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. 2025 Jun 10;10(24):25921–25937. doi: 10.1021/acsomega.5c02394

Synthesis and Evaluation of Novel Bis-Chalcone Derivatives Containing a Thiophene Moiety as Potential Anticancer Agents: In Vitro, In Silico , and Mechanistic Studies

Esraa M Fathi †,⊥, Mostafa E Salem ‡, Farid M Sroor §, Fatma G Mohamed †, Ahmed H M Elwahy ∥, Mohamed Abdel-Megid ‡, Ismail A Abdelhamid ∥, Marwan Emara ⊥,*
PMCID: PMC12198996  PMID: 40584358

Abstract

Chalcone compounds have demonstrated potent anticancer activities in the past few decades with few adverse consequences. Using Claisen–Schmidt condensation, two new series of bis-chalcone derivatives (5a–c and 9a–c) bearing the thiophene moiety have been designed and generated. All compound structures were examined and elucidated by spectroscopic investigations. The MTT assay, gene expression assay, cell cycle analysis, apoptosis assay, Western blotting analysis, zymographic analysis, and molecular docking were used to evaluate the anticancer efficacy of the synthesized compounds against breast, colon, and lung cancer cells. Out of the two synthesized series, four compounds (5a, 5b, 9a, and 9b) showed significant cytotoxic effects against breast (IC50 values of 7.87 ± 2.54 and 4.05 ± 0.96 μM for compounds 5a and 5b, respectively), colon (IC50 values of 18.10 ± 2.51 and 17.14 ± 0.66 μM for compounds 5a and 9a, respectively), and lung (IC50 values of 41.99 ± 7.64 and 92.42 ± 30.91 μM for compounds 5a and 9b, respectively) cancer. These compounds upregulated the proapoptotic genes and caspase-3 and -9 protein, downregulated antiapoptotic and matrix metalloproteinase 2 (MMP-2) gene, and MMP-2 and -9 enzymatic activity. Additionally, these compounds significantly increased early and late apoptosis, necrosis, and induced cell cycle arrest at the subG1 phase with a concomitant decrease in the percentage of cell fractions at G0/G1, S, and G2/M phases. Moreover, the molecular docking was carried out on caspase-3 and -9 and MMP-2 and -9. The results suggest that compounds 5a, 5b, 9a, and 9b are potential and effective anticancer drugs.

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Introduction

According to the statistics of the World Health Organization (WHO), cancer is one of the top causes of death worldwide, accounting for nearly one-sixth of all deaths in 2020, where breast, lung, and colon cancers are the most common. The conventional and most commonly used treatments for cancer include radiotherapy and chemotherapy. Chemotherapy is considered an effective systemic treatment; however, undesired side effects and drug resistance are the major challenges. −

Natural therapies have demonstrated positive anticancer activity and served as the core component of cancer chemotherapy, where flavonoids are the leading candidates. Flavonoids are heat-stable polyphenols present in vegetables, fruits, and plant-based products like chocolate and tea, with protective effects against cancer due to their antioxidant activity. Chalcones are 1,3-diphenyl-2-propane-1-ones and have open-chain precursors for the biosynthesis of flavonoids and isoflavonoids. They are divided into two forms: trans (E) and cis (Z) isomers. These isomers have two aromatic rings connected by a three-carbon α, β-unsaturated carbonyl bridge keto-ethylenic system (−CO–CHCH−). The E isomer is the most common configuration of the chalcones because it is more stable from a thermodynamic perspective. Due to the strong steric interactions between the carbonyl group and the A-ring, the configuration of the Z isomer is unstable. The synthesis of novel chalcone compounds with anticancer moieties is applicable because of their simple chemistry and the availability of chalcone backbones that already have a primary component, a substituent, or a side chain in several biologically active compounds. −

Chalcones are thought to gain biological properties due to the α, β-unsaturated carbonyl motif. Chalcones were found to inhibit angiogenesis, cellular proliferation, the JAK/STAT signaling pathway, histone deacetylases, P53 degradation, and multidrug resistance channels. The chalcones’ multifaceted biological activities are not only restricted to anticancer activities but also include anti-inflammatory, , antiviral, antidiabetic, antimicrobial, and antioxidant. ,

Thiophene is a heterocyclic compound of a five-membered ring containing sulfur. Thiophene and its derivatives are scaffolds used to design and discover numerous thiophene-containing drugs with a wide range of biological activities, including antitumor activity. For example, the commercially available anticancer therapies, such as OSI-930 and Raloxifene, contain thiophene and have strong antiproliferative properties.

Compared to the standard derivatives, the chalcone derivatives containing heterocyclic moieties (e.g., thiophene) have become promising drug candidates because they have shown similar or better biological activities. − Bis-heterocycles in which two bioactive heterocycles are tethered via a flexible linker were reported as antibacterial, fungicidal, anticancer, and plant growth regulators. − They also have numerous applications as chelating agents, electrical conducting materials, and metal ligands. Also, it is expected that the biological activity of bis-chalcones containing thiophene will be better and enhanced than that of monochalcone containing thiophene. In continuation of our interest in the synthesis of bioactive compounds, − herein, we synthesized six novel bis-chalcones containing thiophene. Cell viability and proliferation assays, gene expression assays, cell cycle analyses, apoptosis assays, Western blotting analyses, zymographic analyses, and molecular docking were carried out to study and evaluate the efficacy of the synthesized compounds against A549 lung cancer, HCT116 colon cancer, and MCF7 breast cancer cells.

Results and Discussion

Chemistry

The bis­(ethan-1-one) precursors 3a–c were prepared as previously reported via the bis-alkylation reaction of 4-hydroxyacetophenone 1 with the corresponding dibromo compounds 2 in the presence of KOH (Scheme A and Figure ). The Claisen–Schmidt condensation reaction of 3a–c with two equivalents of thiophene-2-carbaldehyde 4 in ethanol in the presence of KOH at reflux resulted in the formation of bis­(3-(thiophen-2-yl)­prop-2-en-1-one) (5a–c), in which thiophene represents the B-ring, while bis­(4,1-phenylene) represents the A-ring (Scheme A and Figure ). The structures of the generated products were validated by using spectral data. Thus, the IR spectra of compound 5a, as a sample example, revealed the existence of the carbonyl band at 1648 cm–1. The 1H NMR spectrum revealed the methylene linkage as a mutliblet signal at 4.48–4.50 ppm. All other signals appear in their expected placements. In addition, the 13C NMR indicated the carbonyl group at δ 187.3 ppm.

