Partial resistance of E. coli mutants against 2, 4-diamino-5-benzylpyrimidines by interactions with bacterial membrane lipopolysaccharides. Derivation of quantitative structure-binding relationships.

A series of previously synthesized 2,4-diamino-5-benzylpyrimidines, inhibitors of bacterial dihydrofolate reductase (DHFR) showed decreased inhibition of E. coli cultures, despite increased inhibitory activity against DHFR. Preliminary studies using E. coli mutants with different degrees of outer membrane deficiencies suggested that the decrease in activity was partly due to inactivation because of binding to outer membrane constituents. In the present study antibacterial activities of the benzylpyrimidines have been systematically determined as a function of cell membrane defects in E. coli using bacterial growth kinetic techniques. It has been shown that the observed differences in activity were not due to different binding affinities to the target enzyme of the mutants. Lipopolysaccharides have been extracted from the mutants and used in binding studies by ultrafiltration, photometric and NMR techniques. The observed differences in binding affinity to the lipopolysaccharides have been related to the differences in the lipophilic properties and molecular weight of the substituents. Quantitative structure-activity relationships have been derived. The results of the study show the importance of drug-membrane interactions for the rational development of antibacterials.

Inhibition of the conjugation of PABA with glycine in vitro by sulfamoyl benzoic acids, sulfonamides, and penicillins and its relation to tubular secretion.

Elimination of drug molecules via tubular secretion is an important pharmacokinetic parameter especially for oral dosage forms where an extremely short half-life would prevent their application. We have studied the inhibition in vitro of the glycination of p-aminobenzoic acid (PABA) using rat liver microsomal preparations. The I50 values, the concentration of the inhibitor that leads to 50% inhibition of glycine conjugation of PABA as compared to the control, have been determined for sulfamoyl benzoic acids, sulfonamides, and penicillins. Statistically significant regression equations were derived explaining the observed variation in I50 values as a function of lipophilicity and steric bulk of the substituents for the combined set of sulfamoyl benzoic acids and sulfonamides (n = 33). For the penicillins studied, only steric effects seem to be important for the explanation of the I50 values. Finally, regression analysis and the use of neural networks allowed the classification of compounds by their in vitro I50 values as being tubular secreted or not. Therefore, it can be concluded that the inhibition in vitro of the glycine conjugation of PABA is a useful model for the estimation of tubular secretion and drug interaction potential of acidic drug molecules in this process in vivo.

Studies on the inhibitory effects of analogues of dapsone on neutrophil function in-vitro.

We have compared twelve sulphone analogues of dapsone in terms of inhibition both of zymosan-mediated human neutrophil respiratory burst and inhibition of interleukin-1-stimulated neutrophil adhesion to transformed human umbilical vein endothelial cells. Overall, there was a good correlation between the respective rank orders of compound potency in the two test systems. The most effective compounds in terms of respiratory burst and adherence inhibition were the 2-nitro-4-amino-, 2-hydroxy-4-aminopropyl-, and 2-methoxy-4-aminoethyl- derivatives. In general, potency was inversely associated with lipophilicity; compounds with bulky side-chains, e.g. the 2-methyl-4-aminopentyl, 2-methyl-4-aminohexyl and the 2-hydroxymethyl-4-aminoethyl derivatives, were less potent. A 2-hydroxy-4-amino- derivative was the exception, however, with low lipophilicity and relatively low potency. All of the compounds tested showed comparable or greater inhibition in both the neutrophil-mediated assays compared with dapsone. Some of the compounds might, because of their good tissue penetration and lower toxicity than dapsone, have the potential to undergo further development.

Structure-activity relationship studies on benzofuran analogs of propafenone-type modulators of tumor cell multidrug resistance.

A series of benzofurylethanolamine analogs of propafenone (1a) have been prepared and evaluated for multidrug resistance-reversing activity in two in vitro assay systems. As for propafenones, an excellent correlation of biological data with calculated lipophilicity values was found for benzofurans, whereby the latter generally had lower activity/lipophilicity ratios. Almost identical slopes of the regression lines were obtained for both propafenones and benzofurans. Multiple linear regression analysis of the complete data set yielded an equation with excellent predictive power (r2 cross-valid = 0.968). Interaction measurements with artificial membranes indicated that the differences in activity between these two series of compounds are not due to differences in the interaction pattern with biological membranes.

Studies on the toxicity of analogues of dapsone in-vitro using rat, human and heterologously expressed metabolizing systems.

