Recent Discoveries of Nitrogen-Containing Heterocyclic Compounds as InhA Inhibitors against Mycobacterium tuberculosis: An Overview.

It is a formidable challenge to treat tuberculosis as there are increasing cases of multidrugresistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) cases. Moreover, the emergence of totally drug-resistant tuberculosis (TDR-TB) makes it vital and imperative to develop a new generation of anti-tubercular drugs to have control over resistance. The nitrogencontaining heterocyclic class of compounds is being studied extensively to ascertain their anti-TB potentials. Nitrogen-containing compounds have a broad range of targets; wherein, InhA is the most important one. Hence, the primary focus of this review is to summarize the recent developments in the discovery of nitrogen-containing heterocyclic compounds as InhA inhibitors to combat tuberculosis.

Mechanistic Insights into Binding of Ligands with Thiazolidinedione Warhead to Human Histone Deacetylase 4.

Recently, we have reported that non-hydroxamate thiazolidinedione (TZD) analogs are capable of inhibiting human deacetylase 4 (HDAC4). This study aims at the dissection of the molecular determinants and kinetics of the molecular recognition of TZD ligands by HDAC4. For this purpose, a structure activity relationship analysis of 225 analogs was combined with a comprehensive study of the enzyme and binding kinetics of a variety of HDAC4 mutant variants. The experimental data were rationalized by docking to the two major conformations of HDAC4. TZD ligands are competitive inhibitors and bind via a two-step mechanism involving principal molecular recognition and induced fit. The residence time of 24 g is (34 ± 3) min and thus much larger than that of the canonical pan-HDAC inhibitor SAHA ((5 ± 2) min). Importantly, the binding kinetics can be tuned by varying the structure of the CAP group.

Multi-target weapons: diaryl-pyrazoline thiazolidinediones simultaneously targeting VEGFR-2 and HDAC cancer hallmarks.

In anticancer drug discovery, multi-targeting compounds have been beneficial due to their advantages over single-targeting compounds. For instance, VEGFR-2 has a crucial role in angiogenesis and cancer management, whereas HDACs are well-known regulators of epigenetics and have been known to contribute significantly to angiogenesis and carcinogenesis. Herein, we have reported nineteen novel VEGFR-2 and HDAC dual-targeting analogs containing diaryl-pyrazoline thiazolidinediones and their in vitro and in vivo biological evaluation. In particular, the most promising compound 14c has emerged as a dual inhibitor of VEGFR-2 and HDAC. It demonstrated anti-angiogenic activity by inhibiting in vitro HUVEC proliferation, migration, and tube formation. Moreover, an in vivo CAM assay showed that 14c repressed new capillary formation in CAMs. In particular, 14c exhibited cytotoxicity potential on different cancer cell lines such as MCF-7, K562, A549, and HT-29. Additionally, 14c demonstrated significant potency and selectivity against HDAC4 in the sub-micromolar range. To materialize the hypothesis, we also performed molecular docking on the crystal structures of both VEGFR-2 (PDB ID: 1YWN) and HDAC4 (PDB-ID: 4CBY), which corroborated the designing and biological activity. The results indicated that compound 14c could be a potential lead to develop more optimized multi-target analogs with enhanced potency and selectivity.

Development and investigation of thiazolidinedione and pyrazoline compounds as antiangiogenic weapons targeting VEGFR-2.

Background: Angiogenesis deregulation is often linked to cancer and is thus an essential target. Materials & methods: Twenty-nine compounds were developed as VEGFR-2 inhibitors. Compounds were evaluated to determine their antiangiogenic activity. Results: B1, PB11 and PB16 showed HUVEC's IC50 scores in the submicromolar range. B1, B2 and PB16 reduced cellular migration and capillary tube formation of HUVECs. VEGFR-2 inhibitory activity was found in the nanomolar range: 200 nM of B1, 500 nM of B2 and 600 nM of PB16. B1 and PB16 suppressed the formation of new capillaries on growing CAMs. B1 and PB16 occupied the ATP site and allosteric pocket of VEGFR-2 in docking studies. Conclusion: These compounds can target VEGFR-2 and are endowed with in vitro and in vivo antiangiogenic activity.

