Previously, our group synthesized and designed several aryl-substituted 1H-pyrazoles, which showed interesting biological properties [28] - [31] . Among pyrazoles, the pyrazolone moiety is endowed with a wide range of pharmacological activities, such as antimicrobial, neuroactive, anti-inflammatory, antioxidant, and antiviral [32] . Consequently, four aryl-substituted 5-pyrazolone derivatives (compounds 1a-1d) ( Figure 1 ) were obtained as described elsewhere [24] [33] . Among the substituents, we explored the introduction of electron-withdrawing (e.g. chlorine) and electron-donating (e.g., methoxy) groups in the phenyl ring attached to the pyrazolone nucleus. All derivatives were dissolved in sterile dimethylsulfoxide (DMSO) in 50 mM stock. On the other hand, chloroquine (Sigma-Aldrich, Brazil) was prepared in sterile water to a final concentration of 50 mM. All stocks were stored at −20˚C until use. The final concentration of DMSO was <0.1%.

Vero cells (ATCC CCL-81; African green monkey kidney cells) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, Brazil) at 37˚C, supplemented with 5% of fetal bovine serum (FBS) (Sigma-Aldrich, Brazil), 2 g/L of sodium bicarbonate, 100 IU/mL of penicillin, 100 µg/mL of streptomycin, and 2.5 µg/mL of amphotericin B. The Brazilian strain of ZIKV KU49755 was kindly provided by Professor Dr. Ana Maria Pinto from the Biomedical Institute, Fluminense Federal University. This strain was isolated from amniotic fluid samples from a neonate with microcephaly and represents the Asian lineage. Viruses were propagated in Vero cells for five days at 37˚C and 5% CO₂ atmosphere and were harvested and stored at −80˚C until needed. Virus titers were defined by plaque reduction assay.

The cytotoxicity of the compounds on Vero cells was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described elsewhere [34] . Briefly, Vero cells were seeded onto 96-well culture plates at a density of 1 × 10⁴ cells per well and were treated with increasing concentrations of compounds (50, 100, 200, 400, and 800 μM) for 72 h at 37˚C and humidified at 5% CO₂ atmosphere. Then, compounds were removed, and 50 μL of a 5 mg/mL solution of MTT (Sigma-Aldrich, Brazil) was added into each well and incubated for 4 h at 37˚C and humidified 5% CO₂ atmosphere. The MTT solution was then removed and 100 μL of DMSO was added to each well to dissolve formazan crystals. Finally, the resulting absorbance of each well was determined at 545 nm using a microplate reader (TP-Reader Thermo Plate). The relative cell viability was calculated as a percentage comparing the treated to untreated cells and the concentration that reduces cell viability by 50% (CC₅₀) was determined by a linear regression analysis of the dose-response curves. The experiment was undertaken three times independently in triplicates.

Vero cells were seeded onto 24-well plates at a density of 2 × 10⁴ cells per well and were infected with ZIKV at a multiplicity of infection (MOI) of 1.0 for 3 h at 37˚C and humidified 5% CO₂ atmosphere. Then, the inoculum was discarded, and compounds were added. As an initial screening, compounds were added at a final concentration of 50 µM, while the ones that showed antiviral activity at this stage were further evaluated at different concentrations (3.125, 6.25, 12.5, 25, and 50 μM). The treated plates were incubated for 20 h at 37˚C and humidified 5% CO₂ atmosphere. Finally, cells were lysed by three freezing and thawing cycles for viral titer determination by plaque reduction assays.

To determine virus titers, Vero cells maintained in 24-well plates at a cell density of 2 × 10⁵ cells per well were infected with 10-fold serial dilutions of the collected samples for 3 h at 37˚C and humidified 5% CO₂ atmosphere. Afterward, the inoculum was removed, and cell monolayers were covered with DMEM supplemented with 3% of methylcellulose (Sigma-Aldrich, Brazil) and 2% of FBS for 7 - 8 days. Then, cells were fixed and stained with a solution of 0.2% crystal violet and 10% formaldehyde. Virus titers were expressed as the number of plaque-forming units per milliliter (PFU/mL) while inhibition of virus cytopathic effect was calculated comparing the infected treated and untreated cells. These assays were performed in triplicate. Finally, the concentration required to inhibit the virus cytopathic effect by half (EC₅₀) of the most potent compounds was calculated by linear regression.

