Exploring Carboxamide Derivatives as Promising Anticancer Agents: Design, In Vitro Evaluation, and Mechanistic Insights
Abstract
1. Introduction
2. Results and Discussion
3. Materials and Methods
3.1. Synthesis of Target Compounds and Materials
3.2. Spectroscopic and Characterization Techniques
3.3. Synthesis of Target Compounds and Data Analysis
- Compound (2), Indole-2-carbonyl chloride
- Compound (4), N-(4-Chlorophenyl)-1H-indole-2-carboxamide
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- Rf value: 0.60 (cyclohexane/ethyl acetate, 6:4).
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- Melting point: 259 °C.
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- 1H -NMR (300 MHz, DMSO-d6) showed the following peaks, δ ppm: 11.79 (br s, 1H, NH-indole), 10.35 (s, 1H, NHCO), 7.86 (d, J = 9.0 Hz, 2H, Ar-H), 7.69 (d, J = 8.1 Hz, 1H, Ar-H), 7.49 (s, 1H, Ar-H), 7.47–7.39 (m, 3H, Ar-H), 7.23 (dd, J = 8.1, 7.2 Hz, 1H, Ar-H), 7.08 (dd, J = 7.2, 7.8 Hz, 1H, Ar-H).
- ▪
- 13C -NMR (DMSO-d6, δ ppm): 160.26 (CONH), 138.43, 137.36, 131.66, 129.09 (C-3′, 5′), 127.57, 127.44, 124.37 (CH-Ar), 122.26 (CH-Ar), 122.05 (C-2′, 6′), 120.44 (CH-Ar), 112.88 (CH-Ar), 104.60 (CH-Ar).
- ▪
- IR (thin film, cm−1): 3406 (NH-indole), 3318 (NHCO), 1651 (CO), 1593, 1539, 1493, 1400, 1315, 1238, 1192, 1099, 1015, 937, 826, 748.
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- Elemental analysis (C15H11N2OCl):
- ▪
- Theoretical values (calculated): C, 66.55%; H, 4.10%; N, 10.35%.
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- Experimental values (found): C, 66.40%; H, 3.92%; N, 10.12%.
- Compound (6), N-(4-Florophenyl)-1H-indole-2-carboxamide
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- Rf value: 0.54 (cyclohexane/ethyl acetate, 7:3).
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- Melting point: 245 °C (decomposed).
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- 1H -NMR (300 MHz, DMSO-d6, δ ppm): 11.78 (br s, 1H, NH-indole), 10.80 (s, 1H, NHCO), 7.89–7.79 (m, 2H, Ar-H), 7.67 (d, J = 7.8 Hz, 1H, Ar-H), 7.48 (d, J = 8.0 Hz, 1H, Ar-H), 7.44–7.39 (m, 1H, Ar-H), 7.28–7.15 (m, 3H, Ar-H), 7.11–7.01 (m, 1H, Ar-H).
- ▪
- 13C-NMR (DMSO-d6, δ ppm, rotamers observed): 160.28, 157.09 (d, 1J C-F = 283 Hz, C-4′, CH-Ar), 160.14 (CONH), 157.09, 137.30, 135.82, 131.82, 127.47, 124.25 (CH-Ar), 122.44, 122.19 (d, 2J C-F = 19 Hz, C-3′, 5′, CH-Ar), 122.34 (CH-Ar), 120.38 (CH-Ar), 115.89, 115.59 (d, 3J C-F = 22.13 Hz, C-2′, 6’, CH-Ar), 112.86 (CH-Ar), 104.40 (CH-Ar).
- ▪
- IR (thin film, cm−1): 3422 (NH-indole), 3337 (NHCO), 3271, 1639 (CO), 1543, 1508, 1408, 1339, 1315, 1223, 1188, 1096, 833, 818, 748.
- ▪
- HRMS (ESI, negative mode): m/z (M+-H+) 253.07826 (C15H10N2OF) requires 253.07772.
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- Elemental analysis (C15H11N2OF):
- ○
- Theoretical values: C, 70.86%; H, 4.36%; N, 11.02%.
- ○
- Experimental values: C, 70.97%; H, 4.15%; N, 10.90%.
- Compound (8), N-(4-Nitrophenyl)-1H-indole-2-carboxamide
- ▪
- Rf value: 0.47 (CHCl3/CH3OH/CH3CO2H, 94:5:1).
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- Melting point: > 290 °C (decomposed).
