Piperazine-derived small molecules as potential Flaviviridae NS3 protease inhibitors. In vitro antiviral activity evaluation against Zika and Dengue viruses

Bioorganic Chemistry 133 (2023) 106408 Available online 4 February 2023 0045-2068/© 2023 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/). Piperazine-derived small molecules as potential Flaviviridae NS3 protease inhibitors. In vitro antiviral activity evaluation against Zika and Dengue viruses María del Rosario García-Lozano a , b , 1 , Filippo Dragoni c , 1 , Paloma Gallego d , 1 , Sarah Mazzotta e , Alejandro L´ opez-G´ omez a , Adele Boccuto c , f , Carlos Martínez-Cort´ es g , Alejandro Rodríguez-Martínez h , Horacio P´ erez-S´ anchez g , Jos´ e Manuel Vega-P´ erez a , Jos´ e Antonio Del Campo i , * , Ilaria Vicenti c , * , Margarita Vega-Holm a , * , Fernando Iglesias-Guerra a a Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Seville, E-41071 Seville, Spain b SeLiver Group at the Institute of Biomedicine of Seville (IBIS), Virgen del Rocío University Hospital CSIC University of Seville, Seville, Spain c Department of Medical Biotechnologies, Siena University Hospital, Policlinico Le Scotte, Viale Bracci 16, 53100 Siena, Italy d Unit for Clinical Management of Digestive Diseases and CIBERehd, Valme University Hospital, 41014 Seville, Spain e Department of Chemistry, University of Milan, 20133 Milan, Italy f VisMederi Research srl, Siena, Italy g Structural Bioinformatics and High Performance Computing (BIO-HPC) Research Group, UCAM Universidad Cat´ olica de Murcia, 30107 Murcia, Spain h Department of Physical Chemistry and Institute of Biotechnology, University of Granada, Campus Fuentenueva sn, 18071 Granada, Spain i ALGAENERGY S.A., Avda. Europa 19, 28108 Alcobendas, Madrid, Spain ARTICLE INFO Keywords: Flavivirus NS3 protease Small molecules inhibitors Privileged structures Acyl and urea piperazine derivatives Live virus phenotypic assay Molecular docking Molecular modeling ABSTRACT Since 2011 Direct Acting antivirals (DAAs) drugs targeting different non-structural (NS) viral proteins (NS3, NS5A or NS5B inhibitors) have been approved for clinical use in HCV therapies. However, currently there are not licensed therapeutics to treat Flavivirus infections and the only licensed DENV vaccine, Dengvaxia, is restricted to patients with preexisting DENV immunity. Similarly to NS5 polymerase, the NS3 catalytic region is evolutionarily conserved among the Flaviviridae family sharing strong structural similarity with other proteases belonging to this family and therefore is an attractive target for the development of pan-flavivirus therapeutics. In this work we present a library of 34 piperazine-derived small molecules as potential Flaviviridae NS3 protease inhibitors. The library was developed through a privileged structures-based design and then biologically screened using a live virus phenotypic assay to determine the half-maximal inhibitor concentration (IC 50 ) of each compound against ZIKV and DENV. Two lead compounds, 42 and 44, with promising broad-spectrum activity against ZIKV (IC 50 6.6 µM and 1.9 µM respectively) and DENV (IC 50 6.7 µM and 1.4 µM respectively) and a good security profile were identified. Besides, molecular docking calculations were performed to provide insights about key interactions with residues in NS3 proteases’ active sites. Abbreviations: CC 50 , half-maximal cytotoxic concentration; DCM, dichloromethane; DDAs, direct acting antivirals; DENV, Dengue virus; DEPT, distortionless enhancement by polarization transfer; DMF, dimethylformamide; DMSO, dimethylsulfoxide; EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; ELISA, Enzyme- Linked Immunosorbent Assay; HCV, Hepatitis C virus; HIV, Human immunodeficiency virus; HOBt, hydroxybenzotriazole; HRMS, High resolution mass spectrometry; HSQC, heteronuclear single quantum correlation; IC 50 , half maximal inhibitory concentration; MS, mass spectrometry; NMR, nuclear magnetic resonance; SARS–CoV–2, Severe acute respiratory syndrome coronavirus 2; SI, selectivity index; TLC, thin layer chromatography; TMS, tetramethylsilane; TPV, telaprevir; WHO, World Health Organization, ZIKV, Zika virus. * Corresponding authors. E-mail addresses: [email protected] (J. Antonio Del Campo), [email protected] (I. Vicenti), [email protected] (M. Vega-Holm). 1 M.R.G-L, F.D, P.G joint as co-first authors. Contents lists available at ScienceDirect Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg https://doi.org/10.1016/j.bioorg.2023.106408 Received 27 July 2022; Received in revised form 23 January 2023; Accepted 1 February 2023 Bioorganic Chemistry 133 (2023) 106408 2 1. Introduction Flaviviridae family is subdivided into four genera namely, Flavivirus, Hepacivirus, Pegivirus and Pestivirus. Flavivirus genus includes prevalently arboviruses transmitted by infected arthropod, such as Dengue Virus (DENV), Japanese Encephalitis Virus (JEV), Yellow Fever Virus (YFV), Chikungunya Virus (CHIKV), Spondweni Virus (SPOV or SPONV), West Nile Virus (WNV) and Zika Virus (ZIKV) [1,2]. The incidence of Flavivirus infection has grown up to 400 million people infected annually [3] and widely distributed as consequence of globalization and climate changes, causing public health problems worldwide [4,5]. One of the most important pathogens of the flaviviridae family is the Hepatitis C virus (HCV) belonging to the Hepacivirus genus. Globally, an estimated 58 million people have chronic HCV infection, with about 1.5 million new infections per year and approximately 290,000 people died from hepatitis C in 2019, mostly from cirrhosis and hepatocellular carcinoma [6,7]. The absence of a vaccine and the possible emergence of viral resistance toward the drugs approved for HCV treatment, makes necessary the continuous design of novel specific HCV inhibitors which can implement the existing combination therapy. DENV and ZIKV have an outstanding clinical and epidemiological relevance due to their high incidence and lack of effective treatments. DENV infections, has increased its global incidence dramatically (100–400 million infections each year) with about half of the world’s population now at risk, particularly in developing countries [8,9]. Over 80% of infected patients are generally mild and asymptomatic [9] but approximately 5% have severe dengue with hemorrhagic fever [10]. Despite ZIKV infection usually proceeds asymptomatically or with mild symptoms, during the last outbreaks severe ZIKV-associated complications emerged (depending on genetic variant of ZIKV), such as congenital birth defects, including microcephaly, and the Guillain-Barre syndrome in adults [5]. In 2018, WHO has added ZIKV infection to its Research and Development Blueprint (the list of diseases recommended for intensive research) [8]. Currently no effective drugs [11] or vaccines against these diseases are available, the only vaccine approved against DENV, Dengvaxia is restricted to patients with preexisting DENV immunity and for this reason its distribution is limited to endemic area [11,12]. Therefore, at present the therapy for the treatment of DENV and ZIKV infections, includes only symptomatic treatment or supportive care in a hospital setting. All genera of the Flaviviridae family show similarities in the organization of the viral genome, which is characterized by a single stranded positive sense RNA molecule. The whole viral genome is translated to a viral polyprotein which is processed by the host and viral proteases into 9 to 12 mature proteins, consisting of the structural and nonstructural (NS) proteins [1,12]. The structural proteins form the viral particle. The NS proteins participate in the replication of the RNA genome, virion assembly and interaction with innate host immune response [13]. These viral proteins represent relevant targets for the development of novel antiviral therapies. Novel Flaviviridae inhibitors has been designed to target NS3 (protease domain, helicase domain) and NS5 (N-terminal methyltransferase domain and C-terminal RNA-dependent RNA polymerase) viral proteins [14]. The NS3 protease domain belongs to the trypsin/chymotrypsin protease superfamily with a conserved catalytic triad consisted of His- Ser-Asp. During the viral lifecycle in host cell, the NS3 protease cleaves the polyprotein releasing the resulting NS proteins. The majority of NS protein structures are evolutionarily conserved among the Flaviviridae family [15], with strong structural similarity of the NS3 catalytic region [15–18], despite difference in NS3 cofactor usage; for example, NS4A acts as cofactor in HCV whereas NS2B is the cofactor in DENV and ZIKV [18]. In the research of new anti-flavivirus agents, the repurposing of HCV NS3/NS4A inhibitors for the treatment of ZIKV infections is a reported and appealing strategy [19]. In this work we present the design and the synthesis of a small library of 34 piperazine-derived small molecules acting as potential Flaviviridae NS3 protease inhibitors. The compounds were designed using a privileged structure-based approach and their antiviral properties were determined by a live virus cell based phenotypic assay against ZIKV and DENV allowing the identification of lead compounds with promising broad-spectrum activity against flaviviruses. 