Design, Synthesis, and Biological Evaluation of 2-Mercaptobenzoxazole Derivatives as Potential Multi-Kinase Inhibitors

A series of 12 compounds was designed and synthesized, based on 2-mercaptobenzoxazole derivatives containing either the substituted benzenes 4a–d, substituted isatins 5a–f, or heterocycles 6a–b. The in vitro antiproliferative activity of the compounds was evaluated against hepatocellular carcinoma (HepG2), mammary gland cancer (MCF-7), breast cancer (MDA-MB-231), and the epithelioid cervix carcinoma (HeLa) cancer cell lines. Compounds 4b, 4d, 5d, and 6b had the most potent antiproliferative activity, with IC50 values ranging from 2.14 to 19.34 µM, compared to the reference drugs, doxorubicin and sunitinib. Compound 6b revealed a remarkably broad antitumor activity pattern against HepG2 (IC50 6.83 µM), MCF-7 (IC50 3.64 µM), MDA-MB-231 (IC50 2.14 µM), and HeLa (IC50 5.18 µM). In addition, compound 6b showed potent inhibitory activities against EGFR, HER2, VEGFR2, and the CDK2 protein kinase enzymes, with IC50 values of 0.279, 0.224, 0.565, and 0.886 µM, respectively. Moreover, compound 6b induced caspase-dependent apoptosis and cell cycle arrest at the G2/M phase. Finally, a molecular docking simulation was performed for compound 6b to predict the potential ligand–protein interactions with the active sites of the EGFR, HER2, and VEGFR2 proteins.


Introduction
Cancer is a very complex disease, compared to other conditions, and has led to a variety of cellular abnormalities and genetic disorders. Worldwide, cancer may be considered the most widespread health apprehension illness that affects human beings. It is known as a group of illnesses that are characterized by unregulated cell growth and division [1]. Globally speaking, cancer is among the leading causes of mortality and morbidity, affecting almost every region at a socioeconomic level [2]. According to the figures released by the World Health Organization (WHO), the prevalence of cancer disease is anticipated to rise by nearly 22 million cases, with 13 million deaths by 2030 [3]. There are two leading causes of abnormal growth in human cancers: the first is the disturbance of the signaling pathways in the cells and the second is the disruption of the cell cycle [4][5][6]. Drug resistance is considered one of the main difficulties in cancer therapy. It may be developed in all types of cancers and in therapeutic strategies such as immunotherapy, molecularly targeted therapy, and chemotherapy [7].
Protein kinase enzymes have a vital role in the signal transduction processes that control many cellular functions. In the last few decades, kinase enzyme inhibitors have received attention because of their current role in treating cancer. The epidermal growth factor receptor (EGFR) is a tyrosine kinase transmembrane receptor that regulates many signal transduction pathways (Ras/MAPK, Jak/STAT, and PI3K/Akt) that control cell growth, division, and apoptosis [8,9]. The overexpression of EGFR is involved in varied types of cancer, such as breast, ovarian, prostate, and colon cancers, by stimulating neoplastic cell proliferation, metastasis, angiogenesis, and invasiveness [8]. Furthermore, the EGFR-signaling cascades activate the vascular endothelial growth factor (VEGF), which is recognized as the main stimulator of angiogenesis. Conversely, the interaction between vascular endothelial growth factor receptor 2 (VEGFR-2) and VEGF will activate signaling cascades (PI3K/Akt and p38MAPK) that affect many cellular functions, such as migration, survival, vascular permeability, and the proliferation of cancer tissue cells, and, hence, trigger angiogenesis [9]. Moreover, it was determined that VEGFR-2 is highly expressed in tumor cells, particularly in epithelial tissue cells. Additionally, each EGFR, VEGFR-2, and glycoprotein is structurally related; the suppression of EGFR reduces the expression of VEGF, while the targeting of VEGFR-2 induces the antitumor effect of the EGFR inhibitors. Hence, current therapeutic strategies support the twin suppression of each VEGFR-2, and EGFR represents a promising cancer-fighting strategy [8].
CDKs are defined as a large family of serine/threonine kinase enzymes that catalyze the phosphorylation of proteins [10]. More than 30 cyclin proteins have been identified in the human body, based on the box domain of the cyclin protein, which can bind and activate the CDKs. The dysfunction and/or overexpression of cyclins or CDKs have been recognized in many human cancers and various pathologies. Thus, these protein kinase enzymes are considered potential therapeutic goals for drug design and development [11,12].