- ▪
- 1H-NMR (300 MHz, DMSO-d6, δ ppm): 11.84 (br s, 1H, NH-indole), 10.74 (br s, 1H, NHCO), 8.28 (d, J = 9.2 Hz, 2H, H-3′, 5′, Ar-H), 8.11 (d, J = 9.2 Hz, 2H, H-2′, 6′, Ar-H), 7.71 (d, J = 8.0 Hz, 1H, Ar-H), 7.58–7.45 (m, 2H, Ar-H), 7.76 (dd, J = 7.4, 7.5 Hz, 1H, Ar-H), 7.09 (dd, J = 7.6, 7.3 Hz, 1H, Ar-H).
- ▪
- 13C-NMR (DMSO-d6, δ ppm): 160.67 (CONH), 145.84, 142.79, 137.68, 131.15, 127.38, 125.31 (CH-Ar), 124.80 (CH-Ar), 122.45 (CH-Ar), 120.92 (CH-Ar), 120.02 (CH-Ar), 112.97 (CH-Ar), 105.69 (CH-Ar).
- ▪
- Elemental analysis (C15H11N3O3):
- ○
- Theoretical values: C, 64.05%; H, 3.94%; N, 14.94%.
- ○
- Experimental values: C, 63.97%; H, 3.89%; N, 14.78%.
- Compound (10), N-(Pyridin-2-yl)-1H-indole-2-carboxamide
- ▪
- Rf: 0.53 (CHCl3/CH3OH/CH3CO2H, 94:5:1).
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- Melting point: 260 °C (decomposed).
- ▪
- 1H-NMR (300 MHz, DMSO-d6): rotamers, δ = 13.75 (br s, 1H, NH-indole), 11.8 + 10.91 (2 br s, 1H, NHCO), 8.61–7.78 (m, 3H, Ar-H), 7.75–7.35 (m, 3H, Ar-H), 7.30–6.80 (m, 3H, Ar-H) ppm.
- ▪
- 13C-NMR (DMSO-d6): δ = 160.11 (CONH), 144.10, 137.60, 137.24, 133.32, 127.56, 124.76, 113.56, 112.45, 111.90, 108.37, 104.99.
- ▪
- IR (thin film): ν = 3449 (NH-indole), 3422 (NHCO), 3144, 2924, 1701 (CO), 1558, 1450, 1366, 1346, 1200, 1080, 748 cm−1.
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- HRMS (ESI, negative mode): m/z (M+-H+) 236.08294 (C14H11N3O) requires 236.08239.
- ▪
- Elemental analysis (C14H11N3O):
- ○
- Theoretical values (calculated): C, 70.87%; H, 4.67%; and N, 17.71%.
- ○
- Experimental values (found): C, 70.62%; H, 4.88%; and N, 17.55%.
- Compound (12), N-(9,10-Dihydro-9,10-dioxoanthracen-1-yl)-1H-indole-2-carboxamide
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- Rf: 0.59 (cyclohexane/ethyl acetate, 6:4).
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- Melting point: > 350 °C (decomposed).
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- 1H-NMR (300 MHz, DMSO-d6) rotamers: δ = 13.23 (br s, 1H, NH-indole), 12.08 (s, 1H, NHCO), 9.15 + 9.13 (2s, 1H, Ar-H), 8.50–7.76 (m, 5H, Ar-H), 7.62–6.89 (m, 5H, Ar-H) ppm.
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- 13C-NMR (DMSO-d6): δ = 182.60 (CO-ketone), 176.31 (CO-ketone), 163.21 (CONH), 134.37 (CH-Ar), 133.90 (CH-Ar), 132.90, 131.68, 129.37, 128.06 (CH-Ar), 127.69, 127.13, 126.48 (CH-Ar), 125.25, 124.47, 123.85 (CH-Ar), 122.37 (CH-Ar), 121.83, 120.90 (CH-Ar), 118.16 (CH-Ar), 116.20 (CH-Ar), 115.24 (CH-Ar), 112.94 (CH-Ar), 104.18 (CH-Ar) ppm.
- ▪
- IR (thin film): ν = 3314 (NH-indole), 3213 (NHCO), 3125, 1667 (br CO), 1636 (CONH), 1578, 1531, 1412, 1312, 1269, 1238, 1173, 1015, 806, 741, 705 cm⁻¹.
- ▪
- HRMS (ESI, negative mode): m/z (M+-H+) 365.09317 (C23H13N2O3) requires 365.09262.
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- Elemental analysis (C23H₁4N2O3):
- ○
- Theoretical values (calculated): C, 75.40%; H, 3.85%; and N, 7.65%.
- ○
- Experimental values (found): C, 75.19%; H, 4.01%; and N, 7.96%.
- Compound (14), N-(9,10-Dihydro-9,10-dioxoanthracen-2-yl)-1H-indole-2-carboxamide
- ▪
- Rf: 0.41 (cyclohexane/ethyl acetate, 6:4).
