Discovery of New Apoptosis-Inducing Agents for Breast Cancer Based on Ethyl 2-Amino-4,5,6,7-Tetra Hydrobenzo[b]Thiophene-3-Carboxylate: Synthesis, In Vitro, and In Vivo Activity Evaluation

A multicomponent synthesis was empolyed for the synthesis of ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate 1. An interesting cyclization was obtained when the amino-ester 1 reacted with ethyl isothiocyanate to give the benzo[4,5]thieno[2,3-d][1,3]thiazin-4-one 3. Acylation of the amino-ester 1 with chloroacetyl chloride in DCM and Et3N afforded the acylated ester 4. The amino-ester 1 was cyclized to benzo[4,5]thieno[2,3-d]pyrimidin-4(3H)-one 8, which was reacted with some alkylating agents leading to alkylation at nitrogen 9–13. Hydrazide 14 was utilized as a synthon for the synthesis of the derivatives 15–19. Chloro-thieno[2,3-d]pyrimidine 20 was synthesized and reacted with the hydrazine hydrate to afford the hydrazino derivative 21, which was used as a scaffold for getting the derivatives 22–28. Nucleophilic substitution reactions were used for getting the compounds 29–35 from chloro-thieno[2,3-d]pyrimidine 20. In the way of anticancer therapeutics development, the requisite compounds were assessed for their cytotoxicity in vitro against MCF-7 and HepG-2 cancer cell lines. Twelve compounds showed an interesting antiproliferative potential with IC50 from 23.2 to 95.9 µM. The flow cytometric analysis results showed that hit 4 induces the apoptosis in MCF-7 cells with a significant 26.86% reduction in cell viability. The in vivo study revealed a significant decrease in the solid tumor mass (26.6%) upon treatment with compound 4. Moreover, in silico study as an agonist for inhibitors of JAK2 and prediction study determined their binding energies and predicted their physicochemical properties and drug-likeness scores.


Introduction
The development and discovery of high efficacy anticancer agents by many molecular oriented strategies and techniques have increased in the last decades [1]. Globcan in 2018 provided a statistical report worldwide on the global burden of cancer, which shows 18.1 million new cancer incidences and 9.6 million mortalities. The female breast cancer is the second type of cancer, leading to death with 11.6%. The statics show that in every 8-10 women, one gets developed with breast cancer. Moreover, the fourth fatal cancer mortality is liver cancer with 8.2% [2].
There are several reasons for the tremendous growth of cancer mortality and new cases worldwide due to an increasing number of the population, high rate of aging, as well as prevalence changes and distribution of the risk factor for cancer linked with socioeconomic development. More specifically, the risk factors responsible for the development of breast cancer are reproductive factors (e.g., late menopause, multiparty, premature menarche, do not breastfeed), genetic factors (if her mother has breast cancer), lifestyle and dietary related factors (e.g., obesity, drinking, and smoking), and environmental factors (exogenous estrogen exposure for a long time). There are several types of breast cancer treatments including chemotherapy, radiotherapy, surgery, immune, and hormone therapy but still these treatments have diverse side effects. The urgent need to discover and develop a high efficacy anticancer therapy is highly challenged. Several therapeutic potentials have been discovered for cancer treatment with different targets; one of the most promising targets for cancer therapies is the JAK family [3]. JAK family members (JAK1, JAK2, JAK3, and TYK2) play an important role in the pathogenesis of many immunological disorders and cellular malignancies. The activity of JAK kinase was described in a series of abnormal cell proliferation of the hematologic neoplasias, including B-cell non-Hodgkin's and Hodgkin's lymphomas, myeloid leukemias, and lymphoid [4]. One of the most molecular targets and techniques for antiproliferative and pro-apoptotic effects are to design potential JAK inhibitors.

