Antistaphylococcal discovery pipeline; where are we now?

The serious spread of antibiotic-resistant Staphylococcal aureus strains is alarming. This is reflected by the measures governments and health-related bodies are offering to ease antibiotic drug development. Finding new active agents, preferably with novel mechanism of action, or even finding new targets for drug development are essential. In this review, we summarize the current status of novel antistaphylococcal agents undergoing clinical trials. We mainly discuss antistaphylococcal small molecules and peptides in the text with a special focus on their chemistry, while antistaphylococcal immunotherapy (antibodies) are mentioned in a summative table. This review shall serve as a summary that influences future synthetic efforts in the antistaphyloccocals development field.

Synthesis, Biological Evaluation, and In Silico Modeling of N-Substituted Quinoxaline-2-Carboxamides.

Despite the established treatment regimens, tuberculosis remains an alarming threat to public health according to WHO. Novel agents are needed to overcome the increasing rate of resistance and perhaps achieve eradication. As part of our long-term research on pyrazine derived compounds, we prepared a series of their ortho fused derivatives, N-phenyl- and N-benzyl quinoxaline-2-carboxamides, and evaluated their in vitro antimycobacterial activity. In vitro activity against Mycobacterium tuberculosis H37Ra (represented by minimum inhibitory concentration, MIC) ranged between 3.91-500 µg/mL, with most compounds having moderate to good activities (MIC < 15.625 µg/mL). The majority of the active compounds belonged to the N-benzyl group. In addition to antimycobacterial activity assessment, final compounds were screened for their in vitro cytotoxicity. N-(naphthalen-1-ylmethyl)quinoxaline-2-carboxamide (compound 29) was identified as a potential antineoplastic agent with selective cytotoxicity against hepatic (HepG2), ovarian (SK-OV-3), and prostate (PC-3) cancer cells lines. Molecular docking showed that human DNA topoisomerase and vascular endothelial growth factor receptor could be potential targets for 29.

Screening of SIRT6 inhibitors and activators: A novel activator has an impact on breast cancer cells.

Sirtuin 6 (SIRT6), a member of sirtuin family (SIRT1-7), regulates a variety of cellular processes involved in aging, metabolism, and cancer. Dysregulation of SIRT6 is widely observed in different breast cancer subtypes; however, the role and function of SIRT6 in cancer development remain largely unexplored. The aim of this study was to identify novel compounds targeting SIRT6 which may provide a new approach in development of anti-cancer therapy for breast cancer. Virtual screening was utilized to discover potential compounds targeting SIRT6 for in vitro screening. In addition, novel 1,4-dihydropyridine derivatives were synthetized and further subjected for the screening. The impact of the compounds on the deacetylation activity of SIRT6 was determined with HPLC method. The anti-cancer activities were screened for a panel of breast cancer cells. A set of 1,4-dihydropyridine derivatives was identified as SIRT6 inhibitors. A SIRT6 activating compound, (2,4-dihydroxy-phenyl)-2-oxoethyl 2-(3-methyl-4-oxo-2-phenyl-4H-chromen-8-yl)acetate (later called as 4H-chromen), was discovered and it provided 30-40-fold maximal activation. 4H-chromen was proposed to bind similarly to quercetin and place to previously reported SIRT6 activator sites. 4H-chromen was investigated in various breast cancer cells, and it decreased cell proliferation in all cells as well as arrested cell cycle in triple negative cells. Overall, this study describes a highly potent SIRT6 activator and new inhibitors that represent a novel tool to study the mechanism of SIRT6 function.

N-pyridinylbenzamides: an isosteric approach towards new antimycobacterial compounds.