1. Synthesis of Bis­(chalcones) 5a-–c (A) and 9a–c (B).

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Chemical structure of the targeted bis-chalcon derivatives (5a–c) and (9a–c).

Motivated by the results obtained in Scheme , we prepared the isomeric bis­(1-(thiophen-2-yl)­prop-2-en-1-one) 9a–c, in which thiophene represents the A-ring, while bis­(4,1-phenylene) represents the B-ring, via the direct reaction of one-mole equivalent of the appropriate bis­(aldehydes) 7a–c with two-mole equivalents of 1-(thiophen-2-yl)­ethan-1-one 8 in ethanol in the presence of KOH at reflux (Scheme B and Figure ). It is important to note that, the bis­(aldehydes) (7a–c) were prepared via the bis-alkylation of p-hydroxybenzaldehyde (6) with the appropriate dibromo derivatives (2a–c) in ethanol in the presence of KOH (Scheme B and Figure ). As an example for this series, in compound 9a, the IR spectrum revealed the existence of the carbonyl band at 1648 cm–1. The 1H NMR spectrum revealed the methylene linkage as a mutliblet signal at 4.40–4.43 ppm. In addition, the 13C NMR indicated the carbonyl group at 182.0 ppm.

Cell Viability Assay (MTT)

The MTT assay evaluated the cytotoxic effect of the synthesized chalcones containing thiophene and the positive control drug (cisplatin) on the viability and proliferation of A549, HCT116, and MCF7 cells. Following 48 h of treatment, the synthesized compounds (Figure ) and cisplatin (Figure A) showed a differential inhibition of cell viability and proliferation in the tested cell lines. The IC50s of the tested compounds and cisplatin were calculated (Table ). In A549 cells, compounds 5a (IC50 = 41.99 ± 7.64 μM) and 9b (IC50 = 92.42 ± 30.91 μM) showed the strongest cytotoxic effect. In HCT116 cells, compounds 5a (IC50 = 18.10 ± 2.51 μM) and 9a (IC50 = 17.14 ± 0.66 μM) showed the strongest cytotoxic effect. In MCF7 cells, compounds 5a (IC50 = 7.87 ± 2.54 μM) and 5b (IC50 = 4.05 ± 0.96 μM) showed a remarkable cytotoxic effect. Breast cancer cells (MCF7) were the most sensitive to the synthesized compounds, followed by colon (HCT116) and lung (A549) cancer cells. The IC50s of cisplatin were 5.547 ± 0.734, 13.276 ± 0.294, and 27.78 ± 0.929 μM for A549, HCT116, and MCF7 cells, respectively. In MCF7 cells, the IC50s of compounds 5a, 5b, and 9a were lower than the IC50 of cisplatin, indicating a higher potency (Figure A and Table ).

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Cell viability assay and proliferation assay (MTT). The cytotoxic effect of the synthesized compounds on the viability and proliferation of A549, HCT116, and MCF7 cells was determined after 48 h of treatment with the synthesized compounds (A;5a, B;5b, C;5c, D;9a, E;9b, and F;9c).

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Cell viability assay and proliferation assay (MTT). The cytotoxic effect of cisplatin on the viability and proliferation of A549, HCT116, and MCF7 cells (A) and the cytotoxic effect of the synthesized compounds (5a, 5b, 9a, and 9b) on the viability and proliferation of normal CCD-16Lu cells (B) after 48 h.

1. Calculated IC50s (μM) of the Synthesized Compounds (5a–c and 9a–c) and the Positive Control Drug (Cisplatin) .

Cancer Cell lines
 A549
 HCT116
 MCF7
 CCD-16Lu
Compounds IC50 (μM)
5a 41.99 ± 7.64 18.10 ± 2.51 7.87 ± 2.54 52 ± 0.70
5b 114.96 ± 18.86 30.73 ± 2.58 4.05 ± 0.96 NDb
5c ND 356.00 ± 30.71 ND NA
9a 272.55 ± 49.45 17.14 ± 0.66 11.47 ± 1.25 ND
9b 92.42 ± 30.91 120.30 ± 12.90 30.08 ± 4.04 34.5 ± 1.4
9c 232.40 ± 73.59 277.75 ± 53.35 134.13 ± 17.48 NA
Positive Control (cisplatin) 5.547 ± 0.734 13.276 ± 0.294 27.78 ± 0.929 NA
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a

Data are Expressed as Mean ± SE.

b

Nondetectable (ND).

c

Not applicable.

In normal CCD-16Lu cells (Figure B and Table ), the IC50 for compound 5a (52 ± 0.70 μM) was higher than its IC50 in A549, HCT116, and MCF7 cells, while the IC50 for compound 9b (34.5 ± 1.4 μM) was higher than its IC50 in the MCF7 cells. Compounds 5b and 9a have no effect on normal CCD-16Lu cells. This may reflect the selectivity of the synthesized compounds compared with most chemotherapeutics that are cytotoxic for both normal and cancer cells. Compound 5c showed a very low cytotoxic effect and undetectable IC50 on A549 and MCF7 cells, and consequently, it was not applicable to test on CCD-16Lu cells. Similarly, compound 9c had low cytotoxicity and high IC50s on A549, MCF7, and HCT116 cells and hence was not applied to the normal CCD-16Lu cells. The 48 h time point was chosen for all assays to give untreated cells sufficient time for growth and proliferation and to test the effect of the compounds under the same conditions. The 48 h time point was chosen in our previously published chalcone-related articles. , Compounds (5a and 9b), (5a and 9a), and (5a and 5b) showed the best cytotoxic effects against A549, HCT116, and MCF7 cells, respectively. So, these compounds (5a, 5b, 9a, and 9b) were further investigated for their effect on the expression of proapoptotic and antiapoptotic genes, caspase-3 and caspase-9 proteins, apoptosis, cell cycle, and the enzymatic activity of MMP-2 and MMP-9.

