WO2008011588A2 - Glycoconjugates of phosphoramidate alkylators for treatment of cancer - Google Patents

Abstract

Bromoglufosfamide and related compounds are useful in the treatment of cancer and other hyperproliferative diseases. Glufosfamide and bromoglufosfamide are synthesized by reacting the corresponding tetracetyl derivatives with MeOH and a catalytic amount of MeO(-). Tetraacetyl glufosfamide is obtained by reacting a tetraacetyl trichloroacetamidate intermediate of glucose, ifosfamide mustard, and an acid in an anhydrous polar solvent.

Description

GLYCOCONJUGATES OF PHOSPHORAMIDATE ALKYLATORS FOR

TREATMENT OF CANCER

CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Patent Application Nos. 60/832,429 filed 20 July 2006 and 60/878,526 filed 3 January 2007, the content of each of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention provides novel anti-cancer compounds, methods of making them, and methods for treating cancer and other hyperproliferative disease conditions with them, and so relates to the fields of medicine, pharmacology, chemistry, and biology.

Description of Related Art

Glufosfamide, also known as β-D-glucosyl-ifosfamide mustard and glc- IPM, is a glycoconjugated prodrug of the phosphoramidate alkylator ifosfamide mustard and is useful in the treatment of cancer (US Pat. No. 5,662,936; PCT App. Pub. No. WO 05/76888; Niculescu-Duvaz, 2002, Curr. Opin. Investig. Drugs, 3:1527-32; Briasoulis et al., 2000, J. CHn. Oncol., 78(20): 3535-44; Dollner et al., 2004, Anticancer Res., 24(5A):2947-51 ; and Van der Bent et al., 2003, Ann. Oncol., 14{λ 2): 1732-4, each of which is incorporated herein by reference). In glufosfamide, ifosfamide mustard is covalently bonded to the 1 -position of a glucose molecule via a glycoside linkage. Glufosfamide is hydrolyzed in vivo to ifosfamide mustard and glucose. In contrast to ifosfamide, glufosfamide metabolism does not produce the neurotoxin acrolein and so promises to have fewer side effects than ifosfamide.

Glufosfamide can be synthesized starting with 2, 3, 4, 6-tetrabenzyl glucose (tetrabenzyl glucose), via tetrabenzyl glufosfamide and hydrogenolyzing the tetrabenzyl glufosfamide with palladized charcoal (Pd/C) and hydrogen (US Patent No. 5,622,936, supra). Because the hydrogenolysis involves a heterogenous reaction mixture, it is problematic for large scale manufacturing, and requires careful monitoring of temperature and hydrogen pressure to avoid decomposition of glufosfamide. The separation of Pd/C from the reaction mixture is problematic, because the powdery Pd/C clogs the filter pores, and use of filter-aids such as diatomaceous earth (Celite^®, Mallinckrodt Baker) can lead to lower glufosfamide yields.

There is a need for anti-cancer drugs more potent than glufosfamide and for better methods of making glufosfamide and related compounds.

BRIEF SUMMARY OF THE INVENTION In one aspect, the present invention provides bromoglufosfamide having the structure shown below

In another aspect, the present invention provides bromoglufosfamide in substantially pure form and pharmaceutically acceptable formulations comprising bromoglufosfamide and pharmaceutically acceptable diluents or excipients.

In another aspect, the present invention provides a method of treating cancer and other hyperproliferative diseases comprising administering a therapeutically effective amount of bromoglufosfamide or pharmaceutically acceptable formulations thereof to a patient in need of such treatment.

In another aspect, the present invention provides methods of making bromoglufosfamide and glufosfamide. In one embodiment, the present invention provides a method of synthesizing glufosfamide, said method comprising the steps of: (i) reacting 1-hydroxytetraacyl glucose (tetraacyl glucose) having a structure of formula:

H wherein R₆ is selected from CrC₆ alkyl, CrC₆ heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; with trichloroacetonitrile and a base to obtain a tetraacyl trichloroacetamidate intermediate; (ii) reacting said tetraacyl trichloroacetamidate intermediate with ifosfamide mustard and optionally an acid to obtain a tetraacyl-1-β-glufosfamide intermediate having structure of formula:

(iii) reacting said tetraacyl-1-β-glufosfamide obtained in step (ii) with M(OR₈)_n wherein n is 1-3, M is a metal selected from the group consisting of alkali metals, alkaline earth metals, and lanthanide metals, and Re is CrC₆ alkyl, provided that, if M is an alkali metal, then n is 1 , and if M is an alkaline earth metal, then n is 2, and if M is a lanthanide metal, then n is 3; to synthesize said glufosfamide.

These and other aspects and embodiments of the present invention are described below.

DETAILED DESCRIPTION OF THE INVENTION This detailed description of the different aspects and embodiments of the present invention is organized as follows: Section I provides useful definitions; Section Il in parts A and B describes in part A bromoglufosfamide and methods for its synthesis, and in part B a preferred method for making glufosfamide the method also being useful to make bromoglufosfamide upon appropriate substitution of starting material; Section III describes various methods of treatment employing bromoglufosfamide and the novel compounds of the present invention; and Section IV provides illustrative examples of the compounds and methods 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 disclosure elsewhere in the specification. I. Definitions

The following definitions are provided to assist the reader. Unless otherwise defined, all terms of art, notations, and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the chemical and medical arts. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not be construed as representing a substantial difference over the definition of the term as generally understood in the art.

"A" or "an" entity as used herein refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound. As such, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein.

"About" as used herein refers to variation one might see in measurements taken among different instruments, samples, and sample preparations.

"Acid" refers to either a Lewis acid or a Brόnsted acid. A Lewis acid is a molecule or a moiety that can accept a pair of lone electrons from electron pair donors such as nitrogen, oxygen, and sulfur atoms. Examples of Lewis acids include, metal halides and triflates such as aluminum, zinc, iron, hafnium, and lanthanum halides and triflates, trialkylsilyl triflates, and BF₃ (gaseous and etherate). A Brόnsted acid is a molecule or a moiety that can donate a proton. Examples of Brόnsted acids include hydrogen halides, sulfuric acid, phosphoric acid, carboxylic acids, sulfonic acids, and salts of weak bases and strong acids like pyridinium triflates.

"Acidifying or acidification" refers to neutralizing bases present in a reaction mixture or a solution by adding an acid. Acids suitable for neutralizing a reaction mixture are chosen according to the base and a product present in a reaction mixture. Aqueous acids are used for acidifying unless the product is susceptible to aqueous hydrolysis. "Resin bound acids" such as resin bound sulfonic acids are acids useful in acidifying. Water can be used for acidifying depending on the pKa of the base neutralized.

"Alkali metals" refer to Li, Na, K, Rb, and Cs. "Alkaline earth metals" refer to Mg, Ca, Sr, and Ba.

"Administering" or "administration of a drug to a patient (and grammatical equivalents of this phrase) refers to direct administration, which may be administration to a patient by a medical professional or may be self- administration, and/or indirect administration, which may be the act of prescribing a drug. For example, 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.

"Alkoxide" refers to a deprotonated alcohol moiety. In other words an alkoxide is a moiety having structure of formula RO(-), wherein ROH is the corresponding alcohol. An alkoxide can be prepared by reacting the alcohol with a base such as alkali metal hydrides, alkali metals, and alkali metal amides.

"Ci-Cβ alkoxy" refers to a substituted or unsubstituted alkyl group of 1 - 6 carbon atoms covalently bonded to an oxygen atom. In other words, a CrC₆ alkoxy group has the general structure -O-(CrC₆)alkyl. CrCβ alkoxy groups include, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec- butoxy, tert-butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, and 3-methylpentoxy.

