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Synthesis and Characterization of New Dihydronaphthalene Candidates as Potent Cytotoxic Agents against MCF-7 Human Cancer Cells

In the present work, a new series of dihydronaphthalene derivatives were synthesized starting with 6-methoxy-1-tetralone 1, and the corresponding hydrazine derivative 2. Reaction of compound 2 with aryl isothiocyanates produced thiosemicarbazides 3a-d, which were reacted with ethyl chloroacetate to give thiazolidinone derivatives 4a-d. Pyrano thiazolecarbonitrile derivatives 5a-f were prepared by heating a mixture of compounds 4a or 4c, aryl aldehydes, and malononitrile utilizing distilled water in the presence of catalytic amount of potassium hydrogen phthalate. Also, treatment of 4a with DMF-DMA under solvent-free conditions gave enaminone derivative 6, which condensed with ethyl acetoacetate or acetylacetone or malononitrile or cyanothioacetamide to give compounds 7-10, respectively. Finally, reaction of the enaminone 6 with 2-aminoimidazol or 2-aminothiazol in the presence of glacial acetic acid produced derivatives 11 and 12, respectively. Cytotoxic evaluation of eleven compounds, against MCF-7 (human breast adenocarcinoma) cell lines, was estimated. Results revealed that five of the examined compounds 5a, 5d, 5e, 10, and 3d showed potent cytotoxic activities recording, IC50 values; 0.93 ± 0.02, 1.76 ± 0.04, 2.36 ± 0.06, 2.83 ± 0.07, and 3.73 ± 0.09 μM, respectively, which were more potent than the reference used (Saturosporin, IC506.08 ± 0.15 μM). The new products were also examined towards normal epithelial breast cells (MCF10A). All of them showed very good safety profile with different degrees and were safer than the reference drug used. Compound 5a was the most effective against MCF-7 cells and was less toxic than Saturosporin by about 18.45-folds towards MCF01A normal cells. All the new compounds were fully characterized by the different spectral and analytical tools. Herein, detailed syntheses, spectroscopic, and biological data are reported.


Introduction
Breast cancer represents the most common cancer disease among women. It represents the second-highest rate leading cause of women mortality worldwide [1]. Chemotherapy is the most common for cancer treatment. Developing cancer drugs is essential to discover more active products with high potential [2]. This work represents an attempt to develop new therapeutic compounds of high efficacy in treating breast cancer disease. Literature reports confirmed the important diverse types of pharmaceutical activities of thiazole, pyrane, and/or pyridine derivatives. Thiazoles have concerned a great era of attention due to their association with various types of biological activities. Their derivatives exhibited important potency as anticancer [3][4][5], antibacterial [6], anti-inflammatory [7], antioxidant [8], antimalarial [9] agents, and HIV-inhibitors [10]. Also, pyrane containing derivatives have been identified as anticancer [11], antimicrobial [12], anti-inflammatory [13], and antimalarial [14] agents. Furthermore, literature survey reported that compounds containing pyridine ring demonstrated anticancer [15], antimicrobial [16], anti-inflammatory [17], antiviral [18], and antioxidant [19] activities. Based on our recent work which demonstrated that various tetralone-based derivatives verified significant inhibitory activity towards different types of cancer disease, they displayed highly significant cytotoxic activity against MCF-7 human cancer cells (breast cancer), for all the tested compounds, all of them showed activity more potent than that obtained by the reference drug (Doxorubicine) [20]. Also, significant cytotoxic effects on U373 (human glioblastoma cells) were testified [21]. Additionally, cytotoxic activity against HepG-2 (human cancer cells) was established [22]. These facts motivated us to synthesize new compounds derived from coupling of 6-methoxy-1-tetralone with different heterocyclic ring systems such as thiazole, pyrano [2,3-d] thiazole, and dihydrothiazolo [4,5-b] pyridine in one molecule aiming to construct new candidates of enhancing anticancer activity. Herein, 6-methoxy-1-tetralone was used as good building blocks to construct the desired heterocyclic products.

Materials and Methods
2.1. Chemistry. Melting points were uncorrected and were taken in open capillary tubes using Electrothermal apparatus 9100. Infrared spectra were recorded on a Shimadzu FT-IR Affinity-1 Spectrometer, Infrared spectrometer at cm -1 scale using KBr disc technique at Faculty of Pharmacy-Cairo University, Cairo, Egypt. 1 H NMR and 13 C NMR spectra were determined by using a Bruker High-Performance Digital FT-NMR Spectrometer Avance III 400 MHz, Faculty of Pharmacy-Cairo University, Cairo, Egypt. Chemical shifts were expressed in δ (ppm) downfield from TMS as an internal standard. The mass spectra were recorded on a GCMC-QP 1000 EX Shimadzu gas chromatograph-mass spectrometer (GC-MS; Shimadzu Corp. Kyoto, Japan) at electron ionization (EI) of 70 eV. Elemental analyses (C, H, and N) were conducted at the Micro Analytical Center of the Faculty of Science of Cairo University, Cairo, Egypt. All reagents were commercial grade and used without further purification. Reaction progress was monitored by thin-layer chromatography (TLC) on precoated (0.75 mm) silica gel GF254 plates (Merck Group, Darmstadt, Germany). Products were visualized under ultraviolet (UV) light.