Expression of BCL2, BAX, P53, and MMP-2 Transcripts in Different Cells

The antiapoptotic BCL2 gene regulates the activation of the intrinsic mechanism of apoptosis following DNA damage, oncogene activation, or radiation exposure through its BCL2 protein. , BAX is a protein that belongs to the BCL2 gene family and is encoded by the BAX gene. It is essential to the process of apoptosis. BAK and BAX are key modulators of the intrinsic apoptotic process. TP53 is an important tumor suppressor involved in many aspects of the genesis of cancer, and cancer treatment involves the TP53 protein. MMP-2 promotes the migration of cancer cells through tissues by breaking down the components of the extracellular matrix, promoting tumor cell proliferation, strengthening resistance to apoptosis, and boosting angiogenesis. ,

Treatment of A549, HCT116, and MCF7 cells with the IC50s of (5a and 9b), (5a and 9a), and (5a and 5b) compounds, respectively, resulted in a modest decrease in BCL2 and MMP-2 genes, whereas the levels of BAX and P53 genes were increased (Figure ). Compared with normal control cells, upon treatment, the BCL2 transcript was modestly decreased in A549 cells (compounds 5a and 9c; p ≤ 0.05), HCT116, and MCF7 (compound 5a; p ≤ 0.05) (Figure A). Following treatment with the synthesized compounds, the BAX transcript showed a modest increase in A549, HCT116, and MCF7 (compound 5b; p ≤ 0.001) cells (Figure B). Compared with the control cells, the P53 transcript showed a modest increase in A549, HCT116, and MCF7 cells following treatment with the synthesized compounds (Figure C). P53 is known to stimulate both the extrinsic and intrinsic pathways via its protein. P53 was reported to induce the proapoptotic gene BAX and repress the antiapoptotic gene BCL2. , Upregulation of proapoptotic genes (e.g., BAX and P53) and downregulation of antiapoptotic genes (e.g., BCL2) may imply that the synthesized compounds induce apoptotic cell death. At the same time, an increase in P53 may indicate that apoptosis occurs through a P53-dependent pathway. Our results are consistent with other reports , that chalcones containing thiophene have proapoptotic activity. MMP-2 showed a modest decrease in A549, HCT116, and MCF7 treated cells (Figure D). MMP-2 and MMP-9 are implicated in cancer development and progression. In agreement with others, the reduction in MMP-2 expression in treated cells shows the efficacy of the synthesized chalcones containing thiophene in treating different types of cancer.

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Transcript expression of BCL2 (A), BAX, (B), P53 (C), and MMP-2 (D) in A549, HCT116, and MCF7 cells after treatment with the IC50s of compounds (5a and 9b), (5a and 9a), and (5a and 5b), respectively, for 48 h. Data are expressed as fold-change in mRNA relative to the control. c p < 0.05; b p < 0.01; a p < 0.001 [t test or Mann–Whitney rank-sum test (based on the equal variance and normality tests) vs normal control].

Apoptosis Assay

Apoptosis and necrosis are major types of cell death. Cell apoptosis occurs through the intrinsic and/or extrinsic pathways, where both pathways activate the caspase protease family, resulting in cell death. Early apoptosis is characterized by the externalization of phosphatidylserine, followed by late apoptosis, where the plasma membrane becomes permeabilized and then necrosis. Annexin V and propidium iodide (PI) are used to access normal cells (Annexin V–, PI–), early apoptotic cells (Annexin V+, PI–), late apoptotic cells (Annexin V+, PI+), and necrotic cells (Annexin V–, PI+) in A549 cells, HCT116 cells, and MCF7 cells upon treatment with the synthesized chalcones containing thiophene (Figure ). Treatment of A549 cells (Figure A4) with the IC50s of compounds 5a and 9b resulted in a significant decrease in living cells (p ≤ 0.01 and p ≤ 0.05 for compounds 5a and 9b, respectively) and an increase in early apoptotic cells, late apoptotic cells (p ≤ 0.01 and p ≤ 0.05 for compounds 5a and 9b, respectively), and necrotic cells for compounds 5a and 9b (p ≤ 0.05). Treatment of HCT116 cells (Figure B4) with the IC50s of compounds 5a and 9a resulted in a significant decrease in live cells (p ≤ 0.001 for both compounds) and an increase in early apoptotic cells for compounds 5a (p ≤ 0.001) and 9a, late apoptotic cells (p ≤ 0.001 and p ≤ 0.05 for compounds 5a and 9a, respectively), and necrotic cells [compounds 5a and 9a (p ≤ 0.05)]. Treatment of MCF7 cells (Figure C4) with the IC50s of compounds 5a and 5b resulted in a significant decrease in living cells (p ≤ 0.001 for both compounds) and an increase in early apoptotic cells, late apoptotic cells (p ≤ 0.001 for both compounds), and necrotic cells (p ≤ 0.05 and p ≤ 0.01 for compounds 5a and 5b, respectively). Compared to control cells, statistical analysis showed that compound 5a is more potent than compounds 9a, 9b, and 5b in the induction of early and late cell apoptosis and necrosis in A549 cells, HCT116 cells, and MCF7 cells. In agreement with the expression levels of proapoptotic and antiapoptotic genes measured in treated cells, apoptosis assay results may confirm that the synthesized compounds induce cell death through apoptosis. These results align with other reports ,, showing the anticancer and proapoptotic activity of chalcones containing thiophene.

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Apoptosis assay by flow cytometry. The A549 (A), HCT116 (B), and MCF-7 (C) cells were treated with the IC50s of compounds [5a (A2) and 9b (A3)], [(5a (B2) and 9a (B3)], and [5a (C2) and 5b (C3)], respectively, for 48 h. The control and treated cells were stained with Annexin V-Alexa Fluor 488 and propidium iodide. Representative flow cytometric dot plot quadrant plots showing live cells, early apoptosis, late apoptosis, and necrosis in both controls (A1, B1, and C1) and treated cells (A2, A3, B2, B3, C2, and C3). In both control and treated cells, the quantification analysis (mean value ± SE) of live cells, early apoptosis, late apoptosis, and necrosis is shown in Figures A4, B4, and C4 for A549, HCT116, and MCF-7 cells, respectively. c p < 0.05; b p < 0.01; a p < 0.001 [t test or Mann–Whitney rank-sum test (based on the equal variance and normality tests) vs normal control].