"CrCβ alkyl" refers to a substituted or unsusbstituted straight or branched chain alkyl groups having 1-6 carbon atoms. CrC₆ alkyl groups include, for example, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert- butyl, pentyl, 2-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl and 3- methylpentyl. A CrC₆ alkyl substituent may be covalently bonded to an atom within a molecule of interest via any chemically suitable portion of the CrC₆ alkyl group.

"Aryl" refers to a substituted or unsusbstituted moiety that includes one or more monocyclic or fused ring aromatic systems. Such moieties include any moiety that has one or more monocyclic or bicyclic fused ring aromatic systems, including but not limited to phenyl and naphthyl.

"Base" refers to either a Lewis base or a Brόnsted base. A Lewis base is a molecule or a moiety that can donate a pair of lone electrons. A Brόnsted base is a molecule or moiety that can accept a proton. Examples of bases include metal hydrides, metal carbonates, metal amides, metal alkoxides, various trialkyl amines including hindered tertiary amines, amidines, and pyridines.

"Bromoifosfamide mustard" refers to the phosphoramidate mustard having structure of formula HOP(=O)(NHCH₂CH₂Br)₂.

"Compound" refers not only the specified molecular entity but also its pharmaceutically acceptable, pharmacologically active derivatives, including, but not limited to, salts, hydrates, solvates and the like.

"Catalytic amount" of a reactant or a reagent refers to an amount of reactant or reagent that is less than its stoichiometric amount calculated based on the balanced chemical equation for the reaction. For example, if 1 mmol of a reagent A is required, according to a balanced chemical equation, for a reaction with B, and 0.05 mmol of A is actually used in the reaction, then a catalytic amount of, 5 mole% of A, is used in the reaction.

"Deprotection" refers to a chemical reaction wherein a protecting group is removed (see "Protecting group" infra).

"Dialkyl azodicarboxylate" refers to a compound having structure of formula RyO₂CN=NCO₂Ry wherein each R_y is selected from Ci-C₆ alkyl and Ci-C₆ heteroalkyl. Examples of dialkyl azodicarboxylate include, but are not limited to, diethyl azodicarboxylate and diisopropyl azodicarboxylate (DIAD).

"Disaccharide" refers to a covalent dimer of monosaccharides formed upon the removal of one molecule of water from two monosaccharides.

"Halogen or halo" refers to fluorine, chlorine, bromine, and iodine.

"Heteroaryl" refers to a substituted or unsusbstituted monocyclic aromatic system having 5 or 6 ring atoms, or a fused ring bicyclic aromatic system having 8 - 20 atoms, in which the ring atoms are C, O, S, SO, SO₂, or N and at least one of the ring atoms is a heteroatom, i.e., O, S, SO, SO₂, or N. Heteroaryl groups include, for example, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothio-furanyl, benzothiophenyl, benzoxazolyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, NH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, dithiazinyl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazolyl, pyridoimidazolyl, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, quinuclidinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, thiadiazinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl and xanthenyl. Unless indicated otherwise, the arrangement of the hetero atoms within the ring may be any arrangement allowed by the bonding characteristics of the constituent ring atoms.

"Heterocyclyl" refers to a monocyclic or fused ring multicyclic cycloalkyl group at least a portion of which is not aromatic and in which one or more of the carbon atoms in the ring system is replaced by a heteroatom selected from O, S, SO, SO₂, P, or N. Examples of heterocyclyl groups include but are not limited to imidazolinyl, morpholinyl, piperidinyl, piperidin-2-only, piperazinyl, pyrrolidinyl, pyrrolidine-2-onyl, tetrahydrofuranyl, and tetrahydroimidazo [4,5-c] pyridinyl.

"Hydrate" as used herein refers to a compound of the invention or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. Hydrates are formed by the combination of one or more molecules of water with one of the substances in which the water retains its molecular state as H2O, such combination being able to form one or more hydrate.

"Ifosfamide mustard" refers to the phosphoramidate mustard having structure of formula HOP(=O)(NHCH₂CH2CI)2.

"Lanthanide metals" refer to rare earth metals or lanthanides such as La and Hf.

"Monosaccharide" refers to a monomeric carbohydrate having structure of formula [C(H₂O)]_m wherein m is 3-7 or their biologically relevant derivatives.

"Metal carbonates" refer to metal salts of carbonic acid. Examples of metal carbonates include, Li₂CO₃, Na₂COs, K₂CO₃, Cs₂CO₃, and Ag₂CO₃. "Metal hydrides" refer to salts consisting of metal cations and one or more hydride anions depending on the valency of the metal cations. Examples of metal hydrides include, NaH, KH, and CaH₂.

Overall yield" of a compound refers to the yield of the compound obtained after a sequence of reactions. For example, if A reacts to yield B in 50% yield and then B reacts to yield C in 50% yield, then the overall yield of C from A, in the sequence of reactions described, is 25%.

"Pharmaceutically acceptable carrier, excipient, or diluent" refers to a carrier, excipient, or diluent that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes a carrier, excipient, or diluent that is acceptable for veterinary use as well as human pharmaceutical use. A "pharmaceutically acceptable carrier, excipient, or diluent" as used in the specification and claims includes both one and more than one such carrier, excipient, or diluent.

"Prodrug" refers to a compound that, after administration, is metabolized or otherwise converted to an active or more active form with respect to at least one property. To produce a prodrug, a pharmaceutically active compound can be modified chemically to render it less active or inactive, but the chemical modification is such that an active form of the compound is generated by metabolic or other biological processes. A prodrug may 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, incorporated herein by reference).

"Protecting groups" refer to groups or moieties covalently bonded to functional groups wherein the groups or moieties can be removed to yield the functional groups. Protecting groups can be used in a reaction to avoid reaction at one or more functional groups while certain other functional group or groups react. Examples of protecting groups are provided for example in the reference Greene et al., Wiley-lnterscience, 3rd Ed. ,1999 (supra). "Reduction" of a symptom or symptoms (and grammatical equivalents of this phrase) refers to decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s).

"Solvate" as used herein means a compound of the invention or a salt, thereof, that further includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces in an amount of greater than about 0.3% when prepared according to the invention.

"Substituent" refers to an atom or group, including, for example, amino, C-₁-C₆ alkylamino or di(Ci-C₆)alkylamino, C₁-C₆ alkoxy, CrC₆ alkylthio, aryl, - COOH, -CONH₂, cyano, ethenyl, ethynyl, halo, heteroaryl, hydroxy, mono- or di(Ci-C₆)alkylcarboxamido, mono or di(Ci-C₆)alkylsulfonamido, nitro, -OSO₂- (CrC₆)alkyl, and -SO₂NH₂.

"Therapeutically effective amount" of a drug refers to an amount of a drug that, when administered to a subject with cancer or another hyperproliferative disease, will have the intended therapeutic effect, e.g., alleviation, amelioration, palliation or elimination of one or more manifestations of cancer or another hyperproliferative disease in the subject. The full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations.

"Treating" a condition or patient refers to taking steps to obtain beneficial or desired results, including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms of cancer or another hyperproliferative disease, 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.

"Trisubstituted phosphine" refers to a compound having structure of formula P(R_X)₃ wherein each R_x independently is selected from the group consisting of CrC₆ alkyl, CrC₆ heteroalkyl, C₃-Cs cycloalkyl, heterocyclyl, aryl, heteroaryl, and CrC₆ alkoxy. Examples of R_x include, but are not limited to, PPh₃, P((CH₂)₃CH₃)3, and PMe₃. HA. Compounds

In one aspect, the present invention provides bromoglufosfamide having the structure shown below:

.

