Trimethoprim Analogues Research Articles

Design, synthesis, antimicrobial evaluation, and molecular modeling of new sulfamethoxazole and trimethoprim analogs as potential DHPS/DHFR inhibitors

Trimethoprim (TMP) and sulfonamides and are typically utilized to hinder the activities of dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) enzymes, respectively. In this context, we aimed to enhance the understanding of the inhibitory effects on dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) enzymes by synthesizing and evaluating two series of compounds. These compounds, designed as derivatives of p-aminobenzoic acid (related to sulfamethoxazole, SUL) and trimethoprim analogs, were rigorously tested for their in vitro antimicrobial efficacy and their ability to hinder the activities of DHFR and DHPS enzymes.Among the synthesized compounds, compounds 6 and 10 demonstrated exceptional antimicrobial efficacy against a diverse spectrum of bacterial and fungal strains. The assessment of these compounds also included their impact on biofilm formation, evaluated through crystal violet staining. Our in vitro enzyme assays revealed that compound 10 emerged as the most potent inhibitor of DHPS, with an IC50 value of 224.91 μg/mL, surpassing SUL by 1.6 times. Simultaneously, Compound 6 exhibited particularly potent DHFR-inhibitory action, with an IC50 value of 3.731 μg/mL, surpassing TMP by a factor of three.Molecular modeling studies further elucidated the mode of action, illustrating that compound 10 effectively occupied the DHPS pterin-binding site alongside p-aminobenzoic acid. In contrast, compound 6 was adeptly positioned within the DHFR-binding pocket.In summary, our findings underscore the promising prospects of compounds 6 and 10 in the management of bacterial infections. The results not only demonstrate their remarkable antimicrobial efficacy but also position them as potentially effective in a synergistic combination targeting the DHPS/DHFR enzymatic cascade.

Trimethoprim: An Old Antibacterial Drug as a Template to Search for New Targets. Synthesis, Biological Activity and Molecular Modeling Study of Novel Trimethoprim Analogs.

A new series of trimethoprim (TMP) analogs containing amide bonds (1–6) have been synthesized. Molecular docking, as well as dihydrofolate reductase (DHFR) inhibition assay were used to confirm their affinity to bind dihydrofolate reductase enzyme. Data from the ethidium displacement test showed their DNA-binding capacity. Tests confirming the possibility of DNA binding in a minor groove as well as determination of the association constants were performed using calf thymus DNA, T4 coliphage DNA, poly (dA-dT)2 and poly (dG-dC)2. Additionally, the mechanism of action of the new compounds was studied. In conclusion, some of our new analogs inhibited DHFR activity more strongly than TMP did, which confirms, that the addition of amide bonds into the analogs of TMP increases their affinity towards DHFR.

Antifolate Agents Against Wild and Mutant Strains of Plasmodium falciparum.

Plasmodium falciparum dihydrofolate reductase is an important target for antimalarial chemotherapy. The emergence of resistance has significantly reduced the efficacy of the classic antifolate drugs cycloguanil and pyrimethamine. In this paper we report new dihydrofolate reductase inhibitors identified using molecular modelling principles with the goal of designing new antifolate agents active against both wild and tetramutant dihydrofolate reductase strains three series of trimethoprim analogues were designed, synthesised and tested for biological activity. Pyrimethamine and cycloguanil have been reported to loose efficacy because of steric repulsion in the active site pocket produced due to mutation in Plasmodium falciparum dihydrofolate reductase. The synthesised molecules have sufficient flexibility to withstand this steric repulsion to counteract the resistance. The molecules have been synthesised by conventional techniques and fully characterised by spectroscopic methods. The potency of these molecules was evaluated by in vitro enzyme specific assays. Some of the molecules were active in micromolar concentrations and can easily be optimised to improve binding and activity.

