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.

Human placental alkaline phosphatase in benign and malignant ovarian neoplasia.

In benign and malignant ovarian tumor patients, human placental alkaline phosphatase (HPLAP) was determined in serum and extracts from surgical tumor biopsies using a highly specific enzyme-antigen immunoassay based on a mouse monoclonal antibody (E6) to HPLAP. Serum HPLAP levels greater than or equal to 0.1 unit/liter were found in 58% of ovarian cancer patients. Serum carcinoembryonic antigen levels were positive (greater than 5.4 ng/ml) in 17% of these patients. HPLAP was detected in extracts from 13 of the 14 tumors investigated (range, 2.4 to 557 milliunits/g). Only the mixed heterologous Müllerian sarcoma was negative. The highest HPLAP content of normal ovarian tissue was 1.1 milliunits/g. The amount of heat-stable and L-p-bromotetramisole-insensitive alkaline phosphatase was in all cases much higher than the fraction recognized by E6. The neoplastic origin of HPLAP was confirmed immunohistochemically on paraffin sections by an indirect avidin-biotin-peroxidase staining procedure using E6. The staining pattern was compared to the histochemical distribution of total alkaline phosphatase on adjacent sections. A consistency was found between the amount of HPLAP in tissue extracts and its immunohistochemical distribution. In all the tumors, staining for HPLAP was observed mainly on the plasma membranes of carcinoma cells. In 9 of the 10 carcinomas, the histological distribution of HPLAP and also of total alkaline phosphatase was heterogeneous. HPLAP staining, present in one of five normal ovaries, was restricted to germinal inclusion cysts. The present results support the hypothesis that serous ovarian tumors originate from these cysts.

The structure-activity relationship of the papain hydrolysis of N-benzoylglycine esters.

The relationship between structure and the Michaelis-Menten constants (Km) for the papain hydrolysis of a series of 37 N-benzoylglycine esters was investigated. The series studied comprises a wide range of aromatic and aliphatic esters with a 5000-fold variation in their Km constants and essentially constant kcat values. It was found that the variation in the Km constants could be rationalized by the following quantitative structure-activity relationship (QSAR): log 1/Km = 8.13F + 0.33Z + 1.27II3' + 1.95. In this equation F is the field inductive parameter, II3' is the hydrophobic constant for the more lipophilic of the two possible meta substituents and Z is the Van der Waals distance from oxygen through the end of the molecule, in the direction of the 4 position of the aromatic ester moiety.

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.

Selective inhibition of Leishmania dihydrofolate reductase and Leishmania growth by 5-benzyl-2,4-diaminopyrimidines.

The classical anti-microbial antifolates trimethoprim, pyrimethamine, and cycloguanil are poor inhibitors of purified dihydrofolate reductase (DHFR) from Leishmania major. They show no selectivity for Leishmania DHFR relative to the human enzyme, and it is not surprising that they are ineffectual as anti-leishmanial agents. Several 5-(substituted-benzyl)-2,4-diaminopyrimidines have been screened as inhibitors for purified L. major and human DHFRs. These compounds inhibit Leishmania DHFR with I50 values ranging from 0.2 to 11 microM, and show about 5 to greater than 100-fold greater selectivity for the parasite DHFR than the human enzyme. These pyrimidine analogs are more potent inhibitors of Leishmania promastigote and amastigote growth than the classical anti-microbial antifolates, and serve as lead compounds for the development of new selective antileishmanial agents.

On the structure of medicinal chemistry.

The great advances in biochemistry and molecular biology, the development of physical organic chemistry, and the availability of large computers are creating opportunities for restructuring medicinal chemistry. The enormous volume of scientific results relevant to medicinal chemistry which appear with each new round of the journals forces us to make greater efforts to bring the information together in more meaningful patterns. The QSAR paradigm redirects our thinking about structuring medicinal chemistry.