2. Results & discussion 2.1. Design The design of the new antiviral compounds was based on our previous experience in the synthesis of piperazines as relevant scaffolds in anti-adenovirus activity [20,21]. In addition, the role of piperazine derivatives against Flaviviridae viruses such us HCV has also been reported [7,22–27]. Due to the relevance of the piperazine ring in the biological activity of compounds [28], we decided to preserve it as central core. The general skeleton of the first family consisted of two aromatic/ aliphatic rings assembled through amide [29] or urea [14,30] functions. The common structural feature is the 2-substituted piperazine ring as main backbone, with tert-butoxycarbonyl and its isostere 3,3-dimethyl- butanoyl groups as acylating agents at N-4 [21,31]. The structural diversity generation was accomplished at N-1 by the introduction of moieties considered as relevant for the desired antiviral activity. Literature was consulted to identify some structural elements of interest that could be considered privileged structures for this activity (Fig. 1). We have recently employed this designing strategy for developing other antiviral agents based on different amine-propanediol skeletons with promising results [32,33]. Indol-3-yl-2-oxoacetyl, a biologically relevant oxoamide moiety [34–36] having the indole nucleus (privileged scaffold in ligands for diverse viral proteins) [36–38], that has been previously explored in anti-HIV agents [39], was selected for our model. Cinnamoyl and quinoline-3-carbonyl groups were also introduced at N-1 position in our general backbone. They are crucial pharmacophores that exhibit multiple biological activities [40–43], and important structural motifs in antiviral drug discovery against HCV [44,45], ZIKV [46–49], DENV [49–55] and SARS-CoV–2 [56]. In the second type of compounds, the urea spacer connects the privileged moiety with the N-1 of the piperazine ring. To explore the chemical space around the urea scaffold, three different phenyl moieties were introduced. For the design of our library, 4-acetylphenyl [57,58], 2,4-dimethoxyphenyl [14] and 3,4,5-trimethox- yphenyl groups were selected based on their presence in small molecules aryl urea derivatives with potent anti ZIKV activity. 2.2. Synthesis Compounds 8–31 were obtained following the previously described short and high-yielded synthetic methodology that involved two reactions [20,21,59], by employing 2-substituted piperazines as precursors. Firstly, through a chemoselective N-acylation reaction, the acyl function at the less hindered nitrogen was introduced (compounds 3–6) Secondly, to generate chemical diversity at the other nitrogen, it was functionalized as amide and urea groups. Thirdly, compounds 32–34 were prepared in one step from commercial Boc-piperazine by reaction with the appropriate acylating agents: acyl chloride or carboxylic acid. The general synthetic route for these compounds is presented in Scheme 1. Spectral (NMR, Mass Spectrometry) and analytical data of the new synthesized compounds (8–34, Table 1) were in full agreement with the proposed structures (See Experimental Part and Supplementary Information). M. del Rosario García-Lozano et al. Bioorganic Chemistry 133 (2023) 106408 3 2.3. Biological evaluation 2.3.1. In vitro protease inhibition assay In the process of drug discovery, compounds exhibiting antiviral activity in cell based assays have better chance to be efficacious in vivo than in target-based approaches [60]. NS proteins in the Flaviviridae family, specifically the enzymes, are evolutionarily well conserved among different genera [8,61–64], existing a strong structural similarity in the catalytic region between the Flavivirus NS2B/NS3 and the HCV NS3/4A [15–18]. As our main goal was the development of broadly active antiviral compounds acting as pan Flaviviridae inhibitors, we started our antiviral drug discovery study by screening our library in a commercial HCV NS3/4A protease inhibition assay, to rapidly identify those compounds showing inhibitory enzymatic activity. The rationale behind our approach is the fact that ZIKV inhibitors have been obtained by repurposing previously identified HCV NS3/ NS4A inhibitors [19]. The discovery of novel broad spectrum compounds against HCV, Flavivirus and other viruses, by phenotypic screening starts with (i) anti-HCV cell-based viral infection screening, proceed with (ii) the optimizing process of anti HCV activity and it’s completed with (iii) the evaluation of broad antiviral profile of leading compounds [65]. Compounds 8–34 were evaluated to determine their degree of inhibition of the NS3/4A protease activity of the HCV, using Telaprevir (TPV) as reference compound (for details see Experimental Part), obtaining data recorded in Table 1 (Supplementary Information, Graph S31). As can be observed in Table 1, most of the HCV NS3/4A inhibitory values were >70%. Only five compounds gave around 40–50% of inhibition, being 2-phenyl piperazine derivatives having at N-1 a urea function with a 2,4-dimethoxy or 3,4,5-trimethoxy substituted phenyl ring (25, 30 and 31) or an amide (3-quinolein carbonyl group in 29, and cinnamoyl group in 21). 2.3.2. Cytotoxicity and antiviral activity It has been reported that some DENV inhibitors that exhibited inhibitory activities in biochemical protease assays, failed in cell-based viral infection assays [16,24]. This observation is not surprising considering that enzymatic assays evaluate only one activity of virus in a controlled reaction, while the viral replication in cell culture and consequently its inhibition, is a mechanism more complex which involves the interaction between different viral and cellular proteins. Consequently, we screened the compounds with moderate-high percentage of enzymatic inhibitory activity in a cell-based antiviral assay, to determine their potential activity to inhibit ZIKV and DENV replication in vitro. The final goal was to find new lead compounds for hit optimization. Considering the enzymatic inhibition percentage data (Table 1) all compounds from the first family were evaluated. The cell-based antiviral assay has been previously published [66,67] and it is explained in detail in the Experimental Section. Activity / toxicity data for compounds 8–34 are reported in the Table 2. The IC 50 (half maximal inhibitory concentration) values obtained with the reference compound (sofosbuvir) were coherent with those obtained in literature and in previously published papers [66,68,69]. Anti-ZIKV activity was observed in the micromolar range, in 11 out 27 compounds that compose the library with a median [IQR] IC 50 of 24 [8.4–37.0] μ M and a mean SI of 10 [6.4–14.0]. Six of them were 2- methyl piperazine derivatives (8, 11, 14, 15, 16 and 18). Both tertbutoxycarbonyl and 3,3-dimethylbutanoyl groups are present in active compounds at N-4. Related to the different moieties at N-1, amide-linked derivatives with indol-3-yl-2-oxoacetyl (8 and 14), cinnamoyl (15) and quinoline-3-carbonyl (16), together with urea-linked compound with 4- acetylphenyl (11) showed a good SI, with a median SI of 10.2 [13.3–17.1] (Table 2) [70]. Only three 2-phenylpiperazine were active: both 3,4,5-trimethoxy- phenyl urea derivatives (IC 50 24 8.2 μ M and 29 41.2 μ M, with tertbutoxycarbonyl and 3,3-dimethylbutanoyl groups respectively at N-4) and 25, indol-3-yl-2-oxoacetyl derivative with IC 50 3.9 μ M. Of them, only the compound 24 had a low cytotoxic profile (SI =14.6). Three different unsubstituted Boc-piperazine derivatives, 32, 33 and 34, featuring the indol-3-yl-2-oxoacetyl, cinammoyl and indol-3-yl- acetyl respectively at the other N, were also prepared. Compound 32, unsubstituted analogue of 8 was not active, while compound 34, with the indol-3-yl acetyl group showed a poor activity (IC 50 51.9 μ M). Compound 33 showed certain activity (IC 50 24.2 μ M) while its 2- substituted analogues 9 and 21 were inactive. These results suggested that the presence of 2-metylpiperazine core in our model is crucial for the activity against ZIKV. At opposite of compounds active againt ZIKV, most of the compounds containing 2-methylpiperazine moiety were inactive against Fig. 1. Privileged structure-based design strategy for potential piperazine-derived anti-flavivirus agents. M. del Rosario García-Lozano et al. Bioorganic Chemistry 133 (2023) 106408 4 DENV, with the exception of the indol-3-yl-2-oxoacetyl derivatives, compound 8 (IC 50 10.7 µM, SI 14.3) and 14 (IC 50 26.0 µM, SI 7.7) which showed a similar activity profile against ZIKV. The activity profile changed when 2-phenylpiperazine core was present. Six out eleven prepared derivatives displayed anti DENV activity. Indeed, compounds 21, 22, 23, 25, 26 and 29 showed IC 50 values ranging from 7.9 to 31.3 μ M. However, considering their median SI 4.2 [3.1–4.7], probably they require further optimization to reduce their cytotoxicity. Among the three (32–34) unsubstituted piperazine derivatives evaluated, only two presented low activity, compund 32 with indol-3-yl- 2-oxoacetyl group (IC 50 45.0 μ M) and its analogue 34 with indol-3-yl- acetyl (IC 50 38.7 μ M) which was similarly active against ZIKV. Globally, 8 was the most promising tested compound, considering its activity in the low micromolar range against both viruses and its low cytotoxic profile. As our compounds were prepared in racemic form, both enantiomers of compound 8 were evaluated in the cell-based assay to determine their inhibitory activity against ZIKV and DENV replication [(R)-8 [ α ] D = − 8.0 (c 1.04, CH 2 Cl 2 ), (S)-8 [ α ] D =+8.1 (c 0.95, CH 2 Cl 2 )]. Not substantial differences (Fig. 2) were appreciated among their IC 50 values against ZIKV [(R)-8 13.8 ±1.2 μ M, (S)-8 12.7 ±1.4 μ M)] and DENV [(R)-8 12.7 ±1.2 μ M, (S)-8 11.2 ±1.3 μ M)], showing almost similar activities and cytotoxicity (SI >10) to those obtained with the racemic form. As both enantiomers of compound 8 showed similar activity profile in cell-based assay against ZIKV and DENV replication, a docking study was performed to look inside the binding mode of both enantiomers with the active site of Zika NS3/2B and to check the differences between (R) and (S) configurations. The groups in the chiral carbon do not present interactions with any residues, and the interactions in the two enantiomers are similar in both cases with similar binding energy values (Supplementary Information, Fig. S32). 