Benzamide Trimethoprim Derivatives as Human Dihydrofolate Reductase Inhibitors-Molecular Modeling and In Vitro Activity Study.

Human dihydrofolate reductase (hDHFR) is an essential cellular enzyme, and inhibiting its activity is a promising strategy for cancer therapy. We have chosen the trimethoprim molecule (TMP) as a model compound in our search for a new class of hDHFR inhibitors. We incorporated an amide bond, a structural element typical of netropsin, a ligand that binds selectively in the minor groove of DNA, into the molecules of TMP analogs. In this work, we present previously obtained and evaluated eleven benzamides (JW1-JW8; MB1, MB3, MB4). Recently, these compounds were specifically projected as potential inhibitors of the enzymes acetylcholinesterase (AChE) and ß-secretase (BACE1). JW8 was most active against AChE, with an inhibitory concentration of AChE IC50 = 0.056 µM, while the IC50 for donepezil was 0.046 µM. This compound was also the most active against the BACE1 enzyme. The IC50 value was 9.01 µM compared to that for quercetin, with IC50 = 4.89 µM. All the benzamides were active against hDHFR, with IC50 values ranging from 4.72 to 20.17 µM, and showed activity greater than TMP (55.26 µM). Quantitative results identified the derivatives JW2 and JW8 as the most promising. A molecular modeling study demonstrates that JW2 interacts strongly with the key residue Gly-117, while JW8 interacts strongly with Asn-64 and Arg-70. Furthermore, JW2 and JW8 demonstrate the ability to stabilize the hDHFR enzyme, despite forming fewer hydrogen bonds with the protein compared to reference ligands. It can be concluded that this class of compounds certainly holds great promise for good active leads in medicinal chemistry.

New Benzamides as Multi-Targeted Compounds: A Study on Synthesis, AChE and BACE1 Inhibitory Activity and Molecular Docking.

The synthesis of eleven new and previously undescribed benzamides was designed. These compounds were specifically projected as potential inhibitors of the enzymes acetylcholinesterase (AChE) and ß-secretase (BACE1). N,N'-(1,4-phenylene)bis(3-methoxybenzamide) was most active against AChE, with an inhibitory concentration of AChE IC50 = 0.056 µM, while the IC50 for donepezil was 0.046 µM. This compound was also the most active against the BACE1 enzyme. The IC50 value was 9.01 µM compared to that for quercetin, with IC50 = 4.89 µM. Quantitative results identified this derivative to be the most promising. Molecular modeling was performed to elucidate the potential mechanism of action of this compound. Dynamic simulations showed that new ligands only had a limited stabilizing effect on AChE, but all clearly reduced the flexibility of the enzyme. It can, therefore, be concluded that a possible mechanism of inhibition increases the stiffness and decreases the flexibility of the enzyme, which obviously impedes its proper function. An analysis of the H-bonding patterns suggests a different mechanism (from other ligands) when interacting the most active derivative with the enzyme.

s-Triazine Derivatives Functionalized with Alkylating 2-Chloroethylamine Fragments as Promising Antimicrobial Agents: Inhibition of Bacterial DNA Gyrases, Molecular Docking Studies, and Antibacterial and Antifungal Activity.

The spectrum of biological properties of s-triazine derivatives is broad and includes anti-microbial, anti-cancer, and anti-neurodegenerative activities, among others. The s-triazine molecule, due to the possibility of substituting three substituents, offers many opportunities to obtain hybrid compounds with a wide variety of activities. A group of 1,3,5 triazine derivatives containing a dipeptide, 2-ethylpiperazine, and a methoxy group as substituents was screened for their antimicrobial activity. An in vitro study was conducted on pathogenic bacteria (E. coli, S. aureus, B. subtilis, and M. luteus), yeasts (C. albicans), and filamentous fungi (A. fumigatus, A. flavus, F. solani, and P. citrinum) via microdilution in broth, and the results were compared with antibacterial (Streptomycin) and antifungal (Ketoconazole and Nystatin) antibiotics. Several s-triazine analogues have minimal inhibitory concentrations lower than the standard. To confirm the inhibitory potential of the most active compounds against gyrases E. coli and S. aureus, a bacterial gyrases inhibition assay, and molecular docking studies were performed. The most active s-triazine derivatives contained the -NH-Trp(Boc)-AlaOMe, -NH-Asp(OtBu)-AlaOMe, and -NH-PheOMe moieties in their structures.

