Current Pharmaceutical Design - Volume 9, Issue 31, 2003

Antimetabolites are structural analogs of naturally occurring compounds. Antimetabolites interfere with the production of nucleic acids. They work through a variety of mechanisms including competition for binding sites on enzymes and incorporation into nucleic acids. The FdUMP metabolite of 5-FU can even act as a suicide inhibitor of thymidylate synthase. Some antimetabolites are prodrugs, i.e., they must be biochemically converted to their active form. Clinically useful antimetabolites ultimately inhibit DNA synthesis as their major anticancer mechanism although their site of action may be several steps removed. In some cases, e.g., 5-FU, inhibition of RNA processing may also be important. The relative importance of altered DNA versus RNA synthesis and function may be tumor-type dependent. Antimetabolites profoundly inhibit replication of bone marrow cells. Some cause even greater GI toxicity. As their parent category implies, these agents are highly cell cycle-specific when inhibition of DNA synthesis is the dominant effect. Because cells are far more sensitive to some antimetabolites while in the S phase, those antimetabolites are further categorized as phase-specific. There are three categories of antimetabolites: antifolates, purine analogs and pyrimidine antimetabolites. This issue of “Current Pharmaceutical Design” contains the text of five invited review articles. The selection of topics and authors was made with the intention of balancing reports derived from largely chemical expertise with discussions on the biological aspects of new drug development relating to antimetabolites. Science presented in these papers is state of art and will be of interest to those working in the corresponding field. The article by Professor Nair [1] will focus on the major classes of compounds that have been discovered as inhibitors of HIV integrase. The viral enzyme, HIV integrase, is one of three key enzymes of the pol gene of HIV. The various classes include nucleotides, oligonucleotides and dinucleotides. In his review Professor Antonin Holy [2] will discuss the synthetic approaches to acyclic nucleoside phosphonates (ANP). Major focus is placed on structure-activity relationships, biological activities, and mode of action of ANPs, with emphasis to selected drugs or drug candidates. A review on anticancer antifolates, authored by Dr. John McGuire, [3] describes the efforts to design more therapeutically selective antifolates than methotrexate (MTX), the only antifolate anticancer agent in clinical use to this date. Professor Roy Kisliuk, [4] with his article on deaza analogues of folic acid as antitumor agents, highlight on a group of compounds structurally related to deaza derivatives of aminopterin and folic acid. The review focus on deaza antifolates which are presently under clinical development and also on those less developed which represent novel approaches. Compounds are grouped according to their enzyme targets and also according to their membrane transport mechanism into cells. Lastly, the chapter written by me [5] outlines approaches to the development of thioguanine and mercaptopurine structurally related compounds which might find use in treating the cancer and HIV. The aim of this issue was to address the use of antimetabolite agents in drug discovary and development. I am sincerely grateful to Professor Cyril Parkanyi from Florida Atlantic University for his sincere help during the innovative steps of this work and also to the individuals who contributed to this body of work. All are experts in their fields. They devoted a large amount of time to the production of these in-depth reviews. Thanks to them all. References [1] Nair V. Novel Inhibitors of HIV Integrase: The Discovery of Potential Anti-HIV Therapeutic Agents. Curr Pharm Design 2003; 9(31): 2553-2565. [2] Holy A. Phosphonomethoxyalkyl Analogs of Nucleotides. Curr Pharm Design 2003; 9(31): 2567-2592. [3] McGuire JJ. Anticancer Antifolates: Current Status and Future Directions. Curr Pharm Design 2003; 9(31): 2593-2613. [4] Kisliuk RL. Deaza Analogues of Folic Acid as Antitumor Agents. Curr Pharm Design 2003; 9(31): 2615-2625. [5] Elgemeie GH. Thioguanine, Mercaptopurine: Their Analogs and Nucleosides as Antimetabolites. Curr Pharm Design 2003; 9(31): 2627-2642.

The viral enzyme, HIV integrase (MW 32 kDa), is one of the three key enzymes of the pol gene of HIV. HIV integrase is involved in the integration of HIV DNA into host chromosomal DNA. There is apparently no functional equivalent of this enzyme in human cells. Integration of HIV DNA into the host cell genome apparently occurs by a carefully defined sequence of DNA tailoring (3'-processing) and coupling (joining or integration) reactions. In spite of some effort in this area targeted at the discovery of therapeutically useful inhibitors of this viral enzyme, there are no drugs for HIV / AIDS in clinical use where the mechanism of action is inhibition of HIV integrase. It is clear that new knowledge on inhibitors of this enzyme is of critical importance in the anti-HIV drug discovery area. This review focuses on the major classes of compounds that have been discovered as inhibitors of HIV integrase. Some of these compounds are non-specific inhibitors of the enzyme while evidence suggests that others may possess some specificity. The various classes include nucleotides, oligonucleotides, dinucleotides, and miscellaneous small molecules including heterocyclic systems, natural products, diketo acids and sulfones. A major focus of the review is on discoveries from my laboratory in the area of non-natural, nuclease-resistant dinucleotide inhibitors of HIV integrase.

Acyclic nucleoside phosphonates (ANP) are analogs of nucleotides exhibiting various biological (e.g. antiviral, cytostatic, antiparasitic, immunomodulatory) activities. This review describes synthetic approaches to their preparation, biological activities, structure-activity relationships and mode of action of ANPs, with emphasis to selected drugs or drug candidates. Three compounds of the ANP group (cidofovir, adefovir, tenofovir) are active components of potent antivirals approved for therapeutic use in human medicine aimed at hepatitis B, AIDS and various diseases caused by DNA viruses.