2.4. Round of optimization A further round of optimization was performed to obtain a second small family of inhibitors. The goal was to increase the molecular complexity through the assembly of 3 aromatic/aliphatic rings linked by both functions, urea and amide, in the same molecule. In pursuit of improving the antiviral activity against both viruses and reducing the cytotoxicity of those lead compounds, the first decision was to preserve 2-phenylpiperazine ring as the central core, due to its relevance for anti-DENV activity. The following chemical construction process focused on the incorporation of two biologically interesting motifs to both nitrogen atoms of the piperazine ring located at central position The scaffold’s selection Scheme 1. Synthesis of 2-substituted piperazine amide and urea derivatives (8–31) and unsubstituted piperazine derivatives (32–34). M. del Rosario García-Lozano et al. Bioorganic Chemistry 133 (2023) 106408 5 preserved the structural features of the most active compounds from the previous family (Fig. 3): (1) For the amide link, indol-3-yl-2-oxoacetyl and cinammoyl groups carried in compunds 8, 21 and 25. (2) For the urea connection, the 2,4-dimethoxyphenyl ring presents in compounds 18 and 23, and the 4-acetylphenyl, evaluated in 2- methyl piperazine derivatives (11), but not previously incorporated to 2-phenylpiperazine core. The third moiety was (2-chloro- 5-trifluoromethyl) phenyl, based on the fact that urea derivatives having this group have been described as effective anti ZIKV compounds [58]. It is also important to consider the relative position of the substituent at position 2 in the piperazine ring and the privileged scaffolds linked to the nitrogen atoms. Firstly, two indol-3-yl-2-oxoacetyl derivatives were prepared starting from compounds 8 and 20 (2-methyl and phenyl derivatives respectively), which yielded compounds 37 and 38 through the deprotection of the Boc group by treatment with CF 3 COOH (35 and 36), followed by reaction with the appropriate isocyanate (Scheme 2). The groups indol-3-yl-2-oxoacetyl and cinammoyl were introduced at the less hindered nitrogen through a chemoselective N-acylation reaction (39 and 40) and secondly, N-1 was functionalized as urea to introduce the biologically relevant scaffolds mentioned above (41–44). All these compounds were obtained in high yields and their spectral and analytical data confirmed the proposed structure (See Experimental Section) (Scheme 2). The lead compounds, improved with the above-mentioned modifications, were tested in cells-based assay to assess their cytotoxic profile and their antiviral activity (Table 3, Fig. 4). As illustrated in Table 3, compound 37, the only 2-methyl derivative of this family, showed a modest activity against both viruses (mean IC 50 33.5 μ M when considering both ZIKV and DENV). Among the 3-phenyl- derivatives (38, 41 and 43) carrying the indol-3-yl-2-oxoacetyl group the cytotoxicity ranging from moderate to high. Indeed the compound Table 1 Acyl piperazine amide and urea derivatives (8–34). Inhibition of HCV NS3/4A protease. Entry Comp R 1 X R 2 Yield (%) % Enzimatic Inhibition a 1 8 Me O A 64 73.98 ±8.59 2 9 Me O B 67 77.87 ±11.42 3 10 Me O C 94 76.12 ±2.23 4 11 Me O D 79 77.13 ±13.64 5 12 Me O E 68 86.20 ±9.30 6 13 Me O F 61 78.71 ±15.45 7 14 Me CH 2 A 61 76.71 ±14.91 8 15 Me CH 2 B 62 83.18 ±6.06 9 16 Me CH 2 C 88 68.29 ±0.29 10 17 Me CH 2 D 76 79.15 ±9.66 11 18 Me CH 2 E 77 83.76 ±11.74 12 19 Me CH 2 F 68 76.14 ±3.31 13 20 Ph O A 63 75.92 ±4.05 14 21 Ph O B 71 51.05 ±10.05 15 22 Ph O C 90 ND b 16 23 Ph O E 83 79.19 ±7.93 17 24 Ph O F 69 41.90 ±20.19 18 25 Ph CH 2 A 72 81.05 ±1.75 20 26 Ph CH 2 C 79 ND b 21 27 Ph CH 2 D 91 44.92 ±15.71 22 28 Ph CH 2 E 72 47.66 ±23.99 23 29 Ph CH 2 F 65 47.39 ±18.03 24 30 Me O G 74 ND b 25 31 Ph O G 81 61.92 ±4.45 26 32 H O A 94 80.87 ±0.76 27 33 H O B 79 86.90 ±0.79 28 29 34 TVP H O G 78 85.32 ±0.12 – – – – 88.41 ±5.82 a Percent inhibitory activity of compounds on HCV NS3/4A protease (genotype 1b) at 0.5 mM. All results are shown as means ±SD of triplicate samples from three independent experiments. See Experimental Section for details. b Not determined. M. del Rosario García-Lozano et al. Bioorganic Chemistry 133 (2023) 106408 6 43, was completely toxic in cell culture while the 41 had a low SI (1.6) despite its anti DENV activity was in the low micromolar range (IC 50 7.3 μ M). Compound 38, carrying the indol-3-yl-2-oxoacetyl group at N-1 and the 2,4-dimethoxyphenyl urea at N-4 had a good anti-DENV activity (IC 50 2.2 μ M), with a SI of 14.5 which can be improved in future reducing its cytotoxicity (32 μ M). The most promising compounds were the 42 and the 44, carrying the cinammoyl group at N-4. Indeed, they inhibited robustly the ZIKV and DENV replication with IC 50 in the low micromolar range and high SI (60.5/59.7 ZIKV/DENV for compound 42 and 33.5/44.6 ZIKV/DENV for compound 44). Thus, the optimization process led to the development of two promising pan-flavivirus agents with anti-DENV activity higher than that recorded by reference compound sofosbuvir in cell culture. 2.5. Docking studies To further establish whether the inhibitors targeted the viral protease as we hypothesized, molecular docking analyses on 8, 42 and 44, the most promising compounds of both libraries, were performed. 2.5.1. HCV NS3 protease molecular modeling The docking scores and the interactions snapshot for the three compounds were obtained using PLIP [71] and PyMOL [72] software. These were found in the HCV NS3/4A Protease active site [73] with scores of −6.75 kcal/mol, −6.53 kcal/mol and −6.01 kcal/mol, for compounds 8, 42 and 44, respectively. In addition, the compounds were found close to the active site [74] (Fig. 5), and some interactions with the essential catalytic residues (His76, Asp100 and Ser158) [72] were detected (Supplementary Information, Table S33). And additional docking study was performed to evaluate the potential interaction of the ketoamide group present in 8 (indol-3-yl-2- oxoacetyl derivative) with residues of the active site in the protease. This compound was compared to 30 (indol-3-yl-acetyl derivative, analogue to 8). Compound 8 shows two interactions in Ser 158, with both carbonyl groups, amide and ketone. Compound 30 only interacts with this residue once through the carbonyl in the amide function (Supplementary Information Fig. S34). 2.5.2. ZIKV NS3 protease molecular modeling In the case of ZIKV NS3/2B protease, the docking scores for the three compounds were similar to HCV protease scores with values of −5.61, −5.78 and −5.79 kcal/mol for compounds 8, 42 and 44, respectively (Supplementary Information, Table S35). In addition, the interactions obtained were related to critical residues for this active site, which consisted of the following residues: His77, Ser161, Tyr176, Tyr187 [75] (Fig. 6). These results support their expected mechanism of action as protease inhibitors. 