Abstract
Eight Novel chalcones were synthesized and their structures were confirmed by different spectral tools. All the prepared compounds were subjected to SRB cytotoxic screening against several cancer cell lines. Compound 5c exerted the most promising effect against MCF7 and HEP2 cells with IC50 values of 9.5 and 12 µg/mL, respectively. Real-time PCR demonstrated the inhibitory effect of compound 5c on the expression level of Antigen kiel 67 (KI-67), Survivin, Interleukin-1beta (IL-1B), Interleukin-6 (IL-6), Cyclooxygenase-2 (COX-2) and Protein kinase B (AKT1) genes. Flow-cytometric analysis of the cell cycle indicated that compound 5c stopped the cell cycle at the G0/G1 and G2/M phases in MCF7 and HEP2 treated cells, respectively. ELISA assay showed that Caspase 8, Caspase 9, P53, BAX, and Glutathione (GSH) were extremely activated and Matrix metalloproteinase 2 (MMP2), Matrix metalloproteinase 9 (MMP9), BCL2, Malondialdehyde (MDA), and IL-6 were deactivated in 5c treated MCF7 and HEP2 cells. Wound healing revealed that chalcone 5c reduced the ability to close the scrape wound and decreased the number of migrating MCF7 and HEP2 cells compared to the untreated cells after 48 h. Theoretical molecular modeling against P53 cancer mutant Y220C and Bcl2 showed binding energies of -22.8 and -24.2 Kcal/mole, respectively, which confirmed our ELISA results.
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Introduction
Chalcones are found in conjugated form, with the keto-ethylenic system connecting the two rings (A and B) (Lemes et al. 2020). It is believed that these compounds' biological activity results from the double bond's conjugation with the carbonyl group. Research on chalcones is still in progress because of its many biological properties, which include anti-oxidant, (Bandgar et al. 2009; Shenvi et al. 2013) anti-bacterial, (Asiri and Khan 2011; Mohamed et al. 2012) antiviral, (Onyilagha et al. 1997) anti-platelet, (Lin et al. 2001) anti-cancer, (Sashidhara et al. 2010; Shenvi et al. 2013) anti-malarial, (Li et al. 1995) analgesic, (Heidari et al. 2009) and anti-inflammatory properties (Hsieh et al. 2000; Bekhit and Abdel-Aziem 2004). Moreover, thiophenes are sulfur-containing heterocycles that play an essential role in medicinal chemistry due to their wide range of biological applications, including anti-cancer, (Duddukuri et al. 2018) anti-microbial, (Kheder et al. 2008; Bondock et al. 2010) anti-inflammatory, (Helal et al. 2015) activities. Figure 1 shows some anti-cancer natural chalcones and anticancer agents that contain thiophene nuclei. Furthermore, it was noted that compounds having an acetamide linker or its substitutes as essential structures have gained substantial interest due to possible medicinal uses, including anti-oxidant (Ölgen et al. 2013), anti-cancer (Bhavsar et al. 2011; Khazir et al. 2020), analgesic (Yusov et al. 2019; Mikhailovskii et al. 2020), anti-microbial (Gull et al. 2016; Mikhailovskii et al. 2020; Yele et al. 2021), anti-inflammatory (Yusov et al. 2019), anti-urease (Gull et al. 2016), anti-tuberculosis (Borsoi et al. 2020), anti-convulsant (Severina et al. 2020), anti-COVID-19 (Mary et al. 2021), and anti-tubercular agents (Ang et al. 2012). Some acetamide derivatives have been shown to have analgesic or sedative characteristics, such as paracetamol (Rani et al. 2014), which is one of the most commonly used antipyretic and sedative drugs. In addition, AdipoRon, a phenoxyacetamide medication, has attracted great interest as a possible therapy for obesity, heart disease, diabetes, and non-alcoholic fatty liver (Akimoto et al. 2018). In light of these results and our ongoing research interest in the synthesis of bioactive heterocycles (A. Ibrahim et al. 1994; Barsoum et al. 1998; Elwahy and Abbas 2000; Elwahy et al. 2002; Ibrahim et al. 2004; Al-Awadi et al. 2007; Darwish et al. 2010; Mekky and Elwahy 2014; Ghozlan et al. 2015; Sayed et al. 2016; Ibrahim et al. 2018; Mohamed et al. 2018; Sroor et al. 2019; Fathi et al. 2021; Helmy et al. 2022; WalyEldeen et al. 2023; Abdelwahab et al. 2024; Abdullah et al. 2024; Elwahy et al. 2024a; Ragheb et al. 2024a; Saleh et al. 2024; Salem et al. 2024a, b; Ragheb et al. 2024b; Elwahy et al. 2024b) we were motivated to synthesize novel chalcones incorporating 2-phenoxy-N-arylacetamide and thiophene moieties and evaluate their in vitro anti-cancer efficacy against different human cancer cell lines (Scheme 1).
Chemical structures of some naturally occurring chalcones and marketed drug containing thiophene nucleus
Design strategy of the synthesized compounds
Results and Discussion
The 2-(4-formylphenoxy)-N-arylacetamide precursors 3a–d (Omar et al. 2021; Abdelwahab et al. 2023) were produced by the alkylation reaction involving 4-hydroxybenzaldehyde 2 with the corresponding 2-chloro-N-arylacetamide 1 in the presence of KOH, as indicated in Scheme 2. The formation of chalcone incorporating 2-phenoxy-N-arylacetamide, (2-(4-(3-oxo-3-(thiophen-2-yl)prop-1-en-1-yl)phenoxy)-N-arylacetamide 5a–d was due to of the Claisen-Schmidt condensation reaction of precursors 3a–d with the mole equivalent of 1-(thiophen-2-yl)ethan-1-one 4 in ethanol in the presence of KOH at reflux. In the formed chalcones 5a–d, thiophene represents A-ring, while 2-phenoxy-N-arylacetamide represents B-ring (as shown in scheme 3). The constitutions of the resulting products were verified based on spectral data.
