Synthesis and Biological Evaluation of N‐Substituted Sophocarpinic Acid Derivatives as Coxsackievirus B3 Inhibitors

A series of novel N‐substituted sophocarpinic acid derivatives was designed, synthesized, and evaluated for their anti‐enteroviral activities against coxsackievirus type B3 (CVB3) and coxsackievirus type B6 (CVB6) in Vero cells. Structure–activity relationship analysis revealed that the introduction of a benzenesulfonyl moiety on the 12‐nitrogen atom in (E)‐β,γ‐sophocarpinic acid might significantly enhance anti‐CVB3 activity. Among the derivatives, (E)‐12‐N‐(m‐cyanobenzenesulfonyl)‐β,γ‐sophocarpinic acid ( 11 m ), possessing a meta‐cyanobenzenesulfonyl group, exhibited potent activity against CVB3 with a selectivity index (SI) of 107. Furthermore, compound 11 m also showed a good oral pharmacokinetic profile, with an AUC value of 7.29 μM h^?1 in rats, and good safety through the oral route in mice, with an LD₅₀ value of >1000 mg kg^?1; these values suggest a druggable characteristic. Therefore, compound 11 m was selected for further investigation as a promising CVB3 inhibitor. We consider (E)‐β,γ‐N‐(benzenesulfonyl)sophocarpinic acids to be a novel class of anti‐CVB3 agents.     

Synthesis of Albicidin Derivatives: Assessing the Role of N‐terminal Acylation on the Antibacterial Activity

The peptide antibiotic albicidin, which is synthesized by the plant pathogenic bacterium, Xanthomonas albilineans, represents the most prominent member of a new class of antibacterial gyrase inhibitors. It shows remarkable antibacterial activities against Gram‐positive and Gram‐negative microorganisms. Its unique structure potentially represents a new lead structure for the development of an antibacterial drug. Here we report the synthesis of 14 albicidin derivatives with structural variations at the N‐terminus, primarily investigating the effects of variation of cinnamoyl, phenylpropanoyl, and benzoyl residues. Gyrase inhibition in vitro and determination of minimal inhibitory concentrations were assessed in parallel. Activities in a nanomolar range and the importance of N‐acylation were demonstrated.     

Dual Escherichia coli DNA Gyrase A and B Inhibitors with Antibacterial Activity

The emergence of multidrug-resistant bacteria is a global health threat necessitating the discovery of new antibacterials and novel strategies for fighting bacterial infections. We report first-in-class DNA gyrase B (GyrB) inhibitor/ciprofloxacin hybrids that display antibacterial activity against Escherichia coli. Whereas DNA gyrase ATPase inhibition experiments, DNA gyrase supercoiling assays, and in vitro antibacterial assays suggest binding of the hybrids to the E. coli GyrA and GyrB subunits, an interaction with the GyrA fluoroquinolone-binding site seems to be solely responsible for their antibacterial activity. Our results provide a foundation for a new concept of facilitating entry of nonpermeating GyrB inhibitors into bacteria by conjugation with ciprofloxacin, a highly permeable GyrA inhibitor. A hybrid molecule containing GyrA and GyrB inhibitor parts entering the bacterial cell would then elicit a strong antibacterial effect by inhibition of both the GyrA and GyrB subunits of DNA gyrase and potentially slow bacterial resistance development.     

Synthesis of Bicyclic N‐Arylmethyl‐Substituted Iminoribitol Derivatives as Selective Nucleoside Hydrolase Inhibitors

A series of bicyclic N ‐arylmethyl‐substituted iminoribitols were synthesised and evaluated in vitro against T. vivax nucleoside hydrolase. The importance of the N–Asp40 interaction was confirmed and depends on an optimal pK_a value, which can be influenced by substituents. The compounds were active inhibitors of nucleoside hydrolase (IAG‐NH) and are inactive against human purine nucleoside phosphorylase.

     

Design,Synthesis and Evaluation of Triazole‐Pyrimidine Analogues as SecA Inhibitors

SecA, a key component of the bacterial Sec‐dependent secretion pathway, is an attractive target for the development of new antimicrobial agents. Through a combination of virtual screening and experimental exploration of the surrounding chemical space, we identified a hit bistriazole SecA inhibitor, SCA‐21, and studied a series of analogues by systematic dissections of the core scaffold. Evaluation of these analogues allowed us to establish an initial structure–activity relationship in SecA inhibition. The best compounds in this group are potent inhibitors of SecA‐dependent protein‐conducting channel activity and protein translocation activity at low‐ to sub‐micromolar concentrations. They also have minimal inhibitory concentration (MIC) values against various strains of bacteria that correlate well with the SecA and protein translocation inhibition data. These compounds are effective against methicillin‐resistant Staphylococcus aureus strains with various levels of efflux pump activity, indicating the capacity of SecA inhibitors to null the effect of multidrug resistance. Results from studies of drug‐affinity‐responsive target stability and protein pull‐down assays are consistent with SecA as a target for these compounds.     