2.6. In silico prediction of physicochemical and pharmacokinetic properties In the drug development process, most of the new drug candidates fail in clinical trials due to their reduced ADME properties. In early stages of this process is critical to assess pharmacokinetic properties Table 2 Cytotoxic profile and antiviral activity of acyl piperazine amide and urea derivatives (8–34) in vitro. Sofosbuvir, tested in each assay was used as reference control. Experiments were performed in Huh7 cell line. Comp CC 50a ( μ M) IC 50b ( μ M) ZIKV SI c ZIKV IC 50b ( μ M) DENV SI c DENV 8 153 8.9 ±1.2 17.1 10.7 ±2.0 14.3 9 123 NA d – NA – 10 110 NA – NA – 11 200 11.6 ±1.3 17.3 NA – 12 200 NA – NA – 13 200 NA – NA – 14 200 20.6 ±1.2 9.7 26.0 ±3.4 7.7 15 200 15.0 ±1.2 13.3 NA – 16 84 8.0 ±1.3 10.2 NA – 17 >200 NA – NA – 18 200 58.2 ±1.5 3.4 NA – 19 200 NA – NA – 20 15.3 NA – NA – 21 21.5 NA – 7.9 ±3.0 2.7 22 60 NA – 14.8 ±0.2 4.1 23 95 NA – 14.6 ±3.3 6.5 24 115 8.2 ±1.2 14.6 NA – 25 20 3.9 ±0.2 5.1 9.0 ±6.6 2.2 26 112 NA – 22.7 ±7.5 4.9 27 47.3 NA – NA – 28 60 NA – NA – 29 130 41.2 ±11.3 3.2 31.3 ±3.5 4.2 30 100 NA – NA – 31 18.7 NA – NA – 32 130 NA – 45.0 ±1.4 2.9 33 200 24.2 ±1.9 8.3 NA – 34 200 51.9 ±19.4 7.7 38.7 ±2.0 10.3 Sofosbuvir >200 4.0 ±1.3 100.0 13.1 ±1.2 30.5 a Half-maximal cytotoxic concentration (CC 50 ). The results represent means of triplicate experiments. b Half maximal inhibitory concentration (IC 50 ). The results represent means ±standard deviation of 2 independent experiments performed in triplicate. c The selectivity index value was determined as the ratio of CC 50 to IC 50 for each compound. d NA. Not active. Fig. 2. Inhibitory activity of compound 8 and its enantiomers, (R)-8 and (S)-8 against ZIKV and DENV in cell culture. The micromolar compound concentration is expressed as logarithm on x-axis, the viral replication is expressed on yaxis as percentage. The normalized curve for each compound derives from 2 independent experiments performed in triplicate. M. del Rosario García-Lozano et al. Bioorganic Chemistry 133 (2023) 106408 7 prediction, to minimize the risk of drug attrition in late stage, related to poor ADME profiles. In this sense, in silico ADME screens provide a good starting point to select the most promising candidates for development and to reject those with low chance of success [76]. Based on previously discussed results, the 2 lead compounds (42 and 44) and different compounds showing activity against both viruses (8, 14, 34 and 37), or active against only one virus but with a good SI (11, 15, 16, 24 and 38) were submitted to in silico ADME properties evaluation. Compounds were first tested for drug-likeness by Lipinski’s Rule of Five [77–79]. SwissADME was the software used to predict the afore mentioned physicochemical parameters of the selected compounds (Table S36, Supplementary Information) [80]. Excepting compound 44 (with molecular weight >500 and log P > 5), the investigated compounds were satisfying to Lipinski’s rule of five [81,82]. Molsoft was used to predict drug likeness model scores of the compounds (Table S36, Supplementary Information). (Available from: URL: https://www.molsoft.com/) [81]. The more positive drug likeness score values, the more likely the compound was expected to be as a promising therapeutic pharmacophore [83]. A positive model score was attributed for the most promising compounds 38, 42 and 44 (score ranged from 0.59 to 1.09). Prediction of the major pharmacokinetic parameters such as absorption and distribution, were estimated using Pre-ADMET software (Available from: URL: http://preadmet.bmdrc.org) [84,85]. The outcomes of the predicted ADME parameters (Table S37, Supplementary Information) indicate good pharmacokinetic profile and drug likeness. 3. Conclusions In summary, a library of 34 piperazine-derived small molecules have been described. They have been designed employing a privilegedstructures based strategy through the incorporation of a 2-substituted piperazine ring (main backbone) which is considered relevant for exerting antiviral activity as protease inhibitors of viruses belonging to the Flaviviridiae family. The compounds were firstly screened by a commercial HCV NS3/4A protease inhibition assay, recording inhibitory values above 70%, and then, to determine their potential activity to inhibit ZIKV and DENV replication in vitro, they were evaluated in a cell-based antiviral assay. From this library, compound 8 (general structure based on two rings assembled) was the most promising, being active in the low micromolar range against both viruses and poorly cytotoxic. A further round of optimization increasing the molecular complexity, led to the development of two compounds, the 42 and the 44 with a general structure based on three rings assembled. Both compounds had an anti-DENV activity higher than those recorded by reference compound sofosbuvir in cell culture, and a good security profile. The molecular docking analyses on compounds 8, 42 and 44 showed compound residues interacting with both HCV NS3/4A and ZIKV NS3/2B protease active sites, supporting their expected mechanism of action as protease inhibitors. These compounds based on the piperazine scaffold structure can be considered as promising hit candidates for the development of a new class of pan Flavivirus agents. Further chemical exploration of this scaffold will be performed to search potent antiviral agents with broad spectrum activity against flaviviruses. 4. Experimental Section 4.1. Chemistry 4.1.1. General chemistry methods All reagents, solvents, and starting materials were obtained from commercial suppliers and used without further purification. The crude reaction mixtures were concentrated under reduced pressure by removing the organic solvents in a rotary evaporator (BÜCHI R-200). Reactions were monitored by thin layer chromatography (TLC) using Kieselgel 60 F 254 (E. Merck) plates and UV detector (SPECTROLINE MODEL CM-10) for visualization. Flash column chromatography was performed on Silica Gel 60 (E. Merck, 0,040–0,063 mm) and sea sand washed (Panreac, thin grade ~ 0,25–0,30 mm), with the indicated eluent. All reported yields are of purified products. Melting points were obtained on a GALLENKAMP Melting Point Apparatus MPD350.BM2.5 with open capillaries (Kimax-51◦, 1.5–1.8 ×90 mm) and are uncorrected. Mass spectra were recorded on a Orbitrap Elite (Thermo Fig. 3. Privileged structure-based optimization process. M. del Rosario García-Lozano et al. Bioorganic Chemistry 133 (2023) 106408 8 Scientific), a Hybrid Ion trap-orbitrap mass spectrometer capable of acquiring with a resolution higher than 240000, with ESI, HESI, APCI and nanoESI ionization sources. NMR spectra were recorded at 25 ◦C on a Bruker AV500 spectrometer at 500 MHz for 1 H and 125 MHz for 13 C. The chemical shifts (δ) reported are given in parts per million (ppm) and the coupling constants (J) are in hertz (Hz). 1 H chemical shift values (δ) are referenced to the residual non deuterated components of the NMR solvents (δ =2.54 ppm for DMSO‑d 6 ). The 13 C chemical shifts (δ) are referenced to DMSO‑d 6 (central peak, δ =39.5 ppm) as the internal standard. The spin multiplicities are reported as s (singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet), or bs (broad singlet). COSY, DEPT, HSQC experiments were performed to assign the signals in the NMR spectra. The purity of final compounds was evaluated by C, H, N, S elementary analysis on a LECO TruSpec Micro CHNS (Maximum combustion temperature: 1050 ◦C). The purity of the final compounds was confirmed to be ≥95% by combustion. Scheme 2. Synthesis of optimized compounds 37, 38, 41–44. M. del Rosario García-Lozano et al. Bioorganic Chemistry 133 (2023) 106408 9 4.1.2. General procedure 1. Chemoselective N-acylation reaction of 2- substituted piperazines (3–6, 39, 40) 2-Substituted piperazine (5.0 mmol) was dissolved in dry dichloromethane (100 mL) and cooled to 0 ◦C. A solution of the appropriate acylating agent (5.0 mmol) in dichloromethane (20 mL) was added dropwise in 30 min, and then pyridine (10 mmol). The reaction mixture was kept into an ice-water bath with stirring 6 h and left at room temperature until TLC showed that all the starting material had reacted. The reaction mixture was evaporated to dryness to obtain the corresponding monoacyl derivative. Flash column chromatography gave the pure compound in high yield. 4.1.2.1. 1-(tert-Butoxycarbonyl)-3-methylpiperazine (3) [20,21].The product was obtained as an oil and purified by column chromatography using ethyl acetate–methanol (10:1) as eluent (930 mg, 93% yield). 1 H NMR (500 MHz, DMSO‑d 6 ) δ 3.75–3.7 1 (m, 2H), 2.85–2.82 (m, 1H), 2.75–2.69 (m, 1H), 2.60–2.54 (m, 3H), 2.39–2.34 (m, 1H), 1.41 (s, 9H), 0.96 (d, J =6.3 Hz, 3H). 13 C NMR (125 MHz, DMSO‑d 6 ) δ 154.5, 79.3, 51.2, 50.5, 45.5, 44.4, 28.6, 19.3. HRMS (m/z): calcd for C 10 H 20 N 2 O 2 200.1528 [M] +⋅ ; found 200.1525. 4.1.2.2. 1-(3,3-Dimethylbutanoyl)-3-methylpiperazine (4) [21].The product was obtained as an oil and purified by column chromatography using ethyl acetate–methanol (5:1) as eluent (861 mg, 87% yield). 1 H NMR (500 MHz, CDCl 3 ) δ 4.57 (d, J =12.4 Hz, 1H), 3.85 (m, 1H), 3.52–3.08 (m, 2H), 3.06–2.62 (m, 3H), 2.23 (s, 2H), 1.33 (bs, 3H), 1.02 (s, 9H). 13 C NMR (125 MHz, CDCl 3 ) δ 170.3, 51.4, 45.8, 44.6, 44.5, 39.3, 31.5, 30.0, 17.3. HRMS (m/z): calcd for C 11 H 22 N 2 ONa 221.1624 [M + Na] + ; found 221.1619. 4.1.2.3. 1-(tert-Butoxycarbonyl)-3-phenylpiperazine (5) [20,21].The product was obtained as a solid and purified by column chromatography using ethyl acetate–methanol (100:1) as eluent (1.26 g, 96% yield), mp 130–133 ◦C. 1 H NMR (500 MHz, CDCl 3 ) δ 7.4–7.3 (m, 5H), 4.05 (bs, 2H), 3.70 (dd, J =2.4 Hz, J =10.5 Hz, 1H,), 3.07 (m, 1H), 2.9–2.8 (m, 2H), 2.72 (bs, 1H), 1.90 (bs, 1H), 1.47 (s, 9H). 13 C NMR (125 MHz, CDCl 3 ) δ 154.8, 141.5, 128.5, 127.8, 127.0, 79.7, 60.3, 51.5, 46.1, 43.4, 28.5. HRMS (m/z): calcd for C 15 H 23 N 2 O 2 263.1754 [M +H] + ; found 263.1748. 4.1.2.4. 1-(3,3-Dimethylbutanoyl)-3-phenylpiperazine (6) [21].The product was obtained as an oil and purified by column chromatography using ethyl acetate–methanol (20:1) as eluent (1.27 g, 97% yield). 1 H NMR (500 MHz, DMSO‑d 6 ) δ 7.45–7.27 (m, 5H), 4.44–4.39 (m, 1H), 3.94–3.85 (m, 1H), 3.58 (dd, J =2.3 Hz, J =10.3 Hz, 1H), 3.31 (bs, 1H), 3.11–2.99 (m, 2H), 2.91–2.68 (m, 1H), 2.61–2.57 (m, 1H), 2.31–2.20 (m, 2H), 1.02, 1.00 (2 s, 9H). 