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Pharmacological inhibition of dihydrofolate reductase (DHFR) is an established approach for treating a variety of human diseases, including foreign infections and cancer. However, treatment with classic DHFR inhibitors, such as methotrexate (MTX), are associated with negative side-effects and resistance mechanisms that have prompted the search for alternatives. The DHFR inhibitor pyrimethamine (Pyr) has compelling anti-cancer activity in in vivo models, but lacks potency compared to MTX, thereby requiring higher concentrations to induce therapeutic responses. The purpose of this work was to investigate structural analogues of Pyr to improve its in vitro and cellular activity. A series of 36 Pyr analogues were synthesized and tested in a sequence of in vitro and cell-based assays to monitor their DHFR inhibitory activity, cellular target engagement, and impact on breast cancer cell viability. Ten top compounds were identified, two of which stood out as potential lead candidates, 32 and 34. These functionalized Pyr analogues potently engaged DHFR in cells, at concentrations as low as 1 nM and represent promising DHFR inhibitors that could be further explored as potential anti-cancer agents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Masson SAS.)

Since the overexpression of folate receptors (FRs) in certain types of cancers, a variety of FR-targeted fluorescent probes for tumor detection have been developed. However, the reported probes almost all have the same targeting ligand of folic acid with various fluorophores and/or linkers. In the present study, a series of novel tumor-targeted near-infrared (NIR) molecular fluorescent probes were designed and synthesized based on previously reported 6-substituted pyrrolo[2,3-d]pyrimidine antifolates. All newly synthesized probes showed specific FR binding in vitro, whereas GT-NIR-4 and GT-NIR-5 with a benzene and a thiophene ring, respectively, on the side chain of pyrrolo[2,3-d]pyrimidine exhibited better FR binding affinity than that of GT-NIR-6 with folic acid as targeting ligand. GT-NIR-4 also showed high tumor uptake in KB tumor-bearing mice with good pharmacokinetic properties and biological safety. This work demonstrates the first attempt to replace folic acid with antifolates as targeting ligands for tumor-targeted NIR probes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Derivatives with tetrahydrobenzo[h]quinoline chemotype were synthesized via one-pot reactions and evaluated for their antileishmanial, antimalarial and antitubercular activities. Based on a structure-guided approach, they were designed to possess antileishmanial activity through antifolate mechanism, via targeting Leishmania major pteridine reductase 1 (Lm-PTR1). The in vitro antipromastigote and antiamastigote activity are promising for all candidates and superior to the reference miltefosine, in a low or sub micromolar range of activity. Their antifolate mechanism was confirmed via the ability of folic and folinic acids to reverse the antileishmanial activity of these compounds, comparably to Lm-PTR1 inhibitor trimethoprim. Molecular dynamics simulations confirmed a stable and high potential binding of the most active candidates against leishmanial PTR1. For the antimalarial activity, most of the compounds exhibited promising antiplasmodial effect against P. berghei with suppression percentage of up to 97.78%. The most active compounds were further screened in vitro against the chloroquine resistant strain P. falciparum, (RKL9) and showed IC 50 value range of 0.0198-0.096 μM, compared to IC 50 value of 0.19420 μM for chloroquine sulphate. Molecular docking of the most active compounds against the wild-type and quadruple mutant pf DHFR-TS structures rationalized the in vitro antimalarial activity. Some candidates showed good antitubercular activity against sensitive Mycobacterium tuberculosis in a low micromolar range of MIC, compared to 0.875 μM of isoniazid. The top active ones were further tested against a multidrug-resistant strain (MDR) and extensively drug-resistant strain (XDR) of Mycobacterium tuberculosis. Interestingly, the in vitro cytotoxicity test of the best candidates displayed high selectivity indices emphasizing their safety on mammalian cells. Generally, this work introduces a fruitful matrix for new dual acting antileishmanial-antimalarial chemotype graced with antitubercular activity. This would help in tackling drug-resistance issues in treating some Neglected Tropical Diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

