Piperazine-2-carboxylic acid derivatives as MTDLs anti-Alzheimer agents: Anticholinesterase activity, mechanistic aspect, and molecular modeling studies.

Development of Multitarget-Directed Ligands (MTDLs) is a promising approach to combat the complex etiologies of Alzheimer's disease (AD). Herein we report the design, synthesis, and characterization of a new series of 1,4-bisbenzylpiperazine-2-carboxylic acid derivatives 3-5(a-g), 7a-f, 8a-s, and their piperazine-2-yl-1,3,4-oxadiazole analogs 6a-g. In vitro inhibitory effect against Electrophorus electricus acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) from Equine serum was evaluated using modified Ellman's method, considering donepezil and tacrine as reference drugs. Lineweaver-Burk plot analysis of the results proved competitive inhibition of AChE and BChE with Ki values, in low micromolar range. The free carboxylic acid series 4a-g showed enhanced selectivity for AChE. Hence, 4c, 1,4-bis (4-chlorobenzyl)-piperazinyl-2-carboxylic acid), was the most active member of this series (Ki (AChE) = 10.18 ± 1.00 µM) with clear selectivity for AChE (SI âˆ¼ 17.90). However, the hydroxamic acids 7a-f and carboxamides 8a-s congeners were more potent and selective inhibitors of BChE (SI âˆ¼ 5.38 - 21862.5). Extraordinarily, 1,4-bis (2-chlorobenzyl)-piperazinyl-2-hydroxamic acid 7b showed promising inhibitory activity against BChE enzyme (Ki = 1.6 ± 0.08 nM, SI = 21862.5), that was significantly superior to that elicited by donepezil (Ki = 12.5 ± 2.6 µM) and tacrine (Ki = 17.3 ± 2.3 nM). Cytotoxicity assessment of 4c and 7b, on human neuroblastoma (SH-SY5Y) cell lines, revealed lower toxicity than staurosporine and was nearly comparable to that of donepezil. Molecular docking and molecular dynamics simulation afforded unblemished insights into the structure-activity relationships for AChE and BChE inhibition. The results showed stable binding with fair H-bonding, hydrophobic and/or ionic interactions to the catalytic and peripheral anionic sites of the enzymes. In silico predicted ADME and physicochemical properties of conjugates showed good CNS bioavailability and safety parameters. In this regard, compound (7b) might be considered as a promising inhibitor of BChE with an innovative donepezil-based anti-Alzheimer activity. Further assessments of the most potent AChE and BChE inhibitors as potential MTDLs anti-Alzheimer's agents are under investigation with our research group and will be published later.

N4-Substituted Piperazinyl Norfloxacin Derivatives with Broad-Spectrum Activity and Multiple Mechanisms on Gyrase, Topoisomerase IV, and Bacterial Cell Wall Synthesis.

Fluoroquinolones are an important class of antibiotics with broad-spectrum antibacterial and antitubercular activity. Here, we describe the design and synthesis of a series of 38 N4-substituted piperazinyl norfloxacin derivatives. Their activity and mechanism of action were characterized using in silico, in vitro, and in vivo approaches. Several compounds displayed interesting activities against both Gram-negative and Gram-positive bacteria, and few displayed antimycobacterial activity, whereby some were as potent as norfloxacin and ciprofloxacin. Molecular docking experiments suggested that the new derivatives inhibit both DNA gyrase and DNA topoisomerase IV in a similar manner as norfloxacin. Selecting the most promising candidates for experimental mode of action analysis, we confirmed DNA gyrase and topoisomerase IV as targets of all tested compounds using enzymatic in vitro assays. Phenotypic analysis of both Escherichia coli and Bacillus subtilis confirmed a typical gyrase inhibition phenotype for all of the tested compounds. Assessment of possible additional targets revealed three compounds with unique effects on the B. subtilis cell wall synthesis machinery, suggesting that they may have an additional target in this pathway. Comparison with known cell wall synthesis inhibitors showed that the new compounds elicit a distinct and, so far, unique phenotype, suggesting that they act differently from known cell wall synthesis inhibitors. Interestingly, our phenotypic analysis revealed that both norfloxacin and ciprofloxacin displayed additional cellular effects as well, which may be indicative of the so far unknown additional mechanisms of fluoroquinolones.

Rational design, synthesis, molecular modeling, biological activity, and mechanism of action of polypharmacological norfloxacin hydroxamic acid derivatives.