13 C NMR (125 MHz, DMSO‑d 6 ) δ 169.3, 169.2, 142.2, 141.9, 128.2, 128.1, 127.4, 127.3, 127.0, 126.9, 60.1, 59.4, 53.5, 48.1, 46.4, 45.9, 45.5, 43.8, 43.6, 41.1, 40. HRMS (m/z): calcd for C 16 H 25 N 2 O 261.1889 [M +H] + ; found 261.1886. 4.1.2.5. 1-[2-(Indol-3-yl)-2-oxoacetyl]-3-phenylpiperazine (39). The product was obtained as an oil and purified by column chromatography using dicholormethane-methanol (60:1) as eluent (1.1 g, 64% yield). 1 H NMR (500 MHz, DMSO‑d 6 ) δ 12.36 (s, 1H), 8.21 (bs, 1H), 8.17–8.09 (m, 1H), 7.59–7.20 (m, 7H), 4.42–4.34 (m, 1H), 3.85–3.61 (m, 1H), 3.55–3.42 (m, 1H), 3.24–2.66 (m, 5H). 13 C NMR (125 MHz, DMSO‑d 6 ) δ 186.3, 186.2, 165.8, 141.7, 141.1, 137.1, 137.0, 136.9, 128.4, 128.3, 127.5, 127.4, 126.9, 126.7, 124.0, 124.7, 123.6, 122.5, 120.9, 120.8, 113.6, 113.1, 112.6, 60.9, 59.1, 52.9, 47.6, 46.1, 45.8, 45.1, 41.0. HRMS (m/z): calcd for C 20 H 20 N 3 O 2 333.1477 [M +H] + ; found 333.1473. 4.1.2.6. 1-Cinnamoyl-3-phenylpiperazine (40). The product was obtained as an oil and purified by column chromatography using hexane–ethyl acetate (1:7) as eluent (1.4 g, 97% yield). 1 H NMR (500 MHz, DMSO‑d 6 ) δ 7.75–7.30 (m, 12H), 4.52–4.43 (m, 1H), 4.35–4.23 (m, 1H), 3.76–3.63 (m, 2H), 3.56–3.25 (m, 2H), 3.12–0.66 (m, 2H); 13 C NMR (125 MHz, DMSO‑d 6 ) δ 164.5, 141.6, 135.2, 129.5, 128.7, 128.3, 128.2, 128.1, 127.5, 127.3, 126.9, 118.3, 60.2, 59.4, 52.1, 48.9, 46.2, 45.5, 45.3, 41.8. HRMS (m/z): calcd for C 19 H 20 N 2 ONa 315.1473 [M +Na] + ; found 315.1456. 4.1.3. General procedure 2. Synthesis of N,N’-diacyl derivatives (A) By reaction with acyl halide. To a solution of the monoacyl derivatives (3–7) (1.0 mol) in dry dichloromethane (15 mL) was added the corresponding acyl halide (1.2 mmol) and dry pyridine (1.5 mmol). The reaction mixture was stirred at room temperature under argon atmosphere until TLC showed that all the starting material had reacted. The reaction mixture was diluted in dichloromethane and successively washed with hydrochloric acid solution 1 M, saturated solution of sodium bicarbonate, dried (Na 2 SO 4 ), filtered and the filtrate was evaporated to dryness. The compounds obtained were purified by flash chromatography. 4.1.3.1. 4-(tert-Butoxycarbonyl)-1-[2-(indol-3-yl)-2-oxoacetyl]-2-methyl- piperazine (8). The product was obtained as a solid and purified by column chromatography using hexane–ethyl acetate (1:1) as eluent (237 mg, 64% yield), mp 223–225 ◦C. 1 H NMR (500 MHz, DMSO‑d 6 ) δ 12.31, 12.29 (2bs, 1H), 8.22–8.10 (m, 2H), 7.55 (d, J =7.4 Hz, 1H), 7.33–7.25 (m, 2H), 4.67 (m, 1H), 4.31–3.69 (m, 4H), 3.06–2.70 (m, 2H), 1.42 (s, 9H), 1.22, 1.15 (2d, J =6.7 Hz, 3H). 13 C NMR (125 MHz, DMSO‑d 6 ) δ 186.1, 186.0, 166.0, 165.9, 154.2, 137.2, 137.0, 136.9, 124.8, 123.6, 122.5, 120.9, 120.8, 113.0, 112.9, 112.6, 79.2, 49.3, 43.9, 40.7, 35.3, 27.9, 15.6, 14.8. HRMS (m/z): calcd for C 20 H 25 N 3 O 4 Na 394.1737 [M +Na] + ; found 394.1726. Anal. Calcd C 20 H 25 N 3 O 4 : C, 64.67; H, 6.78; N, 11.31. Found: C, 64.66; H, 6.73; N, 11.11. 4.1.3.2. 4-(tert-Butoxycarbonyl)-1-cinnamoyl-2-methylpiperazine (9). The product was obtained as a solid and purified by column chromatography using hexane–ethyl acetate (2.5:1) as eluent (219 mg, 67% yield), mp 130–133 ◦C. 1 H NMR (500 MHz, DMSO‑d 6 ) δ 7.70 (d, J =7.0 Hz, 2H), 7.50 (d, J =15.4 Hz, 1H), 7.46–7.36 (m, 3H), 7.21 (d, J =15.4 Hz, 1H), 4.73–4.44 (m, 1H), 4.29–3.72 (m, 3H), 3.15–2.73 (m, 3H), 1.43 (s, 9H), 1.13 (bs, 3H). 13 C NMR (125 MHz, DMSO‑d 6 ) δ 164.9, 154.3, 141.7, 135.0, 129.6, 128.8, 127.9, 118.3, 79.1, 28.0, 26.8. HRMS (m/z): calcd for C 19 H 26 N 2 O 3 Na 353.1836 [M +Na] + ; found 353.1830. Anal. Calcd C 19 H 26 N 2 O 3 : C, 69.06; H, 7.93; N, 8.48. Found: C, 69.17; H, 7.91; N, 8.40. Table 3 Cytotoxic profile and antiviral activity of optimized compounds 37, 38, 41–44 in vitro. Sofosbuvir, tested in each assay was used as reference control. Experiments were performed in Huh7 cell line. Comp CC 50a ( μ M) IC 50b ( μ M) ZIKV SI c ZIKV IC 50 ( μ M) DENV SI c DENV 37 >200 31.7 ±5.4 12.6 35.2 ±15.1 11.4 38 32 NA d – 2.2 ±1.1 14.5 41 12 NA d – 7.3 ±2.8 1.6 42 >200 6.6 ±1.4 60.5 6.7 ±2.6 59.7 43 Toxic – – – 44 62 1.9 ±0.4 33.5 1.4 ±0.3 44.6 Sofosbuvir >200 4.7 ±1.2 85.1 12.1 ±1.3 33.1 a Half-maximal cytotoxic concentration (CC 50 ). The results represent means of triplicate experiments. b Half-maximal inhibitory concentration (IC 50 ). The results represent means ±Standard deviation of 2 independent experiments performed in triplicate. c The selectivity index value was determined as the ratio of CC 50 to IC 50 for each compound. d NA: Not active. M. del Rosario García-Lozano et al. Bioorganic Chemistry 133 (2023) 106408 16 Luminescent Cell Viability Assay (Promega) in the Huh7 cell line, according to the manufacturer’s protocol. The luminescent signal obtained from cells treated with 5-fold dilution of the test compound, or DMSO as a control, was measured through the GloMax® Discover Microplate Reader (Promega). Raw data were elaborated with the GraphPad PRISM software version 6.01 (La Jolla) to calculate the half-maximal cytotoxic concentration (CC 50 ). The putative inhibitory activity of compounds was tested starting from the concentration causing the 20% of cellular toxicity (CC 20 ). 4.2.4. Antiviral assay Immunodetection assay (IA) using a pan-flavivirus monoclonal antibody recognizing a conserved domain of the envelope protein was the read out to determine the antiviral activity of candidate compounds on the whole viral replication cycle as previously published [66,67] Briefly, 7,000 Huh7 cells per well were infected using 50 TCID 50 (50% tissue culture infectious dose TCID 50 ) of DENV or ZIKV viral stocks, as defined by previous titration according to Reed & Muench [94]. Viral adsorption was performed in 96-well plates for 1 h at 37 ◦C with 5% CO 2 and after virus removal, serial dilutions of each compound tested were added to the cells and incubated at 37 ◦C with 5% CO 2 . After 72 h, culture supernatants were collected, properly diluted, and used to reinfect in triplicate uninfected Huh7 cells in absence of the compounds (infectious virus yield) for 72 h at 37 ◦C with 5% CO 2 . After incubation, cells were fixed for 30 min with 10% formaldehyde (Carlo Erba), rinsed with 1% PBS and permeabilized for 10 min with 1% Triton X-100 (Carlo Erba). After washing, cells monolayer was incubated with monoclonal anti-flavivirus mouse antibody (clone D1-4G2-4–15; Novus Bio) and then with a polyclonal Horseradish Peroxidase (HRP)-coupled antimouse IgG secondary antibody (Novus Bio NB7570). Absorbance values were measured after 10 min incubations with the 3,3′,5,5′-tet- ramethylbenzidine substrate (TMB, Sigma Aldrich) and signals were acquired at 450 nm optical density (OD450) using the absorbance module of the GloMax® Discover Multimode Microplate Reader (Promega). Each IA run was validated by the OD450 value above 1 in the virus control culture. All drug concentrations were tested in triplicate in two independent experiments. In each test, sofosbuvir was used as reference compounds and infected and uninfected cells without drugs were used to calculate the 100% and 0% of viral replication, respectively. The half-maximal inhibitory concentration (IC 50 ) was calculated through a non-linear regression analysis of the dose–response curves generated with GraphPad PRISM software version 6.01. Selectivity Index (SI) of compounds was calculated as ratio between CC 50 and IC 50 . 4.3. Docking studies 4.3.1. Dataset Inactive structural conformation for ZIKV and HCV NS3 Proteases were extracted from the X-ray crystal structure chain A in the PDB Database for each protease (PDB ID: 5LC0 [75] and 6PJ1 [73], respectively). After that, we processed the structure files through Maestro (https://www.schrodinger.com) to obtain mol2 files with assigned charges and prepared for the virtual screening technique using OPLS3e as force field and different tools to fill possible missing side or loops, assign bonds and add hydrogens to the structure. We obtained ligand mol2 files from the generated cdx files through different conversion programs. First, we converted cdx files to smi format via Open Babel [95] converter tool. After that, these smi files were transformed to mol2 files via the Chemaxon tool molconvert to obtain the mol2 files used for molecular modeling. 4.3.2. Molecular modeling When the ligand and protein structures had been prepared, we carried out a molecular modeling protocol with the Lead Finder [96] docking program to obtain docking scores and details about interactions between protein and generated compounds. These calculations were performed for compounds 8, 42 and 44 against 5CL0 and 6PJ1 preprocessed structures via the metascreener tool (https://github. com/bio-hpc/metascreener). Thus, we executed calculations via Lead Finder for each protease with the same compounds as queries in an HPC server and in parallel. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability No data was used for the research described in the article. Acknowledgements This work has been supported by Ministerio de Ciencia, Innovaci´ on y Universidades, Plan Estatal 2017-2020 Retos - Proyectos I +D +i (PID2019-104767RB-I00). MRGL also thanks Ministerio de Economía y Competitividad, Instituto de Salud Carlos III (grants PI19/00589, PI19/ 01404, PI16/01842, PI17/00535 and GLD19/00100) for financial support. This work was supported by funds from MIUR Ministero dell’Istruzione, dell’Universit` a della Ricerca Italiano, project PRIN 2017, ORIGINALE CHEMIAE in Antiviral Strategy—Origin and Modernization of Multi-Component Chemistry as a Source of Innovative Broad- Spectrum Antiviral Strategy (cod. 2017BMK8JR) and from Tuscany region, project Tuscany Antiviral Research Network -TUSCAVIR.NET (Bando Ricerca Salute 2018). We would like to thank Giulietta Venturi for making the ZIKV H/PF/2013 strain available for this study. This work has also been funded by the Fundaci´ on S´ eneca de la Regi´ on de Murcia under Project 20988/PI/18. This research was partially supported by the computer resources and the technical support provided by the supercomputing infrastructure of the NLHPC (ECM-02), Powered@NLHPC and the Extremadura Research Centre for Advanced Technologies (CETA −CIEMAT), funded by the European Regional Development Fund (ERDF). CETA −CIEMAT is part of CIEMAT and the Government of Spain. Authors thank CITIUS (Centro de Investigaci´ on, Tecnología e Innovaci´ on de la Universidad de Sevilla) for its important contribution in recording NMR and Mass spectra and in elemental analysis determination. Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi. org/10.1016/j.bioorg.2023.106408. References [1] N. Vasilakis, S.C. Weaver, Flavivirus transmission focusing on Zika, Curr. Opin. Virol. 22 (2017) 30–35, https://doi.org/10.1016/j.coviro.2016.11.007. [2] N. Alzahrani, M.J. Wu, S. Shanmugam, M.K. Yi, Delayed by design: Role of suboptimal signal peptidase processing of viral structural protein precursors in flaviviridae virus assembly, Viruses. 12 (2020), https://doi.org/10.3390/ v12101090. [3] Z. Weng, X. Shao, D. Graf, C. Wang, C.D. Klein, J. Wang, G.C. Zhou, Identification of fused bicyclic derivatives of pyrrolidine and imidazolidinone as dengue virus-2 NS2B-NS3 protease inhibitors, Eur. J. Med. Chem. 125 (2017) 751–759, https:// doi.org/10.1016/j.ejmech.2016.09.063. [4] T.C. Pierson, M.S. Diamond, The continued threat of emerging flaviviruses, Nat. Microbiol. 5 (2020) 796–812, https://doi.org/10.1038/s41564-020-0714-0. [5] S. Nie, Y. Yao, F. Wu, X. Wu, J. Zhao, Y. Hua, J. Wu, T. Huo, Y.L. Lin, A. R. Kneubehl, M.B. Vogt, J. Ferreon, R. Rico-Hesse, Y. Song, Synthesis, structureactivity relationships, and antiviral activity of allosteric inhibitors of flavivirus NS2B-NS3 protease, J. Med. Chem. 64 (2021) 2777–2800, https://doi.org/ 10.1021/acs.jmedchem.0c02070. M. del Rosario García-Lozano et al. Bioorganic Chemistry 133 (2023) 106408 17 [6] F. Roudot-Thoraval, Epidemiology of hepatitis C virus infection, Clin. Res. Hepatol. Gastroenterol. 45 (2021) 2015–2020, https://doi.org/10.1016/j. clinre.2020.101596. [7] WHO 2022: Hepatitis C (2022) 2022. <https://www.who.int/news-room/factsheets/detail/hepatitis-c>(Accessed: 2022-11-11). [8] M.S. Mehand, F. Al-Shorbaji, P. Millett, B. Murgue, The WHO R&D Blueprint: 2018 review of emerging infectious diseases requiring urgent research and development efforts, Antiviral Res 159 (2018) 63–67, https://doi.org/10.1016/j. antiviral.2018.09.009. [9] WHO 2022: Dengue and severe dengue (2022) 2022. <https://www.who.int/ news-room/fact-sheets/detail/dengue-and- severe-dengue>(Accessed: 2022-11- 11). [10] H. Harapan, A. Michie, R.T. Sasmono, A. Imrie, Dengue: a minireview, Viruses. 12 (2020) 1–35, https://doi.org/10.3390/v12080829. [11] A. Wahaab, B.E. Mustafa, M. Hameed, N.J. Stevenson, M.N. Anwar, K. Liu, J. Wei, Y. Qiu, Z. Ma, Potential role of flavivirus NS2B-NS3 proteases in viral pathogenesis and anti-flavivirus drug discovery employing animal cells and models, A Review (2022) 1–27. [12] H.E. Clapham, B.A. Wills, Implementing a dengue vaccination programme-who, where and how? Trans. R. Soc. Trop. Med. Hyg. 112 (2018) 367–368, https://doi. org/10.1093/trstmh/try070. [13] M.R. Capeding, N.H. Tran, S.R.S. Hadinegoro, H.I.H.M. Ismail, T. Chotpitayasunondh, M.N. Chua, C.Q. Luong, K. Rusmil, D.N. Wirawan, R. Nallusamy, P. Pitisuttithum, U. Thisyakorn, I.K. Yoon, D. Van Der Vliet, E. Langevin, T. Laot, Y. Hutagalung, C. Frago, M. Boaz, T.A. Wartel, N. G. Tornieporth, M. Saville, A. Bouckenooghe, Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observer-masked, placebo-controlled trial, Lancet. 384 (2014) 1358–1365, https:// doi.org/10.1016/S0140-6736(14)61060-6. [14] S. Spizzichino, G. Mattedi, K. Lauder, C. Valle, W. Aouadi, B. Canard, E. Decroly, S. J.F. Kaptein, J. Neyts, C. Graham, Z. Sule, D.J. Barlow, R. Silvestri, D. Castagnolo, Design, synthesis and discovery of N, N’-carbazoyl-aryl-urea inhibitors of Zika NS5 methyltransferase and virus replication, ChemMedChem. 15 (2020) 385–390, https://doi.org/10.1002/cmdc.201900533. [15] N. Palanisamy, D. Akaberi, J. Lennerstrand, Protein backbone flexibility pattern is evolutionarily conserved in the Flaviviridae family: A case of NS3 protease in Flavivirus and Hepacivirus, Mol. Phylogenet. Evol. 118 (2018) 58–63, https://doi. org/10.1016/j.ympev.2017.09.015. [16] S.P. Lim, Q.Y. Wang, C.G. Noble, Y.L. Chen, H. Dong, B. Zou, F. Yokokawa, S. Nilar, P. Smith, D. Beer, J. Lescar, P.Y. Shi, Ten years of dengue drug discovery: progress and prospects, Antiviral Res. 100 (2013) 500–519, https://doi.org/10.1016/j. antiviral.2013.09.013. [17] N. Pathak, Y.P. Kuo, T.Y. Chang, C.T. Huang, H.C. Hung, J.T.A. Hsu, G.Y. Yu, J. M. Yang, Zika virus NS3 protease pharmacophore anchor model and drug discovery, Sci. Rep. 10 (2020) 1–17, https://doi.org/10.1038/s41598-020-65489- w. [18] N. Pathak, M.L. Lai, W.Y. Chen, B.W. Hsieh, G.Y. Yu, J.M. Yang, Pharmacophore anchor models of flaviviral NS3 proteases lead to drug repurposing for DENV infection, BMC Bioinform. 18 (2017), https://doi.org/10.1186/s12859-017-1957- 5. [19] H. Lee, J. Ren, S. Nocadello, A.J. Rice, I. Ojeda, S. Light, G. Minasov, J. Vargas, D. Nagarathnam, W.F. Anderson, M.E. Johnson, Identification of novel small molecule inhibitors against NS2B/NS3 serine protease from Zika virus, Antiviral Res. 139 (2017) 49–58, https://doi.org/10.1016/j.antiviral.2016.12.016. [20] J. S´ anchez-C´ espedes, P. Martínez-Aguado, M. Vega-Holm, A. Serna-Gallego, J. I. Candela, J.A. Marrugal-Lorenzo, J. Pach´ on, F. Iglesias-Guerra, J.M. Vega-P´ erez, New 4-Acyl-1-phenylaminocarbonyl-2-phenylpiperazine derivatives as potential inhibitors of adenovirus infection. synthesis, biological evaluation, and structureactivity relationships, J. Med. Chem. 59 (2016) 5432–5448, https://doi.org/ 10.1021/acs.jmedchem.6b00300. [21] S. Mazzotta, J.A. Marrugal-Lorenzo, M. Vega-Holm, A. Serna-Gallego, J. ´ Alvarez- Vidal, J. Berastegui-Cabrera, J. P´ erez del Palacio, C. Díaz, F. Aiello, J. Pach´ on, F. Iglesias-Guerra, J.M. Vega-P´ erez, J. S´ anchez-C´ espedes, Optimization of piperazine-derived ureas privileged structures for effective antiadenovirus agents, Eur. J. Med. Chem. 185 (2020), https://doi.org/10.1016/j.ejmech.2019.111840. [22] M. Bassetto, P. Leyssen, J. Neyts, M.M. Yerukhimovich, D.N. Frick, M. Courtney- Smith, A. Brancale, In silico identification, design and synthesis of novel piperazine-based antiviral agents targeting the hepatitis C virus helicase, Eur. J. Med. Chem. 125 (2017) 1115–1131, https://doi.org/10.1016/j. ejmech.2016.10.043. [23] Y. Wang, R. Zhou, Y. Quan, S. Chen, X. Shi, Y. Li, S. Cen, Design, synthesis, and evaluation of novel 4-amino-2-(4-benzylpiperazin-1-yl)methylbenzonitrile compounds as Zika inhibitors, Bioorg. Med. Chem. Lett. 30 (2020), 126906, https://doi.org/10.1016/j.bmcl.2019.126906. [24] L. Li, C. Basavannacharya, K.W.K. Chan, L. Shang, S.G. Vasudevan, Z. Yin, Structure-guided discovery of a novel non-peptide inhibitor of dengue virus NS2B- NS3 protease, Chem. Biol. Drug Des. 86 (2015) 255–264, https://doi.org/10.1111/ cbdd.12500. [25] M.X. Dong, L. Lu, H. Li, X. Wang, H. Lu, S. Jiang, Q.Y. Dai, Design, synthesis, and biological activity of novel 1,4-disubstituted piperidine/piperazine derivatives as CCR5 antagonist-based HIV-1 entry inhibitors, Bioorg. Med. Chem. Lett. 22 (2012) 3284–3286, https://doi.org/10.1016/j.bmcl.2012.03.019. [26] D. Dou, G. He, S.R. Mandadapu, S. Aravapalli, Y. Kim, K.O. Chang, W.C. Groutas, Inhibition of noroviruses by piperazine derivatives, Bioorg. Med. Chem. Lett. 22 (2012) 377–379, https://doi.org/10.1016/j.bmcl.2011.10.122. [27] X. Zhang, H. Wang, Y. Li, R. Cao, W. Zhong, Z. Zheng, G. Wang, J. Xiao, S. Li, Novel substituted heteroaromatic piperazine and piperidine derivatives as inhibitors of human enterovirus 71 and coxsackievirus A16, Molecules. 18 (2013) 5059–5071, https://doi.org/10.3390/molecules18055059. [28] M. Shaquiquzzaman, G. Verma, A. Marella, M. Akhter, W. Akhtar, M.F. Khan, S. Tasneem, M.M. Alam, Piperazine scaffold: A remarkable tool in generation of diverse pharmacological agents, Eur. J. Med. Chem. 102 (2015) 487–529, https:// doi.org/10.1016/j.ejmech.2015.07.026. [29] M. Saudi, J. Zmurko, S. Kaptein, J. Rozenski, B. Gadakh, P. Chaltin, A. Marchand, J. Neyts, A. Van Aerschot, Synthetic strategy and antiviral evaluation of diamide containing heterocycles targeting dengue and yellow fever virus, Eur. J. Med. Chem. 121 (2016) 158–168, https://doi.org/10.1016/j.ejmech.2016.05.043. [30] P. Sikka, Role of aryl urea containing compounds in medicinal chemistry, Med. Chem. (Los. Angeles). 