Comparison of triazines as inhibitors of L1210 dihydrofolate reductase and of L1210 cells sensitive and resistant to methotrexate.

The inhibition of dihydrofolate reductase from L1210 leukemia cells as well as the inhibition of intact L1210 cells, both sensitive and resistant, to methotrexate by over 100, 4,6-diamino-2,2-dihydro-2,2-dimethyl-1-(X-phenyl)-s-triazines was studied. Quantitative structure-activity relationships were derived for the three systems. These equations, based on a set of congeners having a range in lipophilicity of about 700,000,000 on the octanol-water scale, delineate the inhibitory potency of the triazines in relation to their hydrophobicity. The data demonstrate that there is a close parallel between the way isolated dihydrofolate reductase and methotrexate sensitive cells respond to the triazines. However, the resistant L1210 cells behave in an entirely different manner, which suggests that the passive diffusion of triazines into the cells dominates the structure-activity relationship. The optimum lipophilicity (pi 0) of triazine substituents for purified L1210 dihydrofolate reductase is 1.76 to 2.11; for sensitive cells, it is 1.45 to 1.83, and for resistant cells, it is approximately 6.

Separation of electronic and hydrophobic effects for the papain hydrolysis of substituted N-benzoylglycine esters.

The role of hydrophobic and electronic effects on the kinetic constants kcat and Km for the papain hydrolysis of a series of 22 substituted N-benzoylglycine pyridyl esters was investigated. The series studied comprises a wide variety of substituents on the N-benzoyl ring, with about a 300,000-fold range in their hydrophobicities, and 2.1-fold range in their electronic Hammet constants (sigma). It was found that the variation in the log kcat and log 1/Km constants could be explained by the following quantitative-structure activity relationships (QSAR): log 1/Km = 0.40 pi 4 + 4.40 and log 1/kcat = 0.45 sigma + 0.18. The substituent constant, pi 4, is the hydrophobic parameter for the 4-N-benzoyl substituents. QSAR analysis of two smaller sets of glycine phenyl and methyl esters produced similar results. A clear separation of the substituent effects indicates that in the case of these particular esters, acylation appears to be the rate limiting catalytic step.

Inhibition of neutrophil elastase and thrombin activity by caffeic acid esters.

Natural and synthetic caffeic acid esters were assayed for their enzymatic activity versus neutrophil elastase (EC 3.4.21.37) and thrombin (EC 3.4.21.5). Lipophilic caffeic acid esters inhibited neutrophil elastase activity and the inhibition rate was enhanced with increasing length of the aliphatic chain of the alcohol component. The geometry of the chain seems to be more important than the number of carbon atoms. The most inhibitory compound was n-octylcaffeic acid ester with an IC50 value of 1.0 microM. Thrombin activity was only weakly inhibited by the caffeic acid esters thus demonstrating a specificity for neutrophil elastase. Because of its critical role in inflammatory processes, inhibition of neutrophil elastase by caffeic acid esters might be of importance in the treatment of inflammation.

QSAR: then and now.

In this review, the evolution of QSAR is traced from the insightful observations of Crum-Brown and Frazier to Hammett's critical equations and finally Hansch's seminal contributions on hydrophobicity and modelling of biological activity based on extrathermodynamic principles. Today's QSAR models can stand alone, augment other graphical approaches or be examined in tandem with equations of a similar mechanistic genre to truly reveal the power of the paradigm. This review will focus on the three standard classifications routinely used in QSAR analysis electronic, hydrophobic, and steric, as well as topological indices. Electronic parameters will focus on Hammett sigma constants and their numerous variations. Dipole moments, hydrogen bond descriptors and quantum chemical indices as well as applications of their utilization will be described. The hydrophobicity parameter will be examined by tracing its early history, its operational definition and its determination by either experimental methods or computational calculations. Steric parameters, which run the gamut from size to shape, will be described by Taft's, Hancock's, Charton's, Fujita's, Verloop's and Simon's contributions. Topological effects, delineated by connectivity indices, kappa shape and electrotopological indices of Kier and Hall are also described. Examples of QSAR models incorporating most of these parameters are reviewed. In cases where the 95% confidence intervals of variables are available, they are listed in parentheses. A brief Comparative QSAR analysis of non-nucleoside reverse transcriptase inhibitors (NNRTI's) is outlined and various models obtained by different groups examining 4, 5, 6, 7-tetrahydro-5-methylimidazo [4, 5,1-j,k][1,4] benzodiazepin-2(1H)-ones (TIBO) and 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)-thymine (HEPT) derivatives are compared for mechanistic insight that could be useful in the process of inhibitor design.