Fluoroquinolones are broad-spectrum antibiotics that target gyrase and topoisomerase IV, involved in DNA compaction and segregation. We synthesized 28 novel norfloxacin hydroxamic acid derivatives with additional metal-chelating and hydrophobic pharmacophores, designed to enable interactions with additional drug targets. Several compounds showed equal or better activity than norfloxacin against Gram-positive, Gram-negative, and mycobacteria, with MICs as low as 0.18 µM. The most interesting derivatives were selected for in silico, in vitro, and in vivo mode of action studies. Molecular docking, enzyme inhibition, and bacterial cytological profiling confirmed inhibition of gyrase and topoisomerase IV for all except two tested derivatives (10f and 11f). Further phenotypic analysis revealed polypharmacological effects on peptidoglycan synthesis for four derivatives (16a, 17a, 17b, 20b). Interestingly, compounds 17a, 17b, and 20b, showed never seen before effects on cell wall synthetic enzymes, including MreB, MurG, and PonA, suggesting a novel mechanism of action, possibly impairing the lipid II cycle.

Design, Synthesis, Molecular Modeling, Biological Activity, and Mechanism of Action of Novel Amino Acid Derivatives of Norfloxacin.

Two series of N4-substituted piperazinyl amino acid derivatives of norfloxacin (24 new compounds) were designed and synthesized to attain structural surrogates with additional binding sites and enhanced antibacterial activity. Synthesized derivatives showed increased antibacterial and antimycobacterial activity compared to their lead structure, norfloxacin. Molecular modeling studies supported the notion that the derivatives can establish additional bonds with the target enzymes gyrase and topoisomerase IV. In vitro enzyme inhibition assays confirmed that the tested compounds were significant inhibitors of these enzymes. Inhibition of gyrase and topoisomerase IV was then confirmed in living bacterial cells using bacterial cytological profiling of both Gram-negative Escherichia coli and Gram-positive Bacillus subtilis, revealing a typical topoisomerase inhibition phenotype characterized by severe nucleoid packing defects. Several derivatives exhibited additional effects on the Gram-positive cell wall synthesis machinery and/or the cytoplasmic membrane, which likely contributed to their increased antibacterial activity. While we could not identify specific cell wall or membrane targets, membrane depolarization was not observed. Our experiments further suggest that cell wall synthesis inhibition most likely occurs outside the membrane-bound lipid II cycle.

Hesperidin hexosomal loaded nanodispersion: insights of its antimycobacterial, cytotoxic and anti-HCoV effects.

Fruits of Citrus sinensis L. Osbeck var. Valencia contain hesperidin as a major flavanone glycoside. Hesperidin (H) was isolated from the peels of Valencia orange and formulated as hexosomal nanodispersions (F1) adopting the hot emulsification method. The antimycobacterial activity(anti-TB) was evaluated through a microplate Alamar blue (MABA) assay where F1 showed significant activity with MIC = 0.19 µM. To unravel the potential mechanism of the anti-TB, a molecular docking study of H using the Mycobacterial Dihydrofolate reductase (Mtb. DHFR) enzyme was performed. Hesperidin exhibited significant interactions with Mtb. DHFR active site. Sulforhodamine B assay was applied to evaluate cytotoxic activity against the lung cancer cell line (A-549). F1 showed a cytotoxic effect at IC50= 33 µM. It also has potent antiviral activity against Human Coronavirus 229E with IC50= 258.8 µM utilising crystal violet assay. Peels of Valencia orange could be a source of bioactive metabolites to control significant diseases.

UPLC-MS/MS and GC-MS based metabolites profiling of Moringa oleifera seed with its anti- Helicobacter pylori and anti-inflammatory activities.

Compared to its leaf, few studies have been reported on the seeds of Moringa oleifera Lam. Metabolites profiling analysis of M. oleifera seed methanolic extract (ME) and its fixed oil (MO) was attempted via LC/MS and GC/MS. LC/MS analysis of M. oleifera seeds annotated 84 peaks of which glucosinolates and their corresponding acetyl isomers were abundant. GC/MS of seed oil revealed the abundance of fatty acids with oleic acid at 34.3%. ME exhibited significant anti-Helicobacter pylori activity with MIC50 0.92 µg/mL, nearly one-half that of Clarithromycin. Fixed oil (MO) showed a nonselective anti-inflammatory effect with IC50= 24.4 ± 0.8 µg/mL correlated to Ibuprofen. To unravel the mechanism of the anti-H. pylori activity a molecular docking study of the principal components of the ME has been performed, using H. pylori urease enzyme. Interactions with Ni2+ ions and amino acid residue in the active site, which are crucial for the enzyme's biochemical role, are evidenced.

