Tricyclic 2,4-diaminopyrimidines with broad antifolate activity and the ability to inhibit Pneumocystis carinii growth in cultured human lung fibroblasts in the presence of leucovorin

A selected number of 1,3-diaminobenzo[f]quinazolines and 1,3-diamino-5,6-dihydrobenzo[f]quinazolines, which may be viewed as tricyclic analogues of the lipid-soluble antifolates pyrimethamine (PM), metoprine (DDMP), and etoprine (DDEP), were tested as inhibitors of purified dihydrofolate reductase (DHFR) from WI-L2 lymphoblasts, and as inhibitors of the growth of Streptococcus faecium ATCC 8043 and L1210 murine leukemia cells in culture. In addition, these tricyclic compounds were tested for antimalarial activity against Plasmodium berghei in mice, and for the ability to inhibit the growth of Pneumocystis carinii trophozoites in WI-38 human lung fibroblast cultures in the presence of leucovorin (LV). The most potent analogues were those with chlorine substitution in the ring distal to the 2,4-diaminopyrimidine moiety. Fully aromatic compounds tended to be more active than those in which the 5,6-bond was reduced, suggesting that planarity favors binding to the DHFR active site and may be favorable for cellular uptake. Several of the 2,4-diaminopyrimidine analogues showed greater potency than PM, DDMP or DDEP, and were more nearly comparable to the bicyclic 2,4-diaminopyrimidine antifolates trimetrexate (TMQ) or piritrexim (BW301U), which are known to be selectively toxic to P. carinii in the presence of LV. Two of the tricyclic compounds, 1,3-diamino-8-chlorobenzo[f]quinazoline and 1,3-diamino-9-chlorobenzo[f]quinazoline, proved to have activity similar to TMQ and BW301U in this system.     

Synthesis and antifolate activity of new diaminopyrimidines and diaminopurines in enzymatic and cellular systems.

The following new 2,4-diamino-6-methylpyrimidines, 5-cyclohexylmethyl, 5-cyclohexylethyl,and 5-(2-naphthyl), as well as 2,6-diaminopurines, 8-adamantyl and8-adamantylmethyl, were synthesized as potential antifolates. Tese, as well as three known compounds, 2,4-diamino-5-cyclohexyl-6-methylpyrimidine, 2,4-diamino-5-(1-naphthyl) -6- methylpyrimidine, and 2,6-diaminopurine, were compared with respect to the inhibition of growth of mammalian cells in culture (TA 3) and with respect to the inhibition of partially purified dihydrofolate reductase. All of the pyrimidines except for the 5-(1maphthyl) derivative were competitive inhivitors of dihydrofolate reductase, with K values ranging from 0.07 to 0.04 pM. They were 2-5 times better as inhibitors of the isolated dihydrofolate reductase than of the cell growth. 2,4-Diamino-5-(1-naphthyl)-6-methylpyrimidine was a noncomptive inhivitor of the enzyme with a Kvalue of 56 pM. This compound was more potent in inhibiting cell growth than the isolated enzyme. indicating that its biological activity was not related to the inhibition of dihydrofolate reductse. All of the purine derivatives were poor growth inhibitors and although some of them inhibited isolated dihydrofolate reductase, their mode of action in cellular system did not seem to concern folate metabolism, as judged by the inability of hypoxanthine, and glycine to provide protection. The implication of these findings as to the structural requirements for inhibition of dihydrofolate reductase is discussed. The implication of these findings as to the structural requirements for inhibition of dihydrofolate reductase is discussed. The pitfalls of the determinition of ID-50 values instead of a complete kinetic analysis in structure-activity studies are emphasized.     

