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Definitions

• the present invention provides compositions and methods for treating cancer and other hyperproliferative disease conditions with phosphoramidate alkylator prodrugs.
• the present invention generally relates to the fields of chemistry, biology, molecular biology, pharmacology, and medicine.
• alkylating agents used in cancer chemotherapy encompass a diverse group of chemicals that have the ability to alkylate biologically vital macromolecules such as DNA under physiological conditions (see Hardman et al., The Pharmacological Basis of Therapeutics, 2001, 1389-1399, McGraw-Hill, New York, USA). DNA alkylation is postulated to be an important mechanism in the antitumor activity of alkylators.
• the chemotherapeutic alkylators act as strong electrophiles, for example, through the formation of neighboring-heteroatorn-stabilized onium intermediates such as an aziridine or an aziridinium cation.
• Phosphoramidate based alkylators used in cancer therapy such as Cyclophosphamide and Ifosfamide
• Cyclophosphamide and Ifosfamide are each activated in the liver and the active alkylator released alkylates nucleophilic moieties such as the DNA within the tumor cells to act as a chemotherapeutic agent. If the active alkylators are released away from the tumor, DNA and other nucleophilic moieties such as the phosphate, amino, sulfhydryl, hydroxyl, carboxyl and imidazo groups of biomolecules of healthy non-cancerous cells, can get alkylated. Such alkylation of healthy cells canand result in unwanted toxic events in patients (see Hardman et al., supra).
• the present invention provides compounds which are hypoxia activated phosphoramidate alkylator prodrugs and methods of their synthesis.
• the phosphoramidate alkylator prodrugs of the present invention can have the formula AIk-T wherein AIk is a phosphoramidate alkylator, T is L-Z 3 wherein L is a linker Z 3 is a bioreductive group.
• the present invention provides phosphoramidate alkylator prodrugs of formula (I):
• Y 1 is O, S, NR 6 , or NSO 2 R 6 wherein each R 6 is independently C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, aryl, or heteroaryl;
• Y 2 is O, S, NR 6 , NCOR 6 , or NSO 2 R 6 ;
• each OfR 1 -R 5 independently is hydrogen, hydroxyl, amino, C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 3 -Cg cycloalkyl, heterocyclyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 1 -C 6 dialkylamino, aryl, heteroaryl, C 1 -C 6 acyl, C!-C 6 heteroacyl, aroyl, or heteroaroyl; or together any two OfR 1 -R 5 form a C 3 -C 10 heterocycle; or each OfR 1 - R 5 independently is a Trigger T wherein T is L-Z 3
• Y 3 is S, O, or NR 7 wherein each R 7 is independently hydrogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 3 -C 8 cycloalkyl, heterocyclyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 1 - C 6 dialkylamino, aryl, heteroaryl, C 1 -C 6 acyl, Q-C ⁇ heteroacyl, aroyl, or heteroaroyl;
• each Z 1 independently is hydrogen, halogen, C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, aryl, heteroaryl, C 3 -C 8 cycloalkyl, heterocyclyl, C 1 -C 6 acyl, C 1 -C 6 heteroacyl, aroyl, or heteroaroyl;
• Z 2 is C 1 -C 6 alkylene, C 1 -C 6 heteroalkylene,
• each X 1 is independently N or CR 8
• each R 8 is independently hydrogen, halogen, nitro, cyano, CO 2 H, C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 1 -C 6 cycloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 1 -C 6 dialkylamino, aryl, CON(R 7 ) 2 , Ci-C 6 acyl, Ci-C 6 heteroacyl, aroyl, or heteroaroyl;
• X 2 is NR 7 , S, or O;
• Z 3 is selected from the group consisting of:
• At least two OfR 1 -R 5 are selected from the group consisting of 2-haloalkyl, 2- alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- heteroalkylsulfonyloxyalkyl;
• At least one OfR 1 -R 5 is selected from the group consisting of 2-haloalkyl, 2-C 1 - C 6 alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- heteroalkylsulfonyloxyalkyl; and at least one OfNR 2 R 3 and NR 4 R 5 is " ⁇ d; or
• Z 3 is a bioreductive group that can accept one or more electrons in an oxidation-reduction reaction.
• the present invention provides phosphoramidate alkylator prodrugs having IC 5O or GI 50 , in cells under hypoxia, of 50 ⁇ M to 0.01 nm.
• the present invention provides phosphoramidate alkylator prodrugs having hypoxic cytotoxicity which are up to a million fold, up to 10,000 fold, and up to 1000 fold less toxic in corresponding normoxic cells.
• the cellular cytotoxicity is measured by antiproliferation assays and using the relative IC 50 value of a compound in hypoxic and normoxic cells.
• the cellular cytotoxicity is measured by clonogenic assays and using the relative C 10 , C 50 , or Cg 0 values of the compounds in hypoxic and normoxic cells.
• the present invention provides phosphoramidate alkylator prodrugs having IC 50 values, in cells under hypoxia, of 50 ⁇ M to 0.01 nM. In another related embodiment, the present invention provides phosphoramidate alkylator prodrugs which are up to 5000 fold less toxic in corresponding normoxic cells as measured by the relative IC 50 values in hypoxic and normoxic cells. In another related embodiment, the present invention provides phosphoramidate alkylator prodrugs having an IC 50 in cells in hypoxia of 50 ⁇ M to 0.01 nM and which is up to 1000 fold less toxic in corresponding normoxic cells as measured by the relative IC 50 values in hypoxic and normoxic cells.
• phosphoramidate alkylator prodrug of the present invention has a hypoxic cytotoxicity of 0.1 nM to 50 ⁇ M and a hypoxia cytotoxicity ratio, HCR, measured by the ratio of normoxic and hypoxic cytotoxicities, and defined in greater detail further later in the application, of 10 to 100,000.
• the phosphoramidate alkylator prodrug of the present invention has a hypoxic cytotoxicity of 0.1 nM to 50 ⁇ M and a HCR of 25 to 100,000.
• phosphoramidate alkylator prodrug of the present invention has a hypoxic cytotoxicity of 0.1 nM to 5 ⁇ M and a HCR of 50 to 10,000.
• the present invention provides novel phosphoramidate alkylators for treatement of cancer and other hyperproliferative diseases.
• the present invention provides a pharmaceutical formulation comprising the phosphoramidate alkylator prodrugs of the invention and a pharmaceutically acceptable excipient, carrier, or diluent.
• the present invention provides a method of treating cancer and other hyperproliferative diseases comprising administering a therapeutically effective amount of a phosphoramidate alkylator prodrug of the invention or one that is known, to a patient in need of such therapy.
• the cancer treated is resistant to first line, second line, or third line therapy, or is a relapsed cancer.
• the cancer treated is a metastatic cancer.
• the phosphoramidate alkylator prodrug of the invention, or one that is known is administered in combination with at least another anticancer agent.
• Figure 1 demonstrates the effect of Compound 25 (50 mg/kg ) on rumor growth in the H460 xenograft mouse model.
• Figure 2 demonstrates the effect of Compound 25 (100 mg/kg ) on tumor growth in the H460 xenograft mouse model.
• Figure 3 demonstrates the effect of Compound 25 (150 mg/kg ) dosed in combination with CDDP on tumor growth in the H460 xenograft mouse model.
• Figure 4 demonstrates the effect of Compound 25 dosed in combination with CDDP on tumor growth in the H460 xenograft mouse model.
• Figures 5, 6 and 7 demonstrate the effect of Compound 25 in combination with Gemcitabine on tumor growth in the H460 xenograft mouse model.
• Section I provides useful definitions
• Section II describes the compounds of the invention and methods for making them
• Section III describes methods of treatment, therapies, administrations, and formulations, employing the compounds of the invention alone or in combination
• Section IV provides examples of synthetic methods and biological assays for the compounds of the invention. This detailed description is organized into sections only for the convenience of the reader, and disclosure found in any section is applicable to any aspect of the invention.
• Alkyl means a linear saturated monovalent hydrocarbon radical or a branched saturated monovalent hydrocarbon radical having the number of carbon atoms indicated in the prefix.
• the prefixes (Ci-C q q), Q- qq , or Q-C qq , wherein qq is an integer from 2-20 have the same meaning.
• (C 1 -Cg) alkyl, C 1 - S alkyl, or C 1 - C 8 alkyl includes methyl, ethyl, n-propyl, 2-propyl, n-butyl, 2-butyl, tert-butyl, pentyl, and the like.
• (C 1 -C 6 ) alkyl can be further optionally be substituted with substituents, including for example, deuterium ("D"), hydroxyl, amino, mono or di(Ci-C 6 ) alkyl amino, halo, C 2 -C 6 alkenyl ether, cyano, nitro, ethenyl, ethynyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, -COOH, -CONH 2 , mono- or di(d-C 6 ) alkylcarbox-amido, -SO 2 NH 2 , -OSO 2 -(C 1 -C 6 ) alkyl, mono or di(Q-C 6 ) alkylsulfonamido, aryl, heteroaryl, alkylsulfonyloxy, heteroalkylsulfonyloxy, arylsulfonyloxy or heteroarylsulfonyloxy
• alkenyl means a linear monovalent hydrocarbon radical or a branched monovalent hydrocarbon radical having the number of carbon atoms indicated in the prefix and containing at least one double bond, but no more than three double bonds.
• (C 2 -C 6 ) alkenyl includes, ethenyl, propenyl, 1,3-butadienyl and the like.
• Alkenyl can be further optionally be substituted with substituents, including for example, deuterium (“D”), hydroxyl, amino, mono or di(C !
• -C 6 alkyl amino, halo, C 2 -C 6 alkenyl ether, cyano, nitro, ethenyl, ethynyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, -COOH, -CONH 2 , mono- or di(CrC 6 ) alkyl- carboxamido, -SO 2 NH 2 , -OSO 2 -(C 1 -C 6 ) alkyl, mono or di(d-C 6 ) alkylsulfonamido, aryl, heteroaryl, alkyl or heteroalkylsulfonyloxy, and aryl or heteroarylsulfonyloxy.
• Alkylator means a reactive moiety capable of forming a covalent alkyl linkage to macromolecules via an electrophillic reaction with a nucleophile on the macromolecule.
• Phosphoramidate alkylator means an alkylator for which an aziridine or aziridinium electrophile is present or generated by intramolecular cyclization.
• Alkylene means a linear saturated divalent hydrocarbon radical having from one to twelve carbon atoms or a branched saturated divalent hydrocarbon radical having from one to twelve carbon atoms optionally substituted with substituents including for example, deuterium ("D"), hydroxyl, amino, mono or di(CrC 6 )alkyl amino, halo, C 2 -C 6 alkenyl ether, cyano, nitro, ethenyl, ethynyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, -COOH, -CONH 2 , mono- or di- -SO 2 NH 2 , -OSO 2 -(C 1 -C 6 ) alkyl, mono or di(CrC 6 ) alkylsulfonamido, aryl, heteroaryl, alkyl or heteroalkylsulfonyloxy, and aryl or heteroarylsulfony
• substituents including
• Heteroalkylene has essentially the meaning given above foran alkylene except that one or more heteroatoms (i.e. oxygen, sulfur, nitrogen and/or phosphorous) may be present in the alkylene biradical.
• heteroalkylene includes, -CH 2 OCH 2 O-,- CH 2 CH 2 OCH 2 CH 2 -, -CH 2 CH 2 N(CH 3 )CH 2 CH 2 -, -CH 2 CH 2 SCH 2 CH 2 -, and the like.
• Aryl refers to a monovalent monocyclic or bicyclic aromatic hydrocarbon radical of 6 to 10 ring atoms which is substituted independently with one to eight substituents, preferably one, two, three, four ot five substituents selected from deuterium ("D"), alkyl, cycloalkyl, cycloalkylalkyl, halo, nitro, cyano, hydroxyl, alkoxy, amino, acylamino, mono- alkylamino, di-alkylamino, haloalkyl, haloalkoxy, heteroalkyl, COR (where R is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, phenyl or phenylalkyl), -(CR'R") n -COOR (where n is an integer from 0 to 5, R' and R" are independently hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl, cycl
• R x and R y together is cycloalkyl or heterocyclyl. More specifically the term aryl includes, but is not limited to, phenyl, biphenyl, 1-naphthyl, and 2- naphthyl, and the substituted forms thereof.
• Cycloalkyl refers to a monovalent cyclic hydrocarbon radical of three to seven ring carbons.
• the cycloalkyl group can have one or more double bonds and can also be optionally substituted independently with one, two, three or four substituents selected from alkyl, optionally substituted phenyl, or -C(O)R 2 (where R z is hydrogen, alkyl, haloalkyl, amino, mono-alkylamino, di-alkylamino, hydroxyl, alkoxy, or optionally substituted phenyl).
• cycloalkyl includes, for example, cyclopropyl, cyclohexyl, cyclohexenyl, phenylcyclohexyl, 4-carboxycyclohexyl, 2-carboxamidocyclohexenyl, 2- dimethylaminocarbonyl-cyclohexyl, and the like.
• Heteroalkyl means an alkyl radical as defined herein with one, two or three substituents independently selected from cyano, -OR W , -NR x R y , and -S(O) P R Z (where p is an integer from 0 to 2 ), with the understanding that the point of attachment of the heteroalkyl radical is through a carbon atom of the heteroalkyl radical.
• R w is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, aryl, aralkyl, alkoxycarbonyl, aryloxycarbonyl, carboxamido, or mono- or di-alkylcarbamoyl.
• R x is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, aryl or araalkyl.
• R y is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, aryl, araalkyl, alkoxycarbonyl, aryloxycarbonyl, carboxamido, mono- or di-alkylcarbamoyl or alkylsulfonyl.
• R z is hydrogen (provided that n is 0), alkyl, cycloalkyl, cycloalkyl-alkyl, aryl, araalkyl, amino, mono- alkylamino, di-alkylamino, or hydroxyalkyl.
• Representative examples include, for example, 2-hydroxyethyl, 2,3-dihydroxypropyl, 2-methoxyethyl, benzyloxymethyl, 2-cyanoethyl, and 2-methylsulfonyl-ethyl.
• R w , R x , R y , and R z can be further substituted by amino, halo, fluoro, alkylamino, di-alkylamino, OH or alkoxy.
• the prefix indicating the number of carbon atoms refers to the total number of carbon atoms in the portion of the heteroalkyl group exclusive of the cyano, -OR W , -NR x R y , or -S(O) P R Z portions.
• R x and R y together is cycloalkyl or heterocyclyl.
• Heteroaryl means a monovalent monocyclic, bicyclic or tricyclic radical of 5 to 12 ring atoms having at least one aromatic ring containing one, two, or three ring heteroatoms selected from N, O, or S, the remaining ring atoms being C, with the understanding that the attachment point of the heteroaryl radical will be on an aromatic ring.
• the heteroaryl ring is optionally substituted independently with one to eight substituents, preferably one, two, three or four substituents, selected from alkyl, cycloalkyl, cycloalkyl-alkyl, halo, nitro, cyano, hydroxyl, alkoxy, amino, acylamino, mono-alkylamino, di-alkylamino, haloalkyl, haloalkoxy, heteroalkyl, -COR (where R is hydrogen, alkyl, phenyl or phenylalkyl, - (CR'R") n -COOR (where n is an integer from 0 to 5, R' and R" are independently hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl, cycloalkyl-alkyl, phenyl or phenylalkyl), or - (CR'R")n-CONR x R y (where n is an integer from 0 to 5, R
• R x and R y together is cycloalkyl or heterocyclyl. More specifically the term heteroaryl includes, but is not limited to, pyridyl, furanyl, thienyl, thiazolyl, isothiazolyl, triazolyl, imidazolyl, isoxazolyl, pyrrolyl, pyrazolyl, pyridazinyl, pyrimidinyl, benzofuranyl, tetrahydrobenzofuranyl, isobenzofuranyl, benzothiazolyl, benzoisothiazolyl, benzotriazolyl, indolyl, isoindolyl, benzoxazolyl, quinolyl, tetrahydroquinolinyl, isoquinolyl, benzimidazolyl, benzisoxazolyl or benzothienyl, indazolyl, pyrrolopyrymidinyl, indolizinyl,
• the heterocyclyl ring can be optionally substituted independently with one, two, three or four substituents selected from alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, cycloalkylalkyl, halo, nitro, cyano, hydroxyl, alkoxy, amino, mono-alkylamino, di-alkylamino, haloalkyl, haloalkoxy, -COR (where R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl), -(CR'R") n -COOR (n is an integer from 0 to 5, R' and R" are independently hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl), or -(CR'R") n -CONR
• heterocyclyl includes, but is not limited to, pyridyl, tetrahydropyranyl, N-methylpiperidin-3-yl, N- methylpyrrolidin-3-yl, 2-pyrrolidon-l-yl, furyl, quinolyl, thienyl, benzothienyl, pyrrolidinyl, piperidinyl, morpholinyl, pyrrolidinyl, tetrahydro furanyl, tetrahydrothiofuranyl, 1,1-dioxo- hexahydro-l ⁇ 6 -thiopyran-4-yl, tetrahydroimidazo [4,5-c] pyridinyl, imidazolinyl, piperazinyl, and piperidin-2-only and the derivatives thereof.
• the prefix indicating the number of carbon atoms refers to the total number of carbon atoms in the portion of the cycloheteroalkyl or heterocyclyl group exclusive of the number of heteroatoms.
• C 1 -C 6 Acyl means -CO-(C 1 -C 6 alkyl), wherein the term alkyl is as defined above.
• C 1 -C 6 Heteroacyl means -CO-(C 1 -C 6 heteroalkyl), wherein the term heteroalkyl is as defined above.
• Aroyl means -CO-aryl, wherein the term aryl is as defined above.
• Heteroaroyl means -CO-heteroayl, wherein the term heteroaryl is as defined above.
• Alkylsulfonyloxy, heteroakylsulfonyloxy, cycloalkylsulfonyloxy, heterocyclylsulfonyloxy, arylsulfonyloxy, and heteroarylsulfonyloxy groups can be leaving groups in phosphoramidate alkylators and can be replaced in a cell by nucleic acids such as DNA or RNA, and imidazoles, carboxylates, or thiols of proteins, causing alkylation and cell death.
• the rate of reaction of various R su isulfonyloxy groups with nucleic acids, proteins or water can be modulated depending on for example the electron withdrawing nature and the steric bulk of the R sul moiety and can provide phosphoramidate alkylators and prodrugs thereof which are more toxic to tumors in general and hypoxic zones of tumor in particular over healthy cells.
• Substituents mean, along with substituents particularly described in the definition of each of the groups above, those selected from: deuterieum, -halogen, -OR', -NR'R", -SR', -SiR'R “R'”, -OC(O)R', -C(O)R', -CO 2 R', -CONR'R", -OC(O)NR 5 R", -NR 55 C(O)R', -NR'-
• R' and R" When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7- membered ring.
• -NR'R is meant to include 1-pyrrolidinyl and 4-morpholinyl.
• Other suitable substituents include each of the above aryl substituents attached to a ring atom by an alkyl ene tether of from 1-4 carbon atoms.
• Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T 2 - C(O)-(CH2)q-U 3 -, wherein T 2 and U 3 are independently -NH-, -O-, -CH 2 - or a single bond, and q is an integer of from 0 to 2.
• two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A- (CH2) r B-, wherein A and B are independently -CH2-, -O-, -NH-, -S-, -S(O)-, -S(O)2-, -S(O) 2NR'- or a single bond, and r is an integer of from 1 to 3.