- ▪
- Melting point: > 350 °C (decomposed).
- ▪
- 1H-NMR (300 MHz, DMSO-d6): δ = 11.88 (br s, 1H, NH-indole), 10.79 (s, 1H, NHCO), 8.68 (d, J = 3.0 Hz, 1H, Ar-H), 8.39 (d, J = 9.0 Hz, 1H, Ar-H), 8.31–8.11 (m, 3H, Ar-H), 8.0–7.83 (m, 2H, Ar-H), 7.75 (d, J = 6.0 Hz, 1H, Ar-H), 7.56 (s, 1H, Ar-H), 7.46 (d, J = 6.0 Hz, 1H, Ar-H), 7.37–7.03 (m, 2H, Ar-H) ppm.
- ▪
- 13C-NMR (DMSO-d6): δ = 183.0 (CO-ketone), 181.86 (CO-ketone), 160.67 (CONH), 145.19, 137.65, 135.03 (CH-Ar), 134.68 (CH-Ar), 134.58, 133.67, 131.20, 128.89 (CH-Ar), 128.50, 127.43, 127.21 (CH-Ar), 127.13 (CH-Ar), 124.94 (CH-Ar), 124.77 (CH-Ar), 122.48 (CH-Ar), 120.58 (CH-Ar), 117.21 (CH-Ar), 112.96 (CH-Ar), 105.59 (CH-Ar) ppm.
- ▪
- IR (thin film): ν = 3402 (NH-indole), 3321 (NHCO), 3117, 3067, 1667 (br CO), 1651 (CONH), 1589, 1535, 1416, 1331, 1292, 1238, 954, 840, 755, 710 cm−1.
- ▪
- HRMS (ESI, negative mode): m/z (M+-H+) 365.09317 (C23H13N2O3) requires 365.09262.
- ▪
- Elemental analysis (C23H14N2O3):
- ○
- Theoretical values (calculated): C, 75.40%; H, 3.85%; and N, 7.65%.
- ○
- Experimental values (found): C, 75.72%; H, 3.74%; and N, 7.87%.
- Compound (16), N-(9,10-Dihydro-9,10-dioxoanthracen-1-yl)-2-furamide
- ▪
- Rf: 0.67 (CHCl3/CH3OH, 9:1).
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- Melting point: 280 °C (decomposed).
- ▪
- 1H-NMR (300 MHz, DMSO-d6) rotamers: δ = 13.18 (br s, 1H, NHCO), 9.15 (br s, 1H, Ar-H, H3-furan), 8.35–8.10 (m, 2H, Ar-H), 7.94–7.72 (m, 4H, Ar-H), 7.46–7.12 (m, 2H, Ar-H), 6.95–6.61 (m, 1H, Ar-H) ppm.
- ▪
- 13C-NMR (DMSO-d6) rotamers: δ = 192.82 (CO-ketone), 186.44 (CO-ketone), 161.22 (CONH), 148.09, 147.51, 147.22, 136.33, 135.25, 127.62, 126.96, 125.71, 122.59, 118.83, 118.18, 116.91, 113.52, 112.77, 112.56 ppm.
- ▪
- Elemental analysis (C19H11NO4):
- ○
- Theoretical values (calculated): C, 71.92%; H, 3.49%; and N, 4.41%.
- ○
- Experimental values (found): C, 71.55%; H, 3.63%; and N, 4.66%.