A series of N-pyridinylbenzamides was designed and prepared to investigate the influence of isosterism and positional isomerism on antimycobacterial activity. Comparison to previously published isosteric N-pyrazinylbenzamides was made as an attempt to draw structure-activity relationships in such type of compounds. In total, we prepared 44 different compounds, out of which fourteen had minimum inhibitory concentration (MIC) values against Mycobacterium tuberculosis H37Ra below 31.25 µg/ml, most promising being N-(5-chloropyridin-2-yl)-3-(trifluoromethyl)benzamide (23) and N-(6-chloropyridin-2-yl)-3-(trifluoromethyl)benzamide (24) with MIC = 7.81 µg/ml (26 µm). Five compounds showed broad-spectrum antimycobacterial activity against M. tuberculosis H37Ra, M. smegmatis and M. aurum. N-(pyridin-2-yl)benzamides were generally more active than N-(pyridin-3-yl)benzamides, indicating that N-1 in the parental structure of N-pyrazinylbenzamides might be more important for antimycobacterial activity than N-4. Marginal antibacterial and antifungal activity was observed for title compounds. The hepatotoxicity of title compounds was assessed in vitro on hepatocellular carcinoma cell line HepG2, and they may be considered non-toxic (22 compounds with IC50 over 200 µm).

N-Pyrazinoyl Substituted Amino Acids as Potential Antimycobacterial Agents-The Synthesis and Biological Evaluation of Enantiomers.

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (Mtb), each year causing millions of deaths. In this article, we present the synthesis and biological evaluations of new potential antimycobacterial compounds containing a fragment of the first-line antitubercular drug pyrazinamide (PZA), coupled with methyl or ethyl esters of selected amino acids. The antimicrobial activity was evaluated on a variety of (myco)bacterial strains, including Mtb H37Ra, M. smegmatis, M. aurum, Staphylococcus aureus, Pseudomonas aeruginosa, and fungal strains, including Candida albicans and Aspergillus flavus. Emphasis was placed on the comparison of enantiomer activities. None of the synthesized compounds showed any significant activity against fungal strains, and their antibacterial activities were also low, the best minimum inhibitory concentration (MIC) value was 31.25 µM. However, several compounds presented high activity against Mtb. Overall, higher activity was seen in derivatives containing ʟ-amino acids. Similarly, the activity seems tied to the more lipophilic compounds. The most active derivative contained phenylglycine moiety (PC-ᴅ/ʟ-Pgl-Me, MIC < 1.95 µg/mL). All active compounds possessed low cytotoxicity and good selectivity towards Mtb. To the best of our knowledge, this is the first study comparing the activities of the ᴅ- and ʟ-amino acid derivatives of pyrazinamide as potential antimycobacterial compounds.

Derivatives of 3-Aminopyrazine-2-carboxamides: Synthesis, Antimicrobial Evaluation, and in Vitro Cytotoxicity.

We report the design, synthesis, and in vitro antimicrobial activity of a series of N-substituted 3-aminopyrazine-2-carboxamides with free amino groups in position 3 on the pyrazine ring. Based on various substituents on the carboxamidic moiety, the series is subdivided into benzyl, alkyl, and phenyl derivatives. The three-dimensional structures of the title compounds were predicted using energy minimization and low mode molecular dynamics under AMBER10:EHT forcefield. Compounds were evaluated for antimycobacterial, antibacterial, and antifungal activities in vitro. The most active compound against Mycobacterium tuberculosis H37Rv (Mtb) was 3-amino-N-(2,4-dimethoxyphenyl)pyrazine-2-carboxamide (17, MIC = 12.5 µg/mL, 46 µM). Antimycobacterial activity against Mtb and M. kansasii along with antibacterial activity increased among the alkyl derivatives with increasing the length of carbon side chain. Antibacterial activity was observed for phenyl and alkyl derivatives, but not for benzyl derivatives. Antifungal activity was observed in all structural subtypes, mainly against Trichophyton interdigitale and Candida albicans. The four most active compounds (compounds 10, 16, 17, 20) were evaluated for their in vitro cytotoxicity in HepG2 cancer cell line; only compound 20 was found to exert some level of cytotoxicity. Compounds belonging to the current series were compared to previously published, structurally related compounds in terms of antimicrobial activity to draw structure activity relationships conclusions.

Design, synthesis and antimycobacterial activity of hybrid molecules combining pyrazinamide with a 4-phenylthiazol-2-amine scaffold.

