Design,synthesis and biological evaluation of novel quinoline‐based carboxylic hydrazides as anti‐tubercular agents

In this study, seventeen novel quinoline‐based carboxylic hydrazides were designed as potential anti‐tubercular agents using molecular hybridization approach and evaluated in‐silico for drug‐likeness behavior. The compounds were synthesized, purified, and characterized using spectral techniques (like FTIR, ¹H NMR, and Mass). The in‐vitro anti‐tubercular activity (against Mycobacterium tuberculosisH37Ra) and cytotoxicity against human lung fibroblast cells were studied. Among the tested hydrazides, four compounds ( 6h , 6j , 6l, and 6m ) exhibited significant anti‐tubercular activity with MIC values below 20 μg/mL. The two most potent compounds of the series, 6j and 6m exhibited MIC values 7.70 and 7.13 μg/mL, respectively, against M. tuberculosis with selectivity index >26. Structure–activity relationship studies were performed for the tested compounds in order to explore the effect of substitution pattern on the anti‐tubercular activity of the synthesized compounds.     

Isoniazid–phytochemical conjugation: A new approach for potent and less toxic anti‐TB drug development

Mycobacterium tuberculosis (Mtb) causes one of the most grievous pandemic infectious diseases, tuberculosis (TB), with long‐term morbidity and high mortality. The emergence of drug‐resistant Mtb strains, and the co‐infection with human immunodeficiency virus, challenges the current WHO‐TB stewardship programs. The first‐line anti‐TB drugs, isoniazid (INH) and rifampicin (RIF), have become extensively obsolete in TB control from chromosomal mutations during the last decades. However, based on clinical trial statistics, the production of well‐tolerated anti‐TB drug(s) is miserably low. Alternately, semi‐synthesis or structural modifications of first‐line obsolete antitubercular drugs remain as the versatile approach for getting some potential medicines. The use of any suitable phytochemicals with INH in a hybrid formulation could be an ideal approach for the development of potent anti‐TB drug(s). The primary objective of this review was to highlight and analyze available INH–phytochemical hybrid research works. The utilization of phytochemicals through chemical conjugation is a new trend toward the development of safer/non‐toxic anti‐TB drugs.     

Synthesis and in vitro investigation of halogenated 1,3‐bis(4‐nitrophenyl)triazenide salts as antitubercular compounds

The diverse pharmacological properties of the diaryltriazenes have sparked the interest to investigate their potential to be repurposed as antitubercular drug candidates. In an attempt to improve the antitubercular activity of a previously constructed diaryltriazene library, eight new halogenated nitroaromatic triazenides were synthesized and underwent biological evaluation. The potency of the series was confirmed against the Mycobacterium tuberculosis lab strain H37Ra, and for the most potent derivative, we observed a minimal inhibitory concentration of 0.85 μm . The potency of the triazenide derivatives against M. tuberculosis H37Ra was found to be highly dependent on the nature of the halogenated phenyl substituent and less dependent on cationic species used for the preparation of the salts. Although the inhibitory concentration against J774A.1 macrophages was observed at 3.08 μm , the cellular toxicity was not mediated by the generation of nitroxide intermediate as confirmed by electron paramagnetic resonance spectroscopy, whereas no in vitro mutagenicity could be observed for the new halogenated nitroaromatic triazenides when a trifluoromethyl substituent was present on both the aryl moieties.     

Design and Development of Mycobacterium tuberculosis Lysine ɛ‐Aminotransferase Inhibitors for Latent Tuberculosis Infection

Lysine ?‐aminotransferase (LAT) is a protein involved in lysine catabolism, and it plays a significant role during the persistent/latent phase of Mycobacterium tuberculosis (MTB), as observed by its up‐regulation by ~40‐fold during this stage. We have used the crystal structure of MTB LAT in external aldimine form in complex with its substrate lysine as a template to design and identify seven lead compounds with IC₅₀ ranging from 18.06 to > 90 μm . We have synthesized 21 compounds based on the identified lead, and compound 21 [2,2′‐oxybis(N′‐(4‐fluorobenzylidene)acetohydrazide)] was found to be the most active with MTB LAT IC₅₀ of 0.81 ± 0.03 μm . Compound 21 also showed a 2.3 log reduction in the nutrient‐starved MTB model and was more potent than standard isoniazid and rifampicin at the same dose level of 10 μg/mL.     

