituted 4-Amtnobenzoic Acid Moiety
[00116} A variety of substituted commercially available 4-aminobenzoic acids may be used as a starting point for the synthesis of methotrexate analog N-oxides in which the benzoic acid is substituted with one, two, three or four substituents. For example, 4-aminobenzoic acid substituted with one or more of, for example, alkyl, halo, cyanό, hydroxy, alkoxy, fluorinated alkoxy, benzyloxy and cyclohexyloxy, which are commercially available, may be used as starting material for the synthesis of methotrexate analogs. The list of the compounds is intended to provide working examples but not intended to limit the invention.
[00117] Those skilled in the art .may synthesize other substituted
4-aminobenzoic acid or its analogs from other commercially available compounds. By way of example, a precursor of the 4-amino group can be a 4-nitro group, which upon catalytic reduction with H2 generates the amino group. Thus virtually any commercially available compound with a nitro group in the desired position may serve as a precursor for the amino group. The nitro group itself can be introduced into an aromatic precursor by way of a nitration reaction. The carboxyl group may be introduced at the correct position by forming a Grignard reagent with a suitable aromatic halide and then quenching the reagent with anhydrous carbon dioxide. Alternatively, position selective metallation of the carboxyl group followed by reaction with a suitable electrophile introduces the electrophile into a position either ortho or meta to the carboxyl group, depending on the reagent used (see: Nguyen, T.-H. Chou, N. T. T.; Castanet, A.-S.; Nguyen, K. P. P.; Mortier, J. Org. Lett., 7:2445 (2005) and references cited therein).
[00118] Further, fluorinated alkyl groups may be introduced to the benzoic acid moiety by choosing appropriately substituted starting material. For example, substituted 4-aminobenzoic acid in which R2 or R3 is a trifluoromethyl group may be prepared by starting with, for example, 4-cyano-2- (trifluoromethyl)acetanilide. Simultaneously hydrolyzing the amide and the cyano groups of 4-cyano-2-(trifluoromethyl)acetanilide will yield 3-trifluromethyl-4-aminobenzoic acid. Similarly, substituted 4-aminobenzoic acid in which R4 or R5 is a trifluoromethyl group may be prepared by starting with 4-amino-2-trifluorornethylbenzonitrile, which, upon hydrolysis of the cyano group, affords 2-trifluormethyl~4-aminobenzoic acid.
[00119] The carboxylic acid group of substituted 4-aminobenzoic acid is protected by transforming it into, for example, an ester (Scheme 2). For example, the substituted 4-aminobenzoic acid is dissolved in anhydrous alcohol followed by catalytic amount of a strong acid. The equilibrium between the ester and the acid can be shifted in favor of the ester if an excess of the alcohol is used, if the ester or water is removed from the reaction mixture by, for example, distillation or if water is removed by a dehydrating agent, silica gel or molecular sieves. See, for example, Smith, M. B. and March, J., "March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure," 5th ed., Chapter 10, John Wiley & Sons, Inc., New York, NY (2001). Scheme 2
Figure imgf000051_0001
[00120] The amine nitrogen of substituted 4-aminobenzoic acid ester may also be protected with, for example, a p-toluenesulfonyl group, a benzyloxycarbonyl group or an acetyl group. For example, a substituted 4-aminobenzoic acid ester may be dissolved in ether in which sodium hydrogen carbonate is suspended. An ether solution of an acid chloride, for example benzyloxycarbonyl chloride, is thereafter added while cooling the reaction mixture with ice. Following stirring for about 2.5 h at room temperature, additional amount of the ether solution of the acid chloride may be added to the reaction mixture while keeping the mixture in ice bath. The stoichiometry of the reaction is such that an excess amount of the acid chloride is used. The reaction mixture will be washed with water and dried with anhydrous sodium sulfate crystals. Evaporation of the ether will yield a residue that can be purified by recrystallization or column chromatography to afford substituted /V-benzyloxycarbonyl-4-aminobenzoic acid ester. See, e.g., 6,559,149, example 12.
[00121] Substituted N-benzyloxycarbonyl-4-amino-benzoic acid ester will then be N-alkylated following methods well known in the art. For example, a solution of the ./V-benzyloxycarbonyl-4-amino-benzoic acid ester in dimethylformamide (DMF) will be added slowly to a suspension of sodium hydride in DMF while maintaining the reaction temperature at or near 0° C. The mixture will be allowed to warm up to room temperature. After stirring the reaction mixture for 1 h at room temperature, an alkyl halide (for example, methyl iodide) will be added slowly and the mixture will be stirred at room temperature for 2 h. Then, the reaction solution will be poured into a saturated aqueous solution of sodium hydrogen carbonate and the mixture will be extracted with toluene. The organic layer will be dried with sodium sulfate and the solvent will be distilled off under vacuum. The resulting residue will be purified, for example, by column chromatography. See, e.g., 6,559,149, example 13.
[00122] The ester group of the substituted /V-methyl-/V-benzyloxycarbonyl-4- aminobenzoic acid ester will be hydrolyzed with a base. For example, treatment Λf-methyl-Λf-benzyloxycarbonyl-4-aminobenzoic acid ester with 2N NaOH solution aqueous/ethanol solution at reflux temperature for two hours will yield N-methyl-7Vr-benzyloxycarbonyl-4-aminobenzoic acid sodium salt. The acid can be isolated by acidifying the mixture with, for example, 4N HCl, a pH below 4, followed by extraction with, for example, chloroform. See, e.g., 6,559,149, example 14.
[00123] The substituted /V-methyl-/V-benzyloxycarbonyl-4-aminobenzoic acid will be coupled with a glutamic acid or analog according to methods known in the art. For example, substituted 7V-methyl-/V-benzyloxycarbonyl-4- aminobenzoic acid will be suspended in a chlorinated hydrocarbon solvent, for example dichloromethane. A large excess of thionyl chloride will be added to the solution while keeping the mixture in an ice path. After stirring the reaction mixture for 2 hours at room temperature, the solution will be concentrated to dryness under vacuum. After redissolving the reaction mixture in dichloromethane and suspending potassium carbonate in the reaction mixture, an equivalent amount of diethyl glutamate (or its hydrochloride salt) or its analog will then be added. The volume of the reaction mixture is approximately doubled with water followed by stirring at about 0° C to about 50° C, more preferably between 15 and 40° C, more preferably at or near room temperature for between about 2 hours to 3 days, more preferably between 4 hours and 24 hours, more preferable 8 to 15 hours. The reaction mixture will be poured into water and extracted with chloroform. The organic layer will be washed with IN HCl and dried with sodium sulfate. The solvent will be distilled off under vacuum and the resulting residue will be purified with a column chromatography, isolating substituted TV-C/V'-benzyloxycarbonyl-iV'- methyl-4-amino-benzoyl)-L-glutamic acid diester or its analog. See, e.g., U.S. Patent No. 6,559,149, example 15.
