RESUMO
Hydrogenation reactions are staple transformations commonly used across scientific fields to synthesise pharmaceuticals, natural products, and various functional materials. However, the vast majority of these reactions require the use of a toxic and costly catalyst leading to unpractical, hazardous and often functionally limited conditions. Herein, we report a new, general, practical, efficient, mild and high-yielding hydrogen-free electrochemical method for the reduction of alkene, alkyne, nitro and azido groups. Finally, this method has been applied to deuterium labelling.
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The major clinical challenge in drug-resistant chronic myelogenous leukemia (CML) is currently represented by the Bcr-Abl T315I mutant, which is unresponsive to treatment with common first and second generation ATP-competitive tyrosine kinase inhibitors (TKIs). Allosteric inhibition of Bcr-Abl represent a new frontier in the fight against resistant leukemia and few candidates have been identified in the last few years. Among these, myristate pocket (MP) binders discovered by Novartis (e.g. GNF2/5) showed promising results, although they proved to be active against the T315I mutant only in combination with first and second generation ATP-competitive inhibitors. Here we used a cascade screening approach based on sequential fluorescence polarization (FP) screening, in silico docking/dynamics studies and kinetic-enzymatic studies to identify novel MP binders. A pyrazolo[3,4-d]pyrimidine derivative (6) has been identified as a promising allosteric inhibitor active on 32D leukemia cell lines (expressing Bcr-Abl WT and T315I) with no need of combination with any ATP-competitive inhibitor.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Proteínas de Membrana/antagonistas & inibidores , Miristatos/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Regulação Alostérica/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas de Membrana/genética , Modelos Moleculares , Estrutura Molecular , Mutação , Miristatos/síntese química , Miristatos/química , Proteínas de Neoplasias/genética , Relação Estrutura-AtividadeRESUMO
The clonidine-like central antihypertensive agent rilmenidine, which has high affinity for I1-type imidazoline receptors (I1-IR) was recently found to have cytotoxic effects on cultured cancer cell lines. However, due to its pharmacological effects resulting also from α2-adrenoceptor activation, rilmenidine cannot be considered a suitable anticancer drug candidate. Here, we report the identification of novel rilmenidine-derived compounds with anticancer potential and devoid of α2-adrenoceptor effects by means of ligand- and structure-based drug design approaches. Starting from a large virtual library, eleven compounds were selected, synthesized and submitted to biological evaluation. The most active compound 5 exhibited a cytotoxic profile similar to that of rilmenidine, but without appreciable affinity to α2-adrenoceptors. In addition, compound 5 significantly enhanced the apoptotic response to doxorubicin, and may thus represent an important tool for the development of better adjuvant chemotherapeutic strategies for doxorubicin-insensitive cancers.
Assuntos
Agonistas alfa-Adrenérgicos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Oxazóis/farmacologia , Apoptose/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Células K562 , Ligantes , Estrutura Molecular , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , RilmenidinaRESUMO
The discovery of a novel class of HCV inhibitors is described. The new amidinourea compounds were designed as isosteric analogues of the antiviral drug moroxydine. The two derivatives 11g and 11h showed excellent HCV inhibition activity and viability and proved to inhibit a step(s) of the RNA replication. The new compounds have been synthesized in only three synthetic steps from cheap building blocks and in high yields, thus turning to be promising drug candidates in the development of cheaper HCV treatments.