Synthesis
The benzo[b]thiophene analogue 1 was synthesized in high yield using the multicomponent strategy from ethyl cyanoacetate, cyclohexanone, sulfur and triethylamine in ethanol. The amino-ester 1 was reacted with phenyl isothiocyanate in ethanol containing Et3N to afford the N,N-disubstituted thiourea derivative 2. Whereas, when it was reacted with ethyl isothiocyanate under the same conditions, surprisingly it afforded the 2-(ethylamino)-5,6,7,8-tetrahydro-4Hbenzo [4,5]thieno [2,3-d] [1,3]thiazin-4-one 3 (Scheme 1). Acylation of the amino-ester 1 with chloroacetyl chloride in DCM and Et3N afforded the acylated ester 4. Compound 4 was used for the alkylation of cyclohexylamine to afford the alkylated product 5. The benzoylation of the benzo[b]thiophene analogue 1 was done by a reaction with benzoyl chloride in benzene containing triethylamine to afford the amide 6. The reaction of 1 with benzene sulfonyl chloride in ethanol yielded the sulfonamide 7 (Scheme 2). Cyclization of the amino-ester 1 was done by reflux with formamide for 3 h to give tetrahydrobenzo [4,5]thieno [2,3-d]pyrimidin-4(3H)-one 8. Michael additions of pyrimidinone 8 to acrylonitrile as the Michael acceptor in ethanol containing Et3N led to an addition at nitrogen to give the Michael adduct 9. Alkylation of 8 with benzyl bromide, phenacyl bromide, 4methoxyphenacyl bromide, and ethyl chloroacetate in DMF and K2CO3, afforded the N-alkylated products 10-13, respectively (Scheme 3). Hydrazinolysis of the ester 13 to hydrazide 14 was done by a reaction with hydrazine hydrate in ethanol. The heating hydrazide 14 with phenyl isothiocyanate in dioxane for 6 h gives the phenyl thiosemicarbazide product 15. The hydrazide 14 was refluxed with benzaldehyde and acetophenone in ethanol to give the hydrazones 16 and 17, respectively. Linking the 4amino-1,2,4-triazolethione moiety to the benzo [4,5]thieno [2,3-d]pyrimidin-4(3H)-one system separated by a methylene spacer was achieved by a reaction with CS2/aq. KOH/EtOH, to give potassium dithiocarbazate 18, which was cyclized by a reaction with hydrazine hydrate and acidification to give 19 (Scheme 4).   Oxygen nucleophiles such as sodium methoxide and sodium ethoxide were used to substitute the chloride of 20 to afford the ethers 29 and 30, respectively. A reaction of the chloro derivative 20 with nitrogen nucleophiles such as p-toluidine, benzyl amine, 4-aminobenzoic acid, and 4-acetylaniline afforded the substituted products 31-34, respectively. The reaction of the chloro derivative 20 with 2-aminobenzoic acid afforded the pyrimido-quinazolinone 35 (Scheme 7).