Recent Advances in the Biological Activity of s-Triazine Core Compounds.

An effective strategy for successful chemotherapy relies on creating compounds with high selectivity against cancer cells compared to normal cells and relatively low cytotoxicity. One such approach is the discovery of critical points in cancer cells, i.e., where specific enzymes that are potential therapeutic targets are generated. Triazine is a six-membered heterocyclic ring compound with three nitrogen replacing carbon-hydrogen units in the benzene ring structure. The subject of this review is the symmetrical 1,3,5-triazine, known as s-triazine. 1,3,5-triazine is one of the oldest heterocyclic compounds available. Because of its low cost and high availability, it has attracted researcher attention for novel synthesis. s-Triazine has a weak base, it has much weaker resonance energy than benzene, therefore, nucleophilic substitution is preferred to electrophilic substitution. Heterocyclic bearing a symmetrical s-triazine core represents an interesting class of compounds possessing a wide spectrum of biological properties such as anti-cancer, antiviral, fungicidal, insecticidal, bactericidal, herbicidal and antimicrobial, antimalarial agents. They also have applications as dyes, lubricants, and analytical reagents. Hence, the group of 1,3,5-triazine derivatives has developed over the years. Triazine is not only the core amongst them, but is also a factor increasing the kinetic potential of the entire derivatives. Modifying the structure and introducing new substituents makes it possible to obtain compounds with broad inhibitory activity on processes such as proliferation. In some cases, s-triazine derivatives induce cell apoptosis. In this review we will present currently investigated 1,3,5-triazine derivatives with anti-cancer activities, with particular emphasis on their inhibition of enzymes involved in the process of tumorigenesis.

1,3,5-Triazine Nitrogen Mustards with Different Peptide Group as Innovative Candidates for AChE and BACE1 Inhibitors.

A series of new analogs of nitrogen mustards (4a-4h) containing the 1,3,5-triazine ring substituted with dipeptide residue were synthesized and evaluated for the inhibition of both acetylcholinesterase (AChE) and ß-secretase (BACE1) enzymes. The AChE inhibitory activity studies were carried out using Ellman's colorimetric method, and the BACE1 inhibitory activity studies were carried out using fluorescence resonance energy transfer (FRET). All compounds displayed considerable AChE and BACE1 inhibition. The most active against both AChE and BACE1 enzymes were compounds A and 4a, with an inhibitory concentration of AChE IC50 = 0.051 µM; 0.055 µM and BACE1 IC50 = 9.00 µM; 11.09 µM, respectively.

Synthesis, Biological Activity, and Molecular Dynamics Study of Novel Series of a Trimethoprim Analogs as Multi-Targeted Compounds: Dihydrofolate Reductase (DHFR) Inhibitors and DNA-Binding Agents.

Eighteen previously undescribed trimethoprim (TMP) analogs containing amide bonds (1-18) were synthesized and compared with TMP, methotrexate (MTX), and netropsin (NT). These compounds were designed as potential minor groove binding agents (MGBAs) and inhibitors of human dihydrofolate reductase (hDHFR). The all-new derivatives were obtained via solid phase synthesis using 4-nitrophenyl Wang resin. Data from the ethidium displacement test confirmed their DNA-binding capacity. Compounds 13-14 (49.89% and 43.85%) and 17-18 (41.68% and 42.99%) showed a higher binding affinity to pBR322 plasmid than NT. The possibility of binding in a minor groove as well as determination of association constants were performed using calf thymus DNA, T4 coliphage DNA, poly (dA-dT)2, and poly (dG-dC)2. With the exception of compounds 9 (IC50 = 56.05 µM) and 11 (IC50 = 55.32 µM), all of the compounds showed better inhibitory properties against hDHFR than standard, which confirms that the addition of the amide bond into the TMP structures increases affinity towards hDHFR. Derivatives 2, 6, 13, 14, and 16 were found to be the most potent hDHFR inhibitors. This molecular modelling study shows that they interact strongly with a catalytically important residue Glu-30.

