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ORIGINAL ARTICLE
Year : 2023 | Volume
: 18
| Issue : 1 | Page : 24-38
Synthesis of oxamide-hydrazone hybrid derivatives as potential anticancer agents
Mina Dehbid1, Raheleh Tahmasvand2, Marzieh Tasharofi3, Fatemeh Shojaie4, Mahmoudreza Aghamaali1, Ali Almasirad3, Mona Salimi2
1 Department of Biology, Faculty of Science, University of Guilan, Rasht, I.R. Iran., I.R. Iran
2 Department of Physiology and Pharmacology, Pasteur Institute of Iran, Tehran, I.R. Iran., I.R. Iran
3 Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran Medical Sciences, Islamic Azad University, Tehran, I.R. Iran., I.R. Iran
4 Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran Medical Sciences, Islamic Azad University, Tehran, I.R. Iran
Correspondence Address:
Ali Almasirad
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran Medical Sciences, Islamic Azad University, Tehran, I.R. Iran. Tel: +98-2122640051, Fax: +9822602059
I.R. Iran
Mona Salimi
Department of Physiology and Pharmacology, Pasteur Institute of Iran, Tehran, I.R. Iran. Tel: +98-2164112264, Fax: +98-2164112834
I.R. Iran
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/1735-5362.363593
Background and purpose: Considering various studies implying anticancer activity of the hydrazone and oxamide derivatives through different mechanisms such as kinases and calpain inhibition, herein, we report the synthesis, characterization, and evaluation of the antiproliferative effect of a series of hydrazones bearing oxamide moiety compounds (7a-7n) against a panel of cancer cell lines to explore a novel and promising anticancer agent (7k).
Experimental approach: Chemical structures of the synthesized compounds were confirmed by FTIR, 1H-NMR, 13C-NMR, and mass spectra. The antiproliferative activity and cell cycle progression of the target compound were investigated using the MTT assay and flow cytometry.
Findings/Results: Compound 7k with 2-hydroxybenzylidene structure was found to have a significant in vitro anti-proliferative influence on MDA-MB-231 (human adenocarcinoma breast cancer) and 4T1 (mouse mammary tumor) cells as the model of triple-negative breast cancer, with the IC50-72h values of 7.73 ± 1.05 and 1.82 ± 1.14 μM, respectively. Following 72-h incubation with compound 7k, it caused MDA-MB-231 cell death through G1/S cell cycle arrest at high concentrations (12 and 16 μM).
Conclusion and implications: Conclusively, this study for the first time reports the anti-proliferative efficacy of compound 7k possessing 2-hydroxyphenyl moiety, which may serve as a potent candidate in triple-negative breast cancer treatment.
Keywords: Anti-proliferation; Cancer; Cell Cycle; Hydrazone; Oxamide
Since the beginning of the twenty-first century, cancer has been a major leading cause of death globally with a significant economic burden in all countries [1]. Breast cancer as one of the most common cancers in women has become a major concern threatening women's health with an annually increasing number of patients [2]. Among all breast cancers, triple-negative breast cancer (TNBC) has a metastatic pattern with a poor prognosis [3]. In other words, it is of particular research interest as triple-negative breast cancer is therapeutically challenging mainly due to its low response to chemotherapeutics and highly aggressive features [4]. Despite the significant progress made in research and the development of breast cancer drugs, drug resistance has remained one of the major clinical obstacles needed to be resolved [5]. Thus, developing novel compounds to fight this fatal disease remains urgent and of great importance.
In the strategy of drug design, connecting various pharmacophores with similar activity and subsequently preparing the hybrid molecules results in the development of novel drugs. In this context, the hydrazone group as a promising pharmacophore presents richly in medicinal molecules and contributes a significant role to discover a wide range of therapeutic agents with several biological and chemotherapeutic activities [6],[7],[8],[9]. Among the various bioactivities reported for hydrazone derivatives including anti-malarial, anti-tuberculosis, and anti-HIV, our attention has been drawn toward their anticancer properties [9]. Besides, oxamide compounds exert their anticancer effects through different mechanisms such as inhibition of calpain, histone deacetylase, protein-tyrosine phosphatase, tyrosine kinase, beta-tubulin inhibition, cyclin-dependent kinase 4 inhibition, upregulation of integrin beta 4, induction of apoptosis [10],[11],[12],[13],[14],[15],[16],[17],[18],[19]. Moreover, the short half-life and low oral bioavailability of the oxamides caused that several compounds possessing this pharmacophore have been noticed [20]. Altogether, oxamide and hydrazone groups are found in a great number of anticancer agents such as compounds 1 and 2 in Scheme 1 [9],[21],[22].
Based on the aforementioned findings, herein, a series of hybrid compounds in which hydrazone and oxamide pharmacophores were covalently associated into a single molecule (7a-7n), were synthesized and assessed for their potential antiproliferative activity. Indeed, the target structures bearing together oxamide and hydrazone moieties may act as dual anticancer functional groups.
Materials and methodsGeneral
Chemicals used in the chemistry experiment were purchased from Merck and Sigma/Aldrich Companies. Melting points were taken on an electrothermal IA 9300 capillary melting-point apparatus (Ontario, Canada) and are uncorrected. Proton and carbon-13 nuclear magnetic resonance (1H-NMR and 13C-NMR) spectra were obtained using Bruker FT-250 or FT-500 spectrometers (Bruker, Rheinstetten, Germany). Mass spectra were obtained using a 5973 Network Mass Selective Detector at 70 eV (Agilent technology, USA). Fourier-transform infrared spectroscopy (FTIR) spectra were recorded by Shimadzu FT-IR 8400S spectrographs (KBr disks, Japan). Elemental analysis was carried out using a Perkin-Elmer Model 240-c apparatus (Perkin Elmer, Norwalk, CT, USA). The results of the elemental analyses (C, H, N) were within ± 0.4% of the calculated amount.
