Synthesis, Biological Evaluation, and Structure−Activity Relationships for 5-[(<i>E</i>)-2-Arylethenyl]-3-isoxazolecarboxylic Acid Alkyl Ester Derivatives as Valuable Antitubercular Chemotypes

Tuberculosis (TB), mostly caused by Mycobacterium tuberculosis (Mtb), is one of the leading causes of death from infectious disease worldwide. Its coinfection with HIV and the emergence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) strains have further worsened the TB pandemic. Despite its global impact, TB is considered a neglected disease and no new anti-TB therapeutics have been introduced over the last four decades. The nonreplicating persistent form of TB (NRP-TB) is responsible for the length of the treatment and is the putative cause of treatment failure. Therefore, new anti-TB agents, which are active against both the replicating form of Mtb (R-TB) and NRP-TB, are urgently needed. Herein, we report the synthesis and structure−activity relationships (SAR) of a series of 5-[(E)-2-arylethenyl]-3-isoxazolecarboxylic acid alkyl esters as potent anti-TB agents. Several compounds had submicromolar minimum inhibitory concentrations (MIC) against R-TB and were active against NRP-TB in the low micromolar range, thus representing attractive lead compounds for the possible development of new anti-TB agents

Rational Design of 5-Phenyl-3-isoxazolecarboxylic Acid Ethyl Esters as Growth Inhibitors of <i>Mycobacterium tuberculosis</i>. A Potent and Selective Series for Further Drug Development

New antituberculosis (anti-TB) drugs are urgently needed to shorten the 6−12 month treatment regimen and especially to battle drug-resistant Mycobacterium tuberculosis (Mtb) strains. In this study, we have continued our efforts to develop isoxazole-based anti-TB compounds by applying rational drug design approach. The biological activity and the structure−activity relationships (SAR) for a designed series of 5-phenyl-3-isoxazolecarboxylic acid ethyl ester derived anti-TB compounds were investigated. Several compounds were found to exhibit nanomolar activity against the replicating bacteria (R-TB) and low micromolar activity against the nonreplicating bacteria (NRP-TB). The series showed excellent selectivity toward Mtb, and in general, no cytotoxicity was observed in Vero cells (IC50 > 128 μM). Notably, selected compounds also retained their activity against isoniazid (INH), rifampin (RMP), and streptomycin (SM) resistant Mtb strains. Hence, benzyloxy, benzylamino, and phenoxy derivatives of 5-phenyl-3-isoxazolecarboxylic acid ethyl esters represent a highly potent, selective, and versatile series of anti-TB compounds and as such present attractive lead compounds for further TB drug development

In Pursuit of Natural Product Leads: Synthesis and Biological Evaluation of 2-[3-hydroxy-2-[(3-hydroxypyridine-2-carbonyl)amino]phenyl]benzoxazole-4-carboxylic acid (A-33853) and Its Analogues: Discovery of <i>N</i>-(2-Benzoxazol-2-ylphenyl)benzamides as Novel Antileishmanial Chemotypes

The first synthesis and biological evaluation of antibiotic 31 (A-33853) and its analogues are reported. Initial screening for inhibition of L. donovani, T. b. rhodesiense, T. cruzi, and P. falciparum cultures followed by determination of IC50 in L. donovani and cytotoxicity on L6 cells revealed 31 to be 3-fold more active than miltefosine, a known antileishmanial drug. Compounds 14, 15, and 25 selectively inhibited L. donovani at nanomolar concentrations and showed much lower cytotoxicity

Integration of Enhanced Sampling Methods with Saturation Transfer Difference Experiments to Identify Protein Druggable Pockets

Saturation transfer difference (STD) is an NMR technique conventionally applied in drug discovery to identify ligand moieties relevant for binding to protein cavities. This is important to direct medicinal chemistry efforts in small-molecule optimization processes. However, STD does not provide any structural details about the ligand–target complex under investigation. Herein, we report the application of a new integrated approach, which combines enhanced sampling methods with STD experiments, for the characterization of ligand–target complexes that are instrumental for drug design purposes. As an example, we have studied the interaction between <i>St</i>OASS-A, a potential antibacterial target, and an inhibitor previously reported. This approach allowed us to consider the ligand–target complex from a dynamic point of view, revealing the presence of an accessory subpocket which can be exploited to design novel <i>St</i>OASS-A inhibitors. As a proof of concept, a small library of derivatives was designed and evaluated in vitro, displaying the expected activity

Integration of Enhanced Sampling Methods with Saturation Transfer Difference Experiments to Identify Protein Druggable Pockets

Saturation transfer difference (STD) is an NMR technique conventionally applied in drug discovery to identify ligand moieties relevant for binding to protein cavities. This is important to direct medicinal chemistry efforts in small-molecule optimization processes. However, STD does not provide any structural details about the ligand–target complex under investigation. Herein, we report the application of a new integrated approach, which combines enhanced sampling methods with STD experiments, for the characterization of ligand–target complexes that are instrumental for drug design purposes. As an example, we have studied the interaction between <i>St</i>OASS-A, a potential antibacterial target, and an inhibitor previously reported. This approach allowed us to consider the ligand–target complex from a dynamic point of view, revealing the presence of an accessory subpocket which can be exploited to design novel <i>St</i>OASS-A inhibitors. As a proof of concept, a small library of derivatives was designed and evaluated in vitro, displaying the expected activity

