According to the World Health Organization, breast cancer is the second leading cause of mortality among women, accounting for 12.5% of all new cancer cases worldwide. By 2030, the estimated number of deaths will escalate to 11.4 million [1,2]. Furthermore, the incidence rates of breast cancer have been rising by 3% each year. Despite the advancement in breast cancer treatment with currently available drugs, the recovery rate is still unacceptably low. A novel heterocyclic scaffold incorporating many natural product structural features provides a new approach to developing safe and effective anti-breast cancer agents. Chromones (4-H-chromen-4-ones) with a benzo-γ-pyrone framework have emerged as privileged structures in drug discovery, showcasing favorable drug-like characteristics [3,4,5]. They exhibit a diverse array of pharmacological activities, including anti-inflammatory [6], anticancer [7,8], antioxidant [9], antibacterial [10,11], antiviral [12], and antimicrobial [13,14,15], monoamine oxidase B inhibitors [16,17,18,19], and possess A3 adenosine receptor activities [20,21]. Numerous investigators have pursued the development of more diverse and complex bioactive molecules based on the chromone template, as they serve as valuable targets for exploring the structure–activity relationship (SAR) studies in new drug discovery projects. Estrogen receptor (ER), progesterone receptor, and their corresponding steroid hormones play a crucial role in the development, differentiation, and function of normal breast and endometrial cells [22,23]. The two subtypes of human ERs, ER-α and ER-β, show different tissue distribution patterns and transcriptional activities. In ER-positive (ER+) breast cancer cells, blocking the binding of estrogen (E2) to the ER receptors with selective antagonists effectively stops cell growth and proliferation. Since both ER-α and ER-β are often over-expressed in breast cancer cells, they represent promising therapeutic targets for treating this type of cancer [24,25,26,27,28]. Tamoxifen TAM has been the leading drug in treating breast cancer for over two decades, showing significant effectiveness in ER(+) breast cancer, particularly in post-menopausal women [29,30,31]. However, a notable limitation of TAM is that it acts as an ER antagonist in the breast tissue while functioning as an agonist in other tissues with an increased risk of developing endometrial cancer [32,33]. Hussien et al. [34] reported that several new coumarin and chromene prototype derivatives have been synthesized and evaluated for their ERα and ERβ selective activities. Coumarin prototype compounds were found to be ERα selective and the most active, exhibiting potential antiproliferative activity against both ER+ and ER− breast cancer cell lines. Similarly, Saquib et al. [35] showed that a few novel coumarin-chalcone chimeric molecules were synthesized and evaluated for in vitro antiproliferative activity against MDA-MB-231, MCF-7, Ishikawa, and Hela cancer cell lines. One compound showed very good activity against MCF-7 (IC50 = 7.42 μM) and MDA-MB-231 (IC50 = 12.58 μM), better than standard drugs tamoxifen and raloxifene. Our approach was to use the information we learned in designing molecular structures that maximize the safety and efficacy of anti-breast cancer activities. The substitution pattern on chromone scaffolds plays a vital role in determining their affinities toward different biologically significant targets, presenting the possibility of new pathways for developing pharmacologically active compounds. In the present study, chromone-2-carboxamides with electron-donating and electron-withdrawing substituents on the rings were synthesized and evaluated for their anticancer activities against breast cancer cell lines. In silico docking analysis and probable binding modes of these compounds were determined using the Glide XP (extra precision) method by mapping the active site of the human ERs ER-α (7KBS) and ER-β (1QKM) crystal structures to estimate the most probable and stable conformation of the molecules.