Design,Synthesis, and in vitro antitubercular activity of 1,2,3‐triazolyl‐dihydroquinoline derivatives

In the quest for new active molecules against Mycobacterium tuberculosis, a series of dihydroquinoline derivatives possessing triazolo substituents were efficiently synthesized using click chemistry. The structure of 6l was evidenced by X‐ray crystallographic study. The newly synthesized compounds were evaluated for their in vitro antitubercular activity against Mycobacterium tuberculosis H37Rv (ATCC27294). The compounds 6a , 6g, and 6j (MIC: 3.13 μg/ml) showed promising activity when compared to the first‐line drug such as ethambutol. In addition, the structure and antitubercular activity relationship were further supported by in silico molecular docking studies of the active compounds against 3IVX.PDB (crystal structure of pantothenate synthetase in complex with 2‐(2‐(benzofuran‐2‐ylsulfonylcarbamoyl)‐5‐methoxy‐1H‐indol‐1‐yl)acetic acid).     

Synthesis,biological evaluation,and molecular docking studies of novel 3‐aryl‐5‐(alkyl‐thio)‐1H‐1,2,4‐triazoles derivatives targeting Mycobacterium tuberculosis

A small library of new 3‐aryl‐5‐(alkyl‐thio)‐1H‐1,2,4‐triazoles was synthesized and screened for the antimycobacterial potency against Mycobacterium tuberculosis H₃₇Ra strain and Mycobacterium bovis BCG both in active and dormant stage. Among the synthesized library, 25 compounds exhibited promising anti‐TB activity in the range of IC₅₀0.03–5.88 μg/ml for dormant stage and 20 compounds in the range of 0.03–6.96 μg/ml for active stage. Their lower toxicity (>100 μg/ml) and higher selectivity (SI = >10) against all cancer cell lines screened make them interesting compounds with potential antimycobacterial effects. Furthermore, to rationalize the observed biological activity data and to establish a structural basis for inhibition of M. tuberculosis, the molecular docking study was carried out against a potential target MTB CYP121 which revealed a significant correlation between the binding score and biological activity for these compounds. Cytotoxicity and in vivo pharmacokinetic studies suggested that 1,2,4‐triazole analogues have an acceptable safety index, in vivo stability and bio‐availability.     

Specific Interaction between Mycobacterium tuberculosis Lipoprotein‐derived Peptides and Target Cells Inhibits Mycobacterial Entry In Vitro

Tuberculosis (TB) continues being one of the diseases having the greatest mortality rates around the world, 8.7 million cases having been reported in 2011. An efficient vaccine against TB having a great impact on public health is an urgent need. Usually, selecting antigens for vaccines has been based on proteins having immunogenic properties for patients suffering TB and having had promising results in mice and non‐human primates. Our approach has been based on a functional approach involving the pathogen–host interaction in the search for antigens to be included in designing an efficient, minimal, subunit‐based anti‐TB vaccine. This means that Mycobacterium tuberculosis has mainly been involved in studies and that lipoproteins represent an important kind of protein on the cell envelope which can also contribute towards this pathogen's virulence. This study has assessed the expression of four lipoproteins from M. tuberculosis H37Rv, that is, Rv1411c (LprG), Rv1911c (LppC), Rv2270 (LppN) and Rv3763 (LpqH), and the possible biological activity of peptides derived from these. Five peptides were found for these proteins which had high specific binding to both alveolar A549 epithelial cells and U937 monocyte‐derived macrophages which were able to significantly inhibit mycobacterial entry to these cells in vitro.     

Design,synthesis and evaluation of a novel series of inhibitors reversing P‐glycoprotein‐mediated multidrug resistance

Multidrug resistance (MDR) is still the main barrier to attaining effective results with chemotherapy. Discovery of new chemo‐reversal agents is needed to overcome MDR. Our study focused on a better way to obtain novel drugs with triazole rings that have an MDR reversal ability through click chemistry. Among 20 developed compounds, compound 19 had a minimal cytotoxic effect compared to tariquidar and verapamil (VRP) and showed a higher reversal activity than VRP through increased accumulation in K562/A02 cells. Compound 19 also played an important role in the P‐gp efflux function of intracellular Rh123 and doxorubicin (DOX) accumulation in K562/A02 cells. Moreover, compound 19 exhibited a long lifetime of approximately 24 hr. These results indicated that compound 19 is a potential lead compound for the design of new drugs to overcome cancer MDR.     