Synthesis, in vitro anticancer activity and in silico studies of certain isoxazole-based carboxamides, ureates, and hydrazones as potential inhibitors of VEGFR2.

The ensuing research presents the results of in vitro anticancer activity of novel 28 compounds of isoxazole-based carboxamides 3(a-d); ureates 4(a-g), 5, 6, 7a,b, 8; and hydrazones 9(a-f), 10(a-d), 11a,b as potential inhibitors of VEGFR2. The carboxamides and ureates were synthesized by converting 5-(aryl)-isoxzaole-3-carbohydrazides 1a,b to the corresponding carbonylazides 2a,b followed by treatment with the appropriate amines. The hydrazones were directly obtained through condensation of the carbohydrazide 1a,b with aldehydes and/or ketones. The structures of the target compounds were confirmed by elemental and spectral analyses. A preliminary in vitro anticancer screening of solutions (10-5M) on 60 cancer cell lines (NCI, USA) revealed that the carboxamide 3c is the most promising growth inhibitor. Explicitly, 3c showed potent anticancer activity at 10µ M against leukemia (HL-60(TB), K-562 and MOLT-4), colon cancer (KM12) and melanoma (LOX IMVI) cell lines with %GI range = 70.79-92.21. Evaluation of growth inhibitory activity of the synthesized compounds against hepatocellular carcinoma (HepG2), that overexpresses VEGFR2, showed superior activity of compounds 8, 10a and 10c with IC50 in sub micromolar concentrations of 0.84, 0.79 and 0.69 µM, respectively, which is better than that of the reference drug, Sorafenib (IC50 = 3.99 µM). Moreover, these compounds displayed high selective cytotoxicity for HepG2 cancer cells over the nontumorigenic THLE2 liver cells (SI range = 26.37-38.60) which reflect their safety. The results of VEGFR2 kinase inhibition assay demonstrate that, compounds 8 and 10a are the most active inhibitors with IC50 = 25.7 and 28.2 nM, respectively, (Sorafenib IC50 = 28.1 nM). Molecular docking of the synthesized derivatives to VEGFR2 (PDB: 3WZE) showed similar binding modes to that of the co-crystallized ligand, sorafenib. Moreover, the results of computational assessment of ADME and drug-likeness characteristics inspire further investigations of the new isoxazole-based derivatives to afford more potent, safe and orally active VEGFR2 inhibitors as potential anticancer drug candidates.

Synthesis, in vitro anticancer activity and in silico studies of certain pyrazole-based derivatives as potential inhibitors of cyclin dependent kinases (CDKs).

New diphenyl-1H-pyrazoles were synthesized and screened for CDK2 inhibition where 8d, 9b, 9c, and 9e exhibited promising activity (IC50 = 51.21, 41.36, 29.31, and 40.54 nM respectively) compared to R-Roscovitine (IC50 = 43.25 nM). Furthermore, preliminary anti-proliferative activity screening of some selected compounds on 60 cancer cell lines was performed at the (NCI/USA). Compounds 8a-c displayed promising growth inhibitory activity (mean %GI; 73.74, 94.32 and 74.19, respectively). Additionally, they were further selected by the NCI for five-dose assay, exhibiting pronounced activity against almost the full panel (GI50 ranges; 0.181-5.19, 1.07-4.12 and 1.07-4.82 µM, respectively) and (Full panel GI50 (MG-MID); 2.838, 2.306 and 2.770 µM, respectively). Screening the synthesized compounds 8a-c for inhibition of CDK isoforms revealed that compound 8a exhibited nearly equal inhibition to all the tested CDK isoforms, while compound 8b inhibits CDK4/D1 preferentially than the other isoforms and compound 8c inhibits CDK1, CDK2 and CDK4 more than CDK7. Flow cytometry cell cycle assay of 8a-c on Non-small cell lung carcinoma (NSCL HOP-92) cell line revealed S phase arrest by 8a and G1/S phase arrest by 8b and 8c. Apoptotic induction in HOP-92 cell line was also observed upon treatment with compounds 8a-c. Docking to CDK2 ATP binding site revealed similar interactions as the co-crystallized ligand R-Roscovitine (PDB code; 3ddq). These findings present compounds 8a-c as promising anti-proliferative agents.

Mechanistic selectivity investigation and 2D-QSAR study of some new antiproliferative pyrazoles and pyrazolopyridines as potential CDK2 inhibitors.