All chemicals and solvents were purchased from Sigma-Aldrich and used without further purification. 1H NMR and 13C NMR spectra were recorded on the Varian Gemini HX 300 MHz instrument in DMSO-d6 and CDCl3. The chemical shifts are expressed in parts per million (δ, ppm) relative to tetramethyl silane as an internal standard. Elemental analyses were carried out by Galbraith Laboratories Inc, Knoxville, TN, USA, and are within ±0.4% of theoretical values unless otherwise noted. Melting points were determined on a Mel-Temp 3.0 melting point apparatus and were uncorrected. Column chromatography was performed on silica gel (200–425 mesh). Analytical thin-layer chromatography was performed on 250 µm-layer flexible plates, and spots were detected under UV light. Human MCF-7 and MDA-MB-231 breast cancer cell lines were purchased from the NCI. The human Ishikawa endometrial cancer cell line was purchased from Sigma Aldrich. All three cell lines were cultured in phenol red-free RPMI-1640 (Hyclone, 500 mL) supplemented with
The substituted chromone-2-carboxamide analogs (5a–n) were synthesized through a four-step process, starting from the readily available 2′,4′,6′-trihydroxyacetophenone, and obtained in moderate to high yields. The synthetic route is depicted in Scheme 1. The dimethoxy-2-hydroxyacetophenones 2a–b were prepared according to the previously described method [36] with an excess of dimethyl sulfate in the presence of anhydrous potassium carbonate in acetone and refluxed for 30 min. The dimethoxy-2-hydroxyacetophenones 2a–b were obtained in good yields. The carboxylic esters 3a–b were prepared by condensation of substituted 2-hydroxy acetophenone 2a–b with diethyl oxalate in the presence of sodium in ethanol. The product underwent cyclization to yield the chromone carboxylic esters [37]. The hydrolysis of the compounds 3a–b with a mixture of conc. HCl and acetic acid (1:2 v/v) gave corresponding chromone-2-carboxylic acids 4a–b in moderate to good yields [38]. The substituted chromone-2-carboxamides 5a–n were obtained by the reaction of 4a–b with differently substituted amines in the presence of diphenylphosphoryl azide (DPPA) and triethylamine (TEA) in moderate to good yields [39]. All the compounds 5a–n were characterized using 1H, 13C NMR, and elemental analyses.
Synthesis of substituted chromone-2-carboxamide derivative 5a–n.
Reaction conditions: (i) DMS, K2CO3, acetone, 60°C, 30 min; (ii) Na, EtOH, diethyl oxalate, conc. HCl, 100°C, 4 h; (iii) acetic acid, conc. HCl, 100°C, 4 h; and (iv) substituted aniline or amine, DMF, DPPA, TEA, R.T. 24 h.
The antiproliferative activities of fourteen substituted chromone-2-carboxamide derivatives (5a–n) were evaluated at the Southern Research Institute (SRI, Birmingham, Alabama, USA). The compounds were screened against human MDA-MB-231 (ER−), MCF-7 (ER+) and Ishikawa (endometrial) cancer cell lines in comparison to Tamoxifen (TAM). The cell lines were cultured and treated with compounds 5a–n, including the standard TAM ranging from 0.01 to 100,000 nM concentration in the presence of 10 nM estradiol using the previously reported method [40,41,42]. The results expressed as IC50 were the average of three data points for each concentration and were calculated using GraphPad Prism 4.0.
The ligands under study (5a–n) were sketched using a 2D sketcher in Schrodinger software’s Maestro interface and were optimized using the LigPrep tool. All possible states at target pH 7.0 ± 2.0 were generated using Epik, and energy was minimized using the OPLS4 force field in Schrodinger’s Small Molecule Drug Discovery Suite [30]. The ER-alpha (ER-α) ligand-binding domain (LBD) in complex with raloxifene (PDB: 7KBS) and the ER-beta (ER-β) LBD in complex with 4-hydroxytamoxifen (2FSZ) were downloaded from RCSB Protein Data Bank using Protein Preparation Workflow in Maestro. The downloaded receptor ligand complexes were prepossessed by capping the terminus, filling in any missed sidechains or loops using Prime. Hydrogen atoms were added to the proteins and ligands, and the entire receptor ligand complex was minimized using the OPLS4 force field. Hydrogen bond assignments were optimized using PROPKA. The water molecules in the crystal structure were removed and later retained close to the bound ligand (5 Å distance) to identify any possible interaction of the compounds under study with water molecules in the active site of the receptors. For thorough experimentation, the centroid of the co-crystallized bound ligand (Raloxifene) was used in receptor grid generation. The size of the grid was subject to small increments to give more room for the ligands to bind as part of the experimentation. Ligand flexibility was also taken into consideration in docking experiments. To validate the docking procedure, re-docking of the co-crystallized ligands (RAL and OHT) was done in the LBD of both the receptors. A Pharmacophore was generated using the PHASE module in Schrodinger Discovery Suite.
