Aminopterin, methylation

Amethopterin (Methotrexate, 4-amino-4-deoxy-Ar -methyIpteroyl-L-glutamic acid) [59-05-2] M 454.4, m 185-204°(dec), [a]p°+19° (c 2, 0.1N aq NaOH). Commonest impurities are 10-methyl pteroylglutamic acid, 4-amino-10-methylpteroylglutamic acid, aminopterin and pteroylglutamic acid. Purified by chromatography on Dowex-1 acetate, followed by filtration through a mixture of cellulose and... 

The tetracyclines (Table 3.3) are a group of broad spectrum, orally active antibiotics produced by cultures of Streptomyces species. Chlortetracycline isolated from Streptomyces aureofaciens was the first of the group to be discovered, closely followed by oxytetracycline from cultures of S. rimosus. Tetracycline was found as a minor antibiotic in S. aureofaciens, but may be produced in quantity by utilizing a mutant strain blocked in the chlorination step b (Figure 3.54). Similarly, the early C-6 methylation step (included in a) can also be blocked, and such mutants accumulate 6-demethyltetracyclines, e.g. demeclocycline (demethylchlorotetracycline). These reactions can also be inhibited in the normal strain of S. aureofaciens by supplying cultures with either aminopterin (which inhibits C-6 methylation) or mercaptothiazole (which inhibits C-7 chlorination). Oxytetracycline from S. rimosus lacks... 

Aminopterin and amethopterin are 4-amino analogues of folic acid (Fig. 11.5) and as such are potent inhibitors of the enzyme dihydrofolate reductase (EC 1.5.1.3) (Blakley, 1969). This enzyme catalyses the reduction of folic acid and dihydrofolic acid to tetrahy-drofolic acid which is the level of reduction of the active coenzyme involved in many different aspects of single carbon transfer. As is clear from Fig. 11.6, tetrahydrofolate is involved in the metabolism of (a) the amino acids glycine and methionine (b) the carbon atoms at positions 2 and 8 of the purine ring (c) the methyl group of thymidine and (d) indirectly in the synthesis of choline and histidine. 

Figure 10.1 also shows the structures of the folate antagonist methotrexate (iV -methyl aminopterin) and the pterin coenzymes tetrahydrobiopterin (Section 10.4) and molybdopterin (Section 10.5). 

These two groups of compounds have different mechanisms for inducing sterility. The antimetabolites have been considered as compounds wherein a metabolite essential to cell development has been changed in one or several ways and, when introduced into an animal, will elicit signs associated with a specific lack of the metabolite (10y 11). Some of the more promising are methotrexate, aminopterin (N- p- [ (2,4-diamino-6-pteridyl)methyl]amino benzoyl glutamic acid), 5-fluorouracil, and S-fluoro-orotic acid. 

Jxiliies. The N -melhyl dcrivutivc of folic acid was found Mbc an antagonist, but it had no antitumor activity. Antitu-5 activity finally was found for the 4-amino-4-deoxy de- uuve.aminopterin. and its N "-methyl homologuc. metho-Ktalc (amcthoptcrin). - ... 

One of the first studies that indicated the possible interference of ametbopterin and aminopterin with purine synthesis was the demonstration by Skipper et al. (S21) that administration of these compounds to mice inhibited the incorporation of labeled formate into nucleic acid purines. Ametbopterin (4-amino-A -methyl-pteroylglutamic acid) has produced some inhibition of uric acid synthesis during the use of this compound for treatment of leukemia (K15, E24). Another inhibitor of... 

C.N.S. have been undertaken with other drugs. N-Methyl-aminopterine (Metho-threxate) has been applied intrathecally, but even then some places of the C.N.S. remain unaffected.This may explain why reports of successful intrathecal instillation with the relatively toxic Vinca alkaloids are still lacking. If local application is possible, good results may be expected for theoretical reasons, and this has been established by clinical tests. 

The isolation in the 1940s of the antianemic factor now known as folic acid and its subsequent synthesis resulted, after many false leads, in the first successful antivitamin antimetabolite aminopterin (Table 4-5). This compound produced sustained remissions in leukemia. The N10-methyl homolog, now designated as MTX (Table 4-5), superseded aminopterin the same year. It is still the only clinically significant antifolate carcinolytic drug. 

Methotrexate and aminopterin, a similar compound, are analogs of dihydro folate (DHF) and inhibitors of dihydrofolate reductase, an enzyme that converts DHF to tetrahydro-folate (THF). The thymidylate synthase reaction converts N, N °-methylenetetrahydro-folate to DHF in the process of methylating dUMP to form dTMP In the presence of one of the inhibitors, this reaction functions as a sink that reduces the THF level of the cell by converting THF to DHF. Since THF derivatives are substrates in two reactions of purine metabolism and one of pyrimidine metabolism, both pathways are affected by the inhibitor. 

It was found in early studies that the folic acid analogues, methotrexate (amethopterin) and aminopterin very effectively blocked the incorporation of labeled deoxyuridine and of labeled formate into DNA thymine however, the incorporation of thymidine was not blocked. It was apparent, therefore, that the analogues interfered with the introduction of the methyl group into thymine, a process known to involve H -folate. When it became established that the antifolic agents were exceedingly potent inhibitors of tetrahydrofolate dehydrogenase (see Chapter 5), the mechanism of their inhibition of DNA synthesis was apparent. 

Tetrahymena can utilize Aminopterin (4-amino-pteroylglutamic acid) and Methopterin (9-methyl pteroylglutamic acid) for growth, the former with about 17% efficiency and the latter with about 2% This was interpreted as showing the presence of enzymes for the deamination of position 4 of... 

The known role of vitamin B12 and folic acid in the formation of labile methyl groups (339) helps to explain the replaceability of folic acid (330) or vitamin B12 (331) by thymidine in certain deficient oi anisms. Also of interest is the role of folic acid compounds in the pathway leading to formation of the thymine methyl group. Low concentrations of a folic acid antimetabolite, aminopterin, blocked the utilization of deoxyiuidine for thymidine synthesis (333). The antimetabolite effects of aminopterin on the utilization of one-carbon donors have been known for some time (333, 334). The details of thymine biosynthetis will be discassed elsewhere in this volume (Chapter 24). 

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