To assess the virucidal effect of the compounds, a cell-free system was employed. 1 × 10⁷ PFU of ZIKV was diluted in DMEM medium in the presence or absence of the compounds (50 µM) and incubated for 2 h at 37˚C and humidified 5% CO₂ atmosphere. Then, Vero cells (2 × 10⁵ cells/well) seeded in a 24-well plate were infected with the incubated virus for 1 h at 37˚C and humidified 5% CO₂ atmosphere. The inoculum was removed, and cell monolayers were covered with DMEM supplemented with 3% of methylcellulose and 2% of FBS for 7 - 8 days. Finally, cells were fixed and stained with a solution of 0.2% crystal violet and 10% formaldehyde. Viral titers were expressed as the number of PFU/mL.

We carried out a time of drug addition assay to investigate at which step of the viral life cycle the compounds act. Vero cells grown in 24-well plates at a cell density of 2 × 10⁵ cells per well were infected with ZIKV at an MOI of 0.1. Compounds were added (50 µM) at different times, as follows: 3 hours before infection (−3 hpi; pretreatment), 0 h (at the time of infection), or at times post-infection, such as 3, 6, 9, and 12 hpi, and incubated for 72 h at 37˚C. After 72 h, the percentage of viral inhibition of each treated sample was calculated by comparing it with virus titers from untreated controls.

The theoretical pharmacokinetic and toxicological properties of the most active compounds (1a, 1b, 1c, and 1d) and chloroquine were analyzed. The admetSAR 2 server [35] was employed to predict human intestinal absorption (HIA) and carcinogenicity, whereas genotoxicity (based on the AMES test), hepatotoxicity, and cardiotoxicity (based on inhibition of hERG I and II) were predicted using the pkCSM server [36] . Additionally, the compounds were also evaluated according to industry rules, like Lipinski’s “rule of five”, GSK 4/400, and Pfizer 3/75 using the FAF-Drugs4 web server [37] .

Molecular docking of the most potent compounds 1a, 1b, 1c, and 1d with the methyltransferase domain of NS5 (NS5 MTase) from ZIKV was carried out to evaluate their putative mechanism of antiviral action. The crystal structure of ZIKV NS5 methyltransferase complexed with its cofactor S-adenosylmethionine (SAM) was retrieved from the Protein Data Bank under the code 5KQR [38] . The three-dimensional structures of the pyrazolone derivatives were constructed and optimized using the Spartan’10 program (Wavefunction Inc., Irvine, CA, USA). Initially, structures were submitted to a conformational analysis in a vacuum using molecular mechanics and the MMFF force field. The lowest-energy conformer was subjected to a geometry optimization step using the semi-empirical RM1 method, followed by an ab initio calculation using the Hartree-Fock method and the 6-31G* basis set.

Molecular docking studies were performed using Autodock Tools 1.5.6 and AutoDock 4.2.6 [39] . First, the co-crystallized structure of SAM was redocked in NS5 methyltransferase to evaluate the docking software accuracy and validate the docking protocol (RMSD 0.61 Å). The protein was prepared by adding polar hydrogens and Gasteiger charges were assigned whereas polar hydrogens were added to the ligand and its rotatable bonds were determined automatically. During docking studies, the protein was kept rigid while the rotatable bonds of ligands were allowed to rotate freely. The grid box, with dimensions of 50 × 50 × 50 points (0.375 Å spacing), was centered on the SAM binding site. Lamarckian genetic algorithm was employed as a search engine with a population size of 50 while other parameters of the search algorithm were kept at default values. After validation, the same protocol was employed for docking studies with the pyrazolopyrimidine derivatives. Finally, the conformer with the lowest binding energy was selected for visual inspection and protein-ligand interaction analysis using Ligplot v. 4.5.3 [40] available at the European Bioinformatics Institute (EMBL-EBI) server.

All the data shown represent the mean ± standard deviation (SD) from triplicates run three independent times. Analysis of variance (ANOVA) was used to analyze differences among group means, and the Tukey test was performed for post hoc analysis to differentiate among experimental groups. Statistical significance was considered when p-value < 0.05. Statistical analysis was performed using GraphPad Prism 7 software (GraphPad Software Inc.).