Human breast cancer cell lines, MDA-MB-231 (C578) and MCF-7 (C135), human colorectal adenocarcinoma cell line HT-29 (C466), hepatocarcinoma cell line Hep-G2 (C158), and normal human skin fibroblast cell line HSF (C192) were provided from the National Cell Bank of Pasture Institute of Iran (NCBI). Dulbecco's modified eagle's medium (DMEM), FBS (fetal bovine serum), trypsin-ethylenediaminetetraacetic acid (EDTA), and penicillin G/streptomycin were purchased from Gibco (Gibco-BRL, Rockville, IN, USA). The 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium (MTT) was obtained from Sigma-Aldrich (Saint Louis, Missouri, USA). The other chemicals were supplied by Merck (Darmstadt, Germany) and Sigma-Aldrich (St Louis, MO, USA).
Chemistry
The fourteen designed compounds were synthesized starting with a reaction of aniline 3 with ethyl oxalyl chloride 4 to give intermediate 5 [22]. Hydrazide 6 was prepared by the treatment of 5 with hydrazine hydrate [23]. Final compounds (7a-7n) were synthesized through acid-catalyzed condensation of 6 with the corresponding aldehydes (Scheme 2). Synthesis of compounds 7a, 7h, 7i, 7j, and 7k has been reported previously; however, ten compounds are novel and reported here for the first time [23],[24],[25],[26],[27],[28],[29].
Synthesis of ethyl oxo(phenylamino)acetate [5]
To a stirring solution of aniline 3 (1.8 mL, 20 mmol) and triethylamine (2.8 mL, 20 mmol) in dichloromethane (60 mL) at 0 °C, a solution of ethyl oxalyl chloride 4 (2.2 mL, 20 mmol) in 20 mL dichloromethane was slowly added and then the reaction was stirred for 1 h in this condition. The mixture was stirred at room temperature for another 1 h and the solvent was evaporated to give 3.74 g of compound 5 with a yield of 98% and the melting point of 66-67 °C used in the next step without further purification. IR (KBr): vcm-1 3346 (NH, stretch), 1704 (C=O, stretch) [26].
Synthesis of 2-hydrazinyl-2-oxo-N-phenyl-acetamide [6]
To a solution of (3.74 g, 19 mmol) compound 5 in 96% ethanol, 135 mL of hydrazine hydrate (1.13 mL, 23 mmol) was added dropwise. The resulting solution was stirred at room temperature for 2 h and the precipitate was filtered off to give 3.12 g of compound 6 with a yield of 90% and a melting point of 217-219 °C [26], which was used in the next step without further purification. IR (KBr): vcm-1 3319, 3284 (NH2, NH, stretch), 1668 (C=O, stretch).
General procedure for the preparation of compounds 7a-7n
A solution of 6 (3.55 mmol), corresponding aldehydes (3.57 mmol), and HCl 37% (2 drops) in 96% ethanol (40 mL) were stirred for 2 h at room temperature. To the resulting mixture, NaHCO3 (10%) was added and the precipitate was filtered off and washed with water, and recrystallized from the suitable solvent.
Cell growth inhibition assay
Synthesized compounds (7a-7n) were dissolved in dimethyl sulfoxide (DMSO, 0.5%), and cells were then treated with the appropriate concentrations (1-50 μM) of the compounds. Untreated cells, 0.5% DMSO- and doxorubicin-treated cells served as the negative, vehicle, and positive control cells, respectively. Viability is defined as the ability of chemical compounds to transform MTT into purple-blue formazan salt. Indeed, the amount of visible formazan crystals in each well is directly proportional to the number of living cells [30]. The number of 1-5 × 103 cells/well for MDA-MB-231, 3-7 × 103 cells/well for MCF7, 3-7 × 103cells/well for HT-29, 4-8 × 103 cells/well for Hep-G2, and 0.5-3.5 × 103 cells/well for 4T1 were cultured in 96-well plates and kept to be attached overnight. In the initial screening, the antiproliferative activity of the compounds was calculated at a unique concentration (50 μM) at 72 h and then the half maximal inhibitory concentration (IC50) values were measured for the selected compound at 24, 48, and 72 h towards MDA-MB-231 cells. Moreover, the number of 2 × 103 cells of HSF was seeded to measure the viability on a normal cell line. To determine cell growth inhibition in each well, 20 μL of MTT solution (5 mg/mL in PBS) was added and incubated at 37 °C for 4 h. Following the formation of the visible formazan crystals by mitochondrial succinate dehydrogenase in the live cells, they were solubilized in DMSO and the optical density was measured at 570 nm using an ELISA plate reader (Epoch2, Biotek, USA). Relative cell growth inhibition was calculated compared to the non-treated cells.