Structural Characterization
The 1 H NMR of 1 displayed the amino group as an exchangeable signal at δ 7.15 ppm. The ethoxy group protons (CH3CH2O) appeared at δ 4.15 and 1.25 ppm. 13 C NMR showed the ester group carbons at δ 165.53 (C=O), 59.03 (OCH2), and 14.79 ppm for CH3. The structure of 2 showed the two exchangeable signals at δ 11.78 and 10.88 ppm. The thiocarbonyl and carbonyl groups were detected at δ 176.36 and 166.02 ppm, respectively. Compound 3 missed the ester group signals and showed only the NH signal at δ 13.43 ppm in addition to the ethyl group signals at 4.40 and 1.20 ppm. The ethyl group carbon signals were found at δ 41.08 and 12.26 ppm, which strongly recommend the cyclic structure of 3. The acetamido group signals in 4 were found at 11.66 ppm for NH and 4.57 ppm for CH2Cl. The two carbonyl groups were identified at δ 165.45 and 164.23 ppm. The benzamide 6 and sulfonamide 7 NMR showed the NH signals at δ 12.03 and 10.36 ppm, respectively. Due to cyclization, the structure of 8 showed the NH as an exchangeable signal at 12.20 ppm and pyrimidine CH at δ 7.96 ppm. The carbonyl group was identified at δ 162.89 ppm. The aza-Michael addition and nitrogen alkylation of 8 to produce 9-13 were deduced from the missing NH signal and illustrated the methylene carbon signals directly attached to the ring nitrogen (NCH2) at δ 41.84, 48.83, 52.05, 51.64, and 47.25 ppm, respectively. The NMR of hydride 14 showed the -CO-NHNH2 group as two exchangeable signals at δ 9.36 and 4.25 ppm, whereas the carbonyl carbon was identified at δ 166.55 ppm. The thiosemicarbazide 15 displayed three NH signals at δ 10.56, 9.77, and 9.33 ppm. The thiocarbonyl and carbonyl carbons were detected at δ 181.06, 167.10, and 162.50 ppm. Hydrazones 16 and 17 were confirmed from the disappearance of the NH2 signal and the detection of benzylidene CH signals around 8.30 ppm. Compound 19 displayed the NH of the ring at δ 13.54 ppm and the NH2 protons at 5.61 ppm. The C=S was found at δ 167.32 ppm. The structure of 20 missed the NH signal, while 21 illustrated the NHNH2 exchangeable signals at δ 7.81 and 4.55 ppm. The benzoylated product 22 showed two NH signals at δ 9.64 and 8.29 ppm. The hydrazones 23 and 24 displayed the NH signal at δ 11.79 and 11.49 ppm, respectively, while the benzylidene CH was observed at 7.80 ppm in the case of 23. The cyclic structures 25 and 26 did not show any NH signal; only CH signals appeared in the aromatic region at 9.48 ppm in the case of 25 and two of the CH signals at 9.55 and 8.60 ppm in the case of 26. The pyrazole ring structure 27 revealed two CH protons at δ 8.97 and 6.17 ppm for the pyrimidine and pyrazole CH. The triazole-thione moiety construction in the case of 28 was identified from the detection of an NH at δ 14.62 ppm and the thiocarbonyl carbon at 160.56 ppm. The ethers 29 and 30 showed the CH signal of the pyrimidine ring at δ 8.55 and 8.52 ppm in addition to the alkoxy group indicating signals. The NH signals appeared in the spectra of 31-34 at δ 8. 34, 8.13, 5.09, and 8.49 ppm, respectively. The five-ring fused system 35 displayed the pyrimidine CH at δ 9.34 ppm, and the four phenyl protons appeared between δ 8.35 and 7.57 ppm.

Antitumor Activity Evaluation
The synthesized compounds were tested for their antiproliferative activity against MCF-7 and HepG-2 cancer cell lines. Twelve compounds displayed a significant activity, which ranged from 23.2 to 95.9 µM. Compounds 4, 24, 29, 30, and 31 were the most active with an IC50 range from 23.2 to 49.9 µM. Compounds 5, 15, 21, 26, 27, 28, and 33 revealed a moderate activity in the range from 52.9 to 95.9 µM. The remaining derivatives showed a lower activity (Table 1). Table 1. The in vitro inhibition % found using (100 µg/mL) of a single dose and IC50 in µg/mL and µM of the tested compounds on MCF-7 and HepG-2 cell lines.

Flow Cytometric Analysis
To demonstrate the apoptotic mechanistic mode of action, the MCF-7 cell line was treated with compound 4 (IC50 = 23.2 µM, 48 h incubation) compared to untreated cells as the control, which include flow cytometric analyses including an FITC/Annexin-V-FITC/PI differential apoptosis/necrosis assessment, DNA content-flow cytometry aided cell cycle analysis, and acridine orange quantitative autophagy assessment. All flow cytometric methodologies were performed according to the standard protocols, as previously described by Kattan et al., 2020 [30].
2.2.2.1. FITC/Annexin-V-FITC/PI differential apoptosis/necrosis assessment Double-staining with annexin-FITC and propidium iodide (PI) on MCF-7 cells treated with compound 4 (IC50 = 23.2 µM), and nontreated cells served as the negative control for 48 h to investigate whether it has an induction of apoptosis and/or cell cycle progression. As displayed in Figure 2, results show that the treatment caused apoptosis in MCF-7 cells with a substantial cell viability reduction of 26.86%. The percentage of cells sub-populating in early apoptosis (AV+/PI−) was 8.73, which is 2.3 times more relative to the untreated regulation. In addition, the average late apoptotic sub-population (AV+/PI+) count was 18.13%, which is 6.6 times higher than the untreated control. Moreover, the tested compound 4 induced an increase with 1.89-fold (6.29%, compared to 3.33% for control) in cell death via necrosis, as shown in Figure 2. The results proved that the tested compound 4 effectively induced apoptosis and necrosis in MCF-7 cells.

