Introduction

Acrylamides exhibit strong antifungal [1], antibacterial [2], antidiabetic [3], anti-inflammatory [4], and anticancer [5] effects. Additionally, the pharmacological actions of some potential thiophenes and their fused derivatives, such as their anti-inflammatory [6], antiviral [7], antibacterial [8, 9], and anticancer [10, 11] effects, have sparked a great deal of interest. Specifically, ATL525 and PD81,723 are examples of aminothiophenes that act as positive allosteric regulators, or enhancers, of the human A1 adenosine receptor (A1AR) (Fig. 1) [12]. Pyrazoles are nitrogen-containing heterocycles that exhibit important bioactivities, such as anticonvulsant [13], antibacterial [14, 15], antioxidant [16, 17], anticancer [18, 19], and anti-inflammatory [15, 20] properties.

Fig. 1
figure 1

The structure of AT525 and PD81,723, of the human A1

Full size image

Based on the aforementioned facts and in continuation towards the synthesis of biologically active heterocycles [21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37] we report herein the synthesis of some novel derivatives of cyanoacrylamides incorporating tetrahydrobenzo[b]thiophene and (pyrazol-4-yl, 1H-indol-3yl, or pyridine-3-yl) moieties and test their cytotoxic activities against various cancer cell lines using MTT assay.

Results and discussion

Chemistry

The newly designed cyanoacrylamide derivatives, 5a-i were synthesized in high yield in two steps as represented in Schemes 1 and 2. In the first step, the ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate 1 was treated with 1-cyanoacetyl-3,5-dimethylpyrazole 2 in toluene at reflux to give the corresponding ethyl 2-(2-cyanoacetamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate 3 in good yield (Scheme 1).

Scheme 1
scheme 1

Synthesis of the targeted cyanoacrylamide derivatives containing pyrazole moiety 5a-i

Full size image

In the second step, the novel cyanoacrylamide derivatives, 5a-i bearing and tetrahydrobenzo[b]thiophene and (pyrazol-4-yl, 1H-indol-3yl, p-dimethylamino, or pyridine-3-yl) moieties were prepared via the Knoevenagel condensation reaction of the ethyl 2-(2-cyanoacetamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate 3 with the appropriately prepared aldehyde derivatives 4a-i (Scheme 1). Thus, heating the molar equivalents of 3 and the appropriate 4a-i at reflux in ethanol in the presence of a few drops of piperidine afforded the targeted cyanoacrylamide derivatives in good to excellent yields.

The structures of all novel cyanoacrylamide derivatives 5a-i were proven based on the elemental analysis and spectral data. IR spectra of the compounds 5a-i depicted the absorption bands in the range of 3411 to 3422 cm-1 which is attributed to NH functionality, while the absorption bands in the range of 1660 to 1667 cm-1 could be assigned to the C = O group. The CN group appeared at 2213 to 2220 cm-1. The 1H NMR spectra exhibited sharp singlets in the downfield region at δ = 9.10–9.24 ppm for pyrazole-H5. The aromatic and aliphatic protons were observed in their expected chemical shifts, while the NH groups were assigned at the range of δ = 11.81–12.57 ppm. Taking compound 5c as a representative example. Thus, the 1H-NMR spectrum (400 MHz, DMSO-d6) of 5c showed the hydrogen of the NH group as a sharp singlet at δ 11.97 ppm, while the aromatic protons were assigned at a range of δ 7.17-9.19 ppm. The CH2 groups were observed as a multiplet at δ 4.36, 2.65, and 1.74 ppm. The methoxy and methyl groups were assigned as a singlet at δ 3.86 and 1.34 ppm, respectively.

Cytotoxic assay

Primary screening

Using MTT assay, we performed screening for the compounds 5a-i at a concentration of 100 µg/mL on cell viability of four human cancer cell lines. Namely, HEPG2 (human hepatocellular carcinoma), MCF7 (human Caucasian breast adenocarcinoma), PACA2 (human pancreatic carcinoma), and PC3 (human prostatic adenocarcinoma). Also, the study was done on BJ1 (normal human skin fibroblast) to compare the results. The best results were demonstrated against MCF7 and PC3 cell lines (Table 1). As compounds 5f and 5i inhibited 100% of PC3 cells at 100 µg/ml. Regarding MCF7, compounds 5b, 5c, 5d, and 5f exerted >70% inhibition (71.8, 79.3, 74.3, and 85.3% respectively). In addition, compound 5a inhibited about 67.2% of MCF7 cells. It was found that compound 5d exerted the most promising effect against HEPG2 (100% inhibition). It was also noticed that only compound 5d had an effect against PACA2 cells (62.5%). Concerning BJ1 normal cells, compounds 5b, 5c, and 5f exerted >50% inhibition of cells (81.2, 33.5, and 42.3% respectively). Secondary screening was performed on the most effective compounds (5b, 5c, 5d, 5f, and 5i) to determine their IC50.