Synthesis of 2-(4-(3-oxo-3-(thiophen-2-yl)prop-1-en-1-yl)phenoxy)-N-arylacetamides 5a–d
Synthesis of N-aryl-2-(4-(3-(thiophen-2-yl)acryloyl)phenoxy)acetamides 9a–d
Motivated by the results obtained in scheme 2, we prepared the isomeric N-aryl-2-(4-(3-(thiophen-2-yl)acryloyl)phenoxy)acetamides 9a–d, in which thiophene represents B-ring, while 2-phenoxy-N-arylacetamide represents A-ring. The 2-(4-acetylphenoxy)-N-arylacetamide precursors 7a–d, were prepared via the alkylation reaction of 4-hydroxyl acetophenone 6 with the corresponding 2-chloro-N-arylacetamides 1 in the presence of KOH as shown in scheme 3. Claisen-Schmidt condensation reaction of 2-(4-acetylphenoxy)-N-arylacetamide precursors 7a–d (Abdullah 2023) with the mole equivalent of thiophene-2-carbaldehyde 8 in ethanol in the presence of KOH at reflux resulted in the formation of 9a–d (as shown in Scheme 3).
A spectrochemical study confirmed the chemical structures of the new compounds 5a–c and 9a–c. The IR spectra of compound 9b, as a sample example, revealed the existence of the carbonyl band at 1691 and 1666 cm-1. The mass spectra for 9b also revealed the right molecular ion peak at m/z 377. Furthermore, in 9b's 1H NMR spectra, two singlet signals at 2.27 ppm and 4.83 ppm, were assigned to tolyl CH3 and OCH2, respectively. The structure of the compound was assigned as trans configuration as it revealed to doublets at 7.57 and 7.90 ppm with coupling constant, J 20 Hz. At 10.03 ppm, amide-NH appeared as a broad signal. The chemical shifts of all other protons and carbons were exactly as expected.
Cytotoxic assay
Primary screening
All the prepared compounds were screened at a concentration of 100 µg/mL against six different human cell lines: human laryngeal carcinoma (HEP2), human colorectal carcinoma (HCT116), human breast carcinoma (MCF7), human Lung carcinoma (A549), human liver carcinoma (HEPG2), and normal African Green monkey kidney cell line (VERO). Doxorubicin was used as a positive control for comparison purposes. Table 1 showed that compounds 5c and 9a exerted the most promising activity against MCF7 and HEP2 cells which showed % inhibition of more than 75%. Also, compounds 5c and 9a showed an inhibition of 58% against the VERO normal cell line. The remaining compounds did not show cytotoxic activity against all the studied cancer cell lines which inhibited lower than 50% of cancer cells (Table 1). So, secondary screening was performed on the most effective compounds (5c and 9a) to determine their IC50 values.
Secondary screening
As shown in Fig. 2 and Table 2, E)-N-(4-Methoxyphenyl)-2-(4-(3-oxo-3-(thiophen-2-yl)prop-1-en-1-yl)phenoxy)acetamide 5c in which the N-arylacetamide has electron-donating methoxy group on B-ring 5c exerted better activity than compound 9a against MCF7 and HEP2 cells. In terms of IC50 values, compound 5c displayed 12 and 9.5 µg/mL against HEP2 and MCF7 cells, which were very comparable to that of doxorubicin (11 and 5.5 µg/mL), respectively. In addition, compound 5c was better than two reported chalcone derivatives ((E)-1-(4-nitrophenyl)-3-(4-(hexyloxy)phenyl) prop-2-en-1-one and (E)-1-(4-cyanophenyl)-3-(4-(hexyloxy)phenyl) prop-2-en-1-one) as they exerted cytotoxic effect against MCF7 with IC50 values of 14.75 and 13.75 µg/mL, respectively (Khairul et al. 2020). While, compound 9a exerted 15.5 and 24.5 µg/mL against HEP2 and MCF7, respectively. Therefore, subsequent molecular studies were conducted on compound 5c against MCF7 and HEP2 cells.
