Exploring Carboxamide Derivatives as Promising Anticancer Agents: Design, In Vitro Evaluation, and Mechanistic Insights

Abstract

Carboxamide derivatives are a promising class of compounds in anticancer drug discovery, owing to their ability to interact with multiple oncogenic targets and their favorable pharmacological profiles. In this study, we report the design, synthesis, and biological evaluation of a series of N-substituted 1H-indole-2-carboxamides as potential anticancer agents. The synthesized compounds were assessed for antiproliferative activity using the MTT assay against MCF-7 (breast cancer), K-562 (leukemia), and HCT-116 (colon cancer) cell lines, with normal human dermal fibroblasts included as a non-cancerous control. Several compounds demonstrated notable cytotoxicity and selectivity. Compounds 12, 14, and 4 exhibited potent activity against K-562 cells, with IC₅₀ values of 0.33 µM, 0.61 µM, and 0.61 µM, respectively. Compound 10 showed the most significant activity against HCT-116 cells (IC₅₀ = 1.01 µM) with a high selectivity index (SI = 99.4). Moderate cytotoxicity was observed against MCF-7 cells. To elucidate the mechanism of action, molecular docking and induced-fit docking studies were conducted against key cancer-related targets, including topoisomerase-DNA (PDB ID: 5ZRF), PI3Kα (4L23), and EGFR (3W32), revealing favorable binding interactions. Additionally, principal component analysis of molecular descriptors indicated that the compounds possess promising drug-like and lead-like properties, particularly compound 10. Overall, this study highlights N-substituted indole-2-carboxamides as promising scaffolds for further optimization. The integration of synthetic chemistry, biological assays, and computational modeling provides a robust foundation for the continued development of these compounds as potential anticancer agents.

Keywords: MTT assay; carboxamides; cheminformatics; molecular docking; principal component analysis.

Scheme 1

The synthetic pathway for the preparation of N-substituted 1H-indole-2-carboxamides, beginning with indole-2-carboxylic acid and leading to the formation of aromatic substitutions 4, 6, 8, 10, 12, and 14. i: Thionyl chloride (SOCl₂), 70–80 °C, chloroform (CHCl₃); ii: triethylamine (TEA), dichloromethane (DCM), RT.

Scheme 2

The synthetic pathway for the preparation of N-substituted 1H-furan-2-carboxamides, highlighting the formation of compound 16 through the reaction of 1-aminoanthraquinone with furan-2-carbonyl chloride. ii: triethylamine (TEA), dichloromethane (DCM), RT.

Figure 1

Determination of IC₅₀ values of carboxamide compounds 4, 6, 8, 10, 12, 14, and 16 against different types of human cancer cells: (A) colon cancer (HCT-119); (B) breast cancer (MCF-7); (C) chronic myeloid leukemia (K-562); and (D) normal fibroblast cell lines. The IC₅₀ value of each compound was calculated using the nonlinear regression of the log concentration as inhibition percentage values using GraphPad Prism 9.0.

Figure 2

(A) The X-ray structure of the topoisomerase II–DNA complex (5ZRF) binding pocket accommodating the co-crystallized ligand EVP (red color) and IF-docked poses of carboxamides 4–16 depicted in yellow, pink, green, purple, and cyan. (B) Superposing of EVP (red color) and ligand 16 (purple color) at 5ZRF binding site. DNA strands are blue colored, some of the key binding residues are depicted, and H atoms are hidden for clarification purposes. Picture visualized using PYMOL [39].

Figure 3

(A) The X-ray structure of the PI3Kα (4L23) binding pocket accommodating the co-crystallized ligand X6K (red color) and IF-docked poses of carboxamides 4–16 depicted in yellow, pink, green, purple, and cyan. (B) Superposing of X6K (red color) and ligand 16 (green color) at 4L23 binding site. Some of the key binding residues are depicted; C atoms are grey colored, O (red), and N (blue). H atoms are hidden for clarification purposes, and the picture is visualized using PYMOL [39].

Figure 4

(A) The X-ray structure of the EGFR (3W32) binding pocket accommodating the co-crystallized ligand W32 (red color) and IF-docked poses of carboxamides 4–16 depicted in yellow, pink, green, purple, and cyan. (B) Superposing of W32 (red color) and ligand 16 (green color) at 3W32 binding site. Some of the key binding residues are depicted; C atoms are grey colored, O (red), and N (blue). H atoms are hidden for clarification purposes, and the picture is visualized using PYMOL [39].

Figure 5

Superposition of the (A) EVP IF-docked pose (purple) and its native geometry (green) in 5ZRF; (B) X6K IF-docked pose (sand) and its native geometry (red) in 4L23; and (C) W32 IF-docked pose (blue) and its native geometry (beige) in 3W32. Picture is visualized using PYMOL.

Figure 6

Molecular descriptor analysis. (A) Graphical 2D representation of the principal component analysis (PCA) of all 2D alvaDesc molecular descriptors for the synthesized active carboxamides 4,6,8,10,12,14, and 16. PC1 explained 77.29% of the variation in descriptor values on the x-axis, and PC2 explained 9.09% of the variation in descriptor values on the y-axis. The dots corresponding to compounds on the figure are colored based on their lead-like scores (LLS_01). (B) Radar plot of drug-like scores (DLSs) and lead-like scores (LLSs) for all synthesized active compounds.