CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims the benefit of U.S. Provisional Patent Application No. 60/608,045, filed September 8, 2004, the disclosure of which is incorporated by reference.

FIELD OF THE INVENTION [0002] The present invention relates to prodrugs of inactivators of 06-alkylguanine-DNA alkyltransferase (AGT). The prodrugs are cleaved by the beta-glucuronidase enzyme found in tumor cells or co-administered to the patient, and are targeted for use in cancer treatment in combination with an antineoplastic alkylating agent such as l,3-bis(2-chloroethyl)-l- nitrosourea (BCNU) or temozolomide.

BACKGROUND OF THE INVENTION [0003] AGT is a DNA repair protein. AGT removes alkyl and aralkyl groups that become attached at the Opposition of guanine in DNA following exposure to mutagenic and/or carcinogenic alkylating agents. It does so by bringing about a stoichiometric transfer of the group attached to the Opposition of a guanine residue in DNA to a cysteine residue within the AGT protein. Pegg, Cancer Research 50: 6119-6129 (1990). Accordingly, AGT is beneficial to a normal cell because it removes the adducts that are formed in DNA by toxic, mutagenic and carcinogenic agents, thereby restoring the DNA to its original state and helping to prevent DNA mutations that can lead to initiation of tumor formation. Unfortunately, AGT is also beneficial to a cancerous cell because it also removes those adducts that are formed at the Opposition of guanine in DNA by antineoplastic alkylating agents, such as monofunctional methylating agents, e.g., procarbazine, dacarbazine and temozolomide, and chloroethylating agents, i.e., chloroethylnitrosoureas (CENUs), such as BCNU, ACNU, CCNU, and MeCCNU. Pegg et al, Prog. Nucleic Acid Research Molec. Biol. 51 : 167-223 (1995). The resulting alkylated AGT molecule is consequently inactivated and is unable to carry out subsequent dealkylation reactions. The presence of more AGT in a cell increases its capacity to repair DNA by this mechanism compared to a cell that has less AGT. [0004] The reduction in the efficacy of cancer chemotherapeutic drugs due to AGT, which acts without requiring the presence of additional enzymes or cofactors, and the existence of a high correlation between AGT activity and reduction in sensitivity of tumor cells to nitrosoureas have led to AGT becoming a prime target for modulation. Modulation has been attempted by two different routes. One route is indirect and involves the use of methylating agents that introduce O^-methylguanine lesions into DNA for subsequent repair by AGT, thereby depleting levels of AGT. The other route is direct and involves the use of an adjuvant, i.e., an inactivator of AGT, such as an (96-aralkylguanine, e.g., Oβ- benzylguanine; see, for example, Moschel et al., U.S. Patent Nos. 5,091,430; 5,352,669; 5,358,952; 5,525,606; 5,691,307; 5,753,668; 5,916,894; 5,958,932; 6,060,458; 6,172,070; 6,303,604; 6,333,331 ; and 6,436,945. It has been shown that such adjuvants can inactivate AGT and that this inactivation can markedly improve the effectiveness of chemotherapeutic drugs that modify the Opposition of DNA guanine residues. Pegg et al., Prog. Nucleic Acid Res. MoL Biol. 51: 167-223 (1995); Kokkinakis et al., Clin. Cancer Res. 5: 3676-3681 (1999); Dolan et al., Biochem. Pharmacol. 46: 285-290 (1993); Felker et al., Cancer Chemo. Pharmacol. 32: 471-476 (1993); Schold, Jr. et al., Cancer Res. 56: 2076-2081 (1996); and Kurpad et al., Cancer Chemo. Pharmacol. 39: 307-316 (1997). In some instances, however, in clinical trials, the adjuvant therapy produces toxic side effects in the patient. Quinn et al., J. Clin. Oncol. 2002, 20, 2277-2283. [0005] There is a desire, therefore, to minimize the toxic side effects of the adjuvant therapy. The foregoing shows that there exists a need for adjuvants that are selective to the tumor cell. The present invention provides such an approach. The advantages of the present invention as well as inventive features will be apparent from the description of the invention provided below.

BRIEF DESCRIPTION OF THE DRAWINGS [0006] Figure IA depicts the concentration of prodrugs 1 (open squares) and 2 (open circles); and of O6-benzylguanine (closed squares) and <26-benzyl-2'-deoxyguanosme (closed circles), which were produced by the enzymatic cleavage of the prodrugs, as a function of time in contact with 10 units/mL bovine liver β-glucuronidase. For formulas of prodrugs 1 and 2, see paragraph 0057. [0007] Figure IB depicts the concentration of prodrugs 1 (open squares) and 2 (open circles); and the concentration of (96-benzylguanme (closed squares) and <96-benzyl-2'- deoxyguanosine (closed circles), which were produced by the enzymatic cleavage of the prodrugs, as a function of time in contact with 0.2 units/mL E. coli β-glucuronidase. [0008] Figure 2A depicts the % remaining activity of AGT as a function of the concentration of prodrug 1 in the absence of β-glucuronidase (closed squares) and in the presence of bovine liver β-glucuronidase: 20 units/mL of β-glucuronidase (closed circles) and 200 units/mL of β-glucuronidase (open squares). [0009] Figure 2B depicts the % remaining activity of AGT as a function of the concentration of prodrug 2 in the absence of β-glucuronidase (closed squares) and in the presence of bovine liver β-glucuronidase: 20 units/mL of β-glucuronidase (closed circles) and 200 units/mL of β-glucuronidase (open squares). [0010] Figure 3 depicts the % survival of HT29 cells as a function of the concentration of prodrug in a two-hour exposure to BCNU (40 μM) after prodrug pretreatment. Prodrug 2 (open squares) and prodrug 1 (open circles) were incubated for 5 hr in cell medium containing no β-glucuronidase; and prodrug 2 was incubated for 14 hr in cell culture containing bovine liver β-glucuronidase at 20 units/mL of medium (closed squares); prodrug 1 was incubated in cell culture containing bovine liver β-glucuronidase at 20 units/mL of medium for 7 hr (closed circles) or 14 hr (closed triangles).

SUMMARY OF THE INVENTION [0011] The present invention provides for ameliorating a disadvantage of some of the known 06-alkylguanine-DNA alkyltransferase inactivators. The present invention provides prodrugs of AGT inactivators. The prodrug includes a glycosyl residue, e.g., glucuronosyl, which is cleavable by the β-glucuronidase enzyme, and which is either administered and/or found on tumor cells. The cleavage by the enzyme releases the inactivator in the vicinity of the tumor cells. As a result, the inactivator is more selective for tumor cells and toxicity to the patient is therefore minimized. [0012] The present invention further provides a pharmaceutical composition comprising a prodrug of the invention and a pharmaceutically acceptable carrier. The present invention further provides a method of enhancing the chemotherapeutic treatment of tumor cells in a mammal with an antineoplastic alkylating agent that causes cytotoxic lesions at the Opposition of guanine. The method comprises administering to the mammal an effective amount of a prodrug and antineoplastic alkylating agent, and optionally the β-glucuronidase enzyme. The present invention also provides a method of inactivating AGT in a tumor cell comprising contacting said tumor cell with an effective amount of a compound or prodrug of the invention and an effective amount of a β-glucuronidase enzyme. [0013] While the invention has been described and disclosed below in connection with certain embodiments and procedures, it is not intended to limit the invention to those specific embodiments. Rather it is intended to cover all such alternative embodiments and modifications as fall within the spirit and scope of the invention.