5 (2015) 479–483, https://doi.org/10.4172/2161- 0444.1000305. [31] E.M.F. Muri, M. Gomes, M.G. Albuquerque, E.F.F. Da Cunha, R.B. De Alencastro, J. S. Williamson, O.A.C. Antunes, Pseudo-peptides derived from isomannide as potential inhibitors of serine proteases, Amino Acids. 28 (2005) 413–419, https:// doi.org/10.1007/s00726-004-0146-9. [32] S. Mazzotta, J. Berastegui-Cabrera, G. Carullo, M. Vega-Holm, M. Carretero- Ledesma, L. Mendolia, F. Aiello, F. Iglesias-Guerra, J. Pach´ on, J.M. Vega-P´ erez, J. S´ anchez-C´ espedes, Serinol-based benzoic acid esters as new scaffolds for the development of adenovirus infection inhibitors: design, synthesis, and in vitro biological evaluation, ACS Infect. Dis. 7 (2021), https://doi.org/10.1021/ acsinfecdis.0c00515. [33] S. Mazzotta, J. Berastegui-Cabrera, M. Vega-Holm, M. del R. García-Lozano, M. Carretero-Ledesma, F. Aiello, J.M. Vega-P´ erez, J. Pach´ on, F. Iglesias-Guerra, J. S´ anchez-C´ espedes, Design, synthesis and in vitro biological evaluation of a novel class of anti-adenovirus agents based on 3-amino-1,2-propanediol, Bioorg. Chem. 114 (2021), https://doi.org/10.1016/j.bioorg.2021.105095. [34] A.K. Timiri, B.N. Sinha, V. Jayaprakash, Progress and prospects on DENV protease inhibitors, Eur. J. Med. Chem. 117 (2016) 125–143, https://doi.org/10.1016/j. ejmech.2016.04.008. [35] C. Steuer, C. Gege, W. Fischl, K.H. Heinonen, R. Bartenschlager, C.D. Klein, Synthesis and biological evaluation of α -ketoamides as inhibitors of the Dengue virus protease with antiviral activity in cell-culture, Bioorg. Med. Chem. 19 (2011) 4067–4074, https://doi.org/10.1016/j.bmc.2011.05.015. [36] M. Robello, E. Barresi, E. Baglini, S. Salerno, S. Taliani, F. Da Settimo, The alpha keto amide moiety as a privileged motif in medicinal chemistry: current insights and emerging opportunities, J. Med. Chem. 64 (2021) 3508–3545, https://doi.org/ 10.1021/acs.jmedchem.0c01808. [37] A. Kling, K. Jantos, H. Mack, W. Hornberger, K. Drescher, V. Nimmrich, A. Relo, K. Wicke, C.W. Hutchins, Y. Lao, K. Marsh, A. Moeller, Discovery of novel and highly selective inhibitors of calpain for the treatment of Alzheimer’s disease: 2-(3- phenyl-1H-pyrazol-1-yl)-nicotinamides, J. Med. Chem. 60 (2017) 7123–7138, https://doi.org/10.1021/acs.jmedchem.7b00731. [38] A. Dorababu, Indole-a promising pharmacophore in recent antiviral drug discovery, RSC Med. Chem. 11 (2020) 1335–1353, https://doi.org/10.1039/ d0md00288g. [39] N.A. Meanwell, M.R. Krystal, B. Nowicka-Sans, D.R. Langley, D.A. Conlon, M. D. Eastgate, D.M. Grasela, P. Timmins, T. Wang, J.F. Kadow, Inhibitors of HIV-1 attachment: the discovery and development of temsavir and its prodrug fostemsavir, J. Med. Chem. 61 (2018) 62–80, https://doi.org/10.1021/acs. jmedchem.7b01337. [40] N. Ruwizhi, B.A. Aderibigbe, Cinnamic acid derivatives and their biological efficacy, Int. J. Mol. Sci. 21 (2020) 1–36, https://doi.org/10.3390/ijms21165712. [41] O.M. Abd El-Raouf, E.S.M. El-Sayed, M.F. Manie, Cinnamic acid and cinnamaldehyde ameliorate cisplatin-induced splenotoxicity in rats, J. Biochem. Mol. Toxicol. 29 (2015) 426–431, https://doi.org/10.1002/jbt.21715. [42] R. Wang, W. Yang, Y. Fan, W. Dehaen, Y. Li, H. Li, W. Wang, Q. Zheng, Q. Huai, Design and synthesis of the novel oleanolic acid-cinnamic acid ester derivatives and glycyrrhetinic acid-cinnamic acid ester derivatives with cytotoxic properties, Bioorg. Chem. 88 (2019), 102951, https://doi.org/10.1016/j.bioorg.2019.102951. [43] S. Guo, Y. Zhen, Z. Zhu, G. Zhou, X. Zheng, Cinnamic acid rescues behavioral deficits in a mouse model of traumatic brain injury by targeting miR-455-3p/ HDAC2, Life Sci. 235 (2019), 116819, https://doi.org/10.1016/j.lfs.2019.116819. [44] R. Amano, A. Yamashita, H. Kasai, T. Hori, S. Miyasato, S. Saito, H. Yokoe, K. Takahashi, T. Tanaka, T. Otoguro, S. Maekawa, N. Enomoto, M. Tsubuki, K. Moriishi, Cinnamic acid derivatives inhibit hepatitis C virus replication via the induction of oxidative stress, Antiviral Res. 145 (2017) 123–130, https://doi.org/ 10.1016/j.antiviral.2017.07.018. [45] A.M. Faucher, M.D. Bailey, P.L. Beaulieu, C. Brochu, J.S. Duceppe, J.M. Ferland, E. Ghiro, V. Gorys, T. Halmos, S.H. Kawai, M. Poirier, B. Simoneau, Y.S. Tsantrizos, M. Llin` as-Brunet, Synthesis of BILN 2061, an HCV NS3 protease inhibitor with proven antiviral effect in humans, Org. Lett. 6 (2004) 2901–2904, https://doi.org/ 10.1021/ol0489907. [46] Y. Chen, Z. Li, P. Pan, Z. Lao, J. Xu, Z. Li, S. Zhan, X. Liu, Y. Wu, W. Wang, G. Li, Cinnamic acid inhibits Zika virus by inhibiting RdRp activity, Antiviral Res. 192 (2021), 105117, https://doi.org/10.1016/j.antiviral.2021.105117. [47] F. Li, E.M. Lee, X. Sun, D. Wang, H. Tang, G.C. Zhou, Design, synthesis and discovery of andrographolide derivatives against Zika virus infection, Eur. J. Med. Chem. 187 (2020), 111925, https://doi.org/10.1016/j.ejmech.2019.111925. [48] G. Barbosa-Lima, A.M. Moraes, A. da S. Araújo, E.T. da Silva, C.S. de Freitas, Y. R. Vieira, A. Marttorelli, J.C. Neto, P.T. Bozza, M.V.N. de Souza, T.M.L. Souza, 2,8- bis(trifluoromethyl)quinoline analogs show improved anti-Zika virus activity, M. del Rosario García-Lozano et al. Bioorganic Chemistry 133 (2023) 106408 18 compared to mefloquine, Eur. J. Med. Chem. 127 (2017) 334–340, https://doi.org/ 10.1016/j.ejmech.2016.12.058. [49] A. Balasubramanian, T. Teramoto, A.A. Kulkarni, A.K. Bhattacharjee, R. Padmanabhan, Antiviral activities of selected antimalarials against dengue virus type 2 and Zika virus, Antiviral Res. 137 (2017) 141–150, https://doi.org/ 10.1016/j.antiviral.2016.11.015. [50] C. Nitsche, M.A.M. Behnam, C. Steuer, C.D. Klein, Retro peptide-hybrids as selective inhibitors of the Dengue virus NS2B-NS3 protease, Antiviral Res. 94 (2012) 72–79, https://doi.org/10.1016/j.antiviral.2012.02.008. [51] C. Nitsche, C. Steuer, C.D. Klein, Arylcyanoacrylamides as inhibitors of the Dengue and West Nile virus proteases, Bioorg. Med. Chem. 19 (2011) 7318–7337, https:// doi.org/10.1016/j.bmc.2011.10.061. [52] C. de la Guardia, D.E. Stephens, H.T. Dang, M. Quijada, O.V. Larionov, R. Lleonart, Antiviral activity of novel quinoline derivatives against dengue virus serotype 2, Molecules. 23 (2018) 1–11, https://doi.org/10.3390/molecules23030672. [53] S.J.F. Kaptein, P. Vincetti, E. Crespan, J.I.A. Rivera, G. Costantino, G. Maga, J. Neyts, M. Radi, Identification of broad-spectrum dengue/zika virus replication inhibitors by functionalization of quinoline and 2,6-diaminopurine scaffolds, ChemMedChem. 13 (2018) 1371–1376, https://doi.org/10.1002/ cmdc.201800178. [54] J. Deng, N. Li, H. Liu, Z. Zuo, O.W. Liew, W. Xu, G. Chen, X. Tong, W. Tang, J. Zhu, J. Zuo, H. Jiang, C.G. Yang, J. Li, W. Zhu, Discovery of novel small molecule inhibitors of dengue viral NS2B-NS3 protease using virtual screening and scaffold hopping, J. Med. Chem. 55 (2012) 6278–6293, https://doi.org/10.1021/ jm300146f. [55] H. Beesetti, P. Tyagi, B. Medapi, V.S. Krishna, D. Sriram, N. Khanna, S. Swaminathan, A quinoline compound inhibits the replication of dengue virus serotypes 1–4 in Vero cells, Antivir. Ther. 23 (2018) 385–394, https://doi.org/ 10.3851/IMP3231. [56] J. Zhao, Y. Zhang, M. Wang, Q. Liu, X. Lei, M. Wu, S. Guo, D. Yi, Q. Li, L. Ma, Z. Liu, F. Guo, J. Wang, X. Li, Y. Wang, S. Cen, Quinoline and quinazoline derivatives inhibit viral RNA synthesis by SARS-CoV-2 RdRp, ACS Infect. Dis. 7 (2021) 1535–1544, https://doi.org/10.1021/acsinfecdis.1c00083. [57] E.D. Micewicz, R. Khachatoorian, S.W. French, P. Ruchala, Identification of novel small-molecule inhibitors of Zika virus infection, Bioorg. Med. Chem. Lett. 28 (2018) 452–458, https://doi.org/10.1016/j.bmcl.2017.12.019. [58] R. Khachatoorian, E.D. Micewicz, A. Micewicz, S.W. French, P. Ruchala, Optimization of 1,3-disubstituted urea-based inhibitors of Zika virus infection, Bioorg. Med. Chem. Lett. 29 (2019), 126626, https://doi.org/10.1016/j. bmcl.2019.126626. [59] S. Mazzotta, T. Cebrero-Cangueiro, L. Frattaruolo, M. Vega-Holm, M. Carretero- Ledesma, J. S´ anchez-C´ espedes, A.R. Cappello, F. Aiello, J. Pach´ on, J.M. Vega- P´ erez, F. Iglesias-Guerra, M.E. Pach´ on-Ib´ a˜ nez, Exploration of piperazine-derived thioureas as antibacterial and anti-inflammatory agents. In vitro evaluation against clinical isolates of colistin-resistant Acinetobacter baumannii, Bioorganic Med, Chem. Lett. 30 (2020), 127411, https://doi.org/10.1016/j.bmcl.2020.127411. [60] J.G. Moffat, J. Rudolph, D. Bailey, Phenotypic screening in cancer drug discoverypast, present and future, Nat. Rev. Drug Discov. 13 (2014) 588–602, https://doi. org/10.1038/nrd4366. [61] S.A. Shiryaev, C. Farhy, A. Pinto, C.T. Huang, N. Simonetti, A.E. Ngono, A. Dewing, S. Shresta, A.B. Pinkerton, P. Cieplak, A.Y. Strongin, A.V. Terskikh, Characterization of the Zika virus two-component NS2B-NS3 protease and structure-assisted identification of allosteric small-molecule antagonists, Antiviral Res. 143 (2017) 218–229, https://doi.org/10.1016/j.antiviral.2017.04.015. [62] L. Papageorgiou, S. Loukatou, K. Sofia, D. Maroulis, D. Vlachakis, An updated evolutionary study of Flaviviridae NS3 helicase and NS5 RNA-dependent RNA polymerase reveals novel invariable motifs as potential pharmacological targets, Mol. Biosyst. 