Korean Red Cross Blood Services provided concentrated human red blood cells for this study using a consent form specifically approved by the exempt review of the Institutional Review Board (IRB) of Seoul National University (IRB No., 2105-005-1205). The experimental methodology used in this study was approved by the Institutional Biosafety Committee of Seoul National University (IBC No., SNUIBC-R211205-2).

Antimalarial drugs, including CQ and atovaquone, were used as control and acquired from Sigma-Aldrich (St. Louis, Missouri, USA). Propidium iodide and SYTOX Green solution were purchased from BD Pharmingen (San Diego, California, USA) and Life Technologies (Grand Island, New York, USA), respectively. The Differential Quik III Stain Kit was purchased from Polysciences, Inc. (Valley Road, Warrington, Pennsylvania, USA).

CQ-sensitive strains (P. falciparum 3D7 (American Type Culture Collection, ATCC PRA-405D) and P. falciparum D6 (ATCC MRA-285)) and Vero cells were purchased from ATCC (Manassas, Virginia, USA).

SKM13-MeO and SKM13-F were synthesized using unnatural amino acids after several modifications of our previously successful antimalarial compound, SKM13 [5]. The synthesis process is shown in Supplementary Fig. S1A and described in the Methods section of the Supplementary Information. The purity of the reaction products and compound structures was monitored and confirmed using proton NMR spectroscopy.

Vero cells were cultured in a monolayer in a 96-well plate using Dulbecco’s modified Eagle’s medium supplemented with 2% fetal bovine serum for 12 h. Then, they were incubated with serially diluted antimalarial compounds for 48 h. After the incubation period, 20 μl of CellTiter 96 AQueous One Solution reagent (Promega, Hollow Road Madison, Wisconsin, USA) was added to each well and incubated at 37°C in a humidifier with 5% CO₂ for 2 h. Absorbance at 490 nm was measured using a Tecan Infinite 200 Pro microplate reader (Tecan Group Ltd., Canton of Zürich, Switzerland).

The blood stage of the P. falciparum was propagated in 5% hematocrit of O+ human RBCs suspended in RPMI 1640 with 25 mM HEPES and L-glutamine and supplemented with 1% AlbuMAX I Lipid-Rich BSA, 25 mM sodium bicarbonate, 25 μg/ml gentamycin, 0.05% hypoxanthine, and 1% penicillin. The cultures were maintained at 37°C under 5% O₂, 5% CO₂, and 90% N₂. The medium was changed daily and the number of parasites in the blood was monitored by Giemsa staining of thin methanol-fixed blood smears.

To synchronize P. falciparum, asexual parasites were treated with 5% D-sorbitol (Sigma-Aldrich) to enrich ring stages. Parasite cultures were centrifuged for 5 min at 500×g. Sterilized and pre-warmed 5% D-sorbitol was added to 10 volumes of the pellet. The cultures were shaken every 2 min with inversion. After 10 min, the cultures were washed twice with RPMI 1640 containing 25 mM HEPES, L-glutamine, and 1% penicillin. The pellet was resuspended in the culture medium to a final hematocrit of 5% for maintenance. The procedure was repeated at least 3 times every 48 h.

In vitro antimalarial activity experiments were performed as described previously [5]. Briefly, parasites were seeded in 48-well cell culture plates at a density of 0.5% in 2% hematocrit. The antimalarial control drugs CQ, SKM13, and 2 novel compounds were serially diluted in a malaria culture medium and incubated with the parasites. After incubation, the RBC pellet was stained with SYTOX Green solution. Parasite-infected erythrocyte populations were determined using a BD FACSCanto II flow cytometer (BD Biosciences, Carlsbad, California, USA). The antimalarial activity of the test drugs was determined by the half-maximal inhibitory concentration on malaria growth (IC₅₀).

All data were analyzed using GraphPad Prism software (version 9.0; GraphPad Software, San Diego, California, USA). All experimental results are expressed as the mean±standard deviation (SD) of 3 biological repeats.

Two final products, SKM13-MeO and SKM13-F, were prepared from commercially available unnatural amino acids. These are synthetic derivatives of SKM13, which exhibited the best antimalarial effect in our previous study. The phenyl group that replaces the methyl group in chloroquine is large. To improve the antimalarial effect, a substituent was introduced on the phenyl group, facilitating the synthesis of substituted phenyl derivatives using unnatural amino acids.

