All the materials employed in this study were procured from E. Merck in pro analysis grade, i.e., 2-methoxyacetophenone, 3-methoxyacetophenone, 4-methoxyacetophenone, 2-pyridinecarbaldehyde, 3-pyridinecarbaldehyde, 4-pyridinecarbaldehyde, methanol, and NaOH. Furthermore, chemicals such as Roswell Park memorial institute (RPMI) solution, DMSO, inoculum solution, human serum, red blood cells, methanol, Giemsa coloring solution, P. falciparum strain 3D7 and FCR3 were used for the in vitro antimalarial assay.

Thin-layer chromatography using Merck pre-coated aluminum F₂₅₄ plates was employed to monitor the progress of the synthesis reactions. The melting point of the synthesized compounds was measured using the Electrothermal 9100 equipment. Analysis of the infrared spectrum of the products was conducted using the Shimadzu Prestige-21 Fourier transform infrared (FTIR), while the purity and mass spectrum of the products were recorded using the Shimadzu QP2010S gas chromatography-mass spectrometry (GC-MS). The nuclear magnetic resonance (¹H- and ¹³C-NMR) spectra were analyzed on JEOL JNMECA with a frequency of 500 and 125 MHz, respectively. The antimalarial assay was carried out using equipment, including laboratory glassware and 96-well microplates, micropipette, microtube, microcentrifuge, ELISA reader (Microplate Reader, Benchmark), CO₂ incubator, object-glass, and microscope (Euromex iScope IS.1152-PLPHi). Molecular docking simulations were performed using a laptop with specifications of a central processing unit (CPU) of AMD Ryzen 7 4800H, a graphic processing unit (GPU) of NVIDIA GTX 1650 Ti, 16 GB of RAM, and a Windows 11 operating system.

The chalcones were synthesized in a one-pot synthesis method. A mixture of 3 mmol pyridinecarbaldehyde derivative in 15 mL methanol and 15 mL 10% (w/v) NaOH solution was added into a round-bottom flask placed in an ice bath with a temperature range of 0–10 °C. The next step involved the addition of methoxyacetophenone derivatives (3 mmol in 5 mL methanol) dropwise for 1 h, and then it was stirred for 5 h with a temperature range of 10±1 °C. The resulting solid was filtered, washed with water, and recrystallized with ethanol. The structure elucidation was performed using FTIR, GC-MS, ¹H- and ¹³C-NMR spectrometers.

Chalcone A (0.39 g) was yielded as a yellowish solid in 53.74%. m.p 60–61 °C. FT-IR (KBr): 3070, 2947, 1658, 1604, 1597, 1473, 1319, 1249, 1018, 763 cm^-1. ¹H-NMR (500 MHz, CDCl₃) δ 3.87 (s, 3H, -OCH₃), 6.96 (d, J 7.4 Hz, 1H, H_Ar-Ph), 7.00 (tod, J 1.1, 7.5 Hz, 1H, H_Ar-Py), 7.24 (dodod, J 1.1, 4.6, 7.5 Hz, 1H, H_Ar-Py), 7.44 (tod, J 1.7, 7.4 Hz, 1H, H_Ar-Ph), 7.46 (dod, J 1.1, 7.5 Hz, 1H, H_Ar-Py), 7.59 (d, J 15.5 Hz, 1H, =CH_α,), 7.61 (dod, J 1.7, 7.4 Hz, 1H, H_Ar-Ph), 7.69 (tod, J 1.7 & 7.4 Hz, 1H, H_Ar-Ph), 7.79 (d, J 15.5 Hz, 1H, =CH_β), 8.63 (dod, J 1.1, 4.6 Hz, 1H, H_Ar-Py). ¹³C-NMR (125 MHz, CDCl₃) δ 55.86 (-OCH₃), 111.68, 120.77, 124.21, 124.72, 129.07, 130.53, 133.27, 136.84, 150.22, 153.77, 158.41 (C_Ar), 130.47, 141.67 (C_alkene), 193.27 (C=O). Mass spectrum (EI, m/z) = 239 (M⁺, 5), 180 (50), 135 (40), 119 (38), 118 (38), 104 (40), 92 (45), 77 (100), 51 (90).