Design and synthesis of 9H‐fluorenone based 1,2,3‐triazole analogues as Mycobacterium tuberculosis InhA inhibitors

We prepared fifty various 9H‐fluorenone based 1,2,3‐triazole analogues varied with NH, –S–, and –SO₂– groups using click chemistry. The target compounds were characterized by routine analytical techniques, ¹H, ¹³CNMR, mass, elemental, single‐crystal XRD ( 8a ) and screened for in vitro antitubercular activity against Mycobacterium tuberculosis (MTB) H37Rv strain and two “wild” strains Spec. 210 and Spec. 192 and MIC₅₀ was determined. Further, the compounds were evaluated for MTB InhA inhibition study as well. The final analogues exhibited minimum inhibitory concentration (MIC) ranging from 52.35 to >295 μm . Among the –NH– analogues, one compound 5p (MIC 58.34 μm ), among –S– containing analogues four compounds 8e (MIC 66.94 μm ), 8f (MIC 74.20 μm ), 8g (MIC 57.55 μm ), and 8q (MIC 56.11 μm ), among –SO₂– containing compounds one compound 10p (MIC 52.35 μm ) showed less than MTB MIC 74.20 μm : Compound 4‐(((9H‐fluoren‐9‐yl)sulfonyl)methyl)‐1‐(3,4,5‐trimethoxyphenyl)‐1H‐1,2,3‐triazole ( 10p ) was found to be the most active compound with 73% InhA inhibition at 50 μm ; it inhibited MTB with MIC 52.35 μm . Further, 10f and 10p were docked to crystal structure of InhA to know binding interaction pattern. Most active compounds were found to be non‐cytotoxic against HEK 293 cell lines at 50 μm .     

In vitro and in silico evaluation of P‐glycoprotein inhibition through 99mTc‐methoxyisobutylisonitrile uptake

P‐glycoprotein (P‐gp) is a multidrug resistance (MDR) transporter with unknown structural details. This macromolecule is normally responsible for extruding xenobiotics from normal cells. Overexpression of P‐gp in tumor cells is a major obstacle in cancer chemotherapy. In this study, human 3D model of P‐gp was built by homology modeling based on mouse P‐gp crystallographic structure and stabilized through 1 ns molecular dynamics (MD) simulation. Stabilized human P‐gp structure was used for flexible docking of 80 drugs into the putative active site of P‐gp. Accordingly, digoxin, itraconazole, risperidone, ketoconazole, prazosin, verapamil, cyclosporine A, and ranitidine were selected for further in vitro assay. Subsequently, cell‐based P‐gp inhibition assay was performed on Caco‐2 cells while ^99mTc‐methoxyisobutylisonitrile (MIBI) was used as a P‐gp efflux substrate for calculating IC50 values. Results of the ^99mTc‐MIBI uptake in drug‐treated Caco‐2 cells were in agreement with the previously reported activities. This study for the first time described the relation between molecular dynamics and flexible docking with cellular experiments using ^99mTc‐MIBI radiotracer for evaluation of potencies of P‐gp inhibitors. Finally, results showed that our radiotracer–cell‐based assay is an accurate and fast screening tool for detecting P‐gp inhibitors and non‐inhibitors in drug development process.     

Evaluation of the inhibitory activity of (aza)isoindolinone‐type compounds: toward in vitro InhA action,Mycobacterium tuberculosis growth and mycolic acid biosynthesis

Inhibitors of the Mycobacterium tuberculosis enoyl‐ACP reductase (InhA) are considered as potential promising therapeutics for the treatment of tuberculosis. Previously, we reported that azaisoindolinone‐type compounds displayed, in vitro, inhibitory activity toward InhA. Herein, we describe chemical modifications of azaisoindolinone scaffold, the synthesis of 15 new compounds and their evaluations toward the in vitro InhA activity. Based on these results, a structure–InhA inhibitory activity relationship analysis and a molecular docking study, using the conformation of InhA found in the 2H7M crystal structure, were carried out to predict a possible mode of interaction of the best (aza)isoindolinone‐type inhibitors with InhA in vitro. Then, the work was extended toward evaluations of these compounds against Mycobacterium tuberculosis (Mtb) growth, and finally, some of them were also investigated in respect of their ability to inhibit mycolic acid biosynthesis inside mycobacteria. Although, some azaisoindolinones were able to inhibit InhA activity and Mtb growth in vitro, they did not inhibit the mycolic acid biosynthesis inside Mtb.     

