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• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/28—Phosphorus compounds with one or more P—C bonds
  • C07F9/30—Phosphinic acids [R2P(=O)(OH)]; Thiophosphinic acids ; [R2P(=X1)(X2H) (X1, X2 are each independently O, S or Se)]
  • C07F9/36—Amides thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
  • C07K5/0205—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)3-C(=0)-, e.g. statine or derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/06—Phosphorus compounds without P—C bonds
  • C07F9/22—Amides of acids of phosphorus
  • C07F9/24—Esteramides
  • C07F9/2404—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic
  • C07F9/2408—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic of hydroxyalkyl compounds
• • C—CHEMISTRY; METALLURGY
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  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/06—Phosphorus compounds without P—C bonds
  • C07F9/22—Amides of acids of phosphorus
  • C07F9/24—Esteramides
  • C07F9/2454—Esteramides the amide moiety containing a substituent or a structure which is considered as characteristic
  • C07F9/2458—Esteramides the amide moiety containing a substituent or a structure which is considered as characteristic of aliphatic amines
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/06—Phosphorus compounds without P—C bonds
  • C07F9/22—Amides of acids of phosphorus
  • C07F9/26—Amides of acids of phosphorus containing P-halide groups
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/28—Phosphorus compounds with one or more P—C bonds
  • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
  • C07F9/44—Amides thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
  • C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06008—Dipeptides with the first amino acid being neutral
  • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
  • C07K5/0606—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing heteroatoms not provided for by C07K5/06086 - C07K5/06139, e.g. Ser, Met, Cys, Thr
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to canfosfamide and its salts, and in particular to their preparation and intermediates in their preparation, pharmaceutical compositions containing some intermediates, and their use as anticancer agents.
• L is an electron withdrawing leaving group
• Y is selected from the group consisting of
• AA C is an amino acid linked through a peptide bond to the remainder of the compound, and their syntheses.
• the compounds are stated to be useful drugs for the selective treatment of target tissues that contain compatible GST isoenzymes, and simultaneously elevate the levels of GM progenitor cells in bone marrow.
• Disclosed embodiments for L include those that generate a drug that is cytotoxic to unwanted cells, including the phosphoramidate and phosphorodiamidate mustards.
• TLK286 is the compound of the formula
• TLK286 The proposed International Nonproprietary Name (pINN) of TLK286 itself is canfosfamide, and the United States Adopted Name (USAN) of TLK286 as the hydrochloride salt is canfosfamide hydrochloride.
• Canfosfamide is an anticancer compound that is activated by the actions of GST Pl-
• canfosfamide has been shown to be more potent in the M6709 human colon carcinoma cell line selected for resistance to doxorubicin and the MCF-7 human breast carcinoma cell line selected for resistance to cyclophosphamide, both of which overexpress GST Pl-I, than in their parental cell lines; and in murine xenografts of M7609 engineered to have high, medium, and low levels of GST Pl-I, the potency of canfosfamide was positively correlated with the level of GST Pl-I (Morgan et al, Cancer Res., 58:2568-2575 (1998)).
• Canfosfamide hydrochloride as a single agent, and in combination with other anticancer agents, is currently being evaluated in multiple clinical trials for the treatment of ovarian, breast, non-small cell lung, and colorectal cancers. It has demonstrated significant single agent antitumor activity and improvement in survival in patients with non-small cell lung cancer and ovarian cancer, and single agent antitumor activity in colorectal and breast cancer.
• Evidence from in vitro cell culture and tumor biopsies indicates that canfosfamide is non-cross-resistant to platinum, paclitaxel, and doxorubicin (Rosario et al., MoI. Pharmacol., 58:167-174 (2000)), and also to gemcitabine. Patients treated with canfosfamide hydrochloride show a very low incidence of clinically significant hematological toxicity.
• N,iV,iV',N'-tetrakis(2-haloethyl)phosphorodiamidates including 2-hydroxyethyl N,N,iV',N'-tetrakis(2-chloroethyl)phosphorodiatnidate and 2-(arylsulfonyloxy)ethyl N,N,N',N -tetrakis(2-chloroethyl)phosphorodiamidates, such as 2-(4-bromobenzenesulfonyloxy)ethyl J N,N,N',N'-tetrakis(2-chloroethyl)phosphorodiamidate. It would be desirable to develop an efficient and economical synthesis of canfosfamide and its salts. Disclosure of the Invention
• this invention is compounds of the formula
• R 1 is an amine-protectiiig group, and their salts.