Hybrid compounds based on a combination of the first-line antitubercular pyrazinamide (PZA) and a formerly identified antimycobacterial scaffold of 4-arylthiazol-2-amine were designed. Eighteen compounds were prepared, characterized and tested for in vitro growth inhibition activity against M. tuberculosis H37Rv, M. kansasii, M. avium and M. smegmatis by Microplate Alamar Blue Assay at neutral pH. Active compounds were tested for in vitro cytotoxicity in the human hepatocellular carcinoma cell line (HepG2). The most active 6-chloro-N-[4-(4-fluorophenyl)thiazol-2-yl]pyrazine-2-carboxamide (9b) also had the broadest spectrum of activity and inhibited M. tuberculosis, M. kansasii, and M. avium with MIC = 0.78 µg mL-1 (2.3 µM) and a selectivity index related to HepG2 cells of SI > 20. Structure-activity relationships within the series are discussed. Based on its structural similarity to known inhibitors and the results of a molecular docking study, we suggest mycobacterial beta-ketoacyl-(acyl-carrier-protein) synthase III (FabH) as a potential target.

Ureidopyrazine Derivatives: Synthesis and Biological Evaluation as Anti-Infectives and Abiotic Elicitors.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) has become a frequently deadly infection due to increasing antimicrobial resistance. This serious issue has driven efforts worldwide to discover new drugs effective against Mtb. One research area is the synthesis and evaluation of pyrazinamide derivatives as potential anti-TB drugs. In this paper we report the synthesis and biological evaluations of a series of ureidopyrazines. Compounds were synthesized by reacting alkyl/aryl isocyanates with aminopyrazine or with propyl 5-aminopyrazine-2-carboxylate. Reactions were performed in pressurized vials using a CEM Discover microwave reactor with a focused field. Purity and chemical structures of products were assessed, and the final compounds were tested in vitro for their antimycobacterial, antibacterial, and antifungal activities. Propyl 5-(3-phenylureido)pyrazine-2-carboxylate (compound 4, MICMtb = 1.56 µg/mL, 5.19 µM) and propyl 5-(3-(4-methoxyphenyl)ureido)pyrazine-2-carboxylate (compound 6, MICMtb = 6.25 µg/mL, 18.91 µM) had high antimycobacterial activity against Mtb H37Rv with no in vitro cytotoxicity on HepG2 cell line. Therefore 4 and 6 are suitable for further structural modifications that might improve their biological activity and physicochemical properties. Based on the structural similarity to 1-(2-chloropyridin-4-yl)-3-phenylurea, a known plant growth regulator, two selected compounds were evaluated for similar activity as abiotic elicitors.

3-Substituted N-Benzylpyrazine-2-carboxamide Derivatives: Synthesis, Antimycobacterial and Antibacterial Evaluation.

A series of substituted N-benzyl-3-chloropyrazine-2-carboxamides were prepared as positional isomers of 5-chloro and 6-chloro derivatives, prepared previously. During the aminolysis of the acyl chloride, the simultaneous substitution of chlorine with benzylamino moiety gave rise to N-benzyl-3-(benzylamino)pyrazine-2-carboxamides as side products, in some cases. Although not initially planned, the reaction conditions were modified to populate this double substituted series. The final compounds were tested against four mycobacterial strains. N-(2-methylbenzyl)-3-((2-methylbenzyl)amino)pyrazine-2-carboxamide (1a) and N-(3,4-dichlorobenzyl)-3-((3,4-dichlorobenzyl)amino)pyrazine-2-carboxamide (9a) proved to be the most effective against Mycobacterium tuberculosis H37Rv, with MIC = 12.5 µg·mL-1. Compounds were screened for antibacterial activity. The most active compound was 3-chloro-N-(2-chlorobenzyl)pyrazine-2-carboxamide (5) against Staphylococcus aureus with MIC = 7.81 µM, and Staphylococcus epidermidis with MIC = 15.62 µM. HepG2 in vitro cytotoxicity was evaluated for the most active compounds; however, no significant toxicity was detected. Compound 9a was docked to several conformations of the enoyl-ACP-reductase of Mycobacterium tuberculosis. In some cases, it was capable of H-bond interactions, typical for most of the known inhibitors.

New Synthetic Pyrazine Carboxamide Derivatives as Potential Elicitors in Production of Secondary Metabolite in In vitro Cultures.