• One of the single bonds of the new ring so formed may optionally be replaced with a double bond.
• two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CH2) s -X 5 -(CH2)t-, where s and t are independently integers of from 0 to 3, and X 5 is -O-, -NR'-, -S-, -S(O)-, -S(O) 2 -, or -S(O) 2 NR'-.
• the substituent R' in -NR'- and -S(O) 2 NR'- is selected from hydrogen or unsubstituted C ⁇ - ⁇ alkyl.
• Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers, regioisomers and individual isomers (e.g., separate enantiomers) are all intended to be encompassed within the scope of the present invention.
• the compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds.
• the compounds may be radiolabeled with radioactive isotopes, such as for example tritium ( 3 H), iodine- 125 ( 125 I) or carbon- 14 ( C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.
• salts are meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein.
• base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent.
• salts derived from pharmaceutically-acceptable inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc and the like.
• Salts derived from pharmaceutically-acceptable organic bases include salts of primary, secondary and tertiary amines, including substituted amines, cyclic amines, naturally- occuring amines and the like, such as arginine, betaine, caffeine, choline, N 5 N'- dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperadine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.
• acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
• pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like.
• salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, e.g., Berge, S.M., et al, "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19).
• Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
• the neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner.
• the parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention.
• Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated fo ⁇ ns.
• the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention.
• Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
• a "glucose analog” includes mono-, di- and tri-saccharides.
• the glucose analog includes sacchrides comprising glucosamine, N-acetyl-glucosamine; fructose; mannose and mannose derivatives; glucose and glucose derivatives, including but not limited to 2-deoxyglucose (2-DG), N-acetyl-2-amino-2-deoxyglucose, 3-amino-3-deoxy-glucose, 2- amino-2-deoxy-glucose; and galactose and galactose derivatives including but not limited to D-2-deoxy-D-galactose, D-4 ⁇ amino-4-deoxy-galactose and D-2-amino-2-deoxy-galactose.
• the glucose analog can differ from glucose or a derivative such as DG and glucosamine in that it is an epimer thereof.
• the glucose analog can be a fluorinated derivative of any of the foregoing compounds.
• the oxygen in the ring of any of the foregoing compounds can be substituted with an isostere selected from the group consisting of S, sulfone, and the like.
• glucose analog can be 5-thio-D-glucose or a derivative thereof.
• a wavy line “ ⁇ ” means the point of attachment of one group or moiety to
• CO 2 and COO are used interchangeably herein.
• SO 2 , S(O) 2 are used interchangeably herein.
• a "bioisostere” of a chemical moiety such as molecule, group, or atom means another chemical moiety having similar size and spatial disposition of electron pair or pairs.
• Bioisosteres and bioisosterism are well-known tools for predicting the biological activity of compounds, based upon the premise that compounds with similar size, shape, and electron density can have similar biological activity.
• a reasonable quantitative prediction of the binding ability or the function of a known molecule can be made based on the spatial arrangement of a small number of atoms or functional groups in the molecule. As used herein, such an arrangement is called a "pharmacophore", and once the pharmacophore or pharmacophores in a molecule have been identified, this information can be used to identify other molecules containing the same or similar pharmacophores.
• Such methods are well known to persons of ordinary skill in the art of medicinal chemistry, and as the structural information described in this application identifies the pharmacophore of phosphoramidate alkylator prodrugs and phosphoramidate alkylators.
• An example of programs available to perform pharmacophore -related searches is the program 3D Pharmacophore search from the Chemical Computing Group (see http ://www. chemcomp .com/ fdept/prodinfo .htm) .
• heterocyclo group optionally mono- or di- substituted with an alkyl group means that the alkyl can, but need not be, present, and the description includes situations where the heterocyclo group is mono- or disubstituted with an alkyl group and situations where the heterocyclo group is not substituted with an alkyl group.
• a combination of substituents or variables is permissible only if such a combination results in a stable or chemically feasible compound.
• a stable compound or chemically feasible compound is one in which the chemical structure is not substantially altered when kept at a temperature of 4 °C or less, in the absence of moisture or other chemically reactive conditions, for at least a week.
• a prodrug means a compound that, after administration, is metabolized or otherwise converted to an active or more active form with respect to at least one biological property, relative to itself.
• a pharmaceutically active compound or a suitable precursor thereof
• a prodrug can have, relative to the drug, altered metabolic stability or transport characteristics, fewer side effects or lower toxicity, or improved flavor, for example ⁇ see the reference Nogrady, 1985, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392).
• Prodrugs can also be prepared using compounds that are not drugs but which upon activation under certain biological conditions generate a pharmaceutically active compound.
• a phosphoramidate alkylator prodrug is a prodrug that upon activation releases the active phosphoramidate alkylator.
• cytotoxic agent is an agent or compound that produces a toxic effect on cells.
• cytostatic agent is an agent that inhibits or suppresses cellular growth and multiplication.
• hypooxic cells are cells residing in a hypoxic environment in vivo such as, for example, in a hypoxic tumor zone, or in vitro.
• normoxic cells are cells residing in a normoxic environment in vivo or in vitro.
• hypooxic cytotoxicity of a compound or agent is its cytotoxicity on hypoxic cells.
• normoxic cytotoxicity of a compound or agent is its cytotoxicity on normoxic cells.
• a “bioreductive group” refers to a group that accepts electrons in an oxidation-reduction reaction.
• the bioreductive group is a group (1) that can be reduced, i.e., a group that can accept electrons, hydrogen, and/or or an hydride ion; (2) that can be reduced in vivo and/or in vitro; (3) that can be reduced in vivo and/or in vitro under hypoxia; (4) that can be reduced in vivo and/or in vitro by DT-diaphorase, thiols, or by photochemical or electrochemical means; or (5) that can be eliminated and/or cleaved by a biological process, such as by enzymatic hydrolysis, metabolism etc.
• one bioreductive group is a nitroimidazole that may be substituted with a variety of groups.
• bioreductive groups include, but are not limited to, groups based on electron deficient nitrobenzenes, electron deficient nitrobenzoic acid amides, nitroazoles, nitroimidazoles, nitrothiophenes, nitrothiazoles, nitrooxazoles, nitrofurans, and nitropyrroles, where each of these classes of moieties may be optionally substituted, such that the redox potential for the bioreductive group lies within a range where the group can undergo reduction in the hypoxic conditions of a tumor, by DT-diaphorase, and/or by a thiol.
• One of skill in the art will understand, in view of the disclosure herein, how to substitute these and other bioreductive groups to provide a a bioreductive group having a redox potential that lies within said range.
• nitrothiophene, nitrofuran, and nitrothiazole groups may be substituted with one or more electron donating groups, including but not limited to methyl, methoxy, or amine groups, to achieve the desired redox potential.
• the nitropyrrole moiety can be substituted with an electron withdrawing group, including but not limited to cyano, carboxamide, -CF 3 , and sulfonamide groups, to achieve the desired redox potential.
• strong electron withdrawing groups such as cyano, sulfone, sulfonamide, carboxamide, or -CF 3
• milder electron withdrawing groups such as -CH 2 -halogen where halogen is -F, -Cl, or -Br, can be used.
• an "anti-neoplastic agent”, “anti-tumor agent”, or “anti-cancer agent” refers to any agent used in the treatment of cancer. Such agents can be used alone or in combination with other compounds and can alleviate, reduce, ameliorate, prevent, or place or maintain in a state of remission of clinical symptoms or diagnostic markers associated with neoplasm, tumor or cancer.
• Anti-neoplastic agents include, but are not limited to, anti- angiogenic agents, alkylating agents or alkylators, antimetabolite, certain natural products, platinum coordination complexes, anthracenediones, substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, certain hormones and antagonists, anti-cancer polysaccharides, chemoprotectants, and certain herb or other plant extracts.
• cancer refers to one of a group of more than 100 diseases caused by the uncontrolled growth and spread of abnormal cells that can take the form of solid tumors, lymphomas, and non-solid cancers such as leukemia.
• malignant cancer refers to cancer cells or cancers that have the capacity of metastasis, with loss of both growth and positional control.
• neoplasm neoplasia
• tumor refers to abnormal new cell or tissue growth, which can be benign or malignant.
• beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms of cancer or other hyperproliferative disease conditions, diminishment of extent of disease, delay or slowing of disease progression, amelioration, palliation or stabilization of the disease state, and other beneficial results described below.
• administering or "administration of a drug to a subject (and grammatical equivalents of this phrase) includes both direct administration, including self- administration, and indirect administration, including the act of prescribing a drug.
• direct administration including self- administration
• indirect administration including the act of prescribing a drug.
• a physician who instructs a patient to self-administer a drug and/or provides a patient with a prescription for a drug is administering the drug to the patient.
• a "therapeutically effective amount" of a drug is an amount of a drug that, when administered to a subject with cancer, will have the intended therapeutic effect, e.g., alleviation, amelioration, palliation or elimination of one or more manifestations of cancer in the subject.
• the full therapeutic effect does not necessarily occur by administration of one dose, and can occur only after administration of a series of doses.
• a therapeutically effective amount can be administered in one or more administrations.
• a prophylactically effective amount of a drug is an amount of a drag that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of disease or symptoms, or reducing the likelihood of the onset (or reoccurrence) of disease or symptoms.
• the full prophylactic effect does not necessarily occur by administration of one dose, and can occur only after administration of a series of doses.
• a prophylactically effective amount can be administered in one or more administrations.
• a "second line” therapy refers to therapy that is given for the treatment of a cancer which has failed to respond to a first chemotherapy regimen or "first line” chemotherapy.
• “Third line” therapy refers to therapy that is given for the treatment of a cancer when both initial treatment, first-line therapy, and subsequent treatment, second-line therapy, don't work, or stop working is called.
• LogP means a measure of lipophilicity of a substance determined based on the partitioning of the substance betwen octanol and water.
• cytotoxic agents selective for dividing cells targeting, for example, their replicating DNA, microtutbule, and various growth factors and growth factor receptors.
• cytotoxic agents selective for dividing cells targeting, for example, their replicating DNA, microtutbule, and various growth factors and growth factor receptors.
• These drugs are effective, because cancer cells generally divide more frequently than normal cells. However, such drugs almost inevitably do not kill all of the cancer cells in the patient. One reason is that cancer cells can mutate and develop drug resistance. Another is that not all cancer cells divide more frequently than normal cells and slowly-dividing cancer cells can be as, or even more, insensitive to such cytotoxic agents as normal cells.
• hypoxic areas of the tumor represent a significant source of metastases and cancer cells resistant to therapy (see for example, De Jaeger et ah, Br J Cancer. 2001, 84(9):1280-5 and Rofstad et al, Br J Cancer. 1999, 80(11):1697-707).
• low tumor oxygen levels are associated with a poor response to therapy, increased metastases, and poor survival.
• the mechanisms of activation and action of Cyclophosphamide and Ifosfamide can exemplify how these agents can not specifically target the difficult to kill hypoxic zone of a tumor.
• Both Cyclophosphamide and Ifosfamide are prodrugs and can be oxidatively activated in the liver via intermediates to yield active phosphoramidate alkylators, Alkylates 1 (cylophosphamide mustard) and 2 (ifosfamide mustard), respectively (see below).
• Alkylates 1 and 2 can have a half life of many minutes and can permeate in and out of the cell.
• the anionic Alkylators 1 and 2 are much less cell membrane permeable and once formed extracellularly inefficiently kills the cell by alkylating cellular
• the phosphoramidate alkylators When the phosphoramidate alkylators reach the tumor, they generally kill cells in the fast growing, well vascularized, normoxic, outer zone of the tumor. However, these phosphoramidate alkylators are not as effective in permeating into the less vascularized, slower growing, progressively hypoxic inner tumor zones and in killing tumor cells therein. Before any of these active alkylators reach the tumor, they can react with healthy cells and result in toxicity and/or cell death.
• hypoxic tumor zone can generate reduced derivatives of a variety of chemical groups (see the reference Workman et al., 1993, Cancer andMetast. Rev. 12: 73-82), and prodrugs of cytotoxins can be developed to exploit such bioreductive environments (PCT Application Nos. US 04/009667 and US 05/08161; PCT/US2005/041959 and PCT/US2005/042095, all Matteucci et al).
• a hypoxia reducable (or hypoxia activated) prodrug can be constructed by employing a bioreductive group (Z 3 ) together with an alkylator.
• the bioreductive group is employed as part of a Trigger moiety covalently bonded or attached to the phosphoramidate alkylator.
• the compounds of the invention can generally be described as phosphoramidate alkylator prodrugs, hi general, the phosphoramidate alkylator prodrugs of the invention have the following structure
• Trigger T has a structure L-Z 3 , wherein the linker L is bonded to a bioreductive group Z 3 .
• the Trigger T is a hypoxia activated trigger.
• the phosphoramidate alkylator prodrugs of the present invention have one or more of the following characteristics: (i) a higher hypoxic toxicity or lower value of IC 50 or IC 90 , in hypoxic tissue, (ii) lower normoxic cytoxicity, and (iii) less toxic side effect profile or some combination of these attributes.
• the phosphoramidate alkylator prodrugs of the present invention differ from known phosphoramidate alkylator derivatives by: (i) the nature of the phosphoramidate alkylator released, (ii) the nature of the linker (L) and/or the bioreductive group Z 3 , (iii) the presence of more than one bioreductive group moiety, or some combination of these attributes (iv) increased hypoxia selective cytoxicity measured by larger HCR values (v) increased aqueous solubility (vi) increased stability to liver microsomal degradation and/or (vii) providing effective phosphoramide alkylator prodrugs that are achiral and avoid enantiomer specific in vivo metabolism.
• prodrug compounds of the present invention represent a significant advance over known anti-cancer phosphoramidate alkylator derivatives, an understanding of tumor biology particularly under hypoxia and pharmacokinetics, and pharmacodynamics of prodrugs provided herein in particular is helpful.
• hypoxia activated prodrug should be much less toxic to healthy normoxic cells compared to hypoxic tumor cells.
• the hypoxia activated prodrugs of the invention are less active and less toxic to normoxic cells than hypoxic cells.
• reduction of the bioreductive group causes dissociation of the the phosphoramidate alkylator or the active cytotoxin.
• the phosphoramidate alkylator is released within the tumor zone and can more easily penetrate the hypoxic region of the solid tumor.
• hypoxia activated prodrugs that are much less toxic to healthy, normoxic cells compared to hypoxic, tumor cells.
• the phosphoramidate alkylator prodrugs of the present invention employ nitro containing aromatic or indole quinone moieties as bioreductive groups in the Trigger T.
• the nitro group is reduced to an hydroxylamino or an amino group and flow of an electron pair from the amino or hydroxylamino group through the conjugated ⁇ electron system of the Trigger T releases the phosphoramidate alkylator.
• an indole quinone is reduced to an indole hydroquinone and flow of an electron pair from the hydroquinone through the Trigger T releases the phosphoramidate alkylator.
• the released phosphoramidate alkylator kills cells in and/or near the hypoxic tumor.
• a number of enzymes can be responsible for the reduction of the bioreductive group Z 3 in the Trigger.
• cytochrome P450 reductase enzymes can reduce the nitro or a quinone moiety in a bioreductive group in a first step respectively to a NO 2 ('-) or a semiquinone radical anion.
• the hypoxic tumor zone can have a higher concentration of the reductase enzyme compared to normoxic tissue.
• normoxia as in well vascularized healthy tissue, in the presence of oxygen, the NO 2 (-) or the semiquinone radical anion formed can react with oxygen to revert back to the bioreductive group and not ultimately generate or release the phosphoramidate alkylator.
• the aryl or heteroaryl moiety covalently bonded to the NO 2 ('-) or the semiquinone radical anion modulates the oxygen sensitivity of the radical anion.
• the oxygen sensitivity of the bioreductive group varies depending partly on the reduction potential of the bioreductive group.
• one bioreductive group can get reduced in a hypoxic tumor zone having 1% oxygen, another in a zone having 0.1% oxygen, and yet another in a zone having 0.01 % oxygen.
• a bioreductive group loses some or all of its hypoxic specificity when it is so easily reduced that the cytochrome P450 reductase enzyme or other reducing agents ("reducing agents") in healthy normoxic tissue can reduce it in the presence of oxygen.
• a NO 2 ( " -) or a semiquinone radical anion in a bioreductive group does not react or reacts slowly with oxygen, the radical anion itself can release the phosphoramidate alkylator, or can be further reduced and release the phosphoramidate alkylator, causing toxicity to healthy normoxic cells and tissue.
• the novel phosphoramidate alkylator prodrugs of the present invention are more toxic to the hypoxic cancer cells and tissue compared to the healthy normoxic cells and tissue.
• the ease or difficulty of reducing the bioreductive group Z 3 can be measured by the reduction potential of the bioreductive group and is influenced by the linker (L), and the phosphoramidate alkylator (AIk-H).
• the linker (L), and the phosphoramidate alkylator (AIk-H) For example, attachment of the bioreductive group to an electron withdrawing linker or an electron withdrawing phosphoramidate alkylator can make the bioreductive group easier to reduce compared to when it is covalently bonded to an electron rich linker or an electron rich phosphoramidate alkylator.
• the Trigger T can be oxidized, hydrolyzed, or thiolyzed and can release the phosphoramidate alkylator in a hypoxia non-senselective manner.
• TelcytaTM a phosphoramidate alkyltor prodrug that is in the clinic, can release an active toxin in absence of hypoxia by the action of glutathione transferase (see, e.g., phophoramidate alkylator If in the "Methods of Treatment" section).
• the chemical nature of the linker and/or the phosphoramidate alkylator can influence the oxidative, hydrolytic, or thiolytic stability of the prodrug with respect to phosphoramidate alkylator release.
• a hypoxia activated phosphoramidate alkylator prodrug does not release the phosphoramidate alkylator in a hypoxia non-specific, oxidation, hydrolysis, or thiolysis.
• a properly employed Trigger in a phosphoramidate alkylator prodrug can be used to "tune" the pharmacokinetic property of the prodrug without altering its cytotoxic properties.
• a high volume of distribution of an anticancer agent ensures that the prodrug is absorbed in the tissue quickly.
• the volume of distribution of a phosphoramidate alkylator prodrug can be modulated by employing a Trigger T containing an amino group capable of forming an ammonium cation under physiological conditions.
• a Trigger T containing a quaternary ammonium group can yield a prodrug compound of the invention having a high volume of distribution while avoiding possible endosomal trapping.
• a Trigger T comprising a carboxyl functionality will exist as the anionic carboxylate anion form.
• the lower pH in tumor extracellular space can convert the CO 2 Q to the uncharged "CO 2 H" form allowing the prodrug to pass through tumor cell membrane.
• a phosphoramidate alkylator containing a hydroxyl, amino, mercapto, and/or a carboxyl group can be transformed into a prodrug by covalently attaching a Trigger T to one or more of these functional groups.
• a hydroxyl group in the phosphoramidate alkylator can be transformed, for example, to an ether or an acetal; an amino to an alkylamino, a carbamate, or an amide; a carboxyl group to an ester; and a mercapto group to a thioether or a thioacyl, as described in greater detail in the Method of Synthesis and the Experimental sections below.
• These transformations can yield a prodrug which is less polar or more lipophilic than the corresponding phosphoramidate alkylator.
• Non polar phosphoramidate alkylator prodrugs may not be readily soluble in aqueous pharmaceutical carriers or diluents.