Recent Design and Structure-Activity Relationship Studies on the Modifications of DHFR Inhibitors as Anticancer Agents.

BACKGROUND: Dihydrofolate reductase (DHFR) has been known for decades as a molecular target for antibacterial, antifungal and anti-malarial treatments. This enzyme is becoming increasingly important in the design of new anticancer drugs, which is confirmed by numerous studies including modelling, synthesis and in vitro biological research. This review aims to present and discuss some remarkable recent advances in the research of new DHFR inhibitors with potential anticancer activity. METHODS: The scientific literature of the last decade on the different types of DHFR inhibitors has been searched. The studies on design, synthesis and investigation structure-activity relationships were summarized and divided into several subsections depending on the leading molecule and its structural modification. Various methods of synthesis, potential anticancer activity and possible practical applications as DHFR inhibitors of new chemical compounds were described and discussed. RESULTS: This review presents the current state of knowledge on the modification of known DHFR inhibitors and the structures and searches for about eighty new molecules, designed as potential anticancer drugs. In addition, DHFR inhibitors acting on thymidylate synthase (TS), carbon anhydrase (CA) and even DNA-binding are presented in this paper. CONCLUSION: Thorough physicochemical characterization and biological investigations highlight the structure-activity relationship of DHFR inhibitors. This will enable even better design and synthesis of active compounds, which would have the expected mechanism of action and the desired activity.

Trimethoprim and other nonclassical antifolates an excellent template for searching modifications of dihydrofolate reductase enzyme inhibitors.

The development of new mechanisms of resistance among pathogens, the occurrence and transmission of genes responsible for antibiotic insensitivity, as well as cancer diseases have been a serious clinical problem around the world for over 50 years. Therefore, intense searching of new leading structures and active substances, which may be used as new drugs, especially against strain resistant to all available therapeutics, is very important. Dihydrofolate reductase (DHFR) has attracted a lot of attention as a molecular target for bacterial resistance over several decades, resulting in a number of useful agents. Trimethoprim (TMP), (2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine) is the well-known dihydrofolate reductase inhibitor and one of the standard antibiotics used in urinary tract infections (UTIs). This review highlights advances in design, synthesis, and biological evaluations in structural modifications of TMP as DHFR inhibitors. In addition, this report presents the differences in the active site of human and pathogen DHFR. Moreover, an excellent review of DHFR inhibition and their relevance to antimicrobial and parasitic chemotherapy was presented.

Synthesis and cellular effects of novel 1,3,5-triazine derivatives in DLD and Ht-29 human colon cancer cell lines.

This study provides new information on the cellular effects of 1,3,5-triazine nitrogen mustards with different peptide groups in DLD and Ht-29 human colon cancer cell lines. A novel series of 2,4,6-trisubstituted 1,3,5-triazine derivatives bearing 2-chloroethyl and oligopeptide moieties was designed and synthesized. The most cytotoxic derivative was triazine with an Ala-Ala-OMe substituent on the ring (compound 7b). This compound induced time- and dose-dependent cytotoxicity in the DLD-1 and HT-29 colon cancer cell lines. The triazine derivative furthermore induced apoptosis through intracellular signaling pathway attenuation. Compound 7b may be a candidate for further evaluation as a chemotherapeutic agent against colorectal cancer.

The cellular effects of novel triazine nitrogen mustards in glioblastoma LBC3, LN-18 and LN-229 cell lines.

1,3,5-triazine is an important heterocyclic skeleton for mono, two or three 2-chloroethylamine groups. The study presented here provides novel information on cellular effects of 1,3,5-triazine with mono, two or three 2-chloroethylamine groups in glioblastoma LBC3, LN-18 and LN-229 cell lines. In our study, the most cytotoxic effect was observed in 1,3,5-triazine with three 2-chloroethylamine groups (12f compound). It has been demonstrated that 12f induce time- and dose-dependent cytotoxicity in all investigated glioma cell lines. Apart from that in glioblastoma cells, treated with 12f compound, we noticed strong induction of apoptosis. In conclusion, this research provides novel information concerning cellular effects of apoptosis in LBC3, LN-18 and LN-229 cell lines. Moreover, we suggest that 12f compound may be a candidate for further evaluation as an effective chemotherapeutic agent for human glioblastoma cells.