Impact of 5c and 9a on the viability of MCF7 and HEP2 cells using SRB assay. GraphPad Prism, version 5, was used to create graphs and analyze the data. The results are expressed as the mean ± SD of 3 separate experiments performed in 5 replicates
RT-PCR
The expression level of the following six genes (Ki-67, Survivin, Interleukin-1 beta (IL-1B), Interleukin-6 (IL-6), Cyclooxygenase-2 (COX-2) and protein kinase B (AKT1)) was determined in MCF7 and HEP2 treated with the IC50 of compound 5c. The untreated cells were used as a negative control for comparison purposes. Data illustrated in Table 3 showed that chalcone 5c had a significant inhibitory effect on the expression of these genes in MCF7 and HEP2-treated cells relative to their untreated control cells. Ki-67 is a popular proliferation marker for human tumor cells. It presents in all active phases of the cell cycle and its cellular distribution substantially alters as the cell cycle advances (Luo et al. 2021) It was down-regulated by 5c in MCF7 and HEP2 cells with fold changes of 0.352±0.0172 and 0.648±0.015, respectively, relative to their controls. Survivin is an inhibitor of apoptotic protein (IAP) family member, which is essential for cell division. The lowering of the expression of survivin inhibited tumor development, triggered apoptosis, and made tumor cells more susceptible to radiation and chemotherapy (Albadari and Li 2023). It was found that 5c decreased the expression level of survivin in MCF7 and HEP2 cells with values (0.632±0.0618 and 0.489±0.07), respectively, relative to their controls. IL-1B is a pleiotropic mediator of inflammation, which is produced in response to a variety of stressors (Aarreberg et al. 2019). Herein, it was down-regulated in 5c treated MCF7 and HEP2 cells with values (0.4008±0.116 and 0.461±0.069) respectively, relative to their controls. IL-6 is a pro-inflammatory cytokine that plays a significant role in the proliferation and differentiation of human cells. It promotes the production of numerous proteins involved in acute inflammation (Uciechowski and Dempke 2020). It was found that 5c lowered the expression level of IL-6 in MCF7 and HEP2 cell lines with values (0.814±0.079 and 0.464±0.08), respectively relative to their untreated control cells. COX-2 is a vital physiological enzyme that is essential for many biological processes, particularly in the mechanisms involved in pain and inflammation. The overexpression of COX-2 is found to be related to inflammatory processes and cancer (Sharma et al. 2019). It was showed that 5c down-regulated COX-2 in MCF7 and HEP2 treated cells with approximately similar values (0.502±0.073 and 0.547±0.0321), respectively, relative to their controls. AKT1 has a critical role in influencing several pathways, including preventing apoptosis, promoting cell proliferation, and altering cellular metabolism (Ghafouri-Fard et al. 2022). Chalcone 5c greatly decreased the expression level of AKT1 in MCF7 and HEP2 treated cells with fold changes (0.402±0.068 and 0.381±0.011), respectively, relative to their controls. So, the inhibitory effect of chalcone 5c on the expression level of KI-67, Survivin, IL-1B, IL-6, COX-2, and AKT1 promoted the apoptotic death and blocked the inflammation in MCF7 and HEP2 cells.
ELISA assay
The concentrations of matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9), Bcl2, BAX, P53, Malondialdehyde (MDA), Glutathione (GSH), caspase 8, caspase 9 and IL-6 were measured quantitatively in 5c treated MCF7 and HEP2 cells using ELISA (Enzyme-Linked Immunosorbent Assay) (Table 4). Gelatinases are a collective word for MMP-2 and MMP-9, where MMP-2 is gelatinase-A and MMP-9 is gelatinase-B. Solid tumor invasion, metastasis, and angiogenesis have long been linked to gelatinases (Das et al. 2021). Our study showed that chalcone 5c decreased the activity of MMP2 and MMP9 in MCF7 and HEP2 cells with values (185.125 and 142.5 pg/mL), respectively, for MMP2 and (271.6 and 236.5 ng/mL), respectively for MMP9 relative to their controls. Bcl2 is a crucial protein that targets apoptosis inhibition. It is regarded as a predictive biomarker or therapeutic target in the diagnosis of cancer due to its broad expression in a variety of cancers (Porter et al. 2009). It was found that Bcl2 was deactivated in MCF7 and HEP2 cells treated with chalcone 5c with values (2089.96 and 1809.34 pg/mL), respectively, relative to their controls. The pro-apoptotic protein BAX permeabilizes the mitochondrial outer membrane by changing from a cytosolic monomer to a hazardous oligomer triggering the apoptosis process (Fulda and Debatin 2006). Herein, 5c increased the concentration of BAX in MCF7 and HEP2 cells with values (1099.36 and 1342.8 pg/mL), respectively, relative to their controls. P53 transcription factor performs an essential tumor suppressor role by coordinating a wide range of physiological responses, including DNA repair, cell cycle arrest, cellular senescence, cell death, cell differentiation, and metabolism (Liebl and Hofmann 2021). It was activated in MCF7 and HEP2-treated cells with values (4.28 and 3.109 ng/mL), respectively, compared to their controls (3.44 and 2.06 ng/mL). MDA is the main biomarker for determining lipid peroxidation. MDA is a final product of polyunsaturated fatty acids (PUFAs) peroxidation, either through healthy or pathological enzyme- or non-enzyme-catalyzed processes (Chakravarty and Rizvi 2011). Our study demonstrated that 5c decreased the concentration of MDA in MCF7 and HEP2 cells with values (8.79 and 11.988 nM), respectively, relative to their controls. GSH is an anti-oxidant that performs a variety of physiological tasks, such as scavenging free radicals, fighting oxidation, and getting rid of electrophiles (Chakravarty and Rizvi 2011). When the ROS/GSH equilibrium is upset, bio-macromolecules are negatively oxidized and chemically modified, which ultimately causes cell cycle arrest, proliferation inhibition, and even cell death. A direct rise in ROS may lead to an imbalanced ROS/GSH ratio. GSH was extremely activated in MCF7 and HEP2 cells treated with 5c with values (0.939 and 0.821 ng/mL), respectively, compared to their controls. The extrinsic apoptotic process is often triggered by caspase-8, a cysteine-aspartate-specific protease when cell surface death receptors (DRs) like FAS, TRAIL-R, and TNF-R are activated (Mandal et al. 2020). In addition to its activities in death receptor-mediated apoptosis, Caspase-8 also inhibits a different type of programmed cell death known as necroptosis, which is an inflammatory cell death (Mandal et al. 2020). It was found that 5c strongly activated caspase-8 in MCF7 and HEP2 cell lines with values (2.13 and 2.27 ng/mL), respectively, relative to their untreated control cells. Caspase-9 is a crucial component of the intrinsic or mitochondrial apoptotic pathway, which is activated by a variety of stimuli such as chemotherapy, stress medications, and radiation (Li et al. 2017). Herein, caspase-9 was up-regulated in MCF7 and HEP2 cell lines treated with 5c with values (28.03 and 27.54 ng/mL), respectively, relative to their controls. Also, it was found that IL-6 was deactivated in MCF7 and HEP2-treated cells with values (74.68 and 54.27 pg/mL), respectively, as compared to their controls. This result supported the downregulating effect of 5c on the expression level of IL-6 as mentioned in the RT-PCR section. So, Caspase 8, Caspase 9, P53, BAX, and GSH were extremely activated and MMP2, MMP9, BCL2, MDA, and IL-6 were deactivated in 5c treated MCF7 and HEP2 cells. From the above results, we could suggest that compound 5c triggered both intrinsic and extrinsic pathways of apoptosis in MCF7 and HEP2 cells. Also, it could inhibit invasion, metastasis, and inflammation and had anti-oxidant activity in treated MCF7 and HEP2 cells.