• this invention is compounds of the formula
• R 1 is an amine-protecting group, and their salts.
• this invention is compounds of the formula
• R 1 is an amine-protecting group, and their salts.
• this invention is compounds of the formula
• this invention is methods of preparing canfosfamide or its salt, comprising deprotecting a compound of the first aspect of this invention, optionally followed by forming a salt of canfosfamide.
• this invention is methods of preparing compounds of the first aspect of this invention, comprising oxidizing a compound of the second aspect of this invention.
• this invention is methods of preparing compounds of the second aspect of this invention, comprising reacting a compound of the third aspect of this invention with a 2-(A-sulfonyloxy)ethyl N,N J N',N'-tetrakis(2-chloroethyl)phosphoro- diamidate under basic conditions.
• this invention is methods of preparing compounds of the third aspect of this invention, comprising deprotecting the sulfur atom of a compound of the fourth aspect of this invention.
• this invention is pharmaceutical compositions comprising a compound of the first aspect of this invention, compounds of the first aspect of this invention for the treatment of cancer and for use in the manufacture of medicaments for the treatment of cancer, and methods of treating cancer by administering a compound of the first aspect of this invention.
• Preferred embodiments of this invention are characterized by the specification and by the features of claims 2-4, 6-8, 10-12, 14-18, 20, 21, 23-27, 29-35, 37-42, and 44-46 of this application as filed.
• Suitable salts [see Berge et al., /. Pharm. Sd., 66:1 (1971) for a nonexclusive list] of canfosfamide and the compounds of this invention are those formed when inorganic bases (e.g. sodium, potassium, and calcium hydroxide) or organic bases (e.g. ethanolamine, diethanolamine, triethanolamine, ethylenediamine, tromethamine, N-methylglucamine) react with the carboxyl groups, and those formed when inorganic acids (e. g hydrochloric, hydrobromic, sulfuric, nitric, and chlorosulfonic acids) or organic acids (e.g.
• inorganic bases e.g. sodium, potassium, and calcium hydroxide
• organic bases e.g. ethanolamine, diethanolamine, triethanolamine, ethylenediamine, tromethamine, N-methylglucamine
• inorganic acids e. g hydrochloric, hydrobromic, sulfuric, nitric
• acetic, propionic, oxalic, malic, maleic, malonic, fumaric, or tartaric acids, and alkane- or arenesulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic, substituted benzenesulfonic such as chlorobenzenesulfonic and toluenesulfonic, naphthalenesulfonic and substituted naphthalenesulfonic, naphthalenedisulfonic and substituted naphthalenedisulfonic, and camphorsulfonic acids) react to form acid addition salts of the amine groups, of canfosfamide and the compounds. These salts are preferably formed with pharmaceutically acceptable acids and bases. Suitable salts for canfosfamide are acid addition salts, especially the hydrochloride salt.
• amine-protecting group is a group capable of protecting the glutamyl ⁇ -amine group of L- ⁇ -glutamyl-L-cysteinyl-2(R)-phenylglycine during the synthesis of the amine- protected canfosfamide, and subsequently removable without affecting the remainder of the canfosfamide molecule.
• a “catalytically removable amine-protecting group” is an amine- protecting group that is removable by catalytic reduction or isomerization. Common such groups are methane-forming groups containing a benzy ⁇ c or allylic carbon atom.
• catalytically removable amine-protecting groups suitable for use in this invention are (optionally substituted benzyl)oxycarbonyl groups, which are conveniently removable by catalytic hydrogenolysis, and (optionally substituted allyl)oxycarbonyl groups, which are conveniently removable by catalytic isomerization.
• a particularly suitable catalytically removable amine-protecting group is benzyloxycarbonyl.
• an "(optionally substituted benzyl) oxycarbonyl” includes benzyloxycarbonyl and benzyloxycarbonyl substituted on the benzene ring with one or two, typically one, electron- withdrawing substituents, such as halo (typically chloro or bromo), nitro, cyano, and trifluoromethyl.
• Examples of (optionally substituted benzyloxycarbonyl include benzyloxycarbonyl, halo-benzyloxycarbonyls such as 2- and 4-bromobenzyloxycarbonyl, 2-, 3-, and 4-chlorobenzyloxycarbonyl, and 2,4-dichlorobenzyloxycarbonyl, 2-, 3-, and 4-nitrobenzyloxycarbonyl, 4-cyanobenzyloxycarbonyl, and the like.
