Amethopterine

Antimetaholites include 5- uorouracil [51-21-8] (153) and amethopterin [59-05-2] (154), a foHc acid antagonist, which produce sterility in female flies when fed at 0.01—0.05% in the diet. 

Amethopterin — see Folic acid, 4-amino-4-deoxy-10-methyl-Amicetin isolation, 3, 147 Amicibone properties, 7, 545 Amide alkaloids synthesis, 1, 470 Amides... 

Itoh, T., et al. Stereoselectivity of the folate transporter in rabbit small intestine studies with amethopterin enantiomers. Chirality 2001, 13, 164-169. 

D. Goldman, The characteristics of the membrane transport of amethopterin and naturally occurring folates. In Folate Antagonists as Chemotherapeutic Agents, Ann. N.Y. Acad. Sci. 186, 400 (1971). 

Amethopterin (Methotrexate, 4-amino-4-deoxy-7V -inethylpteroyl-L-glutaniic acid) [59-05-... 

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... 

Inhibitors. Aside from its role in providing reduced folate coenzymes for cells, this enzyme has attracted a great deal of attention because it appears to be a site of action of the important anticancer drugs methotrexate (amethopterin) and aminopterin.293 364 365 These compounds inhibit dihydrofolate reductase in concentrations as low as 10 8 to 10 9 M. Methotrexate is also widely used as an immunosuppresant drug and in the treatment of parasitic infections. 

Methotrexate (Amethopterin) is a folic acid antagonist that binds to dihydrofolate reductase, thus interfering with the synthesis of the active cofactor tetrahydrofolic acid, which is necessary for the synthesis of thymidylate, purine nucleotides, and the amino acids serine and methionine. 

Fig. 6.1. DNA synthesis in PHA-stimulated horse lymphocytes. Aliquots of 3x10 horse lymphocytes purified by the glass bead column method were incubated for various lengths of time with PHA-M in tubes in 3 ml Eagle s MEM (supplemented with 10 fig serine/ml and 7.5 fig glycine/ml and 10% autologous horse plasma). Before harvesting the cells were labelled for 6 h with [14C]thymidine (3.66 Ci/mol 1 fiCi/ml). o, Amethopterin (10 6M) was added 16 h before harvesting. , no amethopterin added. (Courtesy of Dr. B.S. Zain, 1971.)...

In practice synchronisation at the Gl/S boundary may be achieved by a number of reagents as well as thymidine, e.g. aminopterin, amethopterin, 5-fluorodeoxyuridine or hydroxyurea. 

On addition to cells these inhibitors rapidly bring about inhibition of DNA synthesis. Incorporation of [3H]deoxyuridine into DNA is reduced to 3% of control values within 15 min of addition of 10/xM aminopterin and even O.ljuM reduces incorporation to less than 20% of controls though this takes 45 min (Siegers et a]., 1975). Although concentrations of amethopterin between 0.1 and lfiM block synthesis of thymidylate, DNA synthesis continues at a significant rate and pools of thymidylate are slow to dissipate (Adams et al., 1971 Baumunk and Friedman, 1971 Fridland, 1974 Fridland and Brent, 1975). 

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. 

Siegers et al. (1974) showed that in the presence of amethopterin (10/j.M), hypoxanthine (30/iM) and glycine (100/iM) DNA synthesis could be followed by measuring the incorporation of tritiated thymidine present at 3-30/iM. The rate of incorporation was linear and calculations based on the specific activity of the tritiated thymidine agreed with the amount of DNA made measured by other methods. Moreover, the presence of amethopterin did not affect the cell proliferation rate, the cell cycle time or the duration of S-phase. 

Aminopterin and amethopterin (methotrexate) are 4-amino analogues of folic acid (Fig. 11.3) and interfere with the production of the active folate coenzyme by blocking the enzyme dihydrofolate reductase (reaction b in Fig. 13.1) (see also Fig. 11.6). 

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