Rational Design and Synthesis of Thioridazine Analogues as Enhancers of the Antituberculosis Therapy

Tuberculosis, caused by <i>Mycobacterium tuberculosis</i>, is still one of the leading infectious diseases globally. Therefore, novel approaches are needed to face this disease. Efflux pumps are known to contribute to the emergence of <i>M. tuberculosis</i> drug resistance. Thioridazine has shown good anti-TB properties both in vitro and in vivo, likely due to its capacity to inhibit efflux mechanisms. Here we report the design and synthesis of a number of putative efflux inhibitors inspired by the structure of thioridazine. Compounds were evaluated for their in vitro and ex vivo activity against <i>M. tuberculosis</i> H37Rv. Compared to the parent molecule, some of the compounds synthesized showed higher efflux inhibitory capacity, less cytotoxicity, and a remarkable synergistic effect with anti-TB drugs both in vitro and in human macrophages, demonstrating their potential to be used as coadjuvants for the treatment of tuberculosis

From 6-Aminoquinolone Antibacterials to 6-Amino-7-thiopyranopyridinylquinolone Ethyl Esters as Inhibitors of <i>Staphylococcus aureus</i> Multidrug Efflux Pumps

The thiopyranopyridine moiety was synthesized as a new heterocyclic base to be inserted at the C-7 position of selected quinolone nuclei followed by a determination of antibacterial activity against strains of Staphylococcus aureus. Selected thiopyranopyridinylquinolones showed significant antimicrobial activity, including strains having mutations in gyrA and grlA as well as other strains overexpressing the NorA multidrug (MDR) efflux pump. Most derivatives did not appear to be NorA substrates. The effect of the thiopyranopyridinyl substituent on making these quinolones poor substrates for NorA was investigated further. Several quinolone ester intermediates, devoid of any intrinsic antibacterial activity, were tested for their abilities to inhibit the activities of NorA (MFS family) and MepA (MATE family) S. aureus MDR efflux pumps. Selected quinolone esters were capable of inhibiting both MDR pumps more efficiently than the reference compound reserpine. Moreover, they also were able to restore, and even enhance, the activity of ciprofloxacin toward some genetically modified resistant S. aureus strains

Cyclopropane derivatives as potential human serine racemase inhibitors: unveiling novel insights into a difficult target

<p>d-Serine is the co-agonist of NMDA receptors and binds to the so-called glycine site. d-Serine is synthesized by human serine racemase (SR). Over activation of NMDA receptors is involved in many neurodegenerative diseases and, therefore, the inhibition of SR might represent a novel strategy for the treatment of these pathologies. SR is a very difficult target, with only few compounds so far identified exhibiting weak inhibitory activity. This study was aimed at the identification of novel SR inhibitor by mimicking malonic acid, the best-known SR inhibitor, with a cyclopropane scaffold. We developed, synthesized, and tested a series of cyclopropane dicarboxylic acid derivatives, complementing the synthetic effort with molecular docking. We identified few compounds that bind SR in high micromolar range with a lack of significant correlation between experimental and predicted binding affinities. The thorough analysis of the results can be exploited for the development of more potent SR inhibitors.</p

Identification of Human Alanine–Glyoxylate Aminotransferase Ligands as Pharmacological Chaperones for Variants Associated with Primary Hyperoxaluria Type 1

Primary hyperoxaluria type I (PH1) is a rare kidney disease due to the deficit of alanine:glyoxylate aminotransferase (AGT), a pyridoxal-5′-phosphate-dependent enzyme responsible for liver glyoxylate detoxification, which in turn prevents oxalate formation and precipitation as kidney stones. Many PH1-associated missense mutations cause AGT misfolding. Therefore, the use of pharmacological chaperones (PCs), small molecules that promote correct folding, represents a useful therapeutic option. To identify ligands acting as PCs for AGT, we first performed a small screening of commercially available compounds. We tested each molecule by a dual approach aimed at defining the inhibition potency on purified proteins and the chaperone activity in cells expressing a misfolded variant associated with PH1. We then performed a chemical optimization campaign and tested the resulting synthetic molecules using the same approach. Overall, the results allowed us to identify a promising hit compound for AGT and draw conclusions about the requirements for optimal PC activity

Identification of Human Alanine–Glyoxylate Aminotransferase Ligands as Pharmacological Chaperones for Variants Associated with Primary Hyperoxaluria Type 1

Primary hyperoxaluria type I (PH1) is a rare kidney disease due to the deficit of alanine:glyoxylate aminotransferase (AGT), a pyridoxal-5′-phosphate-dependent enzyme responsible for liver glyoxylate detoxification, which in turn prevents oxalate formation and precipitation as kidney stones. Many PH1-associated missense mutations cause AGT misfolding. Therefore, the use of pharmacological chaperones (PCs), small molecules that promote correct folding, represents a useful therapeutic option. To identify ligands acting as PCs for AGT, we first performed a small screening of commercially available compounds. We tested each molecule by a dual approach aimed at defining the inhibition potency on purified proteins and the chaperone activity in cells expressing a misfolded variant associated with PH1. We then performed a chemical optimization campaign and tested the resulting synthetic molecules using the same approach. Overall, the results allowed us to identify a promising hit compound for AGT and draw conclusions about the requirements for optimal PC activity