• an "(optionally substituted allyl) oxycarbonyl” includes allyloxycarbonyl and allyloxycarbonyl substituted with an electron-withdrawing substituent (such as phenyl optionally substituted with one or two, typically one, electron-withdrawing substituents such as halo (typically chloro or bromo), nitro, cyano, and trifluoromethyl, or pyridyl) on the 3-position of the allyl group, and 1-isopropylallyloxycarbonyl.
• an electron-withdrawing substituent such as phenyl optionally substituted with one or two, typically one, electron-withdrawing substituents such as halo (typically chloro or bromo), nitro, cyano, and trifluoromethyl, or pyridyl) on the 3-position of the allyl group, and 1-isopropylallyloxycarbonyl.
• Examples of (optionally substituted allyl) oxycarbonyl include allyloxycarbonyl, cinnamyloxycarbonyl, 4-nitrocinnamyloxycarbonyl, 3-(3'-pyridyl)allyloxycarbonyl, and 1-isopropylallyloxycarbonyl.
• a “sulfur-protecting group” is a group capable of protecting the sulfur atom of L- ⁇ -glutamyl-L-cysteinyl-2(R)-phenylglycine during the synthesis of compounds of the fourth aspect of this invention, and subsequently removable without affecting the remainder of the molecule of the corresponding compound of the third aspect of this invention (including the amine-protecting group).
• An “acidolytically removable sulfur-protecting group” is a sulfur- protecting group that is removable by acidolysis. Common such groups are (optionally substituted phenyl)-substituted methyl groups.
• acidolytically removable sulfur- protecting groups suitable for use in this invention are triphenylmethyl in which one or more of the benzene rings are optionally substituted with one or more, typically one, electron- donating substituents, such as methoxy, e.g. triphenylmethyl, (4-methoxyphenyl)diphenyl- methyl, and bis(4-methoxyphenyl)phenylmethyl; diphenylmethyl in which one or more of the benzene rings are optionally substituted with one or more, typically one, electron-donating substituents, such as methoxy, e.g.
• diphenylmethyl 4,4-methoxyphenyl)phenylmethyl and bis(4-methoxyphenyl)methyl; diphenylmethyl analogs such as 9H-xanthen-9-yl, 2-methoxy- 9H-xanthen-9-yl, 5H-dibenzo[ ⁇ 2, ⁇ (
• Amine-protecting groups including catalytically removable amine-protecting groups, and sulfur-protecting groups, including acidolytically removable sulfur-protecting groups, are well known in the field of organic synthesis and particularly peptide synthesis. Suitable such groups, and the conditions for their removal, are described in books such as Synthesis of Peptides and Peptidomimetics, Workbench edition, M. Goodman, ed., Georg Thieme Verlag, Stuttgart, Germany, 2004, and Protective groups in organic synthesis, 3 ed., T. W. Greene and P.G.M.
• the compounds of the fourth aspect of this invention contain both an amine-protecting group, especially a catalytically removable amine-protecting group, and a sulfur-protecting group, especially an acidolytically removable sulfur-protecting group, and the eighth aspect of this invention involves deprotecting the sulfur atom of these compounds while leaving the amine-protecting group in place
• amine-protecting group and sulfur-protecting group are not chosen independently and their definitions are to be interpreted in the context of their description in the application and the reaction sequence shown in Reaction Schemes 1 and 2; and that the amine-protecting group and the sulfur-protecting group are to be selected so that the sulfur- protecting group can be removed under conditions that leave the amine-protecting group intact.
• a "2-(A-sulfonyloxy)ethyl N,N,N' J N'-tetrakis(2-chloroethyl)phosphorodiamidate” is a compound of the formula where A is optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted aralkyl, or optionally substituted heteroaralkyl.
• A includes C 1-4 alkyl, optionally substituted with 1 to 3 halogen atoms and further optionally substituted with nitro or cyano; C 640 aryl such as phenyl and naphthyl, especially phenyl, optionally substituted with 1 to 3 groups selected from halogen, nitro, cyano, and C 1-2 alkyl optionally substituted with 1 to 3 halogen atoms (e.g.
• C 540 heteroaryl such as furyl, thienyl, pyrrolyl, pyridinyl, pyrazinyl, and pyrimidinyl, optionally substituted with 1 to 3 groups selected from halogen, nitro, cyano, and C 1-2 alkyl optionally substituted with 1 to 3 halogen atoms; and C 640 aryl-C ⁇ alkyl and C 540 heteroaryl-C j _ 2 alkyl, each optionally substituted on the aryl or heteroaryl group with 1 to 3 groups selected from halogen, nitro, cyano, and C 1-2 alkyl optionally substituted with 1 to 3 halogen atoms, and optionally substituted on the alkyl group with 1 to 3 halogen atoms.