Analysis of cell cycle progression
The impact of compound 7k on cell cycle progression was assessed using propidium iodide (PI) staining. MDA-MB-231 and 4T1 cells with a density of about 3 × 104 and 2.5 × 104 cells/well were subjected to compound 7k at 8, 12, 16 μM, and 2, 4, 8 μM concentrations, respectively, for 72 h. After trypsinization, the cells were fixed using 70% ethanol for 24 h at -20 °C. Then, the samples were stained whit PI (1 mg/mL), Triton™ X-100 (0.1%), and RNAse (100 mg/mL) and incubated for 15 min in a dark place at 37 °C. The percentage of cell populations in the G0/G1, S, and G2/M phases of the cell cycle was determined with the PARTEC flow cytometer (PARTEC GmbH, Munster, Germany) using FlowJo software. Cell cycle phases were determined by recording the peak area of FL3-A on a linear scale. Samples were prepared in triplicates with at least three repetitions for each experiment [9].
Statistical analysis
Experimental data were analyzed statistically using Graph Pad Prism 6 Software (GraphPad Software, La Jolla, CA, United States). The values are expressed as mean ± SEM of at least triplicates. Data were assessed using one-way ANOVA followed by Tukey’s multiple comparison test. P-values < 0.05 were considered statistically significant.
ResultsChemistry
A diverse array of derivatives (7a-7n) was synthesized and then characterized by physical and spectral data (FTIR, 1H-NMR, 13C-NMR, Mass) [Figue 1].
2-[2-Benzylidenehydrazinyl]-2-oxo-N-phenyl-acetamide (7a)
Melting point (m.p) = 274-276 °C (EtOH) [25], yield 85%, IR (KBr): v cm-1 3292, 3249 (NH, stretch), 1656 (C=O, stretch). 1H-NMR (250 MHz, DMSO-d6): δppm 12.33 (s, 1H, NH), 10.81 (s, 1H, NH), 8.62 (s, 1H, N=CH), 7.86 (d, J=7.7 Hz, 2H, H2, H6, 7.73-7.71 (m, 2H, H2, H6), 7.47-7.44 (m, 3H, H3,, H4,, H5,), 7.37 (t, J = 7.7 Hz, 2H, H3, H5), 7.15 (t, J = 7.2, 1H, H4). 13C-NMR (60 MHz, DMSO-d6): δppm 158.77 (C=O), 157.01 (C=O), 151.56 (=C), 137.99 (C-1), 134.29 (C-1'), 131.6 (C-4'), 129.34 (C-2', C-6'), 129.17 (C-3, C-5), 127.88 (C-3', C-5'), 125.10 (C-4), 120.99 (C-2, C-6). Mass: m/z (%) 267 (M+, 33), 147 (100), 119 (39), 120 [26], 92 [24], 93 [28], 77 (44), 51 [15]. Anal. Calcd. for C15H13N3O2: C, 67.40; H, 4.90; N, 15.72. Found: C, 67.19; H, 4.83; N, 15.53.
2-[2-(2-Chlorobenzylidene)hydrazinyl]-2-oxo-N-phenylacetamide (7b)
m.p = 240-241 °C (EtOH), yield 95%, IR (KBr): vcm-1 3292, 3251 (NH, stretch), 1662 (C=O, stretch). 1H-NMR (250 MHz, DMSO-d6): δppm 12.54 (s, 1H, NH), 10.74 (s, 1H, NH), 8.98 (s, 1H, N=CH), 7.94 (d, J = 7.0 Hz, 1H, H6'), 7.77 (d, J = 7.8 Hz, 2H, H2, H6), 7.47-7.26 (m, 5H, H3, H5, H3', H4', H5'), 7.07 (t, J = 7.3 Hz, 1H, H4). 13C-NMR (60 MHz, DMSO-d6): δppm 158.6 (C=O), 157.2 (C=O), 147.6 (=C), 137.9 (C-1), 134.1 (C-2'), 132.4 (C-1'), 131.7 (C-4'), 130.4 (C-6'), 129.2 (C-3 and C-5), 128.1 (C-3'), 127.6 (C-5'), 125.1 (C-4), 120.9 (C-2 and C-6). Mass: m/z (%) 303 (m++2, 6), 301 (M+, 17), 181 (100), 164 (37), 119 (45), 93 [33], 77 (55), 51 [15]. Anal. Calcd. for C15H12C1N3O2: C, 59.71; H, 4.01; N, 13.93. Found: C, 60.02; H, 4.11; N, 13.84.
2-[2-(3-Chlorobenzylidene) hydrazinyl]-2-oxo-N-phenylacetamide (7c)
m.p = 243-245 °C (EtOH), yield 94%, IR (KBr): vcm-1 3303, 3255 (NH, stretch), 1660 (C=O, stretch). 1H-NMR (250 MHz, DMSO-d6): δppm 12.54 (s, 1H, NH), 10.91 (s, 1H, NH), 8.64 (s, 1H, N=CH), 7.91 (d, J = 8.3 Hz, 2H, H2, H6), 7.81 (s, 1H, H2), 7.72 (d, 1H, J = 6.0 Hz, 1H, H6'), 7.57-7.51 (m, 2H, H4', H5'), 7.42 (t, J = 8.0 Hz, 2H, H3, H5), 7.20 (t, J = 7.0 Hz, 1H, H4). 13C-NMR (60 MHz, DMSO-d6): δppm 158.6; (C=O), 157.2 (C=O), 149.8 (=c), 138.0 (C-1), 136.5 (C-1'), 134.1 (C-3'), 131.3 (C-4'), 130.7; (C-2'), 129.2 (C-3 and C-5), 126.9 (C-6'), 126.7 (C-5'), 125.1 (C-4), 120.9 (C-2 and C-6). Mass: m/z (%) 303 (M++2, 5), 301 (M+, 15), 293 [33], 181 (100), 119 (41), 77 (47), 51 [12]. Anal. Calcd. for C15H12ClN3O2: C, 59.71; H, 4.01; N, 13.93. Found: C, 59.98; H, 4.18; N, 13.81.
