Discovery of 2-phenylquinazolines as potent Mycobacterium avium efflux pump inhibitors able to synergize with clarithromycin against clinical isolate.

Nontuberculous mycobacteria (NTM), which include the Mycobacterium avium complex, are classified as difficult-to-treat pathogens due to their ability to quickly develop drug resistance against the most common antibiotics used to treat NTM infections. The overexpression of efflux pumps (EPs) was demonstrated to be a key mechanism of clarithromycin (CLA) resistance in NTM. Therefore, in this work, 24 compounds from an in-house library, characterized by chemical diversity, were tested as potential NTM EP inhibitors (EPIs) against Mycobacterium smegmatis mc2 155 and M. avium clinical isolates. Based on the acquired results, 12 novel analogs of the best derivatives 1b and 7b were designed and synthesized to improve the NTM EP inhibition activity. Among the second set of compounds, 13b emerged as the most potent NTM EPI. At a concentration of 4 µg/mL, it reduced the CLA minimum inhibitory concentration by 16-fold against the clinical isolate M. avium 2373 overexpressing EPs as primary mechanism of CLA resistance.

Small Molecule Drugs Targeting Viral Polymerases.

Small molecules that specifically target viral polymerases-crucial enzymes governing viral genome transcription and replication-play a pivotal role in combating viral infections. Presently, approved polymerase inhibitors cover nine human viruses, spanning both DNA and RNA viruses. This review provides a comprehensive analysis of these licensed drugs, encompassing nucleoside/nucleotide inhibitors (NIs), non-nucleoside inhibitors (NNIs), and mutagenic agents. For each compound, we describe the specific targeted virus and related polymerase enzyme, the mechanism of action, and the relevant bioactivity data. This wealth of information serves as a valuable resource for researchers actively engaged in antiviral drug discovery efforts, offering a complete overview of established strategies as well as insights for shaping the development of next-generation antiviral therapeutics.

Switching the three-component Biginelli-like reaction conditions for the regioselective synthesis of new 2-amino[1,2,4]triazolo[1,5-a]pyrimidines.

Among the eight different triazolopyrimidine isomers existing in nature, 1,2,4-triazolo[1,5-a]pyrimidine (TZP) is one of the most studied and used isomers in medicinal chemistry. For some years, our group has been involved in developing regioselective one-pot procedures for the synthesis of 2-amino-7-aryl-5-methyl- and 2-amino-5-aryl-7-methyl-TZPs of interest in the preparation of antiviral agents. In this work, taking advantage of a Biginelli-like multicomponent reaction (MCR), we report the identification of finely tunable conditions to regioselectively synthesize C-6 ester-substituted amino-TZP analogues, both in dihydro and oxidized forms. Indeed, the use of mild acidic conditions is strongly directed toward the regioselective synthesis of 5-aryl-7-methyl C-6-substituted TZP analogues, while the use of neutral ionic liquids shifted the regioselectivity towards 7-aryl-5-methyl derivatives. In addition, the novel synthesized scaffolds were functionalized at the C-2 position and evaluated for their antiviral activity against RNA viruses (influenza virus, flaviviruses, and SARS-CoV-2). Compounds 25 and 26 emerged as promising anti-flavivirus agents, showing activity in the low micromolar range.

Identification of Anti-Influenza A Compounds Inhibiting the Viral Non-Structural Protein 1 (NS1) Using a Type I Interferon-Driven Screening Strategy.

There is an urgent need to identify efficient antiviral compounds to combat existing and emerging RNA virus infections, particularly those related to seasonal and pandemic influenza outbreaks. While inhibitors of the influenza viral integral membrane proton channel protein (M2), neuraminidase (NA), and cap-dependent endonuclease are available, circulating influenza viruses acquire resistance over time. Thus, the need for the development of additional anti-influenza drugs with novel mechanisms of action exists. In the present study, a cell-based screening assay and a small molecule library were used to screen for activities that antagonized influenza A non-structural protein 1 (NS1), a highly conserved, multifunctional accessory protein that inhibits the type I interferon response against influenza. Two potential anti-influenza agents, compounds 157 and 164, were identified with anti-NS1 activity, resulting in the reduction of A/PR/8/34(H1N1) influenza A virus replication and the restoration of IFN-ß expression in human lung epithelial A549 cells. A 3D pharmacophore modeling study of the active compounds provided a glimpse of the structural motifs that may contribute to anti-influenza virus activity. This screening approach is amenable to a broader analysis of small molecule compounds to inhibit other viral targets.

