2,4-Disubstituted 5-Nitroimidazoles Potent against Clostridium difficile

Metronidazole is one of the first-line treatments for non-severe Clostridium difficile infections (CDI). However, resistance limits its use in cases of severe and complicated CDI. Structure–activity relationships previously described for the 5-nitroimidazole series have shown that functionalization at the 2- and 4-positions can impart better activity against parasites and anaerobic bacteria than metronidazole. Herein we report the synthesis of new 2,4-disubstituted 5-nitroimidazole compounds that show potent antibacterial activity against C. difficile. We used a vicarious nucleophilic substitution of hydrogen (VNS) reaction to introduce a phenylmethylsulfone at the 4-position and a unimolecular radical nucleophilic substitution (S_RN1) reaction to introduce an ethylenic function at the 2-position of the 5-nitroimidazole scaffold.     

Novel 1,4‐Substituted‐1,2,3‐Triazoles as Antitubercular Agents

Tuberculosis (TB) remains a pressing unmet medical need, particularly with the emergence of multidrug‐resistant and extensively drug‐resistant tuberculosis. Here, a series of 1,4‐substituted‐1,2,3‐triazoles have been synthesized and evaluated as potential antitubercular agents. These compounds were assembled via click chemistry in high crude purity and in moderate to high yield. Of the compounds tested, 12 compounds showed promising antitubercular activity with six possessing minimum inhibitory concentration (MIC) values <10 μg mL^?1, and total selectivity for Mycobacterium tuberculosis (Mtb) growth inhibition. A second set of 21 compounds bearing variations on ring C were synthesized and evaluated. This second library gave an additional six compounds displaying MIC values ≤10 μg mL^?1 and total selectivity for Mtb growth inhibition. These compounds serve as an excellent starting point for further development of antitubercular therapies.     

Antichagasic,Leishmanicidal, and Trichomonacidal Activity of 2‐Benzyl‐5‐nitroindazole‐Derived Amines

Three different series of new 5‐nitroindazole derivatives—1‐(ω‐aminoalkyl)‐2‐benzylindazolin‐3‐ones (series A ; ten compounds), 3‐(ω‐aminoalkoxy)‐2‐benzylindazoles (series B ; four compounds) and 3‐alkylamino‐2‐benzylindazoles (series C ; five compounds)—have been synthesized and evaluated against the protozoan parasites Trypanosoma cruzi, Leishmania amazonensis, and Trichomonas vaginalis: etiological agents of Chagas disease, cutaneous leishmaniasis, and trichomoniasis, respectively. Many indazoles of series A , B , and C were efficient against T. cruzi. Some compounds in series A , after successfully passing the preliminary screening for epimastigotes, exhibited activity values against amastigotes of several T. cruzi strains that were better than or similar to those shown by the reference drug benznidazole and displayed low nonspecific toxicity against mammalian cells. On the other hand, preliminary studies against promastigotes of L. amazonensis showed high leishmanicidal activity for some derivatives of series A and C . With regard to activity against T. vaginalis, some indazoles of series B and C were rather efficient against trophozoites of a metronidazole‐sensitive isolate and showed low nonspecific toxicities toward Vero cell cultures. Additionally, some of these compounds displayed similar activity against metronidazole‐sensitive and resistant isolates, showing the absence of cross‐resistance between these derivatives and the reference drug.     

Triazolophthalazines: Easily Accessible Compounds with Potent Antitubercular Activity

Tuberculosis (TB) remains one of the major causes of death worldwide, in particular because of the emergence of multidrug‐resistant TB. Herein we explored the potential of an alternative class of molecules as anti‐TB agents. Thus, a series of novel 3‐substituted triazolophthalazines was quickly and easily prepared from commercial hydralazine hydrochloride as starting material and were further evaluated for their antimycobacterial activities and cytotoxicities. Four of the synthesized compounds were found to effectively inhibit the Mycobacterium tuberculosis (M.tb) H₃₇Rv strain with minimum inhibitory concentration (MIC) values <10 μg mL^?1, whereas no compounds displayed cytotoxicity against HCT116 human cell lines (IC₅₀>100 μm ). More remarkably, the most potent compounds proved to be active to a similar extent against various multidrug‐resistant M.tb strains, thus uncovering a mode of action distinct from that of standard antitubercular agents. Overall, their ease of preparation, combined with their attractive antimycobacterial activities, make such triazolophthalazine‐based derivatives promising leads for further development.     