• a groups include methyl, trifluoromethyl, phenyl, 4-tolyl, 4-nitrophenyl, and 4-halophenyl such as 4-chlorophenyl and 4-bro
• a “therapeutically effective amount” means the amount that, when administered to a mammal, especially a human, for treating a cancer, is sufficient to effect treatment for the cancer.
• Treating" or “treatment” of a cancer in a mammal includes one or more of: (1) inhibiting growth of the cancer, i.e., arresting its development,
• a compound of the first aspect of this invention is administered as part of a combination therapy with one or more other anticancer agents or radiotherapy, the "therapeutically effective amount” may be lower than the amount that would be therapeutically effective if the compound were administered alone.
• “Comprising” and its grammatical variations are terms of inclusion and not of limitation, and specify the presence of the stated features without precluding the presence or addition of other features.
• the compounds of the first aspect of this invention are compounds of the formula
• R 1 is an amine-protecting group, especially a catalytically removable amine-protecting group, and their salts (compounds 4).
• Particular compounds of the first aspect of this invention are compounds where R 1 is an (optionally substituted benzyl)oxycarbonyl or an (optionally substituted allyl)oxycarbonyl.
• Compounds of special interest are the compound where R 1 is benzyloxycarbonyl, i.e. N-a- (benzyloxycarbonyl) -L- ⁇ -glutamyl-3- [[2- [[bis [bis (2-chloroethyl)amino] phosphinyl] oxy] - ethyl] sulfonyl]-L-alanyl-2 (R) -phenylglycine, and its salts, especially the pharmaceutically acceptable salts.
• the compounds of the second aspect of this invention are compounds of the formula
• R 1 is an amine-protecting group, especially a catalytically removable amine-protecting group, and their salts (compounds 3).
• Particular compounds of the second aspect of this invention are compounds where R 1 is an (optionally substituted benzyl)oxycarbonyl or an (optionally substituted alryl)oxy- carbonyl.
• Compounds of special interest are the compound where R 1 is benzyloxycarbonyl, i.e.
• the compounds of the third aspect of this invention are compounds of the formula
• R 1 is an amine-protecting group, especially a catalytically removable amine-protecting group, and their salts (compounds 2).
• Particular compounds of the third aspect of this invention are compounds where R 1 is an (optionally substituted benzyl)oxycarbonyl or an (optionally substituted allyl)oxy- carbonyl.
• Compounds of special interest are the compound where R 1 is benzyloxycarbonyl, i.e. iV- ⁇ -(benzyloxycarbonyl)-L- ⁇ -glutamyl-L-cysteinyl-2(R.)-phenylglycine, and its salts.
• the compounds of the fourth aspect of this invention are compounds of the formula
• R 1 is an amine-protecting group, especially a catalytically removable amine-protecting group
• R 2 is a sulfur-protecting group, especially an acidolytically removable sulfur- protecting group, and their salts (compounds 1).
• Particular compounds of the fourth aspect of this invention are compounds where R 1 is an (optionally substituted benzyloxycarbonyl or an (optionally substituted allyl)oxy- carbonyl.
• Compounds of special interest are compounds where R 1 is benzyloxycarbonyl.
• R 2 is an (optionally substituted phenyl)-substituted methyl.
• R 2 is triphenylmethyl.
• R 1 is benzyloxycarbonyl and R 2 is triphenylmethyl, i.e. iV- ⁇ -(benzyloxycarbonyl)-L- ⁇ -glutarnyl-i'-ttiphenylrnethyl-L-cysteinyl-2(R)-phenylglycine, and its salts.
• the preparation of the compounds of this invention and canfosfamide and its salts by the process of this invention is illustrated in Reaction Scheme 1.
• the naming of a solvent in a process step is not intended to mean that the solvent is necessarily used alone: the solvent may be used with one or more co-solvents provided that the properties of any solvent mixture are determined primarily by the named solvent.
• Compound 1 may be deprotected by any method suitable for removal of the sulfur- protecting group that does not also remove the amine-protecting group.
• the sulfur- protecting group is an acidolytically removable sulfur-protecting group
• compound 1 is conveniently deprotected by acidolysis.
• compound 1 is dissolved in an acid that is strong enough to remove the acidolytically removable sulfur-protecting group but not strong enough to remove the catalytically removable amine-protecting group, optionally in the presence of a scavenger.