Functionalized sulfonyl anthranilic acid derivatives inhibit replication of all the four dengue serotypes.

Dengue virus (DENV), a mosquito-borne flavivirus, continues to be a major public health threat in many countries and no approved antiviral therapeutics are available yet. In this work, we designed and synthesized a series of sulfonyl anthranilic acid (SAA) derivatives using a ligand-based scaffold morphing approach of the 2,1-benzothiazine 2,2-dioxide core, previously used by us to develop DENV polymerase inhibitors resulting devoid of any cell-based antiviral activity. Several derivatives based on the new SAA chemotype exhibited potent inhibition against DENV infection in the cell-based assay but did not inhibit DENV NS5 polymerase activity in the in vitro de novo initiation and elongation assays. Notably, best compounds 26 and 39 showed EC50 values in the range of 0.54-1.36 µM against cells infected with the four dengue serotypes (DENV-1-4). Time-of-drug-addition assay revealed that analogue 26 is a post-entry replication inhibitor that appears to be specific for cells of primate origin, implicating a host target with a high barrier to resistance. In conclusion, SAA derivatives offer a valuable starting point for developing effective Dengue antiviral therapeutics.

Broad-Spectrum Anti-Flavivirus Activity and Chemistry of Compounds Containing Sulfur and Oxygen Chalcogens.

Sulfur and oxygen containing-compounds are a relevant class of derivatives that is constantly growing due to their wide range of pharmacological activity, including the antiviral one. As proof of this, there are several FDA approved antiviral compounds having sulfur and oxygen in their structures. Among RNA viruses, the flavivirus genus (e.g. Dengue, West Nile, Yellow Fever and Zika viruses) holds a relevant place within zoonotic pathogens and thus flavivirus infections are considered a growing risk for the public health. As a consequence, the drug discovery process aimed at identify new anti- flavivirus agents is of great relevance and will help to find effective therapies not available yet. One of the most alarming features of flaviviruses is their ability to co-infect the host, thus aggravating the symptoms of the disease. Therefore, finding compounds endowed with a broad-spectrum anti-flavivirus activity is now becoming a pressing need. In this review, we describe the most promising compounds having both sulfur and oxygen in their structures characterized by a broad-spectrum activity against different flaviviruses. Furthermore, the synthetic procedures applied for the preparation of the described derivatives are also reported. Readers can be inspired by the contents of this review to design and synthesize more effective anti-flavivirus agents as well as to select viral or host targets to achieve an antiviral activity as broadly as possible.

New C-6 functionalized quinoline NorA inhibitors strongly synergize with ciprofloxacin against planktonic and biofilm growing resistant Staphylococcus aureus strains.

Antimicrobial resistance (AMR) represents a global health issue threatening our social lifestyle and the world economy. Efflux pumps are widely involved in AMR by playing a primary role in the development of specific mechanisms of resistance. In addition, they seem to be involved in the process of biofilm formation and maintenance, contributing to enhance the risk of creating superbugs difficult to treat. Accordingly, the identification of non-antibiotic molecules able to block efflux pumps, namely efflux pump inhibitors (EPIs), could be a promising strategy to counteract AMR and restore the antimicrobial activity of ineffective antibiotics. Herein, we enlarge the knowledge about the structure-activity relationship of 2-phenylquinoline Staphylococcus aureus NorA EPIs by reporting a new series of very potent C-6 functionalized derivatives. Best compounds significantly inhibited ethidium bromide efflux in a NorA-overexpressing S. aureus strain (SA-1199B) and strongly synergized at very low concentrations (0.20-0.78 µg/mL) with ciprofloxacin (CPX) against CPX-resistant S. aureus strains (SA-1199B and SA-K2378), as proved by checkerboard and time-kill experiments. In addition, some of these EPIs (9b and 10a) produced a post-antibiotic effect of 1.2 h and strongly enhanced antibiofilm activity of CPX against SA-1199B strain. Interestingly, at the concentrations used to reach synergy with CPX against resistant S. aureus strains, most of the EPI compounds did not show any human cell toxicity. Finally, by exploiting the recent released crystal structure of NorA, we observed that best EPI 9b highlighted a favourable docking pose, establishing some interesting interactions with key residues.

