Quinazoline Antifolate Thymidylate Synthase Inhibitors:  γ-Linked l-d, d-d, and d-l Dipeptide Analogues of 2-Desamino-2-methyl-N¹⁰-propargyl-5,8-dideazafolic Acid (ICI 198583)^,

Abstract

The syntheses of gamma-linked L-D, D-D, and D-L dipeptide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) are described. The general methodology for the synthesis of these molecules involved the preparation of the dipeptide derivatives employing solution phase peptide synthesis followed by condensation of the dipeptide free bases with the appropriate pteroic acid analogue via diethyl cyanophosphoridate (DEPC) activation. In the final step, tert-butyl esters were removed by trifluoroacetic acid (TFA) hydrolysis. Z-L-Glu-OBut-gamma-D-Ala-OBut, for example, was prepared from alpha-tert-butyl N-(benzyloxycarbonyl)-L-glutamate and tert-butyl D-alaninate via isobutyl-mixed anhydride coupling. The Z-group was removed by catalytic hydrogenolysis and the resulting dipeptide free base condensed with 2-desamino-2-methyl-N10-propargyl-5,8-dideazapteroic acid via DEPC coupling. Finally, tert-butyl esters were removed by TFA hydrolysis to give ICI 198583-gamma-D-Ala. The compounds were tested as inhibitors of thymidylate synthase and L1210 cell growth. Good enzyme and growth inhibitory activity were found with gamma-linked L-D dipeptides, the best examples being the Glu-gamma-D-Glu derivative 35 (Ki = 0.19 nM, L1210 IC50 = 0.20 +/- 0.017 microM) and the Glu-gamma-D-alpha-aminoadipate derivative 39 (Ki = 0.12 nM, L1210 IC50 = 0.13 +/- 0.063 microM). In addition, ICI 198583 L-gamma-D-linked dipeptides were resistant to enzymatic degradation in mice.

Inhibition of human folylpolyglutamate synthetase by diastereomeric phosphinic acid mimics of the tetrahedral intermediate

“…Our results show that caution must be used when interpreting the results of studies of the stereospecificity of FPGS, especially when one isomer has an especially low K m value. The occurrence of contaminating diastereomers of antifolate γ-dipeptides has been noted previously [34], but the biochemical consequences of these contaminants, especially with respect to FPGS activity, were not reported. Returning to the phosphinate-containing pseudopeptides, the two stereocenters of the final products were derived from, respectively, commercial L-glutamic acid to yield the 2S (L-) configuration at the N-terminal glutamate and a stereorandom synthesis of the C-terminal moiety.…”

“…Current evidence suggests that FPGS is highly stereospecific for L-Glu (or close L-Glu structural analogs); this evidence is direct for the position occupied by the intrinsic Glu (position 1; [25, 32, 33]), but indirect for the first γ-Glu (position 2; [34]). Thus, the data of Table 1 were unexpected because the low R f species, which presumably mimic the unnatural L-Glu-γ-D-Glu configurations, remain highly potent FPGS inhibitors.…”

Development and Binding Mode Assessment of N-[4-[2-Propyn-1-yl[(6S)-4,6,7,8-tetrahydro-2-(hydroxymethyl)-4-oxo-3H-cyclopenta[g]quinazolin-6-yl]amino]benzoyl]-l-γ-glutamyl-d-glutamic Acid (BGC 945), a Novel Thymidylate Synthase Inhibitor That Targets Tumor Cells

“…Importantly, an L-Glu-γ-D-Glu dipeptide moiety replaces the glutamate moiety present in other antifolates. 33, 34 Further intracellular polyglutamylation of TS antifolate inhibitors by folate polyglutamate synthase increases their affinity for TS. 33,34 The L-Glu-γ-D-Glu dipeptide moiety of 1 serves the same purpose as the polyglutamate tail, increasing affinity for TS through electrostatic interactions with the positively charged polyglutamate-binding groove on the enzyme.…”

Thymidylate synthase inhibition: A structure-based rationale for drug design

“…45,81,110 (b) Modifications to the glutamate portion of the molecule cause it to avoid the FPGS substrate activity but not the use of RFC to pass the cell membrane. [111][112][113][114][115] Two series of compounds have been designed starting from the structure of IC198583, conserving the quinazoline structure. 113 It was observed that modifications to the ␥-position of the glutamic acid residue in the dihydrofolate reductase inhibitor, MTX, resulted in compounds that are poor substrates or nonsubstrates for FPGS.…”