DETAILED DESCRIPTION OF THE INVENTION [0014] The present invention is predicated on the concept that enzymatic conversion of a prodrug to an active drug in the vicinity of a tumor cell provides an approach for delivering an inactivator of AGT more selectively to tumor cells versus normal cells of a host. This approach relies on activation by enzymes that are expressed or released by tumor cells, β- Glucuronidase is an example of a lysosomal hydrolase that is released from necrotic tumor cells found within poorly vascularized regions of tumor masses (Bosslet et al., Tumor Targeting 1995, 1, 45-50; Cancer Res. 1998, 55, 1195-1201; and de Graaf et al., Curr. Pharm. Design 2002, 8, 1391-1403). When the prodrug is cleaved by β-glucuronidase, the released active drug enters nearby tumor cells preferentially, thereby minimizing the more widespread toxicity associated with systemic delivery of some of the unmodified drugs. [0015] In accordance with an embodiment, the present invention provides a compound (or prodrug) of the formula: A — B — C, wherein A is a glucuronosyl residue linked through its 1 -oxygen to the phenyl ring of B; B is a benzyloxycarbonyl group, optionally ring- substituted with one or more electron withdrawing groups; and C is an inactivator of O6- alkylguanine-DNA alkyltransferase (AGT) linked to the carbonyl of B, or a pharmaceutically acceptable salt thereof. [0016] The glucuronosyl residue (A) can be any suitable enzymatically cleavable poly- oligo, or monosaccharide. For example, the glucuronosyl residue is a D-glucuronosyl residue. The hydroxyl groups of the glucuronosyl residue may be free or protected, e.g., by an ester group that is removed enzymatically or hydrolyzed spontaneously, such as an acetyl or mono-, di-, or trihaloacetyl protective group, with the halogen being fluorine or chlorine, or benzyl protective group. The protective group may be released to provide a substrate for the β-glucuronidase enzyme. The acid group of the prodrug may be converted to a salt such as a sodium salt. It has been shown that prodrugs of certain drugs, e.g., anthracyclines and nitrogen mustards, which contain a glucuronic acid residue linked through a self-immolating linker are cleavable by the β-glucuronidase enzyme. Leenders et al., Bioorg. Med. Chem. 1999, 7, 1597-1610; Houba et al., Br. J. Cancer 2001, 84, 550-557; Schmidt et al., Bioorg. Med. Chem. Lett. 1997, 7, 1071-1076; Lougerstay-Madec et al., Anti-Cancer Drug Des. 1998, 13, 995-1007; Florent et al., J. Med. Chem. 1998, 41, 3572-3581; Lougerstay-Madec et al., J. Chem. Soc. - Perkin Trans. 1 1999, 1369-1375; Leu et al., J. Med. Chem. 1999, 42, 3623-3628; and Schmidt et al., Bioorg. Med. Chem. 2003, U, 2277-2283. [0017] The electron withdrawing group can be present on the phenyl ring of B at any suitable position, for example, the meta, or preferably ortho, position relative to the oxygen linking B to the glucuronosyl residue. Any suitable electron-withdrawing group (B) can be employed. Examples of electron withdrawing groups include nitro, halo, alkylsulfonyl, cyano, trifluoroalkoxy, trifluoroalkylthio, alkylcarbonyloxy, nitroso, formyl, alkoxycarbonyl, alkylcarbonyl, thiol, sulfamoyl, alkylsulfamoyl, chloroalkyl, ammonium, hydroxyalkyl, phenyl, N,N-dialkylamino and its ammonium salt, and vinyl. [0018] For example, the electron withdrawing group may be selected from the group consisting of nitro, halo, C1-Cg alkylsulfonyl, cyano, trifluoro Ci-C6 alkoxy, trifluoro C1-C6 alkylthio, C1-C6 alkylcarbonyloxy, nitroso, formyl, C1-C6 alkoxycarbonyl, Ci-C6 alkylcarbonyl, hydroxy, thiol, sulfamoyl, C1-C6 alkylsulfamoyl, chloro C1-C6 alkyl, amino, hydroxy C1-C6 alkyl, phenyl, N5N-C1-C6 dialkylamino, and vinyl, and preferably selected from the group consisting of nitro, halo, methylsulfonyl, cyano, trifluoromethoxy, trifluoromethylthio, acyloxy, nitroso, formyl, methoxycarbonyl, acetyl, thiol, sulfamoyl, methylsulfamoyl, chloromethyl, ammonium, hydroxymethyl, phenyl, N,N-dimethylamino and it ammonium salt, and vinyl. The halo group can be fluoro, chloro, bromo, or iodo. A preferred electron-withdrawing group is nitro. A nitrobenzylphenoxycarbamate linker (Carl et al., J Med. Chem. 1981, 24, 479-480) has been shown for doxorubicin prodrugs (Florent et al., J. Med. Chem. 1998, 41, 3572-3581) and 5-fluoruracil prodrugs ( Lougerstay-Madec et al., J. Chem. Soc. - Perkin Trans. 1 1999, 1369-1375) to self-immolate efficiently once the respective glucuronides are cleaved. [0019] Any suitable inactivator of AGT can be employed in accordance with the present invention. Examples of inactivators of AGT include unsubstituted or substituted O6- benzylguanine (see formula below), C^-benzyl-S-azaguanine, O6-benzyloxy pyrimidine, and 2,4-diamino-Oδ-benzyloxy-5ι-triazine, preferably an unsubstituted or substituted O6- benzylguanine or an unsubstituted or substituted O6~benzyl-2'-deoxyguanosine. The inactivator of AGT is preferably linked through the N2-position to the carbonyl of B. [0020] The benzyl group of the substituted <9δ-benzylguanine, substituted 06-benzyl-2'- deoxyguanosine, substituted O^-benzyloxy pyrimidine, substituted Od-benzyl-8-azaguanine, and substituted 2,4-diamino-05-benzyloxy-,s-triazine may have one or more substituents, in place of hydrogen, selected from the group consisting of halogen, hydroxy, aryl, a C1-C8 alkyl substituted aryl, nitro, a polycyclic aromatic alkyl containing 2-4 aromatic rings wherein the alkyl is a C1-C6, a C3-Cg cycloalkyl, a C2-C6 alkenyl, a C2-C6 alkynyl, a Ci-C6 hydroxyalkyl, a C1-Cg alkoxy, a C2-C8 alkoxyalkyl, aryloxy, acyloxy, an acyloxyalkyl wherein the alkyl is C1-C6, amino, a monoalkylamino wherein the alkyl is C1-C6, a dialkylamino wherein the alkyl is Ci-C6, acylamino, ureido, thioureido, carboxy, a carboxyalkyl wherein the alkyl is C1-C6, cyano, a cyanoalkyl wherein the alkyl is C1-C6, C- formyl, C-acyl, a dialkoxymethyl wherein the alkoxy is C1-C6, an aminoalkyl wherein the alkyl is C1-C6, and SOn Ri wherein n=0, 1, 2 or 3, R1 is H, a Ci-C6 alkyl, or aryl. The term "aryl" stands for an aromatic ring of 6-14 carbon atoms, e.g., phenyl, naphthyl, biphenyl, anthracenyl, and the like. [0021] In accordance with an embodiment, the C^-benzylguanine or substituted O6- benzylguanine may also include a substituent at the 8- and/or 9-position; the R group in the formula below represents a substituent on the phenyl ring. [0022] The 8-position substituent (Ri) may be selected from the group consisting of hydrogen, halo, C1-C4 alkyl, C1-C4 hydroxyalkyl, thiol, C1-C4 alkylthio, trifluoromethylthio, C1- C4 thioacyl, hydroxy, C1-C4 alkoxy, trifluoromethoxy, methanesulfonyloxy, trifluoromethanesulfonyloxy, Ci-C4 acyloxy, amino, C1-C4 aminoalkyl, C1-C4 alkylamino, C1- C4 dialkylamino, trifluoromethylamino, ditrifluoromethylamino, aminomethanesulfonyl, C1-C4 aminoacyl, aminotrifluoromethylcarbonyl, formylamino, nitro, nitroso, C1-C4 alkyldiazo, Cs-C6 aryldiazo, trifluoromethyl, C1-C4 haloalkyl, halomethyl, C1-C4 cyanoalkyl, cyanomethyl, cyano, C1-C4 alkyloxycarbonyl, Ci-C4 alkylcarbonyl, phenyl, phenylcarbonyl, formyl, C1-C4 alkoxymethyl, phenoxymethyl, C2-C4 vinyl, C2-C4 ethynyl, and SOnR' wherein n is 0, 1 , 2, or 3 and R' is hydrogen, C1-C4 alkyl, amino, or phenyl; and the 9-position substituent (R3) may be selected from the group consisting of hydrogen, C1-C4 alkyl, C1-C4 aminoalkyl, C1-C4 hydroxyalkyl, C1-C4 alkylaminoalkyl, C1-C4 dialkylarninoalkyl, Ci-C4 cyanoalkyl, C1-C4 carbamoylalkyl, CrC4 pivaloylalkyl, C1-C6 alkylcarbonyloxy C1-C4 alkyl, C1-C4 alkoxycarbonylalkyl, ribose, 2'-deoxyribose, the conjugate acid form of a C1-C4 carboxyalkyl, and the carboxylate anion of a C1-C4 carboxyalkyl as the sodium salt. Tautomers may also be used, e.g., the substituent at the 8-position may be an oxo group instead of a hydroxy group. [0023] According to another embodiment, the inactivator of AGT may be an unsubstituted or substituted O^-benzyl-S-azaguanine, preferably linked through the N2- position to the carbonyl of B.

