Biological Evaluation of Potent Triclosan‐Derived Inhibitors of the Enoyl–Acyl Carrier Protein Reductase InhA in Drug‐Sensitive and Drug‐Resistant Strains of Mycobacterium tuberculosis

New triclosan (TRC) analogues were evaluated for their activity against the enoyl–acyl carrier protein reductase InhA in Mycobacterium tuberculosis (Mtb). TRC is a well‐known inhibitor of InhA, and specific modifications to its positions 5 and 4′ afforded 27 derivatives; of these compounds, seven derivatives showed improved potency over that of TRC. These analogues were active against both drug‐susceptible and drug‐resistant Mtb strains. The most active compound in this series, 4‐(n‐butyl)‐1,2,3‐triazolyl TRC derivative 3 , had an MIC value of 0.6 μg mL^?1 (1.5 μM ) against wild‐type Mtb. At a concentration equal to its MIC, this compound inhibited purified InhA by 98 %, and showed an IC₅₀ value of 90 nM . Compound 3 and the 5‐methylisoxazole‐modified TRC 14 were able to inhibit the biosynthesis of mycolic acids. Furthermore, mc²4914, an Mtb strain overexpressing inhA, was found to be less susceptible to compounds 3 and 14 , supporting the notion that InhA is the likely molecular target of the TRC derivatives presented herein.     

Mycobacterial Phenolic Glycolipids with a Simplified Lipid Aglycone Modulate Cytokine Levels through Toll‐Like Receptor 2

Phenolic glycolipids (PGLs) are virulence factors present in the cell walls of many pathogenic mycobacteria. PGLs have been implicated in various aspects of mycobacterial disease, but there are limited structure–activity data available for these molecules. We report here the preparation of seven synthetic PGL analogues, differing from the native compounds in the replacement of the complex phenolic lipid moiety with a p‐methoxyphenyl group. The ability of these compounds to stimulate or inhibit the production of cytokines (TNF‐α, IL‐1β, IL‐6, MCP‐1) and nitric oxide (NO) was then evaluated by ELISA‐based assays. None of the compounds stimulated the production of these biological signalling molecules. In contrast, they each displayed concentration‐dependent inhibitory activity, related to the methylation pattern of the molecule and mediated by Toll‐like receptor 2. Additional studies revealed that native PGL‐I from Mycobacterium leprae and a synthetic PGL‐I analogue containing a simplified lipid domain had enhanced inhibitory activities relative to the corresponding analogues containing the p‐methoxyphenyl aglycone; however, the natural lipid phenolthiocerol was only weakly active. These studies reveal that synthetic molecules of this type can be used as probes for PGL function. Moreover, their ease of synthesis relative to the natural glycolipids, as well as their more favourable aqueous solubility, should allow for more thorough structure–activity relationship studies.     

Repurposing the Chemical Scaffold of the Anti‐Arthritic Drug Lobenzarit to Target Tryptophan Biosynthesis in Mycobacterium tuberculosis

The emergence of extensively drug‐resistant strains of Mycobacterium tuberculosis (Mtb) highlights the need for new therapeutics to treat tuberculosis. We are attempting to fast‐track a targeted approach to drug design by generating analogues of a validated hit from molecular library screening that shares its chemical scaffold with a current therapeutic, the anti‐arthritic drug Lobenzarit (LBZ). Our target, anthranilate phosphoribosyltransferase (AnPRT), is an enzyme from the tryptophan biosynthetic pathway in Mtb. A bifurcated hydrogen bond was found to be a key feature of the LBZ‐like chemical scaffold and critical for enzyme inhibition. We have determined crystal structures of compounds in complex with the enzyme that indicate that the bifurcated hydrogen bond assists in orientating compounds in the correct conformation to interact with key residues in the substrate‐binding tunnel of Mtb‐AnPRT. Characterising the inhibitory potency of the hit and its analogues in different ways proved useful, due to the multiple substrates and substrate binding sites of this enzyme. Binding in a site other than the catalytic site was found to be associated with partial inhibition. An analogue, 2‐(2‐5‐methylcarboxyphenylamino)‐3‐methylbenzoic acid, that bound at the catalytic site and caused complete, rather than partial, inhibition of enzyme activity was found. Therefore, we designed and synthesised an extended version of the scaffold on the basis of this observation. The resultant compound, 2,6‐bis‐(2‐carboxyphenylamino)benzoate, is a 40‐fold more potent inhibitor of the enzyme than the original hit and provides direction for further structure‐based drug design.     