Discovery of 2-Phenylquinolines with Broad-Spectrum Anti-coronavirus Activity.

A selection of compounds from a proprietary library, based on chemical diversity and various biological activities, was evaluated as potential inhibitors of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in a phenotypic-based screening assay. A compound based on a 2-phenylquinoline scaffold emerged as the most promising hit, with EC50 and CC50 values of 6 and 18 µM, respectively. The subsequent selection of additional analogues, along with the synthesis of ad hoc derivatives, led to compounds that maintained low µM activity as inhibitors of SARS-CoV-2 replication and lacked cytotoxicity at 100 µM. In addition, the most promising congeners also show pronounced antiviral activity against the human coronaviruses HCoV-229E and HCoV-OC43, with EC50 values ranging from 0.2 to 9.4 µM. The presence of a 6,7-dimethoxytetrahydroisoquinoline group at the C-4 position of the 2-phenylquinoline core gave compound 6g that showed potent activity against SARS-CoV-2 helicase (nsp13), a highly conserved enzyme, highlighting a potentiality against emerging HCoVs outbreaks.

Triazolopyrimidine Nuclei: Privileged Scaffolds for Developing Antiviral Agents with a Proper Pharmacokinetic Profile.

Viruses are a continuing threat to global health. The lack or limited therapeutic armamentarium against some viral infections and increasing drug resistance issues make the search for new antiviral agents urgent. In recent years, a growing literature highlighted the use of triazolopyrimidine (TZP) heterocycles in the development of antiviral agents, with numerous compounds that showed potent antiviral activities against different RNA and DNA viruses. TZP core represents a privileged scaffold for achieving biologically active molecules, thanks to: i) the synthetic feasibility that allows to variously functionalize TZPs in the different positions of the nucleus, ii) the ability of TZP core to establish multiple interactions with the molecular target, and iii) its favorable pharmacokinetic properties. In the present review, after mentioning selected examples of TZP-based compounds with varied biological activities, we will focus on those antivirals that appeared in the literature in the last 10 years. Approaches used for their identification, the hit-to-lead studies, and the emerged structure-activity relationship will be described. A mention of the synthetic methodologies to prepare TZP nuclei will also be given. In addition, their mechanism of action, the binding mode within the biological target, and pharmacokinetic properties will be analyzed, highlighting the strengths and weaknesses of compounds based on the TZP scaffold, which is increasingly used in medicinal chemistry.

From Quinoline to Quinazoline-Based S. aureus NorA Efflux Pump Inhibitors by Coupling a Focused Scaffold Hopping Approach and a Pharmacophore Search.

Antibiotic resistance breakers, such as efflux pump inhibitors (EPIs), represent a powerful alternative to the development of new antimicrobials. Recently, by using previously described EPIs, we developed pharmacophore models able to identify inhibitors of NorA, the most studied efflux pump of Staphylococcus aureus. Herein we report the pharmacophore-based virtual screening of a library of new potential NorA EPIs generated by an in-silico scaffold hopping approach of the quinoline core. After chemical synthesis and biological evaluation of the best virtual hits, we found the quinazoline core as the best performing scaffold. Accordingly, we designed and synthesized a series of functionalized 2-arylquinazolines, which were further evaluated as NorA EPIs. Four of them exhibited a strong synergism with ciprofloxacin and a good inhibition of ethidium bromide efflux on resistant S. aureus strains coupled with low cytotoxicity against human cell lines, thus highlighting a promising safety profile.

1,2,4-Triazolo[1,5-a]pyrimidines: Efficient one-step synthesis and functionalization as influenza polymerase PA-PB1 interaction disruptors.