The Importance of Therapeutically Targeting the Binary Toxin from Clostridioides difficile

Novel therapeutics are needed to treat pathologies associated with the Clostridioides difficile binary toxin (CDT), particularly when C. difficile infection (CDI) occurs in the elderly or in hospitalized patients having illnesses, in addition to CDI, such as cancer. While therapies are available to block toxicities associated with the large clostridial toxins (TcdA and TcdB) in this nosocomial disease, nothing is available yet to treat toxicities arising from strains of CDI having the binary toxin. Like other binary toxins, the active CDTa catalytic subunit of CDT is delivered into host cells together with an oligomeric assembly of CDTb subunits via host cell receptor-mediated endocytosis. Once CDT arrives in the host cell’s cytoplasm, CDTa catalyzes the ADP-ribosylation of G-actin leading to degradation of the cytoskeleton and rapid cell death. Although a detailed molecular mechanism for CDT entry and host cell toxicity is not yet fully established, structural and functional resemblances to other binary toxins are described. Additionally, unique conformational assemblies of individual CDT components are highlighted herein to refine our mechanistic understanding of this deadly toxin as is needed to develop effective new therapeutic strategies for treating some of the most hypervirulent and lethal strains of CDT-containing strains of CDI.     

Targeting Asexual and Sexual Blood Stages of the Human Malaria Parasite P. falciparum with 7-Chloroquinoline-Based 1,2,3-Triazoles

Novel 4-amino-7-chloroquinoline-based 1,2,3-triazole hybrids were synthesised in good yields by Cu^I-catalysed Huisgen 1,3-dipolar cycloaddition reactions of 2-azido-N-(7-chloroquinolin-4-ylaminoalkyl)acetamides with various terminal alkynes. These new hybrids were screened in vitro against asexual blood stages of the chloroquine-sensitive 3D7 strain of P. falciparum. The most active compounds were further screened against asexual and sexual stages (gametocytes) of the chloroquine-resistant RKL-9 strain of P. falciparum. Although all compounds were less potent than chloroquine against the 3D7 strain, the three best compounds were appreciably more active than chloroquine against the RKL-9 strain, displaying IC₅₀ values of <100 nm , with one of them having an IC₅₀ of 2.94 nm . Further, the lead compounds were gametocytocidal with IC₅₀ values in the micromolar range, and were observed to induce morphological deformations in mature gametocytes. Most compounds demonstrated little or no cytotoxicity and exhibited good selectivity indices. The most active compounds represent promising candidates for further evaluation of their schizonticidal and gametocytocidal potential.     

Synthesis and in vitro Evaluation of 3H‐Pyrrolo[3,2‐f]quinolin‐9‐one Derivatives That Show Potent and Selective Anti‐leukemic Activity

A series of new substituted 7‐phenyl‐3H‐pyrrolo[3,2‐f]quinolin‐9‐ones were synthesized and evaluated for their antiproliferative activity. The most active derivatives showed high selectivity against human leukemia cell lines and potently inhibited their growth, with GI₅₀ values in the nanomolar range. The active compounds strongly blocked tubulin assembly and colchicine binding to tubulin. Their activities were equal to or greater than that of the reference compound combretastatin A‐4. Flow cytometry studies showed that the two most active compounds arrested Jurkat cells in the G₂/M cell‐cycle phase in a concentration‐dependent manner. This effect was associated with apoptosis, mitochondrial depolarization, generation of reactive oxygen species, activation of caspase‐3, and cleavage of the enzyme poly(ADP‐ribose) polymerase.     

An Investigation into the Inactivation Kinetics of Hydrogen Peroxide Vapor Against Clostridium difficile Endospores

C. difficile spores are resistant to routine cleaning agents and are able to survive on inanimate surfaces for long periods of time. There is increasing evidence of the importance of the clinical environment as a reservoir for pathogenic agents and as a potential source of healthcare-associated infections (HCAIs). In this context, to reduce the risk of cross-transmission, terminal disinfection of hospital wards and isolation rooms using hydrogen peroxide vapor (HPV) is attracting attention. Spores of C. difficile (ribotype 027) were exposed to constant concentrations of HPV ranging between 11 and 92 mg m^?3 (ppm) for a range of exposure times in a specially designed chamber. The inactivation data thus obtained was fitted using the modified Chick–Watson inactivation model to obtain decimal reduction values (D values). D values ranged from 23 to 1.3 min at HPV concentrations of 11 and 92 ppm, respectively. We present a simple mathematical model based on the inactivation kinetic data obtained here to estimate the efficacy of commercial HPV processes used in healthcare environmental decontamination. C. difficile spores showed linear inactivation kinetics at steady HPV concentrations ranging between 10 and 90 ppm. The data obtained here was used to provide estimates of the inactivation efficacy of commercial HPV process cycles, which employ unsteady HPV concentrations during the decontamination process.     