A solution of 1-(2,4-dihydroxyphenyl) ethanone (1a, 10 mmol), dimethyl sulfate (20 mmol), and anhydrous K2CO3 (20 mmol) in acetone (30 ml) was refluxed at 50–60°C for 30 min. The reaction mixture was filtered, and the solid residue was washed with warm acetone. The solvent was evaporated in vacuo, and the residue was purified by silica gel column chromatography to obtain compound 2a in good yield. The remaining compound 2b was synthesized similarly.
Sodium ethanolate was prepared by dissolving 10 mmol of sodium metal in 15 mL of anhydrous ethanol. At room temperature, the sodium ethanolate solution and diethyl oxalate (1.2 equiv) were added to the corresponding compounds (2a–b, 5 mmol), and the reaction mixture was heated at 90–100°C for 4 h. After this period, 1 mL of conc. HCl was added, and the reaction continued for 30 min. The mixture was then cooled to room temperature, washed with water, and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure using a rotary evaporator. The resulting crude product was purified by silica gel chromatography, yielding compounds 3a–b in 70–90% yields.
Hydrolysis of ethyl esters 3a–b was carried out using the reported method [37,38]. A solution of ethyl ester (3a–b, 9 mmol) was added to a mixture of glacial acetic acid and conc. HCl (2:1 v/v), and the reaction was stirred at 100°C for 4 h. Upon completion, the reaction mixture was cooled to room temperature. The resulting precipitates were separated by filtration, washed with methylene chloride, and the solid was dried under a high vacuum. The compounds 4a–b were obtained in 70–80% yields.
The substituted chromone-2-carboxylic acids (4a–b, 0.2 mmol, 1 eq) were dissolved in DMF (2 mL), and DPPA (82 mg, 1.5 eq), and TEA (30 mg, 1.5 eq). The reaction mixture was cooled to 0°C and stirred for 30 min. The corresponding aniline (28 mg, 1.5 eq) was then added, and the temperature was gradually increased to room temperature. The reaction was allowed to proceed for 24 h. After completion, the reaction mixtures were diluted with ethyl acetate, washed with 5% HCl, and followed with water. The organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residues were purified by silica gel column chromatography and eluted in 20% ethyl acetate/hexane to afford the final product 5a–n in fair to good yields. In the 1H NMR spectra, the –NH proton in compounds 5a–n appeared as a singlet 8.1–10.58 ppm, confirmed by the D2O exchange.
Yield: 90%; solid; m.p. 196–197°C, 1H NMR (CDCl3): δ 3.97 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 7.03 (d, J = 3.0 Hz, 1H), 7.14 (s, 1H), 7.18 (d, J = 3.0 Hz, 1H), 7.36 (t, 2H), 7.64 (d, J = 3.0 Hz, 2H), 7.91 (d, J = 3.0 Hz, 1H), 8.67(s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-D6): δ 56.10, 60.86, 110.07, 111.37, 119.42, 119.43, 120.47, 123.95, 128.96, 135.57, 138.65, 148.51, 153.09, 157.21, 157.93, 177.22. Anal. calcd. for C18H15NO5 (325.32): C, 66.46; H, 4.65; N, 4.31%. Found: C, 66.58; H, 4.63; N, 4.20%.
Yield 49%; solid; m.p. 225°C (decompose); 1H NMR (CDCl3): δ 7.29 (s, 1H), 7.68 (d, J = 4.0 Hz, 2H), 7.42 (t, J = 6.5 Hz, 2H), 7.24 (t, J = 7.1 Hz, 1H), 7.81 (s, 1H), 7.44 (s, 1H), 8.10 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-D6): δ 113.50, 121.47, 122.03, 123.95, 125.66, 127.94, 128.86, 134.42, 138.65, 150.33, 153.86, 157.47, 173.62. Anal. calcd. for C16H9Cl2NO3 0.2 H2O: (337.763): C, 56.90; H, 2.69; N, 4.15%. Found: C, 56.86; H, 2.82; N, 4.07%.