Carbocyclic Analogues of Distamycin and Netropsin.

The DNA as the depository of genetic information is a natural target for chemotherapy. A lot of anticancer and antimicrobial agents derive their biological activity from their selective interaction with DNA in the minor groove and from their ability to interfere with biological processes such as enzyme catalysis, replication and transcription. The discovery of the details of minor groove binding drugs, such as netropsin and distamycin A, oligoamides built of 4-amino-1-methylpyrrole-2-carboxylic acid residues, allowed to develop various DNA sequence-reading molecules, named lexitropsins, capable of interacting with DNA precisely, strongly and with a high specificity, and at the same time exhibiting significant cytotoxic potential. Among such compounds, lexitropsins built of carbocyclic sixmembered aromatic rings occupy a quite prominent place in drug research. This work is an attempt to present current findings in the study of carbocyclic lexitropins, their structures, syntheses and biological investigations such as DNA-binding and antiproliferative activity.

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.

ANTIPROLIFERATIVE EFFECTS ON BREAST CANCER CELLS AND SOME INTERACTIONS OF NEW DISTAMYCIN ANALOGUES WITH DNA, ENDONUCLEASES AND DNA TOPOISOMERASES.

The evaluation of a new group of distamycin analogues 1-6 as potential minor groove binders for the treatment of cancer were investigated. The activity of the new compounds against several restriction enzymes was examined. The studied compounds did not block GC-rich sequences regions of DNA but inhibited catalytic action of endonucleases in AA, AT, TT and AG restriction sites. Determination of association constants using calf thymus DNA, T4 coliphage DNA, poly(dA-dT)2 and poly(dG-dC)2 have confirmed that the tested compounds bind within minor groove of B-DNA. All of the compounds demonstrated activity against DNA topoisomerases II at the concentration 10 µM, but they did not inhibit activity of topoisomerase I. The studied derivatives were evaluated in human MCF-7 breast cancer cells and showed antiproliferative and cytotoxic effects in the range of 81.70 µM and 200.00 µM.

Synthesis of Arylamino-1,3,5-triazines Functionalized with Alkylatin 2-chloroethylamine Fragments and Studies of their Cytotoxicity on the Breast Cancer MCF-7 Cell Line.

Dual action alkyl(aryl)amino-1,3,5-triazines functionalized with nitrogen mustards were obtained by treating 2-alkyl(aryl) amino-4-chloro-6-methoxy-1,3,5-triazines with amines or amino acid methyl esters, followed by reactions with 1,4-diazabicyclo[2.2.2]octane (DABCO) and rearrangement with an opening diazabicyclic fragment, leading to the formation of 2-chloroethylamino moiety. In vitro antitumor activity was tested in the standard human breast cancer MCF-7 and MDA-MB-231cell lines using flow cytometry, based on the detection of apoptosis through qualitative analysis of morphological changes, DNA fragmentation, DNA loss and membrane changes. For all the compounds studied, induced apoptosis was substantially stronger than necrosis at concentrations of both 5 µM and 50 µM, and in some cases there was no increase in necrotic cell death for the estrogen dependent MCF-7 cell line. The most active compounds were derivatives of triazine substituted with phenylamine (IC50 = 12.30 µM) and/or p-tolylamine fragments (IC50 = 7.40 µM).

Semi-automatic synthesis, antiproliferative activity and DNA-binding properties of new netropsin and bis-netropsin analogues.

A general route for the semi-automatic synthesis of some new potential minor groove binders was established. Six four-numbered sub-libraries of new netropsin and bis-netropsin analogues have been synthesized using a Syncore Reactor. The structures of the all new substances prepared in this investigation were fully characterized by NMR ((1)H, (13)C), HPLC and LC-MS. The antiproliferative activity of the obtained compounds was tested on MCF-7 breast cancer cells. The ethidium displacement assay using pBR322 confirmed the DNA-binding properties of the new analogues of netropsin and bis-netropsin.