Cell Cycle Analyses

Cell cycle analyses were carried out to determine the percentage of cells in the different phases of the cell cycle by measuring the intensity of PI bound to the cellular DNA. After 48 h of treatment of different cancer cells with IC50s of all synthesized chalcones containing thiophene resulted in cell cycle arrest in the subG1 phase coupled with a decrease in the percentages of cells in the G0/G1, S, and G2/M phases compared with control cells (Figure ). Treatment of A549 cells with the the IC50s of compounds 5a and 9b caused a significant (p ≤ 0.001 for both 5a and 9b compounds) accumulation of cells in subG1 fraction of cells and decreased the percentage of cells in other phases (G0/G1, S, and G2/M) compared with control cells (Figure A4). In colon cancer cells (HCT116), treatment with the IC50s of 5a and 9a compounds resulted in significant accumulation of the cells in subG1 phase (p ≤ 0.001 for both 5a and 9a compounds) and a decrease in the percentage of cells in other phases [G0/G1 (p ≤ 0.05 for 9a compound), S, and G2/M (p ≤ 0.05 for 9a compound)] compared with control cells (Figure B4). Similar to lung and colon cancer cells, treatment of breast cancer cells with the IC50s of compounds 5a and 5b caused an accumulation of cells in the subG1 phase (p ≤ 0.001 for compounds 5a and 5b) and decreased the percentage of cells in other phases [G0/G1 (p ≤ 0.01 for compound 5b), S (p ≤ 0.01 for compounds 5a and 5b) and G2/M (p ≤ 0.01 for compounds 5a and 5b)] compared with control cells (Figure C4). The subG1 phase represents the number of apoptotic cells characterized by internucleosomal DNA fragmentation, chromatin condensation, and disintegration. , Increased subG1 population by the end of treatment may occur directly or be preceded by transient G2/M blockage as a prerequisite step for the drug-elicited subG1 apoptosis. Our findings are consistent with previous reports. These results agree with other reports ,, showing that chalcone derivatives can induce cell cycle arrest at different phases in various cancer cell lines.

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Cell cycle analyses. The A549 (A), HCT116 (B), and MCF-7 (C) cells were treated with the IC50s of compounds [5a (A2) and 9b (A3)], [5a (B2) and 9a (B3)], and [5a (C2) and 5b (C3)], respectively, for 48 h. The control and treated cells were fixed in ice-cold ethanol (70%) for 30 min. The fixed cells were washed with ice-cold PBS and resuspended in 100 μL of PBS containing 10 μL (100 μg/mL RNase) and 5 μL of PI (50 μg/mL). Representative cell cycle analyses plots showing subG1, G0/G1, S, and G2/M phases in both controls (A1, B1, and C1) and treated cells (A2, A3, B2, B3, C2, and C3). In both control and treated cells, the quantification analysis (mean value ± SE) of subG1, G0/G1, S, and G2/M phases is shown in Figures A4, B4, and C4 for A549, HCT116, and MCF-7 cells, respectively. c p < 0.05; b p < 0.01; a p < 0.001 [t test or Mann–Whitney rank-sum test (based on the equal variance and normality tests) vs normal control].

Expression of Casapase-3 and Caspase-9 Protein

Caspases are a family of 15 cysteine aspartic proteases that mediate apoptosis. Upon treatment of lung, colon, and breast cancer cells with the IC50s of the synthesized chalcones containing thiophene (compounds 5a, 5b, 9a, and 9b) for 48 h, caspase-3 and caspase-9 were quantified using Western blotting analysis (Figure ). Caspase-3 was not detected in MCF7 cells due to a partial deletion in the CASP-3 gene. In A549 cells, caspase-3 and caspase-9 were upregulated significantly upon treatment with compounds 5a (caspase-3: p ≤ 0.05; caspase-9: p ≤ 0.001) and 9b (caspase-3: p ≤ 0.05; caspase-9: p ≤ 0.001) (Figure A1 and A2). In HCT116 cells, a modest increase in caspase-3 and caspase-9 was observed following treatment with compounds 5a and 9a (Figure B1 and B2). In breast cancer cells MCF7, caspase-9 showed a modest increase upon treatment with compound 5a, while treatment with compound 5b showed a significant increase (p ≤ 0.05) (Figure C). Caspases mediate both intrinsic and extrinsic apoptotic pathways. In the intrinsic apoptosis, the activated initiator caspase-9 activates the executioner caspases-3 and caspase-7, which subsequently induce apoptotic cell death. Apoptosis could be initiated through a P53-dependent or P53-independent pathway as well as through intrinsic or extrinsic pathways that activate proapoptotic proteins like caspase-9 and BAX. In this study, increased expression of BAX, P53, caspase-3, and caspase-9, downregulation of BCL2, and cell cycle arrest at the subG1 phase may confirm that the synthesized chalcones containing thiophene induce apoptotic cell death, which is in line with other reports. ,

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Representative Western blots and protein levels of Caspase-3 and -9 in A549 (A1, A2), HCT116 (B1, B2), and MCF-7 (C) cells were assessed by Western blotting analysis. The A549, HCT116, and MCF-7 cells were treated with the IC50s of compounds [5a and 9b], [5a and 9a], and [5a and 5b], respectively, for 48 h. The integrated intensities of caspase-3, −9, and the loading controls (alpha-tubulin and beta-actin) bands were determined and expressed in arbitrary units (AU). All protein levels were normalized to their respective loading controls. Data are expressed in mean value ± SE; c p < 0.05; b p < 0.01; a p < 0.001 [t test or Mann–Whitney rank-sum test (based on the equal variance and normality tests) vs normal control].

Enzymatic Activity of MMP-2 and MMP-9

MMP-2 and MMP-9 are zinc-dependent endopeptidases involved in normal and pathological tissue remodeling. In cancer, MMP-2 and MMP-9 degrade several components in the extracellular matrix, promoting the invasion and metastasis of cancer cells. Therefore, many synthetic MMP inhibitors were developed to target MMPs in cancer. , Following treatment with the IC50s of the synthesized chalcones containing thiophene, gelatin zymographic analysis of MMP-2 and MMP-9 excreted in the culture media of A549, HCT116, and MCF7 cells showed the expression of pro-MMP-2, active MMP-2, and active MMP-9. In the culture media of all cells, the activity levels for pro-MMP-9 were undetectable, while the activity levels for MMP-2 were very low (Figure ). Compared with control cells, treatment of A549 cells with the IC50s of 5a and 9b compounds resulted in a slightly nonsignificant increase in the activity levels of MMP-9 and MMP-2 active forms (Figure A). Treatment of HCT116 cells with the IC50s of compounds 5a and 9a resulted in a modest decrease (Figure B) in the activity levels of the active forms of MMP-9 and MMP-2. Similarly, a modest decrease in the active forms of MMP-9 and MMP-2 was observed in MCF7 cells treated with the IC50s of 5a and 5b compounds (Figure C). The modest inhibitory effect of the synthesized chalcones containing thiophene on the activity levels of active MMP-2 and MMP-9 shown in HCT116 and MCF7 cell lines is consistent with other reports. ,

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Representative zymograms and activity levels of MMP-2 and MMP-9 in A549 (A), HCT116 (B), and MCF7 (C) cells. Activity levels of MMP-2 and MMP-9 were assessed following treatment of A549 (A), HCT116 (B), and MCF-7 (C) cells with the IC50s of compounds [5a and 9b], [5a and 9a], and [5a and 5b], respectively, for 48 h.

