Synthesis, molecular modeling simulations and anticancer activity of some new Imidazo[2,1-b]thiazole analogues as EGFR/HER2 and DHFR inhibitors.

New imidazo[2,1-b]thiazole analogs were designed, synthesized, and biologically evaluated as anticancer agents. In vitro biological evaluation of the anticancer properties of the compounds was performed against different cancer cell lines. Compounds 23 and 39 showed remarkable broad -spectrum cytotoxic potency on most of the tested cell lines. Compounds 23 and 39 exhibited potent activity against the MCF-7 breast cancer cell line, with IC50 values of 1.81 and 4.95 µM, respectively, compared to DOX and SOR (IC50 values of 4.17 and 7.26 µM, respectively). An enzyme inhibition assay was carried out to clarify the possible mode of action of the tested compounds. Compounds 23 and 39 were identified as possible EGFR, HER-2, and DHFR inhibitors. Cell cycle arrest results indicated that compound 23 caused cell cycle arrest at the G0/G1 phase in the MCF-7 cells and at the G2/M phase in the Hep G2 cells. Compound 39 induced cell cycle arrest at the G2/M phase in Hela cells. In vivo testing of the anticancer activity of the two most promising molecules in this study was conducted, and the results indicated that they possess considerable in vivo anticancer activity in mice. Data obtained from the molecular modeling simulation study were consistent with the biological evaluation results.

Synthesis and in vitro antitumor evaluation of new thieno[2,3-d]pyrimidine derivatives as EGFR and DHFR inhibitors.

New thienopyrimidine derivatives 2-16 have been synthesized and their in vitro cytotoxicity was evaluated against five different human cancer cell lines HCT-116, Hela, MDA-MB-231, MCF7 and PC3. Compounds 6e, 7a, 7b, 7d, 10c and 10e displayed the highest antitumor activity against all tested cell lines compared to Doxorubicin. Enzyme inhibition assay revealed that compounds 6e and 10e showed high inhibitory activity against EGFR-TK, with IC50 values of 0.133 and 0.151 µM, compared to Olmutinib (IC50 = 0.028 µM); while the highest DHFR inhibitory activity was shown by compounds 7d and 10e with IC50 values of 0.462 and 0.541 µM, compared to Methotrexate (IC50 = 0.117 µM). Cell cycle analysis following a flow cytometric study using colorectal HCT-116 cancer cell line proved that compound 6e induced cell cycle arrest in G0-G1 phase, while compound 10e arrested the cell cycle at both G0-G1 and S phases. Additionally, both compounds (6e and 10e) were potently able to induce apoptosis in HCT-116 cell line. Docking results of compounds 6e and 10e into the pocket of EGFR active site showed their similar main binding features with Olmutinib, while compounds 7d and 10e showed only moderate fitting into DHFR compared to methotrexate. In silico studies revealed that most of the tested compounds obeyed Lipinski's RO5 and showed positive drug likeness scores.

Human dihydrofolate reductase inhibition effect of 1-Phenylpyrazolo[3,4-d]pyrimidines: Synthesis, antitumor evaluation and molecular modeling study.

A new series of pyrazolo[3,4-d]pyrimidine analogues bearing different amino acid conjugates 10a-m were synthesized with the aim to evaluate their antitumor effect through simultaneous inhibition of human dihydrofolate reductase (hDHFR). All novel compounds were tested to screen their enzyme inhibition activity against (hDHFR) beside their in vitro cytotoxicity against six human MTX resistant cancer cell lines namely, human prostate cancer (PC-3), pancreatic human cancer cell lines (BxPC-3), colorectal carcinoma (HCT-116), human hepatocellular carcinoma (HepG-2), cervical carcinoma (HeLa), and mammary gland breast cancer (MCF-7), besides normal immortalized pancreatic cell line (HPDE). Compounds 10e, 10f, 10g inhibited DHFR at considerable low (IC50 < 1 µM) in comparison to MTX (IC50 = 5.61 µM) beside their characteristic cytotoxic effects on different resistant cancer cell lines. Flow cytometry was done for the most active candidate compound 10e against MCF-7 breast cancer cell line. The results illustrated that compound 10e induced apoptosis and arrested MCF-7 cell cycle in the G1/S phase. Western blot for visualization and quantification was used to confirm the capability of compound 10e to induce the expression of proapoptotic caspases and Bax proteins in MCF-7 breast cancer cell line beside its ability to reduce the expression of antiapoptotic Bcl-2 protein. Molecular modeling studies demonstrated that compound 10e elucidated binding energy of (S= - 8.4390 Kcal/mol) that exceed that of the normal ligand MTX (S= - 8.3951Kcal/mol) in addition to several favorable binding interactions with the active site residues.