[0024] The Oδ-benzyl-8-azaguanine may also include a substituent R1 at the 9-position selected from the group consisting Of C1-C4 alkyl, Ci-C4 alkyloxycarbonyl C1-C4 alkyl, carboxy C1-C4 alkyl, cyano Ci-C4 alkyl, aminocarbonyl C1-C4 alkyl, hydroxy C1-C4 alkyl, and Ci-C4 alkyloxy C1-C4 alkyl. [0025] In another embodiment, the inactivator of AGT may be an unsubstituted or substituted O^-benzyloxy pyrimidine, preferably linked through the N2-position to the carbonyl of B. The C^-benzyloxy pyrimidine may also include a substituent at its 4- and/or 5- position.

[0026] For example, the 4-position substituent (R2) may be selected from the group consisting of hydrogen, halo, amino, C1-C4 alkyl, Ci-C4 hydroxyalkyl, thiol, C1-C4 alkylthio, trifluoromethylthio, C1-C4 thioacyl, hydroxy, Ci-C4 alkyloxy, trifluoromethoxy, methanesulfonyloxy, trifluoromethanesulfonyloxy, Ci-C4 acyloxy, Ci-C4 aminoalkyl, C1-C4 alkylamino, Ci-C4 dialkylamino, trifluoromethylamino, dMfluoromethylarrώio, aminomethanesulfonyl, amino Ci-C4 alkylcarbonyl, aminotrifluoromethylcarbonyl, formylamino, nitro, nitroso, Ci-C4 alkyldiazo, C5-C6 aryldiazo, trifluoromethyl, C1-C4 haloalkyl, cyanomethyl, C1-C4 cyanoalkyl, cyano, C1-C4 alkyloxycarbonyl, C1-C4 alkylcarbonyl, phenyl, phenylcarbonyl, formyl, C1-C4 alkoxymethyl, phenoxymethyl, C2-C4 vinyl, C2-C4 ethynyl, and SOnR' wherein n is 0, 1, 2, or 3 and R' is hydrogen, Ci-C4 alkyl, amino, or phenyl; and the 5-position substituent (Ri) may be NO2 or NO. [0027] A preferred example of the prodrug of the present invention is:

