Analogues of N alpha-(4-amino-4-deoxypteroyl)-N delta-(hemiphthaloyl)-L-
ornithine (
PT523) with 3',5'-dichloro substitution in the p-aminobenzoyl moiety or with one less or one more CH2 group in the
amino acid moiety were synthesized and tested as inhibitors of
dihydrofolate reductase (DHFR) activity and cell growth. Replacement of L-
ornithine in
PT523 by L-2,4-diaminobutanoic
acid or
L-lysine did not decrease binding to human recombinant DHFR but resulted in some loss of activity against SCC25 human and SCC VII murine
squamous cell carcinoma and against MCF-7 human
breast carcinoma in culture.
PT523 was several times more potent than
methotrexate (MTX),
aminopterin (AMT), or
trimetrexate (TMQ). 3',5'-Dichloro substitution did not decrease either DHFR binding or cytotoxicity. A new synthetic route to
PT523 from 2,4-diamino-6-(hydroxymethyl)pteridine and methyl N alpha-(4-aminobenzoyl)-N delta-phthaloyl-L-ornithinate was investigated but was not found superior to previously described methods. In comparative experiments on the ability of
PT523 and MTX to competitively inhibit the influx of (6R)-5,10-dideazatetra-hydrofolate (
DDATHF,
lometrexol), used here as a surrogate for MTX and reduced folates, the Ki of
PT523 was lower than that of MTX in both wild-type CCRF-CEM human leukemic lymphoblasts and the transport- and polyglutamylation-defective subline CEM/MTX. The CCRF-CEM cells were 10-fold more sensitive to
PT523 than to MTX, whereas the CEM/MTX cells were 240-fold more sensitive. However, in contrast to other MTX-resistant cells where collateral sensitivity to
PT523 has been seen. CEM/MTX cells still showed substantial cross resistance to
PT523 which may reflect an unusual heightened ability to utilize exogenous
folic acid. The good correlation observed with both cell lines between the cytotoxicity of
PT523 and MTX and the ability to inhibit
DDATHF influx supported the view that
PT523 and MTX share, at least in part, a common
protein carrier for membrane transport.