Structure‐Guided Design of Thiazolidine Derivatives as Mycobacterium tuberculosis Pantothenate Synthetase Inhibitors

The pantothenate biosynthetic pathway is essential for the persistent growth and virulence of Mycobacterium tuberculosis (Mtb) and one of the enzymes in the pathway, pantothenate synthetase (PS, EC: 6.3.2.1), encoded by the panC gene, has become an appropriate target for new therapeutics to treat tuberculosis. Herein, we report nanomolar thiazolidine inhibitors of Mtb PS developed by a rational inhibitor design approach. The thiazolidine compounds were discovered by using energy‐based pharmacophore modelling and subsequent in vitro screening, which resulted in compounds with a half maximal inhibitory concentration (IC₅₀) value of (1.12±0.12) μM . These compounds were subsequently optimised by a combination of modelling and synthetic chemistry. Hit expansion of the lead by chemical synthesis led to an improved inhibitor with an IC₅₀ value of 350 nM and an Mtb minimum inhibitory concentration (MIC) of 1.55 μM . Some of these compounds also showed good activity against dormant Mtb cells.     

Identification of the Target Protein of Agelasine D,a Marine Sponge Diterpene Alkaloid,as an Anti‐dormant Mycobacterial Substance

One of the major reasons for the wide epidemicity of tuberculosis and for the necessity for extensive chemotherapeutic regimens is that the causative agent, Mycobacterium tuberculosis, has an ability to become dormant. Therefore, new lead compounds that are anti‐bacterial against M. tuberculosis in both active and dormant states are urgently needed. Marine sponge diterpene alkaloids, agelasines B, C, and D, from an Indonesian marine sponge of the genus Agelas were rediscovered as anti‐dormant‐mycobacterial substances. Based on the concept that the transformants over‐expressing targets of antimicrobial substances confer drug resistance, strains resistant to agelasine D were screened from Mycobacterium smegmatis transformed with a genomic DNA library of Mycobacterium bovis BCG. Sequence analysis of the cosmids isolated from resistant transformants revealed that the responsible gene was located in the genome region between 3475.051 and 3502.901 kb. Further analysis of the transformants over‐expressing the individual gene contained in this region indicated that BCG3185c (possibly a dioxygenase) might be a target of the molecule. Moreover, agelasine D was found to bind directly to recombinant BCG3185c protein (K_D 2.42 μm), based on surface plasmon resonance (SPR). This evidence strongly suggests that the BCG3185c protein is the major target of agelasine D, and that the latter is the anti‐mycobacterial substance against dormant bacilli.     

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.     

Optimization of the Antiviral Potency and Lipophilicity of Halogenated 2,6‐Diarylpyridinamines as a Novel Class of HIV‐1 NNRTIS

Nineteen new halogenated diarylpyridinamine (DAPA) analogues modified at the phenoxy C‐ring were synthesized and evaluated for anti‐HIV activity and certain drug‐like properties. Ten compounds showed high anti‐HIV activity (EC₅₀<10 nM ). In particular, (E)‐6‐(2′′‐bromo‐4′′‐cyanovinyl‐6′′‐methoxy)phenoxy‐N²‐(4′‐cyanophenyl)pyridin‐2,3‐diamine ( 8 c ) displayed low‐nanomolar antiviral potency (3–7 nM ) against wild‐type and drug‐resistant viral strains bearing the E138K or K101E mutations, which are associated with resistance to rilvipirine ( 1 b ). Compound 8 c exhibited much lower resistance fold changes (RFC: 1.1–2.1) than 1 b (RFC: 11.8–13.0). Compound 8 c also exhibited better metabolic stability (in vitro half‐life) than 1 b in human liver microsomes, possessed low lipophilicity (clog D: 3.29; measured log P: 3.31), and had desirable lipophilic efficiency indices (LE>0.3, LLE>5, LELP<10). With balanced potency and drug‐like properties, 8 c merits further development as an anti‐HIV drug candidate.     