Table 1 % Inhibition of the tested cancer and normal cell lines at 100 µg/mL
Full size table

Secondary screening

In terms of IC50 values, as shown in Table 2, the most effective and selective compound was 5f against PC3, where IC50 value was 11.7 µM relative to the positive control (doxorubicin) (IC50 = 43.8 µM). Also, compound 5i showed a promising cytotoxic effect against PC3 cells, as IC50 (66.8 µM) was very comparable to that of doxorubicin. Regarding MCF7, compounds 5c, 5d, and 5f exerted moderate activity with IC50 equalled to 92.8, 106.8, and 99.7 µM respectively, as compared to doxorubicin (48 µM). It was noticed that compound 5d exerted a weak cytotoxic effect against PACA2 with IC50 value of 149.2 µM compared to doxorubicin (52.1 µM). While compound 5d had a moderate cytotoxic effect against HEPG2 (IC50 = 78.6 µM) relative to doxorubicin (39.7 µM). So, the most effective compounds 5b, 5c, 5d, 5f, and 5i were chosen for further molecular studies.

Table 2 IC50 ± SD values for compounds (5a- i) (µM)
Full size table

Molecular docking

The molecular simulation studies were done on compound 5i with the active site of Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), while compounds 5c and 5d were studied against STAT1 (Signal transducer and activator of transcription 1) protein. It was noticed in Table S1 (see Supplementary Information) that compound 5i fitted in the active site of KRAS with good binding energy (−20.7 Kcal/mole) relative to the standard ligand (−36.4 Kcal/mole). The binding energy of compound 5c with STAT1 was −26.5 Kcal/mole, which was better than that of the standard ligand (−24.9 Kcal/mole) (Table S1, see Supplementary Information). Also, compound 5d is bound to STAT1 with promising binding energy (−22.7 Kcal/mole) as compared to the standard ligand (−24.9 Kcal/mole). As depicted in Fig. 2a, compound 5i interacted with KRAS through five interactions, two hydrogen bonds between the cyano group, carbonyl of the ester group, and the same amino acid HIS: 95 with bond distances 4.72 A0 and 4.97 A0 respectively, two pi- alkyl hydrophobic interactions between pyridine ring and two amino acids (MET: 72 and ARG: 68), and one amide- pi stacked hydrophobic interaction between thiophene ring and GLN: 61. Figure 2b showed nine interactions between compound 5c and STAT1. Pi-cation electrostatic interaction between the benzene ring and HIS: 629, pi- pi stacked hydrophobic interaction between thiophene ring and TYR: 651, two pi-alkyl hydrophobic interactions with TYR: 634 and VAL: 653 residues, and five hydrogen bonds with GLU: 632, ALA: 630 and HIS: 629 amino acid residues. Compound 5d interacted with STAT1 through six interactions (Fig. 2c). These interactions included a conventional hydrogen bond between the carbonyl of the amide group and IIS: 629 (4.02 A0), a pi-donor hydrogen bond between the thiophene ring and ALA: 630 (4.56A0), pi-sulfur interaction between sulfur of thiophene and IIS: 629, two pi-alkyl hydrophobics between pyrazole ring, chlorobenzene ring, and VAL: 653, and finally alkyl hydrophobic interaction between the methyl group and PRO: 633.

Fig. 2
figure 2

The molecular interactions of a compound 5i with the active site of KRAS, b compound 5c with the active site of STAT1, and c compound 5d with the active site of STAT1 in 2-dimensional (2D) and 3-dimensional (3D) states

Full size image

Gene expression in breast cancer cell lines

Gene expression analysis in breast cancer cell lines (MCF-7) was performed using breast cancer-related genes such as RBL2 (RB transcriptional corepressor like 2) and STAT2: (Signal transducer and activator of transcription 2) genes. The results revealed that the expression levels of RBL2 and STAT2 genes were increased significantly (P < 0.01) in negative samples of breast cancer cell lines compared with treated cell lines (Fig. 3). The expression levels of RBL2 and STAT2 genes were decreased significantly in the groups of 5f-MCF-7, 5b-MCF-7, positive control (Doxorubicin treatment), 5c-MCF-7 and 5d-MCF-7 in a descending manner compared with negative control. So, the expression levels of RBL2 and STAT2 genes were downregulated in 5c-MCF-7 and 5d-MCF-7 groups much lower than those in other treatment groups.