New series of thiazole and imidazo[2,1-b]thiazole derivatives were synthesized and tested for their in vitro anticancer activity. Compounds 27, 34, 39 and 42-44 showed the best anticancer activity against the tested cancer cell lines with high safety profile and selectivity indices, especially MCF-7 breast cancer, compared to sorafenib. As an attempt to reveal their mode of cytotoxicity, EGFR, HER2 kinase and DHFR inhibition assays were performed. Compounds 39 and 43 were the most potent dual EGFR/HER2 kinase inhibitors, with IC 50 values of 0.153 (EGFR), 0.108 (HER2) and 0.122 (EGFR), 0.078 (HER2) μM, respectively. 39 and 42 were the best DHFR inhibitors showing IC 50 0.291 and 0.123 μM, respectively. 39 and 43 induced their cytotoxicity via cell cycle arrest at G1/S and G1 phases, respectively, and apoptosis rather than necrosis in the MCF-7 breast cancer cell line. In vivo anti-breast cancer assay of 39 and 43 showed significant tumor volume reduction with recovered caspase-3 immunoexpression. Modeling study results proved the importance of the 5-(4-substituted phenyl)-imidazo[2,1-b]thiazole moiety and the hydrazide side chain for the anticancer activity. The most potent compounds showed good drug-likeness features and could be used as prototypes for further optimization. 39 could be an example of a multi-targeting anticancer agent that acts by inhibiting EGFR/HER2 kinase, DHFR enzymes and cellular apoptosis., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Malaria is a prevalent fatal disease in tropical and subtropical regions around the world. Combinations of Artemisinin, folate antagonists, quinolines, and antibiotics are major choices in clinics. Currently, wide range of parasite's resistance necessitates the search for chemical compounds with new structures and novel antimalarial targets. The literature review was performed covering the relevant literatures published from 1966 to 2021. On the basis of structural classification (spiro-peroxides, spiroindolones, spirocyclohexadienones et al.), different types of molecules were summarized with resources, anti-malarial activities, structure-activity relationship, target introductions, resistance, development progress, and synthetic strategies, for the purpose of providing comprehensive information for developing antimalarial drugs to overcome drug resistance and highlighting the importance of the spiral structure in medicinal chemistry., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Malaria remains a prevalent infectious disease in developing countries. The first-line therapeutic options are based on combinations of fast-acting artemisinin derivatives and longer-acting synthetic drugs. However, the emergence of resistance to these first-line treatments represents a serious risk, and the discovery of new effective drugs is urgently required. For this reason, new antimalarial chemotypes with new mechanisms of action, and ideally with activity against multiple parasite stages, are needed. We report a new scaffold with dual-stage (blood and liver) antiplasmodial activity. Twenty-six spirooxadiazoline oxindoles were synthesized and screened against the erythrocytic stage of the human malaria parasite P. falciparum. The most active compounds were also tested against the liver-stage of the murine parasite P. berghei. Seven compounds emerged as dual-stage antimalarials, with IC 50 values in the low micromolar range. Due to structural similarity with cipargamin, which is thought to inhibit blood-stage P. falciparum growth via inhibition of the Na  +  efflux pump PfATP4, we tested one of the most active compounds for anti-PfATP4 activity. Our results suggest that this target is not the primary target of spirooxadiazoline oxindoles and further studies are ongoing to identify the main mechanism of action of this scaffold., (Copyright © 2022. Published by Elsevier Masson SAS.)

The shortage of new antibiotics makes infections caused by gram-negative (G - ) bacteria a significant clinical problem. The key enzymes involved in folate biosynthesis represent important targets for drug discovery, and new antifolates with novel mechanisms are urgently needed. By targeting to dihydrofolate reductase (DHFR), a series of 1,3-diamino-7H-pyrrol[3,2-f]quinazoline (PQZ) compounds were designed, and exhibited potent antibacterial activities in vitro, especially against multi-drug resistant G - strains. Multiple experiments indicated that PQZ compounds contain a different molecular mechanism against the typical DHFR inhibitor, trimethoprim (TMP), and the thymidylate synthase (TS) was identified as another potential but a relatively weak target. A significant synergism between the representative compound, OYYF-175, and sulfamethoxazole (SMZ) was observed with a strong cumulative and significantly bactericidal effect at extremely low concentrations (2 μg/mL for SMZ and 0.03 pg/mL for OYYF-175), which could be resulted from the simultaneous inhibition of dihydropteroate synthase (DHPS), DHFR and TS. PQZ compounds exhibited therapeutic effects in a mouse model of intraperitoneal infections caused by Escherichia coli (E. coli). The co-crystal structure of OYYF-175-DHFR was solved and the detailed interactions were provided. The inhibitors reported represent innovative chemical structures with novel molecular mechanism of action, which will benefit the generation of new, efficacious bactericidal compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

To tackle leishmaniasis, search for efficient therapeutic drug targets should be pursued. Dihydrofolate reductase (DHFR) is considered as a key target for the treatment of leishmaniasis. In current study, we are interested in the design and synthesis of selective antifolates targeting DHFR from L. major. We focused on the development of new antifolates based on 3,4-dihydropyrimidine-2-one and 5-(3,5-dimethoxybenzyl)pyrimidine-2,4-diamine motif. Structure activity relationship (SAR) studies were performed on 4-phenyl ring of dihydropyrimidine (26-30) template. While for 5-(3,5-dimethoxybenzyl)pyrimidine-2,4-diamine, the impact of different amino acids (valine, tryptophan, phenylalanine, and glutamic acid) and two carbon linkers were explored (52-59). The synthesized compounds were assayed against LmDHFR. Compound 59 with the IC 50 value of 0.10 μM appeared as potent inhibitors of L. major. Selectivity for parasite DHFR over human DHFR was also determined. Derivatives 55-59 demonstrated excellent selectivity for LmDHFR. Highest selectivity for LmDHFR was shown by compounds 56 (SI = 84.5) and 58 (SI = 87.5). Compounds Antileishmanial activity against L. major and L. donovani promastigotes was also performed. To explore the interaction pattern of the synthesized compounds with biological macromolecules, the docking studies were carried out against homology modelled LmDHFR and hDHFR targets., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Drug-resistant bacteria pose an increasingly serious threat to mankind all over the world. However, the currently available clinical treatments do not meet the urgent demand.Therefore, it is desirable to find new targets and inhibitors to overcome the problems of antibiotic resistance. Dihydrofolate reductase (DHFR) is an important enzyme required to maintain bacterial growth, and hence inhibitors of DHFR have been proven as effective agents for treating bacterial infections. This review provides insights into the recent discovery of antimicrobial agents targeting DHFR. In particular, three pathogens, Escherichia coli (E. coli), Mycobacterium tuberculosis(Mtb) and Staphylococcus aureus(S. aureus), and research strategies are emphasized. DHFR inhibitors are expected to be good alternatives to fight bacterial infections., Competing Interests: Declaration of competing interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the manuscript submitted., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