wherein R is H or , wherein Bn stands for benzyl, which may be optionally substituted as discussed above. [0028] The prodrugs may be used as pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulfonic acids. An example of arylsulphonic acid isp- toluenesulphonic acid. The carboxyl group of the prodrug may be converted to salts known to those skilled in the art, for example, a salt of an alkali metal (e.g., sodium or potassium), alkaline earth metal (e.g., calcium), or ammonium salt. The present invention further provides a pharmaceutical composition comprising a compound (or prodrug) described above and a pharmaceutically acceptable carrier. [0029] Generally, the prodrugs of the present invention as described above will be administered in a pharmaceutical composition to an individual afflicted with a cancer. Those undergoing or about to undergo chemotherapy can be treated with the prodrugs separately or in conjunction with other treatments, as appropriate. In therapeutic applications, compositions are administered to a patient in an amount sufficient to elicit an effective depression of AGT activity thereby potentiating the cytotoxicity of the chemotherapeutic treatment. An amount adequate to accomplish this is defined as a "therapeutically effective dose," which is also an "AGT inactivating effective amount." Amounts effective for a therapeutic or prophylactic use will depend on, e.g., the stage and severity of the disease being treated, the age, weight, and general state of health of the patient, and the judgment of the prescribing physician. The size of the dose will also be determined by the prodrug selected, method of administration, timing and frequency of administration as well as the existence, nature, and extent of any adverse side- effects that might accompany the administration of a particular prodrug and the desired physiological effect. It will be appreciated by one of skill in the art that various disease states may require prolonged treatment involving multiple administrations, perhaps using a series of different prodrugs of AGT inactivators and/or chemotherapeutic agents in each or various rounds of administration. [0030] Suitable chemotherapeutic agents administered in coordination with the prodrugs of the present invention include alkylating agents, such as chloroethylating and methylating agents. Such agents may be administered using techniques such as those described in Wasserman et al., Cancer, 36, pp. 1258-1268 (1975) and Physicians' Desk Reference, 58th ed., Thomson PDR (2004). For example, l,3-bis(2-chloroethyl)-l-nitrosourea (carmustine or BCNU, Bristol-Myers, Evansville, IN) may be administered intravenously at a dosage of about 40 mg/m2 when O6-benzylguanine is employed. Other alkylating agents may be administered in appropriate dosages via routes of administration known to skilled medical practitioners. [0031] Suitable doses and dosage regimens can be determined by conventional range- finding techniques known to those of ordinary skill in the art. Generally, treatment is initiated with smaller dosages that are less than the optimum dose of the prodrug. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. The present inventive method may involve the administration of about 0.1 μg to about 50 mg of one or more of the prodrugs per kg body weight of the individual. For a 70 kg patient, dosages of from about 10 μg to about 200 mg of the prodrug would be more commonly used, depending on a patient's physiological response, as determined by measuring cancer-specific antigens or other measurable parameters related to the tumor load of a patient. [0032] The prodrugs and compositions of the present invention may be employed in many disease states including life-threatening or potentially life-threatening situations. In view of the relatively less toxic nature of the prodrug, it is possible and may be felt desirable by the treating physician to administer some or substantial excess of the prodrug. Single or multiple administrations of the prodrug can be carried out with dose levels and pattern being selected by the treating physician. In any event, the pharmaceutical formulations should provide a quantity of AGT-inactivating prodrugs of the invention sufficient to effectively enhance the cytotoxic impact of the chemotherapy. [0033] The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral or local administration and generally comprise a pharmaceutically acceptable carrier and an amount of the active ingredient sufficient to reduce, and preferably prevent, the activity of the AGT protein. The carrier may be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the prodrug, and by the route of administration. [0034] The pharmaceutically acceptable carrier (or excipient) is preferably one that is chemically inert to the prodrug or the active compound and one that has no detrimental side effects or toxicity under the conditions of use. Such pharmaceutically acceptable carriers preferably include saline (e.g., 0.9% saline), Cremophor EL (which is a derivative of castor oil and ethylene oxide available from Sigma Chemical Co., St. Louis, MO) (e.g., 5% Cremophor EL/5% ethanol/90% saline, 10% Cremophor EL/90% saline, or 50% Cremophor EL/50% ethanol), propylene glycol (e.g., 40% propylene glycol/10% ethanol/50% water), polyethylene glycol (e.g., 40% PEG400/60% saline), and alcohol (e.g., 40% ethanol/60% water). A preferred pharmaceutical carrier is polyethylene glycol, such as PEG 400, and particularly a composition comprising 40% PEG 400 and 60% water or saline. The choice of carrier will be determined in part by the particular prodrug chosen, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention. [0035] The following formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intraarterial, intramuscular, interperitoneal, rectal, and vaginal administration are merely exemplary and are in no way limiting. The pharmaceutical compositions can be administered parenterally, e.g., intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration that comprise a solution of the prodrug dissolved or suspended in an acceptable carrier suitable for parenteral administration, including aqueous and non-aqueous, isotonic sterile injection solutions. [0036] Overall, the requirements for effective pharmaceutical carriers for parenteral compositions are well known to those of ordinary skill in the art. See Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622- 630 (1986). Such compositions include solutions containing anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The prodrug may be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol (for example in topical applications), or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants. [0037] Oils useful in parenteral formulations include petroleum, animal, vegetable, and synthetic oils. Specific examples of oils useful in such formulations include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral oil. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. [0038] Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-β-aminopropionates, and 2-alkyl- imidazoline quaternary ammonium salts, and (e) mixtures thereof. [0039] The parenteral formulations typically will contain from about 0.5% or less to about 25% or more by weight of the active ingredient or prodrug in solution. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit- dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze- dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets. [0040] Topical formulations, including those that are useful for transdermal drug release, are well known to those of skill in the art and are suitable in the context of the present invention for application to skin. [0041] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the prodrug dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the prodrug, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent. Capsule forms can be of the ordinary hard- or soft- shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and cornstarch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising a prodrug in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the prodrug, such excipients as are known in the art. [0042] The prodrugs of the present invention, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. The prodrugs are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of active prodrug may be about 0.01% to about 20% by weight, preferably about 1% to about 10% by weight. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such surfactants are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute from about 0.1% to about 20% by weight of the composition, preferably from about 0.25% to about 5%. The balance of the composition is ordinarily propellant. A carrier can also be included as desired, e.g., lecithin for intranasal delivery. These aerosol formulations can be placed into acceptable pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer. Such spray formulations may be used to spray mucosa. [0043] Additionally, the prodrugs may be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate. [0044] The concentration of the prodrugs of the present invention in the pharmaceutical formulations may vary, e.g., from less than about 1%, usually at or at least about 10%, to as much as 20% to 50% or more by weight, and may be selected primarily by fluid volumes, and viscosities, in accordance with the particular mode of administration selected. [0045] Thus, a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 100 mg of the prodrug. Actual methods for preparing parenterally administrable prodrugs will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science (17th ed., Mack Publishing Company, Easton, PA, 1985). [0046] It will be appreciated by one of ordinary skill in the art that, in addition to the aforedescribed pharmaceutical compositions, the prodrugs of the present inventive method may be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes. Liposomes may serve to target the prodrugs to a particular tissue, such as lymphoid tissue or cancerous hepatic cells. Liposomes can also be used to increase the half- life of the prodrug. Many methods are available for preparing liposomes, as described in, for example, Szoka et al, Ann. Rev. Biophys. Bioeng., 9, 467 (1980) and U.S. Patents 4,235,871, 4,501,728, 4,837,028, and 5,019,369. [0047] The present invention has applicability to the treatment of any type of cancer capable of being treated with an antineoplastic alkylating agent which causes cytotoxic lesions at the Opposition of guanine. Such cancers include, for example, colon tumors, prostate tumors, brain tumors, lymphomas, leukemias, breast tumors, ovarian tumors, lung tumors, Wilms' tumor, rhabdomyosarcoma, multiple myeloma, stomach tumors, soft-tissue sarcomas, Hodgkin's disease, and non-Hodgkin's lymphomas. [0048] The present invention further provides a method of enhancing the chemotherapeutic treatment of tumor cells in a mammal with an antineoplastic alkylating agent that causes cytotoxic lesions at the Opposition of guanine, which method comprises administering to a mammal an effective amount of the prodrug described above and administering to said mammal an effective amount of an antineoplastic alkylating agent which causes cytotoxic lesions at the Opposition of guanine. In a specific embodiment, the tumor cells are necrotic tumor cells which express a β-glucuronidase enzyme. For example, in ovarian, breast, stomach, lung, and bowel carcinomas - necrosis produces β-glucuronidase enzyme. [0049] The prodrug can be administered as part of prodrug monotherapy (PMT); Bosslet et al., Tumor Targeting 1995, /, 45-50; deGroot et al., Curr. Med. Chem. 2001, 8, 1093- 1122. If the tumor cell does not produce a β-glucuronidase enzyme, the enzyme can be administered to the patient, for example, by a targeted delivery. In an embodiment, the β- glucuronidase enzyme can be administered to the patient as a conjugate, such as a conjugate of a humanized F(ab')2 fragment of an anti-CEA antibody and of human β-glucuronidase (see, e.g., U.S. Patent 5,955,100) or alternatively, by the ADEPT system (U.S. Patent 5,935,995; Bagshawe et al., Br. J. Cancer 1988, 58, 700-703), which is a two-step system in which in a first step an antibody-enzyme conjugate (AEC) is injected intravenously. The AEC is retained in the tumor on account of its tumor selectivity, but excreted from healthy tissues in the course of 2-7 days. The prodrug injected intravenously in the second step is activated to give the drug in the tumor by the enzymatic activity of the AEC. As a consequence of the tumor specific activation, increased drug concentrations are observed in tumor and lower drug concentrations are observed in the healthy tissue in comparison with the standard therapy. The FMPA concept (fusion protein-mediated prodrug activation, U.S. Patent 5,935,995) works similarly to the ADEPT system, in which, instead of the xenogeneic and therefore immunogenic AEC, a nonimmunogenic human fusion protein is employed for the tumor selective prodrug activation. In the VDEPT system (vector-dependent enzyme prodrug therapy, U.S. Patent 5,935,995) a two-step recombinant DNA mixture, prodrugs are also activated in a tumor-selective manner after injection of a vector and expression of a structural gene which codes for an enzyme. [0050] The present invention also provides a method of enhancing the effect of an antineoplastic alkylating agent, which alkylates the O6 -position of guanine residues in DNA, in the chemotherapeutic treatment of cancer in a mammal, particularly a human. The method comprises co-administering to the mammal a cancer-treatment effective amount of an antineoplastic alkylating agent and a chemotherapeutic treatment-enhancing amount of a prodrug in accordance with the present invention. [0051] By "enhancing the effect of an antineoplastic alkylating agent" is meant that the antineoplastic alkylating agent has a greater effect in the presence of a prodrug of the present invention than in the absence of the prodrug. When an alkyltransferase acts on the inactivator released from the prodrug, it is inactivated and, therefore, is not able to act on the DNA in a cancerous cell that has been alkylated by the antineoplastic alkylating agent. Given that the alkyltransferase is not able to act on the alkylated DNA in a cancerous cell, the DNA in the cancerous cell is not repaired, thereby leading to death of the cancerous cell. [0052] By "coadministering" is meant administering the antineoplastic alkylating agent and the prodrug sufficiently close in time such that the prodrug can enhance the effect of the antineoplastic alkylating agent. In this regard, the prodrug can be administered first and the antineoplastic alkylating agent can be administered second, or vice versa. Alternatively, the prodrug and the antineoplastic alkylating agent can be administered simultaneously. In addition, a combination of prodrugs can be administered, and one or more of the prodrugs can be administered in combination with another agent useful in the treatment of cancer. [0053] The antineoplastic alkylating agent is administered in a dose sufficient to treat the cancer (e.g., cancer-treatment effective amount of an antineoplastic alkylating agent). Such doses are known in the art (see, for example, the Physicians' Desk Reference (2004)). For example, 1, 3 -bis(2-chloroethyl)-l -nitrosourea (carmustine or BCNU, Bristol-Myers, Evansville, IN) can be administered intravenously to a patient at a dosage of from about 150 to 200 mg/m2 every six weeks. Another alkylating agent, namely l-(2-chloroethyl)-3- cyclohexyl-1 -nitrosourea (lomustine or CCNU, Bristol-Myers), can be administered orally at a dosage of about 130 mg/m2 every six weeks. [0054] The prodrug is administered in a dose sufficient to enhance the effect of the antineoplastic alkylating agent (e.g., chemotherapeutic treatment-enhancing amount). A suitable dosage is that which will result in a concentration of the prodrug in the cancerous cells to be treated sufficient to deplete alkyltransferase activity, e.g., from about 10 nM to 200 nM intracellularly, which may require an extracellular concentration of from about 10 μM to 50 μM. The dose can be adjusted as necessary to enhance the effect of the antineoplastic alkylating agent. [0055] The prodrugs of the present invention are useful in enhancing the effect of any suitable antineoplastic alkylating agent that alkylates the Opposition of guanine residues in DNA. Examples of antineoplastic alkylating agents include chloroethylating agents. The most frequently used chloroethylating agents include l-(2-chloroethyl)-3-cyclohexyl-l- nitrosourea (CCNU, lomustine), l,3-bis(2-chloroethyl)-l -nitrosourea (BCNU, carmustine), l-(2-chloroethyl)-3-(4-methylcyclohexyl)-l-nitrosourea (MeCCNU, semustine), and l-(2- cliloroethyl)-3-(4-amino-2-methyl-5-pyrimidinyl)methyl-l-nit rosourea (ACNU). Such agents have been used clinically against tumors of the central nervous system, multiple myeloma, melanoma, lymphoma, gastrointestinal tumors, and other solid tumors (Colvin and Chabner. Alkylating Agents. In: Cancer Chemotherapy: Principles and Practice. Edited by B.A. Chabner and J. M. Collins, Lippincott, Philadelphia, PA. pp. 276-313 (1990); and McCormick et al., Eur. J. Cancer 26: 207-221 (1990)). Chloroethylating agents, which have fewer side effects and are currently under development include l-(2-chloroethyl)-3-(2- hydroxyethyl)- 1 -nitrosourea (HECNU), 2-chloroethylmethylsulfonylmethanesulfonate (Clomesone), and l-[N-(2- chloroethyl)-N-nitrosoureido]ethylphosphonic acid diethyl ester (Fotemustine) (Colvin and Chabner (1990), supra; and McCormick et al. (1990), supra). Methylating agents include Streptozotocin (2-deoxy-2-(3-methyl-3-nitrosoureido)-D- glucopyranose), Procarbazine (N-(l-methylethyl)-4-[(2- methylhydrazino)methyl]benzamide), Dacarbazine or DTIC (5-(3,3-dimethyl-l-triazenyl)- lH-imidazole-4-carboxamide), and Temozolomide (8-carbamoyl-3-methylimidazo[5.1-d]- 1 ,2,3,5-tetrazin-4-(3H)-one). [0056] Temozolomide is active against malignant melanomas, brain tumors and mycosis fungoides. Streptozotocin is effective against pancreatic tumors. Procarbazine is used to treat Hodgkin's disease and brain tumors. DTIC is used to treat melanoma and lymphomas (Colvin and Chabner (1990), supra; and Longo, Semin. Concol. 17: 716-735 (1990)). [0057] The antineoplastic alkylating agent can be administered by any route. Conventional means of administration are described in Wasserman et al. {Cancer 36: 1258- 1268 (1975)) and in Physicians' Desk Reference (2004). [0058] The inactivators of AGT, e.g., unsubstituted or substituted C^-benzylguanine, unsubstituted or substituted O5-benzyl-2'-deoxyguanosine, O^-benzyl-8-azaguanine, O6- benzyloxy pyrimidine, and (96-benzyloxy-5-triazine can be prepared by methods known to those skilled in the art; see, Moschel et al. patents, e.g., 5,091,430; 5,525,606; and 5,958,932, above. [0059] The prodrugs of the invention can be prepared by any suitable method. By way of illustration, prodrugs 1 and 2 can be prepared in accordance with the reactions shown in Scheme 1.