[00124] Deprotecting the nitrogen amine of /V-(iV'-benzyloxycarbonyl-JV'- methyl-4-aminobenzoyl)-L-glutamic acid diester or its analog will yield N-[N'- methyl-(4-aminobenzoyl)]-L-glutamic acid or analog. For example, about 1 g of Λr-(N'-benzyloxycarbonyl-Λr'-methyl-4-aminobenzoyl)-L-glutamic acid diester or analog will be stirred in a solution of 30% hydrogen bromide/acetic acid (about 10 ml) in the presence of about 1 gram of anisole for about 1 to 10 hours at about 0° C to about 50° C. See, e.g., U.S. Patent No. 6,559,149, example 16. The reaction product will them be extracted and purified according to methods known in the art.
[00125] 7V-[7V'-Methyl-(4-aminobenzoyl)]-L-glutamic acid or analog will be reacted with 2,4,5, 6-tetraaminopyrimidine and 2,3-dibromopropionaldehyde in the presence of Kl followed by air oxidation to yield non-N-oxide analogs of Formula I (i.e., methotrexate and analogs). See Scheme 1. /V-oxidation of the tertiary amine group with MCPBA will yield compounds of Formula 1.
Route to N-oxides of methotrexate analogs of Formula III
[00126] Compounds of formula TIT may be obtained by starting with the commercially available (Apollo Scientific Ltd., U.K.) l,2,3,4~tetrahydro-6- quinolinecarboxylic acid (THQA) and using methods described for compounds of Formula I. The carboxylic acid group of THQA will be esterified followed by protection of N-amine with, for example, benzyloxycarbonyl protecting group. The ester group of
N-benzyloxycarbonyl-l.ZjB^-tetrahydro-ό-quinolinecarboxylic acid ester will be hydrolyzed followed by coupling the resulting 7V-benzyloxycarbonyl~ l,2,3,4-tetrahydro-6-quinolinecarboxylic acid with glutamic acid diester or analog. The resulting product, N-[N'-benzyloxycarbonyl-l,2,3,4-tertahydro-6- quinolyl]glutamic acid diester or its analog, will be reacted with 2,4,5,6-tetraaminopyrimidine and 2,3-dibromopropionaldehyde in the presence of KI to yield the dihydro compound that can be air oxidized to afford non-N-oxide methotrexate analog. See Scheme 1. ./V-oxidation of the tertiary amine group of the methotrexate analog with MCPBA will yield compounds of Formula III.
Route to N-oxides of methotrexate analogs of Formula IV
[00127] Compounds of formula IV may be obtained by coupling iV-benzyloxycarbonyl-5-carboxyindole with glutamic acid or analog followed by reaction with 2,4,5,6-tetraaminopyrimidine and
2,3-dibromopropionaldehyde in the presence of KI to the dihydro compound that can be air oxidized to a methotrexate analog (Scheme 1). 5-Carboxyindole will be obtained by selective reduction of the amide carbonyl of methyl 2-oxindole-5-carboxylate (commercially available from Apollo Scientific Ltd., U.K.) using diborane (B2HO). Diborane reduces amide groups to amines without reducing esters. Walker, E. R. H. Chem. Soc. Rev., 5:23 (1976). The TV-benzyloxycarbonyl-5-carboxyindole (obtained by reaction between benzyloxycarbonyl chloride and methyl 5-indolecarboxylate followed by hydrolysis of the ester) will be coupled with glutamic acid or analog to afford iV^N'-benzyloxycarbonyl-S-carboxyindoly^-L-glutamic acid diester or its analog.
[00128] N-(N'-benzyloxycarbonyI-5-carboxyindolyl)-L-glutamic acid or its analog will be reacted with 2,4,5, 6-tetraaminopyrimidine and 2,3-dibromopropionaldehyde in the presence of KI to afford the dihydro compound of methotrexate analog that can be air oxidized to afford non-N-oxide of compounds of IV (i.e., methotrexate analogs). See Scheme 1. TV-oxidation of the tertiary amine group of the methotrexate analogs with MCPBA will yield compounds of Formula IV.
Introduction of additional tertiary amines into methotrexate analogs
[00129] Primary, secondary or tertiary amines may also be introduced into methotrexate analog by using a modified procedure. For example, mono- or di-fluorinated (e.g., 2-fluoro-4-nitrobenzoic acid, 3-fluoro-4-nitrobenzoic acid, or 2,5-difluoro-4-nirobenzoic acid, which are commercially available) may be reacted with a dialkyl amine (e.g., dimethylamine). For example, reaction between 2-fluoro-4-nitrobenzoic acid and dimethylamine will yield N1N- dimethyl-4-nitro-2-aminobenzoic acid. N,N-dimethyl-4-nitro-3-aminobenzoic acid may similarly be obtained. Reacting 2,5-difluoro-4-nirobenzoic acid with dialkylamine (for example, JV,iV-dimethyl amine, TV.TV-diethyl amine, 7V,N-dipropyl amine, etc.) will allow synthesis of, for example, N1N1N1N'- tetramethyl-4-nitro-2,5-diaminobenzoic acid.
[00130] Reacting ammonia or a primary amine with, for example, 2-fluoro-
4-nitrobenzoic acid, will introduce amino or alkyl amino substituent on the 4-nitrobenzoic acid. Suitable protection scheme may be used to protect the amino groups.
[001311 The nitro group of the substituted benzoic acid may selectively be reduced to amine to afford 4-aminobenzoic acid substituted with one or two amino groups. The nitro group is susceptible to reduction by lithium triethylborohydride in tetrahydrofuran (THF) at 0° C while the carboxylic acid salts are not. See Table 2, Brown, H. C. and Krishnamurthy, S. Tetrahedron, 35:567-601, 591, 604 (1978). Thus, for example, ΛyV-dimethyl-4-nitro-3- aminobenzoic acid tetraethyl ammonium salt in THF will be added into a suspension of lithium triethylborohydride in THF while keeping the pot temperature at between -20° C and 10° C, more preferably at -10° C to 5° C, most preferably at about 0° C. The mixture will be stirred at about 0° C while following the reaction progression using a suitable analytical procedure (e.g., TLC or gas chromatography). The reaction will be quenched by, for example, slowly adding a moist alcohol, which decomposes the excess lithium triethylborohydride, followed by isolation of the reaction product. The product, 3-(iV,N-dimethylamino)-4-aminobenzoic acid, may then be used to synthesize a methotrexate analog according to methods described above.
