4.3.1 History. Antifolates interfere at various points in the process (folic acid metabolism) that provides the one-carbon unit required to convert deoxyuridine monophosphate to thymidylic acid for synthesis of the pyrimidines (Fig. 2.7). They are also key intermediates in the glycinanide ribonucleotide (GAR) -formyltransferase- and aminoimida-zole carboxamide ribonucleotide (AICR)-

formyltransferase-mediated construction cf the purines (276). The first antifolate used clinically was aminopterin (64) and was rapidly followed by methotrexate (65), which was registered for clinical use in 1953. These "classical" (glutamate-containing) antifolates bind tightly to the enzyme dihydrofolate reductase (DHFR; Fig. 2.7). Methotrexate has been very widely used and has been extensively reviewed (277,278). A more recent classical antifolate is the 10-ethyl analog edatrexate (66). This was developed following observations that 1-alkyl analogs showed better relative uptake into tumor tissue, and edatrexate shows enhanced uptake, retention, and polyglutamate formation in tumor cells (279). Whereas edatrexate binds to DHFR similarly to methotrexate, it showed better activity in animal tumor models (280), including models resistant to methotrexate (281). Resistance to methotrexate arises in several ways, the most important of which are elevation of DHFR levels and lowering of both folate transport and polyglu-tamylation activities (282).

The enzyme thymidylate synthase (TS) is also intimately involved in folate metabolism, catalyzing the reductive methylation of deoxyuridine monophosphate (dUMP) to thymidylate (dTMP), a reaction in which N5 fli0-methylenetetrahydrofolate is a cofactor*(Fig. 2.7). Whereas the pyrimidine-binding site on

Table 2.3 Antimetabolites Used in Cancer Chemotherapy

Generic Name (Structure)

Trade Name


Folic acid analogs methotrexate (65) edatrexate (66) raltitrexed (68) permetrexed (69) trimetrexate (70) piritrexim (71) nolatrexted (72)

Pyrimidine analogs

5-fluorouracil (73) cytosine arabinoside (74) gemcitabine (75)

Purine analogs

6-mercaptopurine (76) 6-thioguanine (77) fludarabine (78) 2'-deoxycoformycin (79) 2-chloro-2' deoxyadenosine (80)

Tomudex NeuTrexin



Cytosar Gemzar







US BioScience

Burroughs-Wellcome Zarix


Pharmacia & Upjohn Lilly

Burroughs-Wellcome Glaxo-Wellcome Berlex Laboratories Supergen Inc


Chemical Class folate analog folate analog

Chemical Class folate analog folate analog pyrimidine pyrimidine pyrimidine

pyrimidine pyrimidine pyrimidine

Antifolate Fpgs
N5,N10-methylene-tetrahydrofolic acid

Figure 2.7. Folate biosynthesis.

Figure 2.7. Folate biosynthesis.

the Ts enzyme has been a major target for anticancer drugs such as 5-fluorouracil (see Section 4.4), it also has a folate-binding site that has been a target for drug development. Methotrexate itself binds weakly to this site, and can exercise cytotoxicity through TS inhibition in cells that highly overexpress DHFR The design of highly specific inhibitors of the folate binding site of TS led initially to the quinazoline derivative CB 3717 (67) (284, 285). This proved to be a tight-binding inhibi tor of TS {K{ 4.5 nM), with 10-fold selectivity over DHFR, with the ability to undergo poly-glutamylation in cells to metabolites that are more potent and more selective for TS over DHFR (286). CB 3717 showed some activity in a number of phase 1/11 clinical trials, but severe nephrotoxicity, caused probably by precipitation of drug in the kidneys (287), led to its withdrawal (288).

Raltitrexed (68;tomudex) is another "classical" folic acid derivative that exerts its ther-

apeutic effect through by inhibition of the folate site of TS (289). It is polyglutamylated in cells into metabolites that are more potent inhibitors of TS than the parent drug and are retained in cells. Raltitrexed showed activity in a number of tumor types in phase I/II trials, but a major use may be in colon cancer. Here it shows activity similar to 5-fluorouracil (response rates of 14-19%) but with lesser toxicity (290), although the results of a recent phase II/III trial question this (291).

4.3.2 Mechanism and SAR. Methotrexate (65), introduced in 1953, and the related ami-nopterin (64) bind to DHFR, preventing transfer of the one-carbon unit from dihydro-folic acid to methylenetetrahydrofolic acid and ultimately to thymidine (Fig. 2.7). Methotrexate is taken into cells by the folate transporter and converted in cells to active polyglutamate metabolites by folylpolyglutamate synthase (297); this also has the effect of trapping the drug in cells (279).A large amount of work has


Permetrexed (69; MTA) also has TS as a major target, with DHFR and glycinamide ribonucleotide formyltransferase (GARFT) being important secondary sites of action Permetrexed is an excellent substrate for FPGS, and it and its polyglutamylated metabolites are potent inhibitors for all of the above enzymes (293). Permetrexed performed well in the human tumor-cloning assay against colorectal (32% of cell lines inhibited) and non-small-cell lung cancer (25% of cell lines inhibited) (294). It showed broad antitumor activity in phase II trials with breast, colon, pancreatic, bladder, head-and-neck, and cervical carcinomas, and non-small-cell lung cancer, both as a single agent and in combination with agents such as gem-citabine and cisplatin, and it is in phase III evaluation (295, 296).

been done to delineate the SAR for 2,4-diamin-opteridines binding to DHFR, but no clinical successor to methotrexate as a DHFR inhibitor has yet been found among the "classical" antifolates (287), although edatrexate (66) is still in development. Because there is also a folate site on TS, these compounds have some level of binding to this as well. CB 3717 and raltitrexed were designed specifically to target TS rather than DHFR, whereas permetrexed is closer to a general folate pathway inhibitor.

10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.

Get My Free Ebook

Post a comment