• Suitable such acids include trifluoroacetic acid and other strong acids such as ttifluoromethanesulfonic acid, optionally in the presence of a cosolvent such as dichloromethane;
• suitable scavengers include aromatic ethers and sulfides such as anisole and thioanisole, phenols such as cresol, and, most efficiently, silanes including trialkylsilanes such as triethylsilane and triisopropylsilane and silane polymers such as poly(methylhydrosiloxane); and a particularly suitable deprotection reagent is trifluoroacetic acid in the presence of poly(methylhydrosiloxane).
• Compound 2 can be isolated from the reaction mixture by addition of an anti-solvent, for example an aprotic non-polar solvent such as a hydrocarbon or an ether; and a particularly suitable anti-solvent is a mixture of heptane and methyl tett-butyl ether.
• an anti-solvent for example an aprotic non-polar solvent such as a hydrocarbon or an ether
• a particularly suitable anti-solvent is a mixture of heptane and methyl tett-butyl ether.
• the thiolate anion of compound 2 is alkylated with a 2-(A-sulfonyloxy)ethyl N ) N,JV',iV'-tetrakis(2-chloroethyl)phosphorodiamidate to prepare compound 3.
• compound 2 is dissolved in a solution of a strong base (e.g. an alkali metal or alkaline earth metal hydroxide, carbonate, phosphate, or alkoxide, or ammonium hydroxide; or an organic amine base such as tetramethylguanidine, DBU (l,8-diazabicyclo[5.4.0]undec-7-ene), and the like; especially an alkali metal hydroxide) in a suitable solvent (e.g.
• a strong base e.g. an alkali metal or alkaline earth metal hydroxide, carbonate, phosphate, or alkoxide, or ammonium hydroxide; or an organic amine base such as tetramethylguanidine, DBU (l,8-diazabicyclo[5.4.0]undec-7-ene), and the like; especially an alkali metal hydroxide
• a suitable solvent e.g.
• a C 1-6 alkanol a diol such as 1,2-ethanediol or 1,3-propanediol, an ether such as 2-methoxyethanol, 1,2-dimethoxyethane, or tetrahydrofuran, and the like; especially a C 1-3 alkanol such as methanol) to form the thiolate anion, the phosphorodiamidate is added (typically in excess, for example an excess of at least 1.5-fold, especially at least 2-fold, for example about 2.5-fold, over compound 2), and the reaction mixture is held at an appropriate temperature and time until completion. If the phosphorodiamidate is used in solution in a non-polar aprotic solvent (e.g.
• reaction mixture is neutralized and the aqueous and non-aqueous phases are separated to remove unreacted phosphorodiamidate starting material.
• the solvent e.g. the C 1-6 alkanol
• a non-polar aprotic solvent e.g. an ester such as isopropyl acetate
• Compound 3 can be isolated from the solution by removal of the solvent; but if the solvent is a suitable solvent for the third step of the process, then compound 3 can be carried into that third step in solution. In the third step, the sulfur atom of compound 3 is oxidized to prepare compound 4.
• Suitable solvent/ oxidant combinations include C 2 . 3 alkanoic acid/hydrogen peroxide/peroxyalkanoic acid combinations such as acetic acid/peroxyacetic acid and acetic acid/hydrogen peroxide/peroxyacetic acid, and the use of C 2 _ 3 peroxyalkanoic acids or peroxytrifluoroacetic acid as both solvent and oxidant.
• a reagent such as dimethyl sulfide if desired
• Particularly suitable solvent/ oxidant combinations include combinations where the oxidation takes place in a biphasic system, with compound 3 dissolved in a non-polar aprotic solvent such as an ester (e.g. isopropyl acetate) and the oxidant being in aqueous solution.
• Suitable oxidants for these biphasic oxidations include borates and peroxy compounds such as perborates and persulfates, e.g. ammonium, sodium, or potassium persulfate, and a particularly suitable oxidant is potassium monopersulfate, such as OXONETM (2KHSO 51 KMSO 4 -K 2 SO 4 ).
• the amine group of compound 4 is deprotected to prepare canfosfamide, optionally followed by forming an acid addition salt of canfosfamide.
• Compound 4 may be deprotected by any method suitable for removal of the amine- protecting group that does not also affect the remainder of the canfosfamide molecule.
• the ami ⁇ e-protecting group is a catalytically removable amine-protecting group
• compound 4 is conveniently deprotected by catalytic reduction or isomerization.