Three metabolizing systems (rat, heterologously expressed CYP3A4 and human liver) were used to evaluate 12 analogues of dapsone (4,4'diaminodiphenylsulphone) in-vitro. Methaemoglobin formation in a two-compartment and cytotoxicity in a single-compartment model were studied using human erythrocytes and neutrophils, respectively, as target cells. In the two-compartment system using rat microsomes as a generating system and methaemoglobin as an endpoint, the least potent methaemoglobin formers tested were the 2-methyl-4-propylamino (AXDD14), 2-hydroxy-4-4'amino (ABDD5) derivatives and a sulphone/trimethoprim derivative (K-130). Dapsone itself, a 2-methoxy-4-ethylamino (W10) and a 2-hydroxyl-4-ethylamino compound (ABDD39) were the most toxic. In the single-compartment cytotoxicity test using rat microsomes, AXDD14 was again among the least toxic, as was a 2-methyl 4-cyclopentyl derivative (AXDD17) and surprisingly ABDD39. The most cytotoxic compounds again included dapsone itself as well as two 2-trifluoromethyl derivatives. The only significant methaemoglobin formation and cytotoxicity shown with the heterologously expressed human CYP 3A4 was with AXDD14, which was extensively activated. Interestingly, metabolism of dapsone was low using the expressed CYP 3A4. In the two-compartment system using human liver microsomes, AXDD14, K-130 and ABDD5 were oxidized to a significantly lesser extent compared with dapsone and these preliminary findings indicate that future development of these compounds may be worthwhile.

Syntheses and biological activities of new N1-aryl substituted quinolone antibacterials.

A series of quinolones with a systematically varied substitution at the phenyl ring at N1 has been synthesized. Three lipophilicity descriptors (log K, log P, Rm) and the pKa values have been determined as well as the microbiological activity: The MIC values for eight different strains of three Gram-positive and three Gram-negative species and the inhibitory concentrations of DNA supercoiling (IC90 and IC100) were determined. From a principal component and a QSAR analysis relationships between antibacterial activity concerning the whole-cell system and electronic properties as well as the length of the substituents at the phenyl rings could be derived. The activity in a cell-free system was governed by the lipophilicity and the width of the substituents. It is speculated that the quinolones take a defined place in the DNA gyrase-DNA complex which is characterized by polar amino acids. This is in agreement with findings from studies of mutant gyrases.

New antifolate 4,4'-diaminodiphenyl sulfone substituted 2,4-diamino-5-benzylpyrimidines. Proof of their dual mode of action and autosynergism.

New 4,4'-diaminodiphenylsulfone substituted 2,4-diamino-5-benzylpyrimidines were synthesized. These compounds are highly active inhibitors of both bacterial dihydrofolate reductase (DHFR) and dihydropteroic acid synthase (SYN). The simultaneous inhibition of both enzymes leads to autosynergism in whole cells in the same way as known for combinations of sulfonamides with trimethoprim. The inhibitory activity is demonstrated in cell-free systems of DHFR and SYN derived from various species (M. lufu, E. coli, C. albicans) and in whole cell systems of the mycobacterial strain M. lufu. The compounds are rare examples for the combination of two mechanisms of action in one molecule.

Membrane interactions of some catamphiphilic drugs and relation to their multidrug-resistance-reversing ability.

The multidrug-resistance (MDR)-reversing ability of the catamphiphilic drugs could be mediated through their interaction with the membrane phospholipids. This could lead directly (through changes in membrane permeability and fluidity) and/or indirectly (through inhibition of P-glycoprotein phosphorylation via inhibition of the phosphatidylserine-dependent protein kinase C or changes in the conformation and functioning of the membrane-integrated proteins via changes in the structure organization of the surrounding membrane bilayer) to the reversal of MDR. Using differential scanning calorimetry and NMR techniques and artificial membranes composed of phosphatidylcholine or phosphatidylserines we found a significant correlation between the MDR-reversing activity of the drugs in doxorubicin-resistant human breast carcinoma MCF-7/DOX and murine leukaemia P388/DOX tumour cells (data taken from the literature) and their ability to interact with phosphatidylserines. Trans- and cis-flupentixol were found to interact most strongly with both the phospholipids, followed by trifluoperazine, chlorpromazine, triflupromazine, flunarizine, imipramine, quinacrine and lidocaine. Differences in the interaction of trans- and cis-flupentixol with the phospholipids studied are suggested to be responsible for their different MDR-reversing ability. Verapamil showed moderate membrane activity, assuming that the membrane interactions are not the only reason for its high MDR-reversing ability. Amiodarone showed very strong interactions with phosphatidylserines and is recommended for further MDR-reversal studies.

Preliminary evaluation of the toxicity and efficacy of novel 2,4-diamino-5-benzylpyrimidine-sulphone derivatives using rat and human tissues in vitro.