In the search for new anti-influenza virus (IV) compounds, we have identified the 1,2,4-triazolo[1,5-a]pyrimidine (TZP) as a very suitable scaffold to obtain compounds able to disrupt IV RNA-dependent RNA polymerase (RdRP) PA-PB1 subunits heterodimerization. In this work, in order to acquire further SAR insights for this class of compounds and identify more potent derivatives, we designed and synthesized additional series of analogues to investigate the role of the substituents around the TZP core. To this aim, we developed four facile and efficient one-step procedures for the synthesis of 5-phenyl-, 6-phenyl- and 7-phenyl-2-amino-[1,2,4]triazolo[1,5-a]pyrimidines, and 2-amino-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol. Two analogues having the ethyl carboxylate moiety at the C-2 position of the TZP were also prepared in good yields. Then, the scaffolds herein synthesized and two previous scaffolds were functionalized and evaluated for their anti-IAV activity, leading to the identification of compound 22 that showed both anti-PA-PB1 (IC50 = 19.5 µM) and anti-IAV activity (EC50 = 16 µM) at non-toxic concentrations, thus resulting among the most active TZP derivatives reported to date by us. A selection of the synthesized compounds, along with a set of in-house available analogues, was also tested against SARS-CoV-2. The most promising compound 49 from this series displayed an EC50 value of 34.47 µM, highlighting the potential of the TPZ scaffold in the search for anti-CoV agents.

Synthesis and characterization of 1,2,4-triazolo[1,5-a]pyrimidine-2-carboxamide-based compounds targeting the PA-PB1 interface of influenza A virus polymerase.

Influenza viruses (Flu) are responsible for seasonal epidemics causing high rates of morbidity, which can dramatically increase during severe pandemic outbreaks. Antiviral drugs are an indispensable weapon to treat infected people and reduce the impact on human health, nevertheless anti-Flu armamentarium still remains inadequate. In search for new anti-Flu drugs, our group has focused on viral RNA-dependent RNA polymerase (RdRP) developing disruptors of PA-PB1 subunits interface with the best compounds characterized by cycloheptathiophene-3-carboxamide and 1,2,4-triazolo[1,5-a]pyrimidine-2-carboxamide scaffolds. By merging these moieties, two very interesting hybrid compounds were recently identified, starting from which, in this paper, a series of analogues were designed and synthesized. In particular, a thorough exploration of the cycloheptathiophene-3-carboxamide moiety led to acquire important SAR insight and identify new active compounds showing both the ability to inhibit PA-PB1 interaction and viral replication in the micromolar range and at non-toxic concentrations. For few compounds, the ability to efficiently inhibit PA-PB1 subunits interaction did not translate into anti-Flu activity. Chemical/physical properties were investigated for a couple of compounds suggesting that the low solubility of compound 14, due to a strong crystal lattice, may have impaired its antiviral activity. Finally, computational studies performed on compound 23, in which the phenyl ring suitably replaced the cycloheptathiophene, suggested that, in addition to hydrophobic interactions, H-bonds enhanced its binding within the PAC cavity.

Sustainable, three-component, one-pot procedure to obtain active anti-flavivirus agents.

The mosquito-borne viruses belonging to the genus Flavivirus such as Dengue virus (DENV) and Zika virus (ZIKV) cause human infections ranging from mild flu-like symptoms to hemorrhagic fevers, hepatitis, and neuropathies. To date, there are vaccines only for few flaviviruses while no effective treatments are available. Pyridobenzothiazole (PBTZ) derivatives are a class of compounds endowed with a promising broad-spectrum anti-flavivirus activity and most of them have been reported as potent inhibitors of the flaviviral NS5 polymerase. However, synthesis of PBTZ analogues entails a high number of purification steps, the use of hazardous reagents and environmentally unsustainable generation of waste. Considering the promising antiviral activity of PBTZ analogues which require further exploration, in this work, we report the development of a new and sustainable three-component reaction (3CR) that can be combined with a basic hydrolysis in a one-pot procedure to obtain the PBTZ scaffold, thus reducing the number of synthetic steps, improving yields and saving time. 3CR was significantly explored in order to demonstrate its wide scope by using different starting materials. In addition, taking advantage of these procedures, we next designed and synthesized a new set of PBTZ analogues that were tested as anti-DENV-2 and anti-ZIKV agents. Compound 22 inhibited DENV-2 NS5 polymerase with an IC50 of 10.4 µM and represented the best anti-flavivirus compound of the new series by inhibiting DENV-2- and ZIKV-infected cells with EC50 values of 1.2 and 5.0 µM, respectively, that translates into attractive selectivity indexes (SI - 83 and 20, respectively). These results strongly reaffirm PBTZ derivatives as promising anti-flavivirus agents that now can be synthesized through a convenient and sustainable 3CR in order to obtain more potent compounds for further pre-clinical development studies.