Identification of a novel protein synthesis inhibitor active against gram-positive bacteria

In an effort to identify novel antibacterial chemotypes, we performed a whole‐cell screen for inhibitors of Staphylococcus aureus growth and pursued those compounds with previously uncharacterized antibacterial activity. This process resulted in the identification of a benzothiazolium salt, ABTZ‐1, that displayed potent antibacterial activity against Gram‐positive pathogens. Several clinically desirable qualities were demonstrated for ABTZ‐1 including potent activity against multidrug‐resistant clinical isolates of methicillin‐resistant S. aureus (MRSA) and vancomycin‐resistant enterococci (VRE), retention of this activity in human serum, and low hemolytic activity. The antibacterial activity of ABTZ‐1 was attributed to its inhibition of bacterial translation, as this compound prevented the incorporation of [³⁵S]methionine into S. aureus proteins, and ABTZ‐1‐resistant strains were cross‐resistant to known inhibitors of bacterial translation. ABTZ‐1 represents a promising new class of antibacterial agents.     

Nitrate Esters of Heteroaromatic Compounds as Candida albicans CYP51 Enzyme Inhibitors

Four heteroaromatic compounds bearing nitrate esters were selected using a virtual‐screening procedure as putative sterol 14α‐demethylase (CYP51) Candida albicans inhibitors. Compounds were examined for their inhibition on C. albicans growth and biofilm formation as well as for their toxicity. NMR spectroscopy studies, in silico docking, and molecular dynamics simulations were used to investigate further the selectivity of these compounds to fungal CYP51. All compounds exhibited good antimicrobial properties, indicated with low minimal inhibitory concentrations and ability to inhibit formation of fungal biofilm. Moreover, all of the compounds had the ability to inhibit growth of C. albicans cells. N‐(2‐Nitrooxyethyl)‐1Η‐indole‐2‐carboxamide was the only compound with selectivity on C. albicans CYP51 that did not exhibit cytotoxic effect on cells isolated from liver and should be further investigated for selective application in new leads for the treatment of candidiasis.     

1‐(1H‐Indol‐3‐yl)ethanamine Derivatives as Potent Staphylococcus aureus NorA Efflux Pump Inhibitors

The synthesis of 37 1‐(1H‐indol‐3‐yl)ethanamine derivatives, including 12 new compounds, was achieved through a series of simple and efficient chemical modifications. These indole derivatives displayed modest or no intrinsic anti‐staphylococcal activity. By contrast, several of the compounds restored, in a concentration‐dependent manner, the antibacterial activity of ciprofloxacin against Staphylococcus aureus strains that were resistant to fluoroquinolones due to overexpression of the NorA efflux pump. Structure–activity relationships studies revealed that the indolic aldonitrones halogenated at position 5 of the indole core were the most efficient inhibitors of the S. aureus NorA efflux pump. Among the compounds, (Z)‐N‐benzylidene‐2‐(tert‐butoxycarbonylamino)‐1‐(5‐iodo‐1H‐indol‐3‐yl)ethanamine oxide led to a fourfold decrease of the ciprofloxacin minimum inhibitory concentration against the SA‐1199B strain when used at a concentration of 0.5 mg L ^?1. To the best of our knowledge, this activity is the highest reported to date for an indolic NorA inhibitor. In addition, a new antibacterial compound, tert‐butyl (2‐(3‐hydroxyureido)‐2‐(1H‐indol‐3‐yl)ethyl)carbamate, which is not toxic for human cells, was also found.     

Targeting the Erythrocytic and Liver Stages of Malaria Parasites with s‐Triazine‐Based Hybrids

A diversity‐oriented library of s‐triazine‐based hybrids was screened for activity against the chloroquine‐resistant Plasmodium falciparum W2 strain. The most striking result was sub‐micromolar activity against cultured erythrocytic‐stage parasites of hybrid molecules containing one or two 8‐aminoquinoline moieties. These compounds were not clearly toxic to human cells. The most effective blood‐schizontocidal s‐triazine derivatives were then screened for activity against the liver stage of malaria parasites. The s‐triazine hybrid containing two 8‐aminoquinoline moieties and one chlorine atom emerged as the most potent against P. berghei liver‐stage infection, active in the low nanomolar region, combined with good metabolic stability in rat liver microsomes. These results indicate that s‐triazine‐8‐aminoquinoline‐based hybrids are excellent starting points for lead optimization as dual‐stage antimalarials.     