12 (2016) 2080–2093, https://doi.org/10.1039/c5mb00706b. [63] D. Luo, S.G. Vasudevan, J. Lescar, The flavivirus NS2B-NS3 protease-helicase as a target for antiviral drug development, Antiviral Res. 118 (2015) 148–158, https:// doi.org/10.1016/j.antiviral.2015.03.014. [64] C. Caillet-Saguy, S.P. Lim, P.Y. Shi, J. Lescar, S. Bressanelli, Polymerases of hepatitis C viruses and flaviviruses: structural and mechanistic insights and drug development, Antiviral Res. 105 (2014) 8–16, https://doi.org/10.1016/j. antiviral.2014.02.006. [65] Y. Yang, L. Cao, H. Gao, Y. Wu, Y. Wang, F. Fang, T. Lan, Z. Lou, Y. Rao, Discovery, optimization, and target identification of novel potent broad-spectrum antiviral inhibitors, J. Med. Chem. 62 (2019) 4056–4073, https://doi.org/10.1021/acs. jmedchem.9b00091. [66] I. Vicenti, F. Dragoni, A. Giannini, F. Giammarino, M. Spinicci, F. Saladini, A. Boccuto, M. Zazzi, Development of a cell-based immunodetection assay for simultaneous screening of antiviral compounds inhibiting zika and dengue virus replication, SLAS Discov. 25 (2020) 506–514, https://doi.org/10.1177/ 2472555220911456. [67] A. Brai, A. Boccuto, M. Monti, S. Marchi, I. Vicenti, F. Saladini, C.I. Trivisani, A. Pollutri, C.M. Trombetta, E. Montomoli, V. Riva, A. Garbelli, E.M. Nola, M. Zazzi, G. Maga, E. Dreassi, M. Botta, Exploring the Implication of DDX3X in DENV infection: discovery of the first-in-class DDX3X fluorescent inhibitor, ACS Med. Chem. Lett. 11 (2020) 956–962, https://doi.org/10.1021/ acsmedchemlett.9b00681. [68] I. Vicenti, A. Boccuto, A. Giannini, F. Dragoni, F. Saladini, M. Zazzi, Comparative analysis of different cell systems for Zika virus (ZIKV) propagation and evaluation of anti-ZIKV compounds in vitro, Virus Res. 244 (2018) 64–70, https://doi.org/ 10.1016/j.virusres.2017.11.003. [69] C.Q. Sacramento, G.R. De Melo, C.S. De Freitas, N. Rocha, L.V.B. Hoelz, M. Miranda, N. Fintelman-Rodrigues, A. Marttorelli, A.C. Ferreira, G. Barbosa- Lima, J.L. Abrantes, Y.R. Vieira, M.M. Bastos, E. De Mello Volot˜ ao, E.P. Nunes, D. A. Tschoeke, L. Leomil, E.C. Loiola, P. Trindade, S.K. Rehen, F.A. Bozza, P.T. Bozza, N. Boechat, F.L. Thompson, A.M.B. De Filippis, K. Brüning, T.M.L. Souza, The clinically approved antiviral drug sofosbuvir inhibits Zika virus replication, Sci. Rep. 7 (2017) 1–12, https://doi.org/10.1038/srep40920. [70] H.N. Nagesh, A. Suresh, S.D.S.S. Sairam, D. Sriram, P. Yogeeswari, K.V.G. Chandra Sekhar, Design, synthesis and antimycobacterial evaluation of 1-(4-(2- substitutedthiazol-4-yl)phenethyl)-4-(3-(4-substitutedpiperazin-1-yl)alkyl) piperazine hybrid analogues, Eur. J. Med. Chem. 84 (2014) 605–613, https://doi. org/10.1016/j.ejmech.2014.07.067. [71] M.F. Adasme, K.L. Linnemann, S.N. Bolz, F. Kaiser, S. Salentin, V.J. Haupt, M. Schroeder, PLIP 2021: Expanding the scope of the protein-ligand interaction profiler to DNA and RNA, Nucl. Acids Res. 49 (2021) W530–W534, https://doi. org/10.1093/nar/gkab294. [72] L.L.C. Schr¨ odinger, The PyMOL Molecular Graphics System, Version 1.8, 2015, (n. d.). [73] A.N. Matthew, J. Zephyr, D. Nageswara Rao, M. Henes, W. Kamran, K. Kosovrasti, A.K. Hedger, G.J. Lockbaum, J. Timm, A. Ali, N. Kurt Yilmaz, C.A. Schiffer, Avoiding drug resistance by substrate envelope-guided design: toward potent and Robust HCV NS3/4A protease inhibitors, Mol. Biol. Physiol. 11 (2020) e00172–e220. [74] V.S. Patil, D.R. Harish, U. Vetrivel, S. Roy, S.H. Deshpande, H.V. Hegde, Hepatitis C virus NS3/4A inhibition and host immunomodulation by tannins from terminalia chebula: a structural perspective, Molecules. 27 (2022), https://doi.org/10.3390/ molecules27031076. [75] J. Lei, G. Hansen, C. Nitsche, C.D. Klein, L. Zhang, R. Hilgenfeld, Crystal structure of zika virus NS2b-NS3 protease in complex with a boronate inhibitor, Science (80- .). 353 (2016) 503–505, https://doi.org/10.1126/science.aag2419. [76] C.Y. Jia, J.Y. Li, G.F. Hao, G.F. Yang, A drug-likeness toolbox facilitates ADMET study in drug discovery, Drug Discov. Today. 25 (2020) 248–258, https://doi.org/ 10.1016/j.drudis.2019.10.014. [77] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings, Adv. Drug Deliv. Rev. 64 (2012) 4–17, https://doi.org/10.1016/j.addr.2012.09.019. [78] D.F. Veber, S.R. Johnson, H.-Y. Cheng, B.R. Smith, K.W. Ward, K.D. Kopple, Molecular properties that influence the oral bioavailability of drug candidates, J. Med. Chem. 45 (2002) 2615–2623, https://doi.org/10.1021/jm020017n. [79] R. Wang, Y. Fu, L. Lai, A New Method for Calculating Partition Coefficients of Organic Compounds, Acta Phys. - Chim. Sin. 13 (1997) 615–621, https://doi.org/ 10.3866/pku.whxb19970101. [80] A. Daina, O. Michielin, V. Zoete, SwissADME: A free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules, Sci. Rep. 7 (2017) 1–13, https://doi.org/10.1038/srep42717. [81] N.H. Amin, M.T. El-Saadi, A.A. Ibrahim, H.M. Abdel-Rahman, Design, synthesis and mechanistic study of new 1,2,4-triazole derivatives as antimicrobial agents, Bioorg. Chem. 111 (2021), 104841, https://doi.org/10.1016/j. bioorg.2021.104841. [82] M.M. Morcoss, E.S.M.N. Abdelhafez, R.A. Ibrahem, H.M. Abdel-Rahman, M. Abdel- Aziz, D.A. Abou El-Ella, Design, synthesis, mechanistic studies and in silico ADME predictions of benzimidazole derivatives as novel antifungal agents, Bioorg. Chem. 101 (2020), 103956, https://doi.org/10.1016/j.bioorg.2020.103956. [83] P.F. Lamie, J.N. Philoppes, 2-Thiopyrimidine/chalcone hybrids: design, synthesis, ADMET prediction, and anticancer evaluation as STAT3/STAT5a inhibitors, J. Enzyme Inhib. Med. Chem. 35 (2020) 864–879, https://doi.org/10.1080/ 14756366.2020.1740922. [84] S.K. Lee, Y. Kang, G.S. Chang, I.H. Lee, S.H. Park, J. Park, Bioinformatics and Molecular Design Research Center. Yonsei University, Seoul, (2017). <https:// preadmet. bmdrc.kr>. [85] V. Pogaku, K. Gangarapu, S. Basavoju, K.K. Tatapudi, S.B. Katragadda, Design, synthesis, molecular modelling, ADME prediction and anti-hyperglycemic evaluation of new pyrazole-triazolopyrimidine hybrids as potent α -glucosidase inhibitors, Bioorg. Chem. 93 (2019), 103307, https://doi.org/10.1016/j. bioorg.2019.103307. [86] J. S´ anchez-C´ espedes, M.E. Pach´ on-Ib´ a˜ nez, J. Pach´ on, P. Martínez-Aguado, T. Cebrero-Cangueiro, J.M. ega-Per´ ez, F. Iglesias-Guerra, M. Vega-Holm, J.I. Candela- Lena, S. Mazzotta, Piperazine derivatives as antiviral agents with increased therapeutic activity; year of application. EP16382073.1 (23 February 2016), US 2019/0308956 A1(10 October 2019). [87] B.M. Minrovic, D. Jung, R.J. Melander, C. Melander, New class of adjuvants enables lower dosing of colistin against acinetobacter baumannii, ACS Infect. Dis. 4 (2018) 1368–1376, https://doi.org/10.1021/acsinfecdis.8b00103. [88] P. Gallinari, D. Brennan, C. Nardi, M. Brunetti, L. Tomei, C. Steinkühler, R. De Francesco, Multiple enzymatic activities associated with recombinant NS3 protein of hepatitis C virus, J. Virol. 72 (1998) 6758–6769, https://doi.org/10.1128/ jvi.72.8.6758-6769.1998. [89] G. Barbato, D.O. Cicero, F. Cordier, F. Narjes, B. Gerlach, S. Sambucini, S. Grzesiek, V.G. Matassa, R. De Francesco, R. Bazzo, Inhibitor binding induces active site stabilization of the HCV NS3 protein serine protease domain, EMBO J. 19 (2000) 1195–1206, https://doi.org/10.1093/emboj/19.6.1195. [90] D.L. Sali, R. Ingram, M. Wendel, D. Gupta, C. McNemar, A. Tsarbopoulos, J. W. Chen, Z. Hong, R. Chase, C. Risano, R. Zhang, N. Yao, A.D. Kwong, L. Ramanathan, H.V. Le, P.C. Weber, Serine protease of hepatitis C virus expressed in insect cells as the NS3/4A complex, Biochemistry. 37 (1998) 3392–3401, https://doi.org/10.1021/bi972010r. M. del Rosario García-Lozano et al. Bioorganic Chemistry 133 (2023) 106408 19 [91] X. Tong, R. Chase, A. Skelton, T. Chen, J. Wright-Minogue, B.A. Malcolm, Identification and analysis of fitness of resistance mutations against the HCV protease inhibitor SCH 503034, Antiviral Res. 70 (2006) 28–38, https://doi.org/ 10.1016/j.antiviral.2005.12.003. [92] P. Gallego, ´ A. Rojas, G. Falc´ on, P. Carbonero, M.R. García-Lozano, A. Gil, L. Grande, O. Cremades, M. Romero-G´ omez, J.D. Bautista, J.A. Del Campo, Watersoluble extracts from edible mushrooms (: Agaricus bisporus) as inhibitors of hepatitis C viral replication, Food Funct. 10 (2019) 3758–3767, https://doi.org/ 10.1039/c9fo00733d. [93] U.W. Hawas, R. Al-Farawati, L.T.A. El-Kassem, A.J. Turki, Different culture metabolites of the Red Sea fungus Fusarium equiseti optimize the inhibition of hepatitis C virus NS3/4A protease (HCV PR), Mar. Drugs. 14 (2016), https://doi. org/10.3390/md14100190. [94] H.M.L.J. Reed, A simple method of estimating fifty per cent endpoints, Am. J. Hyg. 27 (1938) 546–558, https://doi.org/10.7723/antiochreview.72.3.0546. [95] N.M. O’Boyle, M. Banck, C.A. James, C. Morley, T. Vandermeersch, G. R. Hutchison, Open babel, J. Cheminform. 3 (2011) 1–14, https://doi.org/ 10.1186/1758-2946-3-33. [96] O.V. Stroganov, F.N. Novikov, V.S. Stroylov, V. Kulkov, G.G. Chilov, Lead finder: an approach to improve accuracy of protein-ligand docking, binding energy estimation, and virtual screening, J. Chem. Inf. Model. 48 (2008) 2371–2385, https://doi.org/10.1021/ci800166p. M. del Rosario García-Lozano et al.