Yield 27%; m.p. 270°C (decompose); 1H NMR (DMSO-D6): δ 6.25 (dd, J = 1.0, 3.0 Hz, 1H), 6.41 (s, 1H), 6.99 (s, 1H), 7.86 (d, J = 3.0Hz, 1H), 7.97 (s, 1H), 8.29 (s, 1H), 9.40 (brs, 2H, OH, D2O exchangeable), 10.30 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-D6): δ 95.5, 98.14, 113.52, 122.03, 125.66, 126.98, 127.95, 134.54, 140.93, 150.33, 154.20, 157.43, 159.34, 173.62. Anal. calcd. for C16H9Cl2NO5 (366.16): C, 52.48; H, 2.48; N, 3.83%. Found: C, 52.38; H, 2.56; N, 3.67%.
Yield 82%; m.p. 271–272°C; 1H NMR (DMSO-d6): δ 3.93 (s, 3H, OCH3), 6.78 (d, J = 3.0 Hz, 2H), 6.85 (s, 1H), 7.12 (dd, J = 1.0,3.0 Hz, 1H), 7.29 (d, J = 1.0 Hz, 1H), 7.55 (d, J = 3.0 Hz, 2H), 7.96 (d, J = 3.0 Hz, 1H), 9.45 (s, 1H, NH D2O exchangeable), 10.50 (s,1H, OH, D2O exchangeable); 13C NMR (DMSO-D6): δ 55.88, 101.06, 112.51, 114.55, 115.80, 117.65, 122.77, 125.91, 131.00, 151.25, 154.67, 157.26, 157.88, 163.77, 176.82. Anal. calcd. for C17H13NO5 (311.29): C, 65.59; H, 4.21; N, 4.50%. Found: C, 65.13; H, 4.25; N, 4.29%.
Yield 26%; m.p. 295–296°C; 1H NMR (DMSO-d6): δ 3.92 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 6.77 (d, J = 3.0 Hz, 2H), 6.88 (s, 1H), 7.32 (d, J = 3.0 Hz, 1H), 7.53 (d, J = 3.0 Hz, 2H), 7.79 (d, J = 3.0 Hz, 1H), 9.43 (s, 1H, NH, D2O exchangeable), 10.36 (s, 1H, OH, D2O exchangeable); 13C NMR (DMSO-D6): δ 56.10, 60.86, 110.07, 111.37, 115.80, 119.43, 122.77, 131.00, 135.57, 148.51, 151.25, 153.20, 157.21, 157.93, 177.22. Anal. calcd. for C18H15NO6 0.2 H2O (344.92): C, 62.68; H, 4.38; N, 4.06%. Found: C, 62.73; H, 4.47; N, 3.97%.
Yield 40%; m.p. 300°C (decompose); 1H NMR (DMSO-d6)): δ 3.83 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 6.24 (dd, J = 2.1, 6.6 Hz, 1H), 6.40 (s, 1H), 6.55 (d, J = 2.1 Hz, 1H), 6.61 (s, 1H), 6.83 (s, 1H), 7.28 (d, J = 8.7 Hz, 1H), 9.35 (s, 2H, OH, D2O exchangeable), 9.70 (s, 1H), 9.76 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-D6): δ 56.38, 56.56, 95.50, 96.45, 98.14, 98.28, 109.03, 110.44, 140.93, 155.03, 153.37, 159.34, 160.04, 164.77, 175.20. Anal. calcd. for C18H15NO7 (357.31): C, 60.50; H, 4.23; N, 3.92%. Found: C, 60.35; H, 4.46; N, 4.12%.