Inhibition of Influenza Virus Polymerase by Interfering with Its Protein-Protein Interactions.

Influenza (flu) virus is a serious threat to global health with the potential to generate devastating pandemics. The availability of broad spectrum antiviral drugs is an unequaled weapon during pandemic events, especially when a vaccine is still not available. One of the most promising targets for the development of new antiflu therapeutics is the viral RNA-dependent RNA polymerase (RdRP). The assembly of the flu RdRP heterotrimeric complex from the individual polymerase acidic protein (PA), polymerase basic protein 1 (PB1), and polymerase basic protein 2 (PB2) subunits is a prerequisite for RdRP functions, such as mRNA synthesis and genome replication. In this Review, we report the known protein-protein interactions (PPIs) occurring by RdRP that could be disrupted by small molecules and analyze their benefits and drawbacks as drug targets. An overview of small molecules able to interfere with flu RdRP functions exploiting the PPI inhibition approach is described. In particular, an update on the most recent inhibitors targeting the well-consolidated RdRP PA-PB1 subunit heterodimerization is mainly reported, together with pioneer inhibitors targeting other virus-virus or virus-host interactions involving RdRP subunits. As demonstrated by the PA-PB1 interaction inhibitors discussed herein, the inhibition of flu RdRP functions by PPI disrupters clearly represents a valid means to identify compounds endowed with a broad spectrum of action and a reduced propensity to develop drug resistance, which are the main issues of antiviral drugs.

Structural Modifications of the Quinolin-4-yloxy Core to Obtain New Staphylococcus aureus NorA Inhibitors.

Tackling antimicrobial resistance (AMR) represents a social responsibility aimed at renewing the antimicrobial armamentarium and identifying novel therapeutical approaches. Among the possible strategies, efflux pumps inhibition offers the advantage to contrast the resistance against all drugs which can be extruded. Efflux pump inhibitors (EPIs) are molecules devoid of any antimicrobial activity, but synergizing with pumps-substrate antibiotics. Herein, we performed an in silico scaffold hopping approach starting from quinolin-4-yloxy-based Staphylococcus aureus NorA EPIs by using previously built pharmacophore models for NorA inhibition activity. Four scaffolds were identified, synthesized, and modified with appropriate substituents to obtain new compounds, that were evaluated for their ability to inhibit NorA and synergize with the fluoroquinolone ciprofloxacin against resistant S. aureus strains. The two quinoline-4-carboxamide derivatives 3a and 3b showed the best results being synergic (4-fold MIC reduction) with ciprofloxacin at concentrations as low as 3.13 and 1.56 µg/mL, respectively, which were nontoxic for human THP-1 and A549 cells. The NorA inhibition was confirmed by SA-1199B ethidium bromide efflux and checkerboard assays against the isogenic pair SA-K2378 (norA++)/SA-K1902 (norA-). These in vitro results indicate the two compounds as valuable structures for designing novel S. aureus NorA inhibitors to be used in association with fluoroquinolones.

Broad-Spectrum Flavivirus Inhibitors: a Medicinal Chemistry Point of View.

Infections by flaviviruses, such as Dengue, West Nile, Yellow Fever and Zika viruses, represent a growing risk for global health. There are vaccines only for few flaviviruses while no effective treatments are available. Flaviviruses share epidemiological, structural, and ecologic features and often different viruses can co-infect the same host. Therefore, the identification of broad-spectrum inhibitors is highly desirable either for known flaviviruses or for viruses that likely will emerge in the future. Strategies targeting both virus and host factors have been pursued to identify broad-spectrum antiflaviviral agents. In this review, we describe the most promising and best characterized targets and their relative broad-spectrum inhibitors, identified by drug repurposing/libraries screenings and by focused medicinal chemistry campaigns. Finally, we discuss about future strategies to identify new broad-spectrum antiflavivirus agents.

Modulating microRNA Processing: Enoxacin, the Progenitor of a New Class of Drugs.