Fig. 3
figure 3

A The alterations of the RBL2 gene in breast cancer cell line lines treated with 5b, 5c, 5d, and 5 f (−ve = untreated cell lines and DOX = doxorubicin). Data are presented as mean ± SD. a,b,c,d,e: Mean values within tissue with unlike superscript letters were significantly different (P < 0.05). B) The alterations of the STAT2 gene in breast cancer cell lines treated with 5b, 5c, 5d, and 5 f. Data are presented as mean ± SD. a,b,c,d: Mean values within tissue with unlike superscript letters were significantly different (P < 0.05). Doxorubicin was used as a positive control

Full size image

Gene expression in prostatic cancer cell lines

Gene expression analysis in prostatic cancer cell lines (PC-3) was performed using prostatic cancer-related genes such as KRAS (Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) and SMAD (Sma and Mad proteins) genes. The results revealed that the expression levels of KRAS and SMAD genes were increased significantly (P < 0.01) in negative samples of prostatic cancer cell lines compared with treated cell lines (Fig. 4). The expression levels of KRAS and SMAD genes were decreased significantly in the groups of 5f-PC-3, positive control (Doxorubicin treatment), and 5i-PC-3 in a descending manner compared with negative control. So, the expression levels of KRAS and SMAD genes were downregulated in 5f-PC-3 and 5i-PC-3 groups much lower than those in other treatment groups.

Fig. 4
figure 4

A The alterations of the KRAS gene in prostatic cancer cell lines treated with 5 f and 5i (−ve = untreated cell lines and DOX = doxorubicin). Data are presented as mean ± SD. a,b,c,d: Mean values within tissue with unlike superscript letters were significantly different (P < 0.05). B The alterations of the STAT2 gene in breast cancer cell lines treated with 5f and 5i. Data are presented as mean ± SD. a,b,c,d: Mean values within tissue with unlike superscript letters were significantly different (P < 0.05)

Full size image

DNA damage in cancer cell lines

The DNA damage in breast (MCF-7) and prostatic (PC-3) cancer cell lines was determined using comet assay as shown in Tables S2 and S3 (see supplementary information) and Fig. 5. The results showed that negative samples of cancer cell lines exhibited a significant decline (P < 0.05) in DNA damage values compared with treated cell lines. However, the DNA damage values were increased significantly (P < 0.01) in treated breast and prostate cancer cell line samples. The percentage of DNA damage values were increased significantly in the groups of 5f-MCF-7, 5f-PC-3, 5b-MCF-7, positive control (Doxorubicin treatment), 5i-PC-3, 5d-MCF-7 and 5c-MCF-7 in an ascending manner (15.5, 18.75, 19.25, 19.77, 20.26, 20.51, 22.27, 23.52% respectively) compared with negative control (11.25 and 11.75% for MCF7 and PC3 respectively). So, the DNA damage values were increased in 5c-MCF-7 and 5d-MCF-7 groups much more than those in other treatment groups. The incidence of DNA damage in our treated MCF7 and PC3 samples means the occurrence of apoptosis (programmed cell death) in MCF7 and PC3 cells.

Fig. 5
figure 5

Visual score of normal DNA (class 0) and damaged DNA (class 1, class 2 and class 3) using comet assay in cancer cell lines. Class 0 = no tail, 1 = tail length< diameter of nucleus, 2 = tail length between 1X and 2X the diameter of nucleus and 3 = tail length> 2X the diameter of nucleus

Full size image

Measurement of DNA fragmentation in cancer cell lines

Determination of the rate of DNA fragmentation in breast (MCF-7) and prostatic (PC-3) cancer cell lines were summarized in Tables S4 and S5 (see supplementary information) and Figs. 6 and 7. It was found that negative cancer cell lines exhibited a significant decrease (P < 0.01) in DNA fragmentation rates compared with those in treated samples (5f-MCF-7, 5f-PC-3, 5b-MCF-7, 5i-PC-3, 5d-MCF-7 and 5c-MCF-7) and positive cancer cell lines. However, the DNA fragmentation values were increased significantly (P < 0.01) in treated prostatic and breast cancer cell lines samples (5f-MCF-7, 5f-PC-3, 5b-MCF-7, positive control (Doxorubicin treatment), 5i-PC-3, 5d-MCF-7 and 5c-MCF-7) compared with negative control. Moreover, the highest values of DNA fragmentation value were observed in 5c-MCF-7 and 5d-MCF-7 much more than those in other treated breast cancer cell lines.