Flow cytometric analysis of cell cycle
Compound 5c induces cell cycle arrest at the G0-G1 phase in MCF-7 cells
The percentage of cells in the G0-G1 phase increased from 45.5% in the untreated control MCF7 cells to 54.3% in the 5c-treated cells, as shown in Table 5 and Fig. 3. In the G2-M and S phases, fewer 5c-treated cells were seen at 2.8% and 42.84%, respectively, compared to the untreated control with 8.9% and 45.4%, and this was compatible with the results obtained by the literature (Gao et al. 2020) where Xanthohumol caused cell cycle arrest at G0-G1phase.
Distribution of cells in different phases of the cell cycle after the treatment of MCF7 and HEP2 cells with the IC50 value of 5c relative to the untreated control cells
Compound 5c induces cell cycle arrest at the G2/M phase in HEP2 cells
According to Table 5 and Fig. 3, the percentage of cells in the G2/M phase increased from 2.2% to 3.56% in the 5c-treated cells compared to the untreated control HEP2 cells, and the percentage of cells in the S phase increased slightly from 40.09% to 40.96%. In the G0-G1 stage, the percentage of 5c-treated cells declined to 55.4% as opposed to 57.6% of untreated control cells. It was found in a previous work that lutein-induced G2/M phase arrest in A549 and PC-9 cells (Di et al. 2019).
Compound 5c inhibits MCF7 and HEP2 cells migration
To examine the effect of chalcone 5c on the migration properties of the MCF7 and HEP2 cells, the wound-healing scratch assay was used. The untreated MCF7 and HEP2 control (C) cells generally displayed wound recovery within 48 h and migration of cells to the wound (Fig. 4 and Table 6). Chalcone 5c at its IC50 reduced the ability to close the scrape wound and decreased the number of migrating MCF7 and HEP2 cells compared to the untreated cells (Fig. 4 and Table 6). The scratch gap percentage in 5c treated MCF7 was 344.684±41.224 relative to the control cells (160.647±61.276) after 48 h of treatment. The scratch gap in HEP2 treated with 5c was 567.281±112.789 compared to the untreated control HEP2 cells (211.604±79.883) after 48 h. This result supported the lowering effect of 5c on the concentration of MMP-2 and MMP-9 in MCF7 and HEP2 cells as shown in the ELISA section. Our result coincided with the result of Luo et al (Luo et al. 2021), where two ligustrazine-chalcone hybrids (compounds 6c and 6f, therein) inhibited significantly the migration of MDA-MB-231 and MCF-7 cells in a concentration-dependent manner.
Evaluation of migration capacity using a wound‑healing assay following compound 5c treatment for 48 h in MCF7 and HEP2 cells
Molecular docking
The molecular docking study was done on the promising compound 5c against P53 cancer mutant Y220C and Bcl2 proteins. As shown in Table 7, the values of the binding energy of the studied compound 5c were -22.8 and -24.2 Kcal/mole, respectively, which were more negative and better than that of the standard co-crystallized ligand (-15.8 and -21.83 Kcal/mole), respectively. The root mean squared deviations (RMSDs) were 0.7 and 2.5 for P53 cancer mutant Y220C and Bcl2, respectively. Compound 5c interacted with the active site of P53 cancer mutant Y220C via 9 interactions (Fig. 5a). These interactions included one carbon-hydrogen bond between the hydrogen of the methylene moiety and CYS:220 with a bond distance of 4.43 A°; a conventional hydrogen bond between the oxygen of the carbonyl group and ARG:202 with a bond distance of 6.37 A°; one pi-cation electrostatic interaction between the benzene ring and ARG:202 with bond distance 7.02 A°; one pi-sulfur electrostatic interaction between the thiophene ring and CYS:220 with bond distance 5.77 A°; and five pi-alkyl hydrophobic interactions with PRO:222, PRO:153, PRO:223, PRO:151 and VAL:147 residues. Compound 5c interacted with the active site of Bcl2 through six interactions (Fig. 5b). These interactions included three carbon-hydrogen bonds with ALA: 100, ASP: 103, and TYR: 108 with bond distances 3.75, 4.39, and 5.40 A°, respectively; and 3 pi- alkyl hydrophobic interactions with LEU: 137, ALA: 149, and ARG: 146 with bond distances 4.53, 7.59, and 4.95 A°, respectively. Figure 6a showed the interaction of 3-iodanyl-2-oxidanyl-5-propylsulfanyl-4-pyrrol-1-yl-benzoicacid (standard ligand) with P53 cancer mutant Y220C which revealed one conventional hydrogen bond with THR: 150 with bond distance 2.04 A°; carbon-hydrogen bond with GLU: 221; and twelve hydrophobic interactions including (amide pi-stacked, alkyl, pi-alkyl and halogen). Figure 6b demonstrated eleven interactions between Bcl2 and its co-crystallized ligand which included four hydrogen bonds; four electrostatic; and three hydrophobic interactions. So, the above results indicated that compound 5c had an activating effect on P53 mutant Y220C and an inhibitory effect against Bcl2 anti-apoptotic protein, and this assumption coincided with our results in the above ELISA assay section.