Scheme 1. Reagents and conditions: (i) phosgene, CH2Cl2/pyridine, O 0C to rt, 20 h; (ii) rt, 2 h; (iii) Me0H/2 N NaOH (1 : 1), O 0C, 30 min, followed by neutralization with 10% acetic acid. [0060] The starting material for prodrug 1 is O6-benzyl-9-[(pivaloyloxy)methyl]guanine (3) and the starting material for prodrug 2 is 3',5'-di-0-acetyl-06-berizyl-2'-deoxyguanosine (4). When 3 or 4 is reacted with an equivalent of phosgene, unstable intermediates, presumably the isocyanates 5 and 6, respectively, are produced. These are reacted individually without isolation with 4-<9-( 2',3',4'-tri-O-acetyl-6'-methyl- β-D- glucopyranuronosyl)-3-nitrobenzyl alcohol (7) in one pot to produce the respective coupled products 8 and 9. Deprotection of these derivatives with methanolic sodium hydroxide and neutralization with 10% acetic acid lead to the formation of viscous colloidal suspensions which are slowly suction-filtered to produce crude samples or either 1 or 2. Analytically pure samples of 1 and 2 are obtained after purification by Sephadex LH-20 column chromatography and lyophilization. [0061] Prodrugs containing other AGT inactivators can be prepared by an analogous method. For example, the inactivator such as a O6-benzyl-9-[(pivaloyloxy)methyl]-8- azaguanine can be reacted with phosgene. The 2-position substituent of the inactivator is preferably an amino group to enable the reaction with phosgene. [0062] The following examples further illustrate the present invention. The examples, of course, should not be construed as in any way limiting the scope of the present invention.