Fig. 6
figure 6

DNA fragmentation detected with Agarose gel in a treated prostate cancer cell line (PC-3). M: represent DNA marker, Lane 1: represents untreated cancer cell lines (negative control), Lane 2: represents 5f-PC-3, Lane 3: represents 5i-PC-3, Lane 4: represents doxorubicin treated cancer cell line (positive control)

Full size image
Fig. 7
figure 7

DNA fragmentation detected with Agarose gel in treated breast cancer cell line (MCF-7). M: represent DNA marker, Lane 1: represents untreated cancer cell lines (negative control), Lane 2: represents 5f-MCF-7, Lane 3: represents 5b-MCF-7; Lane 4: represents 5c-MCF-7; Lane 5: represents 5d-MCF-7; Lane 6: represents doxorubicin treated cancer cell lines (positive control)

Full size image

The lead compound of our series contained ethyl tetrahydrobenzo[b]thiophene-3-carboxylate and acrylonitrile moieties. The literature encouraged us to use this lead compound for its promising cytotoxic effect against different cancer cell lines [38,39,40,41]. In an attempt to enhance the activity of the lead compound, different substituents were added performing nine novel cyanoacrylamide derivatives. According to the structure-activity relationship, we found that compound 5f which contains 1-phenyl-3-(thiophen-2-yl)-1H-pyrazol-4-yl substituent had the best cytotoxic effect against PC3 cells with IC50 = 11.7 µM comparing to doxorubicin (IC50 = 43.8 µM). In addition, compound 5i containing pyridine-3-yl substituent exerted a very comparable effect as the positive control against PC3 cells (IC50 = 66.8 µM). While, compounds 5c, 5d, and 5f containing 4-methoxyphenyl-1-phenyl-1H-pyrazol-4-yl, 4-chlorophenyl-1-phenyl-1H-pyrazol-4-yl, and 1-phenyl-3-(thiophen-2-yl)-1H-pyrazol-4-yl substituents respectively displayed moderate activity against MCF7 cells (IC50 = 92.8, 106.8, and 99.7 µM respectively) as compared to doxorubicin (IC50 = 48 µM). The KRAS gene is an oncogenic signalling protein that encodes a small GTPase transductor protein [42]. It is involved in the regulation of cell division due to its ability to transduce external signals to the nucleus leading to increased resistance to chemotherapy targeting epidermal growth factor receptors. SMADs are a group of signalling proteins transducing extracellular signals directly to the nucleus [43]. SMAD mediates transforming growth factor β (TGFβ) responses. In late phases of cancer, TGFβ can act as a tumor promoter by inducing tumor cell invasion and metastasis. STAT2 is a member of the STAT family that plays an essential role in immune responses to extracellular and intracellular stimuli, including cancer initiation, inflammatory reactions, and invasion of foreign materials [44]. RBL2 is a well-known tumor suppressor gene in the retinoblastoma family, which is mutated in numerous cancers [45]. RBL2 regulates the cell cycle progression at the G1/S transition and its repression causes cell cycle arrest at the G1/S phase. In our study, it was found that compounds 5b, 5c, 5d, and 5f down-regulated the expression of RBL2 and STAT2 in MCF7 cells. While in PC3 cells, compounds 5f and 5i significantly decreased the expression level of KRAS and SMAD genes. So, from the above-mentioned facts about KRAS, SMAD, STAT2, and RBL2, we could conclude that our tested compounds might be used in cancer therapy via down-regulating these genes. Our molecular docking study supported the results of the gene expression section but at the protein level. Compound 5i fitted in the active site of KRAS with promising binding energy (−20.7 kcal/mole). While compounds 5c and 5d interacted with the active site of STAT1 through binding energies of −26.5 and −22.7 kcal/mole respectively. It was known that DNA fragmentation is a late event of apoptosis (programmed cell death) [46]. Herein, DNA fragmentation was studied by two techniques (comet and gel electrophoresis assays). Both techniques revealed the same result in which, the DNA damage values were increased significantly in the groups of 5f-MCF-7, 5f-PC-3, 5b-MCF-7, (Doxorubicin treatment), 5i-PC-3, 5d-MCF-7 and 5c-MCF-7 in an ascending manner compared with negative control.

Conclusions

In summary, the cytotoxicity of a novel series of cyanoacrylamide derivatives bearing pyrazole moiety was studied against MCF7, PC3, HEPG2, and PACA2. The best cytotoxic effect was shown against MCF7 and PC3 carcinomas. Among the series, compounds 5f and 5i exerted the most promising cytotoxic effect against PC3 cells. Whereas, compounds 5b, 5c, 5d, and 5f showed moderate activity against MCF7 cells. In silico study revealed the promising inhibitory effect of compound 5i against KRAS and compounds 5c and 5d against STAT1. Compounds 5f and 5i down-regulated KRAS and SMAD genes and induced DNA fragmentation in PC3 cells. While, compounds 5b, 5c, 5d, and 5f decreased the expression level of STAT2 and RBL2 genes and caused DNA damage in MCF7 cells. So, these compounds might be used as anticancer candidates in prostate and breast carcinomas.

