Results

Synthesis and Characterization of Compounds

Five 4-amino-7-chloroquinoline derivatives were successfully synthesized with yields ranging from 67 to 86%. Structural characterization by 1H and 13C NMR spectroscopy and high-resolution mass spectrometry (HRMS) confirmed the expected chemical structures. The spectral data were consistent with the proposed quinoline framework and corresponding substituents. Detailed characterization data are provided in the Supplementary Information.

Cytotoxicity and Antiviral Activity

The cytotoxicity of the synthesized compounds was initially evaluated in Vero cells to determine CC₅₀ values and define non-toxic concentrations for antiviral assays. The compounds exhibited CC₅₀ values ranging from 13.3 to 98.1 µM (Table 1).

SUBSTANCE | R | VERO CC50(µM) | ZIKV aEC50(µM) | SIb | RPRc | RPCd | CHIKVeEC50(µM) | SI | RPAf | RPC | MAYVgEC50(µM) | SI | RPR | RPC
4 |  | 30.5 ± 1.3 | 19.4 ± 1.8 | 1.6 | 68.4 | 3.7 | NAh | –i | – | – | 25.83 ± 1.56 | 1.2 | 51.2 | 1.6
5 |  | 13.3 ± 1.4 | NA | – | – | – | NA | – | – | – | NA | – | – | –
6 |  | 21.0 ± 1.4 | NA | – | – | – | 4.9 ± 2.6 | 4.26 | 279.69 | 7.4 | NA | – | – | –
7 |  | 81.7 ± 1.2 | NA | – | – | – | NA | – | – | – | NA | – | – | –
8 |  | 98.1 ± 1.3 | NA | – | – | – | NA | – | – | – | NA | – | – | –
Chloroquine(Positive control) |  | 131.1 ± 1.3 | 72.5 ± 3.5 | 1.8 | – | – | 36.4 ± 1.6 | 3.6 | – | – | 83.4 ± 2.3 | 1.6 | – | –
Ribavirin (Positive control) | – | 1516.7 ± 1.2 | 433.2 ± 1.6 | 3.5 | – | – | NT | – | – | – | 431.2 ± 2.2 | 3.5 | – | –
Amantadine(Positive control) | – | 561.3 ± 1.2 | NTj | – | – | – | 419.0 ± 1.7 | 1.3 | – | – | NT | – | – | –

Among the tested derivatives, two compounds demonstrated antiviral activity. Compound 4 showed moderate activity against ZIKV and MAYV, with EC₅₀ values of 19.4 µM and 25.8 µM, respectively, but low selectivity indices. In contrast, compound 6 exhibited selective antiviral activity against CHIKV, with an EC₅₀ of 4.9 µM and a selectivity index of 4.26 (Table 1; Fig. 2). No significant antiviral activity was observed for the remaining derivatives.

Fig. 2: Dose–response curves of chloroquinoline derivative 6 showing (a) cytotoxicity (CC₅₀) and (b) antiviral activity (EC₅₀) against CHIKV in Vero cells, as determined by the MTT assay after 72 h of treatment (n = 3). Data are presented as the mean ± standard deviation of cell viability relative to untreated cell control

Virucidal Activity

The virucidal assay was performed to evaluate whether compound 6 directly inactivates viral particles prior to cell infection. Pre-incubation of CHIKV with compound 6 (6 µM) did not result in a significant reduction in viral titers compared to the viral control (Table 2), indicating the absence of virucidal activity under the tested conditions.

 | Titration media (PFU.mL− 1) | Log10 PFU.mL− 1
CHIKV (viral control) | 1.51 × 105 | 5.16
6 + CHIKV | 1.02 × 105 | 5.01

Inhibition of Cytopathic Effects

The antiviral activity of compound 6 was further assessed by its ability to inhibit CHIKV-induced cytopathic effects (CPE) in Vero cells. At 48 h post-infection, untreated infected cells exhibited pronounced morphological alterations, including cell rounding and detachment. In contrast, infected cells treated with compound 6 (6 µM) showed preservation of the cell monolayer and reduced cytopathic damage (Fig. 3A-D).