Chalcone B (0.56 g) was produced in 78.01% as a yellowish solid. m.p. 63–65 °C. IR (KBr): 3039, 2947, 1658, 1604, 1597, 1473, 1327, 1242, 1018, 756 cm^-1. ¹H-NMR (500 MHz, CDCl₃) δ 3.92 (s, 3H, -OCH₃), 7.02 (d, J 8.0 Hz, 1H, H_Ar-Ph), 7.06 (t, J 8.0 Hz, 1H, H_Ar-Ph), 7.34 (dod, J 4.8, 7.8 Hz, 1H, H_Ar-Py), 7.49 (d, J 15.9 Hz, 1H, =CH_α), 7.51 (tod, J 2.0, 8.0 Hz, 1H, H_Ar-Ph), 7.62 (d, J 15.9 Hz, 1H, =CH_β), 7.67 (dod, J 1.9, 7.8 Hz, 1H, H_Ar-Py), 7.90 (dot, J 2.0, 8.0 Hz, 1H, H_Ar-Ph), 8.60 (dod, J 1.9, 4.8 Hz, 1H, H_Ar-Py), 8.81 (d, J 1.9 Hz, 1H, H_Ar-Py). ¹³C-NMR (125 MHz, CDCl₃) δ 55.84 (-OCH₃), 111.74, 120.96, 123.85, 128.77, 130.67, 131.07, 133.54, 134.75, 149.94, 150.86, 158.41 (C_Ar), 128.86, 138.94 (C_Alkene), 192.15 (C=O). Mass spectrum (EI, m/z) = 239 (M⁺, 3), 135 (45), 120 (20),104 (20), 92 (40), 77 (100), 51 (75).

Chalcone C (0.41 g) was yielded in 57.12% as a yellowish solid. m.p 87–88 °C. IR (KBr): 3070, 2947, 1666, 1604, 1581, 1481, 1303, 1249, 1018, 784, 694 cm^-1. ¹H-NMR (500 MHz, CDCl₃) δ 3.89 (s, 3H, OCH₃), 7.15 (dod, J 2.0, 7.9 Hz, 1H, H_Ar-Py), 7.37 (dod, J 4.7, 7.9 Hz 1H, H_Ar-Py), 7.43 (t, J 8.0 Hz 1H, H_Ar-Ph), 7.55 (dod, J 1.4, 2.3 Hz, 1H, H_Ar-Ph), 7.59 (d, J 15.8 Hz, 1H, =CH_α), 7.61 (dot, J 1.4, 8.0 Hz, 1H, H_Ar-Ph), 7.79 (d, J 15.8 Hz, 1H, =CH_β), 7.96 (dot, J 2.3, 8.0 Hz, 1H, H_Ar-Ph), 8.63 (dod, J 2.0, 4.7 Hz, 1H, H_Ar-Py), 8.86 (d, J 2.0 Hz, 1H, H_Ar-Py). ¹³C-NMR (125 MHz, CDCl₃) δ 55.60 (OCH₃), 112.96, 119.75, 121.19, 123.95, 129.80, 130.75, 134.73, 139.16, 150.07, 151.18, 160.05 (C_Ar), 123.92, 141.03 (C_Alkene), 189.68 (C=O). Mass spectrum (EI, m/z) = 239 (M⁺, 30), 210 (20), 135 (30), 132 (40), 104 (40), 92 (45), 77 (100), 51 (90).

Chalcone D (0.52 g) was yielded in 71.65% as a yellowish solid. m.p 76–78 °C. IR (KBr): 3055, 2970, 1658, 1604, 1597, 1465, 1327, 1257, 1018, 833 cm^-1. ¹H-NMR (500 MHz, CDCl₃) δ 3.85 (s, 3H, OCH₃), 6.95 (dot, J 2.3, 9.1 Hz, 2H, H_Ar-Ph,), 7.25 (dodod, J 1.2, 5.2, 7.5 Hz, 1H, H_Ar-Py), 7.43 (d, J 7.5 Hz, 1H, H_Ar-Py), 7.70 (tod, J 1.2, 7.5 Hz, 1H, H_Ar-Py), 7.73 (d, J 15.0 Hz, 1H, =CH_α), 8.09 (dot, J 2.3, 9.1 Hz, 2H, H_Ar-Ph), 8.10 (d, J 15.0 Hz, 1H, =CH_β), 8.65 (dod, J 1.2, 5.2 Hz, 1H, H_Ar-Py). ¹³C-NMR (125 MHz, CDCl₃) δ 55.60 (OCH₃), 113.95, 124.40, 125.48, 130.47, 131.20, 137.00, 150.21, 153.43, 163.74 (C_Ar), 125.51, 142.06 (C_Alkene), 188.74 (C=O). Mass spectrum (EI, m/z) = 239 (M⁺, 40), 210 (100), 135 (60), 104 (50), 92 (40), 77 (80), 51 (50).