The RNA interference (RNAi) process encompasses the cellular mechanisms by which short-noncoding RNAs posttranscriptionally modulate gene expression. First discovered in 1998, today RNAi represents the foundation underlying complex biological mechanisms that are dysregulated in many diseases. MicroRNAs are effector molecules of gene silencing in RNAi, and their modulation can lead to a wide response in cells. Enoxacin was reported as the first and unique small-molecule enhancer of microRNA (SMER) maturation. Herein, the biological activity of enoxacin as SMER is discussed to shed light on its innovative mode of action, its potential in treating different diseases, and the feasibility of using enoxacin as a chemical template for inspiring medicinal chemists. We debate its mechanism of action at the molecular level and the possible impact on future ligand and/or structure-guided chemical optimizations, as well as opportunities and drawbacks associated with the development of quinolones such as SMERs.

Antitubercular polyhalogenated phenothiazines and phenoselenazine with reduced binding to CNS receptors.

Targeting energy metabolism in Mycobacterium tuberculosis (Mtb) is a new paradigm in the search for innovative anti-TB drugs. NADH:menaquinone oxidoreductase is a non-proton translocating type II NADH dehydrogenase (NDH-2) that is an essential enzyme in the respiratory chain of Mtb and is not found in mammalian mitochondria. Phenothiazines (PTZs) represent one of the most known class of NDH-2 inhibitors, but their use as anti-TB drugs is currently limited by the wide range of potentially serious off-target effects. In this work, we designed and synthesized a series of new PTZs by decorating the scaffold in an unconventional way, introducing various halogen atoms. By replacing the sulfur atom with selenium, a dibromophenoselenazine 20 was also synthesized. Among the synthesized poly-halogenated PTZs (HPTZs), dibromo and tetrachloro derivatives 9 and 11, along with the phenoselenazine 20, emerged with a better anti-TB profile than the therapeutic thioridazine (TZ). They targeted non-replicating Mtb, were bactericidal, and synergized with rifampin and bedaquiline. Moreover, their anti-TB activity was found to be related to the NDH-2 inhibition. Most important, they showed a markedly reduced affinity to dopaminergic and serotonergic receptors respect to the TZ. From this work emerged, for the first time, as the poly-halogenation of the PTZ core, while permitting to maintain good anti-TB profile could conceivably lead to fewer CNS side-effects risk, making more tangible the use of PTZs for this alternative therapeutic application.

Pyridobenzothiazolones Exert Potent Anti-Dengue Activity by Hampering Multiple Functions of NS5 Polymerase.

Treatment of dengue virus (DENV) and other flavivirus infections is an unmet medical need. The highly conserved flaviviral NS5 RNA-dependent RNA polymerase (RdRp) is an attractive antiviral target that interacts with NS3 and viral RNA within the replication complex assembly. Biochemical and cell-based evidence indicate that targeting cavity B may lead to dual RdRp and NS5-NS3 interaction inhibitors. By ligand-based design around 1H-pyrido[2,1-b][1,3]benzothiazol-1-one (PBTZ) 1, we identified new potent and selective DENV inhibitors that exert dual inhibition of NS5 RdRp and NS3-NS5 interaction, likely through binding cavity B. Resistance studies with compound 4 generated sequence variants in the 3'-untranslated region of RNA while further biochemical experiments demonstrated its ability to block also RNA-NS5 interaction, required for correct RNA synthesis in cells. These findings shed light on the potential mechanism of action for this class of compounds, underlying how PBTZs are very promising lead candidates for further evaluation.

New Insights on KCa3.1 Channel Modulation.

The human intermediate conductance calcium-activated potassium channel, KCa3.1, is involved in several pathophysiological conditions playing a critical role in cell secretory machinery and calcium signalling. The recent cryo-EM analysis provides new insights for understanding the modulation by both endogenous and pharmacological agents. A typical feature of this channel is the low open probability in saturating calcium concentrations and its modulation by potassium channel openers (KCOs), such as benzo imidazolone 1-EBIO, without changing calcium-dependent activation. In this paper, we proposed a model of KCOs action in the modulation of channel activity. The KCa3.1 channel has a very rich pharmacological profile with several classes of molecules that selectively interact with different binding sites of the channel. Among them, benzo imidazolones can be openers (positive modulators such as 1-EBIO, DC-EBIO) or blockers (negative modulators such as NS1619). Through computation modelling techniques, we identified the 1,4-benzothiazin-3-one as a promising scaffold to develop new KCa3.1 channel modulators. Further studies are needed to explore the potential use of 1-4 benzothiazine- 3-one in KCa3.1 modulation and its pharmacological application.