Bromoglufosfamide can be synthesized by reacting a suitably protected glucose derivative containing a 1-OH group with

following a Mitsunobu reaction followed by a deprotection. Bromoglufosfamide can also be synthesized by reacting a suitably protected glucose derivative containing a 1-trichloroacetamidate group with

followed by a deprotection. These methods are described in Examples 1 , 2, and 3 in the EXAMPLES section below.

HB. Synthesis of Glufosfamide

Glufosfamide is an anticancer drug and consists of a 1-β-D-glucosyl moiety covalently bonded to ifosfamide mustard and contains four other hydroxy groups. To react the ifosfamide moiety specifically at the 1 -position of glucose, it is beneficial to protect the four other hydroxy groups. After the ifosfamide mustard is covalently bonded to the 1 -position of glucose, the protected hydroxy groups are deprotected to yield glufosfamide.

Glufosfamide has been synthesized by starting with tetrabenzyl glucose via the tetrabenzyl-1-β-glufosfamide intermediate (US Patent No. 5,622,936, supra). The benzyl groups are removed by hydrogenolysis using Pd/C. Because each molecule of protected glufosfamide contains four benzyl groups, up to 40 mole% Pd/C is used in the deprotection. It is expensive to perform the hydrogenolysis and produce glufosfamide due to the high costs of palladium. The hydrogenolysis occurs under heterogenous reaction conditions, and because of the presence of the substantial amount of the insoluble catalyst, controlling the reaction conditions and filtering the reaction mixture after the reaction becomes problematic. Employing diatomaceous earth (CELITE^®) can result in the glufosfamide being absorbed in the CELITE^® and thus reducing reaction yield.

Acetyl and pivaloyl protected glucose ifosfamide mustard conjugates and methods of their synthesis are reported in US Patent No. 5,622,936, supra. In these methods, a 1-bromo group is used as a leaving group and is substituted by the ifosfamide mustard. However, following these methods, tetraacetyl- 1-β- glufosfamide is obtained in only about 5% yield and tetrapivaloyl-1-β- glufosfamide in about 13% yield from the corresponding 1 -bromotetraacyl glucose derivatives (columns 9 and 10, US Patent No. 5,622,936, supra). 1- Bromotetraacyl glucose derivatives are synthesized from, for example, the corresponding tetraacyl glucose derivatives. The yield of synthesizing 1- bromotetrapivaloyl glucose from tetrapivaloyl glucose is reported to be about 55% (column 10, US Patent No. 5,622,936, supra). Therefore, while the yield of synthesizing tetrapivaloyl-1-β-glufosfamide from 1-bromotetrapivaloyl glucose is already low at about 13%, the overall yield of synthesizing tetrapivaloyl-1-β-glufosfamide from tetrapivaloyl glucose is even lower at about 8%. As a result, these methods are not efficient for production of tetraacyl-1 -β-glufosfamides.

The present invention provides methods in which a 1- trichloroacetamidate group is employed as a leaving group (instead of a 1- bromo group), and using the present methods, one can prepare tetraacetyl- 1- β-glufosfamide in an overall yields of at least about 70% (starting from the tetraacetyl glucose). The yield of tetraacetyl-1-β-glufosfamide synthesized by employing a tetraacetyl trichloroacetamidate intermediate according to the present methods therefore is much higher, 70%, than that reported previously using the 1-bromo leaving group (at best about 5% overall yield from tetraacetyl glucose). Accordingly, the present methods offer significant advantage over prior methods used for preparing glufosfamide and its hydroxy protected intermediates.

Without being bound by mechanism, the acetyl group present in the 2- position of the tetraacetyl trichloroacetamidate intermediate provides neighboring group assistance for the ifosfamide mustard to be covalently attached to the 1- position of the glucose moiety with the desired β- stereochemistry. The trichloroacetamidate moiety is removed and the 1- position carbocation is stabilized by an acetyl (or another acyl group) as shown below in Scheme 1 :

Scheme 1

β-attack by ifosfamide mustard

α-attack blocked by the acetyl group

Because an α-attack by the ifosfamide mustard is blocked as shown above, the mustard reacts from the β-face and yields the product with the desired β- stereochemistry.

If ether protecting groups like benzyl are employed for protecting the glucose hydroxyl groups, instead of acyl protecting groups like an acetyl group, the ether moiety at the 2-position cannot direct the incoming ifosfamide mustard to covalently attach at the 1 -position with the desired β- stereochemistry. In spite of using the purified α-isomer of tetrabenzyl 1- trichloroacetamidate, about 5% of the tetrabenzyl glufosfamide is obtained as the undesired 1 -α-isomer necessitating a separation of the desired β-isomer from the undesired α-isomer (US Patent No. 5,622, 936, supra). Because these 1-β and 1-α isomers have similar structure and differ in the stereochemistry only at the glucose 1 -position, and have similar retention factors (R_f) in TLC, separating the desired 1-β isomer from the undesired 1-α isomer by chromatography and/or crystallization can be problematic and reduce the yield of the desired 1-β isomer. As described above, employing a tetraacyl trichloroacetamidate intermediate in the synthesis of the glufosfamide intermediate tetraacyl-1-β-glufosfamide, production of the tetraacyl-1-α-glufosfamide can be lessened if not entirely avoided.

The present invention also provides methods for deprotecting tetraacetyl-1-β-glufosfamide that employ catalytic amounts of NaOMe in MeOH to provide glufosfamide in yields approaching or greater than 90%. After deprotecting the acetyl groups, excess alkoxide is neutralized employing an acidic resin. Because the Na salts produced as a result of the neutralization remain resin bound, a filtration of the neutralized reaction mixture yields a solution of glufosfamide essentially free of inorganic impurities and avoids an aqueous work-up that can decompose glufosfamide. Employing inexpensive alkoxides such as NaOMe for synthesizing glufosfamide from tetraacetyl-1-β-glufosfamide eliminates the use of costly Pd and so reduces the cost of synthesizing glufosfamide. Also, the deprotection involving NaOMe/MeOH according to the present invention occurs under homogenous conditions and can be controlled much easier to produce glufosfamide reproducibly and in consistent yields compared to the method involving Pd/C mediated heterogenous hydrogenolysis.

The present invention also provides methods in which the reaction between the tetraacetyl trichloroacetamidate intermediate and ifosfamide mustard to form a tetraacetyl-1-β-glufosfamide is performed in the presence of acids and/or in a polar solvent. These methods are superior relative to other methods not employing an acid and/or a polar solvent because of cleaner reactions that produce lesser amounts of byproducts if at all. Without being bound by mechanism, acetyl groups are more electron withdrawing compared to benzyl groups, and a tetraacetyl trichloroacetamidate intermediate can react more slowly with ifosfamide mustard than does the corresponding tetrabenzyl trichloroacetamidate intermediate. When an ifosfamide mustard can not replace the trichloroacetamidate group, the ifosfamide mustard can attack the

-O-C(=NH)- carbon and yield byproducts like byproducts like tetraacetyl glucose and bis-ifosfamide mustard pyrophosphoramide (Scheme 2).