Development of novel indolin-2-one derivative incorporating thiazole moiety as DHFR and quorum sensing inhibitors: Synthesis, antimicrobial, and antibiofilm activities with molecular modelling study.

Nowadays, it's imperative to develop novel antimicrobial agents active against both drug-sensitive and drug-resistant bacterial infections with favorable profiles as high efficacy, low toxicity, and short therapy duration. Accordingly, a series of new thiazolo-indolin-2-one derivatives were synthesized based on acid and base catalyzed condensation or reaction of thiosemicarbazone 8 with different electrophilic reagents. The structure of the new compounds was confirmed based on elemental analysis and spectral data. Based on the MIC results, the most active thiazolo-indoline derivatives 2, 4, 7a, and 12 exhibited promising antibacterial activity against gram-positive and gram-negative bacteria with weak to moderate antifungal activities. Surprisingly, the N-(thiazol-2-yl)benzenesulfonamide derivative 4 was found to be most active on antibiofilm activity against both S. aureus (ATCC 29213) with BIC50 (1.95 ± 0.01 µg/mL), while 5-(2-oxoindolin-3-ylidene)-thiazol-4(5H)-one derivative 7a exhibited the strongest antibiofilm activity against P. aeruginosa pathogens with BIC50 (3.9 ± 0.16 µg/mL). Further, the thiazole derivatives 2, 4 and 12 exhibited a significant inhibition activity against the fsr system in a dose-dependent manner without affecting bacterial growth. The target derivatives behaved synergistic and additively effect against MDR p. aeruginosa, and thiazole derivative 12 exhibited a high synergistic effect with most tested antibiotics except Cefepime with FIC value ranging between 0.249 and 1.0, reducing their MICs. Interestingly, the 3-(2-(4-thiazol-2-yl)hydrazono)indolin-2-one derivative 12 displayed the highest selectivity to DHFR inhibitory with IC50 value 40.71 ± 1.86 nM superior to those of the reference Methotrexate. Finally, in silico molecular modeling simulation, some physicochemical properties and toxicity predictions were performed for the most active derivatives.

3-Methyl-imidazo[2,1-b]thiazole derivatives as a new class of antifolates: Synthesis, in vitro/in vivo bio-evaluation and molecular modeling simulations.

Inhibiting the Dihydrofolate reductase (DHFR) enzyme has been validated in multiple clinical manifestations related to bacterial infection, malaria, and multiple types of cancer. Herein, novel series of 3-methyl-imidazo[2,1-b] thiazole-based analogs were synthesized and biologically evaluated for their in vitro inhibitory profile towards DHFR. Compounds 22 and 23 exhibited potent inhibitory profile targeting DHFR (IC50 0.079 and 0.085 µM, respectively comparable to MTX IC50 0.087 µM). Compounds 22 and 23 showed promising cytotoxicity against MCF7 breast cancer cell lines inducing cell cycle arrest and apoptosis. Furthermore, Compound 23 showed its potential to reduce body weight and tumor volume significantly, using Ehrlich ascites carcinoma (EAC) solid tumor animal model of breast cancer, compared to control-treated groups. Further, molecular modeling simulations validated the potential of 22 and 23 to have high affinity binding towards Arg22 and Phe31 residues via π-π interaction and hydrogen bonding within DHFR binding pocket. Computer-assisted ADMET study suggested that the newly synthesized analogs could have high penetration to the blood brain barrier (BBB), better intestinal absorption, non-inhibitors of CYP2D6, adequate plasma protein binding and good passive oral absorption. The obtained model and pattern of substitution could be used for further development of DHFR inhibitors.

