O Antimetabolites

Antimetabolites are compounds closely related in structure to a cellular precursor molecule, yet these imposter substances are capable of preventing the proper use or formation of the normal cellular product. These antimetabolites are similar enough in structure in many cases to interact with the normal cellular process but differ in a manner sufficient to alter the outcome of that pathway. Most an-timetabolites are effective cancer chemotherapeutic agents via interaction with the biosynthesis of nucleic acids. Therefore, several of the useful drugs used in antimetabolite therapy are purines, pyrimidines, folates, and related compounds. The antimetabolite drugs may exert their effects by several individual mechanisms involving enzyme inhibition at active, allosteric, or related sites. Most of these targeted enzymes and processes are involved in the regulatory steps of cell division and cell/tissue growth. Often the administered drug is actually a prodrug form of an antimetabolite and requires activation in vivo to yield the active inhibitor. The administration of many purine and pyrimidine antimetabolites requires the formation of the nucleoside and finally the corresponding nucleotide for antimetabolite activity. An antimetabolite and its transformation products may inhibit several different enzymes involved in tissue growth. These substances are generally cell cycle specific with activity in the S phase.

The purine and pyrimidine antimetabolites are often compounds incorporated into nucleic acids and the nucleic acid polymers (DNA, RNA, etc.). The antifolates are compounds designed to interact at cofactor sites for enzymes involved in the biosynthesis of nucleic acid bases. The biosynthesis of these nucleic acid bases depend heavily on the availability of folate cofactors, hence antimetabolites of the folates find utility as antineoplastic agents. The antitumor effects of all these compounds attempting to masquerade as the natural precursor compound may occur because of a malfunction in the biosynthesis of the corresponding macro-molecules. Classic examples of pyrimidine and purine an-timetabolites are 5-fluorouracil and 6-mercaptopurine, respectively, and the classic antifolate is methotrexate.

The purine analog 6-mercaptopurine and its bioactivation products interact with many enzymes in the various stages of cell division.52 The activity of 6-mercaptopurine requires bioactivation to its ribonucleotide, 6-thioinosinate, by the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT). 6-Thioinosinate is a potent inhibitor of the conversion of 5-phosphoribosylpyrophosphate into 5-phospho-ribosylamine. The ribose diphosphate and triphosphates of 6-mercaptopurine are active enzyme inhibitors, and the triphosphate can be incorporated into DNA and RNA to inhibit chain elongation.

The pyrimidine analog 5-fluorouracil was designed based on the observation that in certain tumors, uracil was used for nucleic acid pyrimidine biosynthesis.53 Fluorine, at the 5-position of uracil, blocks the conversion of uridy-late to thymidylate, thus diminishing DNA biosynthesis. The fluorine induced increase in acidity (inductive effect) was expected to cause the molecule to bind more strongly to enzymes of the various stages of pyrimidine biosynthesis. Thymidine is a logical target, because it is only found in DNA and not in RNA. 5-Fluorouracil is activated by anabolism to 5-fluoro-2-deoxyuridylic acid, and this activated species is a strong inhibitor of thymidylate syn-thetase, the enzyme that converts 2'-deoxyuridylic acid to thymidylic acid.

Methotrexate inhibits the binding of the substrate folic acid to the enzyme dihydrofolate reductase (DHFR), resulting in reductions in the synthesis of nucleic acid bases, perhaps most importantly, the conversion of uridylate to thymidylate as catalyzed by thymidylate synthetase, which requires folate cofactors.

The pathways for the normal biosynthesis of the pyrimi-dine and purine bases are summarized in Schemes 10.15 and 10.16, respectively. The biosynthesis of pyrimidine and purine monomers is a central component for DNA and RNA formation in mammalian cells. The process of cell division and tissue growth is a very complex set of biochemical events with many feedback controls. Thus, antimetabolite drugs based on pyrimidine, purine, and related structures may exert their effects by altering many metabolic processes and pathways. The final step in Figure 10.15 illustrates the conversion of ribonucleotides to deoxyribonucleotides catalyzed by ribonucleotide reductase. The substrate for this enzyme is the ribonucleotide diphosphate, and the process is the same for both purine and pyrimidine bases.