The reaction rate of tetraacetyl trichloroacetamidate with ifosfamide mustard is enhanced by employing an acid catalyst that accelerates the removal of the trichloroacetamidate group relative to a reaction not employing an acid. If a Brόnsted acid is employed in the reaction, the Brόnsted acid can bind to the mustard and increase the acidity of the mustard compared to the unbound mustard. The acidic proton of the bound mustard can protonate the trichloroacetamidate group and facilitate its removal. Once the trichloroacetamidate group is removed, the unbound mustard can attack the 1 -position and form the desired product (Schemes 1 and 2). The use of a polar solvent like THF increases the solubility of ifosfamide mustard in the reacting solvent and accelerates the reaction between the mustard and the tetraacetyl trichloroacetamidate intermediate.

Scheme 2

byproducts

HO-P(=O)(NHCH₂CH₂C1)_:

THF/HO-P(=O)(NHCH₂CH₂C1)₂ *- desired product

In another aspect, the present invention provides a method of synthesizing glufosfamide, said method comprising the steps of: (i) reacting 1- hydroxytetraacyl glucose (tetraacyl glucose) having a structure of formula:

wherein R₆ is selected from CrC₆ alkyl, CrC₆ heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; with trichloroacetonitrile and a base to obtain a tetraacyl trichloroacetamidate intermediate; (ii) reacting said tetraacyl thchloroacetamidate intermediate with ifosfamide mustard and optionally an acid to obtain a tetraacyl-1-β-glufosfamide intermediate having structure of formula:

(iii) reacting said tetraacyl-1-β-glufosfamide obtained in step (ii) with M(OR₈)_n wherein n is 1-3, M is a metal selected from the group consisting of alkali metals, alkaline earth metals, and lanthanide metals, R₈ is CrC₆ alkyl, provided that, if M is an alkali metal, then n is 1 , and if M is an alkaline earth metal, then n is 2, and if M is a lanthanide metal, then n is 3, to synthesize said glufosfamide.

In one embodiment, R₆ is selected from the group consisting of methyl, tert-butyl, ethyl, phenyl, and 4-methylphenyl. In another embodiment, R₆ is methyl, phenyl, and 4-methyl phenyl. In another embodiment, R₆ is methyl.

In another embodiment, the base employed in step (i) is a non- nucleophilic base that can deprotonate the tetraacyl glucose faster than reacting with trichloroacetonitrile. In another embodiment, the base is selected from the group consisting of metal hydrides, metal carbonates, hindered tertiary amines, pyridines, and amidines. In a related embodiment, the base is selected from metal hydrides, and metal carbonates. In another related embodiment, the hindered tertiary amine is selected from the group consisting of triisopropyl amine, diisopropylethylamine, triethyl amine, and 2,2,6,6- tetraalkylpiperidine. In another related embodiment, the amidine is DBU.

In one embodiment, the acid used in step (ii) is a Lewis acid or a Bronsted acid whose conjugate base is non-nucleophilic. In another embodiment, a catalytic amount of the acid is employed. Other reagents that can be used according to the present methods in place of M(ORs)_n in step (iii) include amines in R₈OH solvents. Examples of useful amines include NH₃, amines, alkylamines, dialkylamines, and trialkylamines.

The reacting in the step (iii) is performed in a solvent and the glufosfamide synthesized is in solution in the solvent. The solution of glufosfamide is alkaline due to the presence of M(ORs) and is neutralized by acidifying. Volatiles are removed from the neutralized glufosfamide solution to obtain solid glufosfamide. The glufosfamide obtained can be purified by column chromatography and/or recrystallization. In one embodiment, the acidifying is performed with a resin bound acid. In another embodiment, the acidifying is performed with a resin bound sulfonic acid or a resin bound carboxylic acid.

In another embodiment, the present invention provides a method of synthesizing glufosfamide, said method comprising the steps of: (i) reacting 1-hydroxytetraacetyl glucose (tetraacetyl glucose) having structure of formula:

with trichloroacetonitrile and a base to obtain a tetraacetyl trichloroacetamidate intermediate, wherein said base is selected from metal hydrides and metal carbonates;

(ii) reacting said tetraacetyl trichloroacetamidate intermediate with ifosfamide mustard and optionally an acid to obtain tetraacetyl-1-β-glufosfamide having structure of formula:

(iii) reacting said tetraacetyl-1-β-glufosfamide obtained in step (ii) with M(ORs)_n wherein n, M, and R₈ are defined as above; to synthesize said glufosfamide.

In another embodiment, the metal hydride used in step (i) is selected from the group consisting of NaH, KH, and CaH₂. In another embodiment, the reacting in step (ii) is performed employing tetrahydrofuran as a solvent. In another embodiment, the acid used in step (ii) is selected from the group consisting of trimethylsilyl thflate, triethylsilyl triflate, and silver triflate. In a related embodiment, the acid used in step (ii) is trimethylsilyl triflate. In another embodiment, a catalytic amount of the acid is used in step (ii).

In another embodiment, molecular sieves are employed in step (ii) to keep the reacting moisture free. In another embodiment, the molecular sieve employed is AW 300.

In another embodiment, the tetraacetyl-1-β-glufosfamide obtained in step (ii) is purified before the reacting in step (iii). In another embodiment, the purification is performed by column chromatography. In another embodiment, the column chromatography is performed using silica gel. In another embodiment, the purification is performed by crystallization. In another embodiment, the purified tetraacetyl-1-β-glufosfamide is obtained in an overall yield of at least 10% starting from the tetraacetyl glucose. In another embodiment, the overall yield of the purified tetraacetyl -1-β-glufosfamide is 20% - 80%.

In another embodiment, M is selected from the group consisting of Na, K, and Mg, and R₈ is selected from the group consisting of CH₃, C₂H₅, (CH₃)₂CH, and C(Me)₃. In another embodiment, the M(ORs)_n reacted in step (iii) is a catalytic amount of M(ORs)_n and the reacting in step (iii) is performed in a solvent having a structure of formula R₈OH wherein Re is CrCβ alkyl. In another embodiment, the catalytic amount is 5-30 mole% of the M(ORs)_n- In another embodiment, Rs is Me.

Other reagents that can be used according to the present methods in place of M(ORs)_n in step (iii) include amines in R₈OH solvents. Examples of useful amines include NH₃, amines, alkylamines, dialkylamines, and trialkylamines.

The reacting in the step (iii) is performed in a solvent and the glufosfamide synthesized is in solution in the solvent. The solution of glufosfamide is alkaline due to the presence of M(OR₈) and is neutralized by acidifying. In one embodiment, the acidifying is performed with a resin bound acid. In another embodiment, the acidifying is performed with a resin bound sulfonic acid or a resin bound carboxylic acid. In another embodiment, the resin bound sulfonic acid is Dowex. Volatiles are removed from the glufosfamide solution (pre- or post-acidifying) to obtain glufosfamide in the solid form. In one embodiment, the glufosfamide, in solution and in the solid form, can be purified by column chromatography and/or recrystallization.

In another embodiment, the glufosfamide is synthesized in an overall yield of at least 10% starting from the tetraacetyl glucose. In another embodiment, the glufosfamide is synthesized from the tetraacetyl-1-β- glufosfamide in at least 40% yield.

In another embodiment, the glufosfamide synthesized is up to 95% or more pure. In certain related embodiments, the glufosfamide contains up to 2%, up to 5%, and up to 10% of the undesired α-isomer of glufosfamide. In another embodiment, the glufosfamide obtained is purified by chromatography and/or recrystallization.

The methods for synthesizing glufosfamide as described above is useful for synthesizing bromoglufosfamide and related derivatives upon appropriate substitution of ifosfamide mustard with another mustard. Synthesis of glufosfamide and bromoglufosfamide according to these methods is described in Examples 1 and 2 in the EXAMPLES section below. III. Methods of Treatment

In another aspect, the present invention provides a method of treating cancer and other hyperproliferative diseases comprising administering a therapeutically effective amount of bromoglufosfamide or another novel compound of the present invention to a patient in need of such treatment. Novel compounds of the present invention other than bromoglufosfamide are described in Example 1 in the EXAMPLES section below.