Antifolates as effective antimicrobial agents: new generations of trimethoprim analogs

Over the past six decades, the folate biosynthetic pathway has provided a rich source of drug targets for the treatment of proliferative diseases. Drugs targeting dihydrofolate reductase have been especially successful as anticancer (methotrexate), antibacterial (trimethoprim, TMP) and antiprotozoal (cycloguanil, pyrimethamine) therapeutics. While trimethoprim remains a clinically important antimicrobial DHFR inhibitor, resistance by point mutations in otherwise sensitive strains as well as the natural insensitivity of several species limits its use. In this review, an historical overview of the attempts to develop drugs that target the folate pathway is presented along with a discussion of the basis of insensitivity to trimethoprim. From this vantage, we have developed the propargyl-linked antifolates as potent inhibitors of TMP-insensitive enzymes and strains. The structural basis of the increased affinity is detailed to promote the development of further generations of antifolates.

3D‐QSAR Studies on the Inhibitory Activity of Trimethoprim Analogues against Escherichia coli Dihydrofolate Reductase

Three-dimensional quantitative structure activity relationship (3D-QSAR) study has been carried out on the Escherichia coli DHFR inhibitors 2,4-diamino-5-(substituted-benzyl)pyrimidine derivatives to understand the structural features responsible for the improved potency. To construct highly predictive 3D-QSAR models, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods were used. The predicted models show statistically significant cross-validated and non-cross-validated correlation coefficient of r2 CV and r2 nCV, respectively. The final 3D-QSAR models were validated using structurally diverse test set compounds. Analysis of the contour maps generated from CoMFA and CoMSIA methods reveals that the substitution of electronegative groups at the first and second position along with electropositive group at the third position of R2 substitution significantly increases the potency of the derivatives. The results obtained from the CoMFA and CoMSIA study delineate the substituents on the trimethoprim analogues responsible for the enhanced potency and also provide valuable directions for the design of new trimethoprim analogues with improved affinity.

ChemInform Abstract: Metallation of Diazines. Part 3. New Synthesis of Analogues of Trimethoprim and of Bacimethrin.

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Expression, purification and characterization of recombinant mouse translation initiation factor eIF4E as a dihydrofolate reductase (DHFR) fusion protein

One of the earliest steps in translation initiation is recognition of the mRNA cap structure (m7GpppX) by the initiation factor eIF4E. Studies of interactions between purified eIF4E and its binding partners provide important information for understanding mechanisms underlying translational control in normal and cancer cells. Numerous impediments of the available methods used for eIF4E purification led us to develop a novel methodology for obtaining fractions of eIF4E free from undesired by-products. Herein we report methods for bacterial expression of eIF4E tagged with mutant dihydrofolate reductase (DHFR) followed by isolation and purification of the DHFR–eIF4E protein by using affinity and anion exchange chromatography. Fluorescence quenching experiments indicated the cap-analog, 7MeGTP, bound to DHFR–eIF4E and eIF4E with a dissociation constant ( K d) of 6 ± 5 and 10 ± 3 nM, respectively. Recombinant eIF4E and DHFR–eIF4E were both shown to significantly enhance in vitro translation in dose dependent manner by 75% at 0.5 μM. Nevertheless increased concentrations of eIF4E and DHFR–eIF4E significantly inhibited translation in a dose dependent manner by a maximum at 2 μM of 60% and 90%, respectively. Thus, we have demonstrated that we have developed an expression system for fully functional recombinant eIF4E. We have also shown that the fusion protein DHFR–eIF4E is functional and thus may be useful for cell based affinity tag studies with fluorescently labeled trimethoprim analogs.

Optimized Fluorescent Trimethoprim Derivatives for in vivo Protein Labeling

The combined technologies of optical microscopy and selective probes allow for real-time analysis of protein function in living cells. Synthetic chemistry offers a means to develop specific, protein-targeted probes that exhibit greater optical and chemical functionality than the widely used fluorescent proteins. Here we describe pharmacokinetically optimized, fluorescent trimethoprim (TMP) analogues that can be used to specifically label recombinant proteins fused to E. coli dihydrofolate reductase (eDHFR) in living, wild-type mammalian cells. These improved fluorescent tags exhibited high specificity and fast labeling kinetics, and they could be detected at a high signal-to-noise ratio by using fluorescence microscopy and fluorescence-activated cell sorting (FACS). We also show that fluorescent TMP-eDHFR complexes are complements to green fluorescent protein (GFP) for two-color protein labeling experiments in cells.