2,4-Diamino-5-benzylpyrimidines and analogues as antibacterial agents. 10. 2,4-Diamino-5-(6-quinolylmethyl)- and -[(tetrahydro-6-quinolyl)methyl]pyrimidine derivatives. Further specificity studies

A series of 18 2,4-diamino-5-[(1,2,3,4-tetrahydro-6-quinolyl)methyl]pyrimidines has been prepared by the condensation of 2,4-diamino-5-(hydroxymethyl)pyrimidine with 1,2,3,4-tetrahydroquinolines in acidic medium. Several derivatives were catalytically aromatized; others were synthesized from these by routine aromatic substitution or by condensations of (anilinomethyl)pyrimidines to give quinolinylmethyl analogues. Compounds with 4-methyl-8-methoxy substitution are closely related to trimethoprim (1a) in structure and are excellent inhibitors of bacterial dihydrofolate reductase, with activity at least equivalent to that of 1a. The highest degree of inhibition was achieved with the rigid aromatic series, but greater specificity was accomplished among the tetrahydroquinoline derivatives. This was directly related to N-1 substitution of 4-methyl-8-methoxy derivatives. The spatial relationships around N-1 and protonation at this site may both affect selectivity. Such compounds also had excellent broad-spectrum in vitro antibacterial activity.     

Methotrexate analogues. 16. Importance of the side-chain amide carbonyl group as a structural determinant of biological activity

N-[[[(2,4-Diaminopteridin-6-yl)methyl]amino]benzyl]-L-glutamic acid ("deoxoaminopterin", 1), a new aminopterin analogue containing a CH2 group in the side chain in place of the amide C = O, was synthesized by condensation of 2,4-diamino-6-(bromomethyl)pteridine with diethyl N-(p-aminobenzyl)-L-glutamate, followed by saponification with a stoichiometric amount of barium hydroxide in 50% ethanol. The apparent importance of the amide C = O group as a structural determinant of biological activity was indicated by the finding that 1 has 10- to 20-fold lower affinity for bacterial and mammalian dihydrofolate reductase than aminopterin, is not toxic to L1210 murine leukemia cells in culture at a concentration of up to 1.0 microM, and shows no antitumor effect in L1210 leukemic mice at doses as high as 240 mg/kg (q3d X 3).     

2,4-Diamino-5-benzylpyrimidines as antibacterial agents. 4. 6-Substituted trimethoprim derivatives from phenolic Mannich intermediates. Application to the synthesis of trimethoprim and 3,5-dialkylbenzyl analogues

The preparation of a wide variety of 6-substituted trimethoprim analogues was readily accomplished by the reaction of 2,4-diamino-6-substituted-pyrimidines with 2,6-dimethoxy-4-[(N,N-dimethylamino)methyl]phenol at 120--160 degrees C. The less reactive 2,6-dialkyl-4-[(N,N-dimethylamino)methyl]phenols reacted successfully with 2,4-diamino-6-(alkylthio)pyrimidines to give 5-(substituted benzyl)pyrimidines. The phenolic groups of the products were alkylated in high yield when a nonreactive 6-substituent was present in the pyrimidine ring. 6-(Alkylthio) groups were easily removed with Raney nickel. Trimethoprim was thus obtained in high yield from its 6-(methylthio) counterpart. The 6-substituted trimethoprim analogues all had low activity as inhibitors of Escherichia coli dihydrofolate reductase and as antibacterial agents.     

The inhibition of dihydrofolate reductase by folate analogues: Structural requirements for slow- and tight-binding inhibition

Investigations have been made of the inhibition by 19 folate analogues of the reaction catalysed by dihydrofolate reductase from Streptococcus faecium A. Methotrexate, 7,8-dihydromethotrexate, aminopterin, methyl 4-amino-4-deoxy-10-methylpteroate and trimethoprim act as slow, tight-binding inhibitors, while 1-deazamethotrexate and 1-deaza-4-amino-4-deoxypteroate function as slow-binding inhibitors. Computer analysis of progress curve date indicated that the inhibition by these compounds conforms to a mechanism whereby there is an initial rapid formation of an enzyme-NADPH-inhibitor complex which subsequently undergoes a relatively slow, reversible isomerization reaction. Determinations were made of the dissociation constants for the release of each of the inhibitors from the initial ternary complex as well as of the rate constants associated with the forward and reverse isomerization reactions. The resulting values were used to calculate the overall inhibition constants for the inhibitors. 1-Deaza-2,4-diamino-6-methylpteridine and two derivatives of p-aminobenzoylglutamate also exhibited slow-binding inhibition. As the inhibition was parabolic, it appears that two molecules of each of these compounds combine with the enzyme-NADPH complex. Eight derivatives of 2,4-diaminopteridine and one derivative of 2,4-diaminopyrimidine yielded classical linear competitive inhibition with respect to dihydrofolate and dissociation constants were determined for their interaction with the enzyme-NADPH complex. From the type of inhibition and values for the kinetic parameters, conclusions have been drawn about the structural features of folate analogues which influence the formation of the enzyme-NADPH-inhibitor complex and its subsequent conformation change.     