Fig. 3: Inhibitory effect of synthesized compound 6 (6 µM) on CHIKV-induced cytopathic effect and viral production in Vero cells. Representative images of Vero cell monolayers obtained 48 h post-infection with CHIKV (MOI = 1) under the following conditions: (a) viral control, infected and untreated cells; (b) cell control, uninfected and untreated cells; (c) cells infected with CHIKV and treated with compound 6 (6 µM); and (d) cytotoxicity control, uninfected cells treated with compound 6 (6 µM). Images were acquired under 100× magnification using ZEN 2.3 Lite imaging software. (e) Viral yield quantification by plaque assay of supernatants collected from CHIKV-infected Vero cells following treatment with compound 6 (6 µM) or no treatment (viral control). CHIKV: supernatant from infected and untreated monolayers; 6 + CHIKV: supernatant from infected monolayers treated with compound 6 (6 µM). Experiments were performed in duplicate, and statistical significance was assessed using Student’s t-test with a 95% confidence interval. Data are presented as mean ± standard deviation. **p < 0.0001

Viral titration of the supernatants revealed a marked reduction in viral production in treated cultures. Specifically, a decrease of approximately 7 log₁₀ PFU mL⁻¹ was observed compared to the viral control (Table 3; Fig. 3E).

 | Titration media (PFU.mL− 1) | Log10 PFU.mL− 1
CHIKV viral control | 2.30 × 1010 | 10.36
6 + CHIKV | 2.15 × 103 | 3.33

The active compound 6 (6 µM) was investigated via RT-qPCR to validate its antiviral activity against CHIKV, as observed in the MTT and inhibitory CPE assays. Viral genetic material was quantified, comparing treated and infected Vero cells with viral control at 24-, 36-, and 48-hour post-infection. The results were supported by photographs of cell monolayers, which displayed a clear reduction in viral replication, as evidenced by the preservation of cell structure (Fig. 4).

Fig. 4: Inhibitory effect of synthesized compound 6 (6 µM) on CHIKV-induced cytopathic effect (CPE) in Vero cells. The analysis included uninfected cells, compound 6 cytotoxicity, and virus infected controls. CPE inhibition was evaluated at 24-, 36-, and 48-hours post-infection (MOI = 1). Representative images were obtained using ZEN 2.3 Lite software at 100× magnification. CC: Cell Control, CC50: Cytotoxicity Control (6 µM), VC: viral control, AV: antiviral activity of compound 6 (6 µM)

Viral Load Reduction by RT-qPCR

The effect of compound 6 on viral replication was further quantified by RT-qPCR. Treatment of CHIKV-infected Vero cells resulted in a significant reduction in viral RNA levels (detected in cell culture supernatants), expressed as log₁₀ PFU equivalents mL⁻¹, at 24, 36, and 48 h post-infection compared to untreated controls (Fig. 5). The decrease in viral load was consistent with the results obtained from the cytopathic effect inhibition and plaque reduction assays.

Fig. 5: Viral quantification of CHIKV following the inhibitory cytopathic effect (CPE) assay with compound 6 (6 µM) at 24-, 36-, and 48-hours post-infection (MOI = 1) in Vero cells (n = 3). Viral load was measured by RT-qPCR and expressed as log₁₀ PFU equivalents mL⁻¹ based on a standard curve generated from known CHIKV concentrations. Viral load was measured by RT-qPCR, and statistical analysis was performed using Student’s t-test with a 95% confidence interval. VC: virus control; 6 24 H: anti-CHIKV activity of compound 6 (6 µM) 24 h post-infection; 6 36 H: anti-CHIKV of compound 6 (6 µM) 36 h post-infection; 6 48 H: anti-CHIKV of compound 6 (6 µM) 48 h post infection. Error bars represent standard deviation; *: p value < 0.05