In another embodiment, the daily dose is administered as a pharmaceutically acceptable formulation. In another embodiment, the present invention provides a pharmaceutical formulation comprising bromoglufosfamide or another novel compound of the invention and a pharmaceutically acceptable carrier, excipient, or diluent. In one embodiment, the pharmaceutically acceptable diluent is water, saline, or aqueous dextran. Suitable methods for formulation of drugs generally are known in the art and can be used for formulating the novel compounds of the present invention upon appropriate substitution of the drug. See, e.g., Ansel et al., 1999, Pharmaceutical Dosage Forms and Drug Delivery Systems 7th ed. Lippincott Williams & Wilkins, Philadelphia: pp. 1-562; Marshall, 1979."SoNd Oral Dosage Forms," MODERN PHARMACEUTICS, Vol. 7, (Banker and Rhodes, editors), pp. 359-427.

In one embodiment, the therapeutically effective amount is administered in a daily dose. In another embodiment, the therapeutically effective amount of the compound is administered in daily doses of about 2.5 g/m² - about 8 g/m²; about 3 g/m²- about 7 g/m²; about 4 g/m²- about 6 g/m²; and about 4.5 g/m².

The therapeutically effective daily dose can be administered by employing suitable unit dose forms of the novel compounds of the present invention. In one embodiment, the compound is administered in unit dose forms of about 0.5 g - about 5 g and about 1 g - 2 g.

In another embodiment, the daily dose is administered from once every day, once every two weeks, up to, once every month. In another embodiment, the treatment is continued for a week, a month, a year, or until there is reduction of symptoms, stabilization of the disease state, or slowing of disease progression. In another embodiment, the daily dose is administered parenterally. In another embodiment, the daily dose is administered orally.

In another embodiment, the novel compound of the present invention is administered in combination with one or more anti-cancer agent or anti-cancer therapy. Known anti-cancer agents useful for use in accordance with the present invention and their therapeutically effective administration are provided for example and without limitation, in the product descriptions found in the Physicians' Desk Reference, 2003, 57th Ed., Medical Economics Company, Inc., Oradell, N.J; Goodman & Gilman's The Pharmacological Basis of Therapeutics" 2001 , 10^th Edition, McGraw-Hill, New York; and/or are available from the Federal Drug Administration and/or are discussed in the medical literature.

Treatment of Cancers

According to the methods of the present invention, various cancers can be treated by administering bromoglufosfamide and other novel compounds of the present invention. In one embodiment, the cancer treated is selected from the group consisting of cancer of the adrenal gland, bone, brain, breast, bronchi, colon and/or rectum, gallbladder, head and neck, kidneys, larynx, liver, lung, neural tissue, pancreas, prostate, parathyroid, skin, stomach, and thyroid. In another embodiment, the cancer treated is selected from the group consisting of acute and chronic lymphocytic and granulocytic tumors, adenocarcinoma, adenoma, basal cell carcinoma, cervical dysplasia and in situ carcinoma, Ewing's sarcoma, epidermoid carcinomas, giant cell tumor, glioblastoma multiforma, hairy-cell tumor, intestinal ganglioneuroma, hyperplastic corneal nerve tumor, islet cell carcinoma, Kaposi's sarcoma, leiomyoma, leukemias, lymphomas, malignant carcinoid, malignant melanomas, malignant hypercalcemia, marfanoid habitus tumor, medullary carcinoma, metastatic skin carcinoma, mucosal neuroma, myeloma, mycosis fungoides, neuroblastoma, osteo sarcoma, osteogenic and other sarcoma, ovarian tumor, pheochromocytoma, polycythemia vera, primary brain tumor, small-cell lung tumor, squamous cell carcinoma of both ulcerating and papillary type, hyperplasia, seminoma, soft tissue sarcoma, retinoblastoma, rhabdomyosarcoma, renal cell tumor, topical skin lesion, veticulum cell sarcoma, and Wilm's tumor.

In other embodiments, bromoglufosfamide and other novel compounds of the present invention are administered for the treatment of cancer in combination with other anti-cancer agents or other anti-cancer therapies. Suitable other anti-cancer agents, and their administration, useful according to the present methods to treat cancer is described for example in Physicians' Desk Reference, 2003, 57th Ed. (supra). In other embodiments, bromoglufosfamide and other novel compounds of the present invention are administered to treat pancreatic cancer as described in US patent application nos. 60/910,403 and 60/915,882 (each of which is incorporated herein by reference), upon appropriate substitution of glufosfamide with bromoglufosfamide or another novel compound of the present invention.

Methods for treatment of cancer using glufosfamide are described in the references Niculescu-Duvaz, Briasoulis et al., Dollner et al., and Van der Bent et al.; (each supra) US Pat. No. 5,622,936; and PCT Pat. App. Pub. Nos. WO 07/035961 , WO 06/122227, WO 05/076888 and WO 06/071955 (each of which is incorporated herein by reference); and can be used according to the present methods upon appropriate substitution of glufosfamide by bromoglufosfamide or another novel compound of the present invention.

Treatment of Hyperproliferative Diseases

In another aspect, 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). In one embodiment, the hyperproliferative disease 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 duct diseases, neoplastic vasculitis, pancreatitis, pemphigus vulgaris, primary glomerulonephritis, psoriasis, periodontal disease, polycystic kidney disease, polyarteritis nodosa, polyangitis overlap syndrome, primary sclerosing cholangitis, rheumatoid arthritis, serum sickness, surgical adhesions, stenosis or restenosis, scleritis, scleroderma, strictures of bile ducts, strictures (of duodenum, small bowel, and colon), silicosis and other forms of pneumoconiosis, type I diabetes, ulcerative colitis, ulcerative proctitis, vasculitis associated with connective tissue disorders, vasculitis associated with congenital deficiencies of the complement system, vasculitis of the central nervous system, and Wegener's granulomatosis.

In one embodiment, the hyperproliferative disease treated is psoriasis, a disease characterized by the cellular hyperproliferation of keratinocytes which builds up on the skin to form elevated, scaly lesions. In another embodiment, the hyperproliferative disease treated is multiple sclerosis, a disease characterized by progressive demyelination in the brain. In another embodiment, the hyperproliferative diseases treated is rheumatoid arthritis, a multisystem chronic, relapsing, inflammatory disease that can lead to destruction and ankylosis of joints affected. In another embodiment, 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.

Theranostic Use

In another aspect, the present invention provides methods for treating cancer in a cancer patient comprising a preliminary assessment of the cancer patient to determine the degree of susceptibility of the patient's cancer to drug therapy mediated by bromoglufosfamide and other novel compounds of the present invention. The degree of susceptibility of a cancer to drug therapy mediated by a novel compound of the present invention can be measured by determining the uptake of the compound in cancer cells and comparing the uptake with a predetermined value. Methods for determining cellular uptake of other glycoconjugates are described in PCT Pat. Pub. No. WO 04/081181 (incorporated herein by reference) and can be used in the present methods upon appropriate substitution of the glycoconjugate by the compounds of the present invention.

The invention, having been described in summary and in detail, is illustrated but not limited by the Examples below, which describe the novel compounds and methods of the present invention.

IV. EXAMPLES Example 1. Novel compounds

Example 1 provides novel compounds of the invention having structure of formula selected from the group consisting of Formulas (I), (II), and (III):

Formula (I) Formula (II) Formula (III) wherein R₄ is selected from a monosaccharide and a disaccharide;

X is halo; and each Ri independently is selected from H and methyl.

