Synthesis and in vitro biological activity of new deaza analogues of folic acid, aminopterin, and methotrexate with an L-ornithine side chain.
The 5-deaza and 5,8-dideaza analogues of N alpha-pteroyl-L-ornithine (Pter-Orn), the 5-deaza, 8-deaza, and 5,8-dideaza analogues of N alpha-(4-amino-4-deoxypteroyl)-L-ornithine (APA-Orn), and the N delta-carboxymethyl derivative of N alpha-(4-amino-4-deoxy-N10-methylpteroyl)-L-ornithine (mAPA-Orn) were synthesized and tested as inhibitors of dihydrofolate reductase (DHFR) and as inhibitors of tumor cell growth in culture. Reductive amination of 2-acetamido-6-formylpyrido[2,3-d]pyrimidine-4(3H)-one with methyl N alpha-(4-aminobenzoyl)-N delta-(benzyloxycarbonyl)-L-ornithinate followed by removal of the blocking groups afforded the 5-deaza analogue of Pter-Orn, whereas N-alkylation of methyl N alpha-(4-aminobenzoyl)-N delta-(benzyloxycarbonyl)-L-ornithinate with 2-amino-6-(bromomethyl)quinazolin-4(3H)-one and deprotection gave the corresponding 5,8-dideaza analogue. Reductive coupling of 2,4-diaminopyrido[2,3-d]pyrimidine-6-carbonitrile and 4-aminobenzoic acid followed by reaction with 95-97% formic acid yielded 4-amino-4-deoxy-5-deaza-N10-formylpteroic acid, which on condensation with methyl N delta-(benzyloxycarbonyl)-L-ornithinate and deprotection gave the 5-deaza analogue of APA-Orn. A similar sequence starting from 2,4-diamino-quinazoline-6-carbonitrile led to the corresponding 5,8-dideaza compound, whereas treatment of 2,4-diamino-pyrido[3,2-d]pyrimidine-6-methanol with phosphorus tribromide followed by condensation with methyl N alpha-(4-aminobenzoyl)-N delta-(benzyloxycarbonyl)-L-ornithinate and deprotection afforded the 8-deaza analogue. For the preparation of the N delta-carboxymethyl derivative of mAPA-Orn, N alpha-(benzyloxycarbonyl)-L-ornithine was subjected to N delta-monoalkylation with glyoxylic acid and sodium cyanoborohydride, followed by N delta-acylation with ethyl trifluoroacetate, N alpha-deprotection by hydrogenolysis, condensation with 4-amino-4-deoxy-N10-methylpteroic acid, and N delta-deprotection by gentle treatment with ammonia. The 2,4-diamino derivatives all inhibited the growth of tumor cells in culture, with IC50 values of 0.2-2 microM, and inhibited purified DHFR with IC50 values of 0.02-0.08 microM. Deletion of ring nitrogens and N delta-carboxymethylation both increased potency in the cell growth assay; however, the ornithine derivatives were less potent than aminopterin or methotrexate.
Rosowsky
A
A
Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
Forsch
R A
RA
Bader
H
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Freisheim
J H
JH
D000630:Aminopterin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000138:chemical synthesis*; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004354:Drug Screening Assays, Antitumor; D005492:Folic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology*; D005493:Folic Acid Antagonists / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D015394:Molecular Structure; D009952:Ornithine; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000187:drug effects*
D000970:Antineoplastic Agents; D005493:Folic Acid Antagonists; D007202:Indicators and Reagents; D005492:Folic Acid; D009952:Ornithine; D013762:Tetrahydrofolate Dehydrogenase; D000630:Aminopterin; D008727:Methotrexate