Yield 69 %; m.p. 300°C (decompose); 1H NMR (DMSO-d6): δ 3.83 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 6.55 (d, J = 2.4 Hz, 1H), 6.65 (s, 1H), 6.76 (d, J = 8.7 Hz, 2H), 6.85 (d, J = 2.1 Hz, 1H), 7.53 (dd, J = 6.6, 2.1 Hz, 2H), 9.45 (s, 1H, NH, D2O exchangeable), 10.41 (s, 1H, OH, D2O exchangeable); 13C NMR (DMSO-D6): δ 56.45, 56.63, 94.00, 97.08, 109.33, 112.93, 115.60, 123.39, 129.38, 153.69, 155.11, 157.56, 159.17, 160.81, 164.59, 176.01. Anal. calcd. for C18H15NO6 (341.31): C, 63.34; H, 4.43; N, 4.10%. Found: C, 63.45; H, 4.47; N, 3.97%.
Yield 70%; m.p. 300°C (decompose); 1H NMR (DMSO-d6): δ 3.96 (s, 6H, OCH3), 6.26 (d, J = 3.0 Hz, 1H), 6.43 (s, 1H), 6.82 (s, 1H), 7.32 (d, J = 3.0 Hz, 1H), 7.79 (d, J = 3.0 Hz, 1H), 7.91 (d, J = 3.0 Hz, 1H), 9.34 (s, 2H, OH, D2O exchangeable), 9.42 (s, 1H), 10.32 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-D6): δ 56.10, 60.85, 95.50, 98.14, 111.37, 112.43, 119.43, 135.57, 140.93, 148.51, 153.43, 157.21, 158.02, 159.34, 177.22. Anal. calcd. for C18H15NO7 (357.31): C, 60.50; H, 4.23; N, 3.92%. Found: C, 60.12; H, 4.44; N, 4.02%.
Yield 35%; m.p. 270°C (decompose); 1H NMR (DMSO-D6): δ 6.76 (d, J = 8.7 Hz, 2H), 7.07 (s, 1H), 7.52 (d, J = 2.7 Hz, 2H), 7.96 (d, J = 2.7 Hz, 1H), 8.26 (d, J = 2.4 Hz, 1H), 9.45 (s, 1H, OH, D2O exchangeable), 10.41 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-D6): δ 113.50, 115.8, 122.03, 122.77, 125.66, 127.93, 127.94, 131.00, 134.42, 150.33, 151.25, 153.96, 157.32, 173.62. Anal. calcd. for C16H9Cl2NO4 0.5 H2O (359.17): C, 53.18; H, 2.56; N, 3.86%. Found: C, 53.51; H, 2.53; N, 3.90%.
Yield 37%; m.p. 290°C (decompose); 1H NMR (DMSO-D6): δ 3.74 (s, 6H, OCH3), 6.34 (t, J = 2.4 Hz, 1H), 7.01 (d, J = 3.9 Hz, 2H), 7.13 (s, 1H), 7.96 (dd, J = 0.6, 2.1 Hz, 1H), 8.28 (dd, J = 0.6, 2.1 Hz, 1H), 10.57 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-D6): δ 55.22, 55.31, 93.55, 98.75, 98.81, 113.52, 122.03, 125.66, 127.93, 127.98, 134.42, 140.81, 150.33, 153.88, 157.15, 161.50, 161.57, 173.62. Anal. calcd. for C18H13Cl2NO5 (394.21): C, 54.84; H, 3.32; N, 3.55%. Found: C, 54.56; H, 3.39; N, 3.91%.
Yield 34%; m.p. 250°C (decompose); 1H NMR (CDCl3): δ 2.29 (s, 3H, –CH3), 7.13 (dd, J = 2.1, 4.8 Hz, 2H), 7.24 (s, 1H), 7.69 (dd, J = 2.1, 4.8 Hz, 2H), 7.81 (d, J = 2.4 Hz, 1H), 8.11 (d, J = 2.4 Hz, 1H), 8.56 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-D6): δ 20.85, 113.50, 120.42, 120.85, 121.71, 121.92, 122.18, 15.66, 127.58, 127.94, 134.42, 136.15, 146.85, 150.33, 153.96, 157.31, 169.15, 173.68. Anal. calcd. for C18H11Cl2NO5 (392.19): C, 55.12; H, 2.83; N, 3.57%. Found: C, 54.85; H, 3.15; N, 3.87%.