Two-dimensional and three-dimensional mode of interaction of compound 5c with the active site of a) P53 cancer mutant Y220C; b) Bcl2
Two-dimensional and three-dimensional mode of interaction of the standard ligands with the active site of a) P53 cancer mutant Y220C; b) Bcl2
Experimental
Chemistry
“Melting points were measured with a Stuart melting point apparatus and were uncorrected. The IR spectra were recorded using an FTIR Bruker–vector 22 spectrophotometer as KBr pellets. The 1H and 13C NMR spectra were recorded in DMSO as a solvent on a Brucker spectrometer (400 MHz) using TMS as an internal standard. Chemical shifts are reported as δ values in ppm. Mass spectra were recorded with a Shimadzu GCMS–QP–1000 EX mass spectrometer in an EI (70 eV) model. The elemental analyses were performed at the Microanalytical Center, Cairo University.
General procedures for the synthesis of chalcones 5a-d and 9a-d
A solution consisting of aldehydes (3a–d or 8) or acetyl derivatives (4 or 7a–d) (1 mmol) had been dissolved in ethanol (20 mL). The solution of potassium hydroxide (20%, 5 ml) was then added to this mixture at 0-5 °C. The reaction mixture was stirred regularly for 5 hours at room temperature and then transferred over HCl-containing ice. The resulting yellow solid was filtered, rinsed with water, and dried. The crude product was crystallized using EtOH-Dioxane to produce yellow crystals of chalcones 5a–d and 9a–d.
2-(4-(3-Oxo-3-(thiophen-2-yl)prop-1-en-1-yl)phenoxy)-N-phenylacetamide (5a)
Yellow crystals (85%); mp 194-196 °C; IR (KBr): ν 3366 (NH), 1694 (C=O ketone), 1660 (C=O amide) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 4.80 (s, 2H, CH2), 7.09 – 7.11 (m, 3H, Ar-H + vinyl-H + thiophene-H), 7.31-7.36 (m, 3H, Ar-H + vinyl-H), 7.64 (d, 2H, Ar-H, J = 8Hz), 7.73 (d, 2H, Ar-H, J = 12Hz), 7.87 (d, 2H, Ar-H, J = 12Hz), 8.04 (m, 1H, thiophene-H), 8.30 (m, 1H, thiophene-H),10.14 (s, 1H, NH) ppm; 13C NMR (101 MHz, DMSO-d6): δ 67.6 (-OCH2CO-), 115.6, 120.1, 120.2, 124.2, 128.2, 129.2, 129.3, 131.2, 133.8, 135.7, 138.8, 143.4, 146.2, 160.4 (ArC-O-CH2), 166.6 (-NHCO), 182.0 (-CO) ppm; MS (EI, 70 eV): m/z 363 [M]+; Anal. Calcd for C21H17NO3S: C, 69.40; H, 4.72; N, 3.85%. Found: C, 69.26; H, 4.63; N, 3.77%.
2-(4-(3-Oxo-3-(thiophen-2-yl)prop-1-en-1-yl)phenoxy)-N-(p-tolyl)acetamide (5b)
Yellow crystals (84%); mp 202-204 °C; IR (KBr): ν 3362 (NH), 1697 (C=O ketone), 1661 (C=O amide) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 2.23 (s, 3H, CH3), 4.83 (s, 2H, CH2), 7.13 – 7.21 (m, 5H, Ar-H + thiophene-H), 7.53 (d, 2H, Ar-H, J = 8Hz), 7.56 (d, 1H, vinyl-H, J = 20Hz), 7.69 (d, 1H, thiophene-H, J = 4Hz), 7.77 (d, 1H, thiophene-H, J = 4Hz), 7.87 (d, 1H, vinyl-H, J = 20Hz), 8.12 (d, 2H, Ar-H, J = 8Hz), 10.11 (s, 1H, NH) ppm; MS (EI, 70 eV): m/z 377 [M]+; Anal. Calcd for C22H19NO3S: C, 70.01; H, 5.07; N, 3.71%. Found: C, 69.91; H, 5.02; N, 3.60%.
N-(4-Methoxyphenyl)-2-(4-(3-oxo-3-(thiophen-2-yl)prop-1-en-1-yl)phenoxy)acetamide. (5c)
Green crystals (82%); mp 208-210 °C; IR (KBr): ν 3345 (NH), 1692 (C=O ketone), 1659 (C=O amide) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 3.73 (s, 3H, OCH3), 4.77 (s, 2H, CH2), 6.90 – 6.95 (m, 3H, Ar-H + vinyl-H ), 7.09-7.13 (m, 2H, vinyl-H + thiophene-H), 7.54 (d, 2H, Ar-H, J = 12Hz), 7.73 (d, 2H, Ar-H, J = 12Hz), 7.87 (d, 2H, Ar-H, J = 12Hz), 8.04 (d, 1H, thiophene-H, J = 4Hz), 8.30 (d, 1H, thiophene-H, J = 4Hz), 10.0 (s, 1H, NH) ppm; MS (EI, 70 eV): m/z 393 [M]+; Anal. Calcd for C22H19NO4S: C, 67.16; H, 4.87; N, 3.56%. Found: C, 67.08; H, 4.74; N, 3.49%.