Materials and methods

Chemistry

All reactions were carried out in aerobic conditions at room temperature. Acetonitrile was distilled and kept under an inert atmosphere. All glass was oven-dried at 120 °C for at least 24 h before use. The starting materials of 3 and 4a-i have been prepared as described in the literature [22, 30, 31]. All melting points are uncorrected and measured using Electro‐Thermal IA 9100 apparatus (Shimadzu, Japan). The Infrared spectra were recorded as potassium bromide pellets on a JASCO spectrophotometer between 4000 cm−1 and 400 cm−1. 1H NMR spectra were recorded in deuterated dimethylsulfoxide (DMSO-d6) on a Bruker spectrometer (400 MHz) at 25 °C. The chemical shifts were expressed as part per million (δ values, ppm) using the solvent (DMSO = 2.51, water signal at 3.34) as reference. Low-resolution electron impact mass spectra [MS (EI)] were performed using a high-resolution thermo spectrometer. Microanalyses were operated using Elementar Vario Cube apparatus and the mass spectra were recorded using Shimadzu Qp-2010 plus, Micro Analytical Center, Cairo University.

General procedures for the synthesis of cyanoacrylamide derivatives 5a–5i

To a solution of ethyl 2-(2-cyanoacetamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate 3 (1 mmol) and the corresponding aldehyde derivatives (4a-i) (1 mmol) in ethanol (15 mL) a few drops of piperidine were added. The reaction mixture was then heated at reflux for 30 min. The precipitate was filtered off and washed with hot ethanol (2 × 5 ml) and dried under vacuum to afford the targeted products 5a-i as yellow solids.

Ethyl-2-(2-cyano-3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylamido)-4,5,6,7-tetrahydrobenzo[b]-thiophene-3-carboxylate (5a)

Yield 78%. IR (KBr, cm−1): 3414 (NH), 1667 (C = O), 2219 (CN). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.73 (s, 1H, NH), 9.20 (m, 1H, Ar-H), 7.45-8.22 (m, 11H, Ar-H), 4.35 (br, 2H, CH2), 2.69 (m, 4H, CH2), 1.74 (m, 4H, CH2), 1.34 (br, 3H, CH3). MS (EI, m/z, %): 521.98 (M, 8). Anal. Calcd. for C30H26N4O3S (522.62): C, 68.95; H, 5.01; N, 10.72; Found: C, 68.81; H, 5.21; N, 10.86%.

Ethyl-2-(2-cyano-3-(1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)acrylamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (5b)

Yield 80%. IR (KBr, cm−1): 3416 (NH), 1665 (C = O), 2220 (CN). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.97 (s, 1H, NH), 9.19 (m, 1H, Ar-H), 7.42-8.20 (m, 10H, Ar-H), 4.36 (br, 2H, CH2), 2.67 (m, 4H, CH2), 2.42 (s, 3H, CH3), 1.75 (m, 4H, CH2), 1.34 (br, 3H, CH3). MS (EI, m/z, %): 512 (M-CN, 3). Anal. Calcd. for C31H28N4O3S (536.65): C, 69.38; H, 5.26; N, 10.44; Found: C, 69.45; H, 5.22; N, 10.61%.

Ethyl-2-(2-cyano-3-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)acrylamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (5c)

Yield 91%. IR (KBr, cm−1): 3412 (NH), 1667 (C = O), 2215 (CN). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.97 (s, 1H, NH), 9.19 (m, 1H, Ar-H), 8.21 (m, 1H, Ar-H), 7.94 (m, 2H, Ar-H), 7.60 (m, 5H, Ar-H), 7.17 (m, 2H, Ar-H), 4.36 (br, 2H, CH2), 3.86 (s, 3H, OCH3), 2.65 (m, 4H, CH2), 1.74 (m, 4H, CH2), 1.34 (br, 3H, CH3). MS (EI, m/z, %): 551.8 (M, 11). Anal. Calcd. for C31H28N4O4S (552.65): C, 67.37; H, 5.11; N, 10.14; Found: C, 67.51; H, 5.26; N, 10.23%.