DNA content-flow cytometry aided cell cycle analysis
Cell cycle analysis is an important test that demonstrates the cell proliferation percentage during cell growth in every phase following cytotoxic compound treatment. Therefore, to analyze the cell cycle kinetics of MCF-7 cells treated with compound 4 (IC50 = 23.2 µM, 48h incubation), DNA flow cytometry was carried out to indicate phase interference with the cell cycle of the compound's. As shown in (Figure 3), results showed an induction an increase at G2/M-phase cell-cycle arrest with 1.48-fold (25.56%, compared to 17.23% for control), and at S-phase cell-cycle arrest with 1.39-fold (23.38%, compared to 16.76% for control) in cell population distribution, this may have resulted in genetic material degradation due to the apoptosis induction indicating compound 4 antiproliferative

Acridine Orange quantitative autophagy assessment
Herein, we further investigated the effect of compound 4 (IC50 = 23.2 μM) on the autophagy process within MCF-7 using the acridine orange lysosomal stain coupled with the flow cytometric analysis. The tested compound 4 did not induce any significant cell death by autophagy, it inhibited autophagic cell death (9.59%, compared to 11.24% for control) and this proves the antiproliferative activity of compound 4 through apoptosis and necrosis as a dual activity (Figure 4).

In Silico Molecular Docking Studies
A molecular docking study was made to investigate the binding interactions of hit 4 towards three proteins as JAK2 inhibitors; 3ZMM, 4C62, and 5AEP whose crystal structures complexed with their co-crystallized ligands were easily accessible from the Protein Data Bank. The co-crystallized ligands of the studied proteins form hydrogen bonds with Leu 932 as the key amino acid for interaction.
As seen in (Table 2) with 3D images, compound 4 was docked inside the protein active site of the studied proteins and formed one hydrogen bond with bond length (A º ) through the carbonyl group oxygen as a hydrogen bond acceptor with the key amino acid Leu 932 as their co-crystallized ligand with binding energy −14.32, −13.39, and −11.38 Kcal/mol, respectively. Additionally, compound 4 formed lipophilic interactions with the nonpolar amino acids (Leu 155, Val 24, Ile 140, Leu 90, and Leu 141) inside the receptor pocket. Different models obtained from the docking studies indicated that the designed target 4 showed promising binding activity as JAK2 inhibitors, and this may be the proposed mode of action for the anti-breast cancer activity. Superimposed compound 4 (orange), and the co-crystalized ligand (green) of the three studied 3ZMM, 4C62, and 5AEP proteins.

Bioinformatics Study
Compounds with high binding affinity through ligand-receptor interactions and binding energy towards the investigated target (Jak2/STAT3) were subjected to bioinformatics study to predict the ADME pharmacokinetics properties. Drugs consistent with Lipinski's "five rule" (Ro5) are considered prospective in future [31][32][33][34]. For drug absorption through the intestine, TPSA surface topological polar area values should be 140, and the barrier to blood brain should be as low as 90 Å 2 [30,31] The investigated compounds had good well-permeability and absorption. As shown in (Table 3) and ( Figure 5), compounds had 0-1 Hydrogen-bond donor and 3-4 Hydrogen-bond acceptors. In addition, the tested compounds had log P range from 2.82 to 4.80, so they had strong toleration by cell membranes. For managing conformational changes and for oral bioavailability, the number of rotabile bond (nrotb) should be less than 10 [31,35,36]. All the investigated compounds had 1-5 nrotb. Regarding to drug-likeness scores, compounds with positive values should be considered like drugs; all the tested compounds showed 0.15-0.73 (positive values), so they seem to be drug-like.   At the end of the experiment, animals from different groups were anesthetized, and blood samples were collected for the determination of liver enzymes ALT, AST levels, and CBC parameters, including Hb content, RBCs and WBCs counts. As seen in Table 4, in SEC-bearing mice, liver enzymes ALT and AST were found to be significantly increased to 84.65 and 97.64 (U/L), respectively, as compared to normal mice, this could be contributed to the hepatocellular damage following tumor inoculation. The treatment with compound 4, such as 5-FU, substantially reduced liver enzyme, where elevated transaminases ALT were restored to 64.28, AST to 69.89 close to the normal values measured in normal mice (43.53 and 45.75, respectively), indicating a noticeable improvement in the hepatocellular toxicity induced by SEC proliferation. Regarding hematological parameters in SEC-bearing mice, all CBC parameters were changed upon treatment with compound 4 where the Hb content and RBCs were significantly decreased to be 3.99 (g/dL) and 3.28 (10 6 /µL), respectively, while the WBCs count was significantly increased to be 4.89 (10 3 /µL) compared to the normal control levels. Myelosuppression and anemia are the main issues of cancer chemotherapy. Anemia in a tumor-bearing mouse is primarily due to reductions in the RBC count and hemoglobin, which are either hemolytic or myelopathic [30]. CBC parameters for hemoglobin, RBC, and WBC levels have been greatly improved by compound 4 treatments. CBC parameters improved to 5.99, 4.43, and 4.21, respectively, relative to normal control values 7.9, 5.14, and 3.69, hence demonstrating the ability of the tested compound to cure the change of the hematological parameters.

