Inhibition of dihydrofolate reductase, methotrexate transport, and growth of methotrexate-sensitive and -resistant L1210 leukemia cells in vitro by 5-substituted 2,4-diaminoquinazolines.

A series of eighteen 2,4-diaminoquinazoline analogues of folic, isofolic, pteroic and isopteroic acids having various substituents at position 5 was studied. Each compound was evaluated as an inhibitor of L1210 dihydrofolate reductase, methotrexate influx into L1210 leukemia cells, and growth of methotrexate-sensitive and -resistant L1210 cells in vitro. Bridge reversal at positions 9 and 10 reduced the effectiveness of the classical analogues only with regard to the inhibition of the drug-sensitive cells as compared to methotrexate (MTX). Absence of the glutamate moiety adversely affected the potency of the compounds, particularly when coupled with reversal of the 9,10-bridge. However, the presence of -Cl at position 5 restored significantly the potency of these compounds. The pteroate and isopteroate analogue ethyl esters were generally more effective inhibitors of cell growth than their non-esterified counterparts. Regarding the effects of substituents at position 5, the data suggest that -Cl greater than -CH3 greater than -H for inhibition of methotrexate transport and growth of methotrexate-sensitive L1210 cells. The 5-Cl pteroate analogue and its corresponding ethyl ester were highly effective as growth inhibitors of methotrexate-resistant, transport-defective, L1210 cells in vitro.

Methotrexate analogues. 25. Chemical and biological studies on the gamma-tert-butyl esters of methotrexate and aminopterin.

gamma-tert-Butylaminopterin (gamma-tBAMT), the first example of an aminopterin (AMT) gamma-monoester, was synthesized, and new routes to the known N10-methyl analogue gamma-tert-butyl methotrexate (gamma-tBMTX) were developed. The inhibitory effects of gamma-tBAMT on the activity of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, the growth of L1210 cells and CEM human leukemic lymphoblasts in suspension culture, and the growth of several lines of human squamous cell carcinoma of the head and neck in monolayer culture were compared with the effects of gamma-tBMTX and the parent acids AMT and methotrexate (MTX). Patterns of cross-resistance to gamma-tBAMT, gamma-tBMTX, and AMT among several MTX-resistant cell lines were examined. In vivo antitumor activities of gamma-tBAMT and gamma-tBMTX were compared in mice with L1210 leukemia. While the activity of gamma-tBAMT was very close to that of gamma-tBMTX in the DHFR inhibition assay, the AMT ester was more potent than the MTX ester against cells in culture and against L1210 leukemia in vivo. Only partial cross-resistance was shown against gamma-tBMTX and gamma-tBAMT in cultured cells that were resistant to MTX by virtue of a transport defect or a combination of defective transport and elevated DHFR activity.

Chemistry and antitumor evaluation of selected classical 2,4-diaminoquinazoline analogues of folic acid.

A series of six 2,4-diaminoquinazoline analogues of folic acid which bear close structural resemblance to methotrexate, 1a, were synthesized by unequivocal routes. Three of these have not been described previously, while complete structural characterization of the remaining compounds is presented for the first time. Each of the compounds was a potent inhibitor of dihydrofolate reductase (DHFR) from rat liver or L1210 leukemia cells having I50 values in a range similar to that of 1a. However, a wide divergence in inhibitory activity toward the growth of human gastrointestinal adenocarcinoma or L1210 leukemia cells in vitro was observed. Compounds having a normal folate configuration at positions 9 and 10 were more inhibitory than their isomeric reversed-bridge counterparts. The N-formyl modifications were the least active of the compounds studied. Unsubstituted or N-methyl modifications competed effectively with tritiated 1a for uptake into L1210 leukemia cells, while N-formyl modifications did not. Against an L1210 cell line resistant to 1a by virtue of altered transport and overproduction of DHFR, partial but not complete cross-resistance was observed for certain analogues. Of the three compounds selected for in vivo evaluation against L1210 leukemia in mice, two had a similar level of antitumor activity to that of 1a. The compound 5,8-dideazamethopterin, 2b, however, was slightly more active than 1a but at substantially reduced dose levels.

Regiospecific gamma-conjugation of methotrexate to poly(L-lysine). Chemical and biological studies.