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.

Design, synthesis and biological activity of novel substituted 3-benzoic acid derivatives as MtDHFR inhibitors.

The enzyme dihydrofolate reductase from M.tuberculosis (MtDHFR) has a high unexploited potential to be a target for new drugs against tuberculosis (TB), due to its importance for pathogen survival. Preliminary studies have obtained fragment-like molecules with low affinity to MtDHFR which can potentially become lead compounds. Taking this into account, the fragment MB872 was used as a prototype for analogue development by bioisosterism/retro-bioisosterism, which resulted in 20 new substituted 3-benzoic acid derivatives. Compounds were active against MtDHFR, with IC50 values ranging from 7 to 40 µM, where compound 4e not only had the best inhibitory activity (IC50 = 7 µM), but also was 71-fold more active than the original fragment MB872. The 4e inhibition kinetics indicated an uncompetitive mechanism, which was supported by molecular modeling which suggested that the compounds can access an independent backpocket from the substrate and competitive inhibitors. Thus, based on these results, substituted 3-benzoic acid derivatives have strong potential to be developed as novel MtDHFR inhibitors and also anti-TB agents.

Design, synthesis and biological evaluation of novel pyrrolo[2,3-d]pyrimidine as tumor-targeting agents with selectivity for tumor uptake by high affinity folate receptors over the reduced folate carrier.

Tumor-targeted 6-substituted pyrrolo[2,3-d]pyrimidine benzoyl compounds based on 2 were isosterically modified at the 4-carbon bridge by replacing the vicinal (C11) carbon by heteroatoms N (4), O (5) or S (6), or with an N-substituted formyl (7), trifluoroacetyl (8) or acetyl (9). Replacement with sulfur (6) afforded the most potent KB tumor cell inhibitor, ~6-fold better than the parent 2. In addition, 6 retained tumor transport selectivity via folate receptor (FR) α and -ß over the ubiquitous reduced folate carrier (RFC). FRα-mediated cell inhibition for 6 was generally equivalent to 2, while the FRß-mediated activity was improved by 16-fold over 2. N (4) and O (5) substitutions afforded similar tumor cell inhibitions as 2, with selectivity for FRα and -ß over RFC. The N-substituted analogs 7-9 also preserved transport selectivity for FRα and -ß over RFC. For FRα-expressing CHO cells, potencies were in the order of 8 > 7 > 9. Whereas 8 and 9 showed similar results with FRß-expressing CHO cells, 7 was ~16-fold more active than 2. By nucleoside rescue experiments, all the compounds inhibited de novo purine biosynthesis, likely at the step catalyzed by glycinamide ribonucleotide formyltransferase. Thus, heteroatom replacements of the CH2 in the bridge of 2 afford analogs with increased tumor cell inhibition that could provide advantages over 2, as well as tumor transport selectivity over clinically used antifolates including methotrexate and pemetrexed.

New imidazolone derivatives comprising a benzoate or sulfonamide moiety as anti-inflammatory and antibacterial inhibitors: Design, synthesis, selective COX-2, DHFR and molecular-modeling study.