Double-edged Swords: Diaryl pyrazoline thiazolidinediones synchronously targeting cancer epigenetics and angiogenesis.

In the present study, two novel series of compounds incorporating naphthyl and pyridyl linker were synthesized and biological assays revealed 5-((6-(2-(5-(2-chlorophenyl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethoxy) naphthalene-2-yl)methylene)thiazolidine-2,4-dione (14b) as the most potent dual inhibitors of vascular endothelial growth factors receptor-2 (VEGFR-2) and histone deacetylase 4 (HDAC4). Compounds 13b, 14b, 17f, and 21f were found to stabilize HDAC4; where, pyridyl linker swords were endowed with higher stabilization effects than naphthyl linker. Also, 13b and 14b showed best inhibitory activity on VEGFR-2 as compared to others. Compound 14b was most potent as evident by in-vitro and in-vivo biological assessments. It displayed anti-angiogenic potential by inhibiting endothelial cell proliferation, migration, tube formation and also suppressed new capillary formation in the growing chick chorioallantoic membranes (CAMs). It showed selectivity and potency towards HDAC4 as compared to other HDAC isoforms. Compound 14b (25 mg/kg, i.p.) also indicated exceptional antitumor efficacy on in-vivo animal xenograft model of human colorectal adenocarcinoma (HT-29). The mechanism of action of 14b was also confirmed by western blot.

Thiazolidinedione "Magic Bullets" Simultaneously Targeting PPARγ and HDACs: Design, Synthesis, and Investigations of their In Vitro and In Vivo Antitumor Effects.

Monotargeting anticancer agents suffer from resistance and target nonspecificity concerns, which can be tackled with a multitargeting approach. The combined treatment with HDAC inhibitors and PPARγ agonists has displayed potential antitumor effects. Based on these observations, this work involves design and synthesis of molecules that can simultaneously target PPARγ and HDAC. Several out of 25 compounds inhibited HDAC4, and six compounds acted as dual-targeting agents. Compound 7i was the most potent, with activity toward PPARγ EC50 = 0.245 µM and HDAC4 IC50 = 1.1 µM. Additionally, compounds 7c and 7i were cytotoxic to CCRF-CEM cells (CC50 = 2.8 and 9.6 µM, respectively), induced apoptosis, and caused DNA fragmentation. Furthermore, compound 7c modulated the expression of c-Myc, cleaved caspase-3, and caused in vivo tumor regression in CCRF-CEM tumor xenografts. Thus, this study provides a basis for the rational design of dual/multitargeting agents that could be developed further as anticancer therapeutics.

Pharmacophore hybridization approach to discover novel pyrazoline-based hydantoin analogs with anti-tumor efficacy.

In search for new and safer anti-cancer agents, a structurally guided pharmacophore hybridization strategy of two privileged scaffolds, namely diaryl pyrazolines and imidazolidine-2,4-dione (hydantoin), was adopted resulting in a newfangled series of compounds (H1-H22). Herein, a bio-isosteric replacement of "pyrrolidine-2,5-dione" moiety of our recently reported antitumor hybrid incorporating diaryl pyrazoline and pyrrolidine-2,5-dione scaffolds with "imidazoline-2,4-dione" moiety has been incorporated. Complete biological studies revealed the most potent analog among all i.e. compound H13, which was at-least 10-fold more potent compared to the corresponding pyrrolidine-2,5-dione, in colon and breast cancer cells. In-vitro studies showed activation of caspases, arrest of G0/G1 phase of cell cycle, decrease in the expression of anti-apoptotic protein (Bcl-2) and increased DNA damage. In-vivo assay on HT-29 (human colorectal adenocarcinoma) animal xenograft model unveiled the significant anti-tumor efficacy along with oral bioavailability with maximum TGI 36% (i.p.) and 44% (per os) at 50 mg/kg dose. These findings confirm the suitability of hybridized pyrazoline and imidazolidine-2,4-dione analog H13 for its anti-cancer potential and starting-point for the development of more efficacious analogs.

