Graham M.F. Bisset

The measurement of polyglutamate metabolites of the thymidylate synthase inhibitoR, ICI D1694, in mouse and human cultured cells

A method is described for the measurement of the polyglutamates of the quinazoline thymidylate synthase inhibitor, N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin- 6-ylmethyl)-N-methylamino]-2-theonyl)-L-glutamic acid (ICI D1694). This involved incubation of cells with [5-3H]ICI D1694, extraction of the polyglutamates and their analysis by HPLC using an ion-pairing method. Co-chromatography with ICI D1694 and its synthetic di-hexaglutamate standards (UV detection) aided identification of the [3H]polyglutamates in the fractions recovered from the HPLC. Recovery of the polyglutamates at each stage of extraction and analysis was very good (77-84% overall recovery). Polyglutamates readily accumulated as the tri-, tetra and penta forms and occasionally a small amount of hexaglutamate was found. After mouse L1210 leukemia or human W1L2 lymphoblastoid cells were incubated for 30 min with 0.1 microM [3H]ICI D1694 there was a approximately 6-fold concentration effect intracellularly with most of the 3H associated with polyglutamate forms (approximately 75% and 96% for the L1210 and W1L2, respectively). Even some of the higher chain length tetra- and pentaglutamates could be detected at this time. After 4 hr incubation the total level of intracellular 3H had risen to 2-3 microM, greater than 96% of which was associated with polyglutamates (mainly tetra- and pentaglutamates). Four other human cell lines, two ovarian (CH1 and 41M), the MCF-7 breast and the HT-29 colon, were examined for their ability to form intracellular polyglutamates. A 4 hr incubation with 0.1 microM [3H]ICI D1694 resulted in a substantial intracellular accumulation of the drug (20-100-fold) in its polyglutamate forms with only 2-20% remaining as the parent monoglutamate, depending on the cell line. The major polyglutamate was again cell line dependent, ranging from the tri to the penta form. Prolonging the incubation time to 24 hr allowed a further accumulation of drug with a larger percentage appearing as tri- to hexaglutamates. Although cell lines differed in the total level of polyglutamates formed and the pattern of chain length observed, rapid and extensive polyglutamation of ICI D1694 occurred in all the cell types examined.

Synthesis of novel quinazoline-based antifolates with modified glutamate side chains as potential inhibitors of thymidylate synthase and antitumour agents

Abstract Several novel antifolates, derivatives of 2-desamino-2-methyl- N 10 -propargyl-5,8-dideazafolic acid, were synthesised as inhibitors of thymidylate synthase (TS) and antitumour agents. This was accomplished by first developing routes to the key intermediates Glu-Ome-γ-ψ[CSNH]Glu(OEt)-OEt ( 8 ), Glu-OBu t -γ-ψ[CH 2 NH]Glu(OBu t )-OBu t ( 16 ), Glu-OMe-γ-ψ[CN 4 ]Gly-OMe ( 23 ) and its 2,5-disubstituted regioisomer ( 22 ), followed by DEPC coupling to 4-[ N -[3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)-methyl]- N -prop-2-ynylamino]benzoic acid ( 9 ) or 4-[ N -[(3,4-dihydro-2,7-dimethyl-4-oxo-6-quinazolinyl)-methyl]- N -prop-2-ynylamino]-2-fluorobenzoic acid ( 24 ), and finally removal of the protecting groups. The resulting quinazoline-based antifolates with modified glutamate side chains, and in particular, the tetrazole derivatives 26 and 29 displayed potent TS and L1210 cell growth inhibitory activities ( e.g. , for 26 : TS IC 50 = 2.4 nM, L1210 IC 50 = 1.3 μM).

Carboxylic acid bioisosteres of γ-linked dipeptide analogues of the folate-based thymidylate synthase (TS) inhibitor, 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583)

Abstract Tetrazole carboxylic acid bioisosteres of γ-linked dipeptide derivatives of 2-desamino-2-methyl-N 10 -propargyl-5,8-dideazafolic acid (ICI 198583), a folate-based inhibitor of thymidylate synthase (TS), were synthesised by multistep routes starting from the appropriate pteroic acid analogue and Z- d -Ala or d -Glu. They exhibited excellent TS inhibitory activities which, however, were not accompanied by a parallel improvement in the L1210 cell growth inhibition.

Convenient Preparation of α-Tert-Butyl N-Blocked Glutamates Through γ-Allyl Ester Protection

Abstract Utilisation of γ-allyl ester protection of glutamates provides a convenient route to the synthesis of Z-Glu-OBut, Tr-Glu-OBut and (Boc)2-Glu-OBut.

Quinazoline Antifolates Inhibiting Thymidylate Synthase: Synthesis of γ-Linked Peptide and Amide Analogues of 2-Desamino-2-Methyl-N10-Propargyl-5,8-Dideazafolic Acid (ICI 198583)10-Propargyl-5,8-Dideazafolic Acid (ICI 198583)

Thymidylate Synthase (TS) inhibitors such as ICI D16941 and to lesser extent ICI 1985832 rely on their poly-γ-glutamate metabolites for their antitumour activity. The polyglutamate metabolites are more potent inhibitors of TS than the parent monoglutamate forms and in addition, their polyionic nature leads to prolonged retention within the cells. However, drugs dependent on polyglutamation may have some disadvantages such as a) lack of activity in tumours expressing low levels of, or an altered expression of, folylpolyglutamate synthetase (FPGS) or b) prolonged normal tissue toxicities caused by polyglutamate retention. For these reasons, we were interested in designing and synthesising tight-binding TS inhibitors which would not be dependent on polyglutamation for antitumour activity. Addition of one glutamate residue on the γ-carboxyl of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583), i.e. dipeptide (1), resulted in stronger binding to TS by approximately 30-fold2. This finding was the starting point in our search for a tight TS inhibitor that could not be a substrate for FPGS. We report here the synthesis of 22 dipeptide and 6 γ-amide analogues of ICI 198583-γ-L-Glu.

Synthesis of a tetrafluorinated analogue of the quinazoline antifolate 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid

Abstract A synthesis of a tetrafluoro derivative of the quinazoline antifolate 2-desamino-2-methyl- N 10 -propargyl-5,8-dideazafolic acid is described. 2-Desamino-2-methyl- N 10 -propargyl-2′,3′,5′,6′-tetrafluoro-5,8-dideazafolic acid ( 1 ) has been made in four stages from 4-amino-2,3,5,6-tetrafluorobenzoic acid ( 2 ). The reaction of 2 with thionyl chloride followed by diethyl L -glutamate gave N -(4-amino-2,3,5,6-tetrafluorobenzoyl)- L -glutamate ( 3 ). Condensation of 3 with propargyl bromide using caesium carbonate as base gave diethyl N -[2,3,5,6-tetrafluoro-4-(prop-2-ynylamino)benzoyl]- L -glutamate ( 4 ) which in turn reacted with 6-bromomethyl-3,4-dihydro-2-methyl-4-oxoquinazoline using the same base to give the diethyl ester 5 of 1. Finally, base-catalysed hydrolysis of 5 gave the target product. The sequence differs from other reported syntheses of quinazoline antifolates most notably in the use of an aminobenzoic acid derivative rather than a nitrobenzoic acid derivative as starting material but also in the need for the relatively strongly basic caesium carbonate to effect the two subsequent steps. Evidence for restricted rotation in the intermediate 4 is also presented.