Novel series of diphenyl-1H-pyrazoles (4a-g) and pyrazolo[3,4-b]pyridines (5a-g and 7a-i) were synthesized and evaluated for their antiproliferative activity against breast cancer cell line (MCF7) and Hepatocellular carcinoma cell line (HepG2). The highest MCF7 growth inhibition activity was attained via compounds 4f and 7e (IC50 = 1.29 and 0.93 µM, respectively), while compounds 5b and 7f were the most active ones against HepG2 (IC50 = 1.57 and 1.33 µM, respectively) compared to doxorubicin (IC50 = 1.88 and 7.30 µM, respectively). Cell cycle analysis showed arrest at S and G2-M phases in MCF7 cells treated with 4f and 7e, and at G2-M and G1/S phases in HepG2 cells treated with 5b and 7f, respectively. Apoptotic effect of compounds 4f, 5b, 7e, and 7f was indicated via their pre-G1 early and late apoptotic effects and augmented levels of caspase-9/MCF7 and caspase-3/HepG2. A worthy safety profile was assessed for compounds 4f and 7e on MCF10A and compounds 5b and 7f on THLE2 treated normal cells. Furthermore, compounds 4f, 5b and 7f displayed a promising selective profile for CDK2 inhibition vs. CDK1, CDK4, and CDK7 isoforms as proved from their selectivity index. Docking in CDK2 ATP binding site, co-crystallized with R-Roscovitine, demonstrated analogous interactions and comparable binding energy with the native ligand. 2D QSAR sighted the possible structural features governing the CDK2 inhibition activity elicited by the studied pyrazolo[3,4-b]pyridines. These findings present compounds 4f, 5b, and 7f as selective CDK2 inhibitors with promising antiproliferative activity against MCF7 and HepG2 cancer cells.

Anti-hepatitis-C virus activity and QSAR study of certain thiazolidinone and thiazolotriazine derivatives as potential NS5B polymerase inhibitors.

The present study reports on evaluation of anti-HCV activity and QSAR of certain arylidenethiazolidinone derivatives as potential inhibitors of HCV-NS5B polymerase. The pursued compounds involving, 5-aryliden-3-arylacetamidothiazolidin-2,4-diones 4-6(a-f), 5-arylidine-2-(N-arylacetamido)-iminothiazolidin-4-one (10) and their rigid counterparts 5-arylidinethiazolotriazines 13-15(a-f), were synthesized and their structures confirmed by spectral and elemental analyses. The results of NS5B polymerase inhibition assay revealed compound 4e, as the most active inhibitor (IC50 = 0.035 µM), which is four folds greater than that of the reference agent, VCH-759, (IC50 = 0.14 µM). Meanwhile, compounds 4b, 4c, 5a, and 5c, and 13b, 14e and 15c displayed equipotency to 2 folds higher activity than VCH-759 (IC50 values: 0.085, 0.14, 0.14, 0.10, 0.12, 0.09 and 0.07 µM, respectively). Assessment of the anti-HCV activity (GT1a) using human hepatoma cell line (Huh-7.5) illustrates superior activity of 4e (EC50 = 3.80 µM) relative to VCH-759 (EC50 = 5.29 µM). Cytotoxicity evaluation on, Transformed normal cell lines (Human Liver Epithelial-2, THLE-2 and Proximal Tubular Epithelial, RPTEC/TERT1), demonstrate enhanced safety profile of 4e (CC50 = 102.77, 161.37 µM, respectively) compared to VCH-759 (CC50 = 61.83, 81.28 µM, respectively). Molecular docking of the synthesized derivatives to NS5B polymerase allosteric site (PDB: 2HWH) showed similar binding modes to that of the co-crystallized ligand. Moreover, QSAR models were established for the studied thiazolidinones and thiazolotriazines to investigate the molecular characteristics contributing to the observed NS5B polymerase inhibition activity. The obtained results inspire further investigations of thiazolidinones and thiazolotriazine aiming at affording more potent, safe and orally active non-nucleoside NS5B polymerase inhibitors as anti-HCV drug candidates.

Ambrosin, a potent NF-κß inhibitor, ameliorates lipopolysaccharide induced memory impairment, comparison to curcumin.