Antifolates are the oldest of the antimetabolite class of anticancer agents and were one of the first modern anticancer drugs. The first clinically useful antifolate, described in 1947, was 2,4-diamino-pteroylglutamate (4-amino-folic acid; aminopterin; AMT) which yielded the firstever remissions in childhood leukemia. AMT was soon superseded by its 10-methyl congener, methotrexate (MTX), based on toxicity considerations; MTX remains, with one limited exception, the only antifolate anticancer agent in clinical use to this date. Because of the safety and utility of MTX, considerable effort has been invested in attempting to design more therapeutically selective antifolates or antifolates with a wider tumor spectrum. Initially, the design was based on the burgeoning knowledge of folate-dependent pathways and the determinants of the mechanism of action of MTX. These determinants include transport, the tight-binding inhibition of its target (the folate-dependent enzyme dihydrofolate reductase (DHFR)), and metabolism of MTX to poly-γ-glutamate (Glu n) metabolites. These early studies led to the development of other antifolate DHFR inhibitors of two types: (1) “classical” analogs that use the same cellular transport systems as MTX and are also metabolized to Glun; and (2) “nonclassical” (i.e., lipophilic) analogs that do not require transport systems and that are not metabolized to Glun. Although several of these analogs have undergone clinical trial, none is proved superior to MTX. Detailed examination of the mechanisms of cytotoxicity and selectivity of MTX showed that inhibition of both dTMP synthesis and de novo purine synthesis, secondary to DHFR inhibition, led to DNA synthesis inhibition and subsequent cell death; inhibition of other folate-dependent pathways did not appear necessary for cell death. Further studies showed that the contribution of inhibition of dTMP or purine synthesis to cell death varied in different cell types. These data suggested that inhibition of one of these pathways individually might (at least in some cases) be therapeutically superior to the dual inhibition induced by MTX. Thus in rational design and in structure-based design studies, two new classes of antifolate enzyme inhibitors were elaborated-direct inhibitors of thymidylate synthase (TMPS) and direct inhibitors of one or both of the two folate-dependent enzymes of de novo purine synthesis. Members of each class included both classical and nonclassical types. After preclinical evaluation, several of these have moved into clinical trials. To date only one new TMPS inhibitor has successfully completed clinical trials and been approved for routine use; this drug, Tomudex (D1694, raltitrexed) is currently approved only in Europe and only for the treatment of colon cancer. This still represents a step forward for antifolates, however, since MTX is well-known to be ineffective in colon cancer; thus Tomudex extends the tumor range of antifolates. Antifolate development continues. Based on the immense body of knowledge now extant on antifolates, specific aspects of the mechanism of action have been the focus. Newer antifolates have been described that inhibit more than one pathway in folate metabolism, that have improved delivery, or that inhibit other targets in folate metabolism. These new analogs are in various stages of preclinical and clinical development.

Derivatives of the vitamin folic acid function in the body for the synthesis of thymidylate, purines and amino acids and are necessary for normal metabolism and growth. Methotrexate (MTX), an inhibitor of dihydrofolate reductase (DHFR) is the outstanding example of an antitumor antifolate. MTX is clinically useful in the treatment of childhood leukemia, choriocarcinoma and psoriasis, where it corrects abnormal growth, and in rheumatoid arthritis and other autoimmune diseases where it corrects abnormal immune function. Since 1949, when the chemical synthesis of MTX was reported by workers at the Lederle Laboratories of the American Cyanamid Company, much has been learned about the basis of antifolate cytotoxicity and selectivity. This review will focus on deaza antifolates which are: 1) presently under clinical development and 2) less developed compounds which represent novel approaches. Compounds will be grouped according to their enzyme targets; DHFR , thymidylate synthase (TS) and glycinamide ribonucleotide formyltransferase (GARFT). In addition to inhibition of target enzymes, antifolate membrane transport into cells and conversion to poly-L-γ-glutamate forms are important considerations in drug design along with the reverse processes, cellular hydrolysis of antifolate poly- L-γ-glutamates to monoglutamates and the extrusion of the monoglutamates through the cell membrane. These processes can be modulated by competition with folates.

6-Mercaptopurine (6MP) and 6-thioguanine (6TG) are analogs of the natural purines: hypoxanthine and guanine. Both mercaptopurine and thioguanine are substrates for hypoxanthineguanine phosphoribosyltransferase and are converted into the ribonucleotides 6-thioguanosine monophosphate (6-thioGMP) and 6-thioinosine monophosphate (T-IMP) respectively. The accumulation of these monophosphates inhibits several vital metabolic reactions. Today, these thiopurine bases remain valuable agents for the induction and maintenance of remissions in patients with myelocytic and acute lymphocytic leukemia. Despite their proved clinical importance, 6MP and 6TG have certain therapeutic disadvantages, which have continued to stimulate the search for purine derivatives enhancing therapeutic efficacy. Considerable efforts have been made to prepare other novel mercaptopurine and thioguanine analogs and their nucleosides to improve the antitumor efficacy. The effectiveness of these thiopurines against certain tumor cell lines suggested that some of these mercaptopurine analogs and their nucleosides would be worthy of consideration in order to determine whether they exert a more selective effect against neoplastic cells than against normal cells or they might be useful in patients whose disease has become resistant to 6MP or 6TG. This review will focus on mercaptopurine analogs and their nucleosides as antimetabolite agents.