• Solubility enhancer groups like CO 2 H, amino, alkylamino, dialylamino, and hydroxyl can be employed in the Trigger T to modulate the solubility of the prodrug and overcome any problems encountered in preparing aqueous formulations of the phosphoramidate alkylator prodrugs.
• an aziridine or aziridium species forms which can alkylate DNA (See EXAMPLE section, Example 36).
• the aziridinium formation kinetics can vary. For example, as shown in the reaction sequence below, the rate of alkylation can increase when the NR 2 R 3 moiety is
• the present invention arises in part out of the discovery that phosphoramidate alkylator prodrugs employing 2-nitroimidazole-bioreductive group show unexpectedly high hypoxic cytotoxicity, low normoxic toxicity and high HCR and improved solubility.
• Compounds 24 and 25 were respectively, 400 to 1000 fold more toxic in hypoxic cells than in normoxic cells in an anti-proliferation cytotoxicity assay with a IC 50 of 0.05 ⁇ M in cells under hypoxia. (See EXAMPLE section).
• the present invention provides phosphoramidate alkylators prodrugs of formula (I):
• Y 1 is O 5 S, NR 6 or NSO 2 R 6 wherein each R 6 is independently C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, aryl, or heteroaryl;
• Y 2 is O, S, NR 6 , NCOR 6 , or NSO 2 R 6 ;
• each OfR 1 -R 5 independently is hydrogen, hydroxyl, amino, C 1 -C 6 alky ⁇ Q-Q heteroalkyl, C 3 -C 8 cycloalkyl, heterocyclyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 1 -C 6 dialkylamino, aryl, heteroaryl, C 1 -C 6 acyl, C 1 -C 6 heteroacyl, aroyl, or heteroaroyl; or together any two OfR 1 -R 5 form a C 3 -C 10 heterocycle; or each OfR 1 -R 5 independently is a Trigger T wherein Trigger is L-Z 3 ;
• Y 3 is S, O, or NR 7 wherein each R 7 is independently hydrogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 3 -C 8 cycloalkyl, heterocyclyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 1 - C 6 dialkylamino, aryl, heteroaryl, C 1 -C 6 acyl, Ci-Ceheteroacyl, aroyl, or heteroaroyl;
• Y 4 is O, S, or -NR 7 -C(O)-O-;
• each Z 1 independently is hydrogen, halogen, C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, aryl, heteroaryl, C 3 -C 8 cycloalkyl, heterocyclyl, C 1 -C 6 acyl, CrC ⁇ heteroacyl, aroyl, or heteroaroyl;
• Z 2 is C 1 -C 6 alkylene, C 1 -C 6 heteroalkylene,
• X 2 is NR 7 , S, or O; and [0114] Z 3 is a bioreductive group selected from the group consisting of:
• At least two OfR 1 -R 5 are selected from the group consisting of 2-haloalkyl, 2- alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- heteroalkylsulfonyloxyalkyl;
• At least one OfR 1 -R 5 is selected from the group consisting of 2-haloalkyl, 2- alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- heteroalkylsulfonyloxyalkyl; and at least one OfNR 2 R 3 and NR 4 R 5 is VWN ⁇
• each OfNR 2 R 3 and NR 4 R 5 are """ ⁇ ;
• z is 1.
• R 2 -R 5 are not the same.
• any one of R -R is
• the present invention provides hypoxia activated phosphoramidate alkylator prodrugs each employing two phosphoramidate alkylators.
• the phosphoramidate alkylator prodrug of the present invention employs an 1- N-alkyl-2-nitroimidazol-5-yl moiety or a l-N-methyl-2-nitroimidazol-5-yl moiety as a bioreductive group or Z 3 .
• the phosphoramidate alkylator prodrug of the present invention employs a 2-nitrofuran moiety as a bioreductive group or Z 3 .
• the present invention excludes the compounds:
• R a is H, Br (P14), NMe 2 (Pl 5), CN (P16), or CONH 2 (P17),
• the present invention provides a compound of formula (I) with the proviso that
• At least one OfR 1 -R 5 are selected from the group consisting of 2- alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- heteroalkylsulfonyloxyalkyl and
• At least one OfR 1 -R 5 are selected from the group consisting of 2-haloalkyl, 2- alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- heteroalkylsulfonyloxyalkyl ; or
• At least one OfR 1 -R 5 is selected from the group consisting of 2-haloalkyl, 2-C 1 - C 6 alkylsulfonylox y alkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2-
• the present invention provides a compound of formula (I) with the proviso that the formula (I) excludes R 2 and R 3 together forming a morpholine ring or R 4 and R 5 together forming a morpholine ring.
• the present invention excludes a compound of the following structure:
• Z 1 is hydrogen or C 1 -C 6 alkyl.
• the present invention provides compounds wherein the Trigger T is:
• Z 3 is:
• each -C(ZO 2 - is: -CH 2 -, -CHMe-, -CH(CN)-, -CH(CO 2 H)-, - CH(CONH 2 )-, -CH(CF 3 )-, -CH(CHF 2 )-, -C(Me) 2 -, -C(Et) 2 -, -CH(CH 2 NMe 2 )-, - CH(CH 2 NMe 2 )-, -C(CH 2 NMe 2 ) 2 -, or -C(CH 2 CO 2 H) 2 -.
• -C(Z 1 ) 2 -Y 3 - is: -CH 2 -O-, -CH 2 -S-, -CH 2 -NMe, -CH 2 -NH-, CH(Me)-O-, CH(Me)-S-; -CH(Me)-NMe-, -CH(Me)-NH-; -CMe 2 -NMe-, -CMe 2 -NMe-, or- CMe 2 -NMe-.
• Trigger is:
• Trigger is
• each Z 1 independently is H or C 1 -C 6 alkyl.
• Trigger is:
• the present invention provides compounds of formulas (II) and (III):
• each R 2 -R 5 independently is selected from the group consisting of hydrogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 3 -C 8 cycloalkyl, heterocyclyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 1 -C 6 dialkylamino, aryl and heteroaryl; or together any two OfR 2 -R 5 form a C 3 - C 10 heterocycle; each Y 1 independently is S or O; and each Trigger T is defined as in formula
• At least two OfR 1 -R 5 are selected from the group consisting of 2-haloalkyl, 2- alkylsulfonyloxyalkyl, 2.heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2 heteroalkylsulfonyloxyalkyl; or
• At least one OfR 1 -R 5 is selected from the group consisting of 2-haloalkyl, 2-C 1 - C 6 alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2-
• VW 1 N heteroalkylsulfonyloxyalkyl; and at least one OfNR 2 R 3 and NR 4 R 5 is ⁇ 1 j ; or
• each NR 2 R 3 and NR 4 R 5 are ⁇ 1 J;
• the present invention provides a compound of formula (II) wherein Trigger T is -CH 2 -Z 3 , -CH(Z ⁇ -Z 3 , or -C(Z ⁇ 2 -Z 3 wherein Z 1 is Cralkyl and Z 3 is:
• one of R 2 and R 3 is Q-alkyl and one OfR 4 and R 5 is Ci-alkyl; or
• At least one OfR 2 -R 5 is hydroxyl, amino, C 3 -C 8 cycloalkyl, heterocyclyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 1 -C 6 dialkylamino, aryl, heteroaryl, C 1 -C 6 acyl, Ci-C ⁇ heteroacyl, or aroyl or heteroaroyl.
• the present invention provides a compound of formula (II) wherein Z 3 is a bioreductive group selected from:
• At least one OfR 1 -R 5 are selected from the group consisting of 2- alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- heteroalkylsulfonyloxyalkyl and
• At least one OfR 1 -R 5 are selected from the group consisting of 2-haloalkyl, 2- alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- heteroalkylsulfonyloxyalkyl; or
• At least one OfR 1 -R 5 is selected from the group consisting of 2-haloalkyl, 2-C 1 - C 6 alkylsulfonylox y alkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2-
• heteroalkylsulfonyloxyalkyl and at least one OfNR 2 R 3 and NR 4 R 5 is ⁇ I; or
• the present invention provides phosphoramidate alkylator prodrugs of formula (I):
• each OfR 2 -R 5 independently is hydrogen, hydroxyl, amino, C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 3 -C 8 cycloalkyl, heterocyclyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 1 -C 6 dialkylamino, aryl and heteroaryl, C 1 -C 6 acyl, CrC 6 heteroacyl, aroyl, or heteroaroyl; or together any two OfR 1 -Rs form a C 3 -C 10 heterocycle;
• At least two OfR 2 -R 5 are selected from the group consisting of 2-haloalkyl, 2- alkylsulfonyloxyalkyl, 2,-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- heteroalkylsulfonyloxyalkyl;
• At least one OfR 2 -R 5 is selected from the group consisting of 2-haloalkyl, 2-C 1 - C 6 alkylsulfonylox y alkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2-
• heteroalkylsulfonyloxyalkyl and at least one OfNR 2 R 3 and NR 4 R 5 is ⁇ J; or
• each NR 2 R 3 and NR 4 R 5 are ⁇ ;
• the present invention provides the compounds: Phosphoramidate alylator prodrugs
• the present invention provides the compounds:
• the present invention provides the compounds: .
• R 6 is -(N-CH 2 CH 2 Xi) 2 .
• the present invention provides the compounds:
• R 2 - R 5 are defined as in formula (II).
• Phosphoramidate alkylator prodrug [0176] In another embodiment, the present invention provides the compounds:
• R 2 - R 5 are defined as in formula (II).
• the present invention provides the compounds of the formulas (IV) - (VII)
• each R 9 independently is hydrogen, deuterium, aryl, heteroaryl, C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 3 -C 8 cycloalkyl, heterocyclyl, C 1 -C 6 acyl, CrC 6 heteroacyl, aroyl, heteroaroyl, C 1 -C 6 alkoxycarbonyl, C 1 -C 6 alkylaminocarbonyl, di C 1 -C 6 alkylaminocarbonyl, or C 1 -C 6 alkoxy; or together two R 9 groups form a heterocycle; each R 10 is hydrogen, C 1 -C 6 alkyl, C 1 - C 6 heteroalkyl, C 3 -C 8 cycloalkyl, heterocyclyl aroyl or heteroaroyl, or together two R 10 groups form a heterocycle;
• R 11 is independently is hydrogen, deuterium, aryl, heteroaryl, C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 3 -C 8 cycloalkyl, heterocyclyl, C 1 -C 6 acyl, Q-C ⁇ heteroacyl, aroyl, heteroaroyl, C 1 -C 6 alkoxycarbonyl, C 1 -C 6 alkylaminocarbonyl, di C 1 -C 6 alkylaminocarbonyl, or C 1 -C 6 alkoxy; or together two R 9 groups form a heterocycle with the proviso that when R 11 is C 1 -C 6
• R 11 excludes ; or together two R 11 groups form a heterocycle
• X 4 is Cl, Br, alkylsulfonyloxy, heteroalkylsulfonyloxy, arylsulfonyloxy, or heteroalkylsulfonyloxy;
• each R 9 is independently hydrogen, deuterium, C 1 -C 3 alkyl, C 1 -C 6 heteroalkyl, C 3 -C 6 cycloalkyl, heterocyclyl, aryl or heteroaryl.
• each R 9 is independently hydrogen, deuterium, or C 1 -C 6 alkyl.
• each R 9 is independently methyl, ethyl, propyl, isopropyl, isobutyl, tertiary butyl, or cyclopropyl.
• the present invention provides a compound of formula (IV) wherein one of R 10 is -(CH 2 ) e -Intercalator wherein an Intercalator is an aromatic or heteroaromatic moiety capable of intercalating between a nucleic acid base pair.
• the present invention provides the compound:
• the present invention provides the compound:
• the present invention provides the compound of formula (VIII):
• each R 9 is independently hydrogen, methyl, ethyl, propyl, isopropyl, or cyclopropyl;
• the present invention provides the compound of the formula (IX):
• each R 9 independently is hydrogen, methyl, ethyl, propyl, isopropyl, or cyclopropyl.
• the present invention provides the compound of the formula (X):
• each R 9 independently is hydrogen, methyl, ethyl, propyl, isopropyl, or cyclopropyl
• each R 11 is independently hydrogen, methyl, ethyl, propyl, isopropyl, benzyl, substituted methyl, cyclopropyl, methoxy, and hydroxyl; or together two R 11 form a heterocycle.
• the present invention provides the compounds of the formulas (X-A), (X-B) and (X-C):
• the present invention provides the compounds of the formula (XI) - (XV):
• each R 11 independently is hydrogen, methyl or substituted methyl, benzyl, isopropyl, propyl, cyclopropyl, methoxy, and hydroxyl; and X 1 , X 2 , and Z 3 are defined as above; and X 4 is Cl, Br, alkylsulfonyloxy, heteroalkylsulfonyloxy, cycloalkylsulfonyloxy, heterocycloalkylsulfonyloxy, arylsulfonyloxy, or heteroarylsulfonyloxy.
• Rn excludes isopropyl.
• a compound of formula (X) excludes a compound wherein Z 3 is
• the present invention provides a compound of formula (XII), (XIV), or (XV) wherein each R 11 is hydrogen.
• examples of compounds of formula XII, XIV, or XV include compounds 5, 7, 8, 9, 10, 13, 14, 15, 19, 23, 24, 25, 26, 32, 34, and 36.
• the present invention provides phosphoramidate alkylator prodrugs of formulas XII, XIV, or XV wherein R 11 excludes propyl or isopropyl.
• the present invention excludes the compound:
• the present invention provides a phopsphoramidate alkylator prodrug wherein R 11 is C 3 -C 8 cycloalkyl.
• the cycloalkyl is cyclopropyl.
• a cyclopropyl group can be more stable than an alkyl group to oxidatively metabolizing proteins in the cell, particularly in the liver the prodrug compounds of the invention provide a pharmacokinetically improved phopsphoramidate alkylator prodrug compared to known phosphoramidate alkylator prodrugs.
• the present invention provides the compounds of the formula (XVI)
• K is Q-Csalkylene or C 1 -C 6 heteroalkylene.
• K is (C(R 12 ) 2 ) e , CH 2 CH 2 (-X 6 -CH 2 CH 2 ) f , or CH 2 (-X 6 -CH 2 ) f wherein e is 1-10, f is 0-3, and X 6 is O, S, or NR 12 wherein each R 12 is independently defined as above.
• the present invention provides the compounds of the formula (XVII - (XVIII) Triggers
• e is 0-4, X 4 is Cl or Br, alkylsulfonyloxy, heteroalkylsulfonyloxy, arylsulfonyloxy, or heteroarylsulfonyloxy; X 6 is O, S, or NR 12 wherein R 12 is defined as above.
• the present invention provides the compound of formula (XIX):
• e is 0-4, and X 4 is Cl, Br, alkylsulfonyloxy, heteroalkylsulfonyloxy, arylsulfonyloxy, or heteroarylsulfonyloxy.
• the present invention provides a compound of formula (XIX) wherein e is 1. See EXAMPLE section for examples of compounds of formulas described herein.
• the present invention provides the compound of formula (XX):
• XX wherein R g is glucose or a glucose analog; e is 0-4, and X 4 is Cl, Br, alkylsulfonyloxy, heteroalkylsulfonyloxy, arylsulfonyloxy, or heteroarylsulfonyloxy.
• a glucose analog includes mono, di and tri saccharides.
• the present invention provides a compound of formula XX wherein e is 1.
• the present invention provides the compounds:
• X 4 is Cl, Br, or alkylsulfonyloxy.
• the present invention provides the compounds:
• Rg and X 4 are defined as in formula VI.
• the present invention provides the compound of the formula (XXI)
• Trigger T is defined as in formula (I).
• the present invention provides the oxime-phosphoramidate alkylator conjugate:
• such an oxime-phosphoramidate alkylator conjugate can be hydrolyzed enzymatically to produce
• the present invention provides the compounds of the formula (XXII):
• R 1 - R 5 , Y 1 , and Y 2 are defined as in formula (I);
• each R 1 -R 5 and R 1 + -R 5 * independently is selected from the group consisting of hydrogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 1 -C 6 dialkylamino, aryl, heteroaryl; or together R 2 and R 2 * form a heterocycle; or each Ri-R 5 and R 1 + -R 5 + independently is a Trigger T selected from the group consisting of
• At least two OfR 1 -R 5 and R 1 + -R 5 * are 2-haloalkyl, 2-alkylsulfonyloxyalkyl, 2. heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, or 2 heteroalkylsulfonyloxyalkyl; or
• At least one OfRi-R 5 and R 1 + -R 5 * is 2-haloalkyl, 2-C 1 -C 6 alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, or 2-heteroalkylsulfonyloxyalkyl; and
• At least one OfNR 2 R 3 and NR 2 + R 3 * is
• each Z independently is C, S, or P;
• each t independently is 1 or 2;
• each r independently is 0 or 1 ;
• K is selected from the group consisting of CrQalkylene, C 1 -C 6 heteroalkylene, arylene, or heteroarylene, (C(R 9 ) 2 ) n ; and (Y 5 -(C(R 9 ) 2 ) m -Y 4 -(C(R 9 ) 2 ) in -Y 6 )n wherein n is 1-8;
• each m independently is 1-4;
• each R 9 is independently C 1 -C 6 alkyl or heteroalkyl, or together when covalently bonded to the same carbon atom or adjacent carbon atoms are cycloalkyl or heterocyclyl;
• each Y 4 ,Y 5 , and Y 6 independently is O, S, NR 7 , or a bond; with the proviso that one ofY 4 ,Y 5 , and Y 6 has to be O, S, or NR 7 .
• the present invention provides the compounds of the formula (XXIII):
• R 1 - R 5 , Y 1 , and Y 2 are defined as in formula (I);
• each R 1 -R 5 and R ⁇ -R 5 * independently is selected from the group consisting of hydrogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 1 -C 6 dialkylamino, aryl, heteroaryl; or together R 2 and R 2 * form a heterocycle; or each R 1 -R 5 * independently is a
• Trigger T selected from the group consisting of
• R 2 -R 5 and R 2 *-R 5 * are 2-haloalkyl, 2-alkylsulfonyloxyalkyl, 2- heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, or 2 heteroalkyl-sulfonyloxyalkyl; [0219] (ii) at least one of R 2 -R 5 and R 2 *-R 5 * is 2-haloalkyl, 2-C 1 -C 6 alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, or 2-heteroalkylsulfonyloxyalkyl; and
• the present invention provides the compound of the formula (XXIV):
• R 2 , R 3 , R 4 , R 2 *, R 3 *, R 4 *, Z, K and Trigger are as defined in Formula (XXII).
• the present invention provides the compounds of formula (XXIV) having the structure of formula (XXV) or (XXVI):
• the present invention provides the compound of the formula (XXVI):
• the present invention provides the compound of the formula (XXVII):
• R 2 -R 5 , r, Ic, Y 1 , and Trigger T are defined as in formula (XXIV).
• the present invention provides a compound of Formula: wherein T is L-Z 3 ;
• L 1 is CH 2 , CHMe, C(Me) 2 , CH 2 OCH 2, (CH 2 ) 3 , CH 2 S(CH 2 ) 2 , CH 2 S(CH 2 ) 3 ,
• the present invention provides a Z 3 selected from the group consisting of:
• the present invention provides a moiety having the formula:
• the present invention provides a T selected from the group consisting of:
• Z 1 is hydrogen, methyl, or ethyl
• R 7 is methyl, trifluoroethyl, ethyl, propyl, and cyclohexyl
• R 9 is hydrogen, methyl, ethyl, isopropyl, or isobutyl; and X 4 is chloro, bromo, or methanesulfonyloxy.