Yield 50%; m.p. 135°C; 1H NMR (CDCl3): δ 4.63 (d, J = 2.0 Hz, 2H), 6.30 (t, J = 7.2 Hz, 2H), 7.14 (m, 1H), 7.15 (s, 1H), 7.35 (s, 1H), 7.71 (s, 1H), 8.02 (s, 1H, NH, D2O exchangeable); 13C NMR (CDCl3): δ 35.33, 108.12, 110.56, 113.52, 122.03, 125.39, 127.54, 127.93, 134.42, 141.27, 150.21, 151.78, 152.08, 160.85, 173.56. Anal. calcd. for C15H9Cl2NO4 (338.14): C, 53.28; H, 2.68; N, 4.14%. Found: C, 53.38; H, 2.67; N, 4.04%.
Yield 40%; m.p. 270°C (decompose); 1H NMR (DMSO-D6): δ 7.20 (s, 1H), 7.74 (d, J = 2.0 Hz, 2H), 7.97 (s, 1H), 8.30 (s, 1H), 8.54 (d, J = 2.0 Hz, 2H), 11.00 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-D6): δ 113.50, 114.04, 122.03, 125.66, 127.93, 128.02, 134.42, 146.09, 150.01, 150.35, 154.89, 157.39, 173.62. Anal. calcd. for C15H8Cl2N2O3 (335.14): C, 53.76; H, 2.41; N, 8.36%. Found: C, 54.28; H, 2.90; N, 7.82%.
Yield 44%; m.p. 225°C (decompose); 1H NMR (CDCl3): δ 4.80 (d, J = 2.0 Hz, 2H), 6.94 (dd, J = 1.0, 2.0 Hz, 1H), 7.03 (s, 1H), 7.17 (s, 2H), 7.23 (d, J = 2.0 Hz, 1H), 7.70 (s, 1H), 8.02 (s, 1H, NH, D2O exchangeable); 13C NMR (CDCl3): δ 36.22, 113.32, 122.03, 123.79, 123.79, 125.37, 125.50, 126.85, 127.54, 127.93, 134.42, 141.43, 150.21, 152.74, 160.82, 173.52. Anal. calcd. for C15H9Cl2NO3S (354.21): C, 50.86; H, 2.56; N, 3.95%. Found: C, 50.99; H, 2.71; N, 3.90%.
The in vitro antiproliferative activity of the substituted chromone-2-carboxamide derivatives (5a–n) was evaluated against human MDA-MB-231 (ER−), MCF-7 (ER+), and Ishikawa (endometrial) cancer cell lines to establish a SAR for the development of more potent compounds. The results were expressed as IC50 values, which are the concentrations of the test compounds, where a 50% reduction is observed in cell growth compared to the untreated control after a 72 h period of exposure to the test compounds, as shown in Table 1. Six of the 14 compounds showed antiproliferative activity against at least one cancer cell line with an IC50 value of 25.7–87.8 µM. Five compounds were active against MCF-7, in which compound 5g showed more significant antiproliferative activity with IC50 25.7 µM. Four compounds were active against MDA-MB-231 cell lines, and compound 5h showed the lowest IC50, 43.6 µM compared to other compounds. Only two compounds, 5d and 5g, were active against Ishikawa endometrial cancer cell lines, in which compound 5g showed the best activity with IC50 25.7 µM. A detailed analysis of the antiproliferative activity of the substituted chromone-2-carboxamide derivatives revealed several structure-activity trends. Compounds containing dichloro-substitutions on the chromone benzene ring, with either OCH3 groups (5j) or hydroxyl groups in the para-position (5i, 5c) and compounds with direct five- and six-membered heterocyclic rings attached to carboxamide nitrogen or with one carbon apart (5l, 5m, 5n) exhibited complete inactivity against all three cancer cell lines (>100 μM). However, compound 5b, with no substitution on the carboxamide ring, and compound 5k, with a para-acyl substitution, exhibited good selectivity towards MCF-7 cell lines, with IC50 values of 35.8 and 32.8 µM, respectively. Methoxy-substituted compounds on the chromone ring displayed improved activity and selectivity. In particular, compound 5h, with hydroxyl substitutions at the meta-position of the carboxamide aromatic ring, showed very good selectivity on MDA-MB-231 (IC50 = 43.6 µM), and no activity against MCF-7 (>100 µM) and Ishikawa (>100 µM) cell lines, suggesting a potential candidate for a triple-negative anti-breast cancer agent. Compound (5g) showed the best activity for MCF-7 (IC50 = 25.7 μM), which expresses high levels of ER-α. However, compound (5d) with only one -OCH3 group on the chromone ring with hydroxyl substitutions at the para-position of the carboxamide aromatic ring showed good selectivity towards MCF-7 and Ishikawa cell lines (IC50 = 38.2 and 36.2 μM, respectively), albeit with lower activity than compound (5g). Compound (5a) with no substitutions on the carboxamide benzene ring showed no activity on three cell lines.