Preliminary in vitro Studies on Two Potent, Water‐Soluble Trimethoprim Analogues with Exceptional Species Selectivity Against Dihydrofolate Reductase from Pneumocystis carinii and Mycobacterium avium.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Corrigendum to ?Preliminary in vitro studies on two potent, water-soluble trimethoprim analogues with exceptional species selectivity against dihydrofolate reductase from Pneumocystis carinii and Mycobacterium avium?6Bioorg. Med. Chem. Lett. 14 (2004) 18119

Corrigendum to ?Preliminary in vitro studies on two potent, water-soluble trimethoprim analogues with exceptional species selectivity against dihydrofolate reductase from Pneumocystis carinii and Mycobacterium avium?6Bioorg. Med. Chem. Lett. 14 (2004) 18119

Preliminary in vitro studies on two potent, water-soluble trimethoprim analogues with exceptional species selectivity against dihydrofolate reductase from Pneumocystis carinii and Mycobacterium avium

2,4-Diamino-5-[3′,4′-dimethoxy-5′-(5-carboxy-1-pentynyl)]benzylpyrimidine ( 6) and 2,4-diamino-5-[3′,4′-dimethoxy-5′-(4-carboxyphenylethynyl)benzylpyrimidine ( 7) were synthesized from 2,4-diamino-5-(5′-iodo-3′,4′-dimethoxybenzyl)pyrimidine ( 9) via a Sonogashira reaction with appropriate acetylenic esters followed by saponification, and were tested as inhibitors of dihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), Mycobacterium avium (Ma), and rat in comparison with the widely used antibacterial agent 2,4-diamino-5-(3′,4′,5′-trimethoxybenzyl)pyrimidine (trimethoprim, TMP). The selectivity index (SI) for each compound was calculated by dividing its 50% inhibitory concentration (IC 50) against rat DHFR by its IC 50 against Pc, Tg, or Ma DHFR. The IC 50 of 6 against Pc DHFR was 1.0 nM, with an SI of 5000. Compound 7 had an IC 50 of 8.2 nM against Ma DHFR, with an SI of 11000. By comparison, the IC 50 of TMP was 12000 nM against Pc, 300 nM against Ma, and 180000 against rat DHFR. The potency and selectivity values of 6 and 7 were not as high against Tg as they were against Pc or Ma DHFR, but nonetheless exceeded those of TMP. Because of the outstanding selectivity of 6 against Pc and of 7 against Ma DHFR, these novel analogues may be viewed as promising leads for further structure–activity optimization.

7‐Methyl Trimethoprim Analogues as Inhibitors of the Folate Metabolizing Enzymes.

AbstractFor Abstract see ChemInform Abstract in Full Text.

7‐Methyl Trimethoprim Analogues as Inhibitors of the Folate Metabolizing Enzymes

AbstractA series of 5‐(1‐phenylethyl)pyrimidines 2–10 (Table I) were designed and synthesized as potent and selective inhibitors of Pneumocystis carinii (P. carinii), Toxoplasma gondii (T. gondii) and Mycobacterium avium (M. avium) dihydrofolate reductases (DHFR). The structure of 2–10 incorporates a 7‐methyl group to increase the potency of monocyclic trimethoprim (TMP). The target compounds were synthesized by an acid catalyzed condensation of ethyl cyanoacetate and appropriately substituted benzaldehydes followed by a Michael addition using methyl copper‐lithium. The resulting adduct was cyclocondensed with guanidine to afford 2,6‐diamino‐4‐hydroxy‐5‐(1‐phenylethyl)pyrimidines 2–7. Both amino moieties of 2–4 were protected with pivaloyl groups and their 4‐hydroxy group chlorinated with phosphorus oxychloride. The resulting intermediates were subjected to hydrogenation and deprotection to afford 8–10. Compound 7 was a good inhibitor of DHFR, however the other compounds were poor inhibitors of P. carinii, T. gondii and M. avium DHFR.

2,4-Diamino-6,7-dihydro-5H-cyclopenta[d]pyrimidine analogues of trimethoprim as inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase.