A series of flexible diaminodihydrotriazines or cycloguanil (Cyc) analogues are developed and shown to inhibit P. falciparum dihydrofolate reductase (PfDHFR) of the wild type or those carrying either single (S108N), double (C59R + S108N and A16V + S108T), triple (N51I + C59R + S108N and C59R + S108N + I164L) or quadruple (N51I + C59R + S108N + I164L) mutations, responsible for antifolate resistance. The flexibility of the side chain at position N 1 has been included in the design so as to avoid unfavourable steric interaction with the side chain of residue 108 of the resistant mutants. The inhibition constants of many inhibitors for the mutant enzymes are in the low nanomolar region. Regaining of drug binding efficacies was achieved with both A16V and S108N series of mutants. X-ray studies of some enzyme-inhibitor complexes designed for optimal interaction with the mutant enzymes reveal the modes of binding in line with the K i values. A number of these compounds show excellent antimalarial activities against resistant P. falciparum bearing the mutant enzymes, and exhibit low cytotoxicity to mammalian cells, making them good candidates for further development as antimalarial drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Cobalamin-dependent methionine synthase (MetH) is involved in the process of tumor cell growth and survival. In this study, a novel series of N 5 -electrophilic substituted tetrahydropteroate analogs without glutamate residue were designed as non-classical antifolates and evaluated for their inhibitory activities against MetH. In addition, the cytotoxicity of target compounds was evaluated in human tumor cell lines. With N 5 -chloracetyl as the optimum group, further structure research on the benzene substituent and on the 2,4-diamino group was also performed. Compound 6c, with IC 50 value of 12.1 μM against MetH and 0.16-6.12 μM against five cancer cells, acted as competitive inhibitor of MetH. Flow cytometry studies indicated that compound 6c arrested HL-60 cells in the G 1 -phase and then inducted late apoptosis. The molecular docking further explained the structure-activity relationship., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

A novel series of 6-substituted pyrrolo[2,3-d]pyrimidines with reversed amide moieties from the lead compound 1a were designed and synthesized as nonclassical antifolates and as potential antitumor agents. Target compounds 1-9 were successfully obtained through two sequential condensation reactions from the key intermediate 2-amino-6-(2-aminoethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one. In preliminary antiproliferation assay, all compounds demonstrated submicromolar to nanomolar inhibitory effects against KB tumor cells, whereas compounds 1-3 also exhibited nanomolar antiproliferative activities toward SW620 and A549 cells. In particular, compounds 1-3 were significantly more potent than the positive control methotrexate (MTX) and pemetrexed (PMX) to A549 cells. The growth inhibition induced cell cycle arrest at G1-phase with S-phase suppression. Along with the results of nucleoside protection assays, inhibition assays of dihydrofolate reductase (DHFR) clearly elucidated that the intracellular target of the designed compounds was DHFR. Molecular modeling studies suggested two binding modes of the target compounds with DHFR., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

The emergence of various drug-resistant Mycobacterium tuberculosis (Mtb) strains has necessitated the exploration of new drugs that lack cross-resistance with existing therapeutics. By screening the MedChemExpress bioactive compound library, ceritinib was identified as a compound with activity against Mtb H37Ra. Ceritinib had a MIC value of 9.0 μM in vitro and demonstrated in vivo efficacy in a BALB/c mouse model infected with autoluminescent H37Ra. Then, 32 novel ceritinib derivatives were synthesized, and their antimycobacterial activities were evaluated in vitro. The antimycobacterial activities of the synthesized compounds were drastically affected by substitutions at position 4 of the pyrimidine nucleus and were enhanced by the presence of 2-isopropoxy-5-methyl-4-(piperidin-4-yl)aniline at position 2 of the pyrimidine nucleus. The in vivo antitubercular activities of the three most potent compounds were evaluated. 5-Chloro-N 2 -(2-isopropoxy-5-methyl-4-(piperidin-4-yl) phenyl)-N 4 -(naph thalen-1-yl) pyrimidine-2,4-diamine (16j) remarkably reduced the Mtb burden of mice. This result suggested the potential of 16j as a novel drug with superior antitubercular activities. The results of experiments on the combination of sulfamethoxazole with 16j and in silico modeling suggest that dihydrofolate reductase is the potential molecular target of 16j., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Recently we identified cycloguanil-like dihydrotriazine derivatives, which provided host-factor directed antiviral activity against influenza viruses and respiratory syncytial virus (RSV), by targeting the human dihydrofolate reductase (hDHFR) enzyme. In this context we deemed interesting to further investigate the structure activity relationship (SAR) of our first series of cycloguanil-like dihydrotriazines, designing two novel azaspiro dihydrotriazine scaffolds. The present study allowed the exploration of the potential chemical space, around these new scaffolds, that are well tolerated for maintaining the antiviral effect by means of interaction with the hDHFR enzyme. The new derivatives confirmed their inhibitory profile against influenza viruses, especially type B. In particular, the two best compounds shared potent antiviral activity (4: EC 50  = 0.29 μM; 6: EC 50  = 0.19 μM), which was comparable to that of zanamivir (EC 50  = 0.14 μM), and better than that of ribavirin (EC 50  = 3.2 μM). In addition, these two compounds proved to be also effective against RSV (4: EC 50  = 0.40 μM, SI ≥ 250; 6: EC 50  = 1.8 μM, SI ≥ 56), surpassing the potency and selectivity index (SI) of ribavirin (EC 50  = 5.8 μM, SI > 43). By a perspective of these results, the above adequately substituted azaspiro dihydrotriazines may represent valuable hit compounds worthy of further structural optimization to develop improved host DHFR-directed antiviral agents., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