Structure-activity relationships of antineoplastic agents in multidrug resistance.

Clinical resistance to many antineoplastic agents is a major cause of treatment failure. The well-documented phenomenon addressed as multidrug resistance (MDR) allows cells to withstand exposure to lethal doses of drugs with dissimilar chemical structures, modes of action, and physicochemical properties. In one of the earliest studies on MDR, Biedler and Riehm in an attempt to explain the cross-resistance profile of actinomycin D resistant Chinese hamster cells suggested that molecular weight was an important determinant. Our statistical analysis of their data validates their claim and indeed strongly demonstrates that cross resistance is enhanced by the increased size and hydrophobicity of the antitumor agent. Our preliminary studies with methotrexate-resistant L1210 cells indicates that cross resistance is increased in the case of moderate-sized, hydrophilic drugs. These two studies and others suggest that current chemotherapy regimens may be improved by treating resistant cells with antineoplastic agents displaying physicochemical characteristics opposite to that of the original inducing agent.

Quantitative structure-activity relationships of the inhibition of Pneumocystis carinii dihydrofolate reductase by 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(X-phenyl)-s-triazines.

The inhibitory activities of 60 4,6-diamino-1,2-dihydro-2,2-dimethyl-1- (X-phenyl)-s-triazines versus purified, recombinant Pneumocystis carinii (Pc) dihydrofolate reductase (DHFR) have been determined at pH 7.0. Utilization of these Kiapp values has led to the formulation of appropriate quantitative structure-activity relationships (QSAR's) for both meta- and parasubstituted derivatives. The QSAR's from Pc are compared with other triazine QSAR's derived versus chicken, murine tumor, Escherichia coli, and particularly human DHFR. Selectivity indices indicate that hydrophobic triazines are particularly effective versus Pc DHFR; they have lower Ki values for Pc DHFR than for human DHFR.

On the optimization of hydrophobic and hydrophilic substituent interactions of 2,4-diamino-5-(substituted-benzyl)pyrimidines with dihydrofolate reductase.

The inhibition constants (Kiapp) were obtained from the action of 68 2,4-diamino-5-(substituted-benzyl)pyrimidines on dihydrofolate reductase from an Escherichia coli strain MB 1428. Subsequently, these results were used to formulate appropriate quantitative structure-activity relationships (QSAR). Once again these equations emphasize the paramount importance of steric/dispersion factors in enhancing antibacterial potency. Hydrophobicity also plays a role, albeit a minor one. Comparisons with the QSAR obtained versus prokaryotic dihydrofolate reductase (DHFR) demonstrate subtle differences in binding behavior between meta and para substituents which may be effectively maximized in the design of more efficacious and selective antibacterial agents. The bacterial and avian QSAR equations can be used to calculate selectivity indices for trimethoprim, tetroxoprim, and two other specially designed 2,4-diamino-5-(substituted-benzyl)pyrimidines.

Quantitative structure-activity relationship of triazine-antifolate inhibition of Leishmania dihydrofolate reductase and cell growth.

Quantitative structure-activity relationships have been formulated for the inhibition of Leishmania major dihydrofolate reductase (DHFR) and for inhibition of promastigote cell growth by a series of 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(3-substituted-phenyl)-s-triazine s. The inhibition of DHFR is best correlated by a modified variable for hydrophobicity of the 3-X substituent (pi'3), an alkoxy group indicator variable (IOR), a disposable parameter (beta) obtained by iteration, and a variable that parameterizes steric effects (MR) in the equation, log 1/Ki = 0.65 pi'3 - 1.22 log (beta X 10 pi'3 + 1) - 1.12IOR + 0.58MRY + 5.05 (r = 0.965). The EC50 values for triazine inhibition of L. major cell growth in culture are correlated by the equation log 1/EC50 = 0.21 pi 3 + 0.44 log 1/Ki + 0.53 (r = 0.960). When compared to DHFR from human, other vertebrates, and E. coli, L. major DHFR differs in that it optimally binds triazine congeners that are much more hydrophobic. Furthermore, in contrast to other DHFR's studied, triazine binding to L. major DHFR does not seem to be influenced by the electronic characteristics of the 3-X substituent of the parent triazine molecule. However, L. major DHFR is more sensitive to the steric effects and polarizability of the 3-X substituent. Our results indicate that triazines inhibit L. major promastigote growth via direct inhibition of DHFR as is shown by the good correlation between log 1/Ki values for inhibition of the purified enzyme and log 1/EC50 values for inhibition of cell culture growth. Two lipophilic, sterically large analogues of this triazine series showed selectivity for L. major DHFR over human DHFR. Further optimization of the MR and IOR terms in the above QSAR equations may provide even more selective inhibitors.