α‐Bromoacrylamido N‐Substituted Isatin Derivatives as Potent Inducers of Apoptosis in Human Myeloid Leukemia Cells

A novel series of α‐bromoacryloyl N‐substituted isatin analogues were found to inhibit the growth and viability of human myeloid leukemia HL‐60 and U‐937 cells as well as human lymphoid leukemia MOLT‐3 cells. Cell death induced by these molecules was preceded by a rapid release of cytochrome c from mitochondria into the cytosol and subsequent caspase activation involving caspase‐3, to cleave poly(ADP‐ribose) polymerase (PARP). These findings suggest that these compounds present antiproliferative activity which may be mediated by apoptosis caused by cytochrome c release and caspase activation in human leukemia cells.     

Alanine and Lysine Scans of the LL‐37‐Derived Peptide Fragment KR‐12 Reveal Key Residues for Antimicrobial Activity

The human host defence peptide LL‐37 is a broad‐spectrum antibiotic with immunomodulatory functions. Residues 18–29 in LL‐37 have previously been identified as a minimal peptide (KR‐12) that retains antibacterial activity with decreased cytotoxicity. In this study, analogues of KR‐12 were generated by Ala and Lys scans to identify key elements for activity. These were tested against a panel of human pathogens and for membrane permeabilisation on liposomes. Replacements of hydrophobic and cationic residues with Ala were detrimental for antibiotic potency. Substitutions by Lys increased activity, as long as the increase in cationic density did not disrupt the amphiphilic disposition of the helical structure. Importantly, substitutions showed differential effects against different organisms. Replacement of Gln5 with Lys and Asp9 with Ala or Lys improved the broad‐spectrum activity most, each resulting in up to an eightfold increase in potency against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The improved analogues displayed no significant toxicity against human cells, and thus, KR‐12 is a tuneable template for antibiotic development.     

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.     

Design,Synthesis, and SAR of Naphthyl‐Substituted Diarylpyrimidines as Non‐Nucleoside Inhibitors of HIV‐1 Reverse Transcriptase

A series of 38 2‐naphthyl‐substituted diarylpyrimidine (DAPY) analogues, characterized by various substitution patterns on the pyrimidine and naphthalene rings, was synthesized in a straightforward fashion by means of parallel synthesis and evaluated as inhibitors of the HIV‐1 wild‐type and double mutant (K103N+Y181C) strains. Most of the compounds displayed strong activity against wild‐type HIV‐1. The most active compound, with a cyano group at position C6 on the naphthalene ring, exhibited activity against wild‐type HIV‐1 with an EC₅₀ value of 0.002 μM and against the double mutant strain with an EC₅₀ value of 0.24 μM ; the selectivity index (SI) against wild‐type is >180 000, the highest SI value among DAPY analogues. The structure–activity relationship (SAR) of the newly synthesized DAPYs is presented herein.     

N‐Cinnamoylation of Antimalarial Classics: Effects of Using Acyl Groups Other than Cinnamoyl toward Dual‐Stage Antimalarials

In a follow‐up study to our reports of N‐cinnamoylated chloroquine and quinacrine analogues as promising dual‐stage antimalarial leads with high in vitro potency against both blood‐stage Plasmodium falciparum and liver‐stage Plasmodium berghei, we decided to investigate the effect of replacing the cinnamoyl moiety with other acyl groups. Thus, a series of N‐acylated analogues were synthesized, and their activities against blood‐ and liver‐stage Plasmodium spp. were assessed along with their in vitro cytotoxicities. Although the new N‐acylated analogues were found to be somewhat less active and more cytotoxic than their N‐cinnamoylated counterparts, they equally displayed nanomolar activities in vitro against blood‐stage drug‐sensitive and drug‐resistant P. falciparum, and significant in vitro liver‐stage activity against P. berghei. Therefore, it is demonstrated that simple N‐acylated surrogates of classical antimalarial drugs are promising dual‐stage antimalarial leads.     