Prior to antiviral experiments, the toxicity of pyrazolone derivatives was evaluated in Vero cells. The CC₅₀ of compounds 1a, 1b, 1c, and 1d were approximately 1353 μM ± 17.02, 1472 μM ± 40.42, 1130 μM ± 6.83, and 980 μM ± 18.12, respectively ( Table 1 ). Since the compounds exhibited low cytotoxicity, their antiviral activity against ZIKV replication in Vero cells was assessed at a concentration of 50 µM. Among the four compounds, compound 1d showed antiviral activity of 68.30%, while compound 1c showed moderate activity (virus titer decreased by 50%). On the other hand, compounds 1a, and 1b exhibited stronger inhibitory activity and were able to reduce the virus titer by 71.20% and 79.30%, respectively. Similarly, chloroquine had a moderate inhibitory activity of 58%. Further, compounds were evaluated at different concentrations to determine their EC₅₀ values. All derivatives showed dose-dependent antiviral activity and compound 1b was the most active, with an EC₅₀ value of 4.30 μM, followed by compounds 1a (EC₅₀ = 16.0 μM), 1d (EC₅₀ = 25.4 μM) and 1c (EC₅₀ = 46.63 μM). Compounds 1b and 1b were more potent than chloroquine (EC₅₀ = 16.82 μM), a marketed drug with antiviral activity in vitro against ZIKV. Finally, the selectivity index (SI) of the compounds was calculated, being compound 1b the most selective one with an SI of 342. Despite the lower antiviral activity in comparison to chloroquine (SI = 24.98), the pyrazolone derivatives showed comparable or even higher selectivity than this drug, which indicates the promising profile of these compounds.

^aCC₅₀: Concentration that reduced the cytotoxicity concentration by 50%, ^bInhibition rate (%) of ZIKV replication in Vero cells at 50 µM, ^cEC₅₀: Concentration that reduced 50% of ZIKV replication, ^dSI: Selectivity Index was defined as the ratio between CC₅₀ and EC₅₀.

In order to analyze the inactivation of viral particles by the compounds, ZIKV suspensions were treated with the compounds at 50 µM for 2 h. Then, the remaining infectivity was evaluated by plaque reduction assays. Treatment of virus particles with compounds 1a, 1b, 1c, and 1d reduced virus titers by 38%, 76%, 34%, and 27%, respectively, while chloroquine decreased virus titers by 42% ( Figure 2(A) ). The results obtained in the virucidal assays indicated that only compound 1b has an interesting virucidal potential.

We investigated the antiviral effects of the pyrazolone derivatives when added at different times during ZIKV replication to assess at which step of this process the compounds might act ( Figure 2(B) ). All compounds showed the highest antiviral activity when added 3 h before infection. Compounds 1a, 1b, and 1c still presented high antiviral activity when added 3 h post-infection (higher than 50% inhibition), but their activity decreased significantly when they were added later on in the infection like the drug chloroquine. These findings suggested that all compounds mainly act at early stages of virus replication and may also act at host cells.

Based on the mechanism of action assays, we evaluated whether the most active derivatives (1a, 1b, 1c, and 1d) may bind ZIKV NS5 MTase and interfere with ZIKV replication ( Figure 3 ). Compound 1b showed the highest theoretical affinity with this enzyme, showing a binding energy of −6.23 kcal/mol, followed by compounds 1d (−5.32 kcal/mol), 1c (−4.49 kcal/mol), and 1a (−4.47 kcal/mol). Overall, compounds showed extended binding modes and contacted similar residues ( Figure 3 ). Compound 1b established two hydrogen bond interactions with residues Lys105 and Val132 while the other compounds showed only one hydrogen bond interaction. For instance, compounds 1d and 1c interacted with His110 while compound 1a interacted with Val132.

To evaluate the potential of the pyrazolone derivatives as drug candidates, we evaluated their pharmacokinetic and toxicological properties using computational tools ( Table 2 ). All compounds showed good human intestinal absorption and fulfilled Lipinski’s “rule of five”, indicating good oral bioavailability. In addition, these compounds were approved on the GSK 4/400 rule, indicating satisfactory pharmacokinetic and toxicological properties. However, a warning was raised on the Pfizer 3/75 due to their low polar surface area. Yet, they were not reproved, suggesting they exhibit a low probability of showing pre-clinical toxicity.

Moreover, we evaluated other toxicity risks for these compounds, such as carcinogenicity, hepatotoxicity, and cardiotoxicity ( Table 2 ). None of the compounds showed carcinogenic and cardiotoxic risks, but compounds 1b and 1d showed increased risks for hepatotoxicity. Interestingly, compounds 1a and 1c showed a safer profile than chloroquine, which is a marketed drug, with low toxicity risks for all endpoints evaluated.