Sigma‐2 Receptor Agonists as Possible Antitumor Agents in Resistant Tumors: Hints for Collateral Sensitivity

With the aim of contributing to the development of novel antitumor agents, high‐affinity σ₂ receptor agonists were developed, with 6,7‐dimethoxy‐2‐[4‐[1‐(4‐fluorophenyl)‐1H‐indol‐3‐yl]butyl]‐1,2,3,4‐tetrahydroisoquinoline ( 15 ) and 9‐[4‐(6,7‐dimethoxy‐1,2,3,4‐tetrahydroisoquinolin‐2‐yl)butyl]‐9H‐carbazole ( 25 ) showing exceptional selectivity for the σ₂ subtype. Most of the compounds displayed notable antiproliferative activity in human MCF7 breast adenocarcinoma cells, with similar activity in the corresponding doxorubicin‐resistant MCF7adr cell line. Surprisingly, a few compounds, including 25 , displayed enhanced activity in MCF7adr cells over parent cells, recalling the phenomenon of collateral sensitivity, which is under study for the treatment of drug‐resistant tumors. All of the compounds showed interaction with P‐glycoprotein (P‐gp), and 15 and 25 , with the greatest activity, were able to revert P‐gp‐mediated resistance and reestablish the antitumor effect of doxorubicin in MCF7adr cells. We therefore identified a series of σ₂ receptor agonists endowed with intriguing antitumor properties; these compounds deserve further investigation for the development of alternate strategies against multidrug‐ resistant cancers.     

Bisnaphthalimidopropyl Derivatives as Inhibitors of Leishmania SIR2 Related Protein 1

The NAD⁺‐dependent deacetylases, namely sirtuins, are involved in the regulation of a variety of biological processes such as gene silencing, DNA repair, longevity, metabolism, apoptosis, and development. An enzyme from the parasite Leishmania infantum that belongs to this family, LiSIR2RP1, is a NAD⁺‐dependent tubulin deacetylase and an ADP‐ribosyltransferase. This enzyme's involvement in L. infantum virulence and survival underscores its potential as a drug target. Our search for selective inhibitors of LiSIR2RP1 has led, for the first time, to the identification of the antiparasitic and anticancer bisnaphthalimidopropyl (BNIP) alkyl di‐ and triamines (IC₅₀ values in the single‐digit micromolar range for the most potent compounds). Structure–activity studies were conducted with 12 BNIP derivatives that differ in the length of the central alkyl chain, which links the two naphthalimidopropyl moieties. The most active and selective compound is the BNIP diaminononane (BNIPDanon), with IC₅₀ values of 5.7 and 97.4 μM against the parasite and human forms (SIRT1) of the enzyme, respectively. Furthermore, this compound is an NAD⁺‐competitive inhibitor that interacts differently with the parasite and human enzymes, as determined by docking analysis, which might explain its selectivity toward the parasitic enzyme.     

Design, synthesis, and pharmacological characterization of novel spirocyclic quinuclidinyl-Δ2-isoxazoline derivatives as potent and selective agonists of α7 nicotinic acetylcholine receptors

A set of racemic spirocyclic quinuclidinyl‐Δ²‐isoxazoline derivatives was synthesized using a 1,3‐dipolar cycloaddition‐based approach. Target compounds were assayed for binding affinity toward rat neuronal homomeric (α7) and heteromeric (α4β2) nicotinic acetylcholine receptors. Δ²‐Isoxazolines 3 a (3‐Br), 6 a (3‐OMe), 5 a (3‐Ph), 8 a (3‐OnPr), and 4 a (3‐Me) were the ligands with the highest affinity for the α7 subtype (K_i values equal to 13.5, 14.2, 25.0, 71.6, and 96.2 nM , respectively), and showed excellent α7 versus α4β2 subtype selectivity. These compounds, tested in electrophysiological experiments against human α7 and α4β2 receptors stably expressed in cell lines, behaved as partial α7 agonists with varying levels of potency. The two enantiomers of (±)‐3‐methoxy‐1‐oxa‐2,7‐diaza‐7,10‐ethanospiro[4.5]dec‐2‐ene sesquifumarate 6 a were prepared using (+)‐dibenzoyl‐L ‐ or (?)‐dibenzoyl‐D ‐tartaric acid as resolving agents. Enantiomer (R)‐(?)‐ 6 a was found to be the eutomer, with K_i values of 4.6 and 48.7 nM against rat and human α7 receptors, respectively.     