3.4. Cell Proliferation Study
3.4.1. Cells and Cell Culture Conditions
3.4.2. MTT Assay
3.4.3. Statistical Analysis
3.5. Molecular Modeling and Cheminformatics Methods
3.5.1. Protein Preparation
3.5.2. Ligand Structure Preparation
3.5.3. Induced-Fit Docking (IFD)
3.5.4. Molecular Descriptors
3.5.5. Principal Component Analysis (PCA)
4. Conclusions
Limitation and Future Investigations
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
ANOVA | Analysis of variance |
HCT-116 | Human colorectal carcinoma cell line |
IC50 | 50% Cell proliferation inhibition |
K-562 | Human chronic myeloid leukemia cell line |
MTT | 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide |
MCF-7 | Human breast adenocarcinoma |
OD | Optical density |
PCA | Principal component analysis |
SI | Selectivity index |
RES | Resolution |
PI3K | Phosphoinositide 3-kinase |
EGFR | Epidermal growth factor receptor |
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Compound No. | IC50 (µM) ± S.D. | Selectivity Indices (SIs) | |||
---|---|---|---|---|---|
HCT-116 Colon Cancer | MCF-7 Breast Cancer | K-562 Leukemia | Dermal Fibroblast Normal | ||
4 | 3.94 ± 0.03 | 10.6 ± 0.53 | 0.61 ± 0.03 | >100 | 25.4, 9.4, 164 |
6 | 25.4 ± 0.38 | 80.8 ± 4.04 | 84.6 ± 4.23 | >100 | 3.9, 1.2, 1.2 |
8 | 32.0 ± 0.83 | >100 | >100 | >100 | 3.1, 1.0, 1.0 |
10 | 1.01 ± 0.04 | 70.3 ± 3.52 | >100 | >100 | 99.4, 1.4, 1.0 |
12 | 3.98 ± 0.06 | 9.07 ± 0.45 | 0.33 ± 0.02 | >100 | 25.1, 11.0, 303 |
14 | 2.64 ± 0.07 | 7.16 ± 0.36 | 0.61 ± 0.03 | >100 | 37.9, 14.0, 164 |
16 | 3.11 ± 0.049 | 33.1 ± 1.66 | >100 | >100 | 32.2, 3.0, 1.0 |
Compound No. (Moiety) | Docking Score | Binding Residue | Binding Nucleotide |
---|---|---|---|
4 | −7.35 | NA | DG13 |
6 | −7.83 | Q778 | NA |
8 | −8.88 | NA | DC8, DG13 |
10 | −6.79 | Q778 | NA |
12 | −10.16 | R503 | NA |
14 | −9.35 | NA | DC8, DA12 |
16 | −8.95 | H2O-Q778 | DG13 |
EVP | −13.76 | D479 | DG7, DG13 |
Compound No. (Moiety) | Docking Score | Binding Residue |
---|---|---|
4 | −6.85 | V851 |
6 | −6.71 | Y836 |
8 | −7.11 | E849, V851 |
10 | −7.29 | V851 |
12 | −10.10 | E849, V851, Q859 |
14 | −8.85 | W780, E798, V851, S854 |
16 | −9.11 | Y836, V851 |
X6K | −10.16 | S773, Y836, V851 |
Compound No. (Moiety) | Docking Score | Binding Residue |
---|---|---|
4 | −8.50 | T854 |
6 | −8.60 | T854 |
8 | −9.04 | M793, T854 |
10 | −8.05 | K745, T854 |
12 | −9.99 | K745, D855 |
14 | −11.74 | M793, T854 |
16 | −10.12 | M793, T854 |
W32 | −10.71 | R841 |
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Al-Najdawi, M.M.; Saleh, M.M.; Sabbah, D.A.; Hajjo, R.; Zalloum, H.; Abudoleh, S.M.; Abuarqoub, D.A.; Al-Hiari, Y.M.; Mohammad, M.Y.; ALSalamat, H.; et al. Exploring Carboxamide Derivatives as Promising Anticancer Agents: Design, In Vitro Evaluation, and Mechanistic Insights. Int. J. Mol. Sci. 2025, 26, 5903. https://doi.org/10.3390/ijms26125903
Al-Najdawi MM, Saleh MM, Sabbah DA, Hajjo R, Zalloum H, Abudoleh SM, Abuarqoub DA, Al-Hiari YM, Mohammad MY, ALSalamat H, et al. Exploring Carboxamide Derivatives as Promising Anticancer Agents: Design, In Vitro Evaluation, and Mechanistic Insights. International Journal of Molecular Sciences. 2025; 26(12):5903. https://doi.org/10.3390/ijms26125903
Chicago/Turabian StyleAl-Najdawi, Manal M., Maysaa M. Saleh, Dima A. Sabbah, Rima Hajjo, Hiba Zalloum, Suha M. Abudoleh, Duaa A. Abuarqoub, Yusuf M. Al-Hiari, Mohammad Yasin Mohammad, Husam ALSalamat, and et al. 2025. "Exploring Carboxamide Derivatives as Promising Anticancer Agents: Design, In Vitro Evaluation, and Mechanistic Insights" International Journal of Molecular Sciences 26, no. 12: 5903. https://doi.org/10.3390/ijms26125903
APA StyleAl-Najdawi, M. M., Saleh, M. M., Sabbah, D. A., Hajjo, R., Zalloum, H., Abudoleh, S. M., Abuarqoub, D. A., Al-Hiari, Y. M., Mohammad, M. Y., ALSalamat, H., Mansour, H., Aljbour, N. D., & Mestareehi, A. H. (2025). Exploring Carboxamide Derivatives as Promising Anticancer Agents: Design, In Vitro Evaluation, and Mechanistic Insights. International Journal of Molecular Sciences, 26(12), 5903. https://doi.org/10.3390/ijms26125903