Assuntos
Antivirais/economia , Antivirais/farmacologia , Descoberta de Drogas , Hepacivirus/efeitos dos fármacos , Morfolinas/química , Morfolinas/farmacologia , Antivirais/síntese química , Biguanidas , Bioensaio , Relação Dose-Resposta a Droga , Descoberta de Drogas/tendências , Humanos , Viabilidade Microbiana/efeitos dos fármacos , Estrutura Molecular , Morfolinas/síntese químicaRESUMO
Abl kinase inhibitors targeting the ATP binding pocket are currently used as a front-line therapy for the treatment of chronic myelogenous leukemia (CML), but their use has significant limitation because of the development of drug resistance (especially due to the T315I mutation). Two compounds (GNF-2 and BO1) have been found able to inhibit the Abl activity through a peculiar mechanism of action. Particularly, GNF-2 acts as allosteric inhibitor against Bcr-Abl wild type (wt), but it has no activity against the gatekeeper mutant T315I. Its activity against the last mutant reappears when used together with an ATP-competitive inhibitor such as Imatinib or Nilotinib. A crystal structure of GNF-2 bound to the Abl myristoyl pocket (MP) has been released. On the contrary, BO1 shows an ATP-competitive/mixed mechanism of action against the wt, while it acts as an allosteric inhibitor against T315I. In order to better understand the mechanism of Abl allosteric inhibition, MD simulations and MM/GBSA analysis were performed on Abl wt and T315I in complex with GNF-2 and BO1, and the results were compared to those found for the natural myristoyl ligand. Similarly to that observed for the myristoyl group, the binding of an allosteric inhibitor to the MP promotes the formation of a compact and inhibited conformation of the wt protein, characterized by the stabilization of the intramolecular interactions that occur between SH2-SH3 and kinase domains. Conversely, an overall higher flexibility was observed with the Abl T315I mutant, especially in the case of GNF-2. Our analysis highlighted differences in the dynamic behavior of GNF-2 and BO1 which could explain the different biological profiles of the two allosteric inhibitors against the T315I mutant.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sinergismo Farmacológico , Estabilidade Enzimática/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Inibidores de Proteínas Quinases/metabolismo , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Domínios de Homologia de srcRESUMO
We report the development and application of different strategies for the late-stage functionalization (LSF) of the biologically relevant 4-amino-2-pyridone chemotype. Using the recently discovered PCSK9 inhibitor 5c as a prototype, a series of electrochemical LSF (e-LSF) and multicomponent LSF (MCR-LSF) have been set-up to decorate the 4-amino-2-pyridone scaffold. The usefulness of these methods has been demonstrated by generating a series of novel derivatives in a site-selective and sustainable way.
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The correlation between the CCL20/CCR6 axis and autoimmune and non-autoimmune disorders is widely recognized. Inhibition of the CCL20-dependent cell migration represents therefore a promising approach for the treatment of many diseases, such as inflammatory bowel diseases and colorectal cancer. We report herein our efforts to explore the biologically relevant chemical space around the benzofuran scaffold of MR120, a modulator of the CCL20/CCR6 axis previously discovered by our group. A functional screening allowed us to identify C4 and C5-substituted derivatives as the most effective inhibitors of the CCL20-induced chemotaxis of human peripheral blood mononuclear cells (PBMC). Moreover, selected compounds (16 e and 24 b) also proved to potently inhibit the growth of different colon cancer cell lines, with cytotoxic/cytostatic and antiproliferative activity.
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Respiratory tract infections involving a variety of microorganisms such as viruses, bacteria, and fungi are a prominent cause of morbidity and mortality globally, exacerbating various pre-existing respiratory and non-respiratory conditions. Moreover, the ability of bacteria and viruses to coexist might impact the development and severity of lung infections, promoting bacterial colonization and subsequent disease exacerbation. Secondary bacterial infections following viral infections represent a complex challenge to be overcome from a therapeutic point of view. We report herein our efforts in the development of new bithiazole derivatives showing broad-spectrum antimicrobial activity against both viruses and bacteria. A series of 4-trifluoromethyl bithiazole analogues was synthesized and screened against selected viruses (hRVA16, EVD68, and ZIKV) and a panel of Gram-positive and Gram-negative bacteria. Among them, two promising broad-spectrum antimicrobial compounds (8a and 8j) have been identified: both compounds showed low micromolar activity against all tested viruses, 8a showed synergistic activity against E. coli and A. baumannii in the presence of a subinhibitory concentration of colistin, while 8j showed a broader spectrum of activity against Gram-positive and Gram-negative bacteria. Activity against antibiotic-resistant clinical isolates is also reported. Given the ever-increasing need to adequately address viral and bacterial infections or co-infections, this study paves the way for the development of new agents with broad antimicrobial properties and synergistic activity with common antivirals and antibacterials.