Conformational analysis of antineoplastic antifolates: the crystal structure of trimetrexate and the aminopterine derivative 4-[N-[(2,4-diamino-6-pteridinyl)methyl]amino]benzoic acid

The crystal structures of trimetrexate (TMQ) (2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoli ne) and 4-[N-[(2,4-diamino-6-pteridinyl)methyl]amino]benzoic acid (PMAB) were determined to examine their conformational features with respect to the enzyme-bound form of methotrexate (MTX). TMQ and MTX are antineoplastic drugs that act by inhibiting the enzyme dihydrofolate reductase. The molecular conformation of TMQ is extended with the trimethoxyanilino ring twisted 89 degrees from the quinazoline plane, and the molecular conformation of PMAB is completely planar. The geometry of the 2,4-diaminopteridine and 2,4-diaminoquinazoline rings are sensitive to protonation, and both TMQ and PMAB have geometries characteristic to a free base. TMQ crystallizes as a dimethyl sulfoxide hydrate. The quinazoline ring forms an antiparallel stacking arrangement in the lattice and forms a network of N...O hydrogen bonds with the solvent molecules. In PMAB there are both pteridine-benzoic acid (N...O) hydrogen bonds and pteridine-pteridine (N...N) hydrogen bonds. Although the molecular conformation of TMQ and PMAB differ from enzyme-bound MTX, rotational energy barriers calculated using CAMSEQ indicate that they can adopt a similar conformation to that seen for MTX complexed with dihydrofolate reductase. These energy calculations show that PMAB is quite flexible and further suggest that the 5-methyl in TMQ reduces its conformational flexibility in a different manner than the N(10)-methyl in MTX. These structural data also show that full geometry optimization and proper parameterization of electronic effects at N(10) are required to accurately represent antifolate conformational preferences for enzyme binding.     

Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase inhibitors and antitumor agents: synthesis and biological activities of 2,4-diamino-5-methyl-6-[(monosubstituted anilino)methyl] pyrido[2,3-d]pyrimidines.

Thirteen 2,4-diamino-5-methyl-6-[(monosubstituted anilino)methyl]pyrido[2,3-d]pyrimidines 5-17 were synthesized as potential Pneumocystis carinii (pc) and Toxoplasma gondii (tg) dihydrofolate reductase (DHFR) inhibitors and as antitumor agents. Compounds 5-17 were designed to investigate the structure-activity relationship of monomethoxy and monohalide substitution in the phenyl ring and N10-methylation of the C9-N10 bridge. The synthetic route to compounds 5-12 involved the reductive amination of a common intermediate, 2,4-diamino-5-methylpyrido[2, 3-d]pyrimidine-6-carbonitrile (18), with the appropriate anilines. N10-Methylation was achieved by reductive methylation. In contrast to previous reports of trimethoprim, the removal of methoxy and chloro groups from the phenyl ring in the 2, 4-diamino-5-methyl-6-[(substituted anilino)methyl]pyrido[2, 3-d]pyrimidine series generally did not decrease DHFR inhibitory activity. The monosubstituted phenyl analogues 5-12 were as potent against pcDHFR and tgDHFR as the previously reported disubstituted phenyl analogues. N10-Methylation generally resulted in a marginal increase in potency against both pcDHFR and tgDHFR. Compounds 5, 7, and 9 were evaluated and shown to inhibit the growth of T. gondii cells in culture at nanomolar concentrations. Compounds 6-8, 9, 11, and 16 were selected by the National Cancer Institute for evaluation in an in vitro preclinical antitumor screening program. All six compounds showed GI50 values in the 10(-7)-10(-9) M range in more than 20 cell lines.     