Non‐natural Acetogenin Analogues as Potent Trypanosoma brucei Inhibitors

Neglected tropical diseases remain a serious global health concern. Here, a series of novel bis‐tetrahydropyran 1,4‐triazole analogues based on the framework of chamuvarinin, a polyketide natural product isolated from the annonaceae plant species are detailed. The analogues synthesized display low micromolar trypanocidal activities towards both bloodstream and insect forms of Trypanosoma brucei, the causative agent of African sleeping sickness, also known as Human African Trypanosomiasis (HAT). A divergent synthetic strategy was adopted for the synthesis of the key tetrahydropyran intermediates to enable rapid access to diastereochemical variation either side of the 1,4‐triazole core. The resulting diastereomeric analogues displayed varying degrees of trypanocidal activity and selectivity in structure–activity relationship studies. Together, the biological potency and calculated lipophilicity values indicate that while there is room for improvement, these derivatives may represent a promising novel class of anti‐HAT agents.     

Synthesis and Biological Evaluation of Purine 2′‐Fluoro‐2′‐deoxyriboside ProTides as Anti‐influenza Virus Agents

2′‐Fluoro‐2′‐deoxyguanosine has been reported to have potent anti‐influenza virus activity in vitro and in vivo. Herein we describe the synthesis and biological evaluation of 6‐modified 2′‐fluoro‐2′‐deoxyguanosine analogues and their corresponding phosphoramidate ProTides as potential anti‐influenza virus agents. Whereas the parent nucleosides were devoid of antiviral activity in two different cellular assays, the 5′‐O‐naphthyl(methoxy‐L ‐alaninyl) ProTide derivatives of 6‐O‐methyl‐2′‐fluoro‐2′‐deoxyguanosine, 6‐O‐ethyl‐2′‐fluoro‐2′‐deoxyguanosine, and 2′‐deoxy‐2′‐fluoro‐6‐chloroguanosine, and the 5′‐O‐naphthyl(ethoxy‐L ‐alaninyl) ProTide of 6‐O‐ethyl‐2′‐fluoro‐2′‐deoxyguanosine displayed antiviral EC₉₉ values of ～12 μM . The antiviral results are supported by metabolism studies. Rapid conversion into the L ‐alaninyl metabolite and then 6‐modified 2′‐fluoro‐2′‐deoxyguanosine 5′‐monophosphate was observed in enzymatic assays with yeast carboxypeptidase Y or crude cell lysate. Evidence for efficient removal of the 6‐substituent on the guanine part was provided by enzymatic studies with adenosine deaminase, and by molecular modeling of the nucleoside 5′‐monophosphates in the catalytic site of a model of ADAL1, thus indicating the utility of the double prodrug concept.     

Isocyanides as Influenza A Virus Subtype H5N1 Wild‐Type M2 Channel Inhibitors

Basic bulky amines such as amantadine are well‐characterized M2 channel blockers, useful for treating influenza. Herein we report our surprising findings that charge‐neutral, bulky isocyanides exhibit activities similar to—or even higher than—that of amantadine. We also demonstrate that these isocyanides have potent growth inhibitory activity against the H5N1 virus. The ?NH₂ to ?N≡C group replacement within current anti‐influenza drugs was found to give compounds with high activities at low‐micromolar concentrations. For example, a tenfold improvement in potency was observed for 1‐isocyanoadamantane ( 27 ), with an EC₅₀ value of 0.487 μm against amantadine‐sensitive H5N1 virus as determined by both MTT and plaque‐reduction assays, without showing cytotoxicity. Furthermore, the isocyanide analogues synthesized in this study did not inhibit the V27A or S31N mutant M2 ion channels, according to electrophysiology experiments, and did not exhibit activity against amantadine‐resistant virus strains.     