Hypoglycemic and Hypolipidemic Swords: Synthesis and In-vivo Biological Assessment of 5-benzylidene-2,4-thiazolidinediones.

Thiazolidinedione (TZD), being a privileged scaffold, has been known as a significant structural moiety of antidiabetic drugs. TZD has been known to improve glycaemic control in type 2 diabetes mellitus (T2DM) by increasing insulin sensitivity in the body. A novel series of 5-benzylidene 2,4-thiazolidinedione derivatives were designed, synthesized (V1-V28), and structurally confirmed by different spectroscopic techniques such as FTIR, 1H NMR, 13C NMR, and Mass spectrometry. Upon the safety assessment of the synthesized molecules in non-transformed hepatocytes by MTT reduction assay, these were found non-toxic. These derivatives were then further evaluated for their antihyperglycemic and antihyperlipidemic properties in a high-fat diet and low dose of streptozotocin-induced diabetic rats. Altogether, seven biochemical parameters were analyzed, namely blood glucose, triglycerides, cholesterol, creatinine, blood urea nitrogen, HDL-cholesterol, and glycosylated hemoglobin in serum by standard methods. Four synthetic molecules (V2, V4, V5, and V20) possessed significant hypoglycaemic and hypolipidemic activity as compared to the positive control pioglitazone. Moreover, the histopathological studies of the heart and liver revealed no significant toxicity. Two representative compounds V2 and V4, were evaluated for their PPARγ activation potential, demonstrating that they were partial PPARγ agonists, thus confirming our designing hypothesis. Based on the results obtained, we assume that these compounds have the potential to be developed as future antidiabetic agents.

Power of two: combination of therapeutic approaches involving glucose transporter (GLUT) inhibitors to combat cancer.

Cancer research of the Warburg effect, a hallmark metabolic alteration in tumors, focused attention on glucose metabolism whose targeting uncovered several agents with promising anticancer effects at the preclinical level. These agents' monotherapy points to their potential as adjuvant combination therapy to existing standard chemotherapy in human trials. Accordingly, several studies on combining glucose transporter (GLUT) inhibitors with chemotherapeutic agents, such as doxorubicin, paclitaxel, and cytarabine, showed synergistic or additive anticancer effects, reduced chemo-, radio-, and immuno-resistance, and reduced toxicity due to lowering the therapeutic doses required for desired chemotherapeutic effects, as compared with monotherapy. The combinations have been specifically effective in treating cancer glycolytic phenotypes, such as pancreatic and breast cancers. Even combining GLUT inhibitors with other glycolytic inhibitors and energy restriction mimetics seems worthwhile. Though combination clinical trials are in the early phase, initial results are intriguing. The various types of GLUTs, their role in cancer progression, GLUT inhibitors, and their anticancer mechanism of action have been reviewed several times. However, utilizing GLUT inhibitors as combination therapeutics has received little attention. We consider GLUT inhibitors agents that directly affect glucose transporters by binding to them or indirectly alter glucose transport by changing the transporters' expression level. This review mainly focuses on summarizing the effects of various combinations of GLUT inhibitors with other anticancer agents and providing a perspective on the current status.

Benzylidene thiazolidinediones: Synthesis, in vitro investigations of antiproliferative mechanisms and in vivo efficacy determination in combination with Imatinib.

Thiazolidinedione (TZD) has been an interesting scaffold due to its proven antidiabetic activity and encouraging findings in anticancer drug discovery. We synthesised benzylidene thiazolidinedione derivatives which exhibited excellent antiproliferative effects in chronic myeloid leukemic cells K562 and the most active compounds 3t and 3x had GI50 value of 0.9 and 0.23 µM respectively. Both the compound was found to arrest the growth of K562 cells in G0/G1 phase in a time and dose dependent manner. Further, western blot analysis revealed that 3t and 3x could also inhibit the expression of cell proliferation markers, PCNA and Cyclin D1 and compound 3x up-regulated apoptosis markers, cleaved PARP1 and activated caspase 3, which could be a possible mechanism for the excellent antiproliferative effects exhibited by these compounds. In vitro combination studies of 3t and 3x with Imatinib found to potentiate the antitumor effects of Imatinib. Further in vivo efficacy in K562 xenografts, of 3t and 3x alone and in combination with Imatinib was found to be promising and far better than control group and combination treatment was found to be more effective as compared to only Imatinib treated or test compound treated animals. Thus, our findings suggest that these compounds are promising antitumor agents and could help to enhance the anticancer effects of Imatinib and other tyrosine kinase inhibitors, when used in combination.