Synthesis of N‐Hydroxycinnamides Capped with a Naturally Occurring Moiety as Inhibitors of Histone Deacetylase

Histone deacetylase (HDAC) inhibitors are regarded as promising therapeutics for the treatment of cancer. All reported HDAC inhibitors contain three pharmacophoric features: a zinc‐chelating group, a hydrophobic linker, and a hydrophobic cap for surface recognition. In this study we investigated the effectiveness of osthole, a hydrophobic Chinese herbal compound, as the surface recognition cap in hydroxamate‐based compounds as inhibitors of HDAC. Nine novel osthole‐based N‐hydroxycinnamides were synthesized and screened for enzyme inhibition activity. Compounds 9 d , 9 e , 9 g exhibited inhibitory activities (IC₅₀=24.5, 20.0, 19.6 nM ) against nuclear HDACs in HeLa cells comparable to that of suberoylanilide hydroxamic acid (SAHA; IC₅₀=24.5 nM ), a potent inhibitor clinically used for the treatment of cutaneous T‐cell lymphoma (CTCL). While compounds 9 d and 9 e showed SAHA‐like activity towards HDAC1 and HDAC6, compound 9 g was more selective for HDAC1. Compound 9 d exhibited the best cellular effect, which was comparable to that of SAHA, of enhancing acetylation of either α‐tubulin or histone H3. Molecular docking analysis showed that the osthole moiety of compound 9 d may interact with the same hydrophobic surface pocket exploited by SAHA and it may be modified to provide class‐specific selectivity. These results suggest that osthole is an effective hydrophobic cap when incorporated into N‐hydroxycinnamide‐derived HDAC inhibitors.     

Design,Synthesis, and Biological Evaluation of Pyrazoline‐Based Hydroxamic Acid Derivatives as Aminopeptidase N (APN) Inhibitors

Aminopeptidase N (APN) has been recognized as a target for anticancer treatment due to its overexpression on diverse malignant tumor cells and association with cancer invasion, metastasis and angiogenesis. Herein we describe the synthesis, biological evaluation, and structure–activity relationship study of two new series of pyrazoline analogues as APN inhibitors. Among these compounds, 5‐(2‐(2‐(hydroxyamino)‐2‐oxoethoxy)phenyl)‐3‐phenyl‐4,5‐dihydro‐1H‐pyrazole‐1‐carboxamide (compound 13 e ) showed the best APN inhibition with an IC₅₀ value of 0.16±0.02 μm , which is more than one order of magnitude lower than that of bestatin (IC₅₀=9.4±0.5 μm ). Moreover, compound 13 e was found to inhibit the proliferation of diverse carcinoma cells and to show potent anti‐angiogenesis activity. At the same concentration, compound 13 e presents significantly higher anti‐angiogenesis activity than bestatin in human umbilical vein endothelial cells (HUVECs) capillary tube formation assays. The putative binding mode of 13 e in the active site of APN is also discussed.     

Gyrase ATPase Domain as an Antitubercular Drug Discovery Platform: Structure‐Based Design and Lead Optimization of Nitrothiazolyl Carboxamide Analogues

In this study, we explored the pharmaceutically underexploited mycobacterial gyrase ATPase (GyrB) domain as a template for a structure‐based virtual screening of our in‐house (BITS Pilani) compound collection to discover new inhibitors targeting Mycobacterium tuberculosis (M.tb.) The hit identified was further customized by using a combination of molecular docking and medicinal chemistry strategies to obtain an optimized analogue displaying considerable in vitro enzyme efficacy and bactericidal properties against the M.tb. H₃₇Rv strain. The binding affinity of the ligand toward the GyrB domain was reascertained by differential scanning fluorimetry experiments. Further evaluation of the hERG toxicity (a major limitation among the previously reported N‐linked aminopiperidine analogues) indicated these molecules to be completely devoid of cardiotoxicity, a significant achievement within this class.     