Inhibition by 5-(substituted-benzyl)-2,4-diaminopyrimidines of murine tumor (L5178Y) cell cultures sensitive to and resistant to methotrexate. Further evidence for the sensitivity of resistant cells to hydrophobic drugs.

Forty-three 5-(substituted-benzyl)-2,4-diaminopyrimidines have been studied as inhibitors of murine tumor cell cultures (L5178Y). Two types of cells were used--one resistant to methotrexate and one sensitive to methotrexate. The formulation of quantitative structure--activity relationships showed that the methotrexate-resistant cells are more sensitive to the more hydrophobic congeners. pi 0 for the sensitive cells is about 1.4, while pi 0 for the methotrexate-resistant cells is above 3. These results are similar to those found for 2,4-diaminotriazines (Selassie, C.D.; Guo, Z. R.; Hansch, C.; Khwaja, T. A.; Pentecost, S. J. Med. Chem. 1982, 25, 157).

Comparative structure-activity relationships of antifolate triazines inhibiting murine tumor cells sensitive and resistant to methotrexate.

The inhibitory effect of 108 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(substituted-phenyl)-s-triazines on murine L5178Y tumor cells, resistant and sensitive to methotrexate (MTX), has been studied. From the pI50 values, quantitative structure-activity relationships have been formulated which show that the lipophilic triazines are much more inhibitory against resistant cells than methotrexate or hydrophilic triazines. The results are compared with the behavior of other antifolate drugs that have been used in chemotherapy, as well as with eight antitumor drugs that are not antifolates. The acquired resistance of these cells toward hydrophilic antifolates may be attributed to the combined effect of an impaired active-transport system, a change in the conformation of dihydrofolate reductase in the resistant cells, and an amplified production of dihydrofolate reductase in the resistant cells.

Comparison of quantitative structure-activity relationships of the inhibition of leukemia cells in culture with the inhibition of dihydrofolate reductase from leukemia cells and other cell types.

A set of 2,4-diamino-5-(3-X-phenyl)-s-triazines was used to inhibit the growth of tumor cells (L5178 leukemia) in culture. The molar concentration (C) of triazine causing 50% reduction in the rate of cell growth was used to develop a quantitative structure-activity relationship: log 1/C = 1.32 pi - 1.70 log (beta.10 pi + 1) + 0.44I + 8.10, where pi is the hydrophobic constant for X, beta is a disposable parameter, and I is an indicator variable for congeners containing a -CH2Z-C6H4-Y moiety (Z = O or NH). This equation is compared with similar equations derived for the inhibition of dihydrofolate reductase from leukemia cells and bovine liver.

A comparison of the inhibition of growth of methotrexate-resistant and -sensitive leukemia cells in culture by triazines. Evidence for a new mechanism of cell resistance to methotrexate.

Forty-five 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(3-X-phenyl)-s-triazine inhibitors of dihydrofolate reductase (DHFR) and methotrexate (MTX) were tested on L5178Y/R murine tumor cell culture. The concentration of inhibitor causing a 50% decrease in growth rate was determined, and from these results a quantitative structure-activity relationship (QSAR) was developed. This QSAR is compared with QSAR for the same inhibitors acting on isolated DHFR and on L5178Y cell culture sensitive to MTX. The results show that very potent triazine inhibitors of resistant tumor cell growth can easily be made by making the triazines strongly hydrophobic. The optimum pi value for inhibition of MTX-sensitive cell culture is 0.8, while pi 0 for the resistant cell culture is about 6.0. The QSAR for MTX-sensitive and -resistant tumor cell culture inhibition is compared with the corresponding QSAR for Lactobacillus casei cells. Both the mammalian and bacterial cells appear to protect themselves from the highly hydrophilic MTX by erecting lipophilic barriers.