Ethyl-2-(3-(3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)-2-cyanoacrylamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (5d)

Yield 74%. IR (KBr, cm−1): 3422 (NH), 1664 (C = O), 2217 (CN). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.00 (s, 1H, NH), 9.22 (m, 1H, Ar-H), 7.48-8.18 (m, 10H, Ar-H), 4.38 (br, 2H, CH2), 2.66 (m, 4H, CH2), 1.75 (m, 4H, CH2), 1.35 (br, 3H, CH3). MS (EI, m/z, %): 522 (M-Cl, 2). Anal. Calcd. for C30H25ClN4O3S (557.07): C, 64.68; H, 4.52; N, 10.06; Found: C, 64.82; H, 4.65; N, 10.17%.

Ethyl-2-(2-cyano-3-(1-phenyl-3-(pyridin-4-yl)-1H-pyrazol-4-yl)acrylamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (5e)

Yield 73%. IR (KBr, cm−1): 3420 (NH), 1666 (C = O), 2219 (CN). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.94 (s, 1H, NH), 9.24 (m, 1H, Ar-H), 8.79 (m, 2H, Ar-H), 7.50-8.19 (m, 8H, Ar-H), 4.34 (br, 2H, CH2), 2.72 (m, 4H, CH2), 1.73 (m, 4H, CH2), 1.33 (br, 3H, CH3). MS (EI, m/z, %): 522.88 (M+, 4). Anal. Calcd. for C29H25N5O3S (523.61): C, 66.52; H, 4.81; N, 13.38; Found: C, 66.61; H, 4.93; N, 13.52%.

Ethyl-2-(2-cyano-3-(1-phenyl-3-(thiophen-2-yl)-1H-pyrazol-4-yl)acrylamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (5f)

Yield 62%. IR (KBr, cm−1): 3411 (NH), 1660 (C = O), 2213 (CN). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.91 (s, 1H, NH), 9.19 (m, 1H, Ar-H), 8.38 (m, 2H, Ar-H), 7.29-7.91 (m, 7H, Ar-H), 4.34 (br, 2H, CH2), 2.65 (m, 4H, CH2), 1.73 (m, 4H, CH2), 1.33 (br, 3H, CH3). MS (EI, m/z, %): 527.79 (M+, 18). Anal. Calcd. for C28H24N4O3S2 (528.65): C, 63.62; H, 4.58; N, 10.60; Found: C, 63.77; H, 4.64; N, 10.72%.

Ethyl-2-(2-cyano-3-(1H-indol-3-yl)acrylamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (5g)

Yield 70%. IR (KBr, cm−1): 3417 (NH), 1662 (C = O), 2218 (CN). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.57 (s, 1H, NH), 11.88 (s, 1H, NH), 8.63 (m, 2H, Ar-H), 8.00 (m, 1H, Ar-H), 7.58 (s, 1H, Ar-H), 7.31 (m, 2H, Ar-H), 4.35 (br, 2H, CH2), 2.70 (m, 4H, CH2), 1.74 (m, 4H, CH2), 1.34 (br, 3H, CH3). MS (EI, m/z, %): 418.62 (M+, 3). Anal. Calcd. for C23H21N3O3S (419.50): C, 65.85; H, 5.05; N, 10.02; Found: C, 65.91; H, 5.08; N, 10.11%.

Ethyl-2-(2-cyano-3-(4-(dimethylamino)phenyl)acrylamido)-4,5,6,7-tetrahydrobenzo[b]-thiophene-3-carboxylate (5h)

Yield 64%. IR (KBr, cm−1): 3422 (NH), 1665 (C = O), 2218 (CN). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.81 (s, 1H, NH), 8.19 (m, 1H, Ar-H), 7.97 (m, 2H, Ar-H), 6.89 (m, 2H, Ar-H), 4.35 (br, 2H, CH2), 3.10 (s, 6H, 2CH3), 2.65 (m, 4H, CH2), 1.75 (m, 4H, CH2), 1.34 (br, 3H, CH3). MS (EI, m/z, %): 422.95 (M, 7). Anal. Calcd. for C23H25N3O3S (423.53): C, 65.23; H, 5.95; N, 9.92; Found: C, 65.31; H, 6.05; N, 10.03%.

Ethyl-2-(2-cyano-3-(pyridin-3-yl)acrylamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (5i)

Yield 66%. IR (KBr, cm−1): 3411 (NH), 1666 (C = O), 2219 (CN). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.02 (s, 1H, NH), 9.10 (s, 1H, Ar-H), 8.78 (m, 1H, Ar-H), 8.49 (m, 2H, Ar-H), 7.65 (m, 1H, Ar-H), 4.34 (br, 2H, CH2), 2.67 (m, 4H, CH2), 1.76 (m, 4H, CH2), 1.33 (br, 3H, CH3). MS (EI, m/z, %): 380.90 (M, 85). Anal. Calcd. for C20H19N3O3S (381.45): C, 62.98; H, 5.02; N, 11.02; Found: C, 63.10; H, 5.11; N, 10.98%.