Analog specific aberrancies in antifolate inhibition of L1210 cell dihydrofolate reductase

During studies with L1210 cells and a variety of folate analogs, large discrepancies were revealed between data on membrane transport, on inhibition of dihydrofolate reductase in cell-free extracts, and on inhibition of growth in culture for 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin, and for 3-deaza, 10-methyl-aminopterin. While aminopterin, 10-methyl (methotrexate)-, 10-ethyl- and 10-propyl-aminopterin were tight binding inhibitors (K_i: 2–3 × 10^?12M) of dihydrofolate reductase in cell-free extracts from L1210 cells, the other four analogs were only weak competitive inhibitors (K_i = 3–300 × 10^?8M). Similar differences among analogs were observed for inhibition of dihydrofolate reductase in cell-free extracts from Sarcoma 180 and Ehrlich cells, but not for this enzyme in microbial cell-free extracts. There were only small differences in the transport of all of the analogs by L1210 cells. Inhibition of L1210 cell growth in culture by 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin and by 3-deaza, 10-methyl-aminopterin, in contrast to the other analogs, was several orders of magnitude greater than that predicted from the data on dihydrofolate reductase inhibition. The extent of binding of 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin, and of 3-deaza and 10-methyl-aminopterin to dihydrofolate reductase in intact L1210 cells, in contradistinction to that seen for the cell-free enzyme preparations, approached that observed for methotrexate; these estimates of drug-enzyme interaction in situ were more predictive of the extent of inhibition by these analogs of L1210 cell growth in culture.     

Syntheses and antifolate activity of 5-methyl-5-deaza analogues of aminopterin, methotrexate, folic acid, and N10-methylfolic acid

Evidence indicating that modifications at the 5- and 10-positions of classical folic acid antimetabolites lead to compounds with favorable differential membrane transport in tumor vs. normal proliferative tissue prompted an investigation of 5-alkyl-5-deaza analogues. 2-Amino-4-methyl-3,5-pyridinedicarbonitrile, prepared by hydrogenolysis of its known 6-chloro precursor, was treated with guanidine to give 2,4-diamino-5-methylpyrido[2,3-d]pyrimidine-6-carbonitrile which was converted via the corresponding aldehyde and hydroxymethyl compound to 6-(bromomethyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine. Reductive condensation of the nitrile 8 with diethyl N-(4-amino-benzoyl)-L-glutamate followed by ester hydrolysis gave 5-methyl-5-deazaaminopterin. Treatment of 12 with formaldehyde and Na(CN)BH3 afforded 5-methyl-5-deazamethotrexate, which was also prepared from 15 and dimethyl N-[(4-methylamino)benzoyl]-L-glutamate followed by ester hydrolysis. 5-Methyl-10-ethyl-5-deazaaminopterin was similarly prepared from 15. Biological evaluation of the 5-methyl-5-deaza analogues together with previously reported 5-deazaaminopterin and 5-deazamethotrexate for inhibition of dihydrofolate reductase (DHFR) isolated from L1210 cells and for their effect on cell growth inhibition, transport characteristics, and net accumulation of polyglutamate forms in L1210 cells revealed the analogues to have essentially the same properties as the appropriate parent compound, aminopterin or methotrexate (MTX), except that 20 and 21 were approximately 10 times more growth inhibitory than MTX. In in vivo tests against P388/0 and P388/MTX leukemia in mice, the analogues showed activity comparable to that of MTX, with the more potent 20 producing the same response in the P388/0 test as MTX but at one-fourth the dose; none showed activity against P388/MTX. Hydrolytic deamination of 12 and 20 produced 5-methyl-5-deazafolic acid and 5,10-dimethyl-5-deazafolic acid, respectively. In bacterial studies on the 2-amino-4-oxo analogues, 5-deazafolic acid proved to be a potent inhibitor of Lactobacillus casei DHFR and also the growth of both L. casei ATCC 7469 and Streptococcus faecium ATCC 8043. Its 5-methyl congener 22 is also inhibitory toward L. casei, but its IC50 for growth inhibition is much lower than its IC50 values for inhibition of DHFR or thymidylate synthase from L. casei, suggesting an alternate site of action.     