Molecular Docking Studies

Molecular docking of the new chalcone derivatives (5a, 5b, 9a, and 9b) was performed toward the binding pocket of the Caspase-3, Caspase-9, MMP-2, and MMP-9 active sites. Pose selection was based on improved binding scores and rmsd_refine values, as shown in Table . The scores and binding interactions of each new candidate with the selected targets’ pocket amino acids were promising compared to the experimental studies.

2. Binding Scores, the Mode of Interactions of 5a, 5b, 9a, and 9b Compounds, and the Active Sites of Caspase-3, Caspase-9, MMP-2, and MMP-9.

Macromolecule Drug Interaction Receptor S S(kcal/mol)
Caspase-3 5a H-donor SER 209 –6.19
H-pi HIS 121
pi-cation ARG 207
pi-H ASN 208
5b H-acceptor HOH 2293 –6.2
pi-H TYR 204
pi-H ASN 208
pi-H PHE 250
9a H-pi HIS 121 –6.7
pi-H MET 61
pi-cation ARG 207
9b H-donor GLU 248 –6.6
H-acceptor HOH 2293
pi-H THR 62
Caspase-9 5a H-acceptor HIS 182 –6.6
pi-H GLN 437
5b H-donor SER 60 –6.8
pi-H GLN 437
9a H-acceptor TYR 62 –5.8
pi-H THR 180
pi-H HIS 381
9b H-acceptor TYR 62 –6.2
H-acceptor HIS 99
pi-H SER 434
MMP-2 5a H-donor ALA 137 –8.14
H-donor ALA 140
Metal ZN 201
H-pi HIS 125
pi-H ALA 86
pi-H LEU 138
5b H-donor ALA 137 –8.2
H-donor ALA 140
Metal ZN 201
H-pi HIS 121
H-pi HIS 125
pi-H LEU 82
pi-H ALA 86
pi-H LEU 138
9a pi-H ALA 86 –8.1
pi-H PHE 87
pi-H ALA 88
pi-pi HIS 121
9b H-donor ALA 137 –7.6
H-donor ALA 140
pi-H LEU 138
MMP-9 5a Metal ZN 301 –8.2
pi-H TYR 248
pi-H ARG 249
5b H-donor ALA 189 –8.3
H-donor GLU 227
pi-H GLU 241
pi-H TYR 248
9a H-donor ARG 249 –8.45
pi-H TYR 248
pi-pi HIS 226
9b H-donor ALA 191 –8.48
pi-H HIS 190
pi-H TYR 248
pi-H ARG 249
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For Caspase-3 (Figure ), compound 5a achieved a binding score of −6.19 kcal/mol besides the formation of four interactions: one H-donor with SER 209, one H-pi with HIS 121, one pi-cation with ARG 207, and one pi-H with ASN 208. Compound 5b achieved a binding score of −6.2 kcal/mol, forming four interactions: one H-acceptor with HOH 2293, and three pi–H interactions with TYR 204, ASN 208, and PHE 250. Compound 9a achieved a binding score of −6.7 kcal/mol besides forming three interactions: one H-pi with HIS 121, one pi-H with MET 61, and one pi-cation with ARG 207. Compound 9b achieved a binding score of −6.6 kcal/mol, forming three interactions: one H-donor with GLU 248, one H-acceptor with HOH 2293, and one pi-H with THR 62.

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Three-dimensional and two-dimensional representations of compounds 5a (A), 5b (B), 9a (C), and 9b (D) binding to Caspase-3.

For Caspase-9 (Figure ), compound 5a achieved a binding score of −6.6 kcal/mol, forming two interactions: one H-acceptor with HIS 182 and one pi-H with GLN 437. Compound 5b achieved a binding score of −6.8 kcal/mol in addition to forming two interactions: one H-donor with SER 60 and one pi-H with GLN 437. Compound 9a achieved a binding score of −5.8 kcal/mol besides forming three interactions: one H-acceptor with TYR 62, two pi-H with THR 180 and HIS 381. Compound 9b achieved a binding score of −6.2 kcal/mol besides the formation of three interactions: two H-acceptors with TYR 62 and HIS 99, and one pi-H with SER 434.

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Three-dimensional and two-dimensional representations of compounds 5a (A), 5b (B), 9a (C), and 9b (D) binding to Caspase-9.

For MMP-2 (Figure ), compound 5a achieved a binding score of −8.14 kcal/mol besides the formation of six interactions: two H-donors with ALA 137 and ALA 140, one metal interaction with ZN 201, one H-pi with HIS 125, and two pi–H interactions with ALA 86 and LEU 138. Compound 5b achieved a binding score of −8.2 kcal/mol besides the formation of eight interactions: two H-donors with ALA 137, and ALA 140, one metal interaction with ZN 201, two H-pi with HIS 121 and HIS 125, and three pi–H interactions with LEU 82, ALA 86, and LEU 138. Compound 9a achieved a binding score of −8.1 kcal/mol, forming four interactions: three pi–H interactions with ALA 86, PHE 87, and ALA 88, and one pi–pi interaction with HIS 121. Compound 9b achieved a binding score of −7.6 kcal/mol besides the formation of three interactions: two H-donor interactions with ALA 137 and ALA 140, and one pi–H interaction with LEU 138.

11.

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Three-dimensional and two-dimensional representations of compounds 5a (A), 5b (B), 9a (C), and 9b (D) binding to MMP-2.

For MMP-9 (Figure ), compound 5a achieved a binding score of −8.2 kcal/mol besides the formation of three interactions one metal interaction with ZN 301, two pi-H with TYR 248 and ARG 249, 5b achieved a binding score of −8.3 kcal/mol besides the formation of four interactions two H-donor with ALA 189 and GLU 227, and two pi-H with GLU 241 and TYR 248, 9a achieved a binding score of −8.45 kcal/mol besides the formation of three interactions one H-donor with ARG 249, one pi-H with TYR 248, and one pi-pi with HIS 226, and 9b achieved a binding score of −8.48 kcal/mol besides the formation of four interactions one H-donor with ALA 191 and three pi–H interactions with HIS 190, TYR 248, and ARG 249.

12.

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Three-dimensional and two-dimensional representations of compounds 5a (A), 5b (B), 9a (C), and 9b (D) binding to MMP-9.

The docking results show that our compounds have good binding affinities toward the active sites of Caspase-3, Caspase-9, MMP-2, and MMP-9, which align with the previous experimental results.

Structure–Activity Relationship (SAR)

The structure of the novel bis­(chalcone)-based thiophene can be regarded as an α,β-unsaturated enone group (the hydrophilic region in the targeted structures) linked to two rings (A-ring and B-ring) via an aliphatic linkage (the hydrophobic region in the targeted structures).