One of the most interesting heterocycles in medicinal chemistry is benzoxazole. Benzoxazole derivatives have drawn much attention in the last few decades, due to their utility as intermediates for synthesizing novel, biologically active molecules. Moreover, benzoxazole derivatives display a wide range of pharmacological activities, comprising anticancer, antiviral, antifungal, and anti-inflammatory activities [13,14]. A benzoxzole derivative (I) demonstrated promising antiproliferative activity against various distinct cancer cell lines, with IC50 values in the submicromolar range ( Figure 1).  The strategy to prepare the designed 2-mercaptobenzoxazole derivatives is outlined in Scheme 1. In the initial step, ethyl 2-(2-mercaptobenzoxazole) acetate (2) was synthesized by refluxing 2-mercaptobenzoxazole (1) and ethyl chloroacetate in dry acetone as a solvent for 5-10 h, using anhydrous potassium carbonate as a basic catalyst [13]. This was followed by hydrazide formation through the reaction of the resulting ethyl ester (2) with hydrazine hydrate in ethanol and reflux for 6-10 h, to yield the hydrazide derivative (3). Next, the obtained hydrazide derivative (3) was subjected to Schiff's base formation through the nucleophilic addition-elimination reaction with the appropriate aldehyde or ketone. Finally, to obtain derivatives 4(a-d), the hydrazide derivative (3) was dissolved in the appropriate amount of ethanol, then mixed with the appropriate benzaldehyde derivative. Acetic acid was then added as a catalyst and the mixture was refluxed for around 6-10 h at 90 • C. The same procedure was applied to synthesize compounds 5(a-f), but with the replacement of the benzaldehyde derivatives with isatin derivatives. Thiophene-2carbaldehyde and 1H-pyrrole-2-carbaldehyde were used as the carbonyl part in Schiff's base formation for the synthesis of compounds 6(a-b), according to the procedure mentioned in the experimental section.
The physicochemical characteristics of the newly prepared compounds were determined and reported in the experimental section. The molecular structures of the prepared molecules, 4(a-d), 5(a-f), and 6(a-b), were confirmed by spectroscopic analytical methods, including 1 H-NMR (DMSO-d6, 700 MHz, ppm), 13 C-NMR (DMSO-d6, 176 MHz, ppm), as well as melting point, elemental analysis, and mass spectral analyses. The physicochemical characteristics of the newly prepared compounds were determined and reported in the experimental section. The molecular structures of the prepared molecules, 4(a-d), 5(a-f), and 6(a-b), were confirmed by spectroscopic analytical methods, including 1 H-NMR (DMSO-d6, 700 MHz, ppm), 13 C-NMR (DMSO-d6, 176 MHz, ppm), as well as melting point, elemental analysis, and mass spectral analyses.
In the case of the 1H-NMR spectra, the presence of multiple signals between 7 and 9 ppm reflected the presence of aromatic protons in the prepared derivatives. All the synthesized derivatives, without any exception, have a diastereomeric center at the -N=Cbond; hence, they exist as a E/Z diastereomeric mixture with variable ratios. The synthesized derivatives have a singlet signal at the aliphatic region (~4.25-4.95 ppm) of the methylene -CH2-protons. Compounds 4d, 5b, and 5d have an extra signal at the aliphatic part, due to the presence of the -CH3 group, which appears as a singlet at 3.85, 2.30, and 3.80 ppm, respectively. Compound 4d showed a broad singlet at 9.57 and 9.59 ppm for the E-diastereomer and Z-diastereomer, respectively, because of the aromatic hydroxyl group. Finally, DMSO-d6 was used for recording the 13C-NMR spectra of 2mercaptobenzoxazole derivatives; it was observed that the spectral signals and proposed molecular structure of the synthesized molecules showed good agreement. The reaction times (h), E:Z ratio, the percentage of the yields, and the melting points of the products are given in Table 1 In the case of the 1H-NMR spectra, the presence of multiple signals between 7 and 9 ppm reflected the presence of aromatic protons in the prepared derivatives. All the synthesized derivatives, without any exception, have a diastereomeric center at the -N=Cbond; hence, they exist as a E/Z diastereomeric mixture with variable ratios. The synthesized derivatives have a singlet signal at the aliphatic region (~4.25-4.95 ppm) of the methylene -CH2-protons. Compounds 4d, 5b, and 5d have an extra signal at the aliphatic part, due to the presence of the -CH3 group, which appears as a singlet at 3.85, 2.30, and 3.80 ppm, respectively. Compound 4d showed a broad singlet at 9.57 and 9.59 ppm for the E-diastereomer and Z-diastereomer, respectively, because of the aromatic hydroxyl group. Finally, DMSO-d6 was used for recording the 13 C-NMR spectra of 2-mercaptobenzoxazole derivatives; it was observed that the spectral signals and proposed molecular structure of the synthesized molecules showed good agreement. The reaction times (h), E:Z ratio, the percentage of the yields, and the melting points of the products are given in Table 1.