• the present invention provides a compound of formula:
• T is defined as above or more particularly T is L-Z 3 wherein L CH 2 , CHMe, CMe 2 ,
• the present invention provides deuterated phosphoramidate alkylators and deuterated phosphoramidate alkylator prodrugs of formula
• X 4 is Cl or Br.
• deuterated phosphoramidate alkylators and their prodrugs are equally cytotoxic with respect to hypoxic tumor tissue as their non-deuterated or hydrogenated analogs, such as compounds 25, 36 and the likes,.
• deuterated analogs in vivo can be determined more efficiently compared to their corresponding phosphoramidate alkylators and/or phosphoramidate alkylator prodrugs by nuclear magnetic resonance methods and such deuterated analogs can be useful in determining pharmacokinetic or pharmacodynamic properties of the phosphoramidate alkylators and/or phosphoramidate alkylator prodrugs.
• Pharmacokinetic and or pharmacodynamic information of phosphoramidate alkylators and/or phosphoramidate alkylator prodrugs is used in determining dosage, frequency of dosing, and similar administration related parameters.
• the present invention provides the individual and selective groupings of the compounds of the EXAMPLES.
• Examples of compounds of the invention include:
• the phosphoramidate alkylator prodrug contains
• the present invention provides a novel phosphoramidate alkylator prodrug which upon bioreduction releases the corresponding novel or known phosphoramidate alkylator
• X 4 is defined as in formula (I), and Rg, R 10; and R 11 , are defined as in formulas (IV)- (VII), and ionized forms thereof.
• X 4 is Cl, Br, methanesulfonyloxy, benzenesulfonyloxy, or para-toluenesulfonyloxy.
• the present invention provides a novel phosphoramidate alkylator prodrug which upon bioreduction releases the corresponding novel or known phosphoramidate alkylators
• N(R 10 ) 2 is selected from the group consisting of NH 2 , NHMe, NMe 2 , NEt 2 ,
• each R 11 is independently hydrogen, Me, ethyl, cyclopropyl, isopropyl, propyl, benzyl, substituted methyl, cyclopropyl, methoxy, and hydroxyl; or together two R 11 form a heterocycle.
• the anti-cancer agent Cyclophosphamide metabolizes to Id (R 10 is hydrogen) and Ifosfamide metabolizes to Ie (each R 11 is hydrogen), when used in cancer treatment.
• Glufosfamide which is being evaluated in the clinic for cancer treatment, releases an alkylator of formula Ie (each R 11 is hydrogen, see Wiessler et ai, US Pat. No. 5,622,936; PCT application No. US05/03370 entitled “Anti Cancer Therapies", US Pat. Appl. No. 60/638995 entitled "Glufosfamide Combination Therapy” and Attorney docket No.
• Known phosphoramidate alkylator prodrugs such as ifosfamide and cyclophosphamide metabolize to produce cytotoxic by products such as acrolein and chloroacetaldehyde which cause undesirable patient side-effects such as hemorrhagic cystitis, coma or death.
• the present invention provides a phosphoramidate alkylator prodrug which upon metabolism produces less toxic by products per treatment as compared to those produced by the metabolism of ifosfamide and/or cyclophosphamide.
• the phosphoramidate alkylator prodrugs of the present invention do not produce acrolein by in vivo metabolism.
• Examples of toxic by products resulting from metabolism of the prodrugs of the invention include chloro, bromo, alkylsulfonyloxy, heteroalkylsulfonyloxy, arylsulfonyloxy, or heteroarylsulfonyloxy-acetaldehyde, (for metabolic production of chloroacetaldehyde from ifosfamide see the reference Hardman et ah, supra, page 1396).
• the present invention provides a phosphoramidate alkylator prodrug which upon oxidative metabolism produces 5-95% as much chloroacetaldehyde or an equivalent as defined above, per treatment, as produced by ifosfamide metabolism.
• the phosphoramidate alkylator derivative formed upon the reduction of Z 3 can be different from the phosphoramidate alkylator being protected and the phosphoramidate alkylator prodrug and is termed a modified phosphoramidate alkylator prodrug.
• a phosphoramidate alkylator prodrug can yield a modified phosphoramidate alkylator prodrug Alk-Trigger m0d upon reduction of the bioreductive group (Z 3 ).
• the linker (L) bonded to the phosphoramidate alkylator can undergo degradation to yield either the phosphoramidate alkylator or some other modified phosphoramidate alkylator prodrug.
• the present invention provides a compound which demonstrates a bystander effect upon activation in hypoxic tissue by incorporating a linker (L) as described above.
• the bystander effect allows a modified phosphoramidate alkylator of the present invention to diffuse or penetrate into tumor zones which are not hypoxic enough to activate the prodrug compounds of the invention but reside nearby the hypoxic tumor zone which can activate these prodrugs.
• T M is selected from:
• the TM is selected from:
• the present invention provides a Trigger T which upon bioreduction is modified to TriggerM o d or T M and the phosphoramidate alkylator is separated from T M in less than 0.1 second.
• the phosphoramidate alkylator is separated from T M in between 0.01 to 0.10 second, hi another embodiment, the phosphoramidate alkylator is separated from T M in between 0.1 to 1.0 second.
• the active phosphoramidate is separated from T M in between 1.0 to 10.0 seconds.
• the phosphoramidate alkylator is separated from T M in between 10.0 to 100.0 seconds.
• a phosphoramidate alkylator prodrug upon activation or reduction, yields a prodrug with a modified Trigger T (T M ) which subsequently releases the phosphoramidate alkylator 20 to 500 ⁇ m from the site of activation or reduction; or 20 to 100 ⁇ m from the site of activation or reduction.
• T M Trigger T
• Bystander effect of a phosphoramidate alkylator prodrug of the present invention can be measured using cellular spheroids and multilayer cellular assay (for example of such assays see Kyle et ah, Cancer Res. 2004, 64(17):6304-9 and West et al, Cancer Chemother.
• Tumor cells can be grown in culture as multicellular spheroids to create an in vitro model of the tumor microenvironment in solid tumors containing a hypoxic region and a quiescent cell population responding to the environmental stresses of limited nutrients and increased waste production. These spheroids have the unique property of developing gradients of oxygen and nutrients as the aggregate of cells continue to divide and grow outward. After the viable rim reaches approximately 150 ⁇ m in size, a hypoxic region develops, that drives the cells in this region into a quiescent state and eventually to cell death. A necrotic core develops as a result of the dying cells.
• the spheroid can be divided into 4 distinct compartments for modeling the effectiveness of a hypoxic activated prodrug: 1) the outer aerobic and actively dividing region; 2) a region of intermediate hypoxia; 3) a region of hypoxia where cells are not cycling; 4) and a necrotic core containing dead cells and cellular debris.
• the response of a drug will depend on a number of factors; the ability of compound to penetrate into the deepest regions of the spheroid.
• the assessment of the effectiveness of a compound can therefore be evaluated on a number of different levels.
• the effect of the compound alone can be compared to cells in monolayer culture versus intact spheroids.
• the HAP can used as a monotherapy.
• the hypoxic fraction of the spheroid can be modulated by varying the concentration of O 2 of the equilibrating gas and therefore change the ratio of the aerobic and hypoxic compartments.
• HAP' s can be combined with other chemotherapeutic agents that either target only the outer aerobic cells or are able to target the entire spheroid.
• the expected cell kill can be predicted by knowing the hypoxic fraction and the expected cell kill of each of the monotherapies.
• the present invention provides a phosphoramidate alkylator prodrug which upon activation such as bioreduction releases the phosphoramidate alkylator with a half life of less than 0.1 second; between 0.01 to 0.10 second, between 0.1 to 1.0 second, between 1.0 to 10.0 seconds, and between 10.0 to 100.0 seconds.
• Anti cancers drugs can bind to tissue surrounding the vasculature and/or have high molecular weights that impede diffusion and not reach in therapeutically effective concentrations hypoxic tumor zones that can be up to 150 - 200 ⁇ M away from the vasculature.
• the present invention provides phosphoramidate alkylator prodrugs that can reach hypoxic cancer cells away from the vasculature. Some methods for determining the bystander effect are described in greater detail in Examples 35 and 37.
• the phosphoramidate alkylator used in a hypoxia activated prodrug plays an important role to efficiently kill tumor cells.
• the cytoxicity of the phosphoramidate alkylator and its rate of cellular alkylation, and the cell membrane permeability of the prodrug and the phosphoramidate alkylator impact the hypoxic selectivity and hypoxic cytotoxicity of the phosphoramidate alkylator prodrug.
• the present invention provides phosphoramidate alkylator prodrugs that are safer than the corresponding phosphoramidate alkylators formed in vivo (at least ten and up to one million-fold safer..
• the increased safety results from a modification at the site of attachment of the Trigger T (activation of the phosphoramidate alkylator prodrug releases the alkylator/cytotoxic agent), hi either event, the phosphoramidate alkylator prodrugs are converted into the corresponding alkylator in hypoxic tissues by virtue of the activation or reduction of the bioreductive group (Z 3 ), resulting in its removal and the concomitant or subsequent release or generation of the phosphoramidate alkylator.
• the Trigger T is covalently bonded to the phosphoramidate alkylator, in a manner that masks or reduces the cytotoxic activity of the phosphoramidate alkylator.
• This masking effect can vary and can depend on the cytotoxic activity of the phosphoramidate alkylator.
• the phosphoramidate alkylator prodrug will show at least about 10 fold less cytotoxic activity than the corresponding phosphoramidate alkylator, and can show up to about a million fold or less cytotoxic activity.
• the cytotoxic activity of the phosphoramidate alkylator prodrug is about 100 fold to about 10,000 fold less than the cytotoxic activity of the corresponding phosphoramidate alkylator.
• the IC 50 , IC 90 , or LC 5 o of the corresponding phosphoramidate alkylator prodrug can be 1 ⁇ M or greater.
• compounds provided herein include as phosphoramidate alkylator prodrug, any phosphoramidate alkylator that can be linked to a Trigger T in a manner that yields a phosphoramidate alkylator prodrug that is at least about 10-fold to about 1,000,000- fold, and typically about 100 to about 10,000-fold, less active as a cytotoxic agent than the corresponding phosphoramidate alkylator or modified phosphoramidate alkylator that is released from the compounds under hypoxic conditions.
• a phosphoramidate alkylator prodrug is selectively active under anoxic or hypoxic conditions, cells are exposed to the drug either with air (normoxic) or without oxygen (anoxia) or with very little oxygen (hypoxia).
• cytotoxicity of a phosphoramidate alkylator prodrug as measured in an anti- proliferation assay is expressed by the IC 50 ; and the cytotoxicity of a phosphoramidate alkylator prodrug as measured in a clonogenic survival experiment is expressed as IC 10 or LC 10 , IC 90 or LC 90 , or IC 99 or LC 99 .
• hypoxia cytotoxicity ratio The ratio of cytotoxicity as measured for example by IC 5O , IC 9O , LC 5O , LC 90 , or LC 99 determined in normoxia and hypoxia is called hypoxia cytotoxicity ratio (HCR) and can be a measure of the hypoxia selective cytotoxicity of the prodrugs of the present invention.
• HCR hypoxia cytotoxicity ratio
• the HCR determined based on IC 99 or LC 99 is larger than; that determined based on IC 9 Q or LC 9 Q.
• the phosphoramidate alkylator prodrug of the present invention has a hypoxic cytotoxicity of 0.1 nM to 50 ⁇ M and a HCR of 10 to 100,000. In a related embodiment, the phosphoramidate alkylator prodrug of the present invention has a hypoxic cytotoxicity of 0.1 nM to 50 ⁇ M and a HCR of 25 to 100,000 (see EXAMPLE section). In another related embodiment, the phosphoramidate alkylator prodrug of the present invention has a hypoxic cytotoxicity of 0.1 nM to 5 ⁇ M and a HCR of 50 to 10,000 such as, for example, the compounds as described in Examples 29, 30 and 31.
• the present invention provides a phosphoramidate alkylator prodrug having hypoxic toxicity which is 5 to 1,000,000 folds more than the corresponding normoxic toxicity. In another embodiment, the present invention provides a phosphoramidate alkylator prodrug having hypoxic toxicity which is 10 to 10,000 folds more than the corresponding normoxic toxicity. In another embodiment, the present invention provides a phosphoramidate alkylator prodrug having hypoxic toxicity which is 25 to 5,000 folds more than the corresponding normoxic toxicity.
• Tumors have a gradient of oxygen concentration that can vary from 10%, in tissues adjacent to the vasculature, to 0.5% in tissues about 150 ⁇ M away, and lower in tissues further away from the vasculature and near the necrotic core.
• the present invention provides phosphoramidate alkylator prodrugs that can generate phosphoramidate alkylators, 5-1,000,00; 10-10,00; and 25-5,000 folds more toxic than the corresponding prodrug, under a variety of oxygen concentrations, hi one embodiment, the present invention provides phosphoramidate alkylator prodrugs generate phosphoramidate alkylators, 5- 1,000,00; 10-10,00; and 25-5,000 folds more toxic than the corresponding prodrug, under about 0.5-0.6% oxygen concentrations.
• the Io gP of a phopsphramidate alkylator prodrug of the present invention can measure the lipophilicity or the hydrophilicity of the prodrug.
• the present invention provides a phosphoramidate alkylator prodrug having a logP less than 0.
• Such phosphoramidate alkylator prodrugs can be hydrophilic, such as a prodrug having formula XV wherein each R 11 is H and can be easily formulated as an aqueous formulation for i.v. or i.p. injection.
• Another example of such prodrugs are compounds 24, 25 and 36.
• the present invention provides a phosphoramidate alkylator prodrug having a logP greater than 0.
• the present invention provides a phosphoramidate alkylator prodrug having a logP between 0 and 4 such as those exemplified by formulas XIV; XX and XV wherein each R 11 is methyl or, cyclopropyl, and administered in a patient can pass the cell membrane to penetrate inside cancer cells.
• the present invention provides a method of synthesizing a phosphoramidate compound comprising reacting a phosphoramidic or a phosphordiamidic acid and an alcohol to yield a phosphoramidate.
• the present invention provides methods of synthesizing the novel phosphoramidate alkylator prodrug compounds of the invention or those that are known.
• the present invention provides a method of synthesizing a phosphoramidate alkylator prodrug comprising reacting, a novel or known phosphoramidate alkylator, a Trigger-OH, a trisubstituted phosphine, and a dialkyl azodicarboxylate to yield a novel or known phosphoramidate alkylator prodrug.
• a Trigger-OH in a first step the Trigger-OH is reacted with the trisubstituted phosphine and the dialkyl azodicarboxylate to yield an intermediate
• the phosphoramidate alkylator is added to the intermediate obtained from the first step to yield the product.
• a Mitsunobu type reaction is particularly suitable for synthesis of novel or known phosphoramidate alkylator prodrugs or derivatives, Alk-Trigger, wherein Trigger is
• R 9 is as defined above.
• the present invention provides a method of synthesizing a phosphoramidate alkylator prodrug comprising reacting each of novel or known phosphoramidate alkylators: with a Trigger-OH, a trisubstituted phosphine, and a dialkyl azodicarboxylate to yield respectively,
• the present invention provides a method to synthesize a compound of formula:
• At least two OfR 1 -R 5 are selected from the group consisting of 2-haloalkyl, 2- alkylsulfonyloxyalkyl, 2--heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- heteroalkylsulfonyloxyalkyl; (ii) at least one OfR 1 -R 5 is selected from the group consisting of 2-haloalkyl, 2-C 1 -C 6 alkylsulfonyloxyalkyl, 2-heteroalkylsulfonyloxyalkyl, 2-arylsulfonyloxyalkyl, and 2- heteroalkylsulfonyloxyalkyl; and at least one OfNR 2 R 3 and NR 4 R 5 is -""" N CI; or
• Trigger-OH wherein Trigger is defined as in Formula (I), a trisubstituted phosphine, and
• the reaction includes a solvent such as THF, dioxane, a C 1 - C 6 alkyl acetate, chloroform, dichloromethane, acetonitrile and the like.
• each substituent in the trisubstituted phosphine is independently selected from a C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, heteroaryl, and C 1 -C 6 alkoxy substituent.
• Trigger T- is
• the present invention provides a method to synthesize a phosphoramidate alkylator prodrug comprising [0265] (i) reacting in a solvent selected from THF, dioxane, dichloromethane, chloroform, ethyl acetate, propyl acetate, butyl acetate, or acetonitrile a compoumd of formula:
• a trisubstituted phosphine selected from triphenylphosphine, tributylphosphine, tributylphosphite;
• the present invention provides a method of synthesizing a compound of formula:
• the trisubstituted phosphine is P(R 12 ) 3 wherein each R 12 is H, C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, or heteroaryl.
• the trisubstituted phosphine is a polymer supported trisubstituted phosphine.
• the trisubstituted phosphine is triphenylphosphine, tributylphosphine, tripropylphosphine, triethylphosphine, or trimethylphosphine.
• the trisubstituted phosphine is a polymer supported triphenyl phosphine.
• Polymer supported trisubstituted phosphines are commercially available, for example, from Varian Inc. of Palo Alto, California.
• the present invention provides a method of synthesizing the compounds wherein each R 11 is hydrogen.
• the present invention provides a method of synthesizing the compounds
• the present invention provides the method of making a compound wherein the Trigger selected from the group consisting of:
• the present invention provides a method to synthesize a phosphoramidate alkylator prodrug comprising the steps of:
• step (b) reacting the dichlorophosphoramidate intermediate in step (a) with a N-2- haloethyl-N-(R 13 )ammonium salt, wherein R 13 is hydrogen, C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, heteroaryl, and a base in a solvent to yield a monochlorophosphoramidate intermediate; and
• step (c) reacting the monochlorophosphoramidate intermediate obtained in step (b) with Trigger-OH and a base in a solvent to yield the phosphorarnidate alkylator prodrug.
• the dichlorophosphoramidate intermediate of step (a) is separated from the rest of the reaction mixture before subjecting it to the reaction in step (b).
• the separation is performed by first removing excess POCl 3 in vacuo and then distilling the dichlorophosphoramidate under reduced pressure.
• the phosphoramidate alkylator prodrug of step (c) is separated from the rest of the reaction mixture by flash column chromatography on silica gel.
• the base employed in step (b) is a tertiary amine. Suitable tertiary amines employed in step (b) include trialkyl amines, such as, triethyl amine or diisopropylethylamine.
• the solvent employed in step (b) is tetrahydrofuran (THF) or dioxane.
• the monochlorophosphoramidate intermediate of step (b) is separated from the rest of the reaction mixture by flash column chromatography on silica gel before subjecting it to the reaction in step (c).
• the base useful in step (c) is lithium, sodium, or potassium hexaalkyldisilazide; sodium or potassium hydride; or lithium diisopropylamide.
• the solvent employed in step (c) is dimethoxyethane, diglyme, diethylether, or THF.
• the present invention provides a method to synthesize a phosphoramidate alkylator prodrug comprising the steps of:
• step (b) reacting the dichlorophosphate intermediate in step (a) with a N-2-haloethyl-N- (R 13 )ammonium salt, wherein R 13 is hydrogen, C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 3 -C 8 cycloalkyl, heterocyclyl, aryl, heteroaryl, and a base in a solvent to yield the phosphoramidate alkylator prodrug.