Effect of short peptides containing lysine and epsilon-aminocaproic acid on fibrinolytic activity of plasmin and topoisomerase II action on supercoiled DNA.

Effects of eight short peptides containing lysine and epsilon-aminocaproic acid (EACA) on prolongation of the clot lysis time, as well as hemolytic and antibacterial activities were investigated. Interaction with plasmids pBR322 and pUC19 with the use of ethidium bromide assay and determination of influence on the activity of topoisomerase I and II were also tested. Examined compounds inhibited fibrinolytic activity of plasmin and five of them were more active than EACA. Amides of dipeptides were most active antifibrinolytics (IC50 < 0.2 mM). According to the obtained data, the significant inhibition of fibrinolytic activity of plasmin was not associated with hemolytic effects. Examined compounds did not show antibacterial activity (MIC > 512 mg/L). DNA binding effects determined with the use of ethidium bromide were weak for all peptides and similar to those observed with EACA. Six compounds inhibited topoisomerase II action on supercoiled DNA.

Amino and chlorambucil analogues of pentamidine--synthesis and biological examinations.

The amino analogues of pentamidine with a polymethylene (n = 3 - 6) chain and their chlorambucil derivatives were synthesized. The obtained compounds revealed cytotoxic effect on MCF-7 human breast cancer cell line (IC50 = 22 - 95 +/- 2 pM), mainly by the induction of apoptosis. The topoisomerase I/II inhibition assay and the ethidium displacement assay with the use of pBR322 plasmid DNA were used to the study of mechanism by which the obtained compounds could act. All the compounds are able to bind with DNA and interfere in vitro with the activity of topoisomerase (I and II). The determination of association constants with the use of calf thymus DNA, T4 coliphage DNA, poly(dA-dT)2 and poly(dG-dC)2 showed that the tested compounds bind within minor groove of B-DNA, but not selectively. The alkylating activity of chlorambucil derivatives determined in vitro using a Preussmann test was similar to the activity of chlorambucil. The influence of all the compounds on the amidolytic activity of plasmin and trypsin was also examined. The plasmin activity was inhibited by pentamidine, chlorambucil and aromatic bis-amines (IC50 = 0.1 - 8 mM), whereas the trypsin activity was influenced only by pentamidine.

Synthesis and cytotoxicity studies of bifunctional hybrids of nitrogen mustards with potential enzymes inhibitors based on melamine framework.

The new class of hybrid anticancer drugs were obtained by selective functionalization of the triazine scaffold. These were prepared by rearrangement of mono-, bis- and/or tris-(1,3,5-triazin-2-yl)-1,4-diazabicyclo[2.2.2]octanium chlorides leading to formation of 2-chloroethylamino fragments attached to 1,3,5-triazine via one, two or three piperazine rings respectively. Their inhibitory effect was found strongly dependent on the structure of substituents in triazine ring. The anti-proliferative activity of the hybrids evaluated in vitro by using mammalian tumour cells estimated as IC(50) was in the range 0.62-139,78 µM. Both cytotoxicity and alkylating activity depended on the substituents of triazine ring, however, also the mono-functional analogues of nitrogen mustards, which are unable to form liaisons between two DNA strands, induced apoptosis and necrosis in the tested cells.

New solid phase synthesis of distamycin analogues.

A novel and straightforward solid phase synthesis of distamycin analogues containing benzene units has been developed.

Carbocyclic potential DNA minor groove binders and their biological evaluation.

The biological evaluation of carbocyclic minor groove binders 1-6 is described. The cytotoxicity of the obtained compounds was tested on MDA-MB-231 breast cancer cells. The mechanism of action of compounds 1-6 was studied employing the topoisomerase I/II inhibition assay and ethidium displacement assay using pBR322. Determination of association constants was done using calf thymus DNA, T4 coliphage DNA, poly(dA-dT)(2), and poly(dG-dC)(2). The effect of compounds 1-6 on the amidolytic activity of plasmin, trypsin, thrombin, and urokinase was also examined.