Cytotoxicity Assay
The in vitro cytotoxicity for the final target compounds, 4(a-d), 5(a-f), and 6(a-b), was preliminarily evaluated via the MTT colorimetric assay against a panel of four human cell lines, namely, the hepatocellular carcinoma (HepG2), mammary gland (MCF-7), breast cancer (MDA-MB-231) and epithelioid cervix carcinoma (HeLa) [33][34][35][36]. Doxorubicin and sunitinib were used as reference drugs [37,38]. The activities of the tested compounds are expressed as IC50 values (µ M) and are shown in Table 2 The in vitro cytotoxicity for the final target compounds, 4(a-d), 5(a-f), and 6(a-b), was preliminarily evaluated via the MTT colorimetric assay against a panel of four human cell lines, namely, the hepatocellular carcinoma (HepG2), mammary gland (MCF-7), breast cancer (MDA-MB-231) and epithelioid cervix carcinoma (HeLa) [33][34][35][36]. Doxorubicin and sunitinib were used as reference drugs [37,38]. The activities of the tested compounds are expressed as IC 50 values (µM) and are shown in Table 2. The tested molecules exhibited varying degrees of cytotoxic activities and potencies against the investigated cell lines, HepG2, MCF7, MDA-MB-231, and HeLa cell lines, compared to the positive control reference drugs. As a general pattern, the breast cancer (MDA-MB-231) cell line was the cancer cell line most sensitive to the synthesized compounds. Compound 4b displayed moderate anticancer activity against the tested cell lines, with IC 50 values ranging from 9.72 to 19.34 µM. Moreover, compounds 4d and 5d were very potent and show highly promising activity against all the tested cell lines, with IC 50 values ranging from 2.14 to 12.87 µM. Additionally, compound 6b revealed similar inhibitory activity with the reference drugs doxorubicin and sunitinib, against the HepG2, MCF-7, MDA-MB-231, and HeLa cell lines, with IC 50 values of 6.83, 3.64, 2.14, and 5.18, respectively. On the other hand, the remaining compounds demonstrated modest antiproliferative activities against the tested cell lines.

Structure-Activity Relationship (SAR)
In series 4 and 5, compounds with a methoxy substitution (compounds 4d and 5d) were the most active compounds against the four cell lines. Replacing the substituted phenyl in series 4 or the substituted isatin in series 5 with a pyrrole ring system (compound 6b) resulted in a significant increase in potency against the four cell lines, with slightly better potency than the potency of the reference compound sunitinib, especially against the MDA-MB-231 cell line.