[00132] The following examples are illustrative, but not limiting, of the method and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in clinical therapy and which are obvious to those skilled in the art are within the spirit and scope of the invention.
EXAMPLE 1
N-Oxidation of Methotrexate
[00133] Commercially available m-chloroperbenzoic acid (MCPBA) consists of about 77 wt-% active MCPBA, with m-chlorobenzoic acid and water as impurities. In the experiments described below, the amount of the MCPBA reagent used is adjusted to reflect active MCPBA. Owing to the sensitivity of methotrexate and derivatives toward light, care was taken to minimize exposure of reactants or products to light.
[00134] Procedure 1. MCPBA (0.011 g, 0.064 mmol) was dissolved in 1 mL of methanol. To this solution 4 mL of water was added. In a separate flask 0.020 g (0.044 mmol) of methotrexate was dissolved in 0.3 ml of dimethylformamide (DMF) and then portions were added dropwise to a stirred solution of MCPBA at room temperature. Methotrexate initially precipitates out of the methanol/water/DMF solution after being added. When the mixture becomes clear, another portion of methotrexate/DMF solution was added. The reaction was complete after 1 h. The resulting solution was filtered to remove any traces of solid unreacted methotrexate and then it was extracted with methylene chloride (3 x 5 mL) and ether (1 x 5 mL) to remove m-chiorobenzoic acid. The aqueous phase was lyophilized to give 0.020 g (97%) of methotrexate N-oxide as a yellow-orange powder. The crude N-oxide product is reasonably clean and typically contains about 10% of impurities. The crude product was loaded onto a silica gel column as a suspension in water/acetonitrile (1 :1, 1 mL) and then it was chromatographed using an acetonitrile/water/acetic acid (5:2:2) mixture as eluent. The desired fractions were collected and extracted with methylene chloride (1OmL x 3) and ether (1OmL x 1) to remove some of the acetonitrile and acetic acid. Note that extraction is preferable to evaporation due to sensitivity of the product to light. Lyophilization of the aqueous phase gave 0.01 1 g (52% yield) of methotrexate N-oxide as a greenish-yellow powder with an estimated purity of >90% based on the 1H NMR spectrum.
[00135] Procedure 2. The sodium salt of methotrexate was generated by adding 1.5-1.8 equivalent of aqueous sodium hydroxide to a suspension of methotrexate in water. An aqueous solution of methotrexate sodium salt (0.011 g, 0.022 mmol) in 2 mL of water was added dropwise to a solution of w-chloroperoxybenzoic acid (0.0060 g, 0.035 mmol) dissolved in a mixture of methanol (0.5 mL) and water (0.5 mL). The reaction was complete in 30 min. The m-chlorobenzoic acid byproduct was removed from the reaction mixture by extraction with methylene chloride (5 mL x 3) and ether (5mL x 1). The aqueous phase was lyophilized to give 0.011 g (97%) of methotrexate sodium salt N-oxide as a yellow-orange powder. The crude product was loaded on a silica gel column as a suspension in water/acetonitrile mixture (1 :1, ImL) and then chromatographed using acetonitrile/water/acetic acid (5:2:2) mixture as eluent. The desired fractions were collected and extracted with methylene chloride (10 mL x 3) and ether (1OmL x 1) to remove some of the acetonitrile and acetic acid. Liophilization of the aqueous phase gave 0.006 g (54% yield) of the sodium salt of methotrexate N-oxide as a greenish-yellow powder with an estimated purity of >90% based on the 1H NMR spectrum.
[00136] 1H NMR (D2O) δ 1.95 (m, IH), 2.12 (m, IH), 2.34 (t, J= 7.2, 2H), 3.72
(s, 3H), 4.28-4.33 (m, IH), 5.09 (AB system, J= 12.6, 2H), 7.68-7.79 (m, 4H), 8.48 (s, 0.5H), 8.49 (s, 0.5H). formed
Figure imgf000058_0001
[00137] Procedure 3. A suspension containing 0.010 g (0.022 mmol) of methotrexate, 0.5 mL water and 0.5 mL methanol was stirred for 20 minutes. To this suspension magnesium bis(monoperoxyphthalate) hexahydrate (0.02 g, 0.044 mmol) was added. The methotrexate dissolved over 1 hour. The mixture was stirred additional hour and solvents were evaporated until about 0.5 mL of water remains. This solution was transferred on silica gel column chromatography and eluted with acetonitrile/water/acetic acid mixture. Magnesium phthalate eluted with acetonitrile. Other impurities eluted with 5:0.5:0.5 acetonitrile/water/acetic acid mixture and the desired methotrexate /V-oxide eluted with 5:1:1 acetonitrile/water/acetic acid mixture. Appropriate fractions were collected and solvents were evaporated to give yellow powder (3 mg, 30%) pure by TLC and 1H NMR. MS (m/e): Calculated for methotrexate C20H22N8O5 (M): 454.17; found (M): 454.8. Calculated for methotrexate N-oxide C20H22N8O6 (M): 470.17; found (M): 470.8.
EXAMPLE 2
Cytotoxicity Of Methotrexate And Analog N-Oxides Thereof In Lymphoma, Leukemia, And Multiple Myeloma
[00138] The cytotoxicity of methotrexate and analog N-oxides thereof on different lymphoma, leukemia, and multiple myeloma cell lines will be tested in vitro under normoxic as well as 1% O2 hypoxic conditions. Standard cytotoxicity assays using MTS dye will be run to determine the IC50 for each compound. Cells will be exposed to the compounds for 24 hours and cells will be stained 24-72 hours post-drug exposure. Positive controls will use chemotherapeutic agents at doses shown in the art to be effective. The results should indicate that methotrexate analogs are cytotoxic to many of the cell lines, with IC50 values in the nanomolar to sub-nanomolar range.
Methotrexate analog N-oxides are expected to be less active or inactive compared to non-N-oxide under normoxic conditions. However, under 1% O2 hypoxic conditions, methotrexate analog N-oxides are expected to be converted to the corresponding parent non-N-oxide, which are expected to be cytotoxic with IC50 values in the millimolar to sub-nanomolar range.