For series 5a–c, the A-ring represents the bis­(4,1-phenylene) part of the chalcone and the B-ring is the thiophene ring. On the other hand, for series 9a–c, the A-ring represents the thiophene ring, while bis­(4,1-phenylene) represents the B-ring (Figure ). Generally, it is concluded that B-rings with thiophene connected to the double bond of the enone moiety (compound 3) had greater activity than A-rings with thiophene attached to the carbonyl of the enone (compound 9). A decrease in carbon chain size (n = 0 or 1) improved the antitumor activity of the newly produced compounds (5a,b > 5c; 9a,b > 9c). Moreover, the thiophene moiety is an essential moiety to increase the anticancer activity of the prepared chalcones compared with related compounds. ,,,

13.

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Design strategy of bis­(chalcones) synthesized compounds 5a–c and 9a–c.

Conclusions

Two novel series of bis-chalcones containing thiophene were synthesized using Claisen–Schmidt condensation. The chemical structures were elucidated and confirmed by spectroscopic analysis. The anticancer effect of the synthesized compounds was tested using breast (MCF7), colon (HCT116), and lung (A549) cancer cells. Compared with normal cells, the four compounds (5a, 5b, 9a, and 9b) showed significant cytotoxic effects against breast, colon, and lung cancers. Compound 5a was the most potent cytotoxic compound, and breast cancer cells were the most sensitive to the synthesized compounds. Treatment of A549, HCT116, and MCF7 cells with (5a and 9b), (5a and 9a), and (5a and 5b) compounds, respectively, resulted in a decrease in BCL2 and MMP-2 genes, an increase in BAX and P53 genes, a significant reduction in living cells, and an increase in early apoptotic, late apoptotic, and necrotic cells. Furthermore, the synthesized compounds caused a cycle arrest in the subG1 phase, increased caspase-3 and caspase-9, and decreased MMP-2 and MMP-9 enzymatic activity. These results support that these compounds induce apoptotic cell death and inhibit cancer invasion. The molecular docking study showed very good binding between the novel bis-chalcones and caspase-3, caspase-9, MMP-2, and MMP-9 . Our findings suggest that compounds 5a, 5b, 9a, and 9b are potential and effective anticancer drugs. However, further studies are needed to elucidate these drugs’ specific mechanisms of action and evaluate their safety in the preclinical and clinical phases.

Materials and Methods

Chemistry

All reactions were carried out under aerobic conditions at room temperature. Acetonitrile was distilled and kept under an inert atmosphere. All glasses were oven-dried at 120 °C for at least 24 h before use. The starting materials 3a–c and 7a–c were prepared as described in the literature. All melting points are uncorrected and measured using the Electro-Thermal IA 9100 apparatus (Shimadzu, Japan). The infrared spectra were recorded as potassium bromide pellets on a JASCO spectrophotometer between 4000 and 400 cm–1. The 1H and 13C NMR spectra were recorded in DMSO-d 6 as a solvent on a Varian Gemini NMR spectrometer at 400 and 100 MHz, respectively. Chemical shifts are reported as δ values in parts per million (ppm). APT measurements determined the multiplicities of the signals.

General Procedures for the Synthesis of Bis­(Furan-Substituted) Chalcone Derivatives (5a–c)

To a mixture of thiophene-2-carboxaldehyde 4 (2 mmol) and bis­(acetyl) derivatives 3a–c (1 mmol) in ethanol (20 mL), KOH (2 mmol) was added. The reaction mixture was then refluxed for 30 min. The precipitate was filtered off, washed with hot ethanol (2 × 5 mL), and dried under vacuum to afford the products 5a–c as yellow solids. The newly synthesized compounds were recrystallized from ethanol/ethyl acetate (3:1).

1,1′-((Ethane-1,2-diylbis­(oxy))­bis­(4,1-phenylene))­bis­(3-(thiophen-2-yl)­prop-2-en-1-one) (5a)

m.p = 198–200 °C. Yield 97%. IR (KBr, cm–1): 1648 (CO). 1H NMR (400 MHz, DMSO-d 6): δ (ppm) 8.14 (m, 4H, Ar–H), 7.91 (s, 2H, vinyl–H), 7.78 (m, 2H, Ar–H), 7.69 (m, 2H, Ar–H), 7.60 (m, 2H, Ar–H), 7.15–7.21 (m, 6H, Ar–H+ vinyl–H), 4.48–4.50 (m, 4H, CH2). 13C NMR (100 MHz, DMSO-d 6): δ (ppm) 187.3, 162.7, 140.3, 136.4, 133.0, 131.3, 131.0, 130.9, 130.6, 129.1, 120.8, 115.0, 114.8, 67.1. Anal. Calcd for C28H22O4S2 (486.60): C, 69.11; H, 4.56. Found: C, 69.23; H, 4.49%.

1,1′-((Propane-1,3-diylbis­(oxy))­bis­(4,1-phenylene))­bis­(3-(thiophen-2-yl)­prop-2-en-1-one) (5b)

m.p = 182–184 °C. Yield 90%. IR (KBr, cm–1): 1649 (CO). 1H NMR (400 MHz, DMSO-d 6): δ (ppm) 7.56–8.11 (m, 12H, Ar–H + vinyl–H), 7.11 – 7.20 (m, 6H, Ar–H+ vinyl–H), 4.26–4.28 (m, 4H, CH2), 2.26 (m, 2H, CH2). 13C NMR (100 MHz, DMSO-d 6): δ (ppm) 187.3, 162.9, 140.3, 136.4, 132.9, 131.3, 131.0, 130.8, 130.6, 129.1, 120.8, 114.9, 65.1. Anal. Calcd for C29H24O4S2 (500.63): C, 69.58; H, 4.83; Found: C, 69.71; H, 4.75%.

1,1′-((Butane-1,4-diylbis­(oxy))­bis­(4,1-phenylene))­bis­(3-(thiophen-2-yl)­prop-2-en-1-one) (5c)

m.p = 175–177 °C. Yield 70%. IR (KBr, cm–1): 1647 (CO). 1H NMR (400 MHz, DMSO-d 6): δ (ppm) 7.01–8.11 (m, 18H, Ar–H+ vinyl–H), 4.15–4.17 (m, 4H, CH2), 1.90–1.92 (m, 4H, CH2). 13C NMR (100 MHz, DMSO-d 6): δ (ppm) 187.3, 162.7, 140.3, 136.4, 133.0, 131.3, 131.0, 130.6, 129.1, 120.8, 115.0, 114.8, 67.1. Anal. Calcd for C30H26O4S2 (514.65): C, 70.01; H, 5.09; Found: C, 70.06; H, 5.11%.