Despite its poor bioavailability, curcumin is a promising natural polyphenol targeting NF-κß. NF-κß is a target for new therapeutics because it plays a pivotal role in the pathophysiology of Alzheimer disease (AD). In contrast, ambrsoin, a sesquiterpene lactone which is a potent NF-κß inhibitor, is scarcely studied in AD models. The current work aims to assess the efficacy of ambrosin as a possible remedy for AD. In silico studies showed that bioavailability and BBB permeability could be favorable for ambrosin over curcumin. Memory impairment was induced in mice by single intraperitoneal injection of LPS (0.4 mg/kg). Treated groups received curcumin (100 mg/kg) or ambrosin at doses (5 or 10 mg/kg) for 7 days. Mice in treated groups showed a significant improvement in memory functions during Morris water maze and object recognition tests. Curcumin and ambrosin (10 mg/kg) inhibited the upsurge of NF-κßp65 transcript and protein levels. Consequently, downstream pro-inflammatory and nitrosative mediators were inhibited, namely, TNF-α, IL-1ß, COX-2 and iNOS. BACE1 was inhibited, thereby reducing amyloid plaques (Aß) deposition and eventually reducing inflammation and apoptosis of neurons as revealed by immunohistopathological examination. In conclusion, ambrosin can be repurposed as AD remedy after further pharmacokinetic/pharamacodynamic assessments. It could serve as an additional lead drug for AD therapeutics.

Synthesis, antimicrobial activity and molecular modeling study of 3-(5-amino-(2H)-1,2,4-triazol-3-yl]-naphthyridinones as potential DNA-gyrase inhibitors.

Four series of triazolylnaphthyridinone derivatives were synthesized as structural surrogates of nalidixic acid. The targeted derivatives involve: 3-(5-acylamino-2H-1,2,4-triazol-3-yl)-naphtyridin-4-ones 6(a-e); 3-(5-benzylidineamino-2H-1,2,4-triazol-3-yl)-naphthyridin-4-ones 8(a-g) and their 6-bromonaphthyridin-4-one analogs 7(a-e); 9(a-g). The synthesized compounds were evaluated In vitro for their antimicrobial activity against selected resistant strains of G+ve, G-ve, and Mycobacterium phlei. The results revealed remarkable selectivity, of the tested compounds, against Bacillus subtilis and Aggregatibacter actinomycetemcomitans, which are resistant to nalidixic acid. The growth inhibition zones were ranging from 20 to 40 mm at 10 mg/ml and the respective MIC-values ∼3.68-6.3 µM. The results illustrate that the 6-bromo derivatives 7(a-e) and 9(a-g) were more potent than the non-brominated counterparts 6(a-e) and 8(a-e) respectively. Inhibition of E. coli DNA-gyrase supercoiling activity is also evaluated. The 5-(4-methoxybanzamido)-triazolyl-6-bromonaphthyridinone (7e) exhibits IC50 = 1.94 µg/ml, which is comparable to that of nalidixic acid (IC50: 1.74 µg/ml). In addition, the most prominent IC50-values are displayed by: (7a;IC50: 2.77 µg/ml); (8g; IC50: 3.78 µg/ml); and (9d;IC50: 3.21 µg/ml). Molecular docking to the active site of DNA-gyrase cleavage complex of Acinetobacter baumannii (PDB code: 2xkk) co-crystallized with moxifloxacin revealed similar binding modes in addition to new interactions. Assessment of drug-likeness characteristics illustrate that the synthesized compounds showed agreement to Lipinski's and Veper's parameters. The study could offer an exceptional framework that may lead to the discovery of new potent antimicrobial agents.

Biotransformation studies of prednisone using human intestinal bacteria Part II: Anaerobic incubation and docking studies.

Anaerobic incubation of prednisone 1 with human intestinal bacteria (HIB) afforded nine metabolites: 5beta-androst-1-ene-3,11,17-trione 3, 3alpha-hydroxy-5alpha-androstane-11,17-dione 4, 3beta,17alpha,20-trihydroxy-5alpha-pregnan-11-one 5, 3alpha,17alpha-dihydroxy-5alpha-pregnane-11,20-dione 6, 3alpha,17alpha-dihydroxy-5beta-pregnane-11,20-dione 7, 3beta,17beta-dihydroxy-5alpha-androstan-11-one 8beta, 3beta,17alpha-dihydroxy-5alpha-androstan-11-one 8alpha, 3alpha,17beta-dihydroxy-5alpha-androstan-11-one 9beta, and 3alpha,17alpha-dihydroxy-5alpha-androstan-11-one 9alpha. The structures of these metabolites (3-9) were elucidated using several spectroscopic techniques. Computer-aided prediction of potential biological activities of the isolated prednisone metabolites (3-9) revealed potential inhibition of prostaglandin E2 9-ketoreductase (PGE2 9-KR). Docking studies applied to PGE2 9-KR allowed recommendation of the metabolites 4, 8beta, and 8alpha for further pharmacological study as PGE2 9-KR inhibitors.