EXAMPLE 3
Cytotoxicity Of Methotrexate Analogs And N-Oxides Thereof In Solid
Tumor Lines
[00139] The cytotoxicity of methotrexate and analog N-oxides thereof on different solid tumor cell lines will be tested in vitro under normoxic conditions and 1 % O2 hypoxic conditions. Standard cytotoxicity assays using MTS dye will be ran to determine the ΪC50 for each compound. Cells will be exposed to the compounds for 24 hours and cells will be stained 24-72 hours post-drug exposure. Chemotherapeutic agents at doses shown in the art to be effective will be used as positive controls. The results are expected to indicate that methotrexate analogs are cytotoxic to many of the cell lines, with ΪC50 values in the nanomolar to sub-nanomolar range. Methotrexate and analog N-oxides thereof are expected to be less active or inactive compared to the corresponding methotrexate analogs.
EXAMPLE 4
Anti-Proliferative Activity Of Methotrexate And Analog N-Oxides
Thereof In Cancer Cells
] The anti-proliferative activity of methotrexate and analog N-oxides thereof on established and primary tumor cell lines will be tested in vitro under normoxic and 1% O2 hypoxic conditions at concentrations ranging from 1 nM to 10 mM. The anti-proliferative effect will be measured using the 5-bromo- 2'-deoxyuridine ("BrDU") incorporation technique. The cells will be exposed to the compounds in the presence of BrDU for 24 hours. BrDU is incorporated into the replicating cellular DNA. After cell fixation and washing, the incorporated BrDU is determined in a specific ELlSA using an antibody specific to BrDU coupled to peroxidase. The N-oxides are expected not to have significant anti-proliferative activity in cancer cells at concentrations of up to 10 mM under normoxia. However, the N-oxides are expected to exhibit significant anti-proliferative effect on the cancer cell lines under 1% O2 hypoxia. EXAMPLE 5
Anti-Tumor Activity of Methotrexate and Analog N-Oxide in Murine
Tumor Models
[00141] The in vivo antitumor efficacy of methotrexate and analog Ν-oxides will be evaluated using xenograft murine models. For example, male 5 to 6 week old nude mice will be inoculated subcutaneously in the mammary fat pad on each side with an injection of a human cancer cell line, for example about 1x106 MDA-MB-231 (2LMP) in 0.3 ml serum free medium. The best xenograft recipients will be used. Treatments with methotrexate and analog Ν-oxides will begin when tumors averaged about 5-7 mm in diameter and will be continued for 4 weeks with a 2 month follow up period.
[00142] Test animals will be divided into cohort groups of 5-8 animals into the following treatment groups: Control (Group 1); Vehicle Control (Group 2), daily administration of the vehicle only; Methotrexate and analog Ν-oxide (Group 3), 0.001 mg/kg administered orally per day for 4 weeks; Methotrexate and analog Ν-oxide (Group 4), 0.01 mg/kg administered orally per day for 4 weeks; Methotrexate and analog Ν-oxide (Group 5), 0.05 mg/kg administered orally per day for 4 weeks; Methotrexate and analog Ν-oxide (Group 6), 0.10 mg/kg administered orally per day for 4 weeks; Methotrexate and analog Ν- oxide (Group 7), 0.15 mg/kg administered orally per day for 4 weeks; Methotrexate and analog Ν-oxide (Group 8), 1.0 mg/kg administered orally per day for 4 weeks.
[00143] The test doses may further be increased or decreased upon recognition that such a modification is warranted for a particular methotrexate analog Ν-oxide. For example, a particular methotrexate analog Ν-oxide may exert its own, for example, antiproliferative effect, making it more effective than it would have been had methotrexate analog Ν-oxide been biologically inactive. In such a circumstance, lower doses of the methotrexate analog Ν-oxide may prove effective. More cohort groups may be added to test various doses of methotrexate and analog Ν-oxides. [00144] During the treatment course, tumor sizes and animal weights will be measured periodically, for example 1-3 times per week for each animal. Following treatment, tumor sizes will be measured periodically, for example, 1-3 times per week, and animal weights once per week. Tumor sizes and weight observations will be made without knowledge of the animal's treatment group.
[00145] The data will be modeled using, for example, the natural logarithm of tumor volume versus time. Comparisons of the growth rates of animals administered with varying dosages will be performed. For example, Tumor Growth Delay and Tumor Growth Inhibition for varying dosages will be compared to the control.
[00146] Further, the in vivo antitumor activity of methotrexate and analog
N-oxide in combination with chemotherapeutic agents and/or radiotherapy will be evaluated using a xenograft model in nude mice.
[00147] Having now fully described this invention, it will be understood by those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations and other parameters without affecting the scope of the invention or any embodiment thereof. All patents, patent applications and publications cited herein are fully incorporated by reference herein in their entirety.

Claims

WHAT IS CLAIMED IS:
1. A compound having formula I:
Figure imgf000063_0001
or a pharmaceutically acceptable salt or prodrug thereof, wherein:
X' is hydrogen; deuterium; halo; amino; C(=NH)NH2; C(=O)NH2; C(=S)NH2; NR9R10R11 wherein each of R? and Rio is independently hydrogen, alkyl, haloalkyl, cyanoalkyl, hydroxyalkyl or alkoxyalkyl; carboxyalkyl; phenyl; alkylphenyl; C(=O)R' wherein R' is hydrogen, alkyl, phenyl, or alkylphenyl; or C(=O)OR" wherein R" is hydrogen, alkyl, phenyl or alkylphenyl; each of Y and Z is independently hydrogen or deuterium;
R 1 is a lower alkyl group having 1-4 carbon atoms,
R2 is hydrogen, halo, hydroxy, cyano, a lower alkyl group having 1-4 carbon atoms, NR12R13R14, a lower alkoxy having 1-4 carbon atoms or a trifluoromethyl group, or Ri and R2 together form a 5 or 6 membered saturated or unsaturated heterocycle having one or two hetero atoms where the second heteroatom, when present, is S or O, wherein S is optionally substituted with one or two oxygen atoms;
R3 is hydrogen, halo, cyano, alkoxy, cycloalkyloxy, benzyloxy, alkyl, cycloalkyl, aryl, heteroaryl, aryloxy, aralkyl, NR12R13R14 or haloalkyl, or Ri and R3 together form a 5 or 6 membered saturated or unsaturated heterocycle;
R4 is hydrogen, hydroxy, halo, alkyl, a lower alkoxy or NR12R13R14;
R5 is hydrogen, hydroxy, halo Or NRi2Ri3Ri4; each R5 is independently COORI5or CNHOR16, wherein each of R15 and R16 is independently hydrogen, alkyl, aryl, aralkyl or cycloalkyl; R7 is' COOR17, SO3H, PO3H2, NHCOOR18, NHCOR18, or CONR17R18 wherein R17 is hydrogen, alky], optionally substituted phenyl, carboxyalkyl or alkylsulfonyl and R18 is alkyl, aryl, aralkyl or cycloalkyl; each of Ri2 and Rn is hydrogen or alkyl; each of R8, Ri i and Rj4 is O or is absent provided that at least one of
Figure imgf000064_0001
m is 0-6; and each of n and p is independently 1, 2 or 3; wherein one or more -CKfe- groups of the terminal amino acid is optionally substituted with one or more halogen atoms.