New imidazol-5-one derivatives 12a,b and 12e, f, 14a,b and 16a,b were synthesized and screened for their in vivo anti-inflammatory activity using a standard acute carrageenan-induced rat paw oedema method. All the tested compounds exhibited good anti-inflammatory activity; especially compound 12f which produced the maximum effect of 49.0% compared to the standard drug, celecoxib, (43.1%). The most active anti-inflammatory agents 12a, 12e, and 12f were studied for their interactions with enzyme COX-2 compared to celecoxib. The study showed that, compound 12e exhibited a high selectivity towards COX-2 inhibition with IC50 = 0.087 µM. Moreover, the antibacterial screening indicated that some synthesized compounds showed good antibacterial activity toward the Gram-negative bacteria Escherichia coli. Additionally, compounds 7, 12a, 12f, and 12 showed a good binding affinity with enzyme dihydrofolate reductase (DHFR) whereas compound 12f has a higher inhibitory effect on DHFR than the tested compounds 7, 12a and 12 h. On the other hand, the combination between these tested compounds and sulfadiazine as a reference drug (10 µM compound + 1 µM reference), showed that compound 12 h has higher potency (0.078 ±â€¯0.002) than sulfadiazine (0.135 ±â€¯0.004). In addition, docking analysis was performed and it confirmed the presented results.

Novel heterocyclic hybrids of pyrazole targeting dihydrofolate reductase: design, biological evaluation and in silico studies.

A novel series of pyrazole analogues including hydrazones, pyrazolo[4,3-c]-pyridazines, pyrazolo[3,4-e][1,2,4]triazine and pyrazolo[3,4-d][1,2,3]triazoles was designed, synthesised and screened for their in vitro antimicrobial and DHFR inhibition activity. Compounds bearing benzenesulphonamide moiety incorporated with 3-methyl-5-oxo-1H-pyrazol-4(5H)-ylidene) hydrazine 3a or 6-amino-7-cyano-3-methyl-5H-pyrazolo[4,3-c]pyridazine 6a revealed excellent and broad spectrum antimicrobial activity comparable to ciprofloxacin and amphotericin B as positive antibiotic and antifungal controls, respectively. Furthermore, these derivatives proved to be the most active DHFR inhibitors with IC50 values 0.11 ± 1.05 and 0.09 ± 0.91 µM, in comparison with methotrexate (IC50 = 0.14 ± 1.25 µM). The in silico studies were done to calculate the drug-likeness and toxicity risk parameters of the newly synthesised derivatives. Additionally, the high potency of the pyrazole derivatives bearing sulphonamide against DHFR was confirmed with molecular docking and might be used as an optimum lead for further modification.

Identification of novel selective Mtb-DHFR inhibitors as antitubercular agents through structure-based computational techniques.

Inhibition of dihydrofolate reductase from Mycobacterium tuberculosis-dihydrofolate reductase (Mtb-DHFR) has emerged as a promising approach for the treatment of tuberculosis. To identify novel Mtb-DHFR inhibitors, structure-based virtual screening (SBVS) of the Molecular Diversity Preservation International (MolMall) database was performed using Glide against the Mtb-DHFR and h-DHFR enzymes. On the basis of SBVS, receptor fit, drug-like filters, and ADMET (absorption, distribution, metabolism, excretion, and toxicity) analysis, 16 hits were selected and tested for their antitubercular activity against the H37 RV strain of M. tuberculosis. Five compounds showed promising activity with compounds 11436 and 15275 as the most potent hits with IC50 values of 0.65 and 12.51 µM, respectively, against the H37 RV strain of M. tuberculosis. The two compounds were further tested in the Mtb-DHFR and h-DHFR enzymatic assay for selectivity and were found to be three- to eight-fold selective towards Mtb-DHFR over h-DHFR with minimum inhibitory concentration values of 5.50, 73.89 µM and 42.00, 263.00 µM, respectively. In silico simulation studies also supported the stability of the protein-ligand complex formation. The present study demonstrates the successful utilization of in silico SBVS tools for the identification of novel and potential Mtb-DHFR inhibitors and compound 11436 ((2,4-dihydroxyphenyl)(3,4,5-trihydroxyphenyl)methanone) as a potential lead for the development of novel Mtb-DHFR inhibitors.

Discovery of New Schiff Bases Tethered Pyrazole Moiety: Design, Synthesis, Biological Evaluation, and Molecular Docking Study as Dual Targeting DHFR/DNA Gyrase Inhibitors with Immunomodulatory Activity.