Permuted 2,4-thiazolidinedione (TZD) analogs as GLUT inhibitors and their in-vitro evaluation in leukemic cells.

Cancer is a heterogeneous disease, and its treatment requires the identification of new ways to thwart tumor cells. Amongst such emerging targets are glucose transporters (GLUTs, SLC2 family), which are overexpressed by almost all types of cancer cells; their inhibition provides a strategy to disrupt tumor metabolism selectively, leading to antitumor effects. Here, novel thiazolidinedione (TZD) derivatives were designed, synthesized, characterized, and evaluated for their GLUT1, GLUT4, and GLUT5 inhibitory potential, followed by in-vitro cytotoxicity determination in leukemic cell lines. Compounds G5, G16, and G17 inhibited GLUT1, with IC50 values of 5.4 ± 1.3, 26.6 ± 1.8, and 12.6 ± 1.2 µM, respectively. G17 was specific for GLUT1, G16 inhibited GLUT4 (IC50 = 21.6 ± 4.5 µM) comparably but did not affect GLUT5. The most active compound, G5, inhibited all three GLUT types, with GLUT4 IC50 = 9.5 ± 2.8 µM, and GLUT5 IC50 = 34.5 ± 2.4 µM. Docking G5, G16, and G17 to the inward- and outward-facing structural models of GLUT1 predicted ligand binding affinities consistent with the kinetic inhibition data and implicated E380 and W388 of GLUT1 vs. their substitutions in GLUT5 (A388 and A396, respectively) in inhibitor preference for GLUT1. G5 inhibited the proliferation of leukemia CEM cells at low micromolar range (IC50 = 13.4 µM) while being safer for normal blood cells. Investigation of CEM cell cycle progression after treatment with G5 showed that cells accumulated in the G2/M phase. Flow cytometric apoptosis studies revealed that compound G5 induced both early and late-stage apoptosis in CEM cells.

Synthesis and Biological Evaluation of Pyrazoline and Pyrrolidine-2,5-dione Hybrids as Potential Antitumor Agents.

In search of novel and effective antitumor agents, pyrazoline-substituted pyrrolidine-2,5-dione hybrids were designed, synthesized and evaluated in silico, in vitro and in vivo for anticancer efficacy. All the compounds exhibited remarkable cytotoxic effects in MCF7 and HT29 cells. The excellent antiproliferative activity toward MCF7 (IC50 =0.78±0.01 µM), HT29 (IC50 =0.92±0.15 µM) and K562 (IC50 =47.25±1.24 µM) cell lines, prompted us to further investigate the antitumor effects of the best compound S2 (1-(2-(3-(4-fluorophenyl)-5-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-oxoethyl)pyrrolidine-2,5-dione). In cell-cycle analysis, S2 was found to disrupt the growth phases with increased cell population in G1 /G0 phase and decreased cell population in G2 /M phase. The excellent in vitro effects were also supported by inhibition of anti-apoptotic protein Bcl-2. In vivo tumor regression studies of S2 in HT29 xenograft nude mice, exhibited equivalent and promising tumor regression with maximum TGI, 66 % (i. p. route) and 60 % (oral route) at 50 mg kg-1 dose by both the routes, indicating oral bioavailability and antitumor efficacy. These findings advocate that hybridization of pyrazoline and pyrrolidine-2,5-dioes holds promise for the development of more potent and less toxic anticancer agents.

Structure guided design and synthesis of furyl thiazolidinedione derivatives as inhibitors of GLUT 1 and GLUT 4, and evaluation of their anti-leukemic potential.