Chalcone E (0.54 g) was produced as a yellowish solid with a 75.23% yield. m.p 99–100 °C. IR (KBr): 3039, 2947, 1666, 1604, 1597, 1465, 1311, 1257, 1026, 840 cm^-1. ¹H-NMR (500 MHz, CDCl₃) δ 3.89 (s, 3H, OCH₃), 6.98 (dot, J 2.7, 9.0 Hz, 2H, H_Ar-Ph), 7.35 (dod, J 4.6, 8.0 Hz, 1H, H_Ar-Py), 7.60 (d, J 15.8 Hz, 1H, =CH_α), 7.76 (d, J 15.8 Hz, 1H, =CH_β), 7.93 (dot, J 2.0, 8.0 Hz, 1H, H_Ar-Py), 8.03 (dot, J 2.7, 9.0 Hz, 2H, H_Ar-Ph), 8.61 (dod, J 2.2, 4.6 Hz, 2H, H_Ar-Py), 8.84 (d, J 2.0 Hz, 1H, H_Ar-Py). ¹³C-NMR (125 MHz, CDCl₃) δ 55.65 (OCH₃), 114.04, 123.79, 123.88, 128.58, 130.34, 131.03, 148.31, 149.39, 163.67 (C_Ar), 123.41, 135.54 (C_Alkene), 195.65 (C=O). Mass spectrum (EI, m/z) = 239 (M⁺, 40), 210 (20), 135 (70), 119 (38), 107 (20), 92 (50), 77 (100), 51 (80).

Chalcone F (0.62 g) was yielded as a yellowish solid in 86.37%. m.p 128–130 °C. IR (KBr): 3032, 2970, 1666, 1604, 1597, 1465, 1311, 1234, 1018, 810 cm^-1. ¹H-NMR (500 MHz, CDCl₃) δ 3.80 (s, 3H, OCH₃), 6.83 (d, J 8.6 Hz, 2H, H_Ar-Ph), 6.93 (d, J 5.7 Hz, 2H, H_Ar-Py), 6.99 (d, J 9.2 Hz, 1H, =CH_α), 7.73 (d, J 8.6 Hz, 2H, H_Ar-Ph), 8.03 (d, J 9.2 Hz, 1H, =CH_β), 8.40 (d, J 5.7 Hz, 2H, H_Ar-Py). ¹³C-NMR (125 MHz, CDCl₃) δ 55.67 (OCH₃), 114.07, 123.04, 128.44, 130.35, 140.80, 149.97, 163.75 (C_Ar), 126.06, 147.95 (C_Alkene), 195.35 (C=O). Mass spectrum (EI, m/z) = 239 (M⁺, 40), 210 (10), 135 (100), 107 (20), 92 (40), 77 (80), 51 (70).

The in vitro antimalarial assays were performed using the technique described by Zakiah et al. (2021) with a slight modification. The synthesized product (10 mg) was dissolved in 1 mL DMSO. Then 100 μL of this solution was diluted using 900 μL RPMI solution to obtain a stock solution with a final concentration of 1000 μg/mL. A series of diluted solutions (1, 2, 10, and 20 μg/mL) was prepared by utilizing the stock solution. Next, each diluted solution (100 μL) and inoculum solution (100 μL) with P. falciparum parasites were added into a 96-well microplate. The negative control was prepared in the absence of the sample solution. The 96-well microplate was incubated at 37 °C for 72 h. Afterward, the cultures were used to produce blood smears and stained for 10 min using a 15% Giemsa solution.

The inhibition percentage of P. falciparum was determined by analyzing each group of 1000 erythrocyte cells infected with P. falciparum using a microscope with a magnification of 1000×. The IC₅₀ value was calculated through probit analysis. Triplicate analyses were performed for each sample.