2-[2-(4-Chlorobenzylidene)hydrazinyl]-2-oxo-N-phenylacetamide (7d)
m.p = 299-300 °C (EtOAc), yield 93%, IR (KBr): vcm-1 3301, 3256 (NH, stretch), 1697, 1662 (C=O, stretch). 1H-NMR (250 MHz, DMSO-d6): δppm 12.32 (s, 1H, NH), 10.74 (s, 1H, NH), 8.52 (s, 1H, N=CH), 7.76 (d, J = 7.7 Hz, 2H, H2, H6), 7.66 (d, J = 8.5 Hz, 2H, H2, H6), 7.45 (d, J = 8.5 Hz, 2H, H3', H5'), 7.29 (t, J= 7.7 Hz, 2H, H3, H5), 7.07 (t, J = 7.3 Hz, 1H, H4). 13C-NMR (60 MHz, DMSO-d6): δppm 158.7 (C=O), 157.0 (C=O), 150.1 (=C), 138.0 (C-1), 135.5 (C-4'), 133.2 (C-1'), 129.5 (C-2', C-3', C-5', C-6'), 129.2 (C-3 and C-5), 125.1 (C-4), 121.0 (C-2 and C-6). Mass: m/z (%) 303 (m+ + 2, 11), 301 (M+, 33), 181 (100), 153 [21], 120 [31], 93 [33], 77 (58), 51 [15]. Anal. Caled. for C15H12CIN3O2: C, 59.71; H, 4.01; N, 13.93. Found: C, 59.59.55; H, 9.97; N, 13.13.79.
2-[2-(2-Bromobenzylidene)hydrazinyl]-2-oxo-N-phenylacetamide (7e)
m.p = 237-240 °C (EtOH), yield 86%, IR (KBr): vcm-1 3286, 3247 (NH, stretch), 1660 (c=O, stretch). 1H-NMR (250 MHz, DMSO-d6): δppm 12.65 (s, 1H, NH), 10.81 (s, 1H, NH), 9.02 (s, 1H, N=CH), 7.99 (d, J = 7.5 Hz, 1H, H3'), 7.85 (d, J = 8.0 Hz, 2H, H2, H6), 7.70 (d, J= 7.7 Hz, 1H, H6'), 7.50-7.33 (m, 4H, H3, H5, H4', H5'), 7.15 (t, J = 7.2 Hz, 1H, H4). 13C-NMR (60 MHz, DMSO-d6): δppm 158.61 (C=O), 157.26 (C=O), 149.97 (=C), 137.99 (C-1), 133.68 (C-1'), 133.26 (C-4'), 132.67 (C-3'), 129.17 (C-3, C-5), 128.57 (C-6'), 127.99 (c-5'), 125.11 (C-2'), 124.40 (C-4'), 120.98 (C-2, C-6). Mass: m/z (%) 347 (M++2, 3), 345 (M+, 3), 227 (39), 225 (39), 119 [28], 92 [30], 77 (100), 65 [26], 51 [14]. Anal. Calcd. for C15H12BrN3O2: C, 52.04; H, 3.49; N, 12.14. Found: C, 52.37; H, 3.38; N, 12.40.
2-[2-(3-Bromobenzylidene)hydrazinyl]-2-oxo-N-phenylacetamide (7f)
m.p = 235-237 °C (MeCN), yield 83%, IR (KBr): vcm-1 3268, 3242 (NH, stretch), 1662 (C=O, stretch). 1H-NMR (250 MHz, DMSO-d6): δppm 12.49 (s, 1H, NH), 10.85 (s, 1H, NH), 8.57 (s, 1H, N=CH), 7.9 (s, 1H, H2'), 7.85 (d, J= 8 Hz, 2H, H2, H6), 7.72-7.63 (m, 2H, H4', H6'), 7.46-7.34 (m, 3H, H3, H5, H5'), 7.15 (t, J = 7.0 Hz, 1H, H4). 13C-NMR (60 MHz, DMSO-d6): δppm 158.62 (C=O), 157.15 (C=O), 149.76 (=C), 137.97 (C-1'), 136.69 (C-1), 133.59 (C-4'), 131.56 (C-2'), 129.79 (C-5'), 129.18 (C-3, C-5), 127.08 (C-6'), 125.13 (C-3'), 122.65 (C-4), 120.98 (C-2, C-6). Mass: m/z (%) 347 (M++2, 7), 345 (M+, 7), 227 (42), 225 (42), 120 [19], 92 [30], 77 (100), 65 [25], 51 [14]. Anal. Calcd. for C15H12BrN3O2: C, 52.04; H, 3.49; N, 12.14. Found: C, 52.15; H, 3.62; N, 12.45.