In one embodiment, R₄ is selected from the group consisting of:

In another embodiment, R₄ is selected from the group consisting of:

In another embodiment, R₄ is selected from the group consisting of 1- glucosyl, 1-mannosyl, 1 -galactosyl, 1-fucosyl, and 1-rhamnosyl. In another embodiment, the novel compounds have structures of formula:

The compounds provided in Example 1 can be synthesized as described below. In one embodiment, the present invention provides a method of synthesizing the compound of Formula (I) comprising the steps of:

(i) contacting a protected monosaccharide or disaccharide selected from the group consisting of:

wherein R₃ is selected from the group consisting of CH₂Rs, CH(Rs)₂, C(Rs)₃, and COR₆;

R₇ is selected from the group consisting of CH₂R₅, CH(R₅)₂, and

C(Rs)₃- each R₅ independently is aryl; and

R₆ is selected from the group consisting of C_rC₆ alkyl, C_rC₆ heteroalkyl, C₃-Cs cycloalkyl, hetreocyclyl, aryl, and heteroaryl; a trisubstituted phosphine; and a dialkyl azodicarboxylate;

(ii) contacting the product obtained in step (i) and an alkylator having structure of formula:

Formula (IV) wherein each R₁ independently is selected from H and methyl, to yield an intermediate; and

(iii) converting each of the OR₃ group in the intermediate obtained in step (ii), to an OH group, to yield the compound of Formula (I). In another embodiment, the converting in step (iii) involves a deprotection reaction. In another embodiment, the present present invention provides a method wherein R₃ is benzyl; the trisubstituted phosphine is PPh₃; the dialkyl azodicarboxylate is selected from diisopropyl and diethyl azodicarboxylate; the alkylator has the structure of formula:

and the converting in step (iii) to yield the compound is carried out by reacting the intermediate obtained in step (ii) with palladized charcoal, and hydrogen.

In another embodiment, the present invention provides a method of synthesizing the compound of Formula (II) comprising:

(i) contacting a compound selected from the group consisting of:

wherein each R₃ is independently selected from the group consisting of CH₂R₅, CH(R₅)₂, C(R₅)₃, and COR₆;

R₇ is selected from the group consisting of CH₂Rs, CH(R₅ )₂, C(Rs)₃; each R₅ independently is aryl; and each R₆ is independently selected from the group consisting of CrC₆ alkyl, CrC₆ heteroalkyl, C₃-C₈ cycloalkyl, heterocyclyl, aryl, and heteroaryl; with a trisubstituted phosphine; and a dialkyl azodicarboxylate;

(ii) contacting the product obtained in step (i) and an alkylator having the formula:

Formula (V) wherein each Ri independently is selected from H and methyl; and

(iii) converting each of the OR₃ groups, in the product obtained in step (ii), to an OH group, to yield the compound having the formula of Formula (II). In another embodiment, the present present invention provides a method wherein R₃ is benzyl; the trisubstituted phosphine is PPh₃; the dialkyl azodicarboxylate is selected from diisopropyl and diethyl azodicarboxylate; and the converting in step (iii) to yield the compound is carried out by reacting the intermediate obtained in step (ii) with palladized charcoal, and hydrogen.

In another embodiment, the present invention provides a method of synthesizing a compound having the formula of Formula (III) comprising:

(i) contacting a compound selected from the group consisting of:

wherein each R₃ is independently selected from the group consisting Of CH₂Rs, CH(R₅)₂, C(R₅)₃, and COR₆;

R₇ is selected from the group consisting of CH₂R₅, CH(R₅ )₂, C(R₅)₃; each R₅ independently is aryl; and each R₆ is independently selected from the group consisting of CrC₆ alkyl, CrC-₆ heteroalkyl, C₃-C₈ cycloalkyl, heterocyclyl, aryl, and heteroaryl; with a trisubstituted phosphine; and a dialkyl azodicarboxylate;

(ii) contacting the product obtained in step (i) and an alkylator having the formula:

Formula (Vl) wherein X is halo and each Ri independently is selected from H and methyl,; and

(iii) converting each of the OR₃ groups, in the product obtained in step (ii), to an OH group, to yield the compound having the formula of Formula (III). In another embodiment, the present present invention provides a method wherein R₃ is benzyl; the trisubstituted phosphine is PPh₃; the dialkyl azodicarboxylate is selected from diisopropyl and diethyl azodicarboxylate; and the converting in step (iii) to yield the compound is carried out by reacting the intermediate obtained in step (ii) with palladized charcoal, and hydrogen.

The methods described in Example 1 can be used for synthesizing glufosfamide upon appropriate substitution of starting material.

Methods for reacting alkylators with other alcohols are provided in PCT Pat. Pub. No. WO 07/002931 and can be employed upon appropriate substitution of the alcohol with a monosaccharide or a disaccharide for the synthesis of intermediates and compounds of the present invention. Suitable protecting groups for an OH group, methods for protecting an OH group, and converting a protected OH group to an OH group are described for example in the reference Greene et al., Protective Groups in Organic Synthesis, Wiley- Interscience, 3rd Ed. ,1999.

In one embodiment, the protected monosaccharide or disaccharide is selected from the group consisting of:

wherein R₃ is selected from the group consisting of CH₂Rs, CH(R₅)₂, C(R₅)₃, and COR₆; and R₇ is selected from the group consisting of CH₂Rs, CH(Rs)₂, C(Rs)₃- In another embodiment, each R₃ and R₇ is benzyl. In another embodiment, R₃ is acetyl.

In another aspect, a novel compound of the present invention can be synthesized comprising the steps of, reacting a compound having structure of formula selected from the group consisting of Formulas (IV), (V), and (Vl) and a compound having structure of formula selected from the group consisting of:

wherein R₉ is OCO(=NH)CCI₃; R₃ is selected from the group consisting of CH₂R₅, CH(Rs)₂, C(R₅)₃, and COR₆; and R₇ is selected from the group consisting of CH₂R₅, CH(R₅)₂, C(R₅)₃ wherein R₅ and R₆ are defined as above to yield an intermediate and converting the intermediate to a novel compound of the present invention. In another embodiment, each R₃ and R₇ is benzyl. In another embodiment, R₃ is acetyl. In another embodiment, the reaction further comprises an acid. In another embodiment, the acid is CF₃SO₃H or CF₃CO₂H.

Example 2. Synthesis of bromoglufosfamide

Method A

Novel compounds of the invention, Compounds iv and v, and bromoglufosfamide was synthesized starting from 2,3,4,6-tetra-O-benzyl-D- glucose (or tetrabenzyl glucose) as described below.