Preliminary Evaluation of Artemisinin–Cholesterol Conjugates as Potential Drugs for the Treatment of Intractable Forms of Malaria and Tuberculosis

To evaluate the feasibility of developing drugs that may be active against both malaria and tuberculosis (TB) by using in part putative cholesterol transporters in the causative pathogens and through enhancement of passive diffusion in granulomatous TB, artemisinin–cholesterol conjugates were synthesized by connecting the component molecules through various linkers. The compounds were screened in vitro against Plasmodium falciparum (Pf) and Mycobacterium tuberculosis (Mtb). Antimalarial activities (IC₅₀) against Pf drug‐sensitive NF54, and drug‐resistant K1 and W2 strains ranged from 0.03–2.6, 0.03–1.9, and 0.02–1.7 μm . Although the compounds are less active than the precursor artemisinin derivatives, the cholesterol moiety renders the compounds relatively insoluble in the culture medium, and variation in solubilities among the different compounds may reflect in the range of efficacies observed. Activities against Mtb H37Rv were assessed using a standardized colony‐forming unit (CFU) assay after 24 h pretreatment of cultures with each of the compounds. Percentage inhibition ranged from 3–38 % and 18–52 % at 10 and 80 μm , respectively. Thus, in contrast to the comparator drug artemether, the conjugates display enhanced activities. The immediate aims include the preparation of conjugates with enhanced aqueous solubilities, assays against malaria and TB in vivo, and for TB, assays using an infected macrophage model and assessment of granuloma influx.     

Structure‐Guided Discovery of Antitubercular Agents That Target the Gyrase ATPase Domain

In this study we explored the pharmaceutically underexploited ATPase domain of DNA gyrase (GyrB) as a potential platform for developing novel agents that target Mycobacterium tuberculosis. In this effort a combination of ligand‐ and structure‐based pharmacophore modeling was used to identify structurally diverse small‐molecule inhibitors of the mycobacterial GyrB domain based on the crystal structure of the enzyme with a pyrrolamide inhibitor (PDB ID: 4BAE ). Pharmacophore modeling and subsequent in vitro screening resulted in an initial hit compound 5 [(E)‐5‐(5‐(2‐(1H‐benzo[d]imidazol‐2‐yl)‐2‐cyanovinyl)furan‐2‐yl)isophthalic acid; IC₅₀=4.6±0.1 μm ], which was subsequently tailored through a combination of molecular modeling and synthetic chemistry to yield the optimized lead compound 24 [(E)‐3‐(5‐(2‐cyano‐2‐(5‐methyl‐1H‐benzo[d]imidazol‐2‐yl)vinyl)thiophen‐2‐yl)benzoic acid; IC₅₀=0.3±0.2 μm ], which was found to display considerable in vitro efficacy against the purified GyrB enzyme and potency against the H₃₇Rv strain of M. tuberculosis. Structural handles were also identified that will provide a suitable foundation for further optimization of these potent analogues.     

Synthesis and Anti‐herpetic Activity of Phosphoramidate ProTides

Among the many prodrug approaches aimed at delivering nucleoside monophosphates into cells, the phosphoramidate ProTide approach is one that has shown success, which has made it possible for some of the phosphoramidates to enter into clinical trials. Herein, we report the synthesis and antiviral activity of a series of phosphoramidate ProTides designed to bypass the thymidine kinase (TK) dependence of the parent nucleoside analogues. Phosphoramidate derivatives of (E)‐5‐(2‐bromovinyl)‐2′‐deoxyuridine (BVDU) that contain L ‐alanine or pivaloyloxymethyl iminodiacetate (IDA‐POM) exhibit anti‐HSV‐1 and anti‐VZV activity in cell cultures, but they largely lost antiviral potency against TK‐deficient virus strains. Among deazapurine nucleosides and their phosphoramidate derivatives, the 7‐deazaadenine containing nucleosides and their phosphoramidate triester derivatives showed weak antiviral activity against VZV. Apparently, intracellular nucleotide delivery with these phosphoramidates is partly successful. However, none of the compound prodrugs showed superior activity to their parent drugs.