Design, Synthesis, and Antimalarial Evaluation of New Spiroacridine Derivatives

Abstract

Background/Objectives: Malaria is a tropical disease mainly caused by Plasmodium falciparum and represents a global public health problem, with over 200 million cases and 500 thousand deaths reported worldwide. Considering its treatment limitations, it is essential to develop new compounds against malaria. In this context, acridine derivatives are privileged structures. Methods: Thus, new spiroacridines containing N-acylhydrazone (AMTAC) and N-phenylacetamide (ACMD) were synthesized and evaluated in malaria and cytotoxicity assays, as well as in silico studies. Results: As a result, five spiroacridines showed inhibitory activity over 70% against the P. falciparum 3D7-GFP strain at 10 μM, along with an IC₅₀ range of 2-4 μM. After a brief Structure-Activity Relationship (SAR) analysis, it was observed that the spiroacridine structure must be associated with the hydrazone moiety to successfully inhibit parasite growth. In addition, these molecules presented promising resistance profile, with selectivity for the parasite. After computational studies, spiroacridines showed better affinity with dihydrofolate reductase (DHFR), overcoming the quadruple mutant resistance to pyrimethamine, with more stability in complex with the enzyme. Conclusions: Therefore, the potential of spiroacridines against malaria, with moderate resistance and selectivity profile, as well as DHFR inhibition greater than pyrimethamine, was confirmed.

Keywords: MM-PBSA; acridine; dihydrofolate reductase; malaria; molecular docking; molecular dynamics; synthesis; thymidylate synthase; tropical parasitic diseases.

Figure 1

Design of N-acylhydrazone and acetamide spiroacridines. The acetohydrazide and N-acylhydrazone moieties are represented in blue, whereas acetamide is represented in pink, as well as the compound’s names which contains each group are represented in the corresponding color.

Figure 2

Synthesis of N-acylhydrazone and acetamide compounds. Reaction conditions are as follows: (a) EtOH, AcOH, r.t.; (b) 9-Acridinecarboxaldehyde, EtOH, Et₃N, 78 °C; and (c) DMF, aniline, 160 °C. The acetohydrazide and N-acylhydrazone moieties are represented in blue, whereas acetamide is represented in pink, as well as the compound’s names which contains each group are represented in the corresponding color.

Figure 3

Cytotoxic effects of test compounds AMTAC-01 (A), AMTAC-02 (B), AMTAC-17 (C), AMTAC-21 (D), AMTAC-22 (E), and chloroquine (F) on Vero E6 cells. C, Control: 1% DMSO-treated wells, considered as 100% cell viability. D: 10% DMSO. Results were expressed as mean of cell viability ± standard deviation of four independent experiments performed in triplicates. *** p < 0.001, ** p < 0.01, and * p < 0.05, compared to the corresponding control group. Data were evaluated by ANOVA, followed by Tukey’s post-test using GraphPad Prism 8.0.

Figure 4

Three-dimensional representation of DHFR complex with pyrimethamine (magenta) and AMTAC-01 (cyan) (A). Two-dimensional interaction diagram of DHFR complexes with AMTAC-01 (B) and pyrimethamine (C). Legend: dark green = hydrogen bond; light green = carbon–hydrogen interaction; dark pink = π-π stacking; light pink = π-alkyl and alkyl interactions; and red = unfavorable acceptor–acceptor; All carbon, nitrogen, oxygen and sulfur atoms are represented in gray, blue, red and yellow color, respectively.

Figure 5

Plots of MD simulation in a 100 ns trajectory: RMSD of wtDHFR (A) and qmDHFR (B), and RMSF of wtDHFR (C) and qmDHFR (D), highlighting the free proteins (black lines) and in complex with the standard compound (red line) and AMTAC-01 (green line).

Figure 6

Plots of MD simulation in a 100 ns trajectory: R_g of wtDHFR (A) and qmDHFR (B), and SASA of wtDHFR (C) and qmDHFR (D), highlighting the free proteins (black lines) and in complex with the standard compound (red line) and AMTAC-01 (green line).

Figure 7

Plots of MD simulation in a 100 ns trajectory: H-bond around the trajectory for wtDHFR (A) and qmDHFR (B), highlighting the standard compound (black line) and AMTAC-01 (red line).

Figure 8

Structure–Activity Relationship (SAR) analysis of spiroacridines for antimalarial activity. Blue color represents modifications in the benzene ring with different substituents or replacement with a heterocycle, whereas pink and purple highlights the relevance or not of 2-cyano-N-acylhydrazone moiety and imine group, respectively.