• steps (a) and (b) are performed at temperatures below O 0 C.
• step (b) is performed at a temperature between 20-100 0 C higher than the temperature of step (a).
• the present invention provides a method for synthesizing heterocyclic phopsphoramidate ankylator prodrugs of the present invention as shown below:
• the present invention provides a method to synthesize a phosphoramidate alkylator prodrug comprising the steps of:
• step (c) oxidizing the intermediate in step (b) to yield the phosphoramidate alkylator prodrug.
• the base used in step (b) is triethylamine.
• the solvent used in step (c) is dimethoxyethane, diglyme, or a C 1 -C 6 alkyl acetate.
• Trigger-OH is step (c) is
• l-N-alkyl-2-aminoimidazole-5-carboxylates can be reduced to yield various 1- N-alkyl-2-amino-5-hydroxymethylimidazole derivatives employed in the present invention as bioreductive group Z 3 .
• the present invention provides a method of treating cancer in a patient in need of therapy thereof by administering to the patient a phosphoramidate alkylator prodrug of the present invention or one that is known.
• a phosphoramidate alkylator prodrug of the present invention or one that is known.
• Known phosphoramidate alkylators are provided by the references Borch et al, supra.
• the phosphoramidate alkylator prodrug employed in treating cancer according to the methods provided by the present invention has the formula selected from (I) - (XXVII).
• the phosphoramidate alkylator prodrug employed in treating cancer according to the methods provided by the present invention is selected from the compounds exemplified in the EXAMPLE section.
• Alkylating agents can kill cancer cells in the more rapidly dividing or higher oxygen containing cancer region as compared to the cancer cells in the slower growing hypoxic cancer region. The latter cells survive the treatment by alkylators and can produce cells resistant to such alkylators.
• the prodrugs of the present invention are effective in treating cancers resistant to other therapies. Slowly dividing cancer cells in the hypoxic cancer zone act as a source of resistant cancer cells and strains and are killed by the prodrugs of the present invention.
• the present invention provides a method of treating a cancer resistant to treatment by one or more alkylators by administering the compounds of the present invention alone or in combination with another anticancer agent.
• a phosphoramidate alkylator prodrug of the invention is administered in combination with a drug having substantially no nephrotoxicity.
• the phosphoramidate alkylators prodrug is administered with carboplatin.
• the present invention provides phosphoramidate alkylators prodrugs which are not cross-resistant with known alkylators.
• present invention provides phosphoramidate alkylators prodrugs which are not cross-resistant with the alkylators cyclophosphamide, ifosfamide, glufosfamide, mechlorethamine, melphalan, chlorambucil, dacarbazine, temozolomide, carmustine, streptozocin, bendamustin, busulfan, thiotepa, cisplatin, carboplatin, and oxaliplatin.
• the present invention provides a method of treating cancer by administering as a first line therapy the compounds of the present invention alone or in combination with other anti-cancer agents.
• the present invention provides a method of treating a metastatic cancer by administering as a first line therapy the compounds of the present invention alone or in combination with other anti-cancer agents.
• the present invention provides a method of treating cancer by administering as a second line therapy the compounds of the present invention alone or in combination with other anti-cancer agents.
• the present invention provides a method of treating cancer by administering as a third line therapy the compounds of the present invention alone or in combination with other anti-cancer agents.
• the present invention provides a method of treating cancer by administering after a prior treatment with surgery and/or radiation therapy the compounds of the present invention alone or in combination with other anti-cancer agents.
• the present invention provides a method of treating cancer, the cancer having relapsed after prior chemotherapy, pressurey, radiation or any combination of them, by administering the compounds of the present invention alone or in combination with other anti-cancer agents.
• an effective amount of phosphoramidate alkylator prodrugs is administered to the subject.
• the subject can be any human or non-human mammal.
• the preferred subject is a human subject.
• Other particular subjects include but are not limited to non-human primates, dogs, cats, farm animals and horses.
• the phosphoramidate alkylator prodrug is administered alone.
• the phosphoramidate alkylator prodrug is administered in combination with one or more additional anti-cancer agents.
• the phosphoramidate alkylator prodrug is administered in conjunction with a therapeutic cancer treatment, including but not limited to surgery and radiation.
• the phosphoramidate alkylator prodrug will typically be administered in a pharmaceutical composition.
• Various pharmaceutical compositions that can be used are described in the Formulations section infra.
• the phosphoramidate alkylator prodrug and their pharmaceutical compositions can be used to treat any type of cancer in a subject, particularly in a human subject.
• Cancers that can be treated include but are not limited to leukemia, breast cancer, skin cancer, bone cancer, liver cancer, brain cancer, cancer of the larynx, gallbladder, pancreas, rectum, parathyroid, thyroid, adrenal, neural tissue, head and neck, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papillary type, metastatic skin carcinoma, osteosarcoma, Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lung tumor, gallstones, islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mu
• the phosphoramidate alkylator prodrug can particularly be used in the treatment of cancers containing significant areas of hypoxic tissue.
• cancers include but are not limited to lung cancer, especially non-small cell lung cancer, breast cancer, colon cancer, head and neck cancer, ovarian cancer, pancreatic cancer, and prostate cancer.
• types of cancers that can be treated with the phosphoramidate alkylator prodrugs of the invention are provided in the following references, each of which is incorporated in its entirety herin by reference Tidmarsh et ah, PCT Pat. Appl. No. PCT/US2005/047314 filed on 22 December 2005, and PCT Pat. Appl. entitled "Glufosfamide combination therapy",
• Lung cancer affects more than 100,000 males and 50,000 females in the United States, most of who die within 1 year of diagnosis, making it the leading cause of cancer death.
• Current protocols for the treatment of lung cancer involve the integration of chemotherapy with or without radiotherapy or surgery.
• a phosphoramidate alkylator prodrug can be used as a single agent or in combination with existing combination therapies.
• a variety of combination chemotherapy regimens have been reported for small cell lung cancer, including the combinations consisting of cyclophosphamide, doxorubicin and vincristine (CAV); etoposide and cisplatin (VP-16); and cyclophosphamide, doxorubicin and VP-16 (CAVP- 16). Modest survival benefits from combination chemotherapy (etoposide plus cisplatin) treatment have been reported for non-small cell lung cancer.
• cytotoxic drugs have produced at least temporary regression of ovarian cancer.
• the most active drugs in the treatment of ovarian cancer have been alkylating agents, including cyclophosphamide, ifosfamide, melphalan, chlorambucil, thiotepa, cisplatin, and carboplatin.
• Current combination therapies for ovarian cancer include cisplatin or carboplatin in combination with cyclophosphamide at 3- to 4-week intervals for six to eight cycles.
• the compounds and methods described herein provide prodrug forms and methods for treating ovarian cancer in which a phosphoramidate alkylator prodrug as described herein is used as a single agent or in existing such combination therapy, either to replace an agent or in addition to the agent(s) currently used.
• Cancer of the prostate is the most common malignancy in men in the United States and is the second most common cause of cancer death in men above age 55, and this cancer has been reported to consist primarily of hypoxic tissue.
• Several chemotherapy protocols have been reported for use in late stage disease following relapse after hormonal treatment.
• Agents for the treatment of prostate cancer include the alkylators estramustine phosphate, prednimustine, and cisplatin.
• Combination chemotherapy is also used to treat prostate cancer, including treatment with estramustine phosphate plus prednimustine and cisplatin, and 5-fluorouracil, melphalan, and hydroxyurea.
• the present invention provides methods for treating prostate cancer in which a phosphoramidate alkylator prodrug of the present invention is used in such combinations, either to replace an agent or in addition to the agent(s) currently used.
• a phosphoramidate alkylator prodrug can be used in various known approaches to cancer therapy including but not limited to "antibody-directed enzyme prodrug therapy” (ADEPT), “virus-directed enzyme prodrug therapy (VDEPT), “gene-directed enzyme prodrug therapy” (GDEPT), and “bacteria-directed enzyme prodrug therapy” (BDEPT).
• ADPT antibody-directed enzyme prodrug therapy
• VDEPT virus-directed enzyme prodrug therapy
• GDEPT gene-directed enzyme prodrug therapy
• BDEPT bacteria-directed enzyme prodrug therapy
• the general uses of a phosphoramidate alkylator prodrug are not limited to the foregoing treatment methods.
• the present invention provides a method of treatment of non- cancer hyperproliferative diseases characterized by cellular hyperproliferation (e.g., an abnormally increased rate or amount of cellular proliferation).
• the hyperproliferative disease treated according to the present method is selected from the group consisting of allergic angiitis and granulomatosis (Churg-Strauss disease), asbestosis, asthma, atrophic gastritis, benign prostatic hyperplasia, bullous pemphigoid, coeliac disease, chronic bronchitis and chronic obstructive airway disease, chronic sinusitis, Crohn's disease, demyelinating neuropathies, dermatomyositis, eczema including atopic dermatitis, eustachean tube diseases, giant cell arteritis, graft rejection, hypersensitivity pneumonitis, hypersensitivity vasculitis (Henoch-Schonlein purpura), irritant dermatitis, inflammatory hemolytic anemia, inflammatory neutropenia, inflammatory bowel disease, Kawasaki's disease, multiple sclerosis, myocarditis, myositis, nasal polyps, nasolacrimal
• a compound of the present invention is administered to treat a hyperproliferative disease selected from the group consisting of psoriasis, multiple sclerosis, rheumatoid arthritis, restenosis, and benign prostatic hyperplasia.
• a hyperproliferative disease selected from the group consisting of psoriasis, multiple sclerosis, rheumatoid arthritis, restenosis, and benign prostatic hyperplasia.
• the hyperpriliferative disease treated is psoriasis, a disease characterized by the cellular hyperproliferation of keratinocytes which builds up on the skin to form elevated, scaly lesions.
• the hyperproliferative disease treated is multiple sclerosis, a disease characterized by progressive demyelination in the brain.
• the hyperproliferative diseases treated is rheumatoid arthritis, a multisystem chronic, relapsing, inflammatory disease that can lead to destruction and ankylosis of joints affected.
• the compounds of the present invention are administered to prevent a hyperproliferative disease resulting from cellular proliferation on a prosthesis implanted in a subject by coating the prosthesis with a composition containing a compound of the present invention.
• the hyperproliferative disease treated is benign prostatic hyperplasia, a disease in which prostate epithelial cells grow abnormally and thereby block urine flow.
• a phosphoramidate alkylator prodrug will typically be formulated as pharmaceutical formulations for administration to a subject. Described in this section are modes of administration, formulations, and dosages that can be used when treating cancers using a phosphoramidate alkylator prodrug described herein.
• Administration of a phosphoramidate alkylator prodrug for the treatment of cancer can be effected by any method that enables delivery of the prodrugs to the site of action, the hypoxic region of a tumor.
• Many cancer drugs are administered by intravenous injection, and a phosphoramidate alkylator prodrug can be formulated for such administration, including not only ready-for-injection formulations but also lyophilized or concentrated formulations that must be rehydrated or diluted, respectively, prior to injection.
• a phosphoramidate alkylator prodrug can be formulated for administration by oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion), topical, and rectal routes.
• a phosphoramidate alkylator prodrug may be activated by bacteria in the gut. If such activation is not desired, then the practitioner can employ a route of administration or a formulation that results in absorption of a phosphoramidate alkylator prodrug prior to its entry into the large intestine or colon.
• the actual route of administration and corresponding formulation of the phosphoramidate alkylator prodrug will depend on the type of cancer being treated, the phosphoramidate alkylator prodrug selected for administration, the severity of the cancer, and the age, weight, and condition of the patient, among other factors.
• the amount of a phosphoramidate alkylator prodrug administered and thus the amount of the phosphoramidate alkylator prodrug contained in the dose administered and the product comprising that dose, will be dependent on the subject being treated, the severity of the cancer, localization of the cancer, the rate of administration, the disposition of the prodrug (e.g., molecular weight, solubility and hypoxic and normoxic cytotoxicity), the cytotoxic agent released by a phosphoramidate alkylator prodrug, and the discretion of the prescribing physician.
• the prodrug e.g., molecular weight, solubility and hypoxic and normoxic cytotoxicity
• the present invention provides a method of cancer treatment in a patient wherein an effective dosage is typically in the range of about 0.001 to about O.lg per kg body weight, or about 0.1 to about 35 mg/kg/day in single or divided doses. For a 70 kg human, this would amount to about 0.05 to about 7 g/day, about 0.2 to about 2.5 g/day.
• dosage levels below the lower limit of the aforesaid range can be more than adequate, while in other cases still larger doses can be employed without causing any harmful side effect; larger doses can also be divided into several small doses for administration throughout the day by infusion for an hour or continuously using a peripherally inserted central catheters (PICC line) and portable intravenous bag and pump.
• PICC line peripherally inserted central catheters
• the effective dose of a compound of the present invention for treatement of cancer and other hyperproliferative diseases is in the range of about 0.1 to about 35 mg/kg/day; about 0.5 to about 20 mg/kg/day; about 0.5 to about 15 mg/kg/day; about 0.5 to about 10 mg/kg/day; about 0.5 to about 8 mg/kg/day; and about 1 to about 5 mg/kg/day in single or divided doses.
• the effective dose of a compound of the present invention for treatement of cancer and other hyperproliferative diseases is in the range of about 2 to about 8 mg/kg/day; about 2 to about 4 mg/kg/day; and about 2 mg/kg/day in single or divided doses.
• the effective dose of a compound of the present invention for treatement of cancer and other hyperproliferative diseases is in the range of about 0.25 to about 2.5 mg/kg/day; about 0.25 to about 1 mg/kg/day; and about 0.25 to about 0.5 mg/kg/day in single or divided doses.
• the dose is administered Lv. daily, either as a monotherapy (compound of the present invention alone) or in conjunction (combination) with standard of care therapies.
• the effective dose for treatement of cancer and other hyperproliferative diseases is in the range as described earlier administered once a week.
• a larger dose is administered intermittently (less frequently); a dose in the range of about 3 to about 20 mg/kg; about 6 to about 10 mg/kg; or 8 mg/kg is administered on once every three days for two weeks.
• a dose in the range of about 5 to about 30 mg/kg; about 10 to about 15 mg/kg; or 12.5 mg/kg of the phosphoramidate alkylator prodrug is administered once a week for four weeks.
• a dose in the range of about 0.5 to about 8 mg/kg/day is administered for 5 days over two weekly cycles.
• the maximum daily dose of a phosphoramidate alkylator prodrug is not greater than 500 mg/kg patient weight and, accordingly, a phosphoramidate alkylator prodrug is administered in a daily dose in the range of about 1 mg of a phosphoramidate alkylator prodrug/kg of patient weight to about 500 mg of a phosphoramidate alkylator prodrug/kg of patient weight. In one embodiment, a phosphoramidate alkylator prodrug is administered in a daily dose in the range of about 5 mg/kg to about 500 mg/kg of the body weight of the patient to be treated.
• the therapeutically effective dose is a daily dose of a phosphoramidate alkylator prodrug is about 10 mg/kg to about 250 mg/kg of the body weight of the patient to be treated, hi another embodiment, the therapeutically effective dose of a phosphoramidate alkylator prodrug is about 25 mg/kg to about 150 mg/kg of the body weight of the patient to be treated. In another embodiment, the therapeutically effective dose of a phosphoramidate alkylator prodrug is about 25 mg/kg to about 50 mg/kg of body weight of the patient to be treated. In another embodiment, the therapeutically effective dose of a phosphoramidate alkylator prodrug is about 1.25 mg/kg to about 12.5 mg/kg of body weight of the patient to be treated.
• HED human equivalent dose
• HED human equivalent dose
• a phosphoramidate alkylator prodrug is usually administered multiple times to the patient.
• a phosphoramidate alkylator prodrug is administered daily for a period of time.
• daily administration for at least 3 consecutive days will be employed.
• administration is for at least 5 consecutive days, at least 7 consecutive days, or at least 10 consecutive days.
• the entire daily dose can be administered once daily, or the daily dose can be administered in multiple smaller doses through the course of a day (including by infusion with a pump or intravenous administration).
• the dose can be divided into two smaller doses and administered twice daily, or divided into three smaller doses and administered thrice daily. It will be apparent to one of skill in the art of cancer treatment that, as used herein, "daily" administration is not limited to one administration per day but can include multiple administrations.
• Administration schedules other than consecutive daily administration can also be used. Administration once every other day (qod) is particularly convenient, and administration once every third day, or once a week can be appropiate in some instances, but in any event, a phosphoramidate alkylator prodrug is repeatedly administered over a period of time.
• a phosphoramidate alkylator prodrug is administered at least 2 days per week for at least two, three, four, five or at least six consecutive weeks, or, alternatively, for at least two, three, four, five or at least six weeks within a six-month period, or, alternatively, for at least two, three, four, five or at least six weeks within a twelve-month period.
• a phosphoramidate alkylator prodrug is administered at least 3 days per week for at least two, three, four, five or at least six consecutive weeks, or, alternatively, for at least two, three, four, five or at least six weeks within a six-month period, or, alternatively, for at least two, three, four, five or at least six weeks within a twelve-month period.
• a phosphoramidate alkylator prodrug is administered at least 10 days per month, optionally at least 20 days per month, for at least one month or at least two, three, four, five or at least six consecutive months, or, alternatively, at least one, two, three, four, five or at least six months in a 6-month period.
• the administration of the therapeutically effective dose is continued for multiple days, typically for at least three consecutive days, and often for at least five to ten consecutive days, or for a week, or for several weeks or more.
• a patient can be administered a phosphoramidate alkylator prodrug in accordance with the present methods for several days, a week, a month, two months, three months, six months, or a year or longer.
• a phosphoramidate alkylator prodrug can be administered in multiple "rounds" of administration.
• a phosphoramidate alkylator prodrug can be administered once daily for at least three to ten, or at least five to ten consecutive days, and such three to ten or five to ten day treatments can be repeated once, twice, or three or more times, sometimes with a no-treatment (with a phosphoramidate alkylator prodrug) period ranging from one to several weeks between each multiple-day treatment.
• a phosphoramidate alkylator prodrug is administered every other day for two to ten administrations, more often three to ten administrations, or five to ten administrations, and such two, three or five to ten administrations qod can be repeated once, twice, or three or more times with a no-treatment (with a phosphoramidate alkylator prodrug) period ranging from one to several weeks between each multiple-day treatment.
• a no-treatment with a phosphoramidate alkylator prodrug
• administering a therapeutically effective dose or regimen of a phosphoramidate alkylator prodrug refers to (i) administering a phosphoramidate alkylator prodrug in the ranges stated (e.g., 1 mg to 1 g of a phosphoramidate alkylator prodrug per kg of patient weight, typically 25 to 150 mg of a phosphoramidate alkylator prodrug per kg of patient weight) for a specified minimum number of days within a specified time period, wherein the administration of a phosphoramidate alkylator prodrug has a therapeutic effect on the cancer in the patient.
• ranges stated e.g., 1 mg to 1 g of a phosphoramidate alkylator prodrug per kg of patient weight, typically 25 to 150 mg of a phosphoramidate alkylator prodrug per kg of patient weight
• Illustrative therapeutically effective dose regimens for a phosphoramidate alkylator prodrug include those described herein, such as administration of a phosphoramidate alkylator prodrug for 3 consecutive days, 5 consecutive days, 7 consecutive days, 10 consecutive days, at least 3 days per week, at least 3 days per week for one month, at least 10 days per month, and at least 20 days per month.