Pyrimidine Drugs

The anticancer drugs based on pyrimidine structure are shown in Figure 10.8. The pyrimidine derivative 5-fluo-rouracil (5-FU) was designed to block the conversion of uridine to thymidine. The normal biosynthesis of thymidine involves methylation of the 5-position of the pyrimidine ring of uridine. The replacement of the hydrogen at the 5-posi-tion of uracil with a fluorine results in an antimetabolite drug, leading to the formation of a stable covalent ternary complex composed of 5-FU, thymidylate synthase (TS), and cofactor (a tetrahydrofolate species). The normal pathway for the formation of thymidine from uridine catalyzed by the enzyme TS is shown in Scheme 10.17. Anticancer drugs targeting this enzyme should selectively inhibit the formation of DNA because thymidine is not a normal component of RNA.

TS is responsible for the reductive methylation of de-oxyuridine monophosphate (dUMP) by 5,10-methylenete-trahydrofolate to yield dTMP and dihydrofolate.54 Because thymine is unique to DNA, the TS enzyme system plays an important role in replication and cell division. The tetrahy-drofolate cofactor species serves as both the one-carbon donor and the hydride source in this system. The initial step of the process involves the nucleophilic attack by a sulfhydryl group of a cystine residue at the 6-position of dUMP. The resulting enolate adds to the methylene of 5,10-CH2-THF perhaps activated via the very reactive N-5-iminium ion (see Scheme 10.17). The iminium ion likely forms at N-5 and only after 5,10-CH2-THF binds to TS.54

Deoxyuridine Diphosphate

Deoxyuridine diphosphate (dUDP) Uridine diphosphate (UDP)

Scheme 10.15 • Biosynthetic pathway for pyrimidine nucleotides.

Deoxyuridine diphosphate (dUDP) Uridine diphosphate (UDP)

Scheme 10.15 • Biosynthetic pathway for pyrimidine nucleotides.

The iminium ion is likely formed at N-5 because it is the more basic of the two nitrogens, whereas N-10 is the better leaving group. The loss of the proton at the 5-position of dUMP and elimination of folate yields the exocyclic methylene uracil species. The final step involves hydride transfer from THF and elimination to yield the enzyme, DHF, and dTMP.

5-Fluorouracil is activated by conversion to the corresponding nucleotide species,53 5-fluoro-2-deoxyuridylic acid (see Scheme 10.18). The resulting 5-fluoro-2'-de-oxyuridylic acid is a powerful inhibitor of thymidylate syn-thetase, the enzyme that converts 2'-deoxyuridylic acid to thymidylic acid. In the inhibiting reaction, the sulfhydryl group of TS adds via conjugate addition to the 6-position of the fluorouracil moiety (Scheme 10.19). The carbon at the 5-position then binds to the methylene group of 5,10-methylenetetrahydrofolate following initial formation of the more electrophilic form of folate the N-5-iminium ion. In the normal process, this step is followed by the elimination of dihydrofolate from the ternary complex, regeneration of the active enzyme species, and the product thymidine. Central to this process is the loss of the proton at the 5-position of uracil to form the exocyclic methylene uracil species. The 5-fluorine is stable to elimination, and a terminal product results, involving the enzyme, cofactor, and substrate, all covalently bonded (Scheme 10.19).

The chemical mechanism of inhibition of thymidylate synthetase by 5-fluorouracil is shown in Scheme 10.19. This process clearly shows that in order to inactivate the TS enzyme, both 5-FU and the tetrahydrofolate species are required to form the ternary complex. Some clinical studies have shown that administration of a tetrahydrofolate source prior to treatment with 5-FU results in greater inhibition of total TS activity. The administered source of active 5,10-methylenetetrahydrofolate is leucovorin, N-5-formyl-tetrahydrofolate.