In this study, we investigated the mechanism of action of all synthesized chalcones, chalcone A–F, in inhibiting P. falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) receptor. In addition, we also investigated the mechanism of a well-recognized antimalarial agent, chloroquine, in inhibiting the same receptor as a comparison. AutoDock Vina 1.1.2 software was used for all molecular docking simulations (Trott and Olson 2010). The crystal structure of PfDHFR-TS receptor, complexed with WR99210, NADPH, and dUMP, was retrieved from Protein Data Bank (PDB ID: 1J3K) with the chain A as the model receptor. To prepare the receptor, all solvent molecules and complexed ligands were removed using Discovery Studio Visualizer software and then appended hydrogen atoms and Kollman charges with AutoDock Tools 1.5.6. The active site of PfDHFR-TS was identified by the amino acid residues interacting with the native ligand, WR99210. The molecular docking grid box was focused on the native ligand and sized 12 Å × 12 Å × 12 Å with 1.00 Å grid spacing. The exhaustiveness of the molecular docking protocol was set to 16. To validate the molecular docking protocol, the native ligand was redocked, and the root-mean-square deviation (RMSD) of the docked pose relative to the native ligand crystal structure was calculated using PyMOL software (Schrödinger and DeLano 2020). The molecular docking protocol was considered valid if the RMSD was ≤ 2.00 Å (Diallo et al. 2021).

The preparation of the three-dimensional structure of all chalcones and chloroquine was carried out prior to the molecular docking simulation. The process involved building and geometrically optimizing their structure using GaussView 5.0 and Gaussian 09 package (Frisch et al. 2016) with the DFT/B3LYP method and 6–31G basis set similar to our previous works (Suma et al. 2019; Waskitha et al. 2021). AutoDock Vina was used to carry out the molecular docking simulation of all chalcones and chloroquine, following the same method as the native ligand. The conformation with the least binding affinity was selected as the best orientation, indicating the most stable interaction with the active site (Minnelli et al. 2020).

Six chalcone derivatives have been synthesized from the methoxy-substituted acetophenone and various pyridinecarbaldehyde using a base-catalyzed Claisen-Schmidt condensation reaction. The synthesis scheme is illustrated in Scheme 1. The resulting products were recrystallized from ethanol to obtain yellowish solids with yields ranging from 53.74 to 86.37%. Purity analysis was performed using GC, which showed a single peak at a retention time range of 24–27 min for all products, indicating their high purity level (approximately 100%). In the MS spectra, all chalcones displayed a molecular ion at m/z = 239, corresponding to each chalcone’s molecular mass. FTIR analysis revealed the presence of characteristic enone functional groups at 1604–1666 cm^-1 (HC=CH-C=O) and these findings were consistent with previous reports (Joseph et al. 2014). Both protons of enone were observed in the ¹H-NMR spectra of all products at 6.99–7.73 and 7.62–8.10 ppm, respectively. Furthermore, the HC=CH- and C=O carbons were detected at 123.41–147.95 and 188.74–195.65 ppm, respectively, in the ¹³C-NMR spectra of all products (Marcovicz et al. 2022).

Scheme 1. 

The synthesis scheme of chalcones A–F.

All synthesized compounds have the same molecular formula, i.e., C₁₅H₁₃NO_2, and were examined to assess the effect of methoxy groups and nitrogen atoms on the antimalarial activity of chalcones. The results of the antimalarial activities of chalcones A–F against P. falciparum 3D7 and FCR3 strains are listed in Table 1. According to Basco et al. (1994), the in vitro antimalarial activity has good activity with IC₅₀ < 10 μg/mL, moderate activity with IC₅₀ 10–50 μg/mL, low activity with IC₅₀ 50–100 μg/mL, and inactive with IC₅₀ > 100 μg/mL, so all chalcones could be classified as highly effective antimalarial agents, as their IC₅₀ values were below 5 μg/mL. Even though antimalarial activities from chloroquine against P. falciparum 3D7 and FCR3, respectively, have IC₅₀ of 0.0032 μg/mL and 0.0352 μg/mL (Zakiah et al. 2021).

In terms of antimalarial activity against P. falciparum 3D7, ortho-methoxy groups exhibited an IC₅₀ value range of 0.48–4.16 μg/mL, while meta- and para-methoxy groups demonstrated IC₅₀ value of 0.98 and 1.07–4.46 μg/mL, respectively. These results suggest that ortho-methoxy groups have greater antimalarial activity compared to meta-methoxy groups and are significantly more active than para-methoxy groups of the chalcone’s chemical structure as a P. falciparum 3D7 antimalarial agent. A similar trend was observed for P. falciparum FCR3, with ortho-methoxy groups displaying an IC₅₀ range of 0.31–2.68 μg/mL, whereas meta- and para-methoxy groups exhibited IC₅₀ values of 1.03 and 1.02–2.17 μg/mL, respectively.