Four novel combined dapsone and trimethoprim analogues, K-120, K-150, K-138 and DRS-506, have been compared with dapsone in their methaemoglobin forming abilities as well as their anti-inflammatory properties using rat and human tissues in vitro. All four compounds formed consistently less methaemoglobin compared with dapsone in both the rat and human microsomes. Using human microsomes from five livers, K-120 was significantly less toxic than the other analogues in three of the five livers (P < 0.01). DRS-506 and K-138 both inhibited the human neutrophil respiratory burst to a significantly greater degree compared with dapsone at 0.5 mM (P < 0.01), while K-120 and K-150 showed no significant effect at 0.5 mM. At 1 mM, DRS-506, K-120 and K-138 were more potent than dapsone (P < 0.01), although K-150 appeared to increase the neutrophil activation. All four analogues caused a significant reduction in neutrophil adhesion to human umbilical vein cells at 0.1 mM. In view of its efficacy and low toxicity, K-120 shows considerable promise for future clinical evaluation.

Drug membrane interaction and the importance for drug transport, distribution, accumulation, efficacy and resistance.

Some aspects of drug membrane interaction and its influence on drug transport, accumulation, efficacy and resistance have been discussed. The interactions manifest themselves macroscopically in changes in the physical and thermodynamic properties of "pure membranes" or bilayers. As various amounts of foreign molecules enter the membrane, in particular the main gel to liquid crystalline phase transition can be dramatically changed. This may change permeability, cell-fusion, cell resistance and may also lead to changes in conformation of the embedded receptor proteins. Furthermore, specific interactions with lipids may lead to drug accumulation in membranes and thus to much larger concentrations at the active site than present in the surrounding water phase. The lipid environment may also lead to changes in the preferred conformation of drug molecules. These events are directly related to drug efficacy. The determination of essential molecular criteria for the interaction could be used to design new and more selective therapeutics. This excursion in some aspects of drug membrane interaction underlines the importance of lipids and their interaction with drug molecules for our understanding of drug action, but this is not really a new thought but has been formulated in 1884 by THUDICUM: "Phospholipids are the centre, life and chemical soul of all bioplasm whatsoever, that of plants as well as of animals".

Experimental drugs and combination therapy.

The worldwide increase in tuberculosis, the additional problem of increasing multiple drug resistance (MDR) and the primary resistance of Mycobacterium avium requires new strategies in drug development and in therapy. The reason for development of MDR is manifold. One important factor is the change in cell wall construction which limits the penetration of the drug to the target receptor. This is supported by the observation that within a class of tuberculostatic drugs (identical mode of action) the more lipophilic derivative is more effective. In addition, it has been shown that mycobacteria within macrophages are able to synthesize additional multilamellar cell wall components. Several possibilities exist to overcome MDR. Besides improving the permeation properties of drugs, the development of synergistic drug combinations based on their special mode of action is a promising approach. This is illustrated with the highly synergistic combination of newly developed hydrazones and thiacetazone respectively with rifampicin. Chance combinations which may even lead to antagonism have to be avoided. Examples of antagonistic behavior of the combinations clofazimine-dapsone and ofloxacin-rifampicin are discussed. An optimization procedure has been developed based on the determination of the specific resistance of patient-derived mycobacteria against single drugs and their combinations. With its use, an individual optimal treatment becomes feasible. Preliminary clinical experience is encouraging.

Synthesis and quantitative structure-activity relationships of anticonvulsant 2,3,6-triaminopyridines.

The synthesis of 2,3,6-triaminopyridine derivatives, representing a unique chemical structure for anticonvulsants, is described. The synthetic program was performed (a) to identify more potent analogs, (b) to determine structural properties controlling potency as well as neurotoxicity, and (c) to reduce the requirements for animal testing. As a result, besides other structural properties, the overall molecular lipophilicity (log k', octanol-coated column) explained changes in anticonvulsant potency and neurotoxicity. Mimicking the interaction of the amphiphilic triaminopyridines with biological membranes, NMR experiments in the presence of lecithin vesicles were conducted in order to measure the phospholipid-binding parameter log delta (1/T2). Replacement of log k' with log delta (1/T2) in the correlation analysis afforded a more significant equation describing the anticonvulsant activity of 21 derivatives.

Substituted 2-acylpyridine-alpha-(N)-hetarylhydrazones as inhibitors of ribonucleotide reductase activity and L1210 cell growth.