2-[2-(4-Bromobenzylidene)hydrazinyl]-2-oxo-N-phenylacetamide (7g)
m.p = 311-315 °C (THF), yield 87%, IR (KBr): vcm-1 3299, 3245 (NH, stretch), 1664 (C=O, stretch). 1H-NMR (250 MHz, DMSO-d6): δppm 12.39 (s, 1H, NH), 10.81 (s, 1H, NH), 8.59 (s, 1H, N=CH), 7.85 (d, J = 7.7 Hz, 2H, H2, H6), 7.65 (bs, 4H, H2', H3', H5', H6'), 7.35 (t, J = 7.7 Hz, 2H, H3, H5), 7.14 (t, J = 7.2 Hz, 1H, H4). 13C-NMR (60 MHz, DMSO-d6): δppm 158.67 (C=O), 157.05 (C=O), 150.31 (=C), 137.97 (C-1), 133.55 (C-1'), 132.35 (C-3', C-5'), 129.69 (C-2', C-6'), 129.15 (C-3, C-5), 125.10 (C-4'), 124.37 (C-4), 120.99 (C-2, C-6). Mass: m/z (%) 347 (M++2, 12), 345 (M+, 12), 227 (41), 225 (41), 118 [22], 92 [30], 77 (100), 65 [26], 51; [13]. Anal. Calcd. for C15H12BrN3O2: C, 52.04; H, 3.49; N, 12.14. Found: C, 51.85; H, 3.34; N, 12.30.
2-[2-(2-Nitrobenzylidene)hydrazinyl]-2-oxo-N-phenylacetamide (7h)
m.p = 260-262 °C (EtOAc) [26], yield 96%, IR (KBr): vcm-1 3294, 3255 (NH, stretch), 1699 (C=O, stretch), 1662 (C=O, stretch), 1523, 1348 (NO2). 1H-NMR (250 MHz, DMSO-d6): δppm 12.63 (s, 1H, NH), 10.75 (s, 1H, NH), 8.94 (s, 1H, N=CH), 8.03-7.99 (m, 2H, H3', H6'), 7.75 (d, J = 7.7 Hz, 3H, H2, H6, H5'), 7.66-7.60 (m, 1H, H4'), 7.29 (t, J = 7.8 Hz, 2H, H3, H5), 6.7 (t, J = 7.3 Hz, 1H, H4). 13C-NMR (60 MHz, DMSO-d6): δppm 158.5 (C=O), 157.5 (C=O), 148.8 (C-2'), 147.0 (=C), 137.9 (C-1), 134.3 (C-5'), 131.6 (C-4'), 129.2 (C-3 and C-5), 128.8 (C-1' and C-6'), 125.1 (C-4), 121.0 (C-2 and C-6 and C-3'). Mass: m/z (%) 312 (M+, 13), 192 (100), 164 (40), 120 (66), 92 (59), 77 (80), 51 [24]. Anal. Calcd. for C15H12N4O4: C, 57.69; H, 3.87; N, 17.94. Found: C, 57.38; H, 3.99; N, 17.68.
2-[2-(3-Nitrobenzylidene)hydrazinyl]-2-oxo-N-phenylacetamide (7l)
m.p = 260-264 °C (THF) [25], yield 80%, IR (KBr): vcm-1 3286, 3234 (NH, stretch), 1664 (C=O, stretch), 1520, 1353 (NO2, stretch). 1H-NMR (500 MHz, DMSO-d6) : δppm 12.62 (s, 1H, NH), 8.74 (s, 1H, N=CH), 8.53 (s, 1H, H2'), 8.31 (dd, J = 8.2, 1.3 Hz, 1H, H4'), 8.15 (d, J= 7.7 Hz, 1H, H6'), 7.87 (d, J = 7.9 Hz, 2H, H2, H6), 7.78 (t, J = 8 Hz, 1H, H5'), 7.39 (t, J = 7.9 Hz, 2H, H3, H5), 7.17 (t, J = 7.5 Hz, 1H, H4). 13C-NMR (125 MHz, DMSO-d6): δppm 158.77(C=O),; 157.47(C=O),; 149.34(C-3’), 148.85 (C=N), 138.16 (C-1), 136.25 (C-6'), 134.34 (C-1'), 131.24 (C-5'), 129.37 (C-3, C-5), 125.44, 125.33, 121.88, 121.19. Mass: m/z (%) 312 (M+, 30), 192 (100), 120 (37), 92 (52), 77 (94), 65; [33], 51 [22]. Anal. Calcd. C15H12N4O4: C, 57.69; H, 3.87; N, 17.94. Found: C, 57.87; H, 3.80; N, 17.18.17.
2-[2-(4-Nitrobenzylidene)hydrazinyl]-2-oxo-N-phenylacetamide (7j)
m.p = 317-320 °C (EtOH) [25], yield 86%, IR (KBr): vcm-1 3302, 3256 (NH, stretch), 1660 (C=O, stretch), 1525, 1346 (NO2, stretch). 1H-NMR (500 MHz, DMSO-d6): δppm 12.65 (s, 1H, NH), 10.89 (s, 1H, NH), 8.72 (s, 1H, N=CH), 8.31 (d, J = 8.7 Hz, 2H, H3', H5'), 7.98 (d, J = 8.7 Hz, 2H, H2', H6'), 7.87 (d, 2H, J = 7.9 Hz, H2, H6), 7.39 (t, J=7.9 Hz, 2H, H3, H5), 7.17 (t, J = 7 Hz, 1H, H4). 13C-NMR (125 MHz, DMSO-d6): δppm 158.69 (C=O), 157.53 (C=O), 149.21 (C4'), 148.80 (=C), 140.65 (C-1'), 138.15 (C-1), 129.37 (C-2', C-6'), 129.03 (C-3, C-5), 125.34 (C-4), 124.75 (C-3', C-5'), 121.19 (C-2, C-6 ). Mass: m/z (%) 312 (M+, 33), 192 (100), 120 [30], 92 [30], 77 (41), 65 [12], 51 [7]. Anal. Calcd. for C15H12N4O4: C, 57.69; H, 3.87; N, 17.94. Found: C, 57.81; H, 3.65; N, 17.81.