Design,Synthesis, and Biological Evaluation of 1,4‐dihydropyridine Derivatives as Potent Antitubercular Agents

A series of novel 1,4‐dihydropyridine‐3,5‐dicarbamoyl derivatives bearing an imidazole nucleus at C‐4 position were synthesized in excellent yields via multicomponent Hantzsch reaction. The newly synthesized compounds were characterized by IR, ¹H NMR, ¹³C NMR, and mass spectroscopy. The synthesized compounds 3a‐p were screened for antitubercular activity. Among all the screened compounds, compounds 3j and 3m showed most prominent activity against Mycobacterium tuberculosis with minimum inhibitory concentration of 0.02 μg/mL and SI > 500, making it more potent than first‐line antitubercular drug isoniazid. In addition, these compounds displayed relatively low cytotoxicity.     

Design,synthesis, and in vitro antituberculosis activity of benzo[6,7]cyclohepta[1,2‐b]pyridine‐1,3,4‐oxadiazole derivatives

A new antitubercular agents, benzo[6,7]cyclohepta[1,2‐b]pyridine‐1,3,4‐ oxadiazole hybrids ( 6a–o ), have been designed and synthesized involving oxidative cyclization of hydrazones by use of di(acetoxy)iodobenzene, characterized by IR,¹H NMR,¹³C NMR, and HRMS, and further confirmed by X‐ray analysis. All the newly synthesized compounds 4a–o evaluated for their in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv (ATCC27294). Among the compounds tested, the compounds 4o (MIC: 1.56  μg/ml) and 4l, 4m (MIC: 3.125  μg/ml) are promising lead analogues and have shown lower cytotoxicity.     

In vitro experimental system for evaluating inhibitory effect of investigational drugs on P‐glycoprotein‐mediated transcellular transport of tacrolimus (FK506)

In this study, an in vitro experimental system for evaluating the inhibitory effect of investigational drugs on the P‐glycoprotein (P‐gp, MDR1)‐mediated transport of tacrolimus (FK506) was developed using LLC‐PK1‐MDR1 and LLC‐PK1 wild‐type (control) cells. The amount of tacrolimus (concentrations: 1 and 5 μm ) transported into P‐gp‐expressing and control cells increased with time in both the apical‐to‐basal and basal‐to‐apical directions at incubation times ranging from 40 min to 2 h. The corrected apparent permeability (P_app) ratio, obtained by dividing the P_app ratio in P‐gp‐expressing cells by that in the control cells, ranged from 2.6 to 5.3, showing significant differences in the transport of tacrolimus between the P‐gp‐expressing cells and the control cells. This system was then subsequently used to examine the P‐gp transport of tacrolimus in the presence of verapamil (30 μm ), a model inhibitor for P‐gp‐mediated transport activity. The corrected P_app ratios in the absence and presence of verapamil were 6.9 and 0.8, respectively. Data derived in the present study suggest that our developed system has the ability to detect a sufficient difference in the P‐gp transport of tacrolimus between P‐gp‐expressing and control cells, and we therefore believe our system to be suitable for use in evaluating the inhibitory effects of investigational drugs on the P‐gp‐mediated transport of tacrolimus. Copyright © 2013 John Wiley & Sons, Ltd.     

Preparation and Evaluation of 99mTc‐labeled anti‐CD11b Antibody Targeting Inflammatory Microenvironment for Colon Cancer Imaging

CD11b, an active constituent of innate immune response highly expressed in myeloid‐derived suppressor cells (MDSCs), can be used as a marker of inflammatory microenvironment, particularly in tumor tissues. In this research, we aimed to fabricate a ^99mTc‐labeled anti‐CD11b antibody as a probe for CD11b⁺ myeloid cells in colon cancer imaging with single‐photon emission computed tomography (SPECT). In situ murine colon tumor model was established in histidine decarboxylase knockout (Hdc^?/?) mice by chemicals induction. ^99mTc‐labeled anti‐CD11b was obtained with labeling yields of over 30% and radiochemical purity of over 95%. Micro‐SPECT/CT scans were performed at 6 h post injection to investigate biodistributions and targeting of the probe. In situ colonic neoplasma as small as 3 mm diameters was clearly identified by imaging; after dissection of the animal, anti‐CD11b immunofluorescence staining was performed to identify infiltration of CD11b+ MDSCs in microenvironment of colonic neoplasms. In addition, the images displayed intense signal from bone marrow and spleen, which indicated the origin and migration of CD11b⁺ MDSCs in vivo, and these results were further proved by flow cytometry analysis. Therefore, ^99mTc‐labeled anti‐CD11b SPECT displayed the potential to facilitate the diagnosis of colon tumor in very early stage via detection of inflammatory microenvironment.     