BACKGROUND: Silymarin, an active polyphenolic fraction of Silybum marianum, and high flavonoid content of Fagopyrum possess various interesting biological activities. The substituted pyrazine-2-carboxamides were previously used as effective elicitors of studied secondary metabolites. OBJECTIVE: To study the effect of new synthetic pyrazine carboxamide derivatives, N-(4-chlorobenzyl)-5-tert-butylpyrazine-2-carboxamide (1) and 3-(3-((trifluoromethyl) benzyl) amino) pyrazine-2-carboxamide (2), on flavonolignan and flavonoid production in S. marianum and Fagopyrumes culentum in vitro cultures. MATERIALS AND METHODS: Callus and suspension cultures were cultured on MS medium containing α-naphtaleneacetic acid or 2,4-D. Three elicitor concentrations for different exposure times were tested. Dried and powdered samples of callus and suspension cultures were extracted with methanol and analyzed by DAD-HPLC. RESULTS: Compound 1 showed as a good elicitor of taxifolin production. The effect on silymarin complex was less visible with a maximum between 24 and 48 h after 3.292 ×10(-4) mol/L concentration. The detailed analysis showed that silychristin was the most abundant. Compound 2 was effective in rutin production only in callus culture with maximum 24 h and 168 h after application of 3.3756 ×10(-3) mol/L concentration and 48 and 72 h after 3.3756 ×10(-4) mol/L concentration. CONCLUSION: From the results of the performed experiments, it can be concluded that compound 1 shows to be suitable elicitor for enhanced production of taxifolin and silychristin in S. marianum, mainly when 3.292 ×10(-4) mol/L concentration was used, and compound 2 is suitable for increase rutin production in callus cultures and less appropriate for suspension cultures of F. esculentum. SUMMARY: The influence of two new synthetic pyrazine-2-carboxamidesderivatives on secondary metabolite content of Silybum marianum and Fagopyrum esculentum in vitro cultures was tested.In S. marianum, the derivate N-(4-chlorobenzyl)-5-tert-butylpyrazine-2-carboxamide showed as a good elicitor of taxifolin production and less effective for silymarin complex production with silychristin as the most abundant.The derivate 3-(3-((trifluoromethyl) benzyl) amino) pyrazine-2-carboxamide is suitable for increase rutin production in callus cultures and less appropriate for suspension cultures of F. esculentum.

Pyrazine derivatives: a patent review (June 2012 - present).

INTRODUCTION: Pyrazine is a member of 1,4-diazines, which constitute an important class of heterocycles. Various pyrazine derivatives have been synthesized and successfully evaluated as agents with diverse pharmacological effects (including but not limited to antiproliferative, anti-infective, and effects on cardiovascular or nervous system) and some of them have become clinically used drugs worldwide. AREA COVERED: This review is a survey of important patents on pyrazine derivatives with pharmacological activity published in the period June 2012 - July 2014. The patent databases SciFinder and esp@cenet were used to locate patent applications. EXPERT OPINION: Pyrazine derivatives possess numerous noteworthy pharmacological effects, including antimycobacterial, antibacterial, antifungal, antidiabetic, diuretic, anticancer, antiviral, hypnotic, and analgesic. The class of pyrazine-based candidate drugs has experienced a rapid growth both in absolute numbers of investigated compounds and in the spectrum of diverse biological activities. We expect that several of these compounds will add to existing pharmaceuticals in the very near future. According to the number of compounds and filed patents, the most promising areas are: i) inhibitors of protein kinases (applicable as antiproliferatives); and ii) inhibitors of ß-secretase (applicable for the treatment of Alzheimer's disease).

Benzothiazoles - scaffold of interest for CNS targeted drugs.

Benzothiazole compounds represent heterocyclic systems comprising a benzene ring fused with a thiazole ring containing nitrogen and sulphur in its structure. Besides the presence of a benzothiazole core in naturally occurring molecules, synthesized compounds containing a benzothiazole moiety in their structure proved to be a significant class of potential therapeutics, as they exhibit biological effects such as antitumor, antibacterial, antitubercular, antiviral, anthelmintic, antidiabetic and many others. Apart from the aforementioned peripheral or microbial active sites, benzothiazole analogues are also biologically active compounds in the central nervous system, where some approved drugs containing a benzothiazole moiety have already been identified and are used in the treatment of various neurological disorders. New benzothiazole molecules are currently under development and are being evaluated for several uses including diagnostics and as therapeutic drug candidates for the treatment of epilepsy and neurodegenerative diseases such as Alzheimer's disease, Huntington's disease and amyotrophic lateral sclerosis amongst others.