2-[2-(2-Hydroxybenzylidene)hydrazinyl]-2-oxo-N-phenylacetamide (7k)
m.p = 253-266 °C (EtOH) [24], yield 90%, IR (KBr): vcm-1 3339 (OH, stretch), 3303, 3260 (NH, stretch), 1663 (C=O, stretch). 1H-NMR (500 MHz, DMSO-d6): δppm 12.65 (s, 1H, NH), 11.06 (s, 1H, OH), 10.87 (s, 1H, NH), 8.82 (s, 1H, N=CH), 7.86 (d, J = 7.7 Hz, 2H, H2, H6), 7.56 (dd, J = 7.7, 1.5, Hz, 1H, H6'), 7.39 (t, J = 7.7 Hz, 2H, H3, H5), 7.35-7.31 (m, 1H, H4), 7.17 (t, J = 7.5 Hz, 1H, H4), 6.95-6.92 (m, 2H, H3', H5'). 13C-NMR (125 MHz, DMSO-d6): δppm 158.67 (C=O), 158.23 (C-2'), 157.09 (C=O), 151.61 (=C), 138.18 (C-1), 132.62 (C-4'), 130.04 (C-6'), 129.38 (C-3, C-5), 125.30 (C-4), 121.14 (C-2, C-6), 120.09 (C-6'), 119.24 (C-1'), 117.11 (C-3'). Mass: m/z (%) 283 (M+, 63), 163 (100), 135[16], 120 (36), 93 (36), 77 (41), 65 [12], 51 [7]. Anal. Calcd. C15H13N3O3: C, 63.60; H, 4.63; N, 14.83. Found: C, 63.88; H, 4.75; N, 14.64.
2-[2-(Pyridin-2ylmethylidene)hydrazinyl]-2-oxo-N-phenylacetamide (7l)
m.p = 248-250 °C (EtOH), yield 94%, IR (KBr): vcm-1 3290 (NH, stretch), 1670 (C=O, stretch). 1H-NMR (250 MHz, DMSO-d6): δppm 12.72 (s, 1H, NH), 10.94 (s, 1H, NH), 8.73 (s, 1H, N=Ch), 8.67 (d, J = 4.7 Hz, 1H, H6'), 8.04 (d, J = 7.7 Hz, 1H, H3'), 7.93 (d, J = 7.7 Hz, 2H, H2, H6), 7.89-7.85 (m, 1H, H4), 7.50-7.39 (m, 3H, H3, H5, H5'), 7.19 (t, J = 7.3 Hz, 1H, H4). 13C-NMR (60 MHz, DMSO-d6): δppm 158.5 (C=O), 157.3 (C=O), 153.3 (C-2'), 151.7; (C-6'), 150.0 (=C), 137.9 (C-4'), 137.3 (C-1), 129.2 (C-3 and C-5), 125.2 (C-4), 125.1 (C-5'), 121.0 (C-2 and C-6), 120.1 (C-3'). Mass: m/z (%) 268 (M+, 6), 179 [4], 148 (100), 120 (74), 92 (96), 77 (52), 65 (59), 51 [30]. Anal. Calcd. for C14H12N4O2: C, 62.68; H, 4.51; N, 20.88. Found: C, 62.39; H, 4.14; N, 20.73.
2-[2-(Pyridin-3ylmethylidene)hydrazinyl]-2-oxo-N-phenylacetamide (7m)
m.p = 255-256 °C (EtOH), yield 92%, IR (KBr): vcm-1 3296, 3244 (NH, stretch), 1664 (C=O, stretch). 1H-NMR (250 MHz, DMSO-d6): δppm 12.59 (s, 1H, NH), 10.91 (s, 1H, NH), 8.89 (s, 1H, H2'), 8.72 (s, 1H, N=CH), 8.67 (d, J = 4.5 Hz, 1H, H6'), 8.18 (d, J = 6.5, 1H, H4'), 7.92 (d, J = 8.0 Hz, 2H, H2, H6), 7.54-7.49 (m, 1H, H5'), 7.41 (t, J = 8.0 Hz, 2H, H3, H5), 7.19 (t, J = 7.0 Hz, 1H, H4). 13C-NMR (60MHz, DMSO-d6): δppm 158.6 (c=O), 157.1 (C=O), 151.6 (C-6'), 149.4 (C-2'), 148.8 (=C), 138.0 (C-1), 134.3 (C-4'), 130.2 (C-3'), 129.2 (C-3 and C-5), 125.1 (C-5'), 124.5 (C-4), 121.0 (C-2 and C-6). Mass: m/z (%) 268 (M+, 17), 148 (100), 120 (93), 105 [19], 92 (58), 77 (80), 65 (36), 51 (37). Anal. Calcd. for C14H12N4O2: C, 62.68; H, 4.51; N, 20.88. Found: C, 63.02; H, 4.43; N, 21.05.