Synthesis and Biological Evaluation of Papain‐Family Cathepsin L‐Like Cysteine Protease Inhibitors Containing a 1,4‐Benzodiazepine Scaffold as Antiprotozoal Agents

Novel papain‐family cathepsin L‐like cysteine protease inhibitors endowed with antitrypanosomal and antimalarial activity were developed, through an optimization study of previously developed inhibitors. In the present work, we studied the structure–activity relationships of these derivatives, with the aim to develop new analogues with a simplified and more synthetically accessible structure and with improved antiparasitic activity. The structure of the model compounds was significantly simplified by modifying or even eliminating the side chain appended at the C3 atom of the benzodiazepine scaffold. In addition, a simple methylene spacer of appropriate length was inserted between the benzodiazepine ring and the 3‐bromoisoxazoline moiety. Several rhodesain and falcipain‐2 inhibitors displaying single‐digit micromolar or sub‐micromolar antiparasitic activity against one or both parasites were identified, with activities that were one order of magnitude more potent than the model compounds.     

Multitarget‐Directed Tricyclic Pyridazinones as G Protein‐Coupled Receptor Ligands and Cholinesterase Inhibitors

By following a multitarget ligand design approach, a library of 47 compounds was prepared, and they were tested as binders of selected G protein‐coupled receptors (GPCRs) and inhibitors of acetyl and/or butyryl cholinesterase. The newly designed ligands feature pyridazinone‐based tricyclic scaffolds connected through alkyl chains of variable length to proper amine moieties (e.g., substituted piperazines or piperidines) for GPCR and cholinesterase (ChE) molecular recognition. The compounds were tested at three different GPCRs, namely serotoninergic 5‐HT_1A, adrenergic α_1A, and dopaminergic D₂ receptors. Our main goal was the discovery of compounds that exhibit, in addition to ChE inhibition, antagonist activity at 5‐HT_1A because of its involvement in neuronal deficits typical of Alzheimer’s and other neurodegenerative diseases. Ligands with nanomolar affinity for the tested GPCRs were discovered, but most of them behaved as dual antagonists of α_1A and 5‐HT_1A receptors. Nevertheless, several compounds displaying this GPCR affinity profile also showed moderate to good inhibition of AChE and BChE, thus deserving further investigations to exploit the therapeutic potential of such unusual biological profiles.     

Design,Synthesis, and Biological Evaluation of Enantiomeric β‐N‐Acetylhexosaminidase Inhibitors LABNAc and DABNAc as Potential Agents against Tay‐Sachs and Sandhoff Disease

Combating glycolipid storage disorders : LABNAc was prepared in an efficient 11‐step procedure from D ‐lyxonolactone. The enantiomer DABNAc was also prepared from L ‐lyxonolactone. Preliminary cellular studies indicate that these compounds may find utility as chemical chaperones for the treatment of Tay‐Sachs and Sandhoff diseases.

     

Synthesis and Biological Testing of N‐Aminoimidazole‐Based p38α MAP Kinase Inhibitors

The p38 mitogen‐activated protein (MAP) kinase α plays a central role in the regulation of cellular responses such as differentiation, proliferation, apoptosis, and inflammation. Inhibition of p38 results in decreased synthesis of pro‐inflammatory cytokines. To date, diverse p38α inhibitors are in phase II clinical trials for numerous cytokine‐dependent diseases. 2‐Sulfanylimidazole derivatives offer advantages over the prototype inhibitor SB 203580, including fewer cytochrome P450 interactions and better kinetic properties. The aim of this study was to develop novel 1,2,4,5‐tetrasubstituted pyridinylimidazoles with acyl residues at the imidazole N1 position that can interact with the kinase's hydrophobic region II (HR II) or sugar pocket (SP) to improve both selectivity and activity. The substitution pattern was optimized by variation of the acyl moiety at the N1 position of the N‐aminoimidazole core. Acylation of the amino function was used for optimization and led to potent p38α MAPK inhibitors.     

3‐Heterocycle‐Phenyl N‐Alkylcarbamates as FAAH Inhibitors: Design,Synthesis and 3D‐QSAR Studies

Carbamates are a well‐established class of fatty acid amide hydrolase (FAAH) inhibitors. Here we describe the synthesis of meta‐substituted phenolic N‐alkyl/aryl carbamates and their in vitro FAAH inhibitory activities. The most potent compound, 3‐(oxazol‐2yl)phenyl cyclohexylcarbamate ( 2 a ), inhibited FAAH with a sub‐nanomolar IC₅₀ value (IC₅₀=0.74 nM ). Additionally, we developed and validated three‐dimensional quantitative structure–activity relationships (QSAR) models of FAAH inhibition combining the newly disclosed carbamates with our previously published inhibitors to give a total set of 99 compounds. Prior to 3D‐QSAR modeling, the degree of correlation between FAAH inhibition and in silico reactivity was also established. Both 3D‐QSAR methods used, CoMSIA and GRID/GOLPE, produced statistically significant models with coefficient of correlation for external prediction (R²_PRED) values of 0.732 and 0.760, respectively. These models could be of high value in further FAAH inhibitor design.     