Cell Lines and Cell
Culture. Both MCF-7 and MCF10A cells were purchased from American Type Culture Collection (ATCC). Cells were grown in DMEM culture medium (Invitrogen/Life Technologies) supplemented with 10% FBS (Hyclone, USA), 10 μg/mL insulin (Sigma), and 1% penicillin-streptomycin antibiotic solution. Chemicals used were of cell culture grade and were purchased from Sigma or Invitrogen. Prior to the assay, cells (cells density 1:2 -1:8 × 10,000 cells/well) were plated in 96-well plate with 100 μL medium and were allowed to grow for 24 h.
2.3. In Vitro Cytotoxicity Assay. In vitro cytotoxic activity of the prepared compounds against breast (MCF-7) cancer cells was assessed using MTT assay [24,25]. The assay depends on the mitochondrial reduction of the colorless 3-(4,5-methyl-2thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) within viable cells into a dark blue formazan product. Cells were cultured in DMEM medium supplemented with 10% FBS at a final concentration of 2 × 10 4 cells/mL in 96-well plates and incubated in a 5% CO 2 incubator at 37°C. Twelve hours later, different concentrations (0.39-100 μM) of the tested compound (2 μL) were added to the cells (2 × 10 4 ) in 96-well plates and cultured at 37°C for 3 days. Then, 20 μL of MTT solution was added to the cultured cells and incubated for four hours at 37°C. The supernatant was taken away from each well, and 100 μL of DMSO was added to each well to dissolve the formazan crystals. After mixing with a mechanical plate mixer, a microplate reader was used to measure the absorbance of each well at a wavelength of 570 nm. Data were expressed as IC 50 (μM), i.e., the concentration required to inhibit 50% of viable cell growth. IC 50 values were calculated from the linear regression of the corresponding calibration curves using the Origin® 6.1 software. Each experiment was carried out in triplicate with good reproducibility and standard errors.

Statistical Analysis.
Results were analyzed with the help of SPSS 9.0 and were presented as mean ± SD of three replicates. The mean comparison between different evaluated groups was performed using ANOVA one-way analysis of variance. Statistical significance was defined when p < 0:05.
On the other hand, synthesis of the enaminone, 6 was essential to construct biologically active heterocyclic products. Reaction of the thiazolidinone 4a with dimethylformamidedimethylacetal (DMF-DMA), under solvent-free conditions, produced the enaminone derivative 6 within 1 h., in 87% yield. The enamine 6 was condensed with ethyl acetoacetate or acetyl acetone in the presence of ammonium acetate and glacial acetic acid, to afford the thiazolopyridine carboxalate and thiazolo-pyridine ethanone derivatives 7and 8, respectively, in 83 and 80% yields. While, reaction of compound 6 with cyanothioacetamide or malononitrile in ethanolic sodium ethoxide gave thioxo-pyridine or oxo-pyridine carbonitrile derivatives 9 and 10, respectively, in 83 and 81% yields (Scheme 2).

Cytotoxic
Screening. Eleven compounds were investigated in vitro for their activities against breast cancer cell line MCF-7 using MTT assay. The effect of different concentrations of the newly synthesized compounds was evaluated by determining the percentages of viable cells after being exposed to the applied concentrations, compared to Staurosporin as a reference drug. In this screening, all the tested compounds showed potential cytotoxic activities against MCF-7 cells in a dose-dependent manner (Figure 1). Furthermore, it can be seen that the compounds affected cell viability in different patterns. This can be attributed to the differences of cellular response to each compound, depending on the nature of its terminal and functional   [26,27]. Results demonstrated that five of the tested compounds 5a, 5d, 5e, 10, and 3d showed potential cytotoxic activities against breast cancer cell line MCF-7, recording IC 50 values of 0:93 ± 0:02, 1:76 ± 0:04, 2:36 ± 0:06, 2:83 ± 0:07, and 3:73 ± 0:09 μM, respectively (Table 1). Additionally, it can be seen that these potential compounds had cytotoxic activities higher than the tested positive control (Saturosporin, IC 50 6:08 ± 0:15 μM). Comparing these results with those obtained against normal breast cell line (MCF10A) showed that the tested compounds were less toxic toward normal cells. Compound 5a was the most effective against MCF-7 cells and was less toxic than Saturosporin by about 18.45-folds towards MCF01A normal cells. The acquired data revealed that coupling the pyrano ring in this fused heterocyclic ring system was critically influenced the cytotoxic activity. All the tested pyrane containing compounds (5a, 5d, and 5e) were the most effective cytotoxic agents and were more potent than Staurosporin (IC 50 ; 0.93, 1.76, and 2.36 μM, respectively, IC 50 Staurosporin; 6.08 μM). The size of Nsubstitution in thiourea-imino-linked to hydronaphthaline core directly affected the cytotoxic activity. Accordingly, the N-ethyl thiourea derivative 3d had a very good cytotoxicity (IC 50 ; 3.73 μM). On the other hand, the N-4bromophenyl substitution of thiourea in analog 3a resulted in more than 3 times decrease in cytotoxicity. Also, a closer antiproliferative potency was exhibited in compound 10 (IC 50 ; 2.83 μM), in which our core was imino-linked to 5-thioxo-2,3,4,5-tetrahydrothiazole [4,5-b]pyridine-6-carbonitrile ring system. While, the methylation of C-5 and acetylation of C-6 of the dihydrothiazolo [4,5-b] pyridinyl ring system in compound 8 yielded a significantly weaker cytotoxic activity than compound 10 (IC 50 ; 20 μM). This highlighted the importance of 5-thioxo and 6-carbonitrile functionality for MCF7 antiproliferative activity in this fused heterocyclic ring system.

Data Availability
All data generated in this current work are included in the "Results and Discussion" section.