In vitro antiproliferative activities of substituted chromone-2-carboxamides 5a–n
| Compounds | Structure | Yield (%) | M.P. (°C) | IC50 (µM) | ||
|---|---|---|---|---|---|---|
| MCF-7 | MDA-MB-231 | ISHIKAWA | ||||
| 5a |
| 90 | 196–197 | >100 | >100 | >100 |
| 5b |
| 49 | 225* | 35.8 | 93.1 | >100 |
| 5c |
| 27 | 270* | > 100 | >100 | >100 |
| 5d |
| 82 | 271–272 | 38.2 | >100 | 36.2 |
| 5e |
| 26 | 295–296 | >100 | >100 | >100 |
| 5f |
| 40 | 300* | 87.8 | >100 | >100 |
| 5g |
| 69 | 300* | 25.7 | 48.3 | 25.7 |
| 5h |
| 70 | 300* | >100 | 43.6 | >100 |
| 5i |
| 35 | 270* | >100 | >100 | >100 |
| 5j |
| 37 | 290* | >100 | >100 | >100 |
| 5k |
| 34 | 250* | 32.8 | 81.6 | >100 |
| 5l |
| 50 | 135–136 | >100 | >100 | >100 |
| 5m |
| 40 | 270* | >100 | >100 | >100 |
| 5n |
| 44 | 225* | >100 | >100 | >100 |
| Tamoxifen | — | — | 12.7 | 22.8 | 18.1 | |
*Compound decomposed.
The individual compounds under study (5a–n) were docked into the active site of the ER-α receptor (PDB ID 7KBS) using the Glide XP (extra precision) workflow as implemented in the Schrodinger Small-Molecule Drug Discovery Suite [43]. For the best results, the centroid of the co-crystallized bound ligand (Raloxifene) was used in receptor grid generation, and a room of 20–25 Å was chosen to give more space and conformational flexibility for the compounds to bind in the active site. Binding affinities of the synthesized compounds with the receptor were visually compared using the Ligand Interaction Diagrams.
A close observation of the top-ranked binding pose of the most active compound in the series toward MCF-7 cell lines (5g) reveals strong hydrogen bonding interactions between the phenyl ring OH group with ARG 394 and LEU 387, π–π stacking interactions of the amide phenyl ring with PHE 404, and amide NH with LEU 346. The interaction of phenyl OH with ARG 394 in the active site is considered the key interaction for a biological response [44]. Figures 1 and 2 show the 2-D and 3-D protein–ligand interaction diagram of the active compound 5g under the current study. In another docking experiment, water molecules in the active site close to the co-crystallized ligand (5 Å) were retained to notice any role of water molecules contributing to binding. A three-way interaction between H2O, the phenylic OH group on the compound, and ARG 394 and LEU 387 of the receptor was observed. (Figure 3). These key interactions might have contributed to the tight binding of the compound 5g in the active site of the receptor and hence the best activity in the present study. A close look at the Glide docking score values of the compounds understudy on ER-α active site (7KBS) (Table 2) show that the majority of least active compounds on all cell lines (5a, 5i, 5j, 5l, 5m, 5n) scored very poorly (−4 to −5) whereas some of the most active compounds in the list (5d, 5f, 5g, 5h) scored better (−6 to −7) as compared to the standard compound under study, i.e., Tamoxifen (−8.02).