2. The compound of claim 1, having formula II:
Figure imgf000064_0002
or a pharmaceutically acceptable salt thereof.
3. The compound of claim 1, having formula III:
Figure imgf000064_0003
or a pharmaceutically acceptable salt thereof.
4. The compound of claim 1, having formula IV:
Figure imgf000065_0001
or a pharmaceutically acceptable salt thereof.
5. The compound of claim 1 , having formula V:
Figure imgf000065_0002
or a pharmaceutically acceptable salt thereof.
6. The compound of claim 1, wherein said compound of formula I is selected from the group consisting of NI0-oxides of: methotrexate; dimethyl N- {4-[7V-(2,4-diamino-6-pteridinyl)methyl-7V-methylamino]- 3 -m ethyl } benzoyl-L-2-aminoadipate;
/vr-{4-[yV-(2>4-diamino-6-pteridinyl)methyl-7V-methylamino]-3- melhyl} benzoyl-L-2-aminoadipic acid;
/V-{4-[Λπ-(2,4-diamino-6-pteridinyl)methyl-N'-methylamino]-3- trifluoromethyl}benzoyl-L-2-aminoadipic acid; dimethyl N- {I -[2,4-diamino~6-pteridinyl)methyl]-7-methylindoline-5- carbonyl} -L-2-aminoadipate;
N-{l-[2,4-diamino-6-pteridinyl)methyl]-7-methylindoline-5-carbonyl}- L-2-aminoadipic acid; N-{l-[254-diamino-6-pteridinyl)methyl]indole-5-carbonyl}-L-2- aminoadipic acid;
N-{l~[2,4-diamino-6-pteridinyl)methyl]iπdole-5-carbonyl}-L- homocysteic acid ammonium salt;
N-{l-[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl}-L-glutamic acid;
N- { 1 -[2,4-diamino-6-pteridinyl)methyl]-7-methylindole-5-carbonyl} - L-2-aminoadipic acid; dimethyl N- {4-[ΛP-(234-diamino-6-pteridinyl)methylamino]-3- methyl)benzoyl-L-2-aminoadipate;
Λr-{4-[N'-(234-diamino-6-pteridinyl)methylamino]-3-methyl)benzoyl- L-2-aminoadipic acid. diethyl N-4-[Λ^-(2,4-diamino-6-pteridinyl)methyl-7V-metliylamino]-3- methylbenzoyl-L-glutamate; dimethyl N- {4-[7V-(2,4-diamino-6-pteridinyl)methyl-/V-rnethyIamino]- 3 -methyl } benzoyl-L-2-aminoadipale;
7Vr- {4-[-V-(2,4-diamino-6-pteridinyl)methyI-N'-methylamiπo]-3- methylbeπzoyl}-L-glutamic acid; diethyl Λ^-{4-[N'-(2,4-diamino-6-pteridinyl)methyl-Nrt-ethylamino]-3- methylbenzoyl} -L-glutamate; iV-{4-[Λ"-(2,4-diamino-6-pteridinyl)methyl-Λ/'-ethylamino]-3- methylbenzoyl}-L-glutamic acid; diethyl /V-{4-[iV-(2,4-diamino-6-pteridinyl)methyl-iV-ethylamino]-3- methylbenzoyl} -L-glutamate; iV-{4-[Λ/1-(2,4-diamino-6-pteridinyl)methyl-iV-ethylamino]-3- methyl}benzoyI-L-glutamic acid; dimethyl N-{4-[N'-(2)4-diamino-6-pteridinyl)methyl-Nl-methylamino]- 3-methylbenzoyl}-L-2-aminoadipate;
N- {4-[/V-(2,4-diamino-6-pteridinyl)methyl-A''-methylamino]-3- methyl}benzoyl-L-2-aminoadipic acid; N-(4-[N-(2,4-diamino-6-pteridinyl)methyl-N-methylamino]-3- methylbenzoyl}-L-homocysteic acid ammonium salt; diethyl N- {4-[N-(2,4-diamino-6-pteridinyl)methyl-7V-methylamino] -3- ethylbenzoyl} -L-glutamate;
N- {4-[7V-(2,4-diamino-6-pteridinyl)methyl-N'-methylamino]-3- ethylbenzoyl}-L-glυtamic acid; diethyl N- {4-[N-(2,4-diamino-6-pteridinyI)methyl-NHmethylamino]- 3, 5-dimethylbenzoyl} -L-glutamate;
N-{4-[N-(2,4-diamino-6-pteridinyl)methyl-N-methylamino]-3,5- dimethylbenzoyl}-L-glutamic acid;
N-{4-[N-(2,4-diamino-6-pteridinyl)methyl-N-methylamino]-3- trifluorornethylbenzoyl} -L-2-aminoadipic acid; dimethyl N-{4~[N-(2,4-diamino-6-pteridinyl)methyl-7-methylindoline- 5'-carbonyl} -L-2-aminoadipate;
N- {l-[N-(254-diamino-6-pteridinyl)methyl-7-methylindoline-5'- carbonyl} -L-2-aminoadipic acid;
N- { 1 -[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl } -L- aminoadipic acid;
Λ/-{l-[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl}-L-2- aminoadipic acid;
N- { 1 -[2,4-diamino-6-pteridinyl)methyl]indole-5'-carbonyl} -L- homocysteic acid ammonium salt;
N- { 1 -[(2,4-diamino-6-pteridinyl)methyl]indole-5'-carbonyl} -L- glutamic acid;
N-{l-[(2,4-diamino-6-pteridinyl)methyl-7-methylindole-5'-carbonyl}- L-2-aminoadipic acid; diethyl N- {4-[N-(2,4-diamino-6-pteridinyl)methylamino]-3- methyl} benzoyl-L-glutamate; dimethyl N-{4-[N-(2,4-diamino-6-pteridinyl)methylamino]-3- methyl}benzoyl-L-2-aminoadipate; and Λr-{4-[Λ/'-(2,4-diamino-6-pteridinyl)methylamino]-3-methyl)benzoyl-L- 2-aminoadipic acid; or a pharmaceutically acceptable salt or prodrug thereof.
7. The compound of claim 1 , wherein said compound is methotrexate-Nl0-oxide of formula:
Figure imgf000068_0001
or a pharmaceutically acceptable salt or prodrug thereof.