2-[2-(Pyridin-4ylmethylidene)hydrazinyl]-2-oxo-N-phenylacetamide (7n)
m.p = 288-290 °C (EtOH), yield 91%, IR (KBr): vcm-1 3303, 3255 (NH, stretch), 1697, 1661 (C=O, stretch). 1H-NMR (250 MHz, DMSO-d6): δppm 12.52 (s, 1H, NH), 10.78 (s, 1H, NH), 8.59 (d, J = 4.8 Hz, 2H, H2', H6'), 8.52 (s, 1H, N=CH), 7.76 (d, J = 7.8 Hz, 2H, H2, H6), 7.57 (d, J = 4.8 Hz, 2H, H3', H5'), 7.29 (t, J = 7.5 Hz, 2H, H3, H5), 7.07 (t, J = 7.3 Hz, 1H, H4). 13C-NMR (60 MHz, DMSO-d6): δppm 158.5 (C=O), 157.3 (C=O), 150.8 (C-2' and C-6'), 149.1 (=C), 141.4 (C-4'), 137.9 (C-1), 129.2 (C-3' and C-5'), 125.2 (C-4), 121.7 (C-3' and C-5'), 121.0 (C-2 and C-6). Mass: m/z (%) 268 (M+, 56), 148 (100), 120 (59), 92 (56), 77 (71), 65 [30], 51 (35). Anal. Calcd. for C14H12N4O2: C, 62.68; H, 4.51; N, 20.88. Found: C, 62.57; H, 4.70; N, 21.15.
Antiproliferative activity
The synthesized compounds (7a-7n) were assessed individually for their anti-proliferative activity against a panel of cancer cell lines (MDA-MB-231, Hep-G2, HT-29, and MCF-7) at a concentration of 50 μM following 72 h of incubation. The antiproliferative activity was determined using MTT assay which is based on the ability of viable cells to reduce tetrazolium bromide to formazan. Our preliminary screening demonstrated that almost all compounds exerted a weak to moderate anti-proliferative activity towards the tested cell lines, decreasing cell viability to lower than about 50% [Table 1]. Amongst them, compound 7k displayed remarkable efficacy with cell growth inhibition of more than 90% in MDA-MB-231 and HepG2 cells. Doxorubicin was used as a standard drug. DMSO was applied as the vehicle, which showed no significant effect on the viability of the cells. The MDA-MB-231 cell line was selected for further evaluation because not only it exhibited a desirable sensitivity towards compound 7k, but we could further verify our experiments in the established animal model of breast cancer. In the current study, IC50 values of compound 7k were determined on MDA-MB-231 cells at 24, 48, and 72 h [Table 2]. Since the animal model of breast cancer is established by inoculation of 4T1 cells into the mice, we also obtained the IC50 values of compound 7k on 4T1 cells and reported them in [Table 2]. Furthermore, this compound revealed viability of more than 50% even at the highest concentration used towards normal cells confirming a suitable selectivity index (SI greater than 6.5; [Figure 2].
Figure 2: The effect of compound 7k at different concentrations on the viability of HSF cells, after 72-h treatment, using MTT assay.
Table 1: Cell growth inhibition screening for compounds 7a-7n following treatment at 50 μM for 72 h, towards human cancer cell lines. a Values were determined in at least three independent experiments each performed in triplicate and expressed as mean ± SEM.
Table 2: IC50 values for the antiproliferative activity of compound 7k towards MDA-MB-231 and 4T1 cells. Values were determined in at least three independent experiments each performed in triplicate and expressed as mean ± SEM.Cell cycle arrest occurred by compound 7k
From the above-mentioned results, it was apparent that compound 7k treatment caused significant cell death against both MDA-MB-231 and 4T1 cell lines. Thus, to unveil whether it was owing to the cell cycle arrest, an analysis of the cell cycle distribution was carried out. Data from [Figure 3] indicated that in the MDA-MB-231 untreated control cells, 57.9% of the cells were in G0/G1 phase while treatment with compound 7k at 12 μM resulted in an increase in the cell population up to 67.75%. Notably, the percentage of the cells in this phase progressively augmented at higher concentrations (16 μM, 77.87%). In addition, following treatment with compound 7k, the cell population in the S phase decreased from 32.1% in the control group to 27.3% and 19.6% in the treated cells with 12 μM and 16 μM, respectively. Arrest in the G1/S phase was remarkable at concentrations of 12 and 16 μM. For 4T1 cells, the ratio of the cells in the sub-G1 phase significantly increased from 7.33% in the control/vehicle to 24.36% in the treated group with 8 μM, while no significant increment was observed at other used concentrations. Concomitantly, the number of 4T1 cells in the S phase augmented from 36.18% in the control cells to 46.63% in the 8 μM-treated cells. Although the percentage of cells in the G2/M phase did not alter following treatment with an 8 μM concentration of compound 7k, we speculate a probable S/G2 arrest [Figure 4].
Figure 3: Cell cycle distribution of (A) MDA-MB-231 cells and the cells treated with compound 7k at (B) 8 μM, (C) 12 μM, and (D) 16 μM for 72 h in G0/G1, S, and G2/M phases using flow cytometry after staining with propidium iodide (PI). (E)The data presented in the graph are the mean ± SEM of three independent experiments. **P < 0.01 and ***P < 0.001 indicate significant differences in comparison with the respective control/vehicle group.