Alkylamino derivatives of pyrazinamide: synthesis and antimycobacterial evaluation.

A series of pyrazinamide derivatives with alkylamino substitution was designed, synthesized and tested for their ability to inhibit the growth of selected mycobacterial, bacterial and fungal strains. The target structures were prepared from the corresponding 5-chloro (1) or 6-chloropyrazine-2-carboxamide (2) by nucleophilic substitution of chlorine by various non-aromatic amines (alkylamines). To determine the influence of alkyl substitution, corresponding amino derivatives (1a, 2a) and compounds with phenylalkylamino substitution were prepared. Some of the compounds exerted antimycobacterial activity against Mycobacterium tuberculosis H37Rv significantly better than standard pyrazinamide and corresponding starting compounds (1 and 2). Basic structure-activity relationships are presented. Only weak antibacterial and no antifungal activity was detected.

Synthesis, antimycobacterial activity and in vitro cytotoxicity of 5-chloro-N-phenylpyrazine-2-carboxamides.

5-Chloropyrazinamide (5-Cl-PZA) is an inhibitor of mycobacterial fatty acid synthase I with a broad spectrum of antimycobacterial activity in vitro. Some N-phenylpyrazine-2-carboxamides with different substituents on both the pyrazine and phenyl core possess significant in vitro activity against Mycobacterium tuberculosis. To test the activity of structures combining both the 5-Cl-PZA and anilide motifs a series of thirty 5-chloro-N-phenylpyrazine-2-carboxamides with various substituents R on the phenyl ring were synthesized and screened against M. tuberculosis H37Rv, M. kansasii and two strains of M. avium. Most of the compounds exerted activity against M. tuberculosis H37Rv in the range of MIC = 1.56-6.25 µg/mL and only three derivatives were inactive. The phenyl part of the molecule tolerated many different substituents while maintaining the activity. In vitro cytotoxicity was decreased in compounds with hydroxyl substituents, preferably combined with other hydrophilic substituents. 5-Chloro-N-(5-chloro-2-hydroxyphenyl)pyrazine-2-carboxamide (21) inhibited all of the tested strains (MIC = 1.56 µg/mL for M. tuberculosis; 12.5 µg/mL for other strains). 4-(5-Chloropyrazine-2-carboxamido)-2-hydroxybenzoic acid (30) preserved good activity (MIC = 3.13 µg/mL M. tuberculosis) and was rated as non-toxic in two in vitro models (Chinese hamster ovary and renal cell adenocarcinoma cell lines; SI = 47 and 35, respectively).

Preparation and in vitro screening of symmetrical bis-isoquinolinium cholinesterase inhibitors bearing various connecting linkage--implications for early Myasthenia gravis treatment.

Inhibitors of acetylcholinesterase are compounds widely used in the treatment of various diseases, such as Alzheimer's disease, glaucoma and Myasthenia gravis (MG). Compounds used in the therapy of MG posses a positive charge in the molecule to ensure peripheral effect of action and minimal blood-brain barrier penetration. The most prescribed carbamate inhibitors are however known for many severe side effects related to the carbamylation of AChE. This paper describes preparation and in vitro evaluation of 20 newly prepared bis-isoquinolinium inhibitors of potential concern for MG. The newly prepared compounds were evaluated in vitro on human recombinant AChE and human plasmatic butyrylcholinesterase (BChE). Their inhibitory ability was expressed as IC50 and compared to chosen standards ambenonium dichloride, edrophonium chloride, BW284c51 and ethopropazine hydrochloride. Three novel compounds presented promising inhibition (in nM range) of both enzymes in vitro better or similar to edrophonium and BW284c51, but worse to ambenonium. The novel inhibitors did not present higher selectivity toward AChE or BChE. The kinetic assay confirmed non-competitive inhibition of hAChE by two selected promising novel compounds. Two newly prepared compounds were also chosen for docking studies that confirmed apparent π-π or π-cationic interactions aside the cholinesterases catalytic sites. The SAR findings were discussed.

Design, evaluation and structure-activity relationship studies of the AChE reactivators against organophosphorus pesticides.