• the dose and frequency of a phosphoramidate alkylator prodrug administration can be selected to achieve a maximal sustained area under the plasma concentration curve (AUC) over the course of treatment.
• AUC plasma concentration curve
• the theoretically optimal dosing regimen will result in a maximal exposure of the tumor cells to a phosphoramidate alkylator prodrug, as measured by AUC, while minimizing the maximal plasma concentration (C max ) for any single administration.
• C max maximal plasma concentration
• a higher C max will contribute to toxicity while the AUC will determine efficacy.
• treatment with a phosphoramidate alkylator prodrug can be suspended temporarily if toxicity is observed, or for the convenience of the patient, without departing from the scope of the invention, and then resumed.
• the pharmacokinetics of the phosphoramidate alkylator prodrug of the present invention employed for the treatment of cancer can determine the dose, the method of administration, and the kind of cancer that is treated with the phosphoramidate alkylator prodrug.
• the phosphoramidate alkylator prodrug of the present invention can have a in vivo half life of between 1 to 300 minutes, hi one embodiment, the compounds of the present invention can have a in vivo half life of between 3 to 10 minutes. In one embodiment, the compounds of the present invention can have a in vivo half life of between 10 to 30 minutes.
• a short half life of the phosphoramidate alkylator prodrug can require an infusion time in treatment that is longer than that required for a phosphoramidate alkylator prodrug having a longer half life.
• a short half life of the phosphoramidate alkylator prodrug can increase the maximum tolerated dose (MTD) for that prodrug.
• the present invention provides phosphoramidate alkylator prodrugs that remain up to 20% unchanged when incubated with mouse liver microsomal (update with human example and data if available) protein for 30 minutes.
• the present invention provides phosphoramidate alkylator prodrugs that remain 20-80% unchanged when incubated with mouse liver microsomal protein for 30 minutes.
• the present invention provides phosphoramidate alkylator prodrugs that remain greater than 80% unchanged when incubated with mouse liver microsomal protein for 30 minutes.
• examples of phosphoramidate alkylator prodrugs of the present invention which when incubated with mouse liver microsomal protein for 30 minutes remain greater than 80% unchanged include 1, 25, and 36. The higher the MLM stability of a prodrug of the invention, its therapeutically effective dose and undesirable patient side effects will be lower.
• the bioreductive group of the phosphoramidate alkylator prodrugs of the present invention upon reduction/activation in a hypoxic tumor zone form a phosphoramidate alkylator- T M conjugate.
• the phosphoramidate alkylator- TM conjugate can diffuse and reach other parts of the tumor or other tumors in the case of a metastatic disease.
• Various pharmacokinetic parameters such as volume of distribution under steady state (Vss), clearance (CL), area under curve (AUC), mouse liver microsomal stability (MLM stability), plasma stability, and C max of phosphoramidate alkylator prodrugs of the present invention were measured and listed in the EXAMPLES section (see also Hardman et ah, supra).
• the dose can be adjusted to reflect patient tolerance of the prior treatment.
• dosing can be temporarily stopped as severe symptoms are observed.
• the period of temporary halting of administration can be ended at the time when the first organ of toxicity no longer contains significant concentrations of a phosphoramidate alkylator prodrug or a phosphoramidate alkylator released therefrom (which can be measured or determined indirectly by cessation of symptoms). Therefore, an intermittent dosing period can be defined not only by specific days but individualized by drug holidays that are based on symptoms and normal organ clearance of a phosphoramidate alkylator prodrug or a phosphoramidate alkylators released therefrom.
• a formulation of a phosphoramidate alkylator prodrug can, for example, be in a form suitable for oral administration as a tablet, capsule, pill powder, sustained release formulation, solution, and suspension; for parenteral injection as a sterile solution, suspension or emulsion; for topical administration as an ointment or cream; and for rectal administration as a suppository.
• a formulation of a phosphoramidate alkylator prodrug can be in unit dosage forms suitable for single administration of precise dosages and will typically include a conventional pharmaceutical carrier or excipient.
• Suitable pharmaceutical carriers include inert diluents or fillers, water and various organic solvents.
• the pharmaceutical compositions can, if desired, contain additional ingredients such as flavorings, binders, excipients, and the like.
• excipients such as citric acid
• disintegrants such as starch, alginic acid, and certain complex silicates
• binding agents such as sucrose, gelatin and acacia.
• lubricating agents such as magnesium stearate, sodium lauryl sulfate, and talc can be used to prepare the tablet forms of formulations of a phosphoramidate alkylator prodrug described herein.
• Solid compositions of a similar type can be employed in soft and hard filled gelatin capsules.
• Preferred materials include lactose or milk sugar and high molecular weight polyethylene glycols.
• the prodrug therein can be combined with various sweetening or flavoring agents, coloring matters or dyes and, if desired, emulsifying agents or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin, or combinations thereof.
• Exemplary parenteral administration forms include solutions or suspensions of a phosphoramidate alkylator prodrug in sterile aqueous solutions, for example, aqueous polyethylene glycols, propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired.
• the methods of cancer treatment employing a phosphoramidate alkylator prodrug of the present invention are effective in killing the most difficult to kill cancer cells growing in the hypoxic region of a tumor.
• a phosphoramidate prodrug can diffuse from the hypoxic cells and kill the cancer cells in adjacent regions containing increasing populations of rapidly dividing cells.
• the hypoxic region acts as a drug-factory to produce within a tumor an alkylator for killing adjacent normoxic cancer cells leading to a higher concentration of the phosphoramidate alkylator within the tumor, relative to normal tissues.
• the use of the prodrug to generate the phosphoramidate alkylator within the tumor can reduce toxic side-effects arising due to normal cell toxicity.
• hypoxic region can become normoxic and start to divide.
• such cells can be killed by the phosphoramidate alkylators generated from a phosphoramidate alkylator prodrug of this invention or those known, or by other anticancer agents or cytoxins administered in combination with the phosphoramidate alkylator prodrug, as described in the following section.
• a phosphoramidate alkylator prodrug can be co-administered in combination with other anti-cancer agents ("anticancer agent").
• anticancer agent an anti-cancer agent
• coadministration can in some cases provide one or more of several advantages over known cancer therapies, such as, for example co-administration of a phosphoramidate alkylator prodrug and the anticancer agent has a synergistic effect on induction of cancer cell death.
• Co-administration provides a better therapeutic result than administration of the anticancer agent alone, e.g., greater alleviation or amelioration of one or more symptoms of the cancer, diminishment of extent of disease, delay or slowing of disease progression, amelioration, palliation or stabilization of the disease state, partial or complete remission, prolonged survival or other beneficial therapeutic results.
• a phosphoramidate alkylator prodrug increases the sensitivity of cancer cells to the anticancer agent, allowing lower doses of the anticancer agent to be adminstered to the patient or allowing an anticancer agent to be used for treatment of cells otherwise resistent to the anticancer agent or otherwise refractory to treatment.
• the known anti-cancer agents in general targets the rapidly dividing cells in the normoxic region
• the phosphoramidate alkylator prodrugs of the invention target the hypoxic cells in the regions of tumors that are not efficiently killed by the anticancer agent alone.
• a phosphoramidate alkylator prodrug is "co-administered" with another anticancer agent (also referred to herein as, "Agent") when a phosphoramidate alkylator prodrug and Agent are administered as part of the same course of therapy.
• Agent another anticancer agent
• a phosphoramidate alkylator prodrug is first administered prior to administration of the Agent, (i.e., the initiation of the other cancer therapy), and treatment with a phosphoramidate alkylator prodrug is continued throughout the course of administration of the Agent (i.e., the course of the other therapy).
• a phosphoramidate alkylator prodrug is administered after the initiation or completion of the other cancer therapy. In other embodiments, a phosphoramidate alkylator prodrug is first administered contemporaneously with the initiation of the other cancer therapy. See for example combination therapies as described in EXAMPLE section.
• a phosphoramidate alkylator prodrug is first administered prior to administration of the Agent, and treatment with a phosphoramidate alkylator prodrug is continued after the cessation of administration of the Agent.
• a phosphoramidate alkylator prodrug is first administered prior to administration of the Agent, and treatment with a phosphoramidate alkylator prodrug is continued during part of the period of administration of the Agent.
• a phosphoramidate alkylator prodrug administration can be initiated and completed prior to the administration of the second drug.
• the present invention provides a phosphoramidate alkylator prodrug administered in combination with a chemoprotective agent or a chemoprotectant. Chemoprotective agents protect healthy tissue from the toxic effects of anticancer drugs.
• the chemoprotective agent is a thiol or a disulfide, hi one embodiment, the chemoprotectant can reduce superoxide, hi another embodiment, the the chemoprotectant can react with the "Michael- receptor" generated from a phosphoramidate alkylator prodrug and prevent "Michael- receptor” from reacting with proteins and nucleic acid (see below).
• Anticancer drug therapy today typically involves multiple rounds, or "cycles," of administration of the anti-cancer agent(s).
• cycles of administration of the anti-cancer agent(s).
• each cycle of administration (as well as a complete set of cycles) can be viewed as administration of a second drug.
• a phosphoramidate alkylator prodrug can be administered in any or all of the multiple cycles of treatment with the other Agent; in general, a phosphoramidate alkylator prodrug is administered on a daily basis for at least two or more days during each cycle.
• a phosphoramidate alkylator prodrug is co-administered with the Agent according to a schedule repeated at each round.
• a phosphoramidate alkylator prodrug is administered in combination with an effective amount of one or more chemotherapeutic agents, an effective amount of radiotherapy, an appropriate surgery procedure, or any combination of such additional therapies.
• a phosphoramidate alkylator prodrug and additional therapy can be administered at the same time or can be administered separately.
• a phosphoramidate alkylator prodrug is administered with an additional chemotherapeutic agent
• the two agents can be administered simultaneously or can be administered sequentially with some time between administrations.
• One of skill in the art will understand methods of administering the agents simultaneously and sequentially and possible time periods between administration. See for example combination therapies as described in the EXAMPLE section.
• Agents can be administered as the same or different formulations and can be administered via the same or different routes.
• Chemotherapeutic agents that can be used in combination with the a phosphoramidate alkylator prodrug of the invention include, but are not limited to, busulfan, improsulfan, piposulfan, benzodepa, carboquone, 2-deoxy-D-glucose, lonidamine and analogs thereof (refrence apps), glufosfamide, gemcitibine, erlotinib, meturedepa, uredepa, altretamine, imatinib, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamine, chlorambucil, chlornaphazine, estramustine, ifosfamide, gefitinib, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmus
• a phosphoramidate alkylator prodrug described herein can be used in combination with an antiangeogenisis inhibitor including but not limited to Avastin and similar therapeutics, hi one version of the combination treatment methods, a subject is treated with an antiangeogenisis inhibitor and subsequently treated with a phosphoramidate alkylator prodrug. In one version of the combination treatment methods, a subject is treated with an antiangeogenisis inhibitor and subsequently treated with a phosphoramidate alkylator prodrug with another chemotherapeutic agent, including but not limited to Cisplatin, and carboplatin. hi one version of these combination methods of treatment using an antiangeogenisis inhibitor, the method is used to treat breast cancer.
• a phosphoramidate alkylator prodrug is administered with an anti-cancer agent that acts, either directly or indirectly, to inhibit the epidermal growth factor or EGFR receptor.
• EGFR inhibitors suitable for coadministration with a phosphoramidate alkylator prodrug of the invention include gefitinib and erlotonib.
• a phosphoramidate alkylator prodrug is administered with an anti-cancer agent that acts, either directly or indirectly, to inhibit hypoxia-inducible factor 1 alpha (HIFIa) or to inhibit a protein or enzyme, such as a glucose transporter or VEGF, whose expression or activity is increased upon increased HIFIa levels.
• an anti-cancer agent that acts, either directly or indirectly, to inhibit hypoxia-inducible factor 1 alpha (HIFIa) or to inhibit a protein or enzyme, such as a glucose transporter or VEGF, whose expression or activity is increased upon increased HIFIa levels.
• HIFIa inhibitors suitable for use in this version of the methods and compositions described herein include Pl 3 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
• a phosphoramidate alkylator prodrug is administered with an anti-angiogenic agent, including but not limited to anti-angiogenic agents selected from the group consisting of angiostatin, an agent that inhibits or otherwise antagonizes the action of VEGF, batimastat, captopril, cartilage derived inhibitor, genistein, endostatin, interleukin, lavendustin A, medroxyprogesterone acetate, recombinant human platelet factor 4, Taxol, tecogalan, thalidomide, thrombospondin, TNP -470, and Avastin.
• angiostatin an agent that inhibits or otherwise antagonizes the action of VEGF
• batimastat captopril
• cartilage derived inhibitor genistein
• endostatin interleukin
• lavendustin A lavendustin A
• medroxyprogesterone acetate recombinant human platelet factor 4
• angiogenesis inhibitors for purposes of the combination therapies provided by the present methods and compositions described herein include Cox-2 inhibitors like celecoxib (Celebrex), diclofenac (Voltaren), etodolac (Lodine), fenoprofen (Nalfon), indomethacin (Indocin), ketoprofen (Orudis, Oruvail), ketoralac (Toradol), oxaprozin (Daypro), nabumetone (Relafen), sulindac (Clinoril), tolmetin (Tolectin), rofecoxib (Vioxx), ibuprofen (Advil), naproxen (Aleve, Naprosyn), aspirin, and acetaminophen (Tylenol).
• Cox-2 inhibitors like celecoxib (Celebrex), diclofenac (Voltaren), etodolac (Lodine), fenoprofen
• pyruvic acid plays an important role in angiogenesis
• pyruvate mimics and glycolytic inhibitors like halopyruvates, including bromopyruvate
• a phosphoramidate alkylator prodrug is administered with an anti-angiogenic agent and another anti-cancer agent, including but not limited to a cytotoxic agent selected from the group consisting of alkylators, Cisplatin, Carboplatin, and inhibitors of microtubule assembly, to treat cancer.
• the present invention provides a method of cancer treatment, wherein there is synergy between a phosphoramidate alkylator prodrug and another anticancer agent.
• Two drugs can be said to possess therapeutic synergy if a combination dose regimen of the two drugs produces a significantly better tumor cell kill than the sum of the single Agents at optimal or maximum tolerated doses.
• the "degree of synergy” can be defined as net log of tumor cell kill by the optimum combination regimen minus net log of tumor cell kill by the optimal dose of the most active single Agent. Differences in cell kill of greater than ten-fold (one log) are considered conclusively indicative of therapeutic synergy.
• a phosphoramidate alkylator prodrug When a phosphoramidate alkylator prodrug is used with another anti-cancer agent, a phosphoramidate alkylator prodrug will, at least in some embodiments, be administered prior to the initiation of therapy with the other drug or drugs and administration will typically be continued throughout the course of treatment with the other drug or drugs. In some embodiments, the drug co-administered with a phosphoramidate alkylator prodrug will be delivered at a lower dose, and optionally for longer periods, than would be the case in the absence of a phosphoramidate alkylator prodrug administration.
• Such “low dose” therapies can involve, for example, administering an anti-cancer drug, including but not limited to paclitaxel, docetaxel, doxorubicin, cisplatin, or carboplatin, at a lower than approved dose and for a longer period of time together with a phosphoramidate alkylator prodrug administered in accordance with the methods described herein.
• an anti-cancer drug including but not limited to paclitaxel, docetaxel, doxorubicin, cisplatin, or carboplatin
• the additional anti-cancer agent(s) is dosed using either the standard dosages employed for those Agents (i.e., when used without a phosphoramidate alkylator prodrug) or are less than those standard dosages.
• a phosphoramidate alkylator prodrug in accordance with the methods described herein can therefore allow the physician to treat cancer with existing (or later approved) drugs at lower doses (than currently used), thus ameliorating some or all of the toxic side effects of such drugs.
• the exact dosage for a given patient varies from patient to patient, depending on a number of factors including the drug combination employed, the particular disease being treated, and the condition and prior history of the patient, but can be determined using only the skill of the ordinarily skilled artisan in view of the teachings herein.
• chemotherapeutic agents or antineoplastic agents are known to physicians and are given, for example, in the product descriptions found in the Physician's Desk Reference 2003, (Physicians' Desk Reference, 57th Ed) Medical Economics Company, Inc., Oradell, NJ and/or are available from the Federal Drug Administration.
• Illustrative dosage regimens for certain anti-cancer drugs are also provided below.
• Cancer drugs can be classified generally as alkylators, anthracyclines, antibiotics, aromatase inhibitors, bisphosphonates, cyclo-oxygenase inhibitors, estrogen receptor modulators, folate antagonists, inorganic aresenates, microtubule inhibitors, modifiers, nitrosoureas, nucleoside analogs, osteoclast inhibitors, platinum containing compounds, retinoids, topoisomerase 1 inhibitors, topoisomerase 2 inhibitors, and tyrosine kinase inhibitors.
• a phosphoramidate alkylator prodrug can be co-administered with any anti-cancer drug from any of these classes or can be administered prior to or after treatment with any such drug or combination of such drugs.
• a phosphoramidate alkylator prodrug can be administered in combination with a biologic therapy (e.g., treatment with interferons, interleukins, colony stimulating factors and monoclonal antibodies).
• a biologic therapy e.g., treatment with interferons, interleukins, colony stimulating factors and monoclonal antibodies.
• Biologies used for treatment of cancer are known in the art and include, for example, trastuzumab (Herceptin), tositumomab and 131 I Tositumomab (Bexxar), rituximab (Rituxan).
• Alkylators useful in the practice of the methods described herein include but are not limited to busulfan (Myleran, Busulfex), chlorambucil (Leukeran), ifosfamide (with or without MESNA), cyclophosphamide (Cytoxan, Neosar), glufosfamide, melphalan, L-PAM (Alkeran), dacarbazine (DTIC-Dome), and temozolamide (Temodar).
• busulfan Myleran, Busulfex
• chlorambucil Leukeran
• ifosfamide with or without MESNA
• cyclophosphamide Cytoxan, Neosar
• glufosfamide glufosfamide
• melphalan L-PAM (Alkeran)
• DTIC-Dome dacarbazine
• Temodar temozolamide
• the cancer is chronic myelogenous leukemia, multiple myeloma, or anaplastic astrocytoma.
• the present invention provides a method of treating cancer treatable by administering an alkylator by administering the phosphoramidate alkylator prodrugs of the present invention or those alone or in combination with at least another alkylator or a prodrug thereof.
• Alkylators such as, for example, cyclophosphamide, ifosfamide, glufosfamide, mechlorethamine, melphalan, chlorambucil, dacarbazine, temozolomide, carmustirie, streptozocin, bendamustin, busulfan, thiotepa, cisplatin, carboplatin, and oxaliplatin, and types of cancers treated using any one of such alkylators alone or in combination with other anti cancer or chemoprotective agents are described for example in the reference Hardman et ah, (supra).
• the present invention provides a method of treating cancer by coadministering a phosphoramidate alkylator prodrug with at least the alkylator Cyclophosphamide, in the treatment of Stages III and IV malignant lymphomas, multiple myeloma, leukemia, mycosis fungoides, neuroblastoma, ovarian adenocarcinoma, retinoblastoma, and carcinoma of the breast.
• Cyclophosphamide is administered for induction therapy in doses of 1500-1800 mg/m 2 that are administered intravenously in divided doses over a period of three to five days; for maintenance therapy, 350-550 mg/m 2 are administered every 7-10 days, or 110-185 mg/m 2 are administered intravenously twice weekly.