MTT assay

Cell viability was studied by mitochondrial-dependent reduction of MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) to formazan crystals. The following steps were done in a Laminar flow cabinet biosafety class II level (Baker, SG403INT, Sanford, ME, USA). Cells were suspended in RPMI‐1640 medium for (HEPG2, MCF7, PACA2, and PC3). While the normal cell line (BJ1) was suspended in DMEM-F12 medium. The medium was supplemented with a 1% antibiotic–antimycotic mixture (10,000 U/cm3 potassium penicillin, 25 µg/cm3 amphotericin B, and 10,000 µg/cm3 streptomycin sulfate) and 1% L-glutamine at 37 °C under 5% CO2. After 10 days of culturing the cells, they were seeded at a concentration of 1× 104 cells/well in fresh complete medium in 96-well micro plastic plates for 24 h under 5% CO2 at 37 °C. After that, media were replaced with another fresh medium (without serum) and cells were incubated either without a sample (negative control) or with different concentrations of a sample (100– 50– 25– 12.5– 6.25– 3.125–0.78 and 1.56 µg/ml). Then, after 72 h of incubation, the medium was replaced with 50 μl MTT salt and incubated for a further 4 hs at 37 °C under 5% CO2. 200 µl of sodium dodecyl sulfate (SDS) (10%) was added to each well and incubated overnight at 37 °C to allow the dissolving of the formed formazan crystals and stop the reaction. Doxorubicin (100 µg/cm3) was used as a positive control which gives 100% lethality under the same conditions. The absorbance was then measured using a microplate reader (Bio-Rad Laboratories Inc., model 3350, Hercules, California, USA) at 595 nm and a reference wavelength of 620 nm. A probit analysis was done for IC50 calculation using SPSS 11 program. In our study, the selectivity index (SI) of the synthetic compound is expressed as SI = IC50 of the compound in a normal cell line/ IC50 of the same compound in a cancer cell line, where IC50 is the concentration of the compound required to kill 50% of the cell population.

Molecular docking

The molecular simulation studies were achieved using the Molecular Operating Environment (MOE) version 2009.10 [47]. The target compounds 5c, 5d, and 5i were 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 KRAS and STAT1 proteins complexed with their co-crystallized ligands (PDB ID: 7RT3 and 1YVL) respectively were obtained from the protein database. The co-crystallized ligands and phosphopeptide derived from the alpha chain of interferon-gamma (IFN gamma) receptor respectively. The proteins were modified for molecular simulations as followed; firstly, the hydrogen atoms were added to the proteins with their standard ligands. Afterward, the unwanted co-ligands and water chains were deleted from the target proteins. Then, the MOE alpha site finder was used to determine the active site of selected proteins, and dummy atoms resulted from the alpha spheres. Finally, after the step of self-docking of the modified protein with its co-crystallized ligand, it was then subjected to docking with the target compounds to detect the protein-ligand interactions at the active domain. The final results of the protein-ligand interactions were visualized in 2D and 3D forms through BIOVIA Discovery Studio V6.1.0.15350.

Gene expression analysis

Quantitative Real-Time PCR method

RNA isolation and Reverse Transcription (RT) Reaction

RNeasy Mini Kit (Qiagen, Hilden, Germany) supplemented with DNaseI (Qiagen) digestion step was used to isolate total RNA from the breast (MCF-7)and prostatic (PC-3) cancer cell lines according to the manufacturer’s protocol. Isolated total RNA was treated with one unit of RQ1 RNAse-free DNAse (Invitrogen, Germany) to digest DNA residues, re-suspended in DEPC-treated water, and quantified photospectrometrically at 260 nm. The purity of total RNA was assessed by the 260/280 nm ratio which was between 1.8 and 2.1. Additionally, integrity was assured with ethidium bromide-stain analysis of 28 S and 18 S bands by formaldehyde-containing agarose gel electrophoresis. Aliquots were used immediately for reverse transcription (RT), otherwise, they were stored at -80 °C.

Complete Poly(A)+ RNA isolated from the breast (MCF-7) and prostatic (PC-3) cancer cell lines was reverse transcribed into cDNA in a total volume of 20 µl using RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, Germany). An amount of total RNA (5 µg) was used with a master mix. The master mix consisted of 50 mM MgCl2,10x RT buffer (50 mMKCl; 10 mMTris-HCl; pH 8.3), 10 mM of each dNTP, 50 µM oligo-dT primer, 20 IU ribonuclease inhibitor (50 kDa recombinant enzyme to inhibit RNase activity) and 50 IU MuLV reverse transcriptase. The mixture of each sample was centrifuged for 30 sec at 1000 g and transferred to the thermocycler. The RT reaction was carried out at 25 °C for 10 min, followed by 1 h at 42 °C, and finished with a denaturation step at 99 °C for 5 min. Afterward the reaction tubes containing RT preparations were flash-cooled in an ice chamber until being used for cDNA amplification through quantitative Real-Time- polymerase chain reaction (qRT-PCR).