• a suitable solvent such as a C 1-4 alkanol, or a polar protic solvent, or in a non-polar aprotic solvent, especially in the presence of water, e.g.
• a reduction catalyst typically a palladium catalyst such as palladium black, palladium on barium sulfate, and palladium on carbon.
• a solvent that will become an anti-solvent for the salt, such as an aprotic solvent, e.g.
• a hydrocarbon or halogenated hydrocarbon such as dichloromethane
• the acid chosen to form the salt especially in the form of the anhydrous acid alone or in an aprotic solvent, e.g. hydrogen chloride gas.
• aprotic solvent e.g. hydrogen chloride gas.
• ethers such as diethyl ether, methyl tert-hutyl ether, and tetrahydrofuran, especially methyl tert-butyl ether, may be added if necessary or desired.
• a zerovalent palladium complex such as tetrakis(triphenylphosphine)palladium(0) is used, typically in the presence of a nucleophilic allyl group scavenger such as a secondary amine.
• the canfosfamide is isolated, typically as an acid addition salt, in a suitable manner.
• the compounds of the first, second, third, and fourth aspects of this invention (compounds of formulae 4, 3, 2, and 1, respectively) and the process steps of the fifth, sixth, seventh, and eighth aspects of this invention are useful in the preparation of the known anticancer agent canfosfamide and its salts.
• the compounds of the first aspect of this invention are also active cytotoxic agents. They are therefore useful in the treatment of cancers, especially cancers of types that are treatable with canfosfamide and its salts. These cancers include mammalian cancers, especially human cancers. Cancers that are particularly treatable are cancers with sensitivity to inducers of apoptosis, and more specifically those cancers that express or, particularly, overexpress GST Pl-I. Cancers that express or overexpress GST Pl-I when treated with other anticancer therapies (i.e. those not compounds of the first aspect of this invention) are also especially treatable with compounds of the first aspect of this invention when used in combination with the other anticancer therapy.
• cancers include cancers of the brain, breast, bladder, cervix, colon and rectum, esophagus, head and neck, kidney, lung, liver, ovary, pancreas, prostate, and stomach; leukemias such as ALL, AML, AMML, CLL, CML, CMML, and hairy cell leukemia; Hodgkin's and non- Hodgkin's lymphomas; mesotheliomas, multiple myeloma; and sarcomas of bone and soft tissue.
• Cancers particularly treatable with compounds of the first embodiment of this invention include breast, ovarian, colorectal, and non-small cell lung cancers.
• the ninth aspect of this invention is pharmaceutical compositions comprising a compound of the first aspect of this invention, the use of these compounds for the treatment of cancer and for the manufacture of medicaments for the treatment of cancer, and methods of treating cancer by administering these compounds, typically in a therapeutically effective amount.
• the compounds may be administered by any route suitable to the subject being treated and the nature of the subject's condition.
• Routes of administration include, but are not limited to, administration by injection, including intravenous, intraperitoneal, intramuscular, and subcutaneous injection, by transmucosal or transdermal delivery, through topical applications, nasal spray, suppository and the Like or may be administered orally.
• Pharmaceutical compositions containing these compounds may optionally be liposomal formulations, emulsions, formulations designed to administer the drug across mucosal membranes or transdermal formulations. Suitable formulations for each of these methods of administration may be found, for example, in Remington: The Science and Practice ofPhannacy, 20 ed., A.
• compositions will be either oral or solutions for intravenous infusion and will contain the compound and typically will also contain one or more pharmaceutically acceptable excipients.
• Typical dosage forms will be tablets, solutions for intravenous infusion, and lyophilized powders for reconstitution as solutions for intravenous infusion.
• a therapeutically effective amount of a compound of the first aspect of this invention is about 50 - 3000 mg/m 2 body surface area, especially 500 - 1500 mg/m 2 .
• Administration may be at 1 - 35 day intervals; for example, about 500 - 1000 mg/m 2 at 1 - 5 week intervals, especially at 1, 2, 3, or 4 week intervals, or at higher frequencies including as frequently as once/day for several (e.g. 5 or 7) days, with the administration repeated every 2, 3, or 4 weeks, or constant infusion for a period of 6 — 72 hours, also with the administration repeated every 2, 3, or 4 weeks; and such flexibility of administration will readily enable combination therapy with other anticancer therapies.
• Reaction Scheme 2 shows the preparation of preferred compounds of this invention and canfosfamide hydrochloride by the process of this invention.