The emergence of multi- and extensively-drug resistant tubercular (MDR- and XDR-TB) strains of mycobacteria has limited the use of existing therapies, therefore new drugs are needed. Dihydrofolate reductase (DHFR) has recently attracted much attention as a target for the development of anti-TB agents. This study aimed to develop selective M. tuberculosis DHFR inhibitors using rationale scaffolding design and synthesis, phenotype-oriented screening, enzymatic inhibitory study, whole cell on-target validation, molecular modeling, and in vitro DMPK determination to derive new anti-TB agents. 2,4-diamino-1-phenyl-1,3,5-triazaspiro[5.5]undeca-2,4-dienes 20b and 20c were identified as selective M. tuberculosis DHFR inhibitors, showing promising antimycobacterial activities (MIC 50 : 0.01 μM and MIC 90 : 0.025 μM on M. tuberculosis H37Rv). This study provided compelling evidence that compound 20b and 20c exerted whole cell antimycobacterial activity through DHFR inhibition. In addition, these two compounds exhibited low cytotoxicity and low hemolytic activity. The in vitro DMPK and physiochemical properties suggested their potential in vivo efficacy., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

A series of novel 6-substituted pyrrolo[3,2-d]pyrimidine analogues (10a, 11a-13a, 15a, 17a, 18a, 27a and 28a) have been designed and synthesized as antifolate antitumor agents. The anti-proliferative activities of these compounds against HL60, A549, H1299, Hela, HCT116 and HT29 tumor cells were evaluated. Most of the compounds exhibited micromolar anti-proliferative potencies. Compound 15a, the most potent one, has GI 50 value of 0.73, 1.72, and 8.92 μM against A549, H1299 and HL60 cells, respectively. The cell cycle distribution assay displayed that 15a could increase the accumulation of G2/M-phase cells. 15a showed low potency in induction of apoptosis. However, the inhibition of A549 cell colony formation was observed. These indicated that the tumor cell death relied on the irreversible effect of 15a on clonogenicity and cell proliferation. The identification of targeted pathway of 15a implied that the anti-proliferative potencies of 15a probably act through dual inhibition of thymidylate synthase (TS) and dihydrofolate reductase (DHFR)., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

We have identified a series of 1-aryl-4,6-diamino-1,2-dihydrotriazines, structurally related to the antimalarial drug cycloguanil, as new inhibitors of influenza A and B virus and respiratory syncytial virus (RSV) via targeting of the host dihydrofolate reductase (DHFR) enzyme. Most analogues proved active against influenza B virus in the low micromolar range, and the best compounds (11, 13, 14 and 16) even reached the sub-micromolar potency of zanamivir (EC 50  = 0.060 μM), and markedly exceeded (up to 327 times) the antiviral efficacy of ribavirin. Activity was also observed for two influenza A strains, including a virus with the S31N mutant form of M2 proton channel, which is the most prevalent resistance mutation for amantadine. Importantly, the compounds displayed nanomolar activity against RSV and a superior selectivity index, since the ratio of cytotoxic to antiviral concentration was >10,000 for the three most active compounds 11, 14 and 16 (EC 50 ∼0.008 μM), far surpassing the potency and safety profile of the licensed drug ribavirin (EC 50  = 5.8 μM, SI > 43)., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

An optimization of a modified Grimmel's method for N-heterocyclization of Leucine linked sulphonamide leading to 2,3-disustituted-4-quinazolin-(3H)-ones was accomplished. Further, nineteen hybrid quinazolinone motifs (5a-5s) were synthesized by N-heterocyclization reaction under microwave irradiation using TEAA (IL) as green solvent as well as catalyst. The in vitro screening of the hybrid entities against the plasmodium species P. falciparum yielded five antimalarial potent molecules 5g, 5l, 5m, 5n &5p owing comparable activity to the reference drugs. The active scaffolds were further evaluated for enzyme inhibition efficacy against alleged receptor Pf-DHFR computationally as well as in vitro, proving their candidature as lead dihydrofolate reductase inhibitors. The prediction of the ADMET properties of the potent molecules also indicated their good oral bioavailability., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Dihydrofolate reductase (DHFR) has been a well-recognized target for the treatment of many diseases. Based on 8,10-dideazaminopterins, which are classical antifolates that potently inhibit DHFR, we have designed a series of novel 2,4-diamino-6-substituted pyrido[3,2-d]pyrimidines. By removing the glutamate moiety and introducing lipophilic groups, we hoped to improve passive diffuse through the cell membranes. The target compounds were efficiently synthesized using one-pot procedure and evaluated in vitro for DHFR inhibition and antitumor activity. Compounds 5e, 5h, 5i and 5k were the most potent inhibitors of recombinant human DHFR (rhDHFR) with IC 50 values in the range 0.2-1.0 μM. Analysis using flow cytometric indicated that the effect of compound 5k on cell cycle progression was linked to induction of S phase arrest. Compounds 5g, 5h, 5i and 5k showed broad spectrum antitumor activity against four different tumor cell lines, with IC 50 values in the range 0.07-23 μM. Molecular docking investigations showed that the trimethoyphenyl ring of compound 5k occupied a position near the cofactor-binding site in the rhDHFR-inhibitor complex, with close intermolecular contacts with Asp21, Phe31, Ser59, Ile60 and Pro61., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