Cancer cells increase their glucose uptake and glycolytic activity to meet the high energy requirements of proliferation. Glucose transporters (GLUTs), which facilitate the transport of glucose and related hexoses across the cell membrane, play a vital role in tumor cell survival and are overexpressed in various cancers. GLUT1, the most overexpressed GLUT in many cancers, is emerging as a promising anti-cancer target. To develop GLUT1 inhibitors, we rationally designed, synthesized, structurally characterized, and biologically evaluated in-vitro and in-vivo a novel series of furyl-2-methylene thiazolidinediones (TZDs). Among 25 TZDs tested, F18 and F19 inhibited GLUT1 most potently (IC50 11.4 and 14.7 µM, respectively). F18 was equally selective for GLUT4 (IC50 6.8 µM), while F19 was specific for GLUT1 (IC50 152 µM in GLUT4). In-silico ligand docking studies showed that F18 interacted with conserved residues in GLUT1 and GLUT4, while F19 had slightly different interactions with the transporters. In in-vitro antiproliferative screening of leukemic/lymphoid cells, F18 was most lethal to CEM cells (CC50 of 1.7 µM). Flow cytometry analysis indicated that F18 arrested cell cycle growth in the subG0-G1 phase and lead to cell death due to necrosis and apoptosis. Western blot analysis exhibited alterations in cell signaling proteins, consistent with cell growth arrest and death. In-vivo xenograft study in a CEM model showed that F18 impaired tumor growth significantly.

Discovery of novel N-substituted thiazolidinediones (TZDs) as HDAC8 inhibitors: in-silico studies, synthesis, and biological evaluation.

Epigenetics plays a fundamental role in cancer progression, and developing agents that regulate epigenetics is crucial for cancer management. Among Class I and Class II HDACs, HDAC8 is one of the essential epigenetic players in cancer progression. Therefore, we designed, synthesized, purified, and structurally characterized novel compounds containing N-substituted TZD (P1-P25). Cell viability assay of all compounds on leukemic cell lines (CEM, K562, and KCL22) showed the cytotoxic potential of P8, P9, P10, P12, P19, and P25. In-vitro screening of different HDACs isoforms revealed that P19 was the most potent and selective inhibitor for HDAC8 (IC50 - 9.3 µM). Thermal shift analysis (TSA) confirmed the binding of P19 to HDAC8. In-vitro screening of all compounds on the transport activity of GLUT1, GLUT4, and GLUT5 indicated that P19 inhibited GLUT1 (IC50 - 28.2 µM). P10 and P19 induced apoptotic cell death in CEM cells (55.19% and 60.97% respectively) and P19 was less cytotoxic on normal WBCs (CC50 - 104.2 µM) and human fibroblasts (HS27) (CC50 - 105.0 µM). Thus, among this novel series of TZD derivatives, compound P19 was most promising HDAC8 inhibitor and cytotoxic on leukemic cells. Thus, P19 could serve as a lead for further development of optimized molecules with enhanced selectivity and potency.

Discovery of 5-naphthylidene-2,4-thiazolidinedione derivatives as selective HDAC8 inhibitors and evaluation of their cytotoxic effects in leukemic cell lines.

Histone deacetylases (HDACs) are being explored as a therapeutic target for interventions in different types of cancer. HDAC8 is a class I HDAC that is implicated as a therapeutic target in various indication areas, including different types of cancer and particularly childhood neuroblastoma. Most previously described HDAC8-selective inhibitors contain a hydroxamate function as zinc binding group (ZBG) to confer potency. However, hydroxamate class HDAC inhibitors have raised increasing concerns about their mutagenic character. Therefore, non-hydroxamate based inhibitors could prove to be safer than hydroxamates. In the present work, a series of novel 5-naphthylidene-2,4-thiazolidinedione was designed and evaluated as potential antiproliferative agents targeting selectively HDAC8 enzyme. Eleven novel derivatives were synthesized, purified and characterized by spectroscopic techniques. Compounds 3k and 3h was found to be most potent selective inhibitors of HDAC8 with IC50 values of 2.7 µM and 6.3 µM respectively. 3a to 3i was found to be most cytotoxic in leukemic cell lines. 3a and 3 h both were found to induce apoptosis and cause cell cycle arrest in G2/M phase.

