Methotrexate analogs. 26. Inhibition of dihydrofolate reductase and folylpolyglutamate synthetase activity and in vitro tumor cell growth by methotrexate and aminopterin analogs containing a basic amino acid side chain

Abstract

Analogues of the antitumor antifolate methotrexate (MTX) were synthesized in which the glutamate (Glu) moiety was replaced by ornithine (Orn), 2,4-diaminobutyric acid (Dab), or 2,3-diaminopropionic acid (Dap). An aminopterin (AMT) analogue with Orn in place of Glu was also synthesized. The MTX analogues were obtained by reaction of 4-amino-4-deoxy-N10-methylpteroic acid (mAPA) and N omega-Boc-alpha,omega-diaminoalkanoic acids in the presence of diethyl phosphorocyanidate, followed by deprotection with trifluoroacetic acid (TFA) or by reaction of p-nitrophenyl-mAPA and N omega-Boc-alpha,omega-diaminoalkanoic acids and subsequent treatment with TFA. The AMT analogue (APA-Orn) was synthesized by reaction of p-nitrophenyl 4-amino-4-deoxy-N10-formylpteroate with silylated N delta-Boc-L-ornithine in DMF at 55 degrees C for 3 days (45% yield), saponification (83%), and TFA cleavage (89%). APA-Orn was a potent inhibitor of both dihydrofolate reductase (DHFR) from L1210 mouse leukemia (IC50 = 0.072 microM) and partly purified folylpolyglutamate synthetase (FPGS) from mouse liver (Ki = 0.15 +/- 0.06 microM). The MTX analogue (mAPA-Orn) was likewise active against both enzymes, with an IC50 of 0.160 microM for DHFR and a Ki of 20.4 +/- 7.7 microM for FPGS inhibition. The other MTX analogues and the previously reported lysine derivative (mAPA-Lys) showed DHFR affinity similar to that of mAPA-Orn but lacked activity as FPGS inhibitors. The positively charged amino group appears to be detrimental to cellular uptake, as evidenced by the low cytotoxicity of these compounds (IC50 = 0.40-2.4 microM) in comparison with MTX and AMT (IC50 = 0.002 microM) against wild-type L1210 cells. On the other hand, mAPA-Orn and APA-Orn were both more potent than the corresponding Glu derivatives MTX and AMT against L1210/R81 cells, suggesting that in these MTX-resistant cells there may occur a "self-potentiation" process involving enhanced antifolate activity via interference with the polyglutamylation of reduced folates. APA-Orn is the most potent dual inhibitor of DHFR and FPGS discovered to date, but its effectiveness as a therapeutic agent may require some form of prodrug modification to neutralize the terminal amino group of the side chain.

Synthesis of the Folylpolyglutamate Synthetase Inhibitor Nα-Pteroyl-Lornithine and its Nδ-Benzoyl and Nδ-Hemiphthaloyl Derivatives, and an Improved Synthesis of Nα-( 4-Amino-4-deoxypteroy1)-Nδ-hemiphthaloyl-Lornithine

“…This represents a great improvement over the previous method of synthesis of 4, a compound which is of potential therapeutic interest because of its high activIty against MTXresistant as well as MTX-sensitive tumor cells in culture (51). Removal of the NIO-formyl group and opening of the phthaloyl ring was markedly accelerated by the DMSO, which is known to promote nucleophilic reactions.…”

“…It was recently reported (1) that N"-pteroyl-L ornithine (1), and to an even greater degree the 7 ,8-dihydro and 5,6,7 ,8-tetrahydro derivatives of 1, are inhibitors of purified FPGS from porcine liver. The L-ornithine derivative of 4-amino-4-deoxypteroic acid has likewise been reported to be highly active against mouse (51) and human (55) enzyme, and is more active than the corresponding N 10 _ methyl analogue (54). In this paper we report a straightforward synthesis of 1 for biological and biochemical studies, and, in addition, describe the synthesis of the heretofore unknown N°-benzoyl and N°-hemiphthaloyl derivatives 2 and 3.…”

“…Several laboratories have been actively searching for FPGS inhibitors in the hope of putting this concept into practice (1,(49)(50)(51)(52)(53)(54) . It was recently reported (1) that N"-pteroyl-L ornithine (1), and to an even greater degree the 7 ,8-dihydro and 5,6,7 ,8-tetrahydro derivatives of 1, are inhibitors of purified FPGS from porcine liver.…”

“…Likewise unsuccessful were efforts to condense 6 with O,N°-bis(trimethylsilylated) N°-(tert-butyloxycarbonyl)-L-ornithine in the presence of diethyl phosphorocyanidate even though this method had worked well with 4-amino-4-deoxy-N 1 o-methylpteroic acid (51). On the other hand , when the more soluble silylated amino acid was condensed with 6 by the mixed anhydride method (i-BuOCOCljEt3N), the desired coupling product 7 was isolated in 44% yield after chromatographic purification on silica gel (5: 4 : 1 CHCl r MeOH-NH 4 0H) to separate unreacted 6.…”

Improved Scalable Syntheses of Mono- and Bis-Urethane Derivatives of Ornithine.

“…ref. [10][11][12][13][14]. Recent advances in the area of drug delivery have caused a resurgence of interest in the large scale production of peptides as pharmaceuticals.…”