Krzysztof Pawelczak

Sulfamide antifolates inhibiting thymidylate synthase: synthesis, enzyme inhibition and cytotoxicity

Synthesis and biological evaluation are described of seven new analogues (3-9) of two potent thymidylate synthase inhibitors, 10-propargyl-5,8-dideazafolate (1) and its 2-methyl-2-deamino congener ICI 198583 (2). While the new compunds 3 and 4 were analogues of 1 and 2, respectively, containing a p-aminobenzenesulfonyl residue in place of the p-aminobenzoic acid residue, the remaining 5 new compounds were analogues of 4 with the L-glutamic acid residue replaced by glycine (5), L-valine (6), L-alanine (7), L-phenylglycine (8) or L-norvaline (9). The new analogues were tested as inhibitors of thymidylate synthases isolated from tumour (Ehrlich carcinoma), parasite (Hymenolepis diminuta) and normal tissue (regenerating rat liver) and found to be weaker inhibitors than the parent 10-propargyl-5,8-dideazafolic acid. Selected new analogues, tested as inhibitors of growth of mouse leukemia L 5178Y cells, were less potent than the parent 10-propargyl-5,8-dideazafolic acid. Substitution of the glutamyl residue in compound 4 with L-norvaline (9) resulted in only a 5-fold stronger thymidylate synthase inhibitor, but a 40-fold weaker cell growth inhibitor.

Thymidylate synthases from Hymenolepis diminuta and regenerating rat liver: purification, properties, and inhibition by substrate and cofactor analogues

Comparative studies of thymidylate synthases, isolated from the tapeworm, Hymenolepis diminuta, and regenerating liver of its host, rat, aimed at a possibility of specific inhibition of the helminthic enzyme, are presented. While similar in structure (dimers with monomer molecular masses of 33.7 kDa and 34.9 kDa, respectively) and parameters describing interactions with substrates and products, the tapeworm and rat enzymes differed in the dependences of reaction velocity on temperature (Arrhenius plots biphasic and linear, respectively). The tapeworm, compared with the host, enzyme was less sensitive to the competitive slow-binding inhibition by 5-fluoro-dUMP and its 2-thio congener, but equally sensitive to inhibition by 4-thio-5-fluoro-dUMP, N4-hydroxy-dCMP and N4-hydroxy-5-fluoro-dCMP, the latter being more potent inhibitor of the parasite enzyme than 5-fluoro-dUMP. alpha-Anomer of 5-fluoro-dUMP behaved as a very weak competitive slow-binding inhibitor of both enzymes. Both enzymes differed markedly in sensitivity to inhibition by 10-propargyl-5,8-dideazafolate and its di- and triglutamates (pddPteGlu1-3), with pddPteGlu1 being stronger inhibitor of the mammalian enzyme, but pddPteGlu3 showing opposite specificity. Sulfonamidobenzoylglutamate analogue of pddPteGlu (pddPteSO2Glu) and 2-desamino-2-methyl derivative of this analogue (CH3pddPteSO2Glu) were weaker inhibitors of both enzymes than the parent compound. Substitution of the glutamyl residue in CH3pddPteSO2Glu with either norvaline or alanine increased inhibition potency, whereas similar substitutions with glycine, valine or phenylglycine were without a distinct effect with the host enzyme but weakened inhibition of the tapeworm enzyme.

N-p-Amino- and N-p-nitro-phenylsulfonyl derivatives of dipeptides, a new family of ligands for copper(II). Potentiometric and spectroscopic studies

The co-ordination ability of four dipeptide analogues substituted on the N-terminal amino group with p-nitrophenylsulfonyl (nps-Ala-Ala and nps-Ala-His) and p-aminophenylsulfonyl (aps-Ala-Ala and aps-Ala-His) groups was studied by potentiometric and spectroscopic (UV/VIS absorption, CD and EPR) techniques. The N-terminal sulfonyl substituent drastically changes the acidity of the sulfonamide proton making nitrogen very efficient in binding to CuII. The sulfonamide nitrogen having pK between 9 and 11 does not need any anchoring binding group to form complexes with CuII. The para substituent on the phenyl ring (amino or nitro) influences very strongly the acidity of the sulfonamide proton. The nps or aps moieties change the co-ordination equilibria considerably when compared to the parent dipeptide Ala-His. Both groups enforce the formation of dimeric complexes, whereas in the case of the parent dipeptide the major species are only monomeric.

Binding ability of N-Para-amino-phenylsulfonyl derivatives of amino acids. Potentiometric and spectroscopic studies of Cu(II) complexes

Abstract N-Para-amino-phenylsulfonyl derivatives of amino acids are very effective ligands for Cu(II) ions. Potentiometric and spectroscopic results have shown that Cu(II) ions are able to deprotonate and bind to sulfonamide nitrogen below pH 5 to form stable mono- and bis-[N − , COO − ] chelates. The basicity of sulfonamide nitrogen is lower than peptide amide nitrogen and no distinct anchoring site is necessary to promote the amide nitrogen deprotonation.

Quinazoline Antifolate Thymidylate Synthase Inhibitors: Replacement of Glutamic Acid by Aminophosphonic Acids

The synthesis of six analogues of the potent thymidylate synthase (TS) inhibitor N -[4-[ N -[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinoyl)-methyl]- N -prop-2-ynylamino]benzoyl]- L -glutamic acid 2 is described in which the glutamic acid residue has been replaced by DL -aminophosphonic acids. New antifolates were tested as inhibitors of TS isolated from mouse L1210 leukemic cells as well as inhibitors of growth mouse leukemic L5178Y cells. In general these modifications result in compounds that are considerably less potent than 2 as TS inhibitors with K i s 0.17-1.10 w M. Very poor solubility in water limited their proper assay of growth cells inhibition.

An Improved Synthesis of t-Butyl N-(4-propargylaminobenzoyl)-γ-oligo (L-glutamate )s

Summary A simple synthesis in solution, of the series of six consecutive t-butyl N-(4-propargylaminobenzoyl)-yoligo( L-glutamate)s (8-13) containing from two to seven L-glutamic acid residues (Scheme 1) is described. a-t-Butyl N-(4-propargylaminobenzoyl)-L-glutamate (1), the versatile synthon is coupled with one of the t-butyl y-oligo(L-glutamate)s (2-7) by means of 2-chloro-l-methylpyridinium tosylate to give the title compounds in 91-95% yield subsequent to edulcoration of a post-reaction mixture and crystallization. These compounds were characterized by melting points, elemental analyses and HPLC and are 96.7-100.0% pure according to the last method (Table I).

Synthesis of N -(4-propargylaminobenzoyl)-L-glutamates

Summary To facilitate the rather labour consuming synthesis of antifolate γ-oligo-L-glutamates including those with the propargyl group at position N10 , two synthons possessing this group, α-t-butyl γ-methyl N-(4-propargylaminobenzoyl)- L-glutamate (1) and a-t-butyl N-(4-propargylaminobenzoyl)-L-glutamate (2) have been elaborated (Scheme 2) and characterized by melting points, elemental analysis, NMR, TLC and HPLC. They are of purity >98.5% by the last method.