A series of Bis-pyrazole Schiff bases (6a-d and 7a-d) and mono-pyrazole Schiff bases (8a-d and 9a-d) were designed and synthesized through the reaction of 5-aminopyrazoles 1a-d with aldehydes 2-5 using mild reaction condition with a good yield percentage. The chemical structure of newly formed Schiff bases tethered pyrazole core was confirmed based on spectral and experimental data. All the newly formed pyrazole Schiff bases were evaluated against eight pathogens (Gram-positive, Gram-negative, and fungi). The result exhibited that, most of them have good and broad activities. Among those, only six Schiff bases (6b, 7b, 7c, 8a, 8d, and 9b) displayed MIC values (0.97-62.5 µg/mL) compared to Tetracycline (15.62-62.5 µg/mL) and Amphotericin B (15.62-31.25 µg/mL), MBC values (1.94-87.5 µg/mL) and selectivity to tumor cell than normal cells. Immunomodulatory activities showed that the promising Schiff bases increase the immunomodulator effect of defense cell and the Schiff base 8a is the highest one by (Intra. killing activity = 136.5 ± 0.3%) having a pyrazole moiety as well as amide function (O=C-NH2) and piperidinyl core. Furthermore, the most potent one exhibited broad activity depending on both MIC and MBC values. Moreover, to study the mechanism of these pyrazole Schiff bases, two active Schiff bases 8a and 9b from six derivatives were introduced to study the enzyme assay as dihydrofolate reductase (DHFR) on E. coli organism and DNA gyrase with two different organisms, S. aureus and B. subtilis, to determine the inhibitory activities with lower values in the case of DNA gyrase (8a and 9b) or nearly as DHFR compound 9b, while pyrazole 8a showed excellent inhibitory against all enzyme assay. The molecular docking study against dihydrofolate reductase and DNA gyrase were performed to study the binding between active site in the pocket with the two Schiff bases (8a and 9b) that exhibited good binding affinity with different bond types as H-bonding, aren-aren, and arene-cation interaction as well as study the physicochemical and pharmacokinetic properties of the two active Schiff bases 8a and 9b.

Dihydrofolate reductase inhibition effect of 5-substituted pyrido[2,3-d]pyrimidines: Synthesis, antitumor activity and molecular modeling study.

A new series of pyrido[2,3-d]pyrimidines 3-18 bearing substitution at C-5 position was synthesized. All compounds were tested for their in vitro antitumor activity against five human cancer cell lines namely; hepatocellular carcinoma (HePG2), breast carcinoma (MCF-7), human prostate carcinoma (PC3), colorectal carcinoma (HCT-116), and cervical carcinoma (Hela) using doxorubicin as a positive control. Compounds 3, 4, 9, 11, 13, 14, 15 and 17 exhibited the highest antitumor activity against the tested cell lines and were selected to screen their enzymatic inhibition against dihydrofolate reductase enzyme (DHFR) compared with the reference drug methotrexate (MTX), to explain the probable mechanism of action of the observed anticancer activity. Compound 11 displayed the highest inhibitory activity (IC50 = 6.5 µM) among the tested compounds in comparison with MTX (IC50 = 5.57 µM). Also, compounds 13 and 14 showed high inhibitory activity against DHFR with IC50 values 7.1 and 8.7 µM, respectively. Comparative molecular modeling study was performed between DHFR inhibitors 11, 13 and 14 of the highest activity and 10 of the lowest activity among the eight inhibitors against MTX. Docking studies into the active site of DHFR domain showed good agreement with the obtained biological results. Finally, compound 11 was found to be best antitumor, DHFR inhibitor, and it induced the process of apoptosis at Pre-G phase and cell cycle arrest at G2/M phase in MCF-7 cells.

Synthesis, antitumor testing and molecular modeling study of some new 6-substituted amido, azo or thioureido-quinazolin-4(3H)-ones.