A novel series of dihydro-1,3,5-triazine derivatives bearing a heteroatom spiro-ring were designed and synthesized on the basis of molecular flexible docking work, and their biological activities were evaluated. Compounds A2, A5, B1 and B3 showed potent human dihydrofolate reductase (hDHFR) inhibitory activity with IC 50 values of 7.46 nM, 3.72 nM, 6.46 nM, 4.08 nM, versus reference drug methotrexate (MTX). From the molecular docking result we concluded that the conformation space generated by deformation of the flexible residue Phe31 is favorable for the binding of the spiro-ring, and inserting heteroatom into spiro ring might increase the binding affinity. There were 24 compounds with broadspectrum antiproliferative activity against several tumor cell lines (HCT116, A549, HL-60, HepG2 and MDA-MB-231) with IC 50 values ranging from 0.79 to 0.001 μM. The antitumor activity in vivo of compound A2 was determined in a human alveolar basal epithelial cell line A549 xenograft model. This study offered novel anticancer agents with high inhibitory activity that target hDHFR and have a binding mode of the novel molecular scaffold with hDHFR. This provides potent support for further development of novel hDHFR inhibitors., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

The development of multi-targeting drugs is currently being explored as an attractive alternative to combination therapy, especially for the treatment of complex diseases such as cancer. Dihydrofolate reductase (DHFR) and thioredoxin reductase (TrxR) are enzymes belonging to two unrelated cellular pathways that are known to contribute towards cancer cell growth and survival. In order to evaluate whether simultaneous inhibition of DHFR and TrxR by dihydrotriazines (DHFR-targeting) and chalcones (TrxR-targeting) may be beneficial, breast MCF-7 and colorectal HCT116 carcinoma cells were treated with combinations of selected dihydrotriazines and chalcones at a 1:1 M ratio. Two combinations demonstrated synergy at low-to-moderate concentrations. Based on this result, four merged dihydrotriazine-chalcone compounds were designed and synthesized. Two compounds, 11a [DHFR IC50 = 56.4 μM, TrxR IC50 (60 min) = 12.6 μM] and 11b [DHFR IC50 = 2.4 μM, TrxR IC50 (60 min) = 10.1 μM], demonstrated in vitro inhibition of DHFR and TrxR. The compounds showed growth inhibitory activity against MCF-7 and HCT116 cells, but lacked cytotoxicity. Molecular docking experiments showed 11b to possess rational binding modes to both the enzymes. In conclusion, this study has not only identified the dihydrotriazine and chalcone scaffolds as good starting points for the development of dual inhibitors of DHFR and TrxR, but also demonstrated the synthetic feasibility of producing a chemical entity that could result in simultaneous inhibition of DHFR and TrxR. Future efforts to improve the antiproliferative profiles of such compounds will look at alternative ways of integrating the two pharmacophoric scaffolds., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Gram-negative bacteria are implicated in the causation of life-threatening hospital-acquired infections. They acquire rapid resistance to multiple drugs and available antibiotics. Hence, there is the need to discover new antibacterial agents with novel scaffolds. For the first time, this study explores the 1,3,5-triazine-2,4-diamine and 1,2,4-triazine-2,4-diamine group of compounds as potential inhibitors of Escherichia coli DHFR, a pivotal enzyme in the thymidine and purine synthesis pathway. Using differential scanning fluorimetry, DSF, fifteen compounds with various substitutions on either the 3rd or 4th positions on the benzene group of 6,6-dimethyl-1-(benzene)-1,3,5-triazine-2,4-diamine were shown to bind to the enzyme with varying affinities. Then, the dose dependence of inhibition by these compounds was determined. Preliminary quantitative structure-activity relationship analysis and docking studies implicate the alkyl linker group and the sulfonyl fluoride group in increasing the potency of inhibition. 4-[4-[3-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1-yl)phenyl]butyl]benzenesulfonyl fluoride (NSC120927), the best hit from the study and a molecule with no reported inhibition of E. coli DHFR, potently inhibits the enzyme with a Ki value of 42.50 ± 5.34 nM, followed by 4-[6-[4-(4,6-diamino-2,2-dimethyl-1,3,5-triazin-1-yl)phenyl]hexyl]benzenesulfonyl fluoride (NSC132279), with a Ki value of 100.9 ± 12.7 nM. Detailed kinetic characterization of the inhibition brought about by five small-molecule hits shows that these inhibitors bind to the dihydrofolate binding site with preferential binding to the NADPH-bound binary form of the enzyme. Furthermore, in search of novel diaminotriazine scaffolds, it is shown that lamotrigine, a 1,2,4-triazine-3,5-diamine and a sodium-ion channel blocker class of antiepileptic drug, also inhibits E. coli DHFR. This is the first comprehensive study on the binding and inhibition brought about by diaminotriazines of a gram-negative prokaryotic enzyme and provides valuable insights into the SAR as an aid to the discovery of novel antibiotics., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