Discovery and Biophysical Evaluation of First Low Nanomolar Hits Targeting CYP125 of M. tuberculosis

Tuberculosis, which is predominantly caused by Mycobacterium tuberculosis (Mtb), is still the most lethal bacterial infection with 1.5 million casualties in 2014. Moreover, the fact that the appearance of resistant mutants and long‐term treatment are coupled with economic problems in developing countries hampers an efficient therapy. Interference with the essential cholesterol metabolism of Mtb could be a promising novel strategy to fight Mtb infections. CYP125, a P450 enzyme in Mtb, has been shown to play an important role in this metabolic pathway. For this reason, we used a combined screening approach involving surface plasmon resonance spectroscopy and a heme coordination assay to identify new CYP125 binders by employing a focused P450‐inhibitor library. We identified the first hits with high affinity and favorable ligand efficiencies. Furthermore, frontrunner compounds also showed selectivity toward CYP121 specific to Mtb and required for its survival. To date, these are the first compounds targeting CYP125 with low nanomolar affinity.     

Structural Optimization of Berberine as a Synergist to Restore Antifungal Activity of Fluconazole against Drug‐Resistant Candida albicans

We have conducted systematic structural modification, deconstruction, and reconstruction of the berberine core with the aim of lowering its cytotoxicity, investigating its pharmacophore, and ultimately, seeking novel synergistic agents to restore the effectiveness of fluconazole against fluconazole‐resistant Candida albicans. A structure–activity relationship study of 95 analogues led us to identify the novel scaffold of N‐(2‐(benzo[d][1,3]dioxol‐5‐yl)ethyl)‐2‐(substituted phenyl)acetamides 7 a – l , which exhibited remarkable levels of in vitro synergistic antifungal activity. Compound 7 d (N‐(2‐(benzo[d][1,3]dioxol‐5‐yl)ethyl)‐2‐(2‐fluorophenyl)acetamide) significantly decreased the MIC₈₀ values of fluconazole from 128.0 μg mL^?1 to 0.5 μg mL^?1 against fluconazole‐resistant C. albicans and exhibited much lower levels of cytotoxicity than berberine toward human umbilical vein endothelial cells.     

From Antidiabetic to Antifungal: Discovery of Highly Potent Triazole–Thiazolidinedione Hybrids as Novel Antifungal Agents

In an attempt to discover a new generation of triazole antifungal agents, a series of triazole–thiazolidinedione hybrids were designed and synthesized by molecular hybridization of the antifungal agent fluconazole and rosiglitazone (an antidiabetic). Most of the target compounds showed good to excellent inhibitory activity against a variety of clinically important fungal pathogens. In particular, compounds (Z)‐5‐(2,4‐dichlorobenzylidene)‐3‐(2‐(2,4‐difluorophenyl)‐2‐hydroxy‐3‐(1H‐1,2,4‐triazol‐1‐yl)propyl)thiazolidine‐2,4‐dione) ( 15 c ), (Z)‐3‐(2‐(2,4‐difluorophenyl)‐2‐hydroxy‐3‐(1H‐1,2,4‐triazol‐1‐yl)propyl)‐5‐(furan‐3‐ylmethylene)thiazolidine‐2,4‐dione ( 15 j ), and (Z)‐3‐(2‐(2,4‐difluorophenyl)‐2‐hydroxy‐3‐(1H‐1,2,4‐triazol‐1‐yl)propyl)‐5‐(furan‐3‐ylmethylene)thiazolidine‐2,4‐dione ( 15 r ) were highly active against Candida albicans, with MIC₈₀ values in the range of 0.03–0.15 μM . Moreover, compounds 15 j and 15 r were found to be effective against four fluconazole‐resistant clinical isolates; these two compounds are particularly promising antifungal leads for further optimization. Molecular docking studies revealed that the hydrogen bonding interactions between thiazolidinedione and CYP51 from C. albicans are important for antifungal activity. This study also demonstrates the effectiveness of molecular hybridization in antifungal drug discovery.     

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.     

C2 Arylated Benzo[b]thiophene Derivatives as Staphylococcus aureus NorA Efflux Pump Inhibitors

An innovative and straightforward synthesis of second‐generation 2‐arylbenzo[b]thiophenes as structural analogues of INF55 and the first generation of our laboratory‐made molecules was developed. The synthesis of C2‐arylated benzo[b]thiophene derivatives was achieved through a method involving direct arylation, followed by simple structural modifications. Among the 34 compounds tested, two of them were potent NorA pump inhibitors, which led to a 16‐fold decrease in the ciprofloxacin minimum inhibitory concentration (MIC) against the SA‐1199B strain at concentrations of 0.25 and 0.5 μg mL^?1 (1 and 1.5 μm , respectively). This is a promising result relative to that obtained for reserpine (MIC=20 μg mL^?1), a reference compound amongst NorA pump inhibitors. These molecules thus represent promising candidates to be used in combination with ciprofloxacin against fluoroquinolone‐resistant strains.