N-(4-Chlorophenyl)-2-(4-(3-oxo-3-(thiophen-2-yl)prop-1-en-1-yl)phenoxy)acetamide. (5d)
Yellow crystals (78%); mp 198-200 °C; IR (KBr): ν 3337 (NH), 1695 (C=O ketone), 1655 (C=O amide) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 4.80 (s, 2H, CH2), 1H NMR (400 MHz, DMSO-d6): δ 4.83 (s, 2H, CH2), 7.07 (d, 2H, Ar-H, J = 12Hz), 7.29-7.32 (m, 1H, thiophene-H), 7.40 (d, 2H, Ar-H, J = 12Hz), 7.66-7.73 (m, 4H, Ar-H + 2 vinyl-H), 7.85 (d, 2H, Ar-H, J = 12Hz), 8.04 (d, 1H, thiophene-H, J = 4Hz), 8.28 (d, 1H, thiophene-H, J = 4Hz), 10.24 (s, 1H, NH) ppm; MS (EI, 70 eV): m/z 399 [M+2]+, 397 [M]+; Anal. Calcd for C21H16ClNO3S: C, 63.39; H, 4.05; N, 3.52%. Found: C, 63.22; H, 3.91; N, 3.37%.
N-Phenyl-2-(4-(3-(thiophen-2-yl)acryloyl)phenoxy)acetamide (9a)
Yellow crystals (86%); mp 196-198 °C; IR (KBr): ν 3358 (NH), 1689 (C=O ketone), 1656 (C=O amide) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 4.86 (s, 2H, CH2), 7.08 – 7.21 (m, 4H, Ar-H + thiophene-H), 7.32-7.36 (m, 2H, Ar-H), 7.56 (d, 1H, + vinyl-H, J = 16Hz), 7.64 (d, 2H, Ar-H, J = 8Hz), 7.68 (d, 1H, thiophene-H, J = 4Hz), 7.77 (d, 1H, thiophene-H, J = 4Hz), 7.87 (d, 1H, + vinyl-H, J = 16Hz), 8.12 (d, 2H, Ar-H, J = 8Hz), 10.18 (s, 1H, NH) ppm; 13C NMR (101 MHz, DMSO-d6): δ 67.6 (-OCH2CO-), 115.2, 120.2, 120.8, 124.2, 129.1, 129.2, 130.6, 131.2, 131.4, 133.0, 136.5, 138.8, 140.3, 162.2 (ArC-O-CH2), 166.5 (-NHCO), 187.4 (-CO) ppm; MS (EI, 70 eV): m/z 363 [M]+; Anal. Calcd for C21H17NO3S: C, 69.40; H, 4.72; N, 3.85%. Found: C, 69.29; H, 4.65; N, 3.73%.
2-(4-(3-(Thiophen-2-yl)acryloyl)phenoxy)-N-(p-tolyl)acetamide (9b)
Yellow crystals (85%); mp 200-202 °C; IR (KBr): ν 3354 (NH), 1691 (C=O ketone), 1666 (C=O amide) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 2.27 (s, 3H, CH3), 4.83 (s, 2H, CH2), 7.13 – 7.22 (m, 5H, Ar-H + thiophene-H), 7.55 (d, 2H, Ar-H, J = 8Hz), 7.57 (d, 1H, vinyl-H, J = 20Hz), 7.70 (d, 1H, thiophene-H, J = 4Hz), 7.78 (d, 1H, thiophene-H, J = 4Hz), 7.90 (d, 1H, vinyl-H, J = 20Hz), 8.13 (d, 2H, Ar-H, J = 8Hz), 10.11 (s, 1H, NH) ppm; 13C NMR (101 MHz, DMSO-d6): δ 20.9 (Me), 67.6 (-OCH2CO-), 115.2, 120.2, 120.8, 129.1, 129.6, 130.6, 131.2, 131.3, 133.0, 133.2, 136.3, 136.5, 140.3, 162.2 (ArC-O-CH2), 166.2 (-NHCO), 187.4 (-CO) ppm; MS (EI, 70 eV): m/z 377 [M]+; Anal. Calcd for C22H19NO3S: C, 70.01; H, 5.07; N, 3.71%. Found: C, 69.89; H, 4.95; N, 3.58%.
N-(4-Methoxyphenyl)-2-(4-(3-(thiophen-2-yl)acryloyl)phenoxy)acetamide (9c)
Green crystals (83%); mp 205-207 °C; IR (KBr): ν 3361 (NH), 1697 (C=O ketone), 1667 (C=O amide) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 3.74 (s, 3H, OCH3), 4.81 (s, 2H, CH2), 6.90 (d, 2H, Ar-H, J = 8Hz), 7.14 (d, 2H, Ar-H, J = 8Hz), 7.20 (m, 1H, thiophene-H), 7.54-7.60 (m, 3H, Ar-H + vinyl-H), 7.68 (d, 1H, thiophene-H, J = 4Hz), 7.77 (d, 1H, thiophene-H, J = 4Hz), 7.87 (d, 1H, vinyl-H, J = 16 Hz), 8.12 (d, 2H, Ar-H, J = 8Hz),10.03 (s, 1H, NH) ppm; 13C NMR (101 MHz, DMSO-d6): δ 55.6 (OMe), 67.6 (-OCH2CO-), 114.3, 115.2, 120.8, 121.8, 129.1, 130.6, 131.2, 131.3, 131.8, 133.0, 136.5, 140.3, 156.1, 162.2 (ArC-O-CH2), 166.0 (-NHCO), 187.4 (-CO) ppm; MS (EI, 70 eV): m/z 393 [M]+; Anal. Calcd for C22H19NO4S: C, 67.16; H, 4.87; N, 3.56%. Found: C, 67.06; H, 4.80; N, 3.44%.