Development of 1,2,4-Triazole-5-Thione Derivatives as Potential Inhibitors of Enoyl Acyl Carrier Protein Reductase (InhA) in Tuberculosis.

Tuberculosis (TB) ranks second, next to AIDS making it most formidable disease in the present age. One of the crucial enzymes involved in cell wall synthesis of Mycobacterium tuberculosis, InhA (enoyl acyl carrier protein reductase), one of the crucial enzymes involved in cell wall synthesis of Mycobacterium tuberculosis, has been authenticated as an effective target for anti-mycobacterial drug development. In the current work, novel derivatives of 1,2,4-triazole-5-thione rationally designed, synthesized and spectrally characterized as promising InhA inhibitors. Anti-mycobacterial potential was determined by resazurin microtiter assay using Mtb H37Rv strain. The mechanism of action of these compounds was confirmed by InhA enzyme inhibition studies. 6b, the most active compound of the series displayed MIC of 0.19 µM in resazurin microtiter assay and InhA inhibition with IC50 of 90 nM.

Novel Anthraquinone Derivatives as Dual Inhibitors of Topoisomerase 2 and Casein Kinase 2: In Silico Studies, Synthesis and Biological Evaluation on Leukemic Cell Lines.

BACKGROUND: Cancer being a complex disease, single targeting agents remain unsuccessful. This calls for "multiple targeting", wherein a single drug is so designed that it will modulate the activity of multiple protein targets. Topoisomerase 2 (Top2) helps in removing DNA tangles and super-coiling during cellular replication, Casein Kinase 2 (CK2) is involved in the phosphorylation of a multitude of protein targets. Thus, in the present work, we have tried to develop dual inhibitors of Top2 and CK2. OBJECTIVE: With this view, in the present work, 2 human proteins, Top2 and CK2 have been targeted to achieve the anti-proliferative effects. METHODS: Novel 1-acetylamidoanthraquinone (3a-3y) derivatives were designed, synthesized and their structures were elucidated by analytical and spectral characterization techniques (FTIR, 1H NMR, 13C NMR and Mass Spectroscopy). The synthesized compounds were then subjected to evaluation of cytotoxic potential by the Sulforhodamine B (SRB) protein assay, using HL60 and K562 cell lines. Ten compounds were analyzed for Top2, CK2 enzyme inhibitory potential. Further, top three compounds were subjected to cell cycle analysis. RESULTS: The compounds 3a to 3c, 3e, 3f, 3i to 3p, 3t and 3x showed excellent cytotoxic activity to HL-60 cell line indicating their high anti-proliferative potential in AML. The compounds 3a to 3c, 3e, 3f, 3i to 3p and 3y have shown good to moderate activity on K-562 cell line. Compounds 3e, 3f, 3i, 3x and 3y were found more cytotoxic than standard doxorubicin. In cell cycle analysis, the cells (79-85%) were found to arrest in the G0/G1 phase. CONCLUSION: We have successfully designed, synthesized, purified and structurally characterized 1- acetylamidoanthraquinone derivatives. Even though our compounds need design optimization to further increase enzyme inhibition, their overall anti-proliferative effects were found to be encouraging.

Synthesis and primary cytotoxicity evaluation of new 5-benzylidene-2,4-thiazolidinedione derivatives.

In the present work, ten novel derivatives (3a-3j) of 5-benzylidene-2,4-thiazolidinediones were synthesized and their structures were determined by analytical and spectral (FTIR, (1)H NMR, (13)C NMR) methods. The newly synthesized compounds were evaluated for their antiproliferative activity at Tata Memorial's Advanced Center for Treatment, Research and Education in Cancer (ACTREC), India, in a panel of 7 cancer cell lines using four concentrations at 10-fold dilutions. Sulforhodamine B (SRB) protein assay was used to estimate cell stability or growth. Though the compounds showed varying degrees of cytotoxicity in the tested cell lines, most marked effect was observed by compound 3e in MCF7 (breast cancer), K562 (leukemia) and GURAV (nasopharyngeal cancer) cell lines with log(10) GI(50) values of -6.7, -6.72 and -6.73 respectively.