Carroll Temple

Protonated state of methotrexate, trimethoprim, and pyrimethamine bound to dihydrofolate reductase

13C nuclear magnetic resonance (NMR) of methotrexate, trimethoprim, and pyrimethamine enriched 90% with 13C at C2 has provided a sensitive means of detecting the state of protonation of the heterocyclic rings of these inhibitors. In each case, protonation of N1 causes an upfield movement of the chemical shift of C2 by more than 6 ppm. By this method it has been shown that, at pH values up to 9.2, methotrexate is bound to bovine liver dihydrofolate reductase with N1 of the inhibitor protonated, just as in the case of the complex with reductase from Streptococcus faecium and Lactobacillus casei. Furthermore, trimethoprim bound to reductase from any of the three sources, and pyrimethamine bound to either of the bacterial reductases also have N1 protonated even at pH values up to 10. This implies that in all cases there is a strong interaction between protonated N1 of the inhibitor and the carboxylate group of the active site aspartate or glutamate. In every case pKa of the bound inhibitor is increased by several units, a finding in accord with crystallographic evidence that inhibitor bound to L. casei reductase is in a hydrophobic environment and that N1 is not hydrogen-bonded to water. It was confirmed by titration of protein fluorescence that trimethoprim has greater affinity for bacterial reductase than for vertebrate (bovine) reductase, and that this selectivity is more marked in ternary complexes in which NADPH is also bound to the active site. However, the data cited above indicate that this difference in affinities is not due to a weaker ionic interaction between protonated N1 of trimethoprim and the bovine enzyme. Instead, binding of the trimethoprim side chain to hydrophobic sites on the enzyme must provide less binding energy in the case of the mammalian enzyme.

Human thymidylate synthetase—III: Effects of methotrexate and folate analogs

Abstract The structure-activity relationship of human thymidylate synthetase (EC 2.1.1.45) was studied with two groups of folate analogs: (1) methotrexate (MTX) analogs modified at the glutamate residue and N 10 ; and (2) tetrahydrofolate (H 4 PteGlu) analogs modified at N 5 and N 10 . With respect to MTX analogs, it was found that: (1) substitution of the glutamate side chain by α-aminoadipic acid. α-aminopimelic acid or β-aminoglutaric acid slightly affects its K i ; (2) a free α-carboxyl group on the amino acid side chain of MTX, or any free carboxyl group in that vicinity plays an important role in the inhibitory potency of MTX analogs to the enzyme; (3)esterification or amidation of the α-carboxyl group of MTX decreases the inhibitory potency; and (4) free aspartyl or glutamyl conjugation through a peptide linkage to the γ-carboxyl group of the glutamate side chain decreases its K i to the enzyme by 5- and 8-fold respectively. Tetrahydrofolate analogs formed by inserting an ethylene, iminyl or a carbonyl bridge between the nitrogen at N 5 and N 10 or by substitution at the N 5 position were found to be poor inhibitors under our assay conditions.

A convenient procedure for purification of thymidylate synthase from L1210 cells.

Abstract Several laboratories have described procedures for purification of thymidylate synthase (TMP synthase) that utilize folates or folate analogs covalently attached to a matrix. The principle of separation is the formation of a ternary complex between dUMP, TMP synthase, and the bound ligand and the subsequent elution of the enzyme with buffers that do not contain dUMP. We have successfully used 10-formylfolic acid as the bound ligand for the purification of TMP synthase. As compared to other ligands that have been used, 10-formylfolic acid has the advantages that it can be easily synthesized, it is stable, and the enzyme is eluted as a sharp peak. Application of this procedure to L1210 leukemia cells gave 1765-fold purification of TMP synthase with a recovery of 39%. The native enzyme had a molecular weight of 78,000, which is about the same as that reported.

DEAZA ANALOGUES OF AMETHOPTERIN (METHOTREXATE)

The very strong affinity of 4-amino-4-deoxy analogues ( I and 11) of folic acid ( H I ) for dihydrofolic reductase,l-:’ which has been described as “stoichiometric” or “pseudoirreversible” inhibition of the enzyme,’ has been the subject of numerous and at least three explanations for this phenomenon have been advanced. Since the 2,4-diamino heterocycles are more basic than the 2-amino-4-0x0 heterocycles, Baker proposed that the increase in binding of the diamino compounds is due to protonated specie complexing with a donor cite on the enzyme.x This proposal has been amplified further by Pullman and coworkers,sq l 1 and Baker has presented experimental evidence to support it.“ Mathews and Huennekens believe that the increased binding is due to one additional hydrogen bond of aminopterin to the enzyme, this hydrogen bond being formed through the 4-amino group.12 Zakrzewski suggested that four hydrogen bonds between the enzyme and the four nitrogen functions of the 2,4-diaminopyrimidine moiety of these compounds could account for their strong binding.6* lR

Antitumor drug cross-resistance in vivo in a murine P388 leukemia resistant to ethyl 5-amino-1,2-dihydro-2-methyl-3-phenylpyrido[3,4-b]pyrazin-7-ylcarbamate 2-hydroxyethanesulfonate hydrate (NSC 370 147) 370 147

SummaryEthyl 5-amino-1,2-dihydro-2-methyl-3-phenylpyrido[3,4-b]pyrazin-7-ylcarbamate 2-hydroxyethanesulfonate hydrate (NSC 370 147) is a potent mitotic inhibitor, which has provided the basis for a candidate for clinical trial. As observed with clinically useful drugs, the development of clinical resistance to NSC 370 147 will probably be encountered. Information concerning resistance to NSC 370 147 should aid in the design of strategies for the opitmal clinical use of the drug. A P388 leukemia resistant to NSC 370 147 (P388/NSC 370 147) was isolated and its in vivo cross-resistance profile was determined. The P388/NSC 370 147 line was cross-resistant to vincristine but was not cross-resistant to doxorubicin, etoposide, cisplatin, melphalan, methotrexate, or 5-fluorouracil. This information plus other in vivo cross-resistance data [Waud et al. (1990) Cancer Res 50: 3239] suggests that NSC 370 147 may be useful in non-cross-resistant combinations with doxorubicin, melphalan, cisplatin, or methotrexate. The lack of cross-resistance of P388/NSC 370 147 to doxorubicin and etoposide shows that resistance to NSC 370 147 does not involve multidrug resistance and suggests that themdr1 gene is not involved in resistance to NSC 370 147.

SYNTHESIS OF IMIDAZO(4,5‐B)PYRIDINES AND V‐TRIAZOLO(4,5‐B)PYRIDINES. PREPARATION OF 1‐DEAZA‐6‐THIOGUANINE ANALOGS

Aus den 2,6-Diamino-3-nitro-4-chlorpyridinen (I) entsteht mit Ameisensaure das 4-Pyridon (II), aus dem das Triamin (III) erhalten wird.

Rearrangement of v-triazolo[4,5-d]pyrimidines

The Dimroth and retro-Dimroth rearrangement of a v-triazolo[4,5-d]pyrimidine system is described.