N-(4-Chlorophenyl)-2-(4-(3-(thiophen-2-yl)acryloyl)phenoxy)acetamide (9d)
Yellow crystals (77%); mp 193-195 °C; IR (KBr): ν 3362 (NH), 1699 (C=O ketone), 1661 (C=O amide) cm-1; 1H NMR (400 MHz, DMSO-d6): δ 4.86 (s, 2H, CH2), 7.14 (d, 2H, Ar-H, J = 8Hz), 7.19-7.21 (m, 1H, thiophene-H), 7.39 (d, 2H, Ar-H, J = 8Hz), 7.56 (d, 1H, vinyl-H, J = 16 Hz), 7.76-7.70 (m, 3H, Ar-H), 7.77 (d, 1H, thiophene-H, J = 8Hz), 7.87 (d, 1H, vinyl-H, J = 16 Hz), 8.12 (d, 2H, Ar-H, J = 8Hz),10.31 (s, 1H, NH) ppm; 13C NMR (101 MHz, DMSO-d6): δ 67.5 (-OCH2CO-), 115.2, 120.8, 121.7, 127.9, 129.1, 130.6, 131.2, 131.4, 132.6, 133.0, 136.5, 137.8, 140.3, 162.1 (ArC-O-CH2), 166.7 (-NHCO), 187.3 (-CO) ppm; MS (EI, 70 eV): m/z 399 [M+2]+, 397 [M]+; Anal. Calcd for C21H16ClNO3S: C, 63.39; H, 4.05; N, 3.52%. Found: C, 63.25; H, 4.01; N, 3.41%.
Cytotoxic Sulforhodamine-B (SRB) assay
The human laryngeal carcinoma (HEP2), colorectal carcinoma (HCT116), breast carcinoma (MCF7), Lung carcinoma (A549), liver carcinoma (HEPG2), and normal African Green monkey kidney cell line (VERO) were purchased from American Tissue Culture Collection (Rockville, MD, USA). The cells were treated for 48 h with a single dose (100 µg/mL) of all the tested chalcones. Then the IC50 was calculated for the most active compound 5c against MCF7 and HEP2 cells using different concentrations (50, 25, 12.5, 6.25, 0.0 µg/mL). In brief, the cells were seeded in a 96-well microtiter plate at a concentration of 5×103 cells/well in 100 µL fresh RPMI-1640 medium and left to attach to the plates for 24 h. Then, cells were incubated with 100 μL of different concentrations (50, 25, 12.5, 6.25, and 0.0 µg/mL) of 5c in triplicate at 37 °C for 48 h. After that, the cells were fixed with 10 µL cold 100% Trichloroacetic acid (TCA) for 1h at 4 ºC. The wells were then washed 1 time with distilled water using (automatic Tecan washer, Germany) and stained for 30 min at room temperature with 50 µL 0.4% SRB dissolved in 1% acetic acid. The plates were air-dried and the dye was solubilized with 100 µl/well of 1M tris base (pH 10.5) for 5 min. The optical density (O.D.) of each well was measured spectrophotometrically at 570 nm with an ELISA microplate reader (Sunrise Tecan reader, Germany) with automatic shaking for 30 seconds before reading. The mean background absorbance was subtracted automatically and mean values for each drug concentration were calculated. The percentage of cell survival was calculated as follows: Survival fraction= O.D. (treated cells)/ O.D. (control cells) (Bhat et al. 2023).
Real-time PCR
The expression level of the following six genes (Ki-67, survivin, AKT1, IL-6, COX2, and IL-1B) was examined using real-time polymerase chain reaction (qPCR) (Mohamed et al. 2023a). Total RNA was extracted from the treated and control samples using the RNeasy Mini Kit from Qiagen in Valencia (catalog#74104). cDNA synthesis was carried out using the High-capacity cDNA kit (Applied Biosystem, California, USA, Catalog #4368814) following the manufacturer's instructions. The qPCR was carried out following the manufacturer's instructions using the Promega GoTaq qPCR master mix (Madison, USA, Catalog# A6001). 25 µl of master mix, 0.25 µl of Carboxy-X-Rhodamine (CXR) reference dye, 1 µl of forward and reverse primers, 1µl of cDNA, and 50 µl of total volume was completed. Table 8 shows the sequences of the primers used. All analyses were carried out in triplicate on a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using a protocol that included an initial denaturation step at 95 °C for 10 minutes, followed by 40 cycles of denaturation at 95 °C for 15 seconds, and annealing at 62 °C for 1 minute. The cycle threshold (Ct) was determined automatically. Analysis of data was performed by using the ∆∆Ct method (Livak and Schmittgen 2001). Values were presented as relative expression levels and normalized to GAPDH.
ELISA
ELISA colorimetric assay was used to determine the concentration of the following proteins (BAX, BCL2, P53, Caspase8, Caspase9, MMP2, MMP9, GSH, MDA and IL-6) using the following colorimetric detection kits Cloud-Clone Corp, USA, North America catalog # (SEB343Mu, SEA778Ra, SEH009Hu, MBS452285, MBS2533895, MBS670150, MBS175780, CEA294Ge, CEA597Ge and SEA079Ra, respectively). In brief, the instructions were described in the above kits as followed (Mohamed et al. 2023c): 100 μL of standard, blank, and samples were added into the appropriate wells, covered with the plate sealer, and incubated for 1 hour at 37°C. After removing the liquid from each well, 100 μL of the prepared reagent A working solution was added to each well, and incubated for 1 hour at 37 °C. The solution was aspirated and washed three times with 350 μL of 1X Wash Solution to each well using a squirt bottle, and multi-channel pipette. Then, 100 μL of detection reagent B working solution was added to each well, and incubated for 30 minutes at 37 °C. 90 μL of substrate solution was added to each well, covered with a new plate sealer, and incubated for 10 - 20 minutes at 37 °C in the dark. Finally, 50 μL of stop solution was added to each well. The liquid turned yellow with the addition of a stop solution. The microplate reader was run and conducted the measurement at 450 nm. A standard curve was plotted to calculate the concentrations of the unknown samples and the controls.
