Synthesis of pyrazolo-enaminones, bipyrazoles and pyrazolopyrimidines and evaluation of antioxidant and antimicrobial properties

A novel pyrazolo-enaminones, bipyrazoles and bipyrazolopyridines from 1-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)butane-1,3-dione and 4-methyl-2-phenyl-2H-pyrazolo[3,4-b]pyridine-3,6(3aH,7H)-dione have been synthesized by assisted heating with microwave radiation without any catalyst. The pyridine and pyrazole ring formation has been developed from easily accessible enamino keto esters by formylation followed by intramolecular cyclization. The general applicability for the synthesis of the important pyrazolo-enaminones, bipyrazoles and pyrazolo-pyridines heterocycles was attributed to simplicity of operation, synthesis without catalyst, energy efficiency (shorter reaction time under microwave irradiation), good yields, more environmentally friendly and more cost-effective procedure. The antioxidant activity of new heterocyclic compounds was evaluated by free radical scavenging by DPPH assay. Several of these compounds showed good activity against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria

Inflammasome activation by SARS-CoV-2 accessory protein: Development of novel inhibitors of the SARS-CoV-2 accessory protein ORF9b

1 p.-5 fig.SARS-CoV-2 can activate the inflammasome, which, when unbridled, contributes to pathogenic inflammatory responses and to severe COVID-19. Several SARS-CoV-2 components have been shown to participate in this activation. Here, we have systematically assayed SARS-CoV-2 accessory proteins (ORF3a, ORF3b, ORF6, ORF7,ORF8, ORF9b, ORF9c and ORF10) for their ability to modulate inflammasome activity. We have found that among all accessory proteins, only ORF9b, a protein that locates in mitochondria, triggers a strong activation of caspase-1 activity and cytokine release in A549 lung epithelial cells and THP-1 monocyte-macrophage cells. This induction is observed both by transducing ORF9b alone or upon concomitant transduction of all accessory proteins. Based on the solved structure of ORF9b, we have conducted an in silico drug discovery effort to identify small molecules capable of disrupting the ORF9b homodimer and to attenuate its observed activity. Iterative steps of blind massive docking and molecular dynamics led to the identification of small molecules predicted to prevent ORF9b homodimeric interactions. This prediction was experimentally validated by means of surface plasmon resonance, yielding two active molecules. These molecules showed a potent inhibition of ORF9b-induced caspase-1 activation and cytokine release, and caused a remarkable eviction of ORF9b from mitochondria. These novel first-in-class ORF9b inhibitors are currently being tested for their ability to mitigate viral cytopathogenic effects.Peer reviewe

Dominant induction of the inflammasome by the SARS-CoV-2 accessory protein ORF9b, abrogated by small-molecule ORF9b homodimerization inhibitors

Viral accessory proteins play critical roles in viral escape form host innate immune responses and in viral inflammatory pathogenesis. Here we show that the SARS-CoV-2 accessory protein, ORF9b, but not other SARS-CoV-2 accessory proteins (ORF3a, ORF3b, ORF6, ORF7, ORF8, ORF9c, ORF10), strongly activates inflammasomedependent caspase-1 in A549 lung carcinoma cells and THP-1 monocyte-macrophage cells. Exposure to lipopolysaccharide (LPS) and ATP additively enhanced the activation of caspase-1 by ORF9b, suggesting that ORF9b and LPS follow parallel pathways in the activation of the inflammasome and caspase-1. Following rational in silico approaches, we have designed small molecules capable of inhibiting the homodimerization of ORF9b, which experimentally inhibited ORF9b-ORF9b homotypic interactions, caused mitochondrial eviction of ORF9b, inhibited ORF9b-induced activation of caspase-1 in A549 and THP-1 cells, cytokine release in THP-1 cells, and restored type I interferon (IFN-I) signaling suppressed by ORF9b in both cell models. These small molecules are first-in-class compounds targeting a viral accessory protein critical for viral-induced exacerbated inflammation and escape from innate immune responses, with the potential of mitigating the severe immunopathogenic damage induced by highly pathogenic coronaviruses and restoring antiviral innate immune responses curtailed by viral infection.This work was funded by the Spanish National Research Council (CSIC, project numbers CSIC-COV19-006, CSIC-COV-19-201, 202020E079 and 202320E187), the Catalan Agency for Management of University and Research Grants (AGAUR, 2020PANDE00048, 2021SGR1490, 2021SGR00350), the CSIC’s Global Health Platform (PTI Salud Global), The Networked Center for Biomedical Research in Liver and Digestive Diseases (CIBER-EHD), the Spanish Structures and Excellence María de Maeztu program (CEX2021-001202-M) and the European Commission-Next Generation EU (Regulation EU 2020/2094).N