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Among the strategies to overcome the underperformance of statins in cardiovascular diseases (CVDs), the development of drugs targeting the Proprotein Convertase Subtilisin-like Kexin type 9 (PCSK9) is considered one of the most promising. However, only anti-PCSK9 biological drugs have been approved to date, and orally available small-molecules for the treatment of hypercholesterolemic conditions are still missing on the market. In the present work, we describe the application of a phenotypic approach to the identification and optimization of 4-amino-2-pyridone derivatives as a new chemotype with anti-PCSK9 activity. Starting from an in-house collection of compounds, functional assays on HepG2 cells followed by a chemistry-driven hit optimization campaign, led to the potent anti-PCSK9 candidate 5c. This compound, at 5 µM, totally blocked PCSK9 secretion from HepG2 cells, significantly increased LDL receptor (LDLR) expression, and acted cooperatively with simvastatin by reducing its induction of PCSK9 expression. Finally, compound 5c also proved to be well tolerated in C57BL/6J mice at the tested concentration (40 mg/kg) with no sign of toxicity or behavior modifications.
Assuntos
Inibidores de PCSK9 , Pró-Proteína Convertase 9 , Animais , Humanos , Camundongos , Células Hep G2 , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9/metabolismo , Receptores de LDL/metabolismo , Piridonas/química , Piridonas/metabolismoRESUMO
Targeted molecular dynamics (TMD) simulations allowed for identifying the chemical/structural features of the nucleotide-competitive HIV-1 inhibitor DAVP-1, which is responsible for the disruption of the T-shape motif between Try183 and Trp229 of the reverse transcriptase (RT). DAVP-1 promoted the opening of a connection "gate" between allosteric and catalytic sites of HIV-1 RT, thus explaining its peculiar mechanism of action and providing useful insights to develop novel nucleotide-competitive RT inhibitors.
Assuntos
Transcriptase Reversa do HIV/química , HIV-1/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Pirimidinas/química , Inibidores da Transcriptase Reversa/química , Compostos de Vinila/química , Sítio Alostérico , Ligação Competitiva , Domínio Catalítico , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/enzimologia , Compostos Heterocíclicos com 3 Anéis/química , Humanos , Indóis/química , Nitrilas/química , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Piridonas/química , Relação Estrutura-AtividadeRESUMO
Concerning the growing interest in the role played by the CCL20/CCR6 axis in IBD pathogenesis and in the search for novel anti-IBD small molecules, we have recently discovered the first small-molecule (MR120) endowed with protective action against TNBS-induced colitis and zymosan-induced peritonitis. This protective action occurs through interference with the CCL20/CCR6 signaling. The aim of the present work is to expand the preclinical investigation of MR120, evaluating its beneficial anti-inflammatory effect on a model of chronic colitis obtained by cyclically exposing C57BL/6 mice to 3% DSS. Subcutaneous administration of MR120 at 1 mg/kg, the same dose effective against acute inflammation, helped attenuate several systemic and local inflammatory responses induced by DSS. Besides significantly improving murine health conditions, MR120 counteracted mucosal macroscopic injury, the increase of colonic edema and neutrophils oxidative activity, and mitigated spleen enlargement, while not significantly lowering intestinal IL-6 concentration. Overall, repeated daily treatment with MR120 for approximately 30 days was well tolerated and showed moderate protection in a relevant model of chronic colitis, in line with the beneficial effect previously observed in acute models of intestinal inflammation. Although more potent analogues of MR120 will be needed to more fully evaluate their clinical translatability, the present work provides a valuable example of in vivo efficacy of CCL20/CCR6 modulators in a chronic model of IBD.
Assuntos
Colite , Animais , Camundongos , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/patologia , Colo , Sulfato de Dextrana/efeitos adversos , Modelos Animais de Doenças , Inflamação/patologia , Intestinos/patologia , Camundongos Endogâmicos C57BL , Receptores CCR6RESUMO
The plethora of viral outbreaks experienced in the last decade, together with the widespread distribution of many re-emerging and newly emerging viruses, emphasize the urgent need for novel broad-spectrum antivirals as tools for early intervention in case of future epidemics. Non-natural nucleosides have been at the forefront for the treatment of infectious diseases for many years and still represent one of the most successful classes of antiviral molecules on the market. In the attempt to explore the biologically relevant chemical space of this class of antimicrobials, we describe herein the development of novel base-modified nucleosides by converting previously identified 2,6-diaminopurine antivirals into the corresponding D/L ribonucleosides, acyclic nucleosides and prodrug derivatives. A phenotypic screening against viruses belonging to different families (Flaviviridae, Coronaviridae, Retroviridae) and against a panel of Gram-positive and Gram-negative bacteria, allowed to identify a few interesting molecules with broad-spectrum antimicrobial activities.