Real-Time- PCR (qPCR)

Determination of the breast (MCF-7) and prostatic (PC-3) cancer cell lines cDNA copy number was carried out using StepOne™ Real-Time PCR System from Applied Biosystems (Thermo Fisher Scientific, Waltham, MA USA). PCR reactions were set up in 25 μL reaction mixtures containing 12.5 μL 1× SYBR® Premix Ex TaqTM (TaKaRa, Biotech. Co. Ltd.), 0.5 μL 0.2 μM sense primer, 0.5 μL 0.2 μM antisense primer, 6.5 μL distilled water, and 5 μL of cDNA template. The reaction program was allocated into 3 steps. The first step was at 95.0 °C for 3 min. The second step consisted of 40 cycles in which each cycle was divided into 3 steps: (a) at 95.0 °C for 15 sec; (b) at 55.0 °C for 30 sec; and (c) at 72.0 °C for 30 sec. The third step consisted of 71 cycles which started at 60.0 °C and then increased about 0.5 °C every 10 sec up to 95.0 °C. At the end of each qRT-PCR, a melting curve analysis was performed at 95.0 °C to check the quality of the used primers. Each experiment included a distilled water control. The sequences of specific primers of the breast cancer-related genes such as RBL2 and STAT2 [48] and prostatic (PC-3) cancer-related genes such as KRAS and SMAD4 [49] were designed and listed in Table S6 (see supplementary information). At the end of each qPCR, a melting curve analysis was performed at 95.0 °C to check the quality of the used primers. The relative quantification of the target to the reference was determined by using the 2−ΔΔCT method [50,51,52].

DNA damage using the comet assay

The DNA damage using comet assay was determined using breast (MCF-7) and prostatic (PC-3) cancer cell lines according to Olive et al. [53]. After the trypsin treatment to produce a single-cell suspension, approximately 1.5 × 104 cells were embedded in 0.75% low-gelling-temperature agarose and rapidly pipetted onto a pre-coated microscope slide. Samples were lysed for 4 h at 50 °C in 0.5% SDS, 30 mM EDTA, pH 8.0. After rinsing overnight at room temperature in Tris/borate/EDTA buffer, pH 8.0, samples were electrophoresed for 25 min at 0.6 V/cm, then stained with propidium iodide. Slides were viewed using a fluorescence microscope with a CCD camera, and 150 individual comet images were analyzed from each sample for a tail moment, DNA content, and percentage of DNA in the tail. For each sample, about 100 cells were examined to determine the percentage of cells with DNA damage that appear like comets. The non-overlapping cells were randomly selected and were visually assigned a score on an arbitrary scale of 0–3 (i.e., class 0 = no detectable DNA damage and no tail; class 1 = tail with a length less than the diameter of the nucleus; class 2 = tail with a length between 1× and 2× the nuclear diameter; and class 3 = tail longer than 2× the diameter of the nucleus) based on perceived comet tail length migration and relative proportion of DNA in the nucleus [54].

DNA fragmentation assay

The DNA fragmentation assay in breast (MCF-7) and prostatic (PC-3) cancer cell lines was performed in concordance with the premises established by Yawata et al. [55] with some modifications. Briefly, after 24 h of exposure of MCF-7 and PC-3 cancer cell lines to the tested substances in different Petri-dishes (60 × 15 mm, Greiner), the cells were trypsinized, suspended, homogenized in 1 ml of medium and centrifuged (10 min at 800 rpm). Low-molecular-weight genomic DNA was extracted as described in Yawata et al. [55] Approximately, 1 × 106 cells were plated and treated with the tested substances in various treatments. All the cells (including floating cells) were harvested by trypsinization and washed with Dulbecco’s Phosphate Buffered Saline. Cells were lysed with the lysis buffer containing 10 mMTris (pH 7.4), 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), and 0.5% Triton X-100 for 30 min on ice. Lysates were vortexed and cleared by centrifugation at 10,000 g for 20 min. Fragmented DNA in the supernatant was extracted with an equal volume of neutral phenol: chloroform: isoamyl alcohol mixture (25:24:1) and analysed electrophoretically on 2% agarose gels containing 0.1 μg/ml ethidium bromide.