• a phosphoramidate alkylator prodrug is co-administered with cyclosphosphamide administered at such doses or at lower doses and/or for a longer duration than normal for administration of Cyclosphosphamide alone.
• the present invention provides a method of treating cancer by administering a phosphoramidate alkylator produg of the invention together with a cancer treatment regimen using at least the alkylator Mechlorethamine.
• Mechlorethamine is used in the combination chemotherapy regimen MOPP
• the present invention provides a method of treating cancer by administering a phosphoramidate alkylator produg of the invention with a cancer treatment regimen using at least the alkylator Ifosfamide.
• Ifosfamide is used to treat pediatric and adult sarcomas, carcinomas of cervix and lung, and in combination with other drugs for germ cell testicular cancer.
• Ifosfamide is used as part of the ICE (Ifosfamide, Carboplatin, and Etoposide) ans RICE (Rituxan and ICE) regimens for treating lymphomas (see Hardman et ah, supra).
• the present invention provides a method of treating cancer by administering a phosphoramidate alkylator produg of the invention with a cancer treatment regimen using at least the alkylator Glufosfamide.
• Glufosfamide is in the clinic for the treatment of pancreatic cancer or Gemzar resistant pancreatic cancer.
• Glufosfamide can be used for treating breast cancer, Morbus Hodgkin, gastrointestinal tract cancer, or as part of the GCE (Glufosfamide, Carboplatin, and Etoposide) or RGCE (Rituxan and GCE) regimen, for treating lymphomas.
• GCE Glufosfamide, Carboplatin, and Etoposide
• RGCE Renuxan and GCE
• the present invention provides a method of treating cancer by administering a phosphoramidate alkylator produg of the invention with a cancer treatment regimen using at least an alkylator selected from the group consisting of ethylenimines and methylmelamines.
• the ethylenimine is Triethylenemelamine or Thiotepa.
• Thiotepa can be used to treat adenocarcinomas of the breast, ovary, and bladder, malignant lymphomas, bronchiogenic carcinomas, and Wilms' tumor.
• Thiotepa was used at high doses in combination chemotherapy with cyclophosphamide in patients with refractory malignancies treated with autologous bone transplantation and to treat a variety of cancers including bladder, ovarian, breast, lung, brain, and lymphomas (see, International Agency for Research on Cancer Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans, 1975, 9 : 286, Lyon, France; International Agency for Research on Cancer
• methylmelamine Altretamine is used to treat advanced ovarian cancer after failure of first round therapies.
• the present invention provides a method of treating cancer by administering a phosphoramidate alkylator produg of the invention with a cancer treatment regimen using at least the alkylator Melphalan, Chlorambucil, or Bendamustine.
• Melphalan is used to treat multiple myolema and can be administered orally.
• Chlorambucil is used to treat chronic lyphocytic leukemia and primary macroblobulinemia.
• Bendamustine, developed by Salmedix Inc. can be used to treat hematological malignancies, such as, for example, non-Hodgkin's lymphoma, chronis lymphocytic leukemia, and multiple myeloma.
• the present invention provides a method of treating cancer by administering a phosphoramidate alkylator produg of the invention with a cancer treatment regimen using at least the alkylator Busulfan.
• Busulfan is used to treat chronic granulocytic leukemia and chronic myelogenous leukemia.
• High doses of busulfan can be used in combination with Cyclophosphamide to treat patients with acute myelogenous leukemia before bone marrow transplantation.
• the present invention provides a method of treating cancer by administering a phosphoramidate alkylator produg of the invention with a cancer treatment regimen using at least a nitrosourea alkylator.
• the nitrosourea alkylator is Carmustine. Carmustine can be used to treat Hodgkin's disease, lymphomas, myelomas, malignant astrocytomas, metastatic tumors of the brain, melanoma, and gastrointestinal tumors.
• the nitrosourea is Streptozocin which is used to treat pancreatic islet cell carcinoma.
• the present invention provides a method of treating cancer by administering a phosphoramidate alkylator produg of the invention with a cancer treatment regimen using at least a triazene alkylator.
• the triazene alkylator is
• dacarbazine is used to treat malignant melanoma, Hodgkin's disease, and adult sarcoma.
• the triazene alkylator is Temozolomide. Temozolomide can be used to treat malignant gliomas.
• the present invention provides a method of treating cancer by administering a phosphoramidate alkylator produg of the invention with a cancer treatment regimen using at least a platinum coordination complex alkylator.
• the platinum coordination complex alkylator is Cisplatin.
• Cisplatin can be used to treat cancer of bladder, head and neck, endometrium, small cell carcinoma of the lung, and some neoplasms of childhood.
• Cisplatin alone or with cyclophosphamide is used to treat advanced ovarian cancer.
• Cisplatin with Bleomycin, Etoposide, and Vinblastine is used to treat advanced testicular cancer; and with one of Paclitaxel, Cyclophosphamide, or Doxorubicin to treat ovarian carcinoma.
• Anthracyclines useful in the practice of the methods described herein include but are not limited to, doxorubicin (Adriamycin, Doxil, Rubex), mitoxantrone (Novantrone), idarubicin (Idamycin), valrubicin (Valstar), and epirubicin (Ellence).
• a phosphoramidate alkylator prodrug is co-administered with an anthracycline to treat cancer.
• the cancer is acute nonlymphocytic leukemia, Kaposi's sarcoma, prostate cancer, bladder cancer, metastatic carcinoma of the ovary, and breast cancer.
• Doxorubicin has been used successfully to produce regression in disseminated neoplastic conditions such as acute lymphoblastic leukemia, acute myeloblastic leukemia, Wilm's tumor, neuroblastoma, soft tissue and bone sarcomas, breast carcinoma, ovarian carcinoma, transitional cell bladder carcinoma, thyroid carcinoma, lymphomas of both Hodgkin and non-Hodgkin types, bronchogenic carcinoma, and gastric carcinoma.
• Doxorubicin is typically administered in a dose in the range of 30-75 mg/m 2 as a single intravenous injection administered at 21-day intervals; weekly intravenous injection at doses of 20 mg/m 2 ; or 30 mg/m 2 doses on each of three successive days repeated every four weeks.
• a phosphoramidate alkylator prodrug is co-administered starting prior to and continuing after the administration of doxorubicin at such doses (or at lower doses).
• Cyclic Anthracycline cytotoxin prodrugs useful in the practice of the methods described herein are provided by the reference Matteuci et al, PCT Patent Aplication No. US05/008161.
• Antibiotics useful in the practice of the methods described herein include but are not limited to dactinomycin, actinomycin D (Cosmegen), bleomycin (Blenoxane), daunorubicin, and daunomycin (Cerubidine, DanuoXome).
• a phosphoramidate alkylator prodrug is co-administered with an antibiotic to treat cancer.
• the cancer is a cancer selected from the group consisting of acute lymphocytic leukemia, other leukemias, and Kaposi's sarcoma.
• Aromatase inhibitors useful in the practice of the methods described herein include but are not limited to anastrozole (Arimidex) and letroazole (Femara).
• a phosphoramidate alkylator prodrag is co-administered with an aromatase inhibitor to treat cancer.
• the cancer is breast cancer.
• Bisphosphonate inhibitors useful in the practice of the methods described herein include but are not limited to zoledronate (Zometa).
• a phosphoramidate alkylator prodrug is co-administered with a biphosphonate inhibitor to treat cancer.
• the cancer is a cancer selected from the group consisting of multiple myeloma, bone metastases from solid tumors, or prostate cancer.
• Cyclo-oxygenase inhibitors useful in the practice of the methods described herein include but are not limited to celecoxib (Celebrex).
• a phosphoramidate alkylator prodrug is co-administered with a cyclo- oxygenase inhibitor to treat cancer.
• the cancer is colon cancer or a precancerous condition known as familial adenomatous polyposis.
• Estrogen receptor modulators useful in the practice of the methods described herein include but are not limited to tamoxifen (Nolvadex) and fulvestrant (Faslodex).
• a phosphoramidate alkylator prodrug is coadministered with an estrogen receptor modulator to treat cancer.
• the cancer is breast cancer or the treatment is administered to prevent the occurrence or reoccurrence of breast cancer.
• Folate antagonists useful in the practice of the methods described herein include but are not limited to methotrexate and tremetrexate.
• a phosphoramidate alkylator prodrug is co- administered with a folate antagonist to treat cancer.
• the cancer is osteosarcoma.
• the compound N-[4-[[(2,4-diamino-6-pteridinyl)methyl memylamino]benzoyl]-L-glutamic acid is an antifolate drug that has been used in the treatment of gestational choriocarcinoma and in the treatment of patients with chorioadenoma destruens and hydatiform mole. It is also useful in the treatment of advanced stages of malignant lymphoma and in the treatment of advanced cases of mycosis fungoides. Methotrexate is administered as follows.
• intramuscular injections of doses of 15 to 30 mg are administered daily for a five-day course, such courses repeated as needed with rest period of one or more weeks interposed between courses of therapy.
• twice weekly intramuscular injections are administered in doses of 30 mg/m 2 .
• weekly intramuscular injections of doses of 50 mg or, alternatively, of 25 mg are administered twice weekly.
• a phosphoramidate alkylator prodrug is co-administered with methotrexate administered at such doses (or at lower doses).
• 5-Methyl-6-[[(3,4,5- trimethoxyphenyl)-amino]methyl]-2,4-quinazolinediamine (commonly known as trimetrexate) is another antifolate drug that can be co-administered with a phosphoramidate alkylator prodrug.
• Inorganic arsenates useful in the practice of the methods described herein include but are not limited to arsenic trioxide (Trisenox). Li accordance with the methods described herein a phosphoramidate alkylator prodrug is co-administered with an inorganic arsenate to treat cancer.
• the cancer is refractory acute promyelocytic leukemia (APL).
• Microtubule inhibitors (as used herein, a "microtubule inhibitor” is any agent that interferes with the assembly or disassembly of microtubules) useful in the practice of the methods described herein include but are not limited to vincristine (Oncovin), vinblastine (Velban), paclitaxel (Taxol, Paxene), vinorelbine (Navelbine), docetaxel (Taxotere), epothilone B or D or a derivative of either, and discodermolide or its derivatives.
• vincristine Oncovin
• vinblastine Velban
• paclitaxel Taxol, Paxene
• vinorelbine Vinorelbine
• docetaxel Teaxotere
• epothilone B or D or a derivative of either, and discodermolide or its derivatives.
• Tubulin binding anticancer drugs and prodrugs thereof which can be used in the practice of the methods of the present invention are provided in the reference Matteucci et al, PCT Patent Application No. PCT/US2005/042095; US Patent Applications entitled “Tubulin Binding Anti Cancer Agents and Prodrugs Thereof (Attorney Ref Nos. 021305-008500US, 021305- 008400US and 021305-004520US).
• a phosphoramidate alkylator prodrug is co-administered with a microtubule inhibitor to treat cancer.
• the cancer is ovarian cancer, breast cancer, non-small cell lung cancer, Kaposi's sarcoma, and metastatic cancer of breast or ovary origin.
• the compound 22-oxo-vincaleukoblastine also commonly known as vincristine, is an alkaloid obtained from the common periwinkle plant (Vinca rosea, Linn.) and is useful in the treatment of acute leukemia. It has also been shown to be useful in combination with other oncolytic agents in the treatment of Hodgkin's disease, lymphosarcoma, reticulum-cell sarcoma, rhabdomyosarcoma, neuroblastoma, and Wilm's tumor.
• Vincristine is administered in weekly intravenous doses of 2 mg/m 2 for children and 1.4 mg/m 2 for adults.
• a phosphoramidate alkylator prodrug is co-administered with vincristine administered at such doses.
• a phosphoramidate alkylator prodrug is not administered prior to treatment with a microtubule inhibitor, such as a taxane, but rather, administration of a phosphoramidate alkylator prodrug is administered simultaneously with or within a few days to a week after initiation of treatment with a microtubule inhibitor.
• Modifiers useful in the practice of the methods described herein include but are not limited to Leucovorin (Wellcovorin), which is used with other drugs such as 5-fluorouracil to treat colorectal cancer.
• a phosphoramidate alkylator prodrug is co-administered with a modifier and another anti-cancer agent to treat cancer.
• the cancer is colon cancer
• the modifier is a compound that increases the ability of a cell to take up glucose, including but not limited to the compound N-hydroxyurea.
• N-hydroxyurea has been reported to enhance the ability of a cell to take up 2-deoxyglucose (see the reference Smith et al., 1999, Cancer Letters 141: 85, incorporated herein by reference), and administration of N-hydroxyurea at levels reported to increase 2-deoxyglucose uptake or to treat leukemia together with administration of 2- deoxyglucose and a phosphoramidate alkylator prodrug as described herein is one version of the therapeutic methods provided herein.
• a phosphoramidate alkylator prodrug is co-administered with nitric oxide or a nitric oxide precursor, such as an organic nitrite or a spermineNONOate, to treat cancer, as the latter compounds stimulate the uptake of glucose.
• nitric oxide or a nitric oxide precursor such as an organic nitrite or a spermineNONOate
• Nitrosoureas useful in the practice of the methods described herein include but are not limited to procarbazine (Matulane), lomustine, CCNU (CeeBU), carmustine (BCNU, BiCNU, Gliadel Wafer), and estramustine (Emcyt).
• a phosphoramidate alkylator prodrug is co-administered with a nitrosourea to treat cancer.
• the cancer is prostate cancer or glioblastoma, including recurrent glioblastoma multiforme.
• Nucleoside analogs useful in the practice of the methods described herein include but are not limited to mercaptopurine, 6-MP (Purinethol), fluorouracil, 5-FU (Adrucil), thioguanine, 6-TG (Thioguanine), hydroxyurea (Hydrea), cytarabine (Cytosar-U, DepoCyt), floxuridine (FUDR), fludarabine (Fludara), azacytidine (Vidaza), pentostatin (Nipent), cladribine (Leustatin, 2-CdA), gemcitabine (Gemzar), and capecitabine (Xeloda).
• a phosphoramidate alkylator prodrug is coadministered with a nucleoside analog to treat cancer.
• the cancer is B-cell lymphocytic leukemia (CLL), hairy cell leukemia, adenocarcinoma of the pancreas, metastatic breast cancer, non-small cell lung cancer, or metastatic colorectal carcinoma.
• CLL B-cell lymphocytic leukemia
• hairy cell leukemia adenocarcinoma of the pancreas
• metastatic breast cancer metastatic breast cancer
• non-small cell lung cancer or metastatic colorectal carcinoma.
• the compound 5-fluoro-2,4(lH,3H)-pyrimidinedione also commonly known as 5-fluorouracil
• 5-fluorouracil is an antimetabolite nucleoside analog effective in the palliative management of carcinoma of the colon, rectum, breast, stomach, and pancreas in patients who are considered incurable by surgical or other means.
• 5-Fluorouracil is administered in initial therapy in doses of 12 mg/m 2 given intravenously once daily for 4 successive days with the daily dose not exceeding 800 mg. If no toxicity is observed at any time during the course of the therapy, 6 mg/kg are given intravenously on the 6th, 8th, 10th, and 12th days.
• No therapy is given on the 5th, 7th, 9th, or 1 lth days.
• a daily dose of 6 mg/kg is administered for three days, with the daily dose not exceeding 400 mg. If no toxicity is observed at any time during the treatment, 3 mg/kg can be given on the 5th, 7th, and 9th days.
• No therapy is given on the 4th, 6th, or 8th days.
• a sequence of injections on either schedule constitutes a course of therapy.
• a phosphoramidate alkylator prodrug is co- administered with 5-FU administered at such doses or with the prodrug form Xeloda with correspondingly adjusted doses.
• the compound 2-amino-l,7-dihydro- 6H-purine-6-thione is a nucleoside analog effective in the therapy of acute non-pymphocytic leukemias.
• 6-Thioguanine is orally administered in doses of about 2 mg/kg of body weight per day. The total daily dose can be given at one time. If after four weeks of dosage at this level there is no improvement, the dosage can be cautiously increased to 3 mg/kg/day.
• a phosphoramidate alkylator prodrug is co-administered with 6-TG administered at such doses (or at lower doses).
• Osteoclast inhibitors useful in the practice of the methods described herein include but are not limited to pamidronate (Aredia).
• a phosphoramidate alkylator prodrug is co-administered with an osteoclast inhibitor to treat cancer.
• the cancer is osteolytic bone metastases of breast cancer, and one or more additional anti-cancer agents are also co-administered with a phosphoramidate alkylator prodrug.
• Platinum compounds useful in the practice of the methods described herein include but are not limited to cisplatin (Platinol) and carboplatin (Paraplatin).
• a phosphoramidate alkylator prodrug is co-administered with a platinum compound to treat cancer.
• the cancer is metastatic testicular cancer, metastatic ovarian cancer, ovarian carcinoma, and transitional cell bladder cancer.
• the compound cis-Diaminedichloroplatinum (II), commonly known as cisplatin, is useful in the palliative treatment of metastatic testicular and ovarian tumors, and for the treatment of transitional cell bladder cancer which is not amenable to surgery or radiotherapy.
• Cisplatin when used for advanced bladder cancer, is administered in intravenous injections of doses of 50-70 mg/m 2 once every three to four weeks.
• a phosphoramidate alkylator prodrug is co-administered with cisplatin administered at these doses (or at lower doses).
• One or more additional anti-cancer agents can be co-administered with the platinum compound and a phosphoramidate alkylator prodrug.
• Platinol, Blenoxane, and Velbam can be co-administered with a phosphoramidate alkylator prodrug.
• Platinol and Adriamycin can be coadministered with a phosphoramidate alkylator prodrug.
• Retinoids useful in the practice of the methods described herein include but are not limited to tretinoin, ATRA (Vesanoid), alitretinoin (Panretin), and bexarotene (Targretin).
• a phosphoramidate alkylator prodrug is coadministered with a retinoid to treat cancer.
• the cancer is a cancer selected from the group consisting of APL, Kaposi's sarcoma, and T-cell lymphoma.
• Topoisomerase 1 inhibitors useful in the practice of the methods described herein include but are not limited to topotecan (Hycamtin) and irinotecan (Camptostar).
• a phosphoramidate alkylator prodrug is coadministered with a topoisomerase 1 inhibitor to treat cancer.
• Topoisomerase inhibitors and prodrugs thereof useful in the practice of the methods of the present invention are provided in the reference Matteucci et al., PCT Patent Application No. PCT/US2005/041959.
• the cancer is metastatic carcinoma of the ovary, colon, or rectum, or small cell lung cancer.
• administration of a phosphoramidate alkylator prodrug either precedes or follows, or both, administration of a topoisomerase 1 inhibitor but is not administered concurrently therewith.
• Topoisomerase 2 inhibitors useful in the practice of the methods described herein include but are not limited to etoposide, VP-16 (Vepesid), teniposide, VM-26 (Vumon), and etoposide phosphate (Etopophos).
• a phosphoramidate alkylator prodrug is co-administered with a topoisomerase 2 inhibitor to treat cancer.
• the cancer is a cancer selected from the group consisting of refractory testicular tumors, refractory acute lymphoblastic leukemia (ALL), and small cell lung cancer.
• ALL refractory acute lymphoblastic leukemia
• small cell lung cancer small cell lung cancer.
• Tyrosine kinase inhibitors useful in the practice of the methods described herein include but are not limited to imatinib (Gleevec).