8. A method of treating, ameliorating, or preventing a hyperproliferative disorder comprising administering to an animal in need thereof a therapeutically effective amount of a compound of claim 1.
9. A method of treating, preventing or ameliorating a hyperproliferative disorder in an animal in need thereof, comprising
(a) determining whether said hyperproliferative disorder is characterized by hypoxic tissue, and
(b) treating said animal with an effective amount of a compound of claim 1.
10. The method of claim 8 or 9, wherein said compound is selected from the group consisting of NI0-oxides of: methotrexate; dimethyl N- {4-[N'-(2,4-diamino-6-pteridinyl)methyl-iV'-methylamino]- 3-methyl}benzoyl-L-2-arninoadipate; N-{4-[N-(2,4-diamino-6-pteridinyl)methyl-N'-methylamino]-3- methyI}benzoyl-L-2-aminoadipic acid;
N-{44N-(2,4-diamino-6-pteridinyl)methyl-N'-methylamino]-3- trifluorom ethyl }benzoyl-L-2~aminoadipic acid; dimethyl N-{1 -[2,4-diamino~6-pteridmyl)methyl]-7-methylindoline-5- carbonyl} -L-2-aminoadipate;
N-{l-[2,4-diamino-6-pteridinyl)methyl]-7-methylindoline-5-carbonyl}- L-2-aminoadipic acid;
N~{l-[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl}-L-2- aminoadipic acid;
N-{l-[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl}-L- homocysteic acid ammonium salt;
N-{l-[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl}-L-glutamic acid;
N- { 1 -[2,4-diamino-6-pteridinyl)methyl]-7-methylindole~5-carbonyl}- L-2-aminoadipic acid; dimethyl N- {4-[N-(2,4-diamino-6-pteridinyl)methylamino]-3- methyl)benzoyl-L-2-aminoadipate;
N-{4-[N-(2,4-diamino-6-pteridinyl)methylamino]-3-methyl)benzoyl-L- 2-aminoadipic acid. diethyl N-4-[N-(2,4-diamino-6-pteridinyl)methyl-N'-methylamino]-3- methylbenzoyl-L-glutamate; dimethyl N-{4-[/V-(2,4-diamino-6-pteridinyl)methyl-N'-methylamino]- 3-methyl}benzoyl-L-2-aminoadipate;
N- {4-[N-(2,4-diamino-6-pteridinyl)methyl-N1-methylamino]-3- . methylbenzoyl}-L-glutamic acid; diethyl N-{4-[N'-(2,4-diamino-6-pteridinyl)methyl-N-ethylamino]-3- methylbenzoyl} -L-glutamate;
N-{4-[N-(2,4-diamino-6-pteridinyl)methyl-N'-ethylamino]-3- methylbenzoyl}-L-glutamic acid; diethyl N-{4-[Λ^-(2,4-diamino-6-pteridinyl)methyl-iV-ethylamino]-3- methylbenzoyl} -L-glutamate;
N-{4-[7V-(2,4-diamino-6-pteridinyl)methyl-Λ?t-ethylamino]-3- methyl) benzoyl -L-glutamic acid; dimethyl N- {4-[N-(2s4-diammo-6-pteridinyl)methyl-iV-methylamino]- 3-methylbenzoyl}-L-2-aminoadipate;
Λ/-{4-[Λ/'-(2,4-diamino-6-pteridinyl)methyl-Λ'1-methylamino]-3- methyl}benzoyl-L-2-aminoadipic acid;
N-(4-[ΛP-(2,4-diamino-6-pteridinyl)methyl-ΛT-methylamino]-3- methylbenzoyl}-L-homocysteic acid ammonium salt; diethyl N-{4-[iV-(2,4-diamino-6-pteridinyl)methyl-yV-methylamino]-3- ethylbenzoyl} -L-glutamate;
N-{4-[7V-(2,4-diamino-6-pteridinyl)methyl-Λ'1-methylamino]-3- ethylbenzoyl} -L-glutamic acid; diethyl 7V-{4-[iV-(2,4-diamino-6-pteridinyl)methyl-7Vt-methylamino]- 3, 5-dimethylbenzoyl} -L-glutamate;
N- {4-[A^-(2,4-diamino-6-pteridinyl)methyl-/V-methylamino]-3 ,5- dimethylbenzoyl} -L-glutamic acid;
N-{4-[A/1-(2,4-diamino-6-pteridinyl)methyl-Λ'1-methylamino]-3- trifluoromethylbenzoyl}-L-2-aminoadipic acid; dimethyl JV- {4-[NI-(2,4-diamino-6-pteridinyl)methyl-7-methylindoline- 5'-carbonyl}-L-2-aminoadipate;
N- { 1 -[iV-(2,4-diamino-6-pteridinyl)methyl-7-methylindoline-5l- carbonyl}-L-2-aminoadipic acid;
N- { 1 -[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl } -L- aminoadipic acid;
N-{l-[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl}-L-2- aminoadipic acid;
N- {I -[2,4-diamino-6-pteridinyl)methyl]indole-5'-carbonyl } -L- homocysteic acid ammonium salt; N- { 1 -[(2,4-diamino-6-pteridinyl)methyl]indole-5'-carbonyl}-L- glutamic acid;
Λ/-{l-[(2,4-diamino-6-pteridinyl)methyI-7-methylindole-5'-carbonyl}- L-2-aminoadipic acid; diethyl N- {4-[//-(2,4-diamino-6-pleridinyl)methylamino]-3- methyl} benzoyl-L-glutamate; dimethyl N- {4-[7V-(2,4-diamino-6-pteridinyl)methylamino]-3- methyl}benzoyl-L-2-aminoadipate; and
/V-{4-[iV-(2,4-diamino-6-pteridinyl)methylamino]-3-methyl)benzoyl-L- 2-aminoadipic acid; or a pharmaceutically acceptable salt or prodrug thereof.
11. The method of claim 8 or 9, wherein said compound is methotrexate-N10-oxide of formula:
Figure imgf000071_0001
or a pharmaceutically acceptable diastereoisomer, salt or prodrug thereof.
12. The method of claim 8, further comprising subjecting said animal to an imaging technique selected from the group consisting of computed tomography, magnetic resonance imaging, single photon emission computer tomography and positron emission tomography prior to or during administration of said compound.
13. The method of claim 8 or 9, wherein said hyperproliferative disorder is cancer.
14. The method of claim 13, wherein the cancer is of the bladder, brain, breast, cervix, colon, endometrium, esophagus, head and neck, kidney, larynx, liver, lung, oral cavity, ovaries, pancreas, prostate, skin, stomach, or testis.