EXAMPLE 1 [0063] This example illustrates a method of synthesis of prodrugs in accordance with an embodiment of the invention. [0064] 3',5'-Di-0-acetyl-2'-deoxyguanosine was synthesized by the method of Schaller, et al, J. Amer. Chem. Soc, 1963, 55, 3821-3827. Previously unreported spectroscopic data for this compound are presented below. Unless otherwise stated, all other chemicals were obtained from Sigma or Aldrich and were used without further purification. Melting points were determined using an electrothermal apparatus and are uncorrected. 1H-NMR spectra were recorded in the indicated solvent with a Varian INOVA 400 MHz spectrometer. Chemical shifts are reported as δ values in ppm relative to TMS as internal standard. Mass spectra were obtained on a Thermo Finnigan TSQ Quantum LC mass spectrometer using electrospray ionization (ESI) and measuring either positive or negative ions. Elemental analyses were performed by Atlantic Microlab. [0065] S'^'-Di-O-acetyl-l'-deoxyguanosme. 2'-Deoxyguanosine (Syngen, Inc.) (5.03g, 17.6mmol) was twice evaporated from 3OmL anhydrous pyridine and was suspended in 20OmL dry pyridine. Acetic anhydride (30 mL, 317 mmol) was added and the mixture was stirred at room temperature for 3 days. The resulting suspended solid was collected by filtration and rinsed with ethyl ether to afford 5.73 g (93%) of 3',5'-di-O-acetyl-2'- deoxyguanosine. 1H-NMR δH(DMSO-d6) 10.67 (s, IH, H-I, exchanges with D2O), 7.91 (s, IH, H-8), 6.50 (s, 2H, N2H2 exchange with D2O ), 6.13 (dd, J= 6.0, J'=8.7, IH, H-I1). 5-30" 5.29 (m, IH, H-31), 4.29-4.24(m, IH, H-4'), 4.21-4.16(m, 2H, H-5'), 2.95-2.88(m, IH, H- l'α), 2.48-2.42(m, IH, H-2'β), 2.08 and 2.04 (two s, 6H, 2COCH3). [0066] 3',5'-Di-0~acetyl-06-benzyl-2'-deoxyguanosine (4) was prepared following the method of Zajc et al., Tet. Lett. 1992, 33, 3409-3412. To a mixture of 3',5'-di-0-acetyl-2'- deoxyguanosine (5.7Ig5 16.3mmol), triphenylphosphine (5.91g, 22.5mmol) and benzyl alcohol (2.5mL, 24.1mmol) in 10OmL 1,4-dioxane under argon was slowly added diisopropylazodicarboxylate (4.5mL, 22.8mmol). The mixture was heated to 85 0C for two hr and was then cooled and concentrated to a thick paste on a rotary evaporator. The product was isolated as a light brown solid (1.78g, 24.8%) following silica gel column chromatography with 10% ethyl acetate/chloroform. 1H-NMRδH(DMSO-d6) 8.09 (s, IH, H- 8), 7.51-7.35 (m, 5H3 Ar), 6.54 (s, 2H, N2H2, exchange with D2O)5 6.24 (dd, J= 6.0, J'=8.5, IH, H-I1), 5.50 (s, 2H, OCH2Ar), 5.33-5.32 (m, IH, H-31), 4.32-4.27 (m, IH, H-4'), 4.22-4.18 (m, 2H, H-5'), 3.06-2.98 (m, IH, H-2'α), 2.50-2.44 (m, IH, H-2'β ), 2.09 (s, 3H, COCH3), 2.02 (s, 3H, COCH3); MS, ni/z 442.1 [MH-H]+, 464.1 [M+Na]+; Anal. Calcd. for C21H23N5O6: C, 57.14; H, 5.25; N, 15.86; Found: C, 57.50; H, 5.31; N5 15.40. [0067] O6-Benzyl-7V2-[[[[4'-[(2M,3",4M-tri-O-acetyI-6M-methyI- β-JD- glucopyranuronosyI)oxy]-3'-nitrophenyl]methyI]oxy]carbonyl]- 9- [(pivaloyloxy)methyl] guanine (8). To an ice-cooled solution of O6-benzyl-9- [(pivaloyloxy)methyl]guanine (3) (Chae et al., J. Med. Chem., 1981, 24, 479-480) (1.97 g, 5.5 mmol) in 12OmL anhydrous dichloromethane and 5mL pyridine was added a toluene solution of phosgene (2.8 mL, 5.3 mmol phosgene) and the mixture was stirred for 20 hi while the ice bath was allowed to warm to room temperature. A solution of 4-6>-(2',3',4'-tri- O-acetyl-6'-methyl-β-D-glucopyranuronosyl)-3-nitrobenzyl alcohol (7) (Florent et al., J Med. Chem., 1998, 41, 3572-3581) (2.31 g, 4.8 mmol) in 150 mL dichloromethane was then added and the solution was stirred at room temperature for 2 hr. Purification by flash column chromatography (ethyl acetatexhloroform, 3:7) gave 8 (2.02 g, 42%). 1H-NMR δH(DMSO-d6) 10.63 (s,lH, N2H, exchanges with D2O), 8.29 (s, IH, H-8), 8.03 (d, IH, O2NArH-2'), 7.79 (dd, J= 8.8, J'=2.1, IH, O2NArH-6'), 7.59 (dd, 2H5 Ar), 7.46 (d, J8.7, IH, O2NArH-5'), 7.41-7.35 (m, 3H5 Ai-m,p), 6.08 (s, 2H, 9-CH2), 5.75 (d, J7.8, IH, H-I"), 5.61 (s, 2H, OCH2Ar)5 5.46 (t, J9.6, IH5 H-2"), 5.23 (s, 2H, O2NArCH2), 5.15-5.08 (m, 2H, H- 3",4"), 4.75 (d, J 9.9, IH, H-5"), 3.64 (s, 3H, CH3), 2.02-2.00 (3 s, 9H5 3 COCH3), 1.09 (s, 9H, C(CH3)3); MS m/z 867.5 [M+H]+, 889.4 [M+Na]+; Anal. Calcd. for C39H42N6O17: C5 54.04; H5 4.88; N, 9.70; Found: C, 54.17; H5 5.06; N5 9.64. [0068] 3t,5I-Di-O-acetyl-O6-benzyl-N2-[[[[4M-[(2'M,3'M,4'"-tri-O-ac etyl-6'"-methyl-β- 2)-glucopyranuronosyI)oxy]-3''-nitrophenyl]methyl]oxy]carbon yl]-2'-deoxyguanosine (9). Using the above procedure for compound 8, compound 9 was obtained in 54% yield. 1H-NMR δH(DMSO-d6) 10.56 (s, IH5 N2H, exchanges with D2O)5 8.39 (s, IH5 H-8), 8.02 (d, J= 2.1, IH5 O2NArH-2"), 7.78 (dd, J= 8.7, J'=2.1, IH5 O2NArH-6"), 7.55 (dd5 J= 8.1, J'=1.7, 2H5 Ar-o), 7.46(d5 J= 8.7, IH, O2NAiH-5")5 7.41-7.35(m, 3H, Ax-m,p), 6.35 (dd, J= 6.5, J'=7.5, IH, H-I1), 5.75 (d, J= 7.8, IH, H-I'"), 5.61 (s, 2H, CH2Ar)5 5.50-5.41 (m, 2H5 H- 2'" and H-3'), 5.22 (s, 2H5 O2NArCH2), 5.15-5.08 (m5 2H, H-3'",4"τ), 4.74 (d, J= 9.8, IH, H- 5'"), 4.36-4.31 (m, IH5 H-4')5 4.26-4.22 (m, 2H, H-51), 3.64 (s, 3H5 CH3), 3.29-3.20 (m5 IH, H-21 α), 2.54-2.51 (m, IH, H-2' β), 2.09-1.98(5s, 15H, 5 COCH3); MS m/z 953.2 [M-HH]+, 975.1 [M+Na]+; Anal. Calcd. for C42H44N6O20; C, 52.94; H5 4.65; N, 8.82; Found: C, 52.84; H, 4.70; N5 8.48. [0069] O6-Benxyl-N2-[[[[ 4'-[(- β-Z>-ghicopyranuronosyl)oxy]-3'- nitrophenyl]methyl]oxy] carbonyl] guanine, monosodium salt (1). To a ice-cooled solution of 8 (0.424 g, 0.49 mmol) in 10 mL methanol was added 10 mL of an ice-cooled solution of 2 M sodium hydroxide and the mixture was stirred at 0 0C for 30 min. The solution was neutralized with 10% acetic acid to produce a viscous suspension that was slowly suction filtered (Whatman #50 filter paper) and allowed to dry. The crude product was purified on a Sephadex LH-20 column eluted with H2O methanol (65:35) at a flow rate of 1 mL/min. UV absorption was continuously monitored at 254 nm and fractions (10 mL) were collected. The product (1) eluted in fractions 40-50. Methanol was removed on a rotary evaporator at room temperature. The resulting aqueous solution was lyophilized to provide 1 as a white solid (0.21Og, 64%). UV [0.05M phosphate buffer (pH7.0)] λmax= 267 Qs= 1.32X104 M^-cm"1); 1H-NMR δH(D2O, DSS internal standard) 8.00 (s, IH, H-8), 7.49 - 7.06 (m, 8H, Ax)5 5.36 (s, 2H, OCH2Ar), 5.04 (d, J= 6.9, IH, H-I"), 4.91 (s, 2H, O2NArCH2), 3.86 (d, J9.2, IH, H-5"), 3.68 - 3.61 (m, 3H, H-2",3", 4"); δH(DMSO-d6) 10.34 (s, IH, N2H, exchanges with D2O), 8.15 (s, IH, H-8), 7.97 (d, J= 2.1, IH, O2NArH-2!), 7.71 (dd, J= 9.0, J'=2.1 , IH, O2NArH-6'), 7.57 (dd, J= 8.2, Jf=I.6, 2H, Ar-o), 7.45 (d, J= 8.7, IH, O2NArH- 5'), 7.41-7.35 (m, 3H, Ar-m,p), 7.26 (broad s,lH, H-9, exchanges with D2O), 5.59 (s, 2H, O2NAJ-CH2), 5.23 (d, J= 4.6, IH, OH-2", exchanges with D2O), 5.18 (s, 2H, CH2Ar), 5.08 (d, J= 7.3, IH, H-I"), 5.01 (d, J= 4.5, IH, OH-3"), 3.47 (d, J= 10.0, IH, H-5"), 3.26 - 3.11 (m, 4H, OH-4" and H-2",3",4"); MS [LC (0.1 % HCOOH)] m/z 613.2 [M(acid form)+H]+, 635.2 [M(acid form)+Na]+; Anal. Calcd. for C26H23N5NaO12-2H2O: C, 46.57; H, 4.06; N, 12.53; Found: C, 46.52; H, 3.98; N, 12.33. [0070] 06-Benzyl-iV2-[[[[ 4"-[(β-D-glucopyranuronosyl)oxy]-3M- nitrophenyI]methyl]oxy]carbonyI]-2'-deoxyguanosine, monosodium salt (2). Using the above procedure for 1, compound 2, which eluted from the Sephadex LH-20 column in fractions 35-48 was obtained in 64% yield. UV [0.05M phosphate buffer (pH 7.0)] λmax 267 (ε = 1.97X104 M^-cnf1); 1H-NMR δH(DMSO-d6) 10.49 (s, IH, N2H, exchanges with D2O), 8.40 (s, IH, H-8), 7.97 (d, J= 2.1, IH, O2NArH-2"), 7.73 (dd, J= 8.8, J'=2.2, IH, O2NArH- 6"), 7.56 (dd, J= 8.2, J'=1.7, 2H, Ar-o), 7.48 (d, J= 8.8, IH, O2NArH-5"), 7.41-7.35 (m, 3H, Ar-iM5p), 7.19 (broad, IH, OH-2'", exchanges with D2O), 6.31 (t, J= 7.1, IH5 H-I'), 5.60 (s, 2H, O2NArCH2), 5.43 (d, J= 3.9, IH, OH-3', exchanges in D2O), 5.23 (d, J= 4.7, IH, OH-3'", exchanges with D2O), 5.20 (s, 2H, CH2Ax), 5.08 (d, J= 7.3, IH, H-I'"), 5.01 (d, J= 4.8, IH, OH-4'", exchanges with D2O), 4.89 (t, J= 5.4, IH, OH-5'), 4.41-4.39 (m, IH, H-3'), 3.87-3.84 (m5 IH, H-4'), 3.61 - 3.50 (m, 2H, H-5'), 3.47 (d, J= 9.9, IH, H-5'"), 3.29 - 3.11 (m, 3H, H-2'",3'",4'"), 2.75-2.68 (m, IH, H-2'α), 2.29-2.23 (m, IH, H-2' β); MS [LC (H2O/acetonitrile)] m/z 727.1[M-Na]-; Anal. Calcd. For C31H31N6NaO15-1.5H2O; C, 47.88; H, 4.41; N, 10.81; Found: C, 47.90; H, 4.29; N, 10.73.