A new series of 6-substituted amido, azo or thioureido-quinazolin-4(3H)-one was synthesized and tested for their in-vitro antitumor activity. Compounds 21, 53 and 60 showed broad spectrum antitumor activity with average IC50 values of 6.7, 7.6 and 9.1 µM, respectively compared with methotrexate (1, IC50 19.26 µM). As an attempt to reveal the mechanism of the antitumor potency, cell cycle analysis and DHFR inhibition were performed. Compounds 59 and 61 induced their cytotoxicity in Hela (IC50 10.6 µM) and HCT-116 (IC50 15.5 µM) cell lines, respectively through Pre-G1 apoptosis, inhibiting cell growth at G2-M phase. Compounds 29, 33, 59 and 61 showed DHFR inhibitory potency at IC50 0.2, 0.2, 0.3 and 0.3 µM, respectively. The active DHFR inhibitors showed high affinity binding toward the amino acid residues Thr56, Ser59 and Ser118. The active compounds obeyed Lipinski's rule of five and could be used as template model for further optimization.

TRAPP webserver: predicting protein binding site flexibility and detecting transient binding pockets.

The TRAnsient Pockets in Proteins (TRAPP) webserver provides an automated workflow that allows users to explore the dynamics of a protein binding site and to detect pockets or sub-pockets that may transiently open due to protein internal motion. These transient or cryptic sub-pockets may be of interest in the design and optimization of small molecular inhibitors for a protein target of interest. The TRAPP workflow consists of the following three modules: (i) TRAPP structure- generation of an ensemble of structures using one or more of four possible molecular simulation methods; (ii) TRAPP analysis-superposition and clustering of the binding site conformations either in an ensemble of structures generated in step (i) or in PDB structures or trajectories uploaded by the user; and (iii) TRAPP pocket-detection, analysis, and visualization of the binding pocket dynamics and characteristics, such as volume, solvent-exposed area or properties of surrounding residues. A standard sequence conservation score per residue or a differential score per residue, for comparing on- and off-targets, can be calculated and displayed on the binding pocket for an uploaded multiple sequence alignment file, and known protein sequence annotations can be displayed simultaneously. The TRAPP webserver is freely available at http://trapp.h-its.org.

Design and synthesis of leucine-linked quinazoline-4(3H)-one-sulphonamide molecules distorting malarial reductase activity in the folate pathway.

Optimization of a modified Grimmel's method for N-heterocyclization of a leucine-linked sulfonamide side-arm at position 2 leading to 2,3-disustituted-4-quinazolin-(3H)-ones was accomplished. Further, 22 hybrid quinazolinone motifs (4a-v) were synthesized by N-heterocyclization reaction under microwave irradiation using the ionic liquid [Bmim][BF4 ]-H2 O as green solvent as well as the catalyst. The in vitro screening of the hybrid entities against the malarial species Plasmodium falciparum yielded five potent molecules 4l, 4n, 4o, 4t, and 4u owning antimalarial activity comparable to those of the reference drugs. In continuation, an in silico study was carried out to obtain a pharmacophoric model and quantitative structure-activity relationship. We also built a 3D-QSAR model to procure more information that could be applied to design new molecules with more potent Pf-DHFR inhibitory activity. The designed pharmacophore was recognized to be more potent for the selected molecules, exhibiting five pharmacophoric features. The active scaffolds were further evaluated for enzyme inhibition efficacy against alleged receptor Pf-DHFR computationally and in vitro, proving their candidature as lead dihydrofolate reductase inhibitors, and the selectivity of the test candidates was ascertained by toxicity study against Vero cells. Good oral bioavailability was also proved by studying pharmacokinetic properties.

Antibacterial Evaluation, In Silico Characters and Molecular Docking of Schiff Bases Derived from 5-aminopyrazoles.

A series of Schiff bases 14-25 were designed and synthesized for evaluation of their antibacterial properties against multi-drug resistant bacteria (MDRB). The antibacterial activities of Schiff bases 14-25 showed that most of the synthesized compounds displayed a significant antibacterial activity. Assessment of in silico ADMET properties (absorption, distribution, metabolism, excretion and toxicity) of Schiff bases illustrates that all derivatives showed agreement to the Lipinski's rule of five. Further enzymatic assay aided by molecular docking study demonstrated that compound 18 is a potent inhibitor of staphylococcus aureus DNA gyrase and dihydrofolate reductase kinases. This study could be valuable in the discovery of new potent antimicrobial agents.