• the preparation of N- ⁇ -(benzyloxycarbonyl)-L- ⁇ -glutamyl-i"-triphenylmethyl-L-cysteinyl-2(R)-phenylglyckie, compound IA, is not shown in Reaction Scheme 2, as it will depend on the nature of the protection of its precursor compound, but a process from O- ⁇ -benzyl- N- ⁇ -(benzyloxycarbonyl)-L- ⁇ -glutamyl-i ' -t ⁇ :iphenylmethyl-L-cysteinyl-2(R)-phenylglycine is described in Example 1 below.
• O- ⁇ -benzyl-JV- ⁇ - ⁇ enzyloxycarbony ⁇ -L- ⁇ -glutamyl-i'-triphenylmethyl-L-cysteinyl- 2(R)-phenylglycine may be prepared by standard methods of peptide synthesis.
• a convenient synthesis, using the readily available starting materials JV- ⁇ -(benzyloxycarbonyl)-L- ⁇ -glutamic acid ⁇ -benzyl ester, J'-triphenylmethyl-L-cysteine, and D-phenylglycine, is as follows.
• JV- ⁇ -(benzyloxycarbonyl)-L- ⁇ -glutamic acid ⁇ -benzyl ester is activated as the N-hydroxysuccinimide ester by reaction with IV-hydroxysuccinimide and dicyclohexyl- carbod ⁇ mide in anhydrous 1,4-dioxane.
• JV- ⁇ -(benzyloxycarbonyl)-L- ⁇ -glutamic acid ⁇ -benzyl ester ⁇ -JV-hydroxysuccinirnide ester is dissolved in anhydrous tetrahydtofuran, and added to a solution of ⁇ -triphenylmethyl-L-cysteine and triethylamine in water to give O- ⁇ -benzyl-JV- ⁇ - ⁇ enzyloxycarbony ⁇ -L- ⁇ -glutamyl-i ' -triphenylmethyl-L-cysteine.
• canfosfamide hydrochloride Example 1. Preparation of canfosfamide hydrochloride. N- ⁇ -(benzyloxycarbonyl)4- ⁇ -glutamyl-S-ttiphenykneiiiyl4-cysteinyl-2(R)-phenylglycine, IA.
• the reaction mixture was then cooled to room temperature and stirred for 90 minutes, then the methanol was removed under reduced pressure on a rotary evaporator (40 mbar, final bath temperature 35°C) to provide an aqueous solution of the disodium salt of N- ⁇ -(benzyloxycarbonyl)-L- ⁇ -glutamyl- J'-triphenylmethyl-L-cysteinyl-2(R)-phenylglycine.
• the mixture was charged into a 5-L, three- neck, round-bottom flask equipped with an overhead mechanical stirrer, a thermometer, and a 2-L pressure-equalizing addition funnel, and then diluted with water (1 L).
• Isopropyl acetate (3.2 L) was added, the pH adjusted to 4.8-4.85, the phases separated, and the isopropyl acetate phase (containing die iV- ⁇ -(benzyloxycarbonyl)-L- ⁇ -glutamyl-3- [[2- [[bis [bis (2-chloroethyl)amino] - phosphinyl]oxy]etliyl]d ⁇ io]-L-alanyl-2 (R) -phenylgly cine) retained.
• Isopropyl acetate (0.64 L) was added to the aqueous phase, the pH adjusted to 4.8-4.85, the phases separated, and the isopropyl acetate phase was combined with the previous pH 4.8-4.85 isopropyl acetate phase.
• Water (1.6 L) was added to the combined isopropyl acetate phases, the pH adjusted to 5.25-5.3, the phases separated, and the aqueous phase removed. The water wash was repeated and, after separation of the aqueous phase, the isopropyl acetate phase was distilled under vacuum to reduce the batch volume to about 3.15 L before further processing.
• JV- ⁇ -(benzyloxycarbonyl)-L- ⁇ -glutamyl-3-[[2- [[bis [bis (2-chloroethyl)amino]phosphinyl] oxy] - ethyl]tliio]-L-alanyl-2(R)-phenylglycine can be isolated from the solution by removal of the solvent.
• the aqueous phase was separated and removed, and the isopropyl acetate phase was washed three times with water (885 mL), testing the water for the presence of persulfates with starch/KI paper after each wash.
• Isopropyl acetate (2.65 L) was added to the isopropyl acetate phase, and the combined solution was then distilled to reduce the volume to approximately 1.77 L.
• Isopropyl acetate (1.77 L) was then added, and the solution tested by Karl Fischer titration to ensure that the water content was not more than 0.2%.