EXAMPLE 2 [0071] This example illustrates the stability and enzymatic hydrolysis properties of the prodrugs in accordance with an embodiment of the invention. [0072] Prodrug purity of the prodrugs was determined by HPLC on a Hewlett-Packard LC 1090 Series II system equipped with a Phenomenex 250 x 4 mm column (5 μm particle size) eluted isocratically at 1 mL/min with acetonitrile/0.1 M triethylammonium acetate (TEAA), pH 7.0, (3:7). Aliquots (100 μL) from prodrug solutions were withdrawn and diluted with 100 μL of a solution of j?-nitrobenzyl alcohol (an internal standard) in acetonitrile/0.1 M TEAA (6:4). UV detection was at 254 and 280 run. Retention times for 1, 2, (96-benzylguanine, (96-benzyl-2'-deoxyguanosine and/>-nitrobenzyl alcohol were 5.20, 4.97, 7.13, 7.18 and 8.64 min, respectively. All determinations were carried out in duplicate or triplicate. [0073] The stability of the prodrugs was determined in phosphate buffered saline (pH 7.2) (Life Technologie, Inc), a Tris buffer containing 50 mM Tris-HCl (pH 7.5) (Life Technologies, Inc), 5 mM dithiothreitol and 0.1 mM EDTA, a MOPS buffer (pH 7.0) containing 50 mM morpholinopropane sulfonic acid, 0.01% bovine serum albumin and 0.01% NaCl and a modified Dulbecco's medium prepared by combining 400 mL of Dulbecco's medium with 7 mL of 7.5% NaHCO3, 4 mL of 15 mM glutamine, 2 mL gentamicin and 40 mL of fetal calf serum. Solutions were incubated at 37 0C and prodrug concentrations were analyzed by HPLC at various times as indicated above. [0074] Rate constants for the first-order disappearance of prodrugs were estimated from semi-log plots of the concentration of prodrug as a function of time. Observed first order rate constants for hydrolysis of these prodrugs are presented in Table 1. The spontaneous rate of liberation, for example, in aqueous solutions, of the AGT inactivator is much lower than the rate for β -glucuronidase catalyzed liberation, for example, about 10 times less, and preferably 100 times less. The spontaneous rate of liberation of the AGT inactivator from the prodrug is low, e.g., about 0.2 to about 0.5% by weight per hour.