A series of substituted 2-acylpyridine-alpha-(N)-hetarylhydrazones was prepared and studied for their effects on mammalian ribonucleotide reductase activity using a highly purified enzyme preparation from Ehrlich tumor cells and on mouse leukemia L1210 cell growth in culture. Pyridine-2-aldehyde-2-pyridylhydrazone (PH 22), ethyl-2-pyridylketone-I-phthalazinylhydrazone (PH 22-25) and pyridine-2-aldehyde-2'-quinolylhydrazone (PQ 22) inhibited purified ribonucleotide reductase activity and inhibited L1210 cell growth in culture. PH 22-25 inhibited [3H]thymidine incorporation into DNA and inhibited ribonucleotide reductase activity in situ (as measured bvy [14C]cytidine metabolism and as a result inhibited DNA synthesis. There was no effect on RNA synthesis. These data indicate that these substituted hydrazones are potent inhibitors of tumor cell growth through the inhibition of ribonucleotide reductase.

Effect of hydroxypropyl-beta-cyclodextrin on the antimicrobial action of preservatives.

The interaction between hydroxypropyl-beta-cyclodextrin (HP-beta-CyD) and several preservatives with different chemical structures was investigated in aqueous solution. Complex stability constants of the 1:1 complexes were calculated from differential spectra. Using the serial dilution test the antimicrobial activities of the preservatives and their complexes against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Candida albicans were tested and MIC values determined. For highly water-soluble substances like thimerosal and bronopol, low or no inactivation was found; the more lipophilic substances, such as the phenolic compounds, showed strong inactivation when used in combination with HP-beta-CyD. The loss in activity by complex formation correlated with the bound fraction, thus suggesting that the appropriate antimicrobial substance for the preservation of cyclodextrin solutions can be selected according to the results of this study.

Influence of membrane composition of various parasites on the inhibitory activity of a series of bipyridyl analogues.

In a previous report the dependence of the antimycoplasmal activity of bipyridyl analogues on the presence of Cu+ ions has been shown. The inhibitory activity of these compounds has now been studied against Escherichia coli mycobacteria and Candida albicans in the absence and presence of Cu2+ ions using growth kinetic techniques. It was found that the inhibitory activity against E. coli increases in the presence of Cu2+ ions. In contrast, no additional effect of Cu2+ ions is observed for the inhibitory activity against mycobacteria. Some of the derivatives show very promising activity even against Mycobacterium avium strains. More complicated is the effect of Cu2+ ions on the inhibitory activity of the derivatives against C. albicans. For isoquinolones the observed delay in onset of inhibitors is reduced and no influence on the inhibitory activity is observed. The addition of Cu2+ ions to the phenanthrolines leads in contrast to a decrease in antifungal activity. The possible influence of membrane properties of the studied microorganism on the effect of Cu2+ ions is discussed.

In vitro and in vivo results of brodimoprim and analogues alone and in combination against E. coli and mycobacteria.

New diaminodiphenylsulfone inhibitors of dihydropteroate synthase are described with increased inhibitory activity against mycobacteria and plasmodia, whereas their side effect of methemoglobin formation could be suppressed. The optimization of diaminobenzylpyrimidines, inhibitors of dihydrofolate reductase, led to derivatives with increased inhibitory effect against mycobacteria, especially M. leprae and plasmodia. Some of these derivatives show autosynergism. Finally the combination of brodimoprim (BDP) and dapsone (DDS) was developed for the treatment of leprosy. First clinical trials in Paraguay and Ethiopia show that combinations of BDP/DDS and BDP/DDS plus rifampicin were highly effective and may become an alternative multi-drug therapy for the treatment of leprosy. The tolerance of the regimens used was generally good.

Critical comments on the treatment of leprosy and other mycobacterial infections with clofazimine.

The usefulness of clofazimine (CLO, CAS 2030-63-9) in the treatment of mycobacterial infections with special emphasis on treatment of leprosy is critically discussed. Skin discolouration which decreases compliance, placenta passage, excretion in mother's milk which endanger the embryo or baby respectively, saturation kinetics in absorption and difficulties to determine free drug concentration are severe problems. The observed antagonism in the combination of CLO with other drugs, especially with dapsone, is another argument against its application in the therapy of mycobacterial infections. In Germany CLO has not been approved by the Bundesgesundheitsamt.

A time hierarchy-based model for kinetics of drug disposition and its use in quantitative structure-activity relationships.

By using the time hierarchy of the processes determining the fate of drugs in biosystems (absorption, transport, distribution, protein binding, and elimination), a one-compartment open model is formulated at a subcellular level for the disposition phase of pharmacokinetics. The resulting disposition function describes the kinetics of the intracellular disposition of drugs as determined by their hydrophobicity, acidity or basicity, affinity to proteins, and rate parameters of elimination. Structure-activity relationships, based on the function with incorporated extrathermodynamic relations, fit the literature data well (fixed-time bioactivity-hydrophobicity profiles, kinetics of microbial degradation of organic compounds, and kinetics of analgesic effects of fentanyl derivatives in rats). Application of the approach, creating a basis for the construction of model-based quantitative structure-time-activity relationship, to biosystems of varying complexity is discussed.