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.     

N9-substituted 2,4-diaminoquinazolines: synthesis and biological evaluation of lipophilic inhibitors of pneumocystis carinii and toxoplasma gondii dihydrofolate reductase

N9-substituted 2,4-diaminoquinazolines were synthesized and evaluated as inhibitors of Pneumocystis carinii (pc) and Toxoplasma gondii (tg) dihydrofolate reductase (DHFR). Reduction of commercially available 2,4-diamino-6-nitroquinazoline 14 with Raney nickel afforded 2,4,6-triaminoquinazoline 15. Reductive amination of 15 with the appropriate benzaldehydes or naphthaldehydes, followed by N9-alkylation, afforded the target compounds 5- 13. In the 2,5-dimethoxybenzylamino substituted quinazoline analogues, replacement of the N9-CH 3 group of 4 with the N9-C2H5 group of 8 resulted in a 9- and 8-fold increase in potency against pcDHFR and tgDHFR, respectively. The N9-C2H5 substituted compound 8 was highly potent, with IC50 values of 9.9 and 3.7 nM against pcDHFR and tgDHFR, respectively. N9-propyl and N9-cyclopropyl methyl substitutions did not afford further increases in potency. This study indicates that the N9-ethyl substitution is optimum for inhibitory activity against pcDHFR and tgDHFR for the 2,4-diaminoquinazolines. Selectivity was unaffected by N9 substitution.     

Design and synthesis of classical and nonclassical 6-arylthio-2,4-diamino-5-ethylpyrrolo[2,3-d]pyrimidines as antifolates

The classical antifolate N-{4-[(2,4-diamino-5-ethyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)sulfanyl]benzoyl}-l-glutamic acid (2) and 15 nonclassical analogues (3-17) were synthesized as potential dihydrofolate reductase (DHFR) inhibitors and as antitumor agents. 5-Ethyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (20) served as the key intermediate to which various aryl thiols and a heteroaryl thiol were appended at the 6-position via an oxidative addition reaction. The classical analogue 2 was synthesized by coupling the benzoic acid derivative 18 with diethyl l-glutamate followed by saponification. The classical compound 2 was an excellent inhibitor of human DHFR (IC50 = 66 nM) as well as a two digit nanomolar (<100 nM) inhibitor of the growth of several tumor cells in culture. Some of the nonclassical analogues were potent and selective inhibitors of DHFR from two pathogens (Toxoplasma gondii and Mycobacterium avium) that cause opportunistic infections in patients with compromised immune systems.     

Design, synthesis, and biological evaluation of 2,4-diamino-5-methyl-6-substituted-pyrrolo[2,3-d]pyrimidines as dihydrofolate reductase inhibitors.

2,4-diamino-5-methyl-6-(substituted-phenyl)thiopyrrolo[2,3-d]pyrimidines 4-11 were synthesized as dihydrofolate reductase (DHFR) inhibitors against opportunistic pathogens that afflict patients with AIDS. Synthesis was achieved from 2,4-diamino-5-methypyrrolo[2,3-d]pyrimidine and substituted phenylthiols under modified conditions reported by Gangjee et al. Some of these compounds were potent and selective against DHFR from both Toxoplasma gondii and Mycobacterium avium compared to mammalian DHFR. Compound 11 with a 1-naphthyl substituent is 16-fold more potent and equally selective against Toxoplasma gondii DHFR as the clinically used trimethoprim.     