The procedures for the synthesis of 2 final products were identical, as shown in the scheme in Supplementary Fig. S1A. The amino groups of the unnatural amino acids were protected by the Boc group using excess Boc anhydride under basic conditions to obtain quantitative yields of N-Boc-protected acids (Supplementary Fig. S1A (2a–b)). The conversion of carboxylic acids to N-methoxy-N-methyl amides (Weinreb amides) (Supplementary Fig. S1A (3a and 3b)) was accomplished using dicyclohexylcarbodiimide coupling. After deprotection of the N-Boc group under 5% Trifluoroacetic acid in Dichloromethane, the salts (Supplementary Fig. S1A (4a, 4b)) of amines by TFA were directly used in palladium-mediated coupling reaction with 4,7-dichloroquinoline under the reaction conditions, palladium acetate, cesium carbonate, and BINAP, producing quinolinyl Weinreb’s amides (Supplementary Fig. S1A (5a, 5b)) in moderate yields. Finally, SKM13-F and SKM13-MeO were synthesized by 2 sequential procedures: 1) conversion of Weinreb’s amide to aldehyde by DIBAL-H and 2) Emmons-Horner-Wadsworth olefin synthesis of aldehydes with N-dimethylaminoethyl phosphonate amide using a strong base, otassium tert-butoxide. Purification of the final products was challenging because of the similar polarity of the phosphonate amide and the products, which was overcome by reducing the phosphonate amide. The final structure of each novel compound compared with that of SKM13 is shown in Supplementary Fig. S1B. SKM13 and 2 derivatives had one difference in their chemical structures; the side chains of SKM13-F and SKM13-MeO carried fluorophenyl and methoxyphenyl, respectively.

The cytotoxicity of each compound was assessed by inoculating the drug into Vero cells for 48 h, and the results were evaluated as half-cell cytotoxic concentrations (CC₅₀). CC₅₀ values for CQ and SKM13 were consistent with our previous report [5]. However, SKM13-F and SKM13-MeO had lower CC₅₀ than SKM13 (P<0.001), indicating that 2 novel compounds were more cytotoxic than SKM13 (Fig. 1).

We previously reported the IC₅₀ of P. falciparum (3D7) in the mixed stage [5]. The IC₅₀ of the SKM13 series were measured against different developmental stages (ring, trophozoite, and schizont) of P. falciparum. First, malarial parasites were maintained and synchronized to achieve the same stage of parasites using in vitro culture (Supplementary Fig. S2). P. falciparum strains (3D7 and D6) were chosen to determine the antimalarial efficacy of SKM13 and its derivatives (SKM13-MeO and SKM13-F) by measuring the half-maximal concentration of growth inhibition (IC₅₀).

SKM13 and its derivatives showed different antimalarial activities depending on the P. falciparum strains. SKM13 showed IC₅₀ values of 30.6±1, 29.2±1.6, and 52.7±0.4 (mean±SD) at the ring, trophozoite, and schizont stages, respectively, indicating that it was more effective at the ring and trophozoite stages than at the schizont stage (Fig. 2A-C). SKM13-MeO and -F showed higher IC₅₀ values than SKM13 at all 3 stages, implying that SKM13 was more effective than 2 novel derivatives at inhibiting all stages of P. falciparum 3D7. The final SI (CC₅₀/IC₅₀) showed an approximately 2-folds lower value for 2 derivatives of SKM13 than SKM13 itself, suggesting that 2 novel derivatives are less effective than SKM13. There was no difference in SI values between 2 novel SKM13 derivatives.

D6 strain data suggests that SKM13 might suppress the ring stage better than the other stages because the IC₅₀ value (mean±SD) of SKM13 was increased up to 2 folds in the mixed stage from 11.3±0.2 to 28.5±0.4 (Fig. 3A, B). However, the IC₅₀ of SKM13 was still significantly lower than that of CQ (P<0.001) in D6. SKM13-MeO had a significantly different IC₅₀ than SKM13-F (P<0.001), indicating that the electron-donating group would be more efficient in suppressing P. falciparum than the electron-withdrawing group because CC₅₀ was comparable between 2 modifications. All results of CC₅₀ and IC₅₀ are shown in Table 1.