Three previously unreported (R,S)-2,4-diamino-5-[(3,4,5-trimethoxyphenyl) alkyl]-6,7-dihydro-5H-cyclopenta[d]pyrimidines 15a-c were synthesized as analogues of trimethoprim (TMP) and were tested as inhibitors of Pneumocystis carinii, Toxoplasma gondii, and rat liver dihydrofolate reductase (DHFR). The length of the alkyl bridge between the cyclopenta[d]pyrimidine and trimethoxyphenyl moiety ranged from one in 15a to three carbons in 15c. The products were tested as competitive inhibitors of the reduction of dihydrofolate by Pneumocystis carinii, Toxoplasma gondii, and rat liver DHFR. Compounds 15a-c had IC50 values of > 32, 1.8 and 1.3 microM, respectively, against P. carinii DHFR, as compared to 12 microM for TMP. Against the T. gondii enzyme, 15a-c had IC50 values of 21, 0.14 and 0.14 microM, respectively, as compared to 2.7 microM for TMP. Inhibitors 15b and 15c with two- and three-carbon bridges were significantly more potent than 15a against all three enzymes. Unlike TMP, 15b and 15c were better inhibitors of the rat liver enzyme than of the microbial enzymes. The potency of 15b and 15c against rat liver DHFR was less than has been reported for the corresponding 6,7-dihydro-5H-cyclopenta[d]pyrimidines with a classical p-aminobenzoyl-L-glutamate side chain as inhibitors of bovine, murine, and human DHFR.

ChemInform Abstract: Conformationally Restricted Analogues of Trimethoprim: 2,6‐Diamino‐8‐ Substituted Purines as Potential Dihydrofolate Reductase Inhibitors from Pneumocystis carinii and Toxoplasma gondii.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

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.

New benzylpyrimidines: inhibition of DHFR from various species. QSAR, CoMFA and PC analysis

Summary To further analyze the structural requirements responsible for the enhanced activity of the newly developed, highly active benzylpyrimidine K-130, a series of trimethoprim analogues with various 4-anilinoalkoxy moieties has been synthesized and tested against dihydrofolate reductase (DHFR) derived from various species (Mycobacterium lufu, Escherichia coli, Candida albicans and rats). The importance of the secondary amino group for binding affinity could be shown by varying the substituent in the para position to the sec-amino moiety. This finding could be supported by multiple linear regression and comparative molecular field analysis (CoMFA). The polarized SO2 group, which was thought to be responsible for the increased activity of K-130, seems not to be the only group important for receptor binding. Additionally, a high selectivity of the new compounds for DHFR derived from the various bacteria and C albicans compared with DHFR derived from rat liver is shown by PC analysis.

Drug design by machine learning: the use of inductive logic programming to model the structure-activity relationships of trimethoprim analogues binding to dihydrofolate reductase.

The machine learning program GOLEM from the field of inductive logic programming was applied to the drug design problem of modeling structure-activity relationships. The training data for the program were 44 trimethoprim analogues and their observed inhibition of Escherichia coli dihydrofolate reductase. A further 11 compounds were used as unseen test data. GOLEM obtained rules that were statistically more accurate on the training data and also better on the test data than a Hansch linear regression model. Importantly machine learning yields understandable rules that characterized the chemistry of favored inhibitors in terms of polarity, flexibility, and hydrogen-bonding character. These rules agree with the stereochemistry of the interaction observed crystallographically.

Modelling the structure and function of enzymes by machine learning.

A machine learning program, GOLEM, has been applied to two problems: (1) the prediction of protein secondary structure from sequence and (2) modelling a quantitative structure-activity relationship in drug design. GOLEM takes as input observations and combines them with background knowledge of chemistry to yield rules expressed as stereochemical principles for prediction. The secondary structure prediction was explored on the alpha/alpha class of proteins; on an unrelated test set it yielded 81% accuracy. The rules from GOLEM defined patterns of residues forming alpha-helices. The system studied for drug design was the activities of trimethoprim analogues binding to E. coli dihydrofolate reductase. The GOLEM rules were a better model than standard regression approaches. More importantly, these rules described the chemical properties of the enzyme-binding site that were in broad agreement with the crystallographic structure.

ChemInform Abstract: 2,4‐Diamino‐5‐benzylpyrimidines as Antibacterial Agents. Part 14. 2,3‐ Dihydro‐1‐(2,4‐diamino‐5‐pyrimidyl)‐1H‐indenes as Conformationally Restricted Analogues of Trimethoprim.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.