In Vitro Kinase Inhibitory Activity
Compound 6b, due to its excellent cytotoxic effect, was also subjected to further biological investigations, including enzyme assays against CDK2, EGFR, HER2 and the VEGFR2 protein kinases, in a trial to confirm the mechanism of its cytotoxicity. As shown in Table 3, the results revealed that compound 6b has good inhibitory activity in the nanomolar range on the CDK2, EGFR, HER2, and VEGFR2 protein kinases compared to the reference drugs, with HER2 displaying the most sensitivity, followed by EGFR, CDK2, and VEGFR2. The average IC 50 values of the respective kinases were 0.224, 0.279, 0.886, and 0.565 µM, respectively. These results confirm the previous speculation that these molecules may have multiple cellular targets, including the inhibition of these kinase enzymes, and this may aid in overcoming the drug resistance phenomenon for many of the clinically employed anticancer agents. Consequently, more biological studies should be performed to explore the mechanism of action inside cancer cells. To rationalize the cytotoxic activities and predict the possible types of drug-receptor interactions of the synthesized compounds, the most potent compound, 6b, was docked into the active binding sites of EGFR, VEGVR2, and HER2. The co-crystalized ligands, erlotinib, sorafenib, and lapatinib, were used as reference compounds for EGFR, VEGVR2, and HER2, respectively. First, erlotinib and compound 6b were docked into the ATP-binding site of EGFR. Erlotinib created one hydrogen bond with Met769 and multiple hydrophobic interactions with Lys721, Val702, Ala719, Leu820, Leu694, Thr830, and Met769. Conversely, compound 6b formed two hydrogen bonds with Asp831 and Thr830 and several hydrophobic interactions with Leu694, Ala719, Val702, Leu820, Met742, Leu742, Leu753, and Cys751. It is obvious that compound 6b forms different hydrogen bonds than erlotinib, while the hydrophobic interactions were almost the same; however, compound 6b was superimposed on erlotinib in the active site of EGFR ( Figure 2 and Figure S1 in the Supplementary Materials). The binding energies of erlotinib and compound 6b with the EGFR were −9.5 and −8.0 Kcal/mol, respectively. Second, docking sorafenib into the active site of VEGFR2 resulted in four hydrogen bonds with Cys919 (two hydrogen bonds), Asp1046, and Glu885, and several hydrophobic interactions with the amino acid residues of the active pocket of VEGFR2. The oxygen atom of the benzoxazole ring of compound 6b served as a hydrogen bond acceptor by making a bond with Asp1046, while other functional groups stabilized the compound by making hydrophobic interactions with Leu889, Ala866, Leu1035, Cys919, Leu840, and Val848. In comparing compound 6b with sorafenib, compound 6b was superimposed onto sorafenib in the active site of VEGFR2; however, it then made fewer hydrogen bonds and hydrophobic interactions ( Figure 3 and Figure S2 in the Supplementary Materials). Sorafenib and compound 6b showed binding affinities of −11.9 and −8.4 Kcal/mol, respectively, with VEGFR2. Lastly, lapatinib and compound 6b were docked into the active binding site of the HER2 receptor. Lapatinib superimposed lapatinib and interacted with the amino acid residues of Met801, Thr798, and Thr862 by hydrogen bonds, with Ser783 and Arg784 by halogen bonds, and with Arg784, Leu785, Leu796, Val734, and Lys753 by hydrophobic interaction. Compound 6b formed two hydrogen bonds with Lys753 and The862, in addition to several hydrophobic interactions with Ala751, Leu796, Thr862, val734, and Leu 852 ( Figure 4 and Figure S3 in the Supplementary Materials). The binding energy with HER2 was found to be −9.8 and −8.7 Kcal/mol for lapatinib and compound 6b, respectively. sorafenib, compound 6b was superimposed onto sorafenib in the active site of VEGFR2; however, it then made fewer hydrogen bonds and hydrophobic interactions (Figures 3 and S2 in the Supplementary Materials). Sorafenib and compound 6b showed binding affinities of −11.9 and −8.4 Kcal/mol, respectively, with VEGFR2. Lastly, lapatinib and compound 6b were docked into the active binding site of the HER2 receptor. Lapatinib superimposed lapatinib and interacted with the amino acid residues of Met801, Thr798, and Thr862 by hydrogen bonds, with Ser783 and Arg784 by halogen bonds, and with Arg784, Leu785, Leu796, Val734, and Lys753 by hydrophobic interaction. Compound 6b formed two hydrogen bonds with Lys753 and The862, in addition to several hydrophobic interactions with Ala751, Leu796, Thr862, val734, and Leu 852 (Figures 4 and S3 in the Supplementary Materials). The binding energy with HER2 was found to be −9.8 and −8.7 Kcal/mol for lapatinib and compound 6b, respectively.      Compound 6b was selected to investigate the apoptotic markers against the liver cancer cell line (HepG2) [34]. It exhibited good cytotoxicity toward the tested cell lines, with significantly low IC 50 values ranging from 2 to 6 µM. Typically, Bax, Bcl-2, and caspases contribute to the regulation of apoptotic signaling. Acting as an apoptotic activator (Bax) or inhibitor (Bcl-2), the Bcl-2