A novel series of 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidines were designed and synthesized as potential nonclassical antifolates targeting both thymidylate and purine nucleotide biosynthesis. Condensation of 2,4-diamino-6-hydroxypyrimidine with ethyl-4-chloroacetoacetate and subsequent hydrolysis afforded the key intermediate, 2-amino-4-oxo-pyrrolo[2,3-d]pyrimidin-6-yl-acetic acid. Coupling with various amino acid methyl esters followed by saponification and condensation with 3-(aminomethyl)pyridine provided target compounds 1-9. The new compounds exhibited micromolar to submicromolar antiproliferative potencies against a panel of tumor cell lines including KB, A549 and HepG2. Growth inhibition of compound 2 toward KB cells resulted in cytotoxicity and G1/G2-phase accumulation, and was partially protected by excess thymidine and adenosine, but was completely reversed in the combination of thymidine and adenosine, indicating both thymidylate and de novo purine nucleotide synthesis as the targeted pathway. However, 5-aminoimidazole-4-carboxamide (AICA) protection was incomplete, suggesting inhibition of both glycinamide ribonucleotide formyltransferase (GARFTase) and AICA ribonucleotide formyltransferase (AICARFTase). The results of the docking studies show that 2 could bind and inhibit both thymidylate synthase (TS) and the two folate-dependent purine biosynthetic enzymes (GARFTase and AICARFTase), which is consistent with the results of in vitro metabolic assays. Our studies establish that compound 2 is an excellent lead analog as a multitargeted antifolate for further structure optimization., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Based on our previous results on the ascertained potent growth inhibition effect against a panel of 60 human tumors cell lines at National Cancer Institute of Bethesda (NCI), we have synthesized a novel series of thirty-one 2-[N-methyl(R-phenyl)-aminomethyl]-3-phenyl-7-trifluoromethylquinoxalines (1-31). The lead compound 1 was previously reported to be endowed with significant inhibition against hDHFR enzyme, with a Ki of 0.2 μM. Docking studies were performed on compound 1 and here reported to predict its binding conformation to human dihydrofolate reductase (hDHFR). All compounds (1-31) were assayed versus hDHFR and human thymidylate synthase (hTS). From the screening emerged that all compounds inhibited hDHFR with Ki values included between 0.2 and 11 μM, while only a few (6, 21, 24, 27, 29) showed great activity and selectivity towards hTS. Evaluation of the anticancer activity was performed by NCI, first against the three cell line panel, and only the most active compounds (17, 21, 24, 26, 27) were evaluated on a panel of 60 human tumor cell lines. Compound 21 was the most active against all cell lines with log GI50 equal to -5.49 and log LC50 equal to -4.19 and maintained significant percent of growth inhibition on seven cancer cell lines at the concentration of 1 μM. Compound 17 was the second most active and moreover showed interesting selectivity against some cell lines (Lung cancer: A549/ATCC, Melanoma: UACC-257, Ovarian Cancer: ovcar-8 and Renal cancer: RXF 393) at all concentration examined (100-0.01 μM)., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

A new series of tetrahydro-quinazoline and tetrahydro-1H-dibenzo[b,e][1,4]diazepine analogs were synthesized and tested for their DHFR inhibition and in vitro antitumor activity. Compound 35 showed a remarkable DHFR inhibitory potency (IC₅₀, 0.004 μM) which is twenty fold more active than methotrexate (MTX). Compounds 17 and 23 proved to be fifteen fold more active than the known antitumor 5-FU, with MG-MID GI₅₀, TGI, and LC₅₀ values of 1.5, 46.8, 93.3 and 1.4, 17.4, 93.3 μM, respectively. Computer modeling studies allowed the identification that methoxy and methyl substituents, the π-system of the chalcone core, the nitrogen atoms, on the dibenzodiazepine ring as pharmacophoric features essential for activity. These mark points could be used as template model for further future optimization., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

A new series of compounds possessing 5-(2-aminothiazol-4-yl)-4-phenyl-4H-1,2,4-triazole-3-thiol skeleton was designed, synthesized, and evaluated for their in vitro DHFR inhibition, antimicrobial, antitumor and schistosomicidal activities. Four active compounds were allocated, the antibacterial 22 (comparable to gentamicin and ciprofloxacin), the schistosomicidal 29 (comparable to praziquantel), the DHFR inhibitor 34 (IC₅₀ 0.03 μM, 2.7 fold more active than MTX), and the antitumor 36 (comparable to doxorubicin). Molecular modeling studies concluded that recognition with key amino acid Leu4 and Val1 is essential for DHFR binding. Flexible alignment and surface mapping revealed that the obtained model could be useful for the development of new class of DHFR inhibitors., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

A new series of 2-heteroarylthio-6-substituted-quinazolin-4-one analogs was designed, synthesized, and evaluated for their in vitro DHFR inhibition, antimicrobial, and antitumor activities. Compounds 21, 25, and 39 proved to be active DHFR inhibitors with IC50 range of 0.3-0.8 μM. Compounds 25, 28, 33, 35 and 36 showed broad spectrum antimicrobial activity comparable to the known antibiotic gentamicin. Compound 29 showed broad spectrum antitumor activity toward several tumor cell lines with GI values range of 25.8-41.2%. Molecular modeling studies concluded that recognition with key amino acid Arg38 and Lys31 are essential for binding and biological activities. Flexible alignment; electrostatic and hydrophobic mappings revealed that the obtained model could be useful for the development of new DHFR inhibitors., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