Mg/Fe-LDH: An efficient promoter for the synthesis of tetrahydrospiro[pyrazole-4,5′-pyrazolo[3,4-b]pyridine] derivatives

The synthesis of tetrahydrospiro[pyrazole-4,5′-pyrazolo[3,4-b]pyridine] derivatives using Mg/Fe-LDH as a heterogeneous catalyst was reported. This methodology used for the condensation of pyrazolones with aldehydes in a basic medium and with the presence of the Mg/Fe-LDH catalyst presents many benefits, such as the fact of using a reusable and low-cost catalyst, environmentally friendly conditions, short reaction times and excellent yields. It has been verified and confirmed that the catalyst is relatively stable and allows its reuse without additional treatment. Condensation has been carried out with different aldehydes and bis-pyrazolone derivatives have been obtained with yields ranging from 71 to 91% of purified product. The stereo impedance of the adduct formed prevents subsequent condensation

Evaluation of the cellular protection by novel spiropyrazole compounds in dopaminergic cell death

Simoes, Mario/0000-0001-9071-2252; Neves, Maria da Graca P. M. S./0000-0002-7953-8166; Faustino, Maria Amparo/0000-0003-4423-3802; saso, luciano/0000-0003-4530-8706; Neves, Claudia/0000-0002-3654-5737WOS:000629626600024PubMed: 33454549The loss of neurons is strongly correlated with aging and aging-associated disorders. in this study, cell viability assays and mitochondrial function were performed to evaluate the effect of new spiro-pyrazole derivatives, prepared from aldehydes and 3-amino-1-phenyl-2-pyrazolin-5-one, on neuroprotection in an in vitro model of dopaminergic cell death induced by 1-methyl-4-phenylpyridinium (MPP+). The percentages of neuroprotection by derivatives were found between 21.26% and 52.67% at selected concentrations (10-50 mu M) with compound 4d exerting the best neuroprotective effect. The results show that the studied spiropyrazolones perform important roles in dopaminergic neuroprotection and can be used for potential new therapies in the treatment of neurodegenerative disorders including Parkinson's disease. (C) 2020 Elsevier Masson SAS. All rights reserved.University of AveiroEuropean Commission; Fundacao para a Ciencia e a Tecnologia (FCT, Portugal)/Ministerio da Ciencia, Tecnologia e Ensino Superior (MCTES, Portugal) [UIDB/50006/2020]; FEDER, within the PT2020 Partnership Agreement; Sultan Moulay Slimane University of Beni-Mellal (Morocco); National Center for Scientific and Technical Research (CNRST) -Morocco; FCT-CNRST (Morocco); FCTPortuguese Foundation for Science and TechnologyEuropean Commission [PD/BD/52531/2014]Thanks are due to the University of Aveiro and Fundacao para a Ciencia e a Tecnologia (FCT, Portugal)/Ministerio da Ciencia, Tecnologia e Ensino Superior (MCTES, Portugal) for the financial support for the LAQV-REQUIMTE (UIDB/50006/2020), through national founds and, where applicable, co-financed by the FEDER, within the PT2020 Partnership Agreement, and to the Portuguese NMR Network. The authors also thank the Sultan Moulay Slimane University of Beni-Mellal (Morocco) and the National Center for Scientific and Technical Research (CNRST) -Morocco for the funding the characterization analysis and to the Transnational cooperation programs, FCT-CNRST (Morocco), for financial assistance (2019-2020). C.M.B. Neves is also grateful to FCT for her Ph.D. Grant with the reference PD/BD/52531/2014. Authors also thank the Pharmaceutical Sciences Research Centre (FABAL, Ege University, Faculty of Pharmacy, Turkey) for equipmental support

Evaluation of the cellular protection by novel spiropyrazole compounds in dopaminergic cell death

The loss of neurons is strongly correlated with aging and aging-associated disorders. In this study, cell viability assays and mitochondrial function were performed to evaluate the effect of new spiro-pyrazole derivatives, prepared from aldehydes and 3-amino-1-phenyl-2-pyrazolin-5-one, on neuroprotection in an in vitro model of dopaminergic cell death induced by 1-methyl-4-phenylpyridinium (MPP+). The percentages of neuroprotection by derivatives were found between 21.26% and 52.67% at selected concentrations (10-50 μM) with compound 4d exerting the best neuroprotective effect. The results show that the studied spiropyrazolones perform important roles in dopaminergic neuroprotection and can be used for potential new therapies in the treatment of neurodegenerative disorders including Parkinson's disease