Characterization of cytochrome P450 enzymes and P‐glycoprotein involved in the metabolism and transport of a new anti‐angiogenic agent KR‐31831

The in vitro metabolism and the transport of a novel anti‐angiogenic agent KR‐31831, (2R,3R,4S)‐6‐amino‐4‐[N‐(4‐chlorophenyl)‐N‐(1H‐imidazol‐2‐ylmethyl)amino]‐3‐hydroxy‐2‐dimethyoxymethyl‐3,4‐dihydro‐2‐methyl‐2H‐1‐benzopyran were investigated. Liquid chromatography‐mass spectrometry and tandem mass spectrometry were used for qualitative and quantitative analysis. The bidirectional transport studies of KR‐31831 using Caco‐2 cell monolayers showed the efflux to be significantly higher than influx (29.1 × 10^?6 compared to 11.5 × 10^?6 cm/s). P‐glycoprotein inhibitors significantly increased the influx of KR‐31831 and decreased the efflux of KR‐31831. These data indicate that KR‐313831 is a substrate for an efflux pump, P‐glycoprotein. The incubations of KR‐31831 with human liver microsomes produced three metabolites, M1, M2, and M3. M1 and M2 were identified as N‐(4‐chlorophenyl)‐N‐(1H‐imidazol‐2‐ylmethyl)amine and (2R,3R,4S)‐6‐amino‐4‐[N‐(4‐chlorophenyl)‐N‐(1H‐imidazol‐2‐ylmethyl)amino]‐3‐hydroxy‐2‐hydroxymethyl‐3,4‐dihydro‐2‐methyl‐2H‐1‐benzopyran by comparison with the authentic standards. M3 was tentatively characterized as hydroxy‐KR‐31831. CYP3A4 was identified as the major enzyme responsible for KR‐31831 metabolism to a major metabolite M1 using the combination of correlation analysis, immuno‐inhibition, chemical inhibition in human liver microsomes, and metabolism by cDNA expressed CYP enzymes. There is the possibility of drug–drug interactions when prescribing KR‐31831 concomitantly with known inhibitors or inducers of CYP3A4 and P‐glycoprotein. KR‐31831 was found to inhibit potently the metabolism of CYP2D6 substrate, suggesting that coadministration of KR‐31831 with CYP2D6 substrates may have significant effects on the pharmacokinetics of CYP2D6 substrates. Drug Dev. Res. 66:40–49. 2006. © 2006 Wiley‐Liss, Inc.     

In silico Target Fishing for the Potential Targets and Molecular Mechanisms of Baicalein as an Antiparkinsonian Agent: Discovery of the Protective Effects on NMDA Receptor‐Mediated Neurotoxicity

The flavonoid baicalein has been proven effective in animal models of parkinson's disease; however, the potential biological targets and molecular mechanisms underlying the antiparkinsonian action of baicalein have not been fully clarified. In the present study, the potential targets of baicalein were predicted by in silico target fishing approaches including database mining, molecular docking, structure‐based pharmacophore searching, and chemical similarity searching. A consensus scoring formula has been developed and validated to objectively rank the targets. The top two ranked targets catechol‐O‐methyltransferase (COMT) and monoamine oxidase B (MAO‐B) have been proposed as targets of baicalein by literatures. The third‐ranked one (N‐methyl‐d ‐aspartic acid receptor, NMDAR) with relatively low consensus score was further experimentally tested. Although our results suggested that baicalein significantly attenuated NMDA‐induced neurotoxicity including cell death, intracellular nitric oxide (NO) and reactive oxygen species (ROS) generation, extracellular NO reduction in human SH‐SY5Y neuroblastoma cells, baicalein exhibited no inhibitory effect on [³H]MK‐801 binding study, indicating that NMDAR might not be the target of baicalein. In conclusion, the results indicate that in silico target fishing is an effective method for drug target discovery, and the protective role of baicalein against NMDA‐induced neurotoxicity supports our previous research that baicalein possesses antiparkinsonian activity.