TS is the most obvious and well-documented mechanism of action for 5-FU cytotoxic activity. However, other mechanisms may play a role in the overall value of this drug in the treatment of human cancer. The triphosphate of 5-FU

Tetrahydrofolate Pathway
Scheme 10.16 • Biosynthetic pathway for purine nucleotides. (THF, tetrahydrofolate.)
Pyrimidine Drugs
Figure 10.8 • Anticancer drugs based on pyrimidine and related compounds.

nucleotide is a substrate for RNA polymerases, and 5-FU is incorporated into the RNA of some cell lines. The incorporation of 5-FU into DNA via DNA polymerase occurs in some tissue lines even though uracil is not a common component of human DNA. The 5-FU in DNA likely serves as substrate for the editing and repair enzymes involved in DNA processing for cell division and tissue growth. The actual addition of 5-FU into RNA and/or DNA may not be the direct cytotoxic event, but the incorporation may lead to less efficient utilization of cellular resources. The significance of these various mechanisms on the overall cytotoxic effects of 5-FU may vary with cell line and tissue.

The metabolic activation (anabolism) of 5-FU required to produce the anticancer effects accounts for no more than 20% of the administered amount of drug in most patients. Catabolic inactivation via the normal pathways for uracil consumes the remaining approximate 80% of the dose. The major enzyme of pyrimidine catabolism is dihydropyrimidine dehy-drogenase (DPD), and 5-FU is a substrate for this enzyme.55 The DPD catabolism of 5-FU is shown in Scheme 10.20. The formation of dihydro-5-FU (5-FU-H2) occurs very rapidly and accounts for the majority of the total 5-FU dose in most patients. Thus, a-fluoro-^-alanine is the major human metabolite of 5-FU.56 Uracil is a substrate for this enzyme system also and has been dosed with 5-FU and 5-FU prodrugs in an attempt to saturate DPD and conserve active drug species. Variability in the levels of DPD activity among the patient population is a major factor in the bioavailability of 5-FU. Inhibitors of DPD such as uracil or 5-chloro-2,4-dihydroxypyridine (CDHP)53 increase the plasma concentration-time curve of 5-FU by preventing 5-FU catabolism. One mechanism of drug resistance in 5-FU-treated patients may be caused by increased levels of DPD in the target tissue. The observed low bioavailability of 5-FU as a result of the cata-bolic efficiency of DPD and other enzymes has lead to the development of unique dosing routes and schedules as well as the development of prodrug forms of 5-FU.53

Attempts at chemical modification of 5-FU to protect from catabolic events have produced several prodrug forms, which are converted via in vivo metabolic and/or chemical transformation to the parent drug 5-FU.57 Figure 10.8 shows the structure of the more common prodrug forms of 5-FU. The carbamate derivative of 5'-deoxy-5-fluorocytidine is known as capecitabine, and it is converted to 5-FU through a series of activation steps. The activation sequence is shown in Scheme 10.21. The initial step is carbamate hydrolysis followed by deamination, then hydrolysis of the sugar moiety to yield 5-FU. Some of these activation steps take place at a higher rate in tumor tissue leading to selective accumulation in those cells.53 The last step in the sequence shown in Scheme 10.21 is catalyzed by phosphoro-lases, and these enzymes occur in higher levels in colorectal tumors. Despite this complex activation process, capecitabine still exhibits some of the significant toxicities of 5-fluorouracil. The tetrahydrofuran derivative tegafur is slowly converted to 5-FU but requires quite high doses to reach therapeutic plasma concentrations. Esters of the N-hy-droxymethyl derivative of tegafur show greater anticancer activity than tegafur.58

Pyrimidine analogs as antimetabolites for cancer therapy have been developed based on the cytosine structure as well. Modification of the normal ribose or deoxyribose moiety has produced useful drug species such as cytarabine (ara-C) and gemcitabine, see Figure 10.8. Cytosine arabinoside (ara-C or cytarabine) is simply the arabinose sugar instead

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Chemistry Structure

Cys—Thymidylate Synthase

Scheme 10.17 • Biochemical conversion of uridine to thymidine.

Cytarabine Gemcitabine Mechanism
Scheme 10.18 • Metabolic activation of 5-fluorouracil to 5-FdUMP.
Fluorouracil Mechanism
Scheme 10.19 • Mechanism of inhibition of TS by 5-fluorouracil.
Dihydropyrimidine Dehydrogenase And

H2N H ot-fluoro-(3-alanine

Scheme 10.20 • Catabolic inactivation of 5-FU by dihydropyrimidine dehydrogenase.

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