Regarding the nitrogen atoms, the 2-pyridine moiety demonstrated an IC₅₀ value range of 0.48–4.46 μg/mL against P. falciparum 3D7, while the 3- and 4-pyridine moieties exhibited IC₅₀ values of 0.98–4.16 and 1.11 μg/mL, respectively. These results indicate that the 2-pyridine moiety possesses higher antimalarial activity than 3-pyridine and is significantly more active than 4-pyridine within the chalcone chemical structure as a P. falciparum 3D7 antimalarial agent. Similar observations were made for P. falciparum FCR3, with the IC₅₀ range for the 2-pyridine moiety being 0.31–2.17 μg/mL, while the IC₅₀ values for 3- and 4-pyridine moieties were 1.03–2.68 and 1.02 μg/mL, respectively. These findings indicate that the presence of 2-methoxy and 2-pyridine substituents play a crucial role in the antimalarial activity of chalcones against both P. falciparum 3D7 and FCR3.

Furthermore, all chalcones had a low resistance index (0.49–1.06) compared with chloroquine, which has a resistance index of 11 (Zakiah et al. 2021), thus demonstrating no-cross resistance with compounds like chloroquine, which share a similar mechanism of action (Sinha et al. 2019). These findings emphasize the potential of these chalcones as antimalarial agents.

A molecular docking study was conducted on the PfDHFR-TS receptor as the target, which is one of a kind important target of antimalarial drugs, to investigate the possibility of the inhibitory mechanism of chalcone A–F and chloroquine as antimalarial drugs. The redocking results revealed that the native ligand had a binding affinity of -6.8 kcal/mol and closely matched the X-ray crystallographic conformation, with an RMSD value of 1.828 Å. This demonstrates the molecular docking protocol’s reliability and validity (Diallo et al. 2021).

The molecular docking results of chalcone A–F, chloroquine, as well as the native ligand are tabulated in Table 2. The binding affinity of all chalcones ranged from -6.9 to -7.3 kcal/mol, showing their relatively strong interactions with the active site of PfDHFR-TS whereas the binding affinity of chloroquine was -7.4 kcal/mol. Notably, even though chloroquine had the lowest binding affinity, all chalcones exhibited lower binding affinity than the native ligand, indicating higher stability of the ligand-receptor complex within the active site of the PfDHFR-TS receptor.

The findings indicated that chloroquine was stabilized by a hydrogen bond with one of the crucial amino acid residues, Asn108, and mostly by hydrophobic interactions along with van der Waals interactions. We also found that all chalcones established interaction with Asn108 through the formation of a hydrogen bond as well. The carbonyl group of the chalcones played a crucial role in binding to PfDHFR active site, and there were no steric clashes with Asn108, which is associated with resistance (Adane et al. 2010). A previous study has also highlighted the significance of the carbonyl group of chalcones in PfDHFR binding (Ahmed and Abdullah 2019). Additionally, interactions with hydrophobic amino acid residues such as Met55, Phe58, and Ile112 contributed to the stability of ligand-receptor interactions (Kyei et al. 2022). These hydrophobic interactions were reinforced by the phenyl and pyridine group, enhancing the overall stability of the interactions in the active site of PfDHFR-TS. Fig. 1 illustrates the two-dimensional molecular interactions of chalcones A–F, chloroquine, and the native ligand within the active site of PfDHFR.

Figure 1. 

The two-dimensional molecular interactions of a. Native ligand; b–g. Chalcones A–F, and h. Chloroquine in the active site of PfDHFR-TS receptor.

The molecular docking study of chloroquine against PfDHFR-TS has been also reported (Melaku et al. 2022). Similar to the chalcone-pyridine hybrid compound, chloroquine also interacted with Ala16, Leu46, Phe58, Asn108, and Ser111 in the active site of PfDHFR-TS since both chloroquine and our synthesized compounds contain pyridine moiety. This result also agreed with the molecular docking result of pyridine derivatives that interacted with Ala16, Leu46, Phe58, Asn108, and Ser111 key amino acid residues on PfDHFR-TS receptor (Bekhit et al. 2012). These findings show that the antimalarial mechanism of action of the chalcone-pyridine hybrid compounds is similar to the chloroquine, as well as to the reported chalcones and pyridine derivatives.