Discovery of Mono‐ and Disubstituted 1H‐Pyrazolo[3,4]pyrimidines and 9H‐Purines as Catalytic Inhibitors of Human DNA Topoisomerase IIα

Human DNA topoisomerase IIα (htIIα) is a validated target for the development of anticancer agents. Based on structural data regarding the binding mode of AMP‐PNP (5′‐adenylyl‐β,γ‐imidodiphosphate) to htIIα, we designed a two‐stage virtual screening campaign that combines structure‐based pharmacophores and molecular docking. In the first stage, we identified several monosubstituted 9H‐purine compounds and a novel class of 1H‐pyrazolo[3,4]pyrimidines as inhibitors of htIIα. In the second stage, disubstituted analogues with improved cellular activities were discovered. Compounds from both classes were shown to inhibit htIIα‐mediated DNA decatenation, and surface plasmon resonance (SPR) experiments confirmed binding of these two compounds on the htIIα ATPase domain. Proposed complexes and interaction patterns between both compounds and htIIα were further analyzed in molecular dynamics simulations. Two compounds identified in the second stage showed promising anticancer activities in hepatocellular carcinoma (HepG2) and breast cancer (MCF‐7) cell lines. The discovered compounds are suitable starting points for further hit‐to‐lead development in anticancer drug discovery.     

Design and Synthesis of 4‐(2,4,5‐Trifluorophenyl)butane‐1,3‐diamines as Dipeptidyl Peptidase IV Inhibitors

The worldwide prevalence of diabetes has spurred numerous studies on the development of new antidiabetic medicines. As a result, dipeptidyl peptidase IV (DPP4) has been recognized as a validated target. In our efforts to discover new DPP4 inhibitors, we analyzed the complexed structures of DPP4 available in Protein Data Bank and designed a series of triazole compounds. After enzyme activity assays and crystallographic verification of the binding interaction patterns, we found that the triazole compounds can inhibit DPP4 with micromolar IC₅₀ values. Liver microsome stability and cytochrome P450 metabolic tests were performed on this series, revealing undesirable pharmacokinetic profiles for the triazole compounds. To overcome this liability, we substituted the triazole ring with an amide or urea group to produce a new series of DPP4 inhibitors. Based on its enzyme activity, metabolic stability, and selectivity over DPP8 and DPP9, we selected compound 21 r for further study of its in vivo effects in mice using an oral glucose tolerance test (OGTT). The results show that 21 r has efficacy similar to that of sitagliptin at a dose of 3 mg kg^?1. The crystal structure of 21 r bound to DPP4 also reveals that the trifluoromethyl group is directed toward a subpocket different from the subsite bound by sitagliptin, providing clues for the design of new DPP4 inhibitors.     

Synthesis and Biological Evaluation of 4‐Sulfamoylphenyl/Sulfocoumarin Carboxamides as Selective Inhibitors of Carbonic Anhydrase Isoforms hCA II,IX, and XII

With the aim to develop potent and selective human carbonic anhydrase inhibitors (hCAIs), we synthesized 4‐sulfamoylphenyl/sulfocoumarin benzamides (series 5 a – r and series 7 a – q ) and evaluated their inhibition profiles against five isoforms of the zinc‐containing human carbonic anhydrase (hCA, EC 4.2.1.1): cytosolic hCA I and II, and the transmembrane isozymes hCA IV, IX, and XII. Compounds 5 a – r were found to selectively inhibit hCA II in the nanomolar range, while being less effective against the other hCA isoforms. As noted from the literature, sulfocoumarin (1,2‐benzoxathiine 2,2‐dioxide) acts as a “prodrug” inhibitor and is hydrolyzed by the esterase activity of hCA to form 2‐hydroxyphenylvinylsulfonic acid, which thereafter binds to the enzyme in a manner similar to that of coumarins and sulfoxocoumarins. All these sulfocoumarins (compounds 7 a – q ) were found to be very weak or ineffective as inhibitors of the housekeeping off‐target hCA isoforms I and II, and effectively inhibited the transmembrane tumor‐associated isoforms IX and XII in the high nanomolar to micromolar ranges. Further structural modifications of these molecules could be useful for the development of effective hCA inhibitors used for the treatment of glaucoma, epilepsy, and cancer.     