• a phosphoramidate alkylator prodrug is co-administered with a tyrosine kinase inhibitor to treat cancer.
• the cancer is CML or a metastatic or unresectable malignant gastrointestinal stromal tumor.
• Lonidamine analogs useful in the practice of the present invention are provided in the Matteucci et al. U.S. Pat. Appl. Nos. 11/346632; 60/764,427; 60/764,438; and applications entitled "Heterocyclic Lonidamine Analogs" (Attorney Docket No. 021305-007220US; 021305-007900US) and PCT Publication Nos. WO 2006/015191, WO 2006/015263 and WO 2006/01007 A2. .
• phosphoramidate alkylator prodrug or a pharmaceutically acceptable salt thereof and one or more additional anti-cancer agents are administered to a patient.
• additional anti-cancer agents include without limitation 5-methyl-6-[[(3,4,5-trimethoxyphenyl)amino]-methyl]-2,4- quinazolinediamine or a pharmaceutically acceptable salt thereof, (8S,10S)-10-(3-amino- 2,3,6-trideoxy-alpha-L-lyxo-hexopyranosyl)oxy]-8-glycoloyl-7,8,9, 10-tetrahydro-6,8, 11- trihydroxy-l-methoxy-5,12-naphthacenedione or a pharmaceutically acceptable salt thereof; 5-fluoro-2,4(lH,3H) ⁇ pyrimidinedione or a pharmaceutically acceptable salt thereof; 2-amino- l,7-dihydro-6
• a particular eluent can depend, among other factors, on the polar nature of the compound, existence of other closely eluting compounds, type of stationary phase such as silica gel or alumina used, and the amount of pressure used to elute the solvent through the stationary phase. In practice, different compositions of solvents can be used to separate the same compound.
• N-methyl-2-nitroimidazole-5-methanol (76.8 mg, 0.489 mmol) was partially dissolved in dry THF (2 ml) under argon.
• the reaction mixture was cooled to -78°C and a solution of lithium bis(trimethylsilyl)amide in THF (1.6M, 0.306 ml, 0.489 mmol) was added. After 15 minutes, a solution of 7iii (172 mg, 0.538 mmol) in 2 ml THF was added. After 15 minutes the reaction mixture was slowly warmed to rt, stirred for 2 hours, poured into 25 ml water and extracted 3 times with ethyl acetate (30 ml).
• N-methyl-2-nitroimidazole-5-methanol 50 mg, 0.318 mmol was dissolved in dry THF (2 ml) under nitrogen. The solution was cooled to -78°C and a solution of lithium bis(trimethylsilyl) amide (IM in toluene, 0.35 ml, 0.35 mmol) was added by syringe. After 5 minutes a solution of bis(chloroethyl) phosphoramidic dichloride (91 mg, 0.35 mmol) in THF (2 ml) was added.
• the following compounds 37 - 105 were synthesized employing the Mitsunobu type coupling described for the synthesis of 25 or 36 above, and upon appropriate substitution of the Trigger-OH and the ifosfamide mustard analog employed.
• Trigger-OH compounds employed in the synthesis of Compounds 37 - 105 included the following Trigger-OH compounds: l-N-methyl-2-nitroimidazole-5- methanol, l-N-methyl-5-nitroimidazole-2-methanol, 5-nitrofuran-2 -methanol, 5- nitrothiophene-2-methanol;
• Examples 10 - 26 describes the synthesis of various Trigger-OH compounds employed in the synthesis of phosphoramidate alkylator prodrugs of the invention.
• reaction mixture was diluted with EA, washed with brine, the organic layer separated, dried, and concentrated to yield a residue which was separated by column chromatography on silica gel employing as eluent EA ⁇ Hex (0-70%) to yield compound 5Si.
• reaction mixture was stirred at -20 0 C for 0.5 h, and at rt for 2 h, poured into water, and extracted twice with DCM (30 mL). The combined organic layers were washed with brine, dried over Na 2 SO 4 , and concentrated under reduced pressure to yield a residue which was separated by column chromatography on silica gel employing as eluent Hexane/EA (100:70(v/v)) to yield 7.0 g of compound 64ii.
• reaction mixture was stirred at rt for 1.5 h, NaBH 4 (25 mg) added to it in portions, and after stirring for 15 min the reaction poured into water, extracted with EA, the EA layer dried and concentrated to yield a residue which was separated by silica gel column chromatography employing as eluent EA/Hexane (0-100%) to yield compound 2i (16 mg).
• the reaction mixture was stirred at 90-100 0 C for 1.5 h to yield a clear solution which was cooled, its pH adjusted to 1 using concentrated HCl and the resulting solution concentrated to 1/5 its original volume using a rotary evaporator at a temperature not more than 45 0 C.
• the concentrated reaction mixture was carefully neutralized by addition of K 2 CO 3 to a pH of 8-9 and extracted with EA (5 x 200 mL followed by 3 x 50 mL). The combined ethyl acetate layers were dried over MgSO 4 , filtered, and volatiles removed to yield 48 g of l-N-methyl-2-amino imidazole-5-carboxylic acid ethyl ester.
• Ethyl formate (850 niL) was added to sarcosine methyl ester HCl salt (205 g, 1.46 mol, grounded into powder prior to use), potassium carbonate (205 g, 1.48 mol), and EtOH (800 mL,), stirred overnight at rt, and filtered. The filtrate was concentrated in a rotary evaporator during which the residue separated into two layers. The upper layer was separated and the lower layer was extracted with EA. Combined EA layers and the upper layer was dried over MgSO 4 , filtered, and concentrated to yield 185 g (81%) of N-formyl sarcosine methyl ester which was used for the following reaction.
• reaction mixture was then extracted with EA (8 x 100 mL), the combined EA layers dried, filtered, and concentrated to yield a residue which was purified by recrystallization to yield 1 -N-methyl-2-amino imidazole-5-carboxylic acid ethyl ester ("amino ester").
• This method described in Example 24 and employing aqueous acetic acid is an improvement of the method using about 7% sulfuric acid (v/v) for the diazonium ion formation from the amino ester.
• aqueous sulfuric acid the reaction volume becomes large causing difficulty in stirring the reaction mixture effectively.
• a reaction involving 150 g of the amino ester required a reaction mixture volume of about 12 L.
• the sticky nitro ester formed as product in aqueous sulfuric acid and disrupted the stirring of the reaction mixture.
• reaction mixture was warmed up to O 0 C.
• the solid was filtered off and washed with THF.
• the combined THF portions were evaporated to yield l-N-methyl-2- nitroimidazole-5-methanol as an orange solid (25 g) which was recrystallized from ethyl acetate.
• NCI-H460 cells ATCC HTB-177, RPMI medium (Gibco Products, Invitrogen Corporation, Carlsbad, CA)
• HT29 cells ATCC HTB- 38, RPMI medium (Gibco)
• MES-SA cells ATCC CRL- 1976, McCoy's 5a medium (ATCC)
• MES-SA/Dx5 cells (ATCC CRL-1977), McCoy's 5a medium (ATCC)
• ACHN cells ATCC CRL-1611, Minimum essential medium, Eagle (ATCC)
• PC3 cells ATCC CRL-1435, Ham's F12K medium (ATCC)).
• the cells were seeded in glass inserts placed in each well of a 24- well plate in the density and medium as specified above one day prior to compound testing. After 24 hours, these plates were divided into two groups - anoxia group and air group.
• a test compound was added to each well (200 ⁇ L volume) in the treatment groups at concentrations varying from 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, to 0.01 ⁇ M. All test compounds were serially diluted in complete medium with final DMSO concentrations less than or equal to 1% in each well.
• the cells in the anoxia treatment group were incubated for 2 hours in a Bactron II anaerobic chamber.
• the cells in the air treatment group were incubated for 2 hours in standard tissue-culture incubators.
• test compound was removed from each well, cells were washed with 500 ⁇ L medium, and incubated for 3 days in 500 ⁇ L fresh medium. After 3 days, cells were stained with 10% Alamar Blue for 2 hours after which the capacity of cells to proliferate was measured (as mentioned above), and the 50% growth inhibitory concentration (GI 50 (also referred to IC 50 herein)) of test compounds was calculated and tabulated in Table X below.
• NCI-H460 cells ATCC HTB-177, RPMI medium (Gibco)
• HT29 ATCC HTB-38, RPMI medium (Gibco)
• the cells were incubated for 2 hours in a Bactron II anaerobic chamber flushed with gasses of the desired oxygen concentrations varying from anoxia, 0.1%, 0.3%, 0.6%, 1%, 10% oxygen, and air.
• the calculated IC 50 values ( ⁇ M) are tabulated in Table Yl (H460 cells) or Table Y2 (HT29 cells) below.
• Table Y2 IC 50 values ( ⁇ M) in HT29 cells
• a clonogenic survival assay was performed.
• Cells were plated in 60 mm glass dishes (5x10 5 cells per dish in 5mL of medium) 2 days prior to compound testing.
• the following cell lines were tested: NCI-H460 cells (ATCC HTB- 177, RPMI medium (Gibco)), HT29 cells (ATCC HTB-38, RPMI medium (Gibco)), PC3 cells (ATCC CRL-1435, Ham's F12K medium (ATCC)).
• a solution of the test compound was made in complete medium immediately before the test and added directly to cells (2mL volume).
• Anoxia or hypoxia was achieved by exposing the glass dishes in a Bactron II anaerobic chamber or in aluminum vessels (see Example 33) for 2 hours.
• desired levels of oxygenation between 200ppm and air were achieved by flushing the anaerobic chamber with pre-calibrated gasses prior to experimentation.
• aluminum vessels anoxia or hypoxia was achieved by exposing the glass dishes in pre- warmed, air tight aluminum jigs to a series of five rapid evacuations and flushings with 95% nitrogen plus 5% carbon dioxide in a 37 0 C water bath on a shaking platform (controls are flushed as well).
• the platform (with water bath and jigs) was shaken for 5 minutes, after which one more evacuation and flushing were performed, and the jigs were transferred to a shaker in a 37 degree C incubator for the remainder of the 1 to 2 hour drug exposure.
• Levels of oxygenation between 200 ppm and air were achieved by varying the degree and number of evacuations.
• the oxygen concentrations in the medium and gas phases were checked using an oxygen electrode (Anima, Phoenixville, PA) in a specially modified aluminum jig that allowed for monitoring of both gas and liquid phases.
• the glass dishes were removed from the chamber or aluminum vessels and the drug was washed off the cells by rinsing with medium.
• the cells were then trypsinized and plated for clonogenic survival in plastic Petri dishes. Ten to 14 days later, the dishes were stained with crystal violet (0.25% in 95% ethanol), and colonies containing more than 50 cells were counted (see Example 33). The 90% growth inhibitory concentration (IC 90 , 90% killing, 10% survival) of test compounds was calculated and tabulated in Table Y3 below.
• Electrochemistry To determine the electrochemical properties and reduction potentials of phosphoramidate alkylator prodrugs, cyclic voltammograms of these compounds were generated by Bioanalytical Systems, Inc. AU experiments were conducted with glassy carbon (3.0mm diameter) working electrodes, Ag/ AgCl reference electrodes, and platinum wire auxiliary electrodes. Compounds were dissolved in ImL methanol to make final drug concentrations between 0.5 and 1.5mM after the addition of 9mL Phosphate Buffered Saline (PBS). The solution was added to an electrochemical cell vial and sparged with Argon for 5 minutes to remove most of the oxygen.
• PBS Phosphate Buffered Saline
• Cyclic voltammetry was performed at 100 mV/sec and at 10,000 mV/sec scan rates at a glassy carbon working electrode.
• One test run was performed at a CGME mercury electrode (CGME in SMDE mode, 150 ⁇ m bore capillary, size 8 drop), but little difference was observed between mercury and glassy carbon voltammograms, so the mercury electrode was not used further.
• the single electron or multiple electron reduction potentials of compounds were generated at each scan rate and are tabulated in the table below.
• the phosphoramidate alkylator prodrugs of the invention were tested in the assay as follows. Exponentially growing human H460 cells (obtained from the ATCC) were seeded into 60mm notched glass plates at a density of between 2.5 and 5 xlO 5 cells per plate and grown in RPMI medium supplemented with 10 % fetal bovine serum for 2 days prior to initiating drug treatment. On the day of the test, drug stocks of known concentrations were prepared in complete medium, and 2 ml of the desired stock added to each plate. To achieve complete equilibration between the surrounding gas phase and the liquid phase, the lid of the glass plate was removed and the plate shaken for 5 minutes on an orbital shaker. The plates were recovered and stored inside a glove-box.
• the glove-box was evacuated and gassed with either a certified anoxic gas mixture (95% nitrogen and 5% carbon dioxide) or with an aerobic (normoxic) gas mixture (95% air and 5% carbon dioxide). Cells were then incubated with the drug for 2 hours at 37°C.
• HT 29 and cell based clonogenic assays were performed in the same way as described above and in Example 31.
• Cytotoxicity of compounds were determined in hypoxia and in normoxia by clonogenic assay employing H460 and HT29 cell lines as provided in Examples 31 and 33 and expressed as IC 90 in ⁇ M, and by anti-proliferation assay performed by modifying a multi-well assay described by Hay et ah, J. Med. Chem., 2003, 46:169-82 employing H460, HT29, HCTl 16, and DX-5 cell lines and expressed as IC 50 in ⁇ M (see
• Example 29 The ratio of IC 50 or IC 90 determined in normoxia and hypoxia is called hypoxia cytotoxicity ration (HCR) and can be a measure of the hypoxia selective cytotoxicity of the prodrugs of the present invention.
• HCR hypoxia cytotoxicity ration
• Cells (H60, PC3 and HT29) were seeded at a density of 1.0 x 10 6 cells/3ml medium per 60mm dish. After 24 h attachment, cells were exposed to Compound 25 at the indicated concentrations for 2 h under either normoxia (air) or anoxia (nitrogen). Cells were washed twice, and incubated for additional 22 h in fresh medium. Cells were trypsinized, centrifuged, and fixed in 75% ethanol at least for 24 h at -2O 0 C. Cell cycle distribution was determined using Guava Cell Cycle reagent (Guava, Hayward, CA) by flow cytometry (Guava, Hayward, CA). The data demonstrate that Compound 25 induces cell cycle arrest in oxygen- and concentration-dependent manner in multiple human cancer cell lines.
• HT29 colorectal adenocarcinoma colon carcinoma
• H460 lung carcinoma cells were seeded into a flask coated with a non-adherent surface to form small balls of cells that can be seeded into a spinner flask.
• H460 cell seeds 150 cm 2 tissue culture flasks were coated with 1% agarose and then 10,000 cells per flask were added and allowed to grow in RPMI medium supplemented with 10% FBS and antibiotics for 3 to 5 days before seeding into spinner cultures. For both cell lines, growth medium was changed every day after the spheroids became visible to the eye.
• the dishes were placed in sealed aluminum vessels and exposed to a series of evacuations and gassings with certified gases containing 5% CO 2 and a defined amount of O 2 (0% O 2 , 3% O 2 , 10% O 2 or air).
• Spheroids were incubated in a shaking water bath to ensure both the equilibrium of the dissolved O 2 in solution and the integrity of the spheroids in solution for 2 h.
• the test compound was removed and the spheroids were washed before being completely digested with trypsin. Since the necrotic core contains cellular debris a treatment with DNase I was required to yield a uniform single cell-suspension. Cells were resuspended at 10 /mL and plated for clonogenic survival.
• Daunorabicin was used to penetrate the outer layers of a multicellular spheroid due to its high affinity toward cells and cisplatin was used at doses appropriate kill only the outer aerobic cancer cells.
• Tirapazamine was used both in monolayer based experments and in spheroids as tabulated below for H460 cells exposed for 2 h.
• IC 90 for H460 cells exposed as monolayers or spheroids to phosporamidate prodrugs for 2 h.
• Phosphoramidate alkylator prodrugs demonstrate the ability to penetrate into the inner lying cells in the spheroid and kill hypoxic cancer cells alone and in combination with another agent that targets aerobic cancer cells.
• Example 36 Antiproliferation Assay - DNA Mutant Repair Cells
• Chinese hamster ovary cells mutant to specific DNA repair pathways were obtained from ATCC. The following cell lines were tested with 2,500 or 3,000 cells/well/500 ⁇ L Dulbecco's Modified Eagle Medium (Gibco) supplemented with 10% fetal bovine serum and antibiotics: AA8 cells (ATCC CRL-1859), EM9 cells (ATCC CRL-1861), UV41 cells (ATCC CRL-1860), UV135 cells (ATCC CRL-1867), IRSlSF cells.
• AU cell lines were initially screened with an anti-proliferation assay and those demonstrating sensitivity were retested with the clonogenic assay (as previously described) to confirm the proliferation results.
• MCC multilayered cell culture
• Compound 25 penetrates effectively through relatively well oxygenated tissue.
• Compound 25 is capable of killing hypoxic cells located about 150 ⁇ m from functional blood vessels. Approximately 3 -fold reduction in exposure to Compound 25 to the hypoxic side was observed under 0% O 2 relative to the exposure under 5% O 2 conditions. Bystander effect was observed only at the highest concentration.
• Compound 25 An in vitro assessment of metabolic stability of a phosphoramidate alkylator prodrug (Compound 25) was performed using human (HLM), rat (RLM) and mouse (MLM) liver microsomal proteins containing cytochrome P450 enzymes.
• HLM human
• RLM rat
• MLM mouse
• a solution of Compound 25 500 ⁇ L, 5 ⁇ M was prepared by diluting a DMSO stock solution 100 fold in a water:methanol bridge solution, adding microsomal protein (1 mg/mL) in PBS/MgCl 2 , and enzymatic reactions initiated by adding an NADPH solution.
• Cytochrome P450 Inhibition of the Metabolism of Compound 25 Eight reaction wells with 100 ⁇ L of a solution containing 50 mM potassium phosphate, pH 7.4, 2.6 mM NADP+, 6.6 mM glucose-6-phosphate, 0.8 U/mL of glucose-6- phosphate dehydrogenase, andl:3 serial dilutions of the test compound (such as Compound 25) were prepared along with eight wells of 1 :3 serial dilutions of a suitable positive control inhibitor (such as furafylline for CYPl A2, sulfaphenazole for CYP2C9, N-benzylnirvanol for CYPC219, quinidine for CYP2D6 and ketoconazole for CYP3A4).
• a suitable positive control inhibitor such as furafylline for CYPl A2, sulfaphenazole for CYP2C9, N-benzylnirvanol
• test compound ranges from 0.0229 ⁇ M to 200 ⁇ M.
• the reactions were initiated by adding 100 ⁇ L of a pre- warmed enzyme/substrate solution.
• a zero time-point control reaction was prepared by adding 50 mL of 10% formic acid (400 mL of acetonitrile for 2C19) in water to 100 mL of cofactor solution to inactivate the enzymes, then adding 100 mL of enzyme/substrate solution.
• a control reaction with no inhibitor was also prepared. After a suitable incubation at 37 0 C, the reactions were terminated by the addition of 50 mL of 10% formic acid in water (400 mL of acetonitrile for 2Cl 9).
• the reactions were prepared and analyzed for the metabolite forms of the probe substrate (phenacetin for CYPl A2, diclofenac for CYP2C9, (S)-mephenytoin for CYPC219, dextromethorphan for CYP2D6 and midazolam, testosterone and nifedipine for CYP3A4) using HPLC/MS/MS. Each assay was performed in duplicate. A summary of the IC50 values are listed below.

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