• the three-cycle nitrogen purge was repeated, followed by two cycles of hydrogen addition and release, then the autoclave was pressurized with hydrogen to 3.4 bar with hydrogen, heated to 30°C, and held at that temperature for 7.7 hours, monitoring the reaction for completion by HPLC.
• the reaction mixture was filtered through diatomaceous earth (CELITETM) to remove the catalyst, and the filter washed with isopropyl acetate (2 x 175 mL).
• the combined filtrate and washings were dehydrated by vacuum distillation and re-addition of isopropyl acetate, then re-concentrated to a final solution mass of 485.7 g.
• hematoma (breast carcinoma) were obtained from the National Cancer Institute, Bethesda, Maryland, U.S.A.
• the CellTiter-Glo assay kit was obtained from Promega Corporation, Madison, Wisconsin, U.S.A, and was used in accordance with the manufacturer's directions. All assays were conducted in triplicate wells, with dimethyl sulfoxide (DMSO) solvent control. The extent of cell growth was expressed as a percentage of the signal from the solvent control wells.
• DMSO dimethyl sulfoxide
• Log-phase cells were trypsinized, collected by centrifugation, and resuspended in a small volume of fresh medium, and the density of viable cells was determined after Trypan Blue staining.
• Cells were diluted in fresh media (5 X 10 3 cells/mL for HL-60 and 3 X 10 3 cells/mL for MX-I cells), Compound 4A (concentrations between 0.1 and 200 ⁇ M, dissolved in DMSO, 50 ⁇ L) added immediately after dilution to achieve a final DMSO concentration of 0.5%, then the suspensions added at 150 ⁇ L/well to 96-well plates, and incubated for several hours to allow attachment in the case of adherent cells. The cells were cultured for approximately three doubling times (3 days).
• the cells were then collected by centrifugation, and 100 ⁇ L of the culture supernatant was replaced by the CellTiter-Glo reagent. After incubation for 15 minutes at room temperature, the plate was read with a luminometer.
• the activity of compound 4A, expressed as IC 50 , against the cell lines was:HL-60, 2.2 ⁇ M, and MX-I: 34.6 ⁇ M. The compound is approximately as potent as canfosfamide in this assay.
• a solid formulation for oral administration is prepared by combining the following: Compound 4A 25.0% w/w
• Microcrystalline cellulose 71.5% w/w and the mixture is compressed to form tablets or filled into hard gelatin capsules containing, for example, 250 mg of compound 4A.
• Tablets may be coated, if desired, by applying a suspension of a film-forming agent (for example, hydroxypropylmethylcellulose), pigment (for example, titanium dioxide), and plasticizer (for example, diethyl phthalate), and drying the film by evaporation of the solvent.
• a film-forming agent for example, hydroxypropylmethylcellulose
• pigment for example, titanium dioxide
• plasticizer for example, diethyl phthalate
• a formulation for IV administration is prepared by dissolving compound 4A, for example as a pharmaceutically acceptable salt, to a concentration of 1% w/v in phosphate- buffered saline; and the solution is sterilized, for example by sterile filtration, and sealed in sterile containers containing, for example, 250 mg of a compound of this invention.
• a lyophilized formulation is prepared by dissolving compound 4A, again for example as a pharmaceutically acceptable salt, in a suitable buffer, for example the phosphate buffer of the phosphate-buffered saline mentioned above, sterilizing the solution and dispensing it into suitable sterile vials, lyophilizing the solution to remove the water, and sealing the vials.
• a suitable buffer for example the phosphate buffer of the phosphate-buffered saline mentioned above
• sterilizing the solution and dispensing it into suitable sterile vials sterilizing the solution and dispensing it into suitable sterile vials, lyophilizing the solution to remove the water, and sealing the vials.
• the lyophilized formulation is reconstituted by the addition of sterile water, and the reconstituted solution may be further diluted for administration with a solution such as 0.9% sodium chloride intravenous infusion or 5% dextrose intravenous infusion.
• Compound 4A diluted in 5% dextrose intravenous infusion, is administered intravenously over 30 minutes to a patient suffering from metastatic ovarian carcinoma at an initial dose of 100 mg/m 2 ; and this dose is increased to 250 mg/m 2 , 500 mg/m 2 , 750 mg/m 2 , and 1000 mg/m 2 .
• the compound is administered at 1-week intervals.
• the same dose escalation is administered at 2- and 3-week intervals to other patients suffering from the same cancer.

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