General Procedures for the Synthesis of Bis-Furan-Substituted Chalcone Derivatives (9a–c)

To a solution of 2-acetylthiophene 8 (2 mmol) and bis-aldehyde derivatives 7a–c (1 mmol) in ethanol (15 mL), KOH (2 mmol) was added. The reaction mixture was then heated at reflux for 30 min. The precipitate was filtered off, washed with hot ethanol (2 × 5 mL), and dried under vacuum to afford the products 9a–c as yellow solids. The newly synthesized compounds were recrystallized from ethanol/ethyl acetate (3:1).

3,3′-((Ethane-1,2-diylbis­(oxy))­bis­(4,1-phenylene))­bis­(1-(thiophen-2-yl)­prop-2-en-1-one) (9a)

m.p = 202–204 °C. Yield 85%. IR (KBr, cm–1): 1648 (CO). 1H NMR (400 MHz, DMSO-d 6): δ (ppm) 8.31 (s, br, 2H, Ar–H), 8.03 (d, 2H, vinyl–H, J = 4.2 Hz), 7.87 (d, 4H, Ar–H, J = 8.04 Hz), 7.74 (d, 4H, Ar–H, J = 15.6 Hz), 7.28–7.31 (m, 2H, vinyl–H), 7.10 (d, 4H, Ar–H, J = 8.04 Hz), 4.40–4.43 (s, 4H, CH2). 13C NMR (100 MHz, DMSO-d 6): δ (ppm) 182.0, 160.9, 146.2, 143.5, 135.7, 133.8, 131.3, 129.3, 127.9, 120.0, 115.4, 66.9. Anal. Calcd for C28H22O4S2 (486.60): C, 69.11; H, 4.56; Found: C, 69.22; H, 4.41%.

3,3′-((Propane-1,3-diylbis­(oxy))­bis­(4,1-phenylene))­bis­(1-(thiophen-2-yl)­prop-2-En-1-one) (9b)

m.p = 184–186 °C. Yield 85%. IR (KBr, cm–1): 1649 (CO). 1H NMR (400 MHz, DMSO-d 6): δ (ppm) 8.30 (s, 2H, Ar–H), 8.03 (m, 2H, vinyl–H), 7.85 (m, 4H, Ar–H), 7.70 (m, 4H, Ar–H), 7.31 (m, 2H, vinyl–H), 7.02–7.06 (m, 4H, Ar–H), 4.40–4.43 (m, 4H, CH2), 2.20–2.23 (m, 2H, CH2). 13C NMR (100 MHz, DMSO-d 6): δ (ppm) 182.0, 161.1, 146.2, 143.5, 135.6, 133.7, 131.3, 129.3, 127.7, 119.9, 115.4, 64.9, 28.9. Anal. Calcd for C29H24O4S2 (500.63): C, 69.58; H, 4.83; Found: C, 69.71; H, 4.77%.

3,3′-((Butane-1,4-diylbis­(oxy))­bis­(4,1-phenylene))­bis­(1-(thiophen-2-yl)­prop-2-en-1-one) (9c)

m.p = 178–180 °C. Yield 88%. IR (KBr, cm–1): 1643 (CO). 1H NMR (400 MHz, DMSO-d 6): δ (ppm) 8.30 (m, 2H, Ar–H), 8.04 (m, 2H, vinyl–H), 7.86 (m, 4H, Ar–H), 7.73 (m, 4H, Ar–H), 7.30 (m, 2H, vinyl–H), 7.00–7.03 (m, 4H, Ar–H), 4.10–4.13 (m, 4H, CH2), 1.88–1.91 (m, 4H, CH2). 13C NMR (100 MHz, DMSO-d 6): δ (ppm) 187.3, 163.0, 140.3, 136.3, 132.9, 131.2, 131.0, 130.9, 130.7, 130.5, 130.0, 129.1, 120.8, 114.9, 68.0, 25.7. Anal. Calcd for C30H26O4S2 (514.65): C, 70.01; H, 5.09; Found: C, 70.21; H, 5.02%.

Cell Lines and In Vitro Culture Conditions

The cancer (A549, HCT116, and MCF7) and normal (CCD-16Lu) cell lines were purchased from the American Type Culture Collection (VA, USA). The cells were grown as monolayer cultures in a culture medium containing 10% fetal bovine serum (Biowest, France, Cat. No. S1810-500) at 37 °C in a humidified 5% CO2 incubator. HCT116 and MCF7 cell lines were cultured in RPMI-1640 medium (Biowest, Cat. No. L0498-500). The CCD-16Lu and A549 cells were cultured in MEM (Biowest, France, Cat. No. L0470-500) medium and DMEM High Glucose (Biowest, France, Cat. No. L0103-500) medium, respectively. Plasticwares of cell culture were purchased from SPL (Life Sciences, Korea).

Cell Viability and Proliferation Assay

Cell viability and proliferation were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Cat. No. 20395.02, Serva, Germany). Briefly, cells were cultured in 96-well tissue culture plates and treated with different concentrations of the synthesized compounds. After 48 h, 110 μL of medium containing 10 μL of MTT (5 mg/mL) was added to each well. Plates were then incubated at 37 °C in a 5% CO2 incubator for 4 h, and then the media were discarded and replaced with 100 μL of DMSO (Molekula, UK). The plates were kept for 15 min on an orbital shaker at room temperature. The absorbance was measured at 570 nm (FLUOstar Omega, BMG LABTECH GmbH, Germany).

RNA Extraction and cDNA Generation

The A549, HCT116, and MCF7 cells were cultured and treated with the IC50s of the synthesized compounds. Total RNA was extracted as per the manufacturer’s protocol (TRIsure kit, Bioline GmbH, Germany). Reverse transcription was carried out according to the manufacturer’s instructions using a High Capacity cDNA Reverse Transcription Kit (Cat. No. 4368814; Applied Biosystems; Thermo Fisher Scientific, Inc.) with an RNase Inhibitor (Cat. No. N8080119; Applied Biosystems; Thermo Fisher Scientific, Inc.).

Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR)

RT-qPCR analysis was performed using a QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). TaqMan Fast Universal PCR Master Mix (Cat. No. 4366072; Applied Biosystems; Thermo Fisher Scientific, Inc.) and validated TaqMan Gene Expression Assays (Applied Biosystems; Thermo Fisher Scientific, Inc.) were used for human MMP-2 (assay no. Hs01548727_m1), BCL2 (assay no. Hs00608023_m1), BAX (assay no. Hs00180269_m1), and P53 (assay no. Hs01034249_m1). The human β-actin gene (assay no. Hs99999903_m1) was used as an endogenous housekeeping control. Using the 2–ΔΔCT method, the fold change in the expression of the target gene, normalized to the endogenous housekeeping gene and relative to the control, was quantified.