Further studies on substituted quinazolines and triazines as inhibitors of a methotrexate-insensitive murine dihydrofolate reductase

Data are presented on the systematic analysis of thirty-five quinazoline and substituted triazine compounds as inhibitors of a methotrexate-insensitive form of dihydrofolate reductase purified from methotrexate-resistant L5178Y murine leukemia cells. Several of the compounds were found to be more potent inhibitors of this enzyme activity than was methotrexate. Two of the triazine compounds had IC50 values approaching 10nM, which is close to that of methotrexate for the normal drug-sensitive dihydrofolate reductase. In addition, some of these compounds, especially the triazines, exhibit a specificity of inhibition for the methotrexate-insensitive enzyme as compared to the normal methotrexate-sensitive dihydrofolate reductase derived from the same cell line. These compounds may, therefore, be potentially useful in the treatment of those methotrexate-resistant tumours which express an altered, methotrexate-insensitive dihydrofolate reductase.     

Methotrexate analogues. 31. Meta and ortho isomers of aminopterin, compounds with a double bond in the side chain, and a novel analogue modified at the alpha-carbon: chemical and in vitro biological studies

Five heretofore undescribed analogues of methotrexate (MTX) and aminopterin (AMT) were synthesized and tested as dihydrofolate reductase (DHFR) inhibitors and tumor cell growth inhibitors. The meta isomer of AMT was obtained from 2,4-diamino-6-(bromomethyl)pteridine and m-(aminobenzoyl)-L-glutamic acid, while the ortho isomer was obtained via the same route by using alpha-methyl gamma-tert-butyl o-(aminobenzoyl)-L-glutamate instead of the free acid. Analogues of MTX and AMT containing a double bond in the side chain were prepared from dimethyl D,L-2-amino-4-hexenedioate and 4-amino-4-deoxy-N10-methylpteroic acid and 4-amino-4-deoxy-N10-formylpteroic acid, respectively. Finally, a positional isomer of MTX with the CH2CH2COOH moiety moved from the alpha-carbon to the adjacent carboxamide nitrogen was synthesized from 3-[N-(carboxymethyl)amino]propanoic acid diethyl ester and 4-amino-4-deoxy-N10-methylpteroic acid. The positional isomers of AMT were weak DHFR inhibitors and showed very little growth-inhibitory activity against L1210 murine leukemia cells or the MTX-resistant L1210/R81 mutant line in culture. The MTX and AMT analogues with the CH2CH2COOH moiety replaced by a CH2CH = CHCOOH side chain showed anti-DHFR activity similar to that of the previously described saturated compound N-(4-amino-4-deoxy-N10-methylpteroyl)-L-2-aminoadipic acid, but were less potent than the parent drugs. The MTX analogue with the CH2CH2COOH side chain displaced from C to N was weakly bound to DHFR, confirming the importance of an intact CONH moiety, and showed greatly diminished cell growth inhibitory potency relative to MTX. None of the compounds was a substrate for folylpolyglutamate synthetase (FPGS) from mouse liver. Furthermore, inhibition of folic acid polyglutamylation in vitro at equimolar 500 microM concentrations of drug and substrate was negligible. The structural changes embodied in these five novel compounds are therefore too great for binding to the FPGS active site.     

Quinazoline antifolate thymidylate synthase inhibitors: 2'-fluoro-N10-propargyl-5,8-dideazafolic acid and derivatives with modifications in the C2 position

The synthesis of 2'-fluoro-10-propargyl-5,8-dideazafolic acid and its 2-desamino, 2-desamino-2-hydroxymethyl, and 2-desamino-2-methoxy analogues is described. In general the synthetic route involved the coupling of diethyl N-[2-fluoro-4-(prop-2-ynylamino)benzoyl]-L-glutamate with the appropriate 6-(bromomethyl)quinazoline followed by deprotection with mild alkali. These four compounds together with the 2-desamino-2-methyl analogue were tested for their activity against L1210 thymidylate synthase (TS). They were also examined for their inhibition of the growth of the L1210 cell line and of two mutant L1210 cell lines, the L1210:R7A that overproduces dihydrofolate reductase (DHFR) and the L1210:1565 that has impaired uptake of reduced folates. Compared with their non-fluorinated parent compounds, the 2'-fluoro analogues were all approximately 2-fold more potent as TS inhibitors. Similarly, they also showed improved inhibition of L1210 cell growth (1.5-5-fold), and this activity was prevented by co-incubation with thymidine. All had retained or improved activity against both the L1210:R7A and L1210:1565 cell lines.     