Figure 4: Cell cycle distribution of (A) 4T1 cells and the cells treated with compound 7k at (B) 2 μM, (C) 4 μM, and (D) 8 μM for 72 h in G0/G1, S, and G2/M phases using flow cytometry after staining with propidium iodide (PI). (E) The data presented in the graph are the mean ± SEM of three independent experiments. *P < 0.05 and ***P < 0.001 indicate significant differences in comparison with the respective control/vehicle group. Discussion Currently, anticancer agents with different molecular mechanisms are applied in cancer treatment, though developing novel, effective, and promising agents are urgently required. In this context, the combination of hydrazone and α-ketoamide is expected to provide a novel design for the anticancer agents. Based on our literature survey, these hybrids have been linked to multiple biological functions such as anticancer effects [9],[21],[22]. Hence, to ascertain our speculation, a series of compounds (7a-7n) were synthesized and investigated in vitro to achieve an efficacious compound possessing an antiproliferative effect.
Structures of the synthesized hybrid molecules were determined by spectral analysis data. In the FT-IR spectra, two NH absorptions over 3000 cm-1 were detected. Due to the similarity of the carbonyl groups; i.e., both are amide types, two carbonyl peaks were seen in most of the compounds as one broad absorbance between 1650-1700 cm-1[31]. In 1H-NMR, two NH hydrogens were found at about 10.00 and 12.00 ppm and imine hydrogen was discovered close to 8.5 ppm implying the formation of an E isomer. In 13C-NMR, the carbonyl moieties were found in the range of 156-158 ppm and imine carbon was detected at about 147 to 151 ppm. At the outset of our project, we employed an MTT test on a panel of cancer cells to drive our optimization effort. Our preliminary screening returned compound 7k as a potent anti-proliferative agent. It showed proliferation inhibition of more than 90% at 72 h against the Hep-G2 and MDA-MB-231 cells at 50 μM as an initial concentration to elude the false-negative and false-positive effects at the lower and higher concentrations. By contrast, HT-29 and MCF-7 cell lines were substantially less sensitive to this compound. Interestingly, this compound demonstrated reasonable viability toward normal cells. To present a detailed structure-activity description, we introduced a number of substituents at various positions at the aromatic ring attached to the hydrazone moiety in order to improve the potency. Comparison of compound 7a with 7b, 7e, and 7h showed that insertion of electron-withdrawing groups at position 2 of the phenyl ring resulted in increasing the activity. The introduction of Cl at the meta position improves the activity but no advantage was observed by replacing it with Br. However, employing the polar and strong electron-accepting groups at the meta position led to a significant decrease in cell viability. The existence of electron-accepting moieties like Cl, Br, and NO2 at position 4 of the phenyl ring demonstrated a deteriorative effect on the potency. Furthermore, the bioisosteric replacement of phenyl with the pyridyl substituents gave rise to the formation of compounds 71 and 7m with negligible changes in potency and 7n with a dramatic reduction in activity when compared to 7a. Interestingly, compounds 7j and 7n bearing 4-nitrophenyl and 4-pyridyl and as bioisosteres exhibited the weakest potency in cell proliferation. These findings are in contrast to other studies indicating that the replacement of phenyl ring with heteroaryl groups like pyridine in hydrazone derivatives increased the antitumor potential [32]. Surprisingly, the presence of a strong polar electron donating group at position 2 led to compound 7k as the most active derivative. Overall, electron density at the phenyl ring of the hydrazone group or H-bond donating ability decreased the proliferative activity of the compounds. This conclusion is in agreement with the reports of Wu et al. and Cui et al. implying the anticancer-enhancing effect of 2-hydroxyl phenyl moiety in N-acyl hydrazone derivatives [33],[34]. Thus, we determined the IC50 value of this compound on MDA-MB-231 cells at 24, 48, and 72 h due to the concentration-dependent effect of the compound. It is noteworthy that a significant cell growth inhibition was also shown for compound 7k on 4T1 cells. These data not only verified the IC50 values on MDA-MB-231 cells but also provided a suitable background for our ongoing in vivo studies.
Seeking to understand the role of cell cycle arrest as an appealing approach in cancer therapy, we applied flow cytometry analysis to evaluate the mechanism of action of cell death induced by compound 7k. MDA-MB-231 cell subpopulations behaved differently based on the concentration of the compound. The histogram of 8 μM-treated cells was the same as that of the vehicle-treated cells, while treatment with 12 and 16 μM led to an increase in cell subpopulation at G1-phase and a decrease at S-phase, in comparison with vehicle-treated cells suggesting a G1/S arrest. Therefore, we propose that cell death induced by compound 7k is an event linked to cell cycle arrest.
ConclusionAltogether, we herein report the percentage of proliferation inhibition of a new series bearing oxamide and hydrazone moieties (7a- 7n) towards human cancer cell lines, among which compound 7k possessing 2-hydroxyphenyl moiety showed the greatest anti-proliferative effect against MDA-MB-231 cells at 72 h. Also, we suggest that cell death is also correlated with G1/S cell cycle arrest. Interestingly, this synthesized compound exhibited a concentration-dependent mode of action, making it likely to have great potency to treat cancer cells.
Acknowledgments
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
Conflict of interest statement
The authors declared no conflicts of interest in this study.
Authors’ contributions
M. Dehbid and R. Tahmasvand contributed to the biological experiments and data collection. M. Tasharofi and F. Shojaie synthesized the compounds. M. Aghamaali contributed to analyzing the data. A. Almasirad designed the structures, supervised the synthesis of the compounds and interprete the spectra of the compounds. M. Salimi wrote the manuscript, provided technical support, and supervised the whole project. The final version of the manuscript was approved by all authors.
References
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