2-D protein–ligand interaction of compound 5g in the active site of 7KBS.
3-D protein–ligand interaction of compound 5g in the active site of 7KBS. Reference ligand (raloxifene) in green.
2-D protein–ligand interaction of compound 5g in the active site of 7KBS with waters retained in the active site.
Glide docking scores of substituted chromone-2-carboxamides 5a–n at the active site of ER-α (PDB ID: 7KBS) and ER-β (PDB ID: 2FSZ)
| Compounds | Structure | Glide docking scores of ER-α (7KBS) | Glide docking scores of ER-β (2FSZ) |
|---|---|---|---|
| 5a |
| −5.25 | −5.17 |
| 5b |
| −5.22 | −6.16 |
| 5c |
| −6.80 | −4.24 |
| 5d |
| −6.02 | −6.16 |
| 5e |
| −6.12 | −7.54 |
| 5f |
| −7.10 | −7.09 |
| 5g |
| −6.44 | −6.16 |
| 5h |
| −7.10 | −7.05 |
| 5i |
| −4.76 | −5.12 |
| 5j |
| −4.82 | −5.11 |
| 5k |
| −1.485 | −2.70 |
| 5l |
| −4.71 | −6.68 |
| 5m |
| −4.88 | −6.87 |
| 5n |
| −4.83 | −5.84 |
| Tamoxifen | −8.02 | −10.55 |
Based on the docking poses of raloxifene (7KBS ligand) and compound 5g in the active site of the ER-α receptor, the pharmacophore hypothesis was generated using PHASE [43]. The best hypothesis generated is ADHRR, where acceptor, donor, hydrophobic groups, and aromatic rings were hypothesized to be essential for the biological activity (Figure 4).
Overlay of raloxifene (7KBS ligand, green) and compound 5g to generate PHASE hypothesis.
Similar to the human ER-α docking procedures, the individual compounds under study (5a–n) were docked into the active site of the ER-β receptor (PDB ID: 2FSZ) using the Glide XP (extra precision) workflow as implemented in the Schrodinger Small-Molecule Drug Discovery Suite [43]. The binding affinities of the synthesized compounds with the receptor were compared using the Ligand Interaction Diagrams. The docking scores of the compounds range from −2.7 to −7.4 (Table 2). The crucial π–π stacking interactions between one of the phenyl rings in the compounds under study with PHE 356 of the receptor were not observed. This interaction was believed to be crucial for biological activity, as was observed in Tamoxifen. Also, no good correlation of the docking scores with the activity data was noticed. Interestingly, the crystal structure of the receptor 2FSZ showed to have an alternative binding site, and hence, the possibility of the compounds binding in that active site was explored. Here, the binding scores were very low (−1 to −3) and, hence, the compounds binding there were deemed to be ruled out.
In this study, 14 chromone-2-carboxamide derivatives were synthesized in moderate to high yields. The antiproliferative activities of these compounds, 5a–n, against MCF-7, MDA-MB-231, and Ishikawa cell lines were evaluated by CellTiter-Glo assay. Seven compounds showed IC50 values of 25.7–87.8 µM against at least one cancer cell line. Compound 5g showed more significant activity against all three cell lines, with methoxy groups on chromone and a hydroxyl group at the para position of the phenyl carboxamide. Molecular docking was carried out on receptors ER-α and ER-β to identify the key interactions of the compounds understudy with the receptor amino acids. The study on the ER-α showed strong hydrogen bonding interactions between the phenyl ring OH group with ARG 394 and LEU 387, which might be responsible for the biological activity. Good docking score correlations were noticed in this study. Docking studies of the compounds on ER-b did not show any significant interactions or docking score correlations. During this study, we have identified a few molecules that are selective toward MCF-7 over MDA-MB-231 cell lines (5d and 5f) and MDA-MB-231 over MCF-7 cell lines (5h). Efforts are on to synthesize more analogs based on the above data to improve the biological activity of these molecules over those cell lines.