Regiospecific syntheses of gamma- and alpha-conjugates of methotrexate and poly(L-lysine) are described. The alpha- and gamma-t-butyl esters, respectively, of methotrexate were coupled to poly(L-lysine) with diphenylphosphoryl azide in N,N-dimethylformamide, the ester-protecting group was cleaved with 15% hydrogen bromide in acetic acid, and small molecules were removed by dialysis. Poly(L-lysine) of Mr = 1,500-8,000 and 8,000-30,000 was used to prepare six different conjugates, which were characterized by ultraviolet absorbance measurement and quantitative amino acid analysis. The degree of substitution varied from one methotrexate per 4.7 lysines to one methotrexate per 10.2 lysines. Dihydrofolate reductase inhibition in a cell-free assay was observed with alpha- and gamma-conjugates, but the latter had the greater affinity (only 3-fold less than that of methotrexate itself). The binding of the conjugates exhibited a slight pH dependence, with affinity being greater at pH 7.2 than at pH 8.5 for both alpha- and gamma-conjugates. Toxicity to cultured rat hepatoma cells (H35) was also greater for the gamma-conjugates, and showed some dependence on the chain-length and degree of substitution of the poly(L-lysine) carrier. Cells resistant to methotrexate by virtue of a transport defect (H35R0.3 line) retained their sensitivity to the gamma-conjugate, but less so to the alpha-conjugate. There was also some retention of sensitivity in a more highly resistant cell line (H35R10) with impaired methotrexate transport and a concomitant increase in dihydrofolate reductase activity. gamma-Conjugation was likewise more favorable in cytotoxicity assays against L1210 murine leukemia cells, and there was partial retention of activity against highly methotrexate-resistant lines (L1210/R71 and L1210/R81) with a transport defect and/or an elevation of dihydrofolate reductase content. In antitumor assays against intraperitoneal L1210 leukemia in mice, a gamma-conjugate with Mr = 8,000-30,000 and one methotrexate per 5.5 lysines produced a 35-75% increase in lifespan when administered intraperitoneally at single doses equivalent to 10-20 mg/kg of methotrexate. A similar increase in lifespan with methotrexate alone on the single-dose regimen required 50-150 mg/kg. An alpha-conjugate of similar Mr and degree of substitution was inactive at nontoxic doses, as were other gamma-conjugates of lower Mr and/or degree of substitution.(ABSTRACT TRUNCATED AT 400 WORDS)

A fluorescent analogue of methotrexate as a probe for folate antagonist molecular receptors.

A dansyl-L-lysine analogue of methotrexate, N alpha-(4-amino-4-deoxy-10- methylpteroyl )-N epsilon-(5-[N,N-dimethylamino]-1-naphthalenesulfonyl)-L-lysine, is a potent inhibitor of murine L1210 dihydrofolate reductase. The dansyl fluorescence emission was enhanced approximately 3-fold with a 10 nm blue shift upon binding to L1210 dihydrofolate reductase. The fluorescent analogue was only 10-fold less potent than methotrexate in inhibiting the growth of methotrexate-sensitive and -resistant L1210 cells and competes effectively for [3H]methotrexate transport with a Ki of 7.02 microM, a value virtually identical to the Kt for methotrexate in both cell lines. In addition, strong dansyl fluorescence was found to be associated with dihydrofolate reductase from methotrexate-resistant, dihydrofolate reductase-overproducing L1210 cells following incubation of viable cells with the fluorescent methotrexate analogue for 4 hr. The results demonstrate that the dansyl-L-lysine analogue of methotrexate was rapidly transported into L1210 cells where it formed a high-affinity, fluorescent complex with intracellular dihydrofolate reductase.

Inhibition of human dihydrofolate reductase by 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(substituted-phenyl)-s-triazine s. A quantitative structure-activity relationship analysis.

The inhibitory activity of 101 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(substituted-phenyl)-s-triazines against purified dihydrofolate reductase from human lymphoblastoid cell (WIL 2) has been studied. From the obtained Kiapp values, quantitative structure-activity relationships (QSAR) have been derived. The QSAR from human dihydrofolate reductase are compared with QSAR for triazines inhibiting bovine and murine tumor DHFR, as well with QSAR for their inhibitory action on murine tumor cell culture.

Purification and characterization of dihydrofolate reductase from methotrexate-resistant human lymphoblastoid cells.

Dihydrofolate reductase has been isolated from methotrexate-resistant WIL2 human lymphoblastoid cells. This subline produces ca. 150 times more enzyme than the parental drug-sensitive line. The reductase has been purified to homogeneity by methotrexate affinity chromatography, gel filtration, and preparative isoelectric focusing in a yield of 65%. The enzyme has a pI = 7.7 and a molecular weight of ca. 22000. The amino-terminal 27 amino acid residues have been determined, revealing the location of the single cysteine residue at position 6. Reaction of this cysteine with p-(hydroxy-mercuri)benzoate in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) results in a 5-fold increase in enzyme activity. Other agents including KCl, urea, and thiourea also cause enzyme activation. The Km values for NADPH and dihydrofolate are 0.25 and 0.036 microM, respectively. Mercurial activation of the enzyme results in a 27-fold increase in the Km for NADPH and a 35-fold increase in the Km for dihydrofolate.

Influence of different levels of dietary pyridoxine on milk composition in the rat.

The effects of five different levels of dietary pyridoxine on milk composition were studied in the rat. Sprague-Dawley strain rats received diets containing 1.2, 2.4, 4.8, 9.6 or 19.2 mg pyridoxine-HCl/kg diet throughout growth, gestation and lactation. Milk samples obtained on days 10, 11, and 12 of lactation were similar in concentrations of total fat, solids-not-fat, carbohydrate, casein and non-casein protein for rats fed the five levels of pyridoxine. The level of vitamin B-6 in milk was significantly higher for rats fed 9.6 or 19.2 mg pyridoxine.HCl/kg diet compared to values for rats fed the three lower levels of vitamin. A higher level of dietary pyridoxine (9.6 mg/kg diet) was required to increase the levels of the vitamin in mammary gland and milk than was needed for maternal liver (4.8 mg/kg) or muscle tissue (2.4 mg/kg). The findings indicated that as the level of pyridoxine was decreased in the diet from an apparently adequate level, the concentration of the vitamin in milk decreased before that in liver or muscle tissue. This suggested that the concentration of the vitamin in milk was an indicator of marginal deficiency of vitamin B-6.