Apoptosis Assay

Early and late apoptosis were quantified using a dead cell apoptosis kit with Annexin V Alexa Fluor 488 and Propidium Iodide (Cat. No. V13245, Thermo Fisher Scientific, Inc.). In brief, A549, HCT116, and MCF7 cells were cultured and treated with the IC50s of the synthesized compounds for 48 h. Cells were trypsinized, washed, and resuspended in 1X Annexin-binding buffer. Alexa Fluor 488 Annexin V (5 μL) and 5 μL of PI (20 μg/mL) were added to assay tubes (105 cells/100 μL/assay). Cells were incubated at room temperature for 15 min, then 390 μL of 1X Annexin -binding buffer, and then analyzed using flow cytometry (Attune Acoustic Focusing Cytometer, Thermo Fisher Scientific, Inc.).

Cell Cycle Analysis

The cells were cultured and treated with the IC50s of the synthesized compounds for 48 h. The cells (106) were fixed in ice-cold ethanol (70%) for 30 min. The fixed cells were washed with ice-cold PBS and resuspended in 100 μL of PBS containing 10 μL (100 μg/mL RNase) and 5 μL of PI (50 μg/mL). The cells were incubated in the dark at room temperature for 30 min and topped up to 0.5 mL with PBS. The cell cycle was analyzed using a CytoFLEX flow cytometer (Beckman Coulter, USA).

Gelatin Zymography

The activity levels for MMP-2 and MMP-9 in A549, HCT116, and MCF7 cells were determined by gelatin zymographic analysis as previously reported. In brief, cells were cultured and treated with the IC50s of the synthesized compounds for 48 h. Following treatment, the cell culture media were aspirated, mixed with 6X sample buffer, and electrophoresed on 10% SDS-PAGE containing gelatin (2 mg/mL, Sigma, Cat. No. G-8150). Each sample was analyzed in duplicate. Prestained protein markers were used. Following electrophoresis, the gels were washed three times (20 min each) in 2.5% Triton X-100 and then incubated in Tris buffer containing 0.01 M CaCl2, 0.2 M NaCl, and 0.05% NaN3 at 37 °C for 20 h. The gels were stained with 0.05% Coomassie Brilliant Blue G-250 (Sigma) in 25% methanol and 10% acetic acid for 2 h at room temperature and then destained with 4% methanol and 8% acetic acid for 1 h. The gels were rinsed in distilled water containing 10% glycerol and placed between two sheets of cellophane (Cat. No. Z377600, Sigma). The zymograms were scanned using a CanoScan LiDE 700F (Canon Inc., Japan), and the gelatinolytic bands of MMP-2 and MMP-9 were determined and analyzed using ImageJ software (NIH, USA). The activity of the gelatinolytic bands was expressed in arbitrary units.

Western Blotting Analysis

The cells were cultured and treated with the IC50s of the synthesized compounds for 48 h. The whole-cell lysate was prepared in complete Lysis-M buffer (Cat. No. 04719956001, Roche Diagnostics GmbH, Germany), and protein content was quantified using the Pierce BCA Protein Assay Kit (Cat. No. 23225, Thermo Fisher Scientific, Inc.). Under reducing conditions, equal protein concentrations of 20 μg were resolved in 12% SDS-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked in 5% nonfat dry milk for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies against caspase-3 (1:1000, Cell Signaling Technologies, Cat. No. 9662S) and caspase-9 (1:1000, Cell Signaling Technologies, Cat. No. 9508S). Alpha-tubulin antibody (1:2000, Invitrogen, Cat. No. PA1-38814) and beta-actin antibody (1:500, Santa Cruz, Cat. No. sc-8432) at dilution were used as loading controls. The membranes with caspase-3 and beta-actin were incubated with goat antirabbit IRDye 680LT secondary antibody (1:15000, Cat. No. 926–68021, LI-COR Biotechnology, USA), while the membranes with caspase-9 and alpha-tubulin were incubated with donkey antimouse IRDye 800 secondary antibody (1:15000, Cat. No. 926-32212, LI-COR Biotechnology) for 1 h at room temperature. The membranes were scanned using the Odyssey Infrared Imaging System (LI-COR Biotechnology, USA). Bands were analyzed using LI-COR software, and the integrated areas of the bands were determined and expressed in arbitrary units (AU).

Molecular Docking Studies

Molecular docking studies and modeling calculations were carried out using the Molecular Operating Environment (MOE) program, version 2014.0901, to explain the suggested mechanism of action for the newly synthesized chalcone derivatives in the active sites of the X-ray structures of caspase-3, caspase-9, MMP-2, and MMP-9 were obtained from the Protein Data Bank (PDB) website (http://www.rcsb.org/- PDB codes: 2xyg, 6ysa, 8h78, and 5cuh, respectively). The target receptor was prepared for the docking process, as mentioned in the steps in detail. In addition, the structure of synthesized chalcones 5a, 5b, 9a, and 9b was drawn using the ChemDraw Professional 16.0 program, and it was put into the MOE and prepared alone for docking according to the aforementioned steps. Then, all of the prepared compounds were introduced into one database to be uploaded in a general docking process. The best pose for each studied compound was selected based on the score and rmsd_refine values.

Statistical Analyses

Data are expressed as the mean ± SE of three experimental replicates with 2–3 technical replicates each. The IC50s of the studied compounds was calculated using GraphPad Prism 6 software (GraphPad Software, Boston, MA). Statistical analyses were conducted using SigmaPlot 13 software (Systat Software Inc.). Comparison between untreated and treated cells was measured using a t-test or Mann–Whitney rank-sum test based on the equal variance and normality tests.

Supplementary Material

ao5c02394_si_001.pdf (6MB, pdf)

Acknowledgments

This work was supported and funded by the Deanship of Scientific Research at Imam Mohammad Ibn Saud Islamic University (IMSIU) (Grant No . IMSIU-DDRSP2501).

The data supporting this article have been included in the Supporting Information.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c02394.

  • 1H NMR and 13C NMR spectrum of compounds 5a, 5b, 5c, 9a, 9b, 9c, and 9c (Figure S1), Western blots (Figures S2–S11) and zymograms (Figures S12–S14) (PDF)

The authors declare no competing financial interest.

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