Methotrexate Analogues. 33. Nδ-Acyl-Nα-(4-amino-4-deoxypteroyl)-L-ornithine Derivatives: Synthesis and in Vitro Antitumor Activity

Article abstract of DOI:10.1021/jm00402a013

N^δ-Acyl derivatives of the potent folylpolyglutamate synthetase (FPGS) inhibitor N^α-(4-amino-4-deoxypteroyl)-L-ornithine (APA-L-Orn) were synthesized from N^α-(4-amino-4-deoxy-N¹⁰-formylpteroyl)-L-ornithine by reaction with an N-(acyloxy)succinimide or acyl anhydride, followed by deformylation with base.The N^δ-hemiphthaloyl derivative was also prepared from 4-amino-4-deoxy-N¹⁰-formylpteroic acid by reaction with persilylated N^δ-phthaloyl-L-ornithine, followed by simultaneous deformylation and ring opening of the N^δ-phthaloyl moiety with base.The products were potent inhibitors of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, with IC 50's ranging from 0.027 and 0.052 μM as compared with 0.072 μM for APA-L-Orn.Several of the N^δ-acyl-N¹⁰-formyl intermediates also proved to be good DHFR inhibitors.One of them, N^α-(4-amino-4-deoxy-N¹⁰-formylpteroyl)-N^δ-(4-chlorobenzoyl)-L-ornithine, had a 2-fold lower IC 50 than its deformylated product, confirming that the N¹⁰-formyl group is well tolerated for DHFR binding.While N^δ-acylation of APA-L-Orn did not significantly alter anti-DHFR activity, inhibition of FPGS was dramatically diminished, supporting the view that the basic NH2 on the end of the APA-L-Orn side chain is essential for the activity of this compound against FPGS.N^δ-Acylation of APA-L-Orn markedly enhanced toxicity to cultured tumor cells.However, N^δ-acyl derivatives also containing an N¹⁰-formyl substituent were less cytotoxic than the corresponding N¹⁰-unsubstituted analogues even though their anti-DHFR activity was the same, suggesting that N¹⁰-formylation may be unfavorable for transport.Two compounds, the N^δ-benzoyl and N^δ-hemiphthaloyl derivatives of APA-L-Orn, with IC 50's against L1210 cells of 0.89 and 0.75 nM, respectively, were more potent than either methotrexate (MTX) or aminopterin (AMT) in this system.These compounds were also more potent than MTX against CEM human lymphoblasts and two human head and neck squamous cell carcinoma cell lines (SCC15, SCC25) in culture.Moreover, in assays against SCC15/R1 and SCC25/R1 sublines with 10 - 20-fold MTX resistance, the N^δ-hemiphthaloyl derivative of APA-L-Orn showed potency exceeding that of MTX itself against the parental cells.Although other mechanisms cannot be ruled out, a possible expalanation for the fact that these N^δ-acyl derivatives of APA-L-Orn are much more potent in the cell growth assay than in the DHFR assay is that they are efficientyl taken up by the cell and are then cleaved to APA-L-Orn, which can simultaneously inhibit DHFR and FPGS, thereby acting as a "self-potentiating antifolate".According to this view, blockade of cellular FPGS activity should complement DHFR inhibition by diminishing the cell's ability to convert tetrahydrofolate monoglutamate cofactors to polyglutamates, which are the most efficiently used species for DNA precursor synthesis.