Assuntos
Antivirais , Vírus , Humanos , Antivirais/química , Nucleosídeos/química , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias Gram-Negativas , Bactérias Gram-PositivasRESUMO
Efficacy of currently approved anti-HIV drugs is hampered by mutations of the viral enzymes, leading invariably to drug resistance and chemotherapy failure. Recent data suggest that cellular co-factors also represent useful targets for anti-HIV therapy. Here we describe the identification of the first small molecules specifically designed to inhibit the HIV-1 replication by targeting the RNA binding site of the human DEAD-Box RNA helicase DDX3. Optimization of a easily synthetically accessible hit (1) identified by application of a high-throughput docking approach afforded the promising compounds 6 and 8 which proved to inhibit both the helicase and ATPase activity of DDX3 and to reduce the viral load of peripheral blood mononuclear cells (PBMC) infected with HIV-1.
Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/metabolismo , HIV-1/efeitos dos fármacos , RNA Viral/metabolismo , Adenosina Trifosfatases/metabolismo , Sítios de Ligação/efeitos dos fármacos , RNA Helicases DEAD-box/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/enzimologia , HIV-1/fisiologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
A combined targeted/phenotypic approach for the rapid identification of novel antiangiogenics with in vivo efficacy is herein reported. Considering the important role played by the tyrosine kinase c-Src in the regulation of tumour angiogenesis, we submitted our in-house library of c-Src inhibitors to a sequential screening approach: in silico screening on VEGFR2, in vitro screening on HUVEC cells, ADME profiling, formulation and in vivo testing on a zebrafish model. A promising antiangiogenic candidate able to interfere with the vascular growth of a zebrafish model at low micromolar concentration was thus identified.
Assuntos
Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Descoberta de Drogas/métodos , Embrião não Mamífero/irrigação sanguínea , Neovascularização Patológica/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Ciclodextrinas/química , Portadores de Fármacos/química , Embrião não Mamífero/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Modelos Animais , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidores , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Peixe-Zebra/embriologiaRESUMO
The CCL20/CCR6 axis is implicated in the migration of CCR6+ immune cells towards CCL20, its sole ligand, whose expression is increased during inflammatory processes and is known to play a pivotal role in triggering different autoimmune-mediated inflammatory diseases. Herein, we report a drug discovery effort focused on the development of a new pharmacological approach for the treatment of inflammatory bowel diseases (IBDs) based on small-molecule CCR6 antagonists. The most promising compound 1b was identified by combining in silico studies, sustainable chemistry and in vitro functional/targeted assays, and its efficacy was finally validated in a classic murine model of colitis (TNBS-induced) and in a model of peritonitis (zymosan-induced). These data provide the proof of principle that a pharmacological modulation of the CCL20/CCR6 axis may indeed represent the first step for the development of an orally bioavailable drug candidate for the treatment of IBD and, potentially, other diseases regulated by the CCL20/CCR6 axis.
Assuntos
Doenças Autoimunes , Doenças Inflamatórias Intestinais , Camundongos , Humanos , Animais , Receptores CCR6/metabolismo , Quimiocina CCL20/metabolismo , Doenças Inflamatórias Intestinais/tratamento farmacológicoRESUMO
Cystic fibrosis (CF) is a genetic disease caused by loss-of-function mutations in the CFTR gene, which codes for a defective ion channel. This causes an electrolyte imbalance and results in a spiral of negative effects on multiple organs, most notably the accumulation of thick mucus in the lungs, chronic respiratory tract infections and inflammation leading to pulmonary exacerbation and premature death. Progressive decline of lung function is mainly linked to persistent or recurring infections, mostly caused by bacteria, which require treatments with antibiotics and represent one of the major life-limiting factors in subjects with CF. Treatment of such a complex disease require multiple drugs with a consequent therapeutic burden and complications caused by drug-drug interactions and rapid emergence of bacterial drug resistance. We report herein our recent efforts in developing innovative multifunctional antibiotics specifically tailored to CF by a direct action on bacterial topoisomerases and a potential indirect effect on the pulmonary mucociliary clearance mediated by ΔF508-CFTR correction. The obtained results may pave the way for the development of a simplified therapeutic approach with a single agent acting as multifunctional Antibacterial-Corrector.