Quantitative structure-activity relationships of 2, 4-diamino-5-(2-X-benzyl)pyrimidines versus bacterial and avian dihydrofolate reductase.

Quantitative structure-activity relationships (QSAR) have been formulated for a set of 15 2,4-diamino-5-(2-X-benzyl)pyrimidines versus dihydrofolate reductase from Lactobacillus casei and chicken liver. QSARs were also developed for comprehensive data sets containing mono-, di-, and trisubstituted benzyl derivatives. Particular emphasis was placed on the role played by ortho substituents in the overall binding process and subsequent inhibition of the catalytic process in both the prokaryotic and eucaryotic DHFRs. Comparisons between the two QSARs reveal subtle differences at specific positions which can be optimized to design more selective antibacterial agents.

Quantitative structure-activity relationship of antifolate inhibition of bacteria cell cultures resistant and sensitive to methotrexate.

Sets of 5-(substituted benzyl)-2,4-diaminopyrimidines and 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(3-substituted phenyl)-s-triazines as well as several other antifolates were tested as inhibitors of Escherichia coli dihydrofolate reductase and E. coli cell cultures both sensitive and resistant to methotrexate. From the results quantitative structure-activity relationships (QSAR) were formulated. The triazines were found to inhibit sensitive and resistant cell cultures to the same degree, but the benzylpyrimidines showed marked differences against the two types of cells. Increased hydrophobicity produced benzylpyrimidines more active against the resistant E. coli cell. Metroprine did not discriminate between the two types of cells cultures, but pyrimethamine and 2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyrimidin e (BW 301U) did. The results are compared with triazines and benzylpyrimidines acting on Lactobacillus casei and murine tumor cells sensitive and resistant to methotrexate. QSAR is shown to be an effective means for detecting receptor differences.

On the structure selectivity problem in drug design. A comparative study of benzylpyrimidine inhibition of vertebrate and bacterial dihydrofolate reductase via molecular graphics and quantitative structure-activity relationships.

Quantitative structure-activity relationships (QSAR) have been derived for the action of 68 5-(substituted benzyl)-2,4-diaminopyrimidines on dihydrofolate reductase (DHFR) from Lactobacillus casei and chicken liver. The QSAR are analyzed with respect to the stereographics models of the active sites of the enzymes and found to be in good agreement. Using these QSAR equations, we have attempted to design new trimethoprim-type antifolates having higher selectivity for the bacterial enzyme. The general problem of developing selective inhibitors is discussed.

Inhibition of chicken liver dihydrofolate reductase by 5-(substituted benzyl)-2,4-diaminopyrimidines. A quantitative structure-activity relationship and graphics analysis.

The inhibition of chicken liver dihydrofolate reductase by a series of substituted benzylpyrimidines has been investigated. From the inhibition constants a quantitative structure-activity relationship has been formulated. This mathematical model is compared with molecular graphics models constructed from the X-ray crystallographic coordinates of trimethoprim and 5-(3,4-dimethoxy-4-isopropenylbenzyl)-2,4- diaminopyrimidine bound to the enzyme. There is good correspondence between the two types of models.

Thiopurine methyltransferase: structure-activity relationships for benzoic acid inhibitors and thiophenol substrates.

Twenty-seven substituted benzoic acids have been studied as inhibitors of partially purified human renal thiopurine methyltransferase (TPMT). Quantitative structure-activity relationship (QSAR) analysis resulted in the following equation: pI50 = 1.25( +/- 0.53)pi'3 + 0.73( +/- 0.38)MR3,4 + 2.92( +/- 0.39). In this equation pI50 is the -log of the concentration of compound that inhibits the enzyme activity by 50% (IC50);pi'3 is the relative hydrophobicity of the more hydrophobic of the two meta substituents; and MR3,4 is the molar refractivity of the more hydrophobic of the two meta substituents and of the para substituent on the phenyl ring. In addition, 14 substituted thiophenols were tested as substrates for the enzyme. All 14 thiophenols tested were excellent substrates with Km constants (0.8-7.8 microM) that were at least 2 orders of magnitude lower than those of any known thiopurine substrate for TPMT. However, there was no discernible relationship between the activities of thiophenol substrates and their physicochemical parameters. These results suggest that benzoic acid inhibitors of and thiophenol substrates for TPMT may interact with different sites on the enzyme.