A series of substituted 2,4-diaminopyrimidines 1 has been prepared and evaluated for activity against Bacillus anthracis using previously reported (±)-3-{5-[(2,4-diamino-5-pyrimidinyl)methyl]-2,3-dimethoxyphenyl}-1-(1-propyl-2(1H)-phthalazinyl)-2-propen-1-one (1a), with a minimum inhibitory concentration (MIC) value of 1-3 μg/mL, as the standard. In the current work, the corresponding isobutenyl (1e) and phenyl (1h) derivatives displayed the most significant activity in terms of the lowest MICs with values of 0.5 μg/mL and 0.375-1.5 μg/mL, respectively. It is likely that the S isomers of 1 will bind the substrate-binding pocket of dihydrofolate reductase (DHFR) as in B. anthracis was found for (S)-1a. The final step in the convergent synthesis of target systems 1 from (±)-1-(1-substituted-2(1H)-phthalazinyl)-2-propen-1-ones 6 with 2,4-diamino-5-(5-iodo-3,4-dimethoxybenzyl)pyrimidine (13) was accomplished via a novel Heck coupling reaction under sealed-tube conditions., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Cancer-targeting drug delivery can be based on the rational design of a therapeutic platform. This approach is typically achieved by the functionalization of a nanoparticle with two distinct types of molecules, a targeting ligand specific for a cancer cell, and a cytotoxic molecule to kill the cell. The present study aims to evaluate the validity of an alternative simplified approach in the design of cancer-targeting nanotherapeutics: conjugating a single type of molecule with dual activities to nanoparticles, instead of coupling a pair of orthogonal molecules. Herein we investigate whether this strategy can be validated by its application to methotrexate, a dual-acting small molecule that shows cytotoxicity because of its potent inhibitory activity against dihydrofolate reductase and that binds folic acid receptor, a tumor biomarker frequently upregulated on the cancer cell surface. This article describes a series of dendrimer conjugates derived from a generation 5 polyamidoamine (G5 PAMAM) presenting a multivalent array of methotrexate and also demonstrates their dual biological activities by surface plasmon resonance spectroscopy, a cell-free enzyme assay, and cell-based experiments with KB cancer cells., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Serine hydroxymethyltransferase (SHMT), a ubiquitous representative of the family of fold-type I, pyridoxal 5'-phosphate (PLP) dependent enzymes, catalyzes the reversible conversion of tetrahydrofolate (H4PteGlu) and serine to 5,10-CH2-H4PteGlu and glycine. Together with thymidylate synthase (TS) and dihydrofolate reductase (DHFR), SHMT participates to the thymidylate (dTMP) biosynthetic process. Elevated SHMT activity has been coupled to the increased demand for DNA synthesis in tumour cells. However, SHMT is the only enzyme of the thymidylate cycle yet to be targeted by chemotherapeutics. In this study, the interaction mode of SHMT with pemetrexed, an antifolate drug inhibiting several enzymes involved in folate-dependent biosynthetic pathways, was assessed. The mechanism of SHMT inhibition by pemetrexed was investigated in vitro using the human recombinant protein. The results of this study showed that pemetrexed competitively inhibits SHMT with respect to H4PteGlu with a measured Ki of 19.1±3.1 μM; this value was consistent with a Kd of 16.9±5.0 μM, measured by isothermal titration calorimetry. The binding mode of pemetrexed to SHMT was further investigated by molecular docking. The calculated interaction energy of pemetrexed in the active site of SHMT was -7.48 kcal/mol, and the corresponding predicted binding affinity was 36.3 μM, in good agreement with Kd and Ki values determined experimentally. The results thus provide insights into the mechanism of action of this antifolate drug and constitute the basis for the rational design of more selective inhibitors of SHMT., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

A series of novel 8-fluoro Norfloxacin derivatives and the hybrids of its piperazinyl derivatives incorporated with 1,3,5-triazine and pyrimidine were synthesized. All the above compounds were evaluated for their antibacterial activity against Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus and methicillin & vancomycin-resistant S. aureus. Among all, compounds having Morpholine, N-methyl/phenyl/benzyl/pyrimidinyl piperazines and n-butylamine substitution at C-7 position, have shown increased potency in comparison to norfloxacin and ciprofloxacin., (Copyright © 2011. Published by Elsevier Masson SAS.)

Thymidylate synthase (TS) is a crucial enzyme for DNA biosynthesis and many nonclassical lipophilic antifolates targeting this enzyme are quite efficient and encouraging as antitumor drugs. Herein, we report some 3D-QSAR analyses using CoMFA and CoMSIA on quinozoline antifolates in order to have a better understanding of the mechanism of action and structure-activity relationship of these compounds. By applying leave-one-out (LOO) cross-validation study, we obtained cross-validated q(2) value of 0.573 for CoMFA and 0.445 for CoMSIA, while the non-cross-validated r(2) values for them were found to be 0.935 and 0.893, respectively. The models were graphically interpreted using CoMFA and CoMSIA contour plots. The results obtained from this study could be used for rational design of potent inhibitors against thymidylate synthase., (Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.)

We report, for the first time, the synthesis and biological activities of 8-deaza-5,6,7,8-tetrahydroaminopterin 9, and the 5-substituted and 5,10-disubstituted analogues 11, 13, 15, and 17. The analogues were obtained from key compound diethyl 8-deaza-5,6,7,8-tetrahydroaminopterin 8 following the catalytic reduction of the pyridine ring of diethyl 8-deaza aminopterin 5. The five novel 8-deaza-5,6,7,8-tetrahydroaminopterin derivatives were assayed in vitro for their cytotoxicity on BGC-823, HL-60, Bel-7402 and Hela tumor cell lines, and inhibition on recombinant human dihydrofolate reductase (DHFR), among which the most potent molecule (compound 9) was about 4- to 10-fold poorer than MTX on the four kinds of tumor cell lines, and its effect on DHFR was about 17-fold poorer than MTX. The docking studies were followed to explain the biological testing results.