Materials and Methods
All general information about the equipment used in this text, biological activities assays (in vitro MTT assay, flow cytometric analysis, in silico molecular docking, bioinformatics study, and in vivo SEC model) of full protocols have been amended in the Supplementary material.

Synthesis
Synthesis of (1): Sulpher (0.02 mol) was added to a mixture of ethyl cyanoacetate (0.03 mol) and cyclohexanone (0.03 mol) in 30 mL of ethanol followed by the addition of Et3N (4.3 mmol), the mixture kept on stirring at an ambient temperature for 2 h, then refluxed for further 2 h. The reaction progress was monitored by TLC then, cooled, added to ice water, and kept overnight to complete the precipitation. The ppt was filtered, washed with water, dried, and recrystallized from ethanol to give yellow needle crystals. Synthesis of (2) and (3): A mixture of amino-ester 1 (2.2 mmol) and the appropriate isothiocyanate (2.2 mmol) and Et3N 0.5 mL was refluxed in 30 mL of absolute ethanol for 3 h then left to cool. Acidified with concentrated HCl, the formed ppt was collected by filtrations, washed with water, dried, and recrystallized from ethanol.

In Vitro
In vitro work regarding cell culturing, cytotoxic screening using the MTT assay, and the IC50 calculations were made according to Mosmann 1983 (see Supplementary materials) [38].

Flow Cytometry
All flow cytometry including FITC/Annexin-V-FITC/PI differential apoptosis/necrosis assessment, DNA content-flow cytometry aided cell cycle analysis, and acridine orange quantitative autophagy assessment were made according to Nafie et al., 2020 [39].

In Silico Molecular Docking
All in silico studies including ligand optimization, protein preparation, and molecular docking calculation were investigated followed by the reported [40].

Bioinformatics
Bioinformatics study (in silico and bioactivity prediction) of the most active compounds were calculated using a set of software's including MolSoft, Molinspiration [41], and SwissADME [42] websites as previously described by Youssef et al., 2020 [37].

In vivo (SEC) Model
Experiment design and methodology including tumor volume and percentage of tumor inhibition were summarized in Figure 7.

Statistical Analysis
Data were represented by the value of the mean for three different replicates, with the standard error of the mean (mean ± SEM). All statistical analysis was done as using GraphPad Prism software version 7.0. The statistical difference between two groups was examined using the unpaired t-test. The significance level was set at P < 0.05.

Conclusions
In conclusion, approximately 33 compounds were synthesized and characterized. The target compounds were evaluated in vitro against HepG-2 and MCF-7 cancer cell lines. Hit 4 was found to be the most active, and the cell cycle analysis showed that this lead induces apoptosis. Moreover, the in vivo study demonstrates that our target significantly reduces the tumor mass. The in silico molecular docking shows the binding of 4 as an agonist for JAK2 inhibitors.
Supplementary Materials: The following are available online, Full protocols for the biological assays; Figure  S1-S87 copies of the HNMR and CNMR spectrum of the synthesized compounds.

Conflicts of Interest:
The authors declare no conflict of interest.