Targeting species specific amino acid residues: Design, synthesis and biological evaluation of 6-substituted pyrrolo[2,3-d]pyrimidines as dihydrofolate reductase inhibitors and potential anti-opportunistic infection agents.

To combine the potency of trimetrexate (TMQ) or piritrexim (PTX) with the species selectivity of trimethoprim (TMP), target based design was carried out with the X-ray crystal structure of human dihydrofolate reductase (hDHFR) and the homology model of Pneumocystis jirovecii DHFR (pjDHFR). Using variation of amino acids such as Met33/Phe31 (in pjDHFR/hDHFR) that affect the binding of inhibitors due to their distinct positive or negative steric effect at the active binding site of the inhibitor, we designed a series of substituted-pyrrolo[2,3-d]pyrimidines. The best analogs displayed better potency (IC50) than PTX and high selectivity for pjDHFR versus hDHFR, with 4 exhibiting a selectivity for pjDHFR of 24-fold.

Inhibitors of dihydrofolate reductase as antitumor agents: design, synthesis and biological evaluation of a series of novel nonclassical 6-substituted pyrido[3,2-d]pyrimidines with a three- to five-carbon bridge.

Bridge homologation of the previously reported nonclassical two-carbon-bridged antifolate, 2,4-diamino-6-phenethylpyrido[3,2-d]pyrimidine (wm-5a), afforded the three-, four- and five-carbon-bridged antifolate analogues 3.1-3.5, 4.1-4.2 and 5.1-5.5. The target compounds, with substituents at various positions on the carbon bridges, were efficiently synthesized by aldol condensation or Wittig reaction and followed by reduction. Elongation of the two-carbon bridge to three-, four- or five-carbon bridges, and also saturation of the carbon bridges, provided compounds with good inhibitory activity against recombinant human DHFR (rhDHFR). Analogue 3.5, which has a three-carbon bridge, inhibited the proliferation of HL-60 and HCT116 cells to a greater extent than the other analogues. Compound 3.5 was also the most potent inhibitor of rhDHFR (IC50 = 0.06 µM), and was approximately 38-fold more potent than the two-carbon-bridged lead compound. Docking studies revealed that both the length and flexibility of the saturated carbon bridge in 3.5 were important for high potency. Flow cytometry studies indicated that compound 3.5 arrested HL-60 cells in the S-phase and induced apoptosis. Western blot analysis of HL-60 cells treated with 3.5 showed a dose-dependent upregulation of DHFR protein levels.

Imidazo[2',1':2,3]thiazolo[4,5-d]pyridazinone as a new scaffold of DHFR inhibitors: Synthesis, biological evaluation and molecular modeling study.

New series of thiazolo[4,5-d]pyridazin and imidazo[2',1':2,3]thiazolo[4,5-d]pyridazin analogues were designed, synthesized and evaluated for their invitro DHFR inhibition and antitumor activity. Compounds 13 and 43 proved to be DHFR inhibitors with IC50 0.05 and 0.06 µM, respectively. 43 proved lethal to OVCAR-3 Ovarian cancer and MDA-MB-435 Melanoma at IC50 0.32 and 0.46 µM, respectively. The active compounds formed hydrogen bond at DHFR binding site between N1-nitrogen of the pyridazine ring with Glu30; the carbonyl group with Trp24, Arg70 or Lys64; π-cation interaction with Arg22 and π-π interaction with Phe31 residues. Ring annexation of the active 1,3-thiazole ring analogue 13 into the bicyclic thiazolo[4,5-d]pyridazine (18,19) or imidazo[2,1-b]thiazoles (23-25) decreased the DHFR inhibition activity; while the formation of the tricyclic imidazo[2',1':2,3]-thiazolo[4,5-d]pyridazine (43-54) increased potency. The obtained model could be useful for the development of new class of DHFR inhibitors.