Assuntos
Fibrose Cística , Microbiota , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Fibrose Cística/tratamento farmacológico , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Pulmão , MutaçãoRESUMO
Neuroblastoma (NB) represents the most common extracranial paediatric solid tumor for which no specific FDA-approved treatment is currently available. The tyrosine kinase c-Src has been reported to play an important role in the differentiation, cell-adhesion and survival of NB cells. Starting from dual Src/Abl inhibitors previously found active in NB cell lines (1-3), small modification of the original structures almost abolished the Abl activity with a contemporary improvement of affinity and specificity for c-Src. Among the synthesized compounds, the most potent c-Src inhibitor (10a) showed a very interesting antiproliferative activity in SH-SY5Y cells with an IC(50) of 80 nM and a favourable ADME profile. A 3D SAR analysis was also attempted and may guide the design of more potent c-Src inhibitors as potential agents for NB treatment.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Desenho de Fármacos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Quinases da Família src/antagonistas & inibidores , Antineoplásicos/química , Antineoplásicos/farmacocinética , Adesão Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Terapia de Alvo Molecular , Neuroblastoma/tratamento farmacológico , Neuroblastoma/patologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Pirazóis/síntese química , Pirimidinas , Relação Quantitativa Estrutura-Atividade , Especificidade por Substrato , Quinases da Família src/metabolismoRESUMO
Targeting the binding site of 14-3-3 proteins lets the release of partner proteins involved in cell cycle progression, apoptosis, cytoskeletal rearrangement and transcriptional regulation and may therefore be regarded as an alternative strategy to integrate conventional therapeutic approaches against cancer. In the present work, we report the identification of two new small molecule inhibitors of 14-3-3σ/c-Abl protein-protein interaction (BV01 and BV101) discovered by means of computational methods. The most interesting compound (BV01) showed a lethal dose (LD(50)) in the low micromolar range against Ba/F3 murine cell lines expressing the Imatinib (IM)-sensitive wild type Bcr-Abl construct and the IM-resistant Bcr-Abl mutation T315I. BV01 interaction with 14-3-3σ was demonstrated by NMR studies and elucidated by docking. It blocked the binding domain of 14-3-3σ, hence promoting the release of the partner protein c-Abl (the one not involved in Bcr rearrangement), and its translocation to both the nuclear compartment and mitochondrial membranes to induce a pro-apoptotic response. Our results advance BV01 as a confirmed hit compound capable of eliciting apoptotic death of Bcr-Abl-expressing cells by interfering with 14-3-3σ/c-Abl protein-protein interaction.
Assuntos
Proteínas 14-3-3/metabolismo , Biomarcadores Tumorais/metabolismo , Desenho de Fármacos , Exonucleases/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-abl/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas 14-3-3/antagonistas & inibidores , Proteínas 14-3-3/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/química , Linhagem Celular , Exonucleases/antagonistas & inibidores , Exonucleases/química , Exorribonucleases , Humanos , Camundongos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidoresRESUMO
The worldwide circulation of different viruses coupled with the increased frequency and diversity of new outbreaks, strongly highlight the need for new antiviral drugs to quickly react against potential pandemic pathogens. Broad-spectrum antiviral agents (BSAAs) represent the ideal option for a prompt response against multiple viruses, new and re-emerging. Starting from previously identified anti-flavivirus hits, we report herein the identification of promising BSAAs by submitting the multi-target 2,6-diaminopurine chemotype to a system-oriented optimization based on phenotypic screening on cell cultures infected with different viruses. Among the synthesized compounds, 6i showed low micromolar potency against Dengue, Zika, West Nile and Influenza A viruses (IC50 = 0.5-5.3 µM) with high selectivity index. Interestingly, 6i also inhibited SARS-CoV-2 replication in different cell lines, with higher potency on Calu-3 cells that better mimic the SARS-CoV-2 infection in vivo (IC50 = 0.5 µM, SI = 240). The multi-target effect of 6i on flavivirus replication was also analyzed in whole cell studies (in vitro selection and immunofluorescence) and against isolated host/viral targets.