Organophosphate pesticides (OPPs; e.g. chlorpyrifos, diazinon, paraoxon) are a wide and heterogeneous group of organophosphorus compounds. Their biological activity of inhibiting acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) ranks them as life endangering agents. The necessary treatment after OPP exposure involves the use of parasympatolytics (e.g. atropine), oxime reactivators (e.g. obidoxime), and anticonvulsive drugs (e.g. diazepam). Therefore, the reactivators of AChE are essential compounds in the treatment of OPP intoxications. Commercial AChE reactivators (e.g. pralidoxime, HI-6, obidoxime, trimedoxime, methoxime) were originally developed for other members of the organophosphate family, such as nerve agents (e.g. sarin, soman, tabun, VX). Pralidoxime, HI-6, and methoxime were found to be weak reactivators of OPP-inhibited AChE. Obidoxime and trimedoxime showed satisfactory reactivation against various OPPs with minor toxicity issues. During the last two decades, the treatment of OPP exposure has become more widely discussed because of growing agricultural production, industrialization, and harmful social issues (e.g. suicides). In this review is the summarized design, evaluation, and structure-activity relationship studies of recently produced AChE reactivators. Since pralidoxime, over 300 oximes have been produced or tested against OPP poisoning, and several novel compounds show very promising abilities as comparable (or higher) to commercial oximes. Some of these are highlighted for their further testing of OPP exposure and, additionally, the main structure-activity relationship of AChE reactivators against OPP is discussed.

Preparation and in vitro screening of symmetrical bispyridinium cholinesterase inhibitors bearing different connecting linkage-initial study for Myasthenia gravis implications.

Reversible inhibitors (e.g., pyridostigmine bromide, neostigmine bromide) of carbamate origin are used in the early treatment of Myasthenia gravis (MG) to block acetylcholinesterase (AChE) native function and conserve efficient amount of acetylcholine for decreasing number of nicotinic receptors. Carbamate inhibitors are known for many undesirable side effects related to the reversible inhibition of AChE. In contrast, this paper describes 20 newly prepared bispyridinium inhibitors of potential concern for MG. Although some compounds from this series have been known before, they were not assayed for cholinesterase inhibition yet. The newly prepared compounds were evaluated in vitro on human erythrocyte AChE and human plasmatic butyrylcholinesterase (BChE). Their inhibitory ability was expressed as IC(50) and compared to standard carbamate drugs. Three compounds presented promising inhibition (in muM range) of both enzymes in vitro similar to the used standards. The novel inhibitors did not present selectivity between AChE and BChE. Two newly prepared compounds were chosen for docking studies and confirmed apparent pi-pi or pi-cationic interactions aside enzyme's catalytic sites. The kinetics assay confirmed non-competitive inhibition of AChE by two best newly prepared compounds.

Substituted pyrazinecarboxamides as abiotic elicitors of flavolignan production in Silybum marianum (L.) gaertn cultures in vitro.

Substituted pyrazinecarboxamides markedly influenced production of flavonolignans in Silybum marianum callus and suspension cultures. In this study the effect of two compounds, N-(3-iodo-4-methylphenyl)pyrazine-2-carboxamide (1) and N-(3-iodo-4-methylphenyl)-5-tert-butyl-pyrazine-2-carboxamide (2), as abiotic elicitors on flavono-lignan production in callus culture of S. marianum was investigated. Silymarin complex compounds have hepatoprotective, anticancer and also hypocholesterolemic activity. In vitro flavonolignan concentration in cells is very low and the elicitation is one of the methods to increase production. Elicitors were tested at three concentrations and at different culture times. In the case of elicitation with 1, the greatest increase of flavonolignan and taxifoline production was observed at concentration c(1a) after 6-hours of elicitation and after 24 and 72-hours at concentration c(1b). However, increased production of silychristin, one of the compounds in the silymarin complex, was achieved after only 6-hours elicitation with c(1a) (2.95 x 10(-4) mol/L). The content of silychristin was 2-times higher compared to the control sample. An increased production of silychristin was reached with compound 2 at the concentration c(2) (2.53 x 10(-3) mol/L) after 72 h of elicitation. The production of silychristin in this case was increased 12-times compared to control.