Scheme 3

Phenyldichlorophosphate (1.66 ml, 11.15 mmol) was added to a suspension of R-2-chloro-1-methyl-ethylamine hydrobromide (2.9 g, 22.3 mmol) in dichloromethane (DCM, 60 ml) at O ⁰C, followed by the drop wise addition of triethylamine (TEA, 7.8 ml, 55.8 mmol), and the reaction mixture was stirred vigorously. The reaction mixture was warmed up to room temperature (rt), stirred for 2 hour (h), poured into brine, and the DCM layer separated. The aqueous layer was extracted with DCM and the combined DCM layers were dried over anhydrous MgSO₄ and concentrated to yield a residue. The residue was separated by silica gel flash column chromatography (ethyl acetate/hexane, 20 - 80%) to yield Compound i. A mixture of Compound i (1 g) in ethanol (EtOH, 20 ml) and platinum(IV) oxide (200 mg) was evacuated and purged with nitrogen and the evacuation- purging cycle repeated thrice followed by evacuation and purging with hydrogen thrice, and the mixture stirred vigorously under a hydrogen atmosphere for 1 h. The reaction mixture was filtered, the filtrate concentrated under vacuum at temperatures below 2O⁰C, and the residue co- evaporated with toluene to yield Compound ii. DIAD (0.264 ml, 1.365 mmol) was added drop wise to a solution of Compound iii (170 mg, 0.683 mmol), 2,3,4,6-tetra-O-benzyl-D-glucose (740 mg, 1.365 mmol), and PPh₃ (358 mg, 1.365 mmol) in anhydrous tetrahydrofuran (THF, 10 ml) maintained at O⁰C. The reaction mixture was warmed up to rt, stirred overnight, and concentrated to yield a residue. The residue was separated by silica gel flash chromatography (acetone/toluene, 0- 100%) to yield Compound iii. Palladized charcoal (Pd/C, 30 mg) was added to a solution of Compound iii (100 mg) in EtOH (5 ml). The reaction mixture was evacuated and purged with nitrogen, this cycle repeated thrice followed by evacuation and purging thrice with hydrogen and the reaction carried out under a hydrogen atmosphere with vigorous stirring for 1 h. The reaction mixture was filtered, the filtrate concentrated under vacuum to yield a residue and the residue separated by silica gel flash chromatography (methanol/DCM, 10 - 30%) to yield Compound iv.

Compound v, having a structure of formula:

was synthesized and separated following the same method as that described for the synthesis of compound iv and substituting R-2-chloro-1-methyl- ethylamine hydrobromide with S-2-chloro-1-methyl-ethylamine hydrobromide. Bromoglufosfamide, having a structure of formula:

was synthesized and separated according to the method described above, by reacting 2,3,4,6-tetra-O-benzyl-D-glucose (349 mg) PPh₃ (169 mg), DIAD (125 μL) and bromoifosfamide mustard (100 mg) to yield tetrabenzylbromoglufosfamide having a structure of formula:

and converting the tetrabenzylbromoglufosfamide to yield bromoglufosfamide.

Bromoglufosfamide was also synthesized starting from 2,3,4,6- tetraacetyl glucose as described below. Method B

DIAD (220 μl_)was added to a solution of 2,3,4,6-tetraacetyl glucose (200 mg), bromoifosfamide mustard (178 mg), and PPh₃ (300 mg) maintained at rt and the mixture stirred for 16 h. Then, the volatiles were evaporated and the residue separated by column chromatography in silica gel using acetone/ toluene (0-80%) to yield β-tetraacetylbromoglufosfamide (86 mg) containing trace amounts of the α-isomer that was used for deprotection without further separation. The β-tetraacetylbromoglufosfamide thus obtained was dissolved in anhydrous methanol and a catalytic amount of NaOMe added to it. The pH of the reaction mixture was measured to be about 10. After 20 min, the reaction mixture was filtered though a bed of Amberlite resin (acidic form). The filtrate was concentrated, and the residue separated by column chromatography in silica gel using methanol/ DCM (10-20%) to yield bromoglufosfamide.

Another synthesis of bromoglufosfamide using the trichloroacetamidate of 2,3,4,6-tetraacetyl glucose as described below. Method C

NaH (4 mg) was added to a solution of 2,3,4,6-tetraacetyl glucose (100 mg) in THF (3 ml_) maintained at O⁰C and stirred for 10 min followed by the addition of CCI₃CN (115 μl_). After 30 min, alumina (100 mg) was added maintaining the temperature at O⁰C. The reaction mixture was stirred for 30 min, filtered, and the residue washed with anhydrous THF. Molecular sieves (AW-300, 500 mg) were added to the filtrate and stirred for 30 min at rt followed by the sequential addition of bromoifosfamide mustard (90 mg) and trimethylsilyl triflate (TMSOTf, 10 μl_). The reaction mixture was stirred at rt for 30 min and poured into a saturated NaHCO₃ solution and extracted with ethyl acetate (EtAc). The EtAc portion was concentrated in vacuo and the residue separated by column chromatography in silica gel using acetone/ toluene (0- 90%) to yield bromoglufosfamide.

Example 3: Synthesis of qlufosfamide Synthesis of tetraacetyl glucose

This example describes the synthesis of tetraacetyl glucose, a tetraacyl glucose intermediate employed in the present invention, starting from pentaacetyl glucose which can be readily synthesized by reacting glucose with acetic anhydride and pyridine. In method A, benzyl amine (BnNH₂) is employed and in method B, NaOMe/MeOH, for the selective deprotection of the 1 -acetyl group. Method A

BnNH₂ (2.1 ml, 19.2 mmol) was added to a solution of pentaacetyl glucose (5 g, 12.8 mmol) in THF (30 ml) maintained at rt and the reaction mixture was stirred at rt overnight. The reaction mixture was poured into brine (100 ml) and extracted with EtAc (150 ml). The organic layer was washed with HCI (1% aqueous solution, 100 ml), saturated NaHCO₃ (100 ml), brine (100 ml), dried over MgSO₄, and concentrated. The resulting residue was separated by column chromatography on silica gel (EtAc/hexane 0-80%) to yield tetraacetyl glucose (3.5 g, 91 %). Method B

NaOMe (0.07 g, 1.28 mmol) was added to a solution of pentaacetyl glucose (1 g, 2.56 mmol) and MeOH (0.32 ml, 7.68 mmol) in THF (10 ml) maintained at O⁰C and the reaction mixture was stirred at rt for 3 h. The reaction mixture was poured into brine (100 ml) and extracted with EtAc (50 ml), dried with MgSO₄, and concentrated. TLC demonstrated a clean reaction containing the product and about 20% of the starting material.

Efficient synthesis of tetraacetyl-1-3-glufosfamide Tetraacetyl-1-β-glufosfamide, a tetraacyl-1-β-glufosfamide intermediate employed in the present methods, is synthesized in high yields by reacting tetraacetyl glucose with trichloroacetonitrile and NaH to yield the tetraacetyl trichloroacetamidate intermediate, and reacting without further purification the trichloroacetamidate intermediate with ifosfamide mustard and trimethylsilyl triflate in anhydrous THF as described in Method A below to obtain tetraacetyl-1 -β-glufosfamide. Method A

Trichloroacetonitrile (0.6 ml_, 5.7 mmol) was added to a solution of tetraacetyl glucose (Compound vi, 0.53 g, 1.5 mmol) in anhydrous THF (4 ml_) maintained at -1O⁰C followed by the addition of NaH (21.3 mg). The temperature of the reaction mixture was raised to rt and stirred for 30 min. The temperature of the reaction mixture was reduced to O⁰C, alumina (0.8 g) added to the reaction mixture, the temperature of the reaction mixture raised to rt and stirred for 30 min. The reaction mixture was filtered, and the residue washed with anhydrous THF (2mL). Molecular sieves (AW 300, 0.8 g) and ifosfamide mustard (420 mg) were added to the filtrate containing the trichloroacetamidate intermediate, and the reaction mixture stirred at rt for 30 min followed by the addition of a catalytic amount of trimethylsilyl triflate (34 μl_, 0.15 mmol, ~10 mole%). After 10 min, the reaction mixture was filtered, poured into a solution of aqueous NaHCO₃, and extracted with EtAc. The EtAc solution was dried, volatiles removed in a rotary evaporator, and the residue separated from impurities (purified) by flash column chromatography in silica gel employing acetone/toluene (0-90%) as eluent to obtain, tetraacetyl-1 -β-glufosfamide (Compound vii, 560 mg, 67% yield from Compound vi). Thus, this example demonstrates an efficient synthesis of the glufosfamide intermediate, tetraacetyl-1 -β-glufosfamide. Method B Tetraacetyl-1-β- glufosfamide was also synthesized by reacting glufosfamide (1 g) with acetic anhydride (3 ml_) and pyridine (6 ml_) at rt for 1 h. After removing the volatiles and coevaporating the residue with toluene, the residue was separated by flash column chromatography in silica gel employing acetone/toluene (0-80%) as eluent to obtain 1.2 of the tetraacetyl- 1-β-glufosfamide.

