Materials and Methods

Chemicals and Synthesis of Compounds

All reagents and solvents were of analytical grade and used without further purification unless otherwise stated. The 4-amino-7-chloroquinoline derivatives were synthesized through a three-step synthetic route. Structural characterization of the compounds was performed by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HRMS). Detailed synthetic procedures and spectral data are provided in the Supplementary Information. The synthetic route is summarized in Fig. 1.

Fig. 1: Synthetic route scheme of the 4-amino-7-chloroquinoline derivatives

Cell Lines and Viruses

Vero cells (ATCC CCL-81) were maintained in Dulbecco’s Modified Eagle Medium (DMEM, high glucose) supplemented with 5% fetal bovine serum (FBS) and antibiotics under standard conditions (37 °C, 5% CO₂). The mosquito-derived C6/36 cell line was cultured in Leibovitz’s L-15 medium supplemented with 5% FBS.

Zika virus (ZIKV, strain PE243/2015) was propagated in C6/36 cells. Chikungunya virus (CHIKV, strain 27-African) and Mayaro virus (MAYV, strain Acre27) were propagated in Vero cells. Viral stocks were obtained from infected cell culture supernatants, clarified by centrifugation, aliquoted, and stored at − 80 °C. Viral titers were determined prior to use.

Cytotoxicity Assay

Cell viability was evaluated using the MTT assay. Vero cells (1 × 10⁴ cells/well) were seeded in 96-well plates and incubated with serial dilutions of the synthesized compounds (0.78–100 µg mL⁻¹) prepared in dimethyl sulfoxide (DMSO). After incubation, MTT reagent was added and absorbance was measured spectrophotometrically. The median cytotoxic concentration (CC₅₀) was determined from dose response curves [10, 11].

In Vitro Antiviral Assay

Antiviral activity was assessed using an MTT-based assay in Vero cells (1 × 10⁴ cells/well) infected with CHIKV, ZIKV, or MAYV at defined viral titers. Cells were treated with non-cytotoxic concentrations of the compounds [12, 13]. The median effective concentration (EC₅₀), defined as the concentration required to protect 50% of infected cells from virus-induced cytopathic effects, was calculated [14]. The selectivity index (SI) was determined as the ratio CC₅₀/EC₅₀. Amantadine and chloroquine were used as positive controls for CHIKV, while ribavirin was used for ZIKV and MAYV [15].

Virucidal Assay

The virucidal activity of the most active compound was evaluated by pre-incubating the compound (6 µM) with CHIKV prior to infection. After incubation, residual infectivity was determined using a plaque reduction assay, and viral titers were expressed as plaque forming units per milliliter (PFU mL⁻¹) [15–17].

Inhibitory Cytopathic Effect Assay

The inhibitory effect on virus induced cytopathic effects (CPE) was assessed in Vero cells (1 × 10⁶ cells/well) seeded in 6-well plates. Cells were infected with CHIKV (MOI = 1) and treated with the selected compound (6 µM). After 48 h, cell monolayers were examined microscopically to evaluate morphological changes [18]. Supernatants were collected for viral titration by plaque assay.

Viral Load Quantification by RT-qPCR

Viral RNA was extracted from supernatants using a commercial extraction kit according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized using a reverse transcription kit. Quantitative PCR (RT-qPCR) was performed using SYBR Green chemistry to determine viral RNA levels at 24, 36, and 48 h post-infection.

Relative viral quantification was based on standard curves generated from serial dilutions of known viral titers. Results were expressed as log₁₀ PFU mL⁻¹ equivalents.

Viral RNA Extraction

Viral RNA was extracted using the Bio Gene Viral DNA/RNA Extraction kit (Bioclin/Quibasa) following the manufacturer’s instructions. The quality and quantity of the extracted RNA were assessed by measuring the A₂₆₀/₂₈₀ and A₂₆₀/₂₃₀ ratios, as well as the concentration (ng µL⁻¹), using a NanoDrop™ Lite Plus spectrophotometer (Thermo Fisher), according to Loaiza-Cano et al. with modifications [19].

cDNA Synthesis

Complementary DNA (cDNA) was obtained from the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, Massachusetts, USA) according to Monsalve-Escudero et al. and Souza et al., with modifications [20, 21]. The amplification program consisted of a cycle of 25 °C for 10 min, 37 °C for 120 min, 85 °C for 5 min and holding at 4 °C, following the manufacturer’s recommendations.

RT-qPCR

For the relative quantification of CHIKV viral RNA, quantitative PCR (RT-qPCR) was performed using GoTaq® qPCR Master Mix kit (Promega) containing SYBR™ Green, according to Cuellar-Quimbaya et al., with modifications [22]. To estimate the relative concentration of viral genetic material, serial dilutions with a dilution factor of 1:10 were performed. Six points were generated in duplicates on the standard curve with 1 × 10⁹, 1 × 10⁸, 1 × 10⁷, 1 × 10⁶, 1 × 10⁵, and 1 × 10⁴ PFU mL⁻¹ equivalents. The primer sequences for CHIKVF were 5’-GACAATGCGCGCGGTACC-3’ and CHIKVR were 5’-TGTTGTTTTGTGGCGCCT-3’40. The amplification program consisted of a 95 °C hold cycle for 2 min and 40 cycles of 95 °C for 15 s, 51 °C for 15 s, and 72 °C for 1 min. The melting curve was standardized at 95 °C for 15 s, 60 °C for 1 min, 95 °C for 30 s (dissociation), and 60 °C for 15 s.

The relative quantification of the viral load in the sample was performed by analyzing the quantification cycle (Cq). The Cq value was inserted into a straight-line equation constructed from a standard curve generated with known concentrations of the CHIKV genome (converted to Log₁₀ PFU mL⁻¹). Subsequently, the result was compared to the log₁₀ PFU mL⁻¹ values of the virus control samples (VC), allowing the determination of the relative viral load of CHIKV [22–24].

Statistical Analysis

All experiments were performed in at least duplicate or triplicate. Data were expressed as mean ± standard deviation (SD). Statistical analyses were conducted using Student’s t-test, and differences were considered statistically significant at p ≤ 0.05.