NEW APPROACHES TO ANTIFOLATE CHEMOTHERAPY

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This laboratory has been engaged in a program of design, synthesis, and biological evaluation of derivatives and structural analogs of classical and nonclassical antifolates with the aim of generating new agents with improved pharmacological and therapeutic properties or a qualitatively altered spectrum of antitumor activity. The research involves collaborative interactions with a number of other groups sharing an interest in innovative approaches to antifolate drug discovery. A major goal of the work is to find compounds with increase ability to accumulate in tumor cells resistant to classical antifolates like methotrexate (MTX) by virtue of a defect in drug uptake. It is expected that cross resistance between such compounds and MTX will be low, and that they will have potential clinical utility against tumors with either natural or acquired MTX resistance based on defects in transport and/or polyglutamylation. Secondly, since an important form of MTX resistance is known to involve structural mutations in dihydrofolate reductase (DHFR) resulting in weak binding of classical antifolates of the MTX type, another goal of the work is to find compounds that will bind more tightly than MTX to these DHFR variants. Finally, since it has become widely recognized that inhibition of folate pathway enzymes other than DHFR, especially thymidylate synthase (TS) and glycinamide ribotide formyltransferase (GART), offers a powerful alternative approach to the selective killing of tumor cells and the circumvention of MTX resistance based on DHFR mutation or increased production of wild-type enzyme, a third goal of research is to generate compounds that inhibit these enzymes instead of, or in addition to, DHFR. An important lead already uncovered during this project has been the water-soluble nonclassical DHFR inhibitor N-alpha-(4-amino-4- deoxypteroyl)-N-delta-hemiphthaloyl-L-ornithine (PT523, NSC633713 ). This structurally unique antifolate is more potent than MTX against a broad range of cultured human solid tumor cells, including cells 10- to 30-fold resistant to MTX by virtue of either impaired transport or increased DHFR activity -- a level of MTX resistance typically associated with acquired resistance in patients. Greater potency and decreased growth has also been shown with PT523 in vivo against murine tumors and human tumor xenografts in athymic nude mice. Because PT523 lacks a glutamate side chain it cannot form polyglutamates; thus, unlike classical antifolates, it is not a prodrug and does not rely on polyglutamation to manifest its full impact on one-carbon metabolism. Moreover, unlike lipophilic nonclassical antifolates such as trimetrexate and piritrexim, PT523 retains activity against cells with the classical multidrug resistance (MDR) phenotype based on high P-glycoprotein expression; thus it is unlikely to be cross- resistant to a number of widely prescribed antineoplastic natural products such as anthracyclines, vinca alkaloids, podophyllotoxins camptothecins, and taxanes. On the basis of these very exciting results, a major focus of the next grant cycle will be the synthesis of second-generation analogs and targeted prodrugs of PT523 for in vitro/in vivo testing and SAR analysis. Structural modification of PT523 will feature changes in the side chain, aryl moiety, bridge, and B-ring. Targeted prodrugs will include peptide derivatives that are designed to release the parent drug in vivo after cleavage by tumor-targeted MoAb-conjugated peptidases. Efforts will also be made to discover analogs that bind well to mutant DHFRs that are insensitive to standard classical and nonclassical antifolates. The focus of this effort will be on the synthesis of molecules in which the bridge has been moved from the 6- to the 5- position of the B-ring. The longterm goal of this program is to discover and develop new and novel antifolates for the treatment of human cancer.