Correlation analysis of Baker's studies on enzyme inhibition. 2. Chymotrypsin, trypsin, thymidine phosphorylase, uridine phosphorylase, thymidylate synthetase, cytosine nucleoside deaminase, dihydrofolate reductase, malate dehydrogenase, glutamate dehydrogenase, lactate dehydrogenase, and glyceraldehyde-phosphate dehydrogenase.

The inhibitory activity of 1058 inhibitors of the title enzymes has been formulated in 13 equations correlating chemical structure with inhibitory potency. Two types of regions in enzymes have been defined by means of pi and molar refractivity constants. The use of indicator variables has been extensively developed to suggest special enzyme-ligand interactions. Several examples are given of the use of correlation equations in comparing structural features of different systems.

Correlation analysis of Baker's studies on enzyme inhibition. 1. Guanine deaminase, xanthine oxidase, dihydrofolate reductase, and complement.

Five correlation equations are presented which relate inhibitory activity of 578 inhibitors of guanine deaminase, xanthine oxidase, dihydrofolate reductase, and complement to their chemical structures. The use of correlation analysis in enzyme studies for drug development is discussed. The importance of indicator variables in such studies is emphasized.

Structure-activity relationship of the ficin hydrolysis of phenyl hippurates. Comparison with papain, actinidin, and bromelain.

A study of the hydrolysis of 30 substituted-phenyl hippurates by the enzyme ficin has been made. From the results the following quantitative structure--activity relationship (QSAR) has been derived: log 1/Km = 0.79 pi'3 + 0.58 sigma + 0.28 MR4,5 + 3.70. In this expression Km is the Michaelis constant, pi'3 refers to the more hydrophobic of the two meta substituents, and MR4,5 is the molar refractivity of substituents in the 4- and 5-positions of the phenyl ring. This QSAR is compared with those from papain, actinidin, bromelain B, and bromelain D.

Structure-activity relationship of aniline mustards acting against B-16 melanoma in mice.

A set of 23 aniline mustards [X-C6H4N(CH2CH2Cl)2] have been tested for their activity against B-16 melanoma in mice. The following quantitative structure-activity relationship (QSAR) correlates the data well: log 1/C = -2.06 sigma - 0.15 pi - 0.13 pi2 + 4.13 (r = 0.936). When this equation is compared with those formulated for aniline mustards acting against leukemia, it is found that log P0 (ideal lipophilicity) is higher for solid tumors. The QSAR brings out the unique activity of phenylalanine aniline mustard.

Structure-activity relationships in antitumor aniline mustards.

Quantitative structure-activity relationships (QSAR) have been formulated for the hydrolysis of aniline mustards and their antitumor activity against Walker 256 tumor and L1210 and P388 leukemia. In general, the antitumor activity parallels hydrolysis under the conditions defined by Ross; toxicity (LD50) parallels antitumor efficacy. Chlorambucil is an exception. A most important finding is that ideal lipophilicity for effectiveness against Walker tumor appears to be much higher than for the leukemias which suggests that solid tumors may, in general, require more lipophilic drugs than leukemias.

Quantitative structure-activity relationship of chymotrypsin-ligand interactions.

Quantitative structure-activity relationships (QSAR) have been formulated for the interactions of a variety of ligands with chymotrypsin. The parameters Km, k2, k3, kcat, and Ki are found to be strongly dependent on molar refractivity as well as steric and electronic character of the substituents in structures of the type R2CH(COOR3)NHCOR1 where R may be H. A model for binding of D and L esters is presented which gives a consistent view of the binding step, acylation, and deacylation. The model suggests new avenue for exploration.

Quantitative structure-selectivity relationships. Comparison of the inhibition of Escherichia coli and bovine liver dihydrofolate reductase by 5-(substituted-benzyl)-2,4-diaminopyrimidines.