15. The method of claim 13, wherein the cancer is selected from the group consisting of acute and chronic lymphocytic leukemia, acute granulocytic leukemia, adrenal cortex carcinoma, bladder carcinoma, breast carcinoma, cervical carcinoma, cervical hyperplasia, choriocarcinoma, chronic granulocytic leukemia, chronic lymphocytic leukemia, colon carcinoma, endometrial carcinoma, esophageal carcinoma, essential thrombocytosis, genitourinary carcinoma, hairy cell leukemia, head and neck carcinoma, Hodgkin's disease, Kaposi's sarcoma, lung carcinoma, lymphoma, malignant carcinoid carcinoma, malignant hypercalcemia, malignant melanoma, malignant pancreatic insulinoma, medullary thyroid carcinoma, melanoma, multiple myeloma, mycosis fungoides, myeloid and lymphocytic leukemia, neuroblastoma, non-Hodgkin's lymphoma, osteogenic sarcoma, ovarian carcinoma, pancreatic carcinoma, polycythemia vera, primary brain carcinoma, primary macroglobulinemia, prostatic carcinoma, renal cell carcinoma, rhabdomyosarcoma, skin cancer, small-cell lung carcinoma, soft-tissue sarcoma, squamous cell carcinoma, stomach carcinoma, testicular carcinoma, thyroid carcinoma, and Wilms' tumor.
16. The method of claim 8 or 9, wherein said hyperproliferative disorder is age-related macular degeneration, Crohn's disease, cirrhosis, chronic inflammatory-related disorders, diabetic retinopathy, granulomatosis, immune hyperproliferation associated with organ or tissue transplantation, an immunoproliferative disease or disorder, e.g., inflammatory bowel disease, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, vascular hyperproliferation secondary to retinal hypoxia, or vasculitis.
17. The method of claim 8 or 9, further comprising administering one or more other active agents or treatments to the animal.
18. The method of claim 17, wherein said one or more other active agents or treatments are independently selected from the group consisting of a chemotherapeutic agent, a radiotherapeutic agent/treatment, an anti-angiogenesis agent, a vascular targeting agent, an HIFl inhibitor, an Hsp90 inhibitor, a tyrosine kinase inhibitor, a serine/threonine kinase inhibitor, a proteasome inhibitor, an HDAC inibitor, a caspase inducer, a CDK inhibitor, and a proapoptotic molecule.
19. The method of claim 18, wherein the chemotherapeutic agent is selected from the group consisting of abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, asparaginase, BCG live, bevaceizumab, bexarotene, bleomycin, bortezomib, busulfan, calusterone, camptothecin, capecitabine, carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cinacalcet, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, darbepoetin alfa, daunorubicin, denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone, Elliott's B solution, epirubicin, epoetin alfa, estramustine, etoposide, exemestane, filgrastim, floxuridine, fludarabine, fluorouracil, fulvestrant, gemcitabine, gemtuzumab ozogamicin, gefitinib, goserelin, hydroxyurea, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib, interferon alfa-2a, interferon alfa-2b, irinotecan, letrozole, leucovorin, levamisole, lomustine, meclorethamine, megestrol, melphalan, mercaptopurine, mesna, methotrexate, methoxsalen, methylprednisolone, mitomycin C, mitotane, mitoxantrone, nandrolone, nofetumomab, oblimersen, oprelvekin, oxaliplatin, paclitaxel, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed, pentostatin, pipobroman, plicamycin, polifeprosan, porfimer, procarbazine, quinacrine, rasburicase, rituximab, sargramostim, streptozocin, talc, tamoxifen, tarceva, temozolomide, teniposide, testolactone, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, and zoledronate.
20. The method of claim 18, wherein said anti-angiogenesis agent is selected from the group consisting of bevacizumab, angiostatin, endostatin, batimastat, captopril, cartilage derived inhibitor, gcnistein, interleukin 12, lavendustin, medroxypregesterone acetate, recombinant human platelet factor 4, tecogalan, thrombospondin, TNP -470, anti-VEGF • monoclonal antibody, soluble VEGF-receptor chimaeric protein, anti-VEGF receptor antibodies, anti-PDGF receptors, inhibitors of integrins, tyrosine kinase inhibitors, serine/threonine kinase inhibitors, antisense oligonucleotides, antisense oligodexoynucleotides, siRNAs, anti-VEGF aptamers and pigment epithelium derived factor.
21. The method of- claim 17, wherein said compound is administered prior to the administration of said active agents or treatments.
22. The method of claim 17, wherein said compound is administered concurrently with the administration of said active agents or treatments.
23. The method of claim 22, wherein the administration of said compound is continued beyond the administration of said active agents or treatments.
24. The method of claim 17, wherein said compound is administered after the administration of said active agents or treatments.