Table 1. Observed First Order Rate Constants (x 106, min"1) for Hydrolysis of Prodrugs in Aqueous Buffers Prodrug feulbecco

1 3.86 4.06 3.77 4.29

2 0.646 0.155 0.188 0.468

a PBS = Phosphate buffered saline, pH 7.2; b Tris = 5OmM Tris-HCl (pH 7.5), 5mM dithiothreitol and 0.ImM EDTA, pH 7.5; c MOPS = 5OmM morpholinopropane sulfonic acid, pH 7.0, 0.1% bovine serum albumin, 0.01% NaCl; d Dulbecco = modified Dulbecco's medium.

[0075] The decomposition of 1 was faster than that of 2 in all buffers although decomposition rates for both compounds are fairly low. For example, under these aqueous conditions, the average half-time for decomposition of 1 is of the order of 100 days. [0076] The enzymatic hydrolysis of the prodrugs was investigated with both E. coli and bovine liver β-glucuronidase in MOPS buffer at pH 7.0. Prodrugs and p-nitrobenzyl alcohol (HPLC internal standard) were dissolved in the MOPS buffer (pH 7.0) at 37 0C. Enzymatic cleavage was initiated by adding 0.2 Fishman units of E. coli β-glucuronidase (Sigma type IX-A) (42.8 units/mg of protein) or 10 Fishman units of bovine liver β-glucuronidase (Sigma type B-IO) (10.2 units/mg of protein) to the incubation buffer. Aliquots (100 μL) of the reaction mixture were withdrawn at various times and were mixed with 100 μL of acetonitrile/O.lM TEAA (6:4) to quench the enzymatic reaction. Prodrug and product concentrations were determined by HPLC. For determination of enzyme kinetic parameters, prodrug solutions at concentrations between 5 and 300 μM were incubated with a fixed amount of enzyme. Aliquots were withdrawn at time intervals varying from 1 to 20 min and the reactions were quenched as described above. Initial reaction velocities were determined at each substrate concentration. Non-linear regression methods were used to determine KM and Vmax values. Data were processed with Prism 3.0 software. [0077] Representative data for hydrolysis of 1 and 2 with the bovine liver enzyme are presented in Figure IA. Hydrolysis by the E. coli enzyme is shown in Figure IB. As indicated, hydrolysis by both the bovine liver enzyme (10 units/mL) or the E. coli enzyme (0.2 unit/mL) led to rapid disappearance of both 1 and 2 accompanied by the formation of <96-benzylguanine and O6-benzyl-2'-deoxyguanosine, respectively. Although 1 and 2 were cleaved more rapidly by the E. coli protein than the bovine liver protein, and 1 was hydrolyzed more rapidly than 2 by both proteins, the hydrolyses followed Michaelis-Menten kinetics (Table 2) in all cases. These data (Table 2) are comparable to data for other glucuronic acid conjugates such as DOX-GA3 (Houba et al., Biochem. Pharmacol. 1996, 52, 455-463) and epicurubicin-glucuronide (Haisma et al., Br. J. Cancer 1992, 66, 474-478) although the testing conditions are different.

Table 2. Enzyme Kinetic Parameters for Prodrugs^ bovine liver β-glucuronidase Prodrug KM(μM) Vmax(μmol-mg ,-'l --IuT-K) V KM( Λμ. ΛM/T)l Λ V7max(μmol-m .g~-"l. --hi.-'h ) 1 17.0 4.66 xlO5 244 1.62xlO4 2 41.1 6.77x105 362 2.36xlO4

"Data obtained at 37 0C in MOPS buffer, pH 7.0.

EXAMPLE 3 [0078] This example illustrates certain properties of the prodrugs of the present invention, namely, their ability to inactivate AGT in the presence of β-glucuronidase, and cell killing. [0079] Purified recombinant human alkyltransferase was incubated with different concentrations of prodrugs in 0.5 mL of reaction buffer (50 mM Tris-HCl, pH 7.6, 0.1 mM EDTA, 5.0 mM dithiothreitol) containing 50 μg of hemocyanin for 30 min at 37 0C. For experiments involving β-glucuronidase, the bovine liver protein, prodrugs and alkyltransferase were incubated together in the above hemocyanin-containing buffer for 30 min at 37 0C. The remaining alkyltransferase activity was determined after incubation with a [3H] -methylated calf thymus DNA substrate for 30 min at 37 °C by measuring the [3H]- methylated protein formed, which was collected on nitrocellulose filters. The results were expressed as the percentage of the alkyltransferase activity remaining. The concentration of inhibitor that led to a 50% loss of alkyltransferase activity (EDs0) was calculated from graphs of the percentage of remaining alkyltransferase activity against inactivator concentration. [0080] For inactivation of the human O6-alkylguanine-DNA alkyltransferase, prodrugs 1 and 2 were inactive up to a concentration of 50 μM (Figure 2) while the ED50 for Oβ- benzylguanine is 0.2 μM (Pegg et al., Prog. Nucleic Acid Res. MoI Biol. 1995, 51, 167-223). This indicates that both 1 and 2 are intrinsically very poor alkyltransferase inactivators compared to <96-benzylguanine. However, incubation of these drugs in the presence of bovine liver β-glucuronidase for 30 min led to efficient alkyltransferase inactivation due to liberation of O6-benzylguanine from 1 (Figure 2A) or O6-benzyl~2'-deoxyguanosine from 2 (Figure 2B). [0081] HT29 cells were grown in RPMI 1640 medium in the presence of 10% fetal bovine serum. The effect of alkyltransferase inactivators on the sensitivity of cells to BCNU was determined using a colony-forming assay. Cells were plated at a density of 106 in 25 cm2 flasks and 24 h later were incubated with different concentrations of prodrugs for the time indicated before exposure to 40 μM BCNU for 2 hr. For experiments involving β- glucuronidase, the bovine liver protein was added to the cell cultures at 20 units/mL of medium and incubated along with the prodrug. BCNU was dissolved in absolute ethanol at a concentration of 8 mM. It was diluted with the same volume of ice-cold phosphate-buffered saline, and was immediately administered to cells. After two hr, the medium was replaced with fresh medium and the cells were left to grow for an additional 16-18 h. The cells were then replated at densities of 250 cells per 25 cm2 flask and grown for 8 days until discrete colonies had formed. The colonies were washed with 0.9% saline solution, stained with 0.5% crystal violet in ethanol, and counted. The plating efficiency of cells not treated with drugs was about 50%. [0082] HT29 cell killing by BCNU in combination with the prodrugs 1 and 2 is illustrated in Figure 3. Cells treated for 5 hr with increasing concentrations of prodrugs 1 and 2 were quite resistant to killing after a two-hour exposure to 40 μM BCNU. However, when cultures containing 1 were treated with bovine liver β-glucuronidase at 20 units/mL of medium for either 7 or 14 hr, the cells were greatly sensitized to killing by BCNU. Similarly, incubation of 2 in cell cultures containing β-glucuronidase at 20 units/mL for 14 hr also led to much greater cell killing by BCNU. These results were again a consequence of liberation of 06-benzylguanine from 1 or <96-benzyl-2'-deoxyguanosine from 2, respectively. [0083] These data suggest that if levels of β-glucuronidase secreted by necrotic human tumor cells are sufficiently high, the prodrugs 1 and 2 will be useful for selectively delivering <96-benzylguanine and <96-benzyl-2'-deoxyguanosine to tumor cells compared to normal cells. This would greatly improve chemotherapy for human tumors with the combination of alkyltransferase inactivators and either chloroethylating or methylating drugs since possible side effects associated with widespread systemic alkyltransferase inactivation would be significantly reduced.

[0084] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. [0085] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non- claimed element as essential to the practice of the invention. [0086] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.