In our previous publication (Blaney, J. M.; Dietrich, S. W.; Reynolds, M. A.; Hansch, C. J. Med. Chem. 1979, 22, 614), correlation equations were presented for the inhibition of bovine liver and Escherichia coli dihydrofolate reductase (DHFR) by 5-(substituted benzyl)-2,4-diaminopyrimidines. These equations brought out differences in the way these two enzymes interact with substituents, which explain the high selectivity of drugs like trimethoprim. We have tested and further developed these equations in this report. It is of particular interest that our previously published correlation equation for E. coli DHFR accurately predicted the potency of a commercial competitor of trimethoprim (tetroxoprim) now in clinical use. We believe that new and effective competitors for trimethoprim can be designed by means of the two correlation equations.

A comparison of the inhibitory action of 5-(substituted-benzyl)-2,4-diaminopyrimidines on dihydrofolate reductase from chicken liver with that from bovine liver.

Forty-four 5-(substituted-benzyl)-2,4-diaminopyrimidines have been tested as inhibitors of chicken and bovine liver dihydrofolate reductase. The chicken enzyme is, on the average, about 10 times less easily inhibited than bovine enzyme. Substituents which show the greatest selectivity are 4-NHCOCH3, 3-OC4H9, 3-I, 3-CF3-4-OCH3, and 3,4,5-(OCH3)3. The inhibition constants have been used to formulate quantitative structure-activity relationships for comparative purposes.

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.

Mutagenicity of substituted (o-phenylenediamine)platinum dichloride in the Ames test. A quantitative structure-activity analysis.

A set of 13 substituted (o-phenylenediamine)platinum dichlorides has been studied in the Ames test using Salmonella typhimurium (TA-92). These cis-platinum compounds are mutagenic without activation by microsomes. The following correlation equation shows that the most important determinant of mutagenicity by substituents (X) is electron withdrawal via through resonance: log 1/C = 2.23 sigma sigma minus + 5.78. C in this expression is the molar concentration of compound producing 30 mutations/10(8) bacteria initially delivered above background mutation, and sigma minus is the Hammett constant obtained from substituted anilines.

Structure-activity relationship in synthetic fibrinolytics. 2-Phenethynylcyclopropanecarboxylates.

The fibrinolytic activity of nine 2-phenethynylcyclopropanecarboxylates was measured in the hanging clot test. The structure-activity relationship is given by log 1/C equals 0.54 log P + 2.01 where P is the octanol-water partition coefficient of the carboxylate ion pair and C is the molar concentration of the drug. The equation obtained for the cyclopropanecarboxylates is compared with similar equations for benzoates, salicylates, and N-phenylanthranilates.

Inhibition of dihydrofolate reductase. Structure-activity correlations of quinazolines.

A quantitative structure-activity relationship (QSAR) has been formulated for quinazolines causing 50% inhibition of liver dihydrofolate reductase. The QSAR for the quinazolines is compared with QSAR for triazine and pyrimidine inhibitors. The three QSAR suggest new possibilities for the design of inhibitors of mammalian dihydrofolate reductase.

Dependence of hydrophobicity of apolar molecules on their molecular volume.

Cavity size is the primary determinant of the partition coefficient (P) of apolar solutes between octanol and water. Although the energy of cavity formation would be expected to be related to cavity area, older methods of area calculation give a poorer correlation with log P than does volume. Apolar solutes clearly fall into two classes based on their log P/volume relationship, the distinction possibly being whether the solute exposes mostly hydrogen atoms or unbonded electrons.

Quantitative structure-activity relationships of antimalarial and dihydrofolate reductase inhibition by quinazolines and 5-substituted benzyl-2,4-diaminopyrimidines.

A quantitative structure-activity relationship (QSAR) for the inhibition of dihydrofolate reductase from S. faecium by quinazolines has been formulated. This is compared with a QSAR for inhibition of E. coli dihydrofolate reductase by 2,4-diamino-5-benzylpyrimidines. The QSAR for inhibition of bacterial enzyme is compared with QSAR for mammalian enzyme inhibition. A QSAR has been formulated for the antimalarial action of quinazolines against P. berghei in mice. The antimalarial QSAR is consistent with that of the in vitro bacterial study.