Flow cytometric assay of cell cycle
In DMEM-supplemented media, cells were plated in 12-well plates at a cell density of 6–8×105 per well. After twenty-four hours, cells were cultured for an additional 48 hours with the IC50 of the target compound. The untreated cells were used as a negative control. After 48 h of incubation, the treated cells were centrifuged at 1,200 rpm and 4 °C for 10 min. After discarding the supernatant, the wells were then given a single phosphate-buffered saline (PBS) wash and then centrifuged for 10 min at 1,200 rpm. Trypsin/EDTA was used to collect the cells, and after one PBS wash, they were resuspended in 0.5 mL of 0.05% Triton X-100 for 10 min at room temperature. Each cell suspension was stained by being given 1 mL of 50 g/mL propidium iodide (PI) to be left at room temperature in the darkness for 20 minutes. The analysis of the cell cycle was done by a flow cytometer (Becton Dickenson (BD) FACSCalibur, USA) (Michalkova et al. 2022).
Wound healing assay
MCF7 and HEP2 treated and untreated cells were detached from the tissue culture plate using 0.25% Trypsin-EDTA solution. Cells pellet was prepared in a 15 mL conical tube by centrifugation. The supernatant was aspirated, and the cells were re-suspended in culture media. The appropriate number of cells was platted in a 6-well plate for 100% confluence in 24 hours. In a sterile environment (typically a biosafety hood), a 200 μL pipette tip was used to press firmly against the top of the tissue culture plate and swiftly made a vertical wound down through the cell monolayer. Carefully, the media and cell debris were aspirated. Slowly, enough culture media was added against the good wall to cover the bottom of the well and avoid detaching additional cells. Following the generation and inspection of the wound an initial picture was taken. The tissue culture plate was placed in an incubator set at the appropriate temperature and CO2 concentration (typically 37 °C and 5% CO2). After 48 hours, the plate was removed from the incubator and placed under an inverted microscope to take a snapshot picture and check for wound closure (Justus et al. 2014).
Molecular docking
The molecular simulation studies were achieved using the Molecular Operating Environment (MOE) version 2009.10 (Mohamed et al. 2023b). The target compound 5c was drawn using the program builder interface and then subjected to local energy minimization using the included MOPAC. Afterward, the model was subjected to global energy minimization using systematic conformational search where RMS gradient and RMS distance were set at 0.01 kcal/mole and 0.1 Ao, respectively. The X-ray crystallographic structure of P53 cancer mutant Y220C and Bcl2 proteins complexed with their co-crystallized ligands (PDB ID: 5O1H and 6QGG), respectively, were obtained from the protein database. The proteins were modified for molecular simulations as follows; firstly, the hydrogen atoms were added. Afterward, the unwanted co-ligands and water chains were deleted. Then, the MOE alpha site finder was used to determine the active site of selected proteins. Finally, after the step of self-docking of the modified protein with its co-crystallized ligand, it was then subjected to be docked with the target compound to detect the protein-ligand interactions at the active domain. The final result of the protein-ligand interactions was visualized in 2D and 3D forms through BIOVIA Discovery Studio V6.1.0.15350.
Conclusion
Among the synthesized chalcones, 5c and 9a had the most promising cytotoxic activity against MCF7 and HEP2 cells. Compound 5c was chosen for further molecular studies for its lower IC50 values than Chalcone 9a. It was found that compound 5c had anti-proliferative activity in MCF7 and HEP2 cells by downregulating the expression level of AKT1, Ki-67, and survivin genes. Also, it had an anti-inflammatory effect in MCF7 and HEP2 cells which was shown by decreasing the expression of IL-1B, COX-2 genes, and IL-6 (at the gene and protein levels). In addition, the anti-invasive and anti-metastatic effects of chalcone 5c were exerted in MCF7 and HEP2 cells via lowering the activity of MMP-2 and MMP-9 and these results were confirmed by wound healing assay. Chalcone 5c decreased the concentration of MDA and enhanced the activity of GSH demonstrating the anti-oxidant activity in MCF7 and HEP2 cells. Chalcone 5c triggered intrinsic and extrinsic pathways of apoptosis in MCF7 and HEP2 cells by lowering the concentration of Bcl2 and increasing the concentration of BAX, P53, and caspases-8 and -9. Our molecular docking study against P53 mutant Y220C and Bcl2 supported our results in the ELISA studies. Chalcone 5c caused cell cycle arrest at the G0-G1 phase in MCF7 cells and G2-M in HEP2 cells. So, the above-mentioned results predicted that chalcone 5c might be used as a chemotherapeutic agent for the treatment of breast and laryngeal cancer.
Data availability
No datasets were generated or analysed during the current study.
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The authors declare that all data were generated in-house and that no paper mill was used. NSI and M.S. shared in conceptualization, methodology, writing the experimental part related to biochemistry and writing - Original Draft. HAS shared in methodology and writing the experimental part related to biochemistry. HMD, AHME and IAA shared in Conceptualization, Writing - Review & Editing. All authors read and approved the manuscript, and all data were generated in-house and that no paper mill was used.
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Ibrahim, N.S., Sayed, H.A., Sharaky, M. et al. Synthesis, cytotoxicity, anti-inflammatory, anti-metastatic and anti-oxidant activities of novel chalcones incorporating 2-phenoxy-N-arylacetamide and thiophene moieties: induction of apoptosis in MCF7 and HEP2 cells. Naunyn-Schmiedeberg's Arch Pharmacol (2024). https://doi.org/10.1007/s00210-024-03255-9
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DOI: https://doi.org/10.1007/s00210-024-03255-9