Several diamino quinoxalines were designed, synthesized and evaluated as anti-tumor agents. Two compounds showed the most potent cytotoxic activities against the leukemia CCRF-CEM cell line (GI(50)<0.01microM) and the ovarian cancer cell line OVCAR-4 (GI(50)=0.03microM), respectively, with comparable/better activities than Methotrexate (MTX). Docking calculations of the complexes of hDHFR with the most active compounds identified the binding mode of the described molecules with respect to MTX.

Piritrexim (PTX) (1), a lipophilic inhibitor of the human dihydrofolate reductase, has been evaluated as an anticancer agent. The synthesis of four structural variations (2-5) of PTX is reported. The PTX analogues 2-5 were obtained by reaction of suitable C3-building blocks with pyrimidine-2,4,6-triamine (14) or with cyanacetamide (7) and guanidine (10). The evaluation of 2-4 for antitumor activity against a panel of 60 human cancer cell lines showed inhibitory effects on the growth of the cell lines. These data are supported by molecular modeling and docking studies, which show that compounds 2-4 share the same binding mode within the DHFR active site. Moreover, the estimated ligand binding energies are in good agreement with the experimental activity data.

In a program to design and develop mechanism-based compounds active as substrates and inhibitors of dihydrofolate reductase (DHFR), we report the synthesis and physical properties of the 6-methyl- (7), 8-methyl- (8a), and 8-ethyl- (8b) derivatives of the parent 2-aminopyrimido[4,5-d]pyrimidin-4-(3H)-one (6). These compounds are the first members of a class of heterocycles related to 8-alkylpterins (N8-alkyl-2-aminopteridin-4(8H)-ones) (2a-2c), which have been shown to be novel substrates for DHFR. Three methods were developed for the synthesis of target compounds 7, 8a and 8b; however, the optimum yields (1-8%) could not be improved because the products decomposed by ring opening (e.g. to 2,4-diamino-5-methyliminomethylpyrimidin-6(1H)-one (9)) under the reaction conditions. The marked pi-electron deficiency of compounds 7, 8a and 8b is the likely cause for the susceptibility of the quaternised pyrimidine ring in the related cations 10, 15a and 15b, respectively, to add nucleophiles, thus promoting the opening of the pyrimidopyrimidine ring system. 1H-NMR spectroscopic studies of compounds 7, 8a and 8b revealed a fast and reversible covalent hydration of the associated cations across the C7z.sbnd;N8 bond for the N6-methyl derivative 7 and across the N6z.sbnd;C7 bond for the N8-methyl derivative 8a. UV spectroscopic studies of methyl derivatives 7 and 8a as well as the parent heterocycle 6 showed that protonation of the latter occurred at N1, while methylation with iodomethane proceeded at N6 and N8. The basicities of the N-methyl derivatives 7 and 8a (pK(a) ca. 5.5) are similar to those of 8-alkylpterins 2; this is an essential element of the design to promote binding to DHFR in their protonated form. Enzyme kinetics of 7, 8a and 8b with chicken DHFR confirmed our predictions that they are substrates, with apparent K(m) values of 3.8, 0.08, and 0.65 mM, and apparent V(max) values of 0.47, 2.27, and 0.30 nmol L(-1) min(-1) (for enzyme concentration 0.122 micro M), respectively. The parent compound 6 was not a substrate.

There is an urgent need for the development of new drugs to treat Chagas' disease, which is caused by the protozoan parasite Trypanosoma cruzi. The enzyme dihydrofolate reductase (DHFR) has been a very successful drug target in a number of diseases and we decided to investigate it as a potential drug target for Chagas' disease. A homology model of the enzyme was used to search the Cambridge Structural Database using the program DOCK 3.5. Compounds were then tested against the enzyme and the whole parasite. Compounds were also screened against the related parasite, Trypanosoma brucei.

N-[4-[[2,4-diamino-6-pteridinyl)methyl]amino]bicyclo[2.2.2]octane-1-carbonyl]-L-glutamic acid (1) was synthesized and tested for antifolate activity. N-(4-Aminobicyclo[2.2.2]octane-1-carbonyl-L-glutamic acid dimethyl ester (6), the side chain precursor to subject compound 1, was synthesized readily via reported bicyclo[2.2.2]octane-1,4-dicarboxylic acid monoethyl ester (2). The side chain precursor 6 was alkylated by 6-(bromomethyl)-2,4-pteridinediamine (7). Subsequent ester hydrolysis then afforded 1. Antifolate and antitumor evaluation of 1 verses L1210 dihydrofolate reductase (DHFR) and three tumor cell lines (L1210, S180, and HL60) showed it to be ineffective. Although compound 1 was very similar to aminopterin structurally, the bicyclo[2.2.2]octane ring system in place of the phenyl ring in the p-aminobenzoate moiety effectively negates the stoichiometric binding displayed by many classical DHFR inhibitors bearing appropriate aromatic ring systems in the side chain.

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.

Our continuing program to develop new antifolate drugs useful against rheumatoid arthritis led us to modify the pteridine ring of gamma-fluoromethotrexate. Pyrrolopyrimidine derivatives 1e and 1t were found to exhibit potent suppressive effects on the responses of both T and B cells to mitogens, although tetrahydropyridopyrimidine derivatives 2e and 2t and quinazoline derivatives 3e, 3t and 4e showed very weak suppressive activities. Thus, conversion of the pteridine ring of gamma-fluoromethotrexate to a pyrrolopyrimidine ring led to a new potential antirheumatic compound.