25. The method of claim 17, wherein the method is repeated at least once.
26. A pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1.
27. The pharmaceutical composition of claim 26, wherein said compound is selected from the group consisting of N10-oxides of: methotrexate; dimethyl N- {4-[N'-(2,4-diamino-6-pteridinyl)methyl-N'-methylamino]- 3-methyl}benzoyl-L-2-aminoadipate; iV-{4-[A''-(2,4-diamino-6-pteridinyl)methyI-N'-methylamino]-3- methyl} benzoyl-L-2-aminoadipic acid;
Λr-{4-[7V-(2,4-diamino-6-pteridinyl)methyl-N'-methylamino]-3- trifluoro methyl} benzoyl-L-2-aminoadipic acid; dimethyl N- { 1 -[2,4-diamino-6-pteridinyl)methyI]-7-methylindoline-5- carbonyl) -L-2-aminoadipate;
N- { 1 -[2,4-diamino-6-pteridinyl)methyl]-7-methylindoline-5-carbonyl}- L-2-aminoadipic acid;
N- {I -[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl} -L-2- aminoadipic acid;
N- { 1 -[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl } -L- homocysteic acid ammonium salt;
N-{l-[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl}-L-glutamic acid;
N-{l-[2,4-diamino-6-pteridinyl)methyl]-7-methylindole-5-carbonyl}- L-2-aminoadipic acid; dimethyl N- {4-[7V1-(2,4-diamino-6-pteridinyl)methylamino]-3- methyl)benzoyl-L-2-aminoadipate; 7V-{4-[Λ''-(2,4-diainino-6-pteridinyl)methylamino]-3-methyl)benzoyl-L- 2-aminoadipic acid. diethyl N-4-[Λp-(2,4-diamino-6-pteridinyl)methyl-Λ/'-methylamino]-3- methylbenzoyl-L-glutamate; dimethyl A^-{4-[Λ?'-(2,4-dianiino-6-pteridinyl)methyl-N'-methylamino]- 3-methyl}-benzoyl~L-2-aminoadipate;
N-{4-[7V-(2,4-diamino-6-pteridinyl)methyl-Λ/1-methylamino]-3- methylbenzoyl}-L-glutamic acid; diethyl N-{4-[ΛP-(2,4-diamino-6-pteridinyl)methyl-Λ/I-ethylammo]-3- methylbenzoyl } -L-glutamate;
N-{4-[Λ/'-(2,4-diamino-6-pteridinyl)methyl-Λ'1-ethylamino]-3- methylbenzoyl}-L-glutamic acid; diethyl N-{4-[iV-(2,4-diamino-6-pteridinyl)methyl-Λ/'-ethylamino]-3- methylbenzoyl } -L-glutamate;
J/V-{4-[Λ/'-(2,4-diamino-6-pteridinyl)methyl-Λ/'-ethylamino]-3- methyl}benzoyl-L-glutamic acid; dimethyl //-{4-[iV-(2,4-diamino-6-pteridinyl)methyl-7V-methylamino]- 3-methylbenzoyl}-L-2-aminoadipate;
Λr-{4-[yV-(2,4-diamino-6-pteridinyl)methyl-iV-methylamino]-3- methyl}benzoyl-L-2-aminoadipic acid;
N-(4-[Λ/'-(2,4-diamino-6-pteridinyl)τnethyl-iV-methylamino]-3- methylbenzoyl}-L-homocysteic acid ammonium salt; diethyl N-{4-[N'-(2,4-diamino-6-pteridinyl)methyl-Λ/'-methylamino]-3- ethylbenzoyl } -L-glutamate;
N-{4-[Λ^-(2,4-diamino-6-pteridinyl)methyl-Λ/'-methylamino]-3- ethylbenzoyl}-L-glutamic acid; diethyl N-{4-[N'-(2,4-diamino-6-pteridinyl)methyI-N'-methylamino]- 3,5-dimethylbenzoyl}-L-glutamate;
N-{4-[NI-(2,4-diamino-6-pteridinyl)methyl-N'-methylamino]-3,5- dimethylbenzoyl} -L-glutamic acid; N-{4-[7V-(2,4-diamino-6-pteridinyl)methyl-/V-methylamino]-3- trifluoromethylbenzoyl}-L-2-aminoadipic acid; dimethyl N- {4-[7V-(2,4-diaraino-6-pteridinyl)methyl-7-methylindoline- 5'-carbonyl}-L-2-aminoadipate;
N- { 1 -[Λ/1-(2,4-diamino-6-pteridinyl)methyl-7-methylindoline-5l- carbonyl}-L-2-aminoadipic acid;
7V-{l-[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl}-L- aminoadipic acid;
N- { 1 -[2,4-diamino-6-pteridinyl)methyl]indole-5-carbonyl} -L-2- aminoadipic acid;
Λr-{l-[2,4-diamino-6-pteridinyl)methyl]indole-5'-carbonyl}-L- homocysteic acid ammonium salt; iV-{l-[(2,4-diamino-6-pteridinyl)methyl3indole-5'-carbonyl}-L- glutamic acid;
Λr-{l-[(2,4-diamino-6-pteridinyl)methyl-7-methylindole-5'-carbonyl}- L-2-aminoadipic acid; diethyl N- {4-[Λ'"-(2,4-diamino-6-pteridinyl)methylamino]-3- methyt} benzoyl-L-glutamate; dimethyl Λ^-{4-[Λ?I-(2,4-diamino-6-pteridinyl)methyIamino]-3- methyl) benzoyl-L-2-aminoadipate; and
N- {4- [N1 -(2 ,4-di am ino-6-pter i dinyl)methylam ino] - 3 -methyl)benzoyl -L- 2-aminoadipic acid; or a pharmaceutically acceptable salt or prodrug thereof.
28. The pharmaceutical composition of claim 26, wherein said compound is methotrexate-N10-oxide of formula:
Figure imgf000078_0001
or a pharmaceutically acceptable diastereoisomer, salt or prodrug thereof.
29. The pharmaceutical composition of claim 26, further comprising one or more active agents independently selected from the group consisting of chemo therapeutic agents, anti-angiogenesis agents, vascular targeting agents, HIFl inhibitors, Hsp90 inhibitors, a tyrosine kinase inhibitor, a serine/threonine kinase inhibitor, a proteasome inhibitor, an HDAC inibitor, a caspase inducer, a CDK inhibitor, and a proapoptotic molecule.
30. The pharmaceutical composition of claim 29, wherein said chemotherapeutic agent is selected from the group consisting of abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, asparaginase, BCG live, bevaceizumab, bexarotene, bleomycin, bortezomib, busulfan, calusterone, camptothecin, capecitabine, carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cinacalcet, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, darbepoetin alfa, daunorubicin, denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone, Elliott's B solution, epirubicin, epoetin alfa, estramustine, etoposide, exemestane, filgrastim, floxuridine, fludarabine, fluorouracil, fulvestrant, gemcitabine, gemtuzumab ozogamicin, gefitinib, goserelin, hydroxyurea, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib, interferon alfa-2a, interferon alfa-2b, irinotecan, letrozole, leucovorin, levamisole, lomustine, meclorethamine, megestrol, melphalan, mercaptopurine, mesna, methotrexate, methoxsalen, methylprednisolone, mitomycin C, mitotane, mitoxantrone, nandrolone, nofetumomab, oblimersen, oprelvekin, oxaliplatin, paclitaxel, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed, pentostatin, pipobroman, plicamycin, polifeprosan, porfimer, procarbazine, quinacrine, rasburicase, rituximab, sargramostim, streptozocin, talc, tamoxifen, tarceva, temozolomide, teniposide, testolactone. thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, and zoledronate.
31. The pharmaceutical composition of claim 29, wherein said anti-angiogenesis agent is selected from the group consisting of bevacizumab, angiostatin, endostatin, batimastat, captopril, cartilage derived inhibitor, genistein, interleukin 12, lavendustin, medroxypregesterone acetate, recombinant human platelet factor 4, tecogalan, thrombospondin, TNP-470, anti-VEGF monoclonal antibody' soluble VEGF-receptor chimaeric protein, anti-VEGF receptor antibodies, anti-PDGF receptors, inhibitors of integrins, tyrosine kinase inhibitors, serine/threonine kinase inhibitors, antisense oligonucleotides, antisense oligodexoynucleotides, siRNAs, anti-VEGF aptamers and pigment epithelium derived factor.
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