Multivariate analysis and quantitative structure-activity relationships. Inhibition of dihydrofolate reductase and thymidylate synthetase by quinazolines.

Quantitative structure-activity relationships (QSAR) have been established for the inhibition of dihydrofolate reductase and thymidylate synthetase by 2,4-diaminoquinazoline-glutamic acid analogues. For dihydrofolate reductase from both human acute lymphocytic leukemia cells and murine L1210R cells, QSAR's obtained with 50 quinazolines were similar. On the other hand, for the inhibition of thymidylate synthetase from murine L1210S cells and from Lactobacillus casei, QSAR's formulated on the basis of data measured with 33 compounds were different, indicating that the two enzymes are dissimilar. The use of multivariate statistics including cluster analysis, factor analysis, and discriminant analysis is shown to facilitate the formulation of a satisfactory correlation equation. The procedure is demonstrated by the development of QSAR for the inhibition of thymidylate synthetase.     

Studies with a 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP)-resistant L1210 leukemia cell line without cross-resistance to methotrexate

An L1210 leukemia cell line resistant to 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP) (L1210/DDMP) was developed in vivo by treatment of tumor-bearing mice. Resistance to DDMP was confirmed by subsequent in vivo survival experiments and by in vitro dose-response curves. The L1210/DDMP line demonstrated little cross-resistance to another folate analog, methotrexate (MTX). This was confirmed both in vivo, with survival experiments, and in vitro, using dose-response curves. A statistical analysis of the in vivo data confirmed DDMP resistance with lack of MTX cross-resistance. Dihydrofolate reductase (DHFR) activity in the L1210/DDMP/R₅ line was no greater than in the parent cell line (L1210/S). and the K_m of DHFR for dihydrofolate was the same in the L1210/DDMP/R₅ and L1210/S lines. The K_i for DHFR of the L1210/DDMP/R₅ cell line versus the L1210/S cell line was increased 3.0-fold for MTX and 3.5-fold for DDMP. Total accumulation of [¹⁴C]DDMP was identical in the two cell lines. The explanation for the lack of MTX cross-resistance in the L1210/DDMP/R₅ line is unknown.     

Novel dihydrofolate reductase inhibitors. Structure-based versus diversity-based library design and high-throughput synthesis and screening

Novel 2,4-diaminopyrimidines bearing N,N-disubstituted aminomethyl residues at the 5-position were designed as dihydrofolate reductase (DHFR) inhibitors. These compounds were obtained by treatment of 1-[(2,4-diamino-5-pyrimidinyl)methyl]pyridinium bromide with secondary amines in a polar solvent and in the presence of triethylamine at room temperature. The procedure was found to be very efficient and suitable for application in high-throughput synthesis. In addition, we found that high-throughput screening for enzymatic and in vitro antibacterial activity could be performed on crude reaction mixtures, thus avoiding any purification step. Over 1200 proprietary secondary amines were selected for high-throughput synthesis, based on structural and diversity-related criteria, and the resulting products were submitted to high-throughput screening. A greater number of hits, and significantly more active compounds, were obtained through structure-based library design than through diversity-based library design. Different classes of inhibitors of DHFR were identified in this way, including compounds derived from di-, tri-, and tetracyclic amines. In general, these products showed high activity against the enzymes derived from both TMP-sensitive and TMP-resistant Streptococcus pneumoniae. Some compounds possessed appreciable selectivity for the bacterial over the human enzyme, whereas other compounds were not at all selective. In most cases, active enzyme inhibitors also displayed antibacterial activity.