Rational Design,Synthesis, and Potency of N‐Substituted Indoles,Pyrroles, and Triarylpyrazoles as Potential Fructose 1,6‐Bisphosphatase Inhibitors

By using computer modeling and lead structures from our earlier SAR results, a broad variety of pyrrole‐, indole‐, and pyrazole‐based compounds were evaluated as potential fructose 1,6‐bisphosphatase (FBPase) inhibitors. The docking studies yielded promising structures, and several were selected for synthesis and FBPase inhibition assays: 1‐[4‐(trifluoromethyl)benzoyl]‐1H‐indole‐5‐carboxamide, 1‐(α‐naphthalen‐1‐ylsulfonyl)‐7‐nitro‐1H‐indole, 5‐(4‐carboxyphenyl)‐3‐phenyl‐1‐[3‐(trifluoromethyl)phenyl]‐1H‐pyrazole, 1‐(4‐carboxyphenylsulfonyl)‐1H‐pyrrole, and 1‐(4‐carbomethoxyphenylsulfonyl)‐1H‐pyrrole were synthesized and tested for inhibition of FBPase. The IC₅₀ values were determined to be 0.991 and 1.34 μM , and 575, 135, and 32 nM , respectively. The tested compounds were significantly more potent than the natural inhibitor AMP (4.0 μM ) by an order of magnitude; indeed, the best inhibitor showed an IC₅₀ value toward FBPase more than two orders of magnitude better than that of AMP. This level of activity is virtually the same as that of the best currently known FBPase inhibitors. This work shows that such indole derivatives are promising candidates for drug development in the treatment of type II diabetes.     

Direct Staudinger–Phosphonite Reaction Provides Methylphosphonamidates as Inhibitors of CE4 De‐N‐acetylases

De‐N‐acetylases of β‐(1→6)‐D ‐N‐acetylglucosamine polymers (PNAG) and β‐(1→4)‐D ‐N‐acetylglucosamine residues in peptidoglycan are attractive targets for antimicrobial agents. PNAG de‐N‐acetylases are necessary for biofilm formation in numerous pathogenic bacteria. Peptidoglycan de‐N‐acetylation facilitates bacterial evasion of innate immune defenses. To target these enzymes, transition‐state analogue inhibitors containing a methylphosphonamidate have been synthesized through a direct Staudinger–phosphonite reaction. The inhibitors were tested on purified PgaB, a PNAG de‐N‐acetylase from Escherichia coli, and PgdA, a peptidoglycan de‐N‐acetylase from Streptococcus pneumonia. Herein, we describe the most potent inhibitor of peptidoglycan de‐N‐acetylases reported to date (K_i=80 μM ). The minimal inhibition of PgaB observed provides insight into key structural and functional differences in these enzymes that will need to be considered during the development of future inhibitors.     

Synthesis and Biological Evaluation of 1‐Methyl‐1H‐indole–Pyrazoline Hybrids as Potential Tubulin Polymerization Inhibitors

A series of 1‐methyl‐1H‐indole–pyrazoline hybrids were designed, synthesized, and biologically evaluated as potential tubulin polymerization inhibitors. Among them, compound e19 [5‐(5‐bromo‐1‐methyl‐1H‐indol‐3‐yl)‐3‐(3,4,5‐trimethoxyphenyl)‐4,5‐dihydro‐1H‐pyrazole‐1‐carboxamide] showed the most potent inhibitory effect on tubulin assembly (IC₅₀=2.12 μm ) and in vitro growth inhibitory activity against a panel of four human cancer cell lines (IC₅₀ values of 0.21–0.31 μm ). Further studies confirmed that compound e19 can induce HeLa cell apoptosis, cause cell‐cycle arrest in G₂/M phase, and disrupt the cellular microtubule network. These studies, along with molecular docking and 3D‐QSAR modeling, provide an important basis for further optimization of compound e19 as a potential anticancer agent.     

Design and Synthesis of A‐Ring Simplified Pyripyropene A Analogues as Potent and Selective Synthetic SOAT2 Inhibitors

Currently, pyripyropene A, which is isolated from the culture broth of Aspergillus fumigatus FO‐1289, is the only compound known to strongly and selectively inhibit the isozyme sterol O‐acyltransferase 2 (SOAT2). To aid in the development of new cholesterol‐lowering or anti‐atherosclerotic agents, new A‐ring simplified pyripyropene A analogues have been designed and synthesized based on total synthesis, and the results of structure–activity relationship studies of pyripyropene A. Among the analogues, two A‐ring simplified pyripyropene A analogues exhibited equally efficient SOAT2 inhibitory activity to that of natural pyripyropene A. These new analogues are the most potent and selective SOAT2 inhibitors to be used as synthetic compounds and attractive seed compounds for the development of drug for dyslipidemia, including atherosclerotic disease and steatosis.