Crystallography, quantitative structure-activity relationships, and molecular graphics in a comparative analysis of the inhibition of dihydrofolate reductase from chicken liver and Lactobacillus casei by 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(substituted-phenyl)-s-triazine s.

The inhibition of dihydrofolate reductase from chicken liver and from Lactobacillus casei has been studied with 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(substituted-phenyl)-s-triazines. It was found that for the chicken enzyme, inhibitor potency for 101 triazines was correlated by the following equation: log 1/Kiapp = 0.85 sigma tau' - 1.04 log (beta X 10 sigma tau' + 1) + 0.57 sigma + 6.36. The parameter tau' indicates that for certain substituents, tau = 0. In the case of the L. casei DHFR results, meta and para derivatives could not be included in the same equation. For 38 meta-substituted compounds, it was found that log 1/Kiapp = 0.38 tau'3-0.91 log (beta X 10 tau'3 + 1) + 0.71I + 4.60 and for 32 para-substituted phenyltriazines log 1/Kiapp = 0.44 tau'4-0.65 log (beta tau'4 + 1') - 0.90 upsilon + 0.69I + 4.67. In the L. casei equation, I is an indicator variable for substituents of the type CH2ZC6H4-Y and ZCH2C6H4-Y, where Z = O, NH, S, or Se. The parameter upsilon is Charton's steric parameter, which is similar to Taft's Es. The mathematical models obtained from correlation analysis are compared with stereo color graphics models.

Trypsin hydrolysis of X-phenyl hippurates.

The kinetics of hydrolysis of eight substituted phenyl hippurates by trypsin at pH values 6, 7 and 8 were investigated and the kinetic constants Km, kcat (kcatalysis) and kcat/Km were shown to fit the Hammett equation. The rho (rho) value obtained from the correlation of trypsin binding with this class of esters was compared with that obtained with other serine and cysteine proteases. The rho value for trypsin was similar to that obtained for alpha-chymotrypsin in that both enzymes reveal a pronounced dependence on through resonance (sigma-) in the formation of the Michaelis complex and the acyl-enzyme. It is apparent that through resonance facilitates the leaving of the phenoxy moiety during catalysis in the serine proteases but not in the case of the cysteine or bacterial serine proteases.

Phenol toxicity in leukemia cells: a radical process?

The multiple functions of the phenol moiety that are widely present in disparate sources such as drugs, pesticides, teas, fuel additives and surfactants have not been clearly delineated. The differences in behavior of phenols, which run the gamut from aberrations in DNA/chromosomes to suppression of genotoxic activity of carcinogenic compounds, merit further attention. In this study, a through examination of the growth inhibition patterns of 37, simple 3- and 4-substituted phenols in mouse leukemia cells was carried out and the following quantitative structure-activity relationship (QSAR) was obtained for the 23 electron releasing substituents in X-phenols: log 1/IC50 = -1.58 sigma(+) +0.21 log P + 3.10. In this QSAR, IC50 is the concentration of phenol that induces 50% inhibition of growth. P is a measure of the hydrophobicity of each phenol and Brown's electronic parameter, sigma+, represents the electronic effect of the substituent. The negative dependence on sigma+ is strongly reminiscent of what is observed in the developmental toxicity of phenols on rat embryos as well as for the radical abstraction of a hydrogen atom from phenolic groups. The other 15 electron-attracting substituted X-phenols clearly show a linear dependence on hydrophobicity alone: Log 1/IC50 = 0.62 log P + 2.35. The bifurcation in mechanism of action of this large set of diverse phenols is novel and unusual. It suggests that two distinct processes are operative. In the case of electron releasing substituted phenols, the observations are not inconsistent with a radical mediated process while with electron attracting substituted phenols, non-specific toxicity as modulated by hydrophobicity, appears to predominate.