Example 4. Efficient deprotection of tetraacetyl-i-3-glufosfamide for the synthesis of glufosfamide

This example demonstrates an efficient method of deprotecting acetyl groups in particular and acyl groups in general by employing a catalytic amount of MeO(-)/MeOH for the synthesis of glufosfamide.

To a solution of tetraacetyl-1-β-glufosfamide (900 mg, obtained by method B) maintained at O⁰C was added 0.05 molar NaOMe (27 mg, 30 mole% of the tetraacetyl glufosfamide) dissolved in MeOH (10 ml_). The temperature of the reaction mixture was raised to rt and stirred for 30 min followed by acidifying with DOWEX acidic resin. The reaction mixture was filtered, adsorbed on silica gel, and volatiles were removed. Glufosfamide adsorbed on the silica was separated by flash column chromatography in silica gel employing water/MeCN (0-15%) as eluent to obtain 560 mg of glufosfamide in 89% yield starting from tetraacetyl-1-β-glufosfamide.

Example 5. Antiproliferation activity

The antiproliferation activity of bromoglufosfamide was tested in a multi-well Alamar Blue based assay. H460 cells (ATCC HTB-177 (NCI-H460), 20,000 cells/well/500 μl) were seeded in a 24 well plate in RPMI medium (Invitrogen Corporation, Carlsbad, CA). After 24 h, these plates were divided into 2 groups - a "control group" and a 2 h "treatment group" where the cells were kept in contact with the test compound for 2 h. In the treatment group, after 2 h the cells were rinsed to remove the test compound and incubated for 3 days and stained with AlamarBlue. In the control group, AlamarBlue was added to the plate at day 3 and the fluorescence measured to establish the control reading. In the treatment group, cell proliferation in the presence and absence of the test compound was determined 2 h after addition of AlamarBlue by measuring fluorescence using a fluorescence plate reader and the IC₅₀ of the compound determined to be 100 μM (λ_ex = 550 nm and λ_em = 590 nm, see Biosource International Inc., Tech Application Notes, Use of Alamar Blue in the measurement of Cell Viability and Toxicity, Determining IC₅₀). Employing a similar method, Compounds iv and v were tested to have IC₅₀ values >100 μM. This method can be used to screen compounds of the present invention based on their in vitro antiproliferation activity.

_***

Although the present invention has been described in detail with reference to specific embodiments, those of skill in the art will recognize that modifications and improvements are within the scope and spirit of the invention, as set forth in the claims that follow. All publications and patent documents (patents, published patent applications, and unpublished patent applications) cited herein are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any such document is pertinent prior art, nor does it constitute any admission as to the contents or date of publication of the same. The invention having now been described by way of written description and example, those of skill in the art will recognize that the invention can be practiced in a variety of embodiments and that the foregoing description and examples are for purposes of illustration and not limitation of the following claims.

Claims

CLAIMS 1. A compound having the structure:

2. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier, excipient, or diluent.

3. A method of treating cancer comprising administering a therapeutically effective amount of a compound having the structure:

to a patient in need of such treatment.

4. A method of synthesizing a compound having the formula of:

Formula (I) wherein each Ri independently is selected from H and methyl and R₄ is selected from a group consisting of:

the method comprising:

(i) contacting a compound selected from the group consisting of:

wherein each R₃ is independently selected from the group consisting of CH₂Rs, CH(R₅)₂, C(R₅)₃, and COR₆;

R₇ is selected from the group consisting of CH2R5, CH(Rs)₂,

C(Rs)₃; each R₅ independently is aryl; and each Re is independently selected from the group consisting of CrC₆ alkyl, CrC₆ heteroalkyl, C₃-C₈ cycloalkyl, heterocyclyl, aryl, and heteroaryl; with a trisubstituted phosphine; and a dialkyl azodicarboxylate;

(ii) contacting the product obtained in step (i) and an alkylator having the formula of:

Formula (IV) wherein each R₁ independently is selected from H and methyl; and (iii) converting each of the OR₃ groups, in the product obtained in step (ii), to an OH group, to yield the compound having the formula of Formula (I).

5. The method of claim 4 wherein R₃ and R₇ is benzyl; the trisubstituted phosphine is PPh₃; the dialkyl azodicarboxylate is selected from diisopropyl azodicarboxylate and diethyl azodicarboxylate; the alkylator has structure of formula:

and the converting in step (iii) to yield the compound of Formula (I) is carried out by contacting the product obtained in step (ii) with palladized charcoal and hydrogen.

6. A method of synthesizing glufosfamide, said method comprising the steps of:

(i) reacting tetraacetyl glucose with trichloroacetonitrile and a base to obtain a tetraacetyl trichloroacetamidate intermediate wherein, said base is selected from metal hydrides and metal carbonates;

(ii) reacting said tetraacetyl trichloroacetamidate intermediate with ifosfamide mustard, and optionally an acid, in a polar solvent to obtain tetraacetyl-1 -β-glufosfamide; and

(iii) reacting said tetraacetyl-1 -β-glufosfamide obtained in step (ii) with M(ORs)_n wherein n is 1-3, M is selected from the group consisting of alkali metals, alkaline earth metals, and lanthanide metals, Rs is CrC₆ alkyl, provided that, if M is an alkali metal, then n is 1 , if M is an alkaline earth metal, then N is 2, and if M is a lanthanide metal, then n is 3; to synthesize said glufosfamide.

7. The method of claim 6, further comprising the step of purifying said tetraacetyl-1-β-glufosfamide obtained in step (ii) before said reacting in step (iii).

8. The method of claim 6 wherein, said M is selected from the group consisting of Na, K, and Mg and said Re is selected from the group consisting of CH₃, C₂H₅, (CHa)₂CH, and C(Me)₃.

9. The method of claim 6 wherein said metal hydride reacted in said step (i) is selected from the group consisting of NaH, KH, and CaH₂.

10. The method of claim 6, wherein said glufosfamide is synthesized in an overall yield of at least 10% starting from said tetraacetyl glucose.

11. The method of claim 6, wherein said glufosfamide is synthesized from said tetraacetyl-1-β-glufosfamide in at least 40% yield.

12. The method of claim 6, wherein said M(ORs)_n reacted in said step (iii) is a catalytic amount of M(ORs)_n and wherein said reacting is performed in a solvent, R₈OH.

13. The method of claim 6, wherein said solvent in said step (ii) is tetrahydrofuran.

14. The method of claim 6, wherein said acid reacted in said step (ii) is trimethylsilyl triflate.

15. The method of claim 6, wherein said acid is a resin bound acid.

16. The method of claim 7, wherein said purified tetraacetyl-1-β- glufosfamide is obtained in an overall yield of at least 10% starting from said tetraacetyl glucose.

17. The method of claim 12, wherein said catalytic amount is 5 mole%.

18. The method of claim 14, wherein R₈ is Me.

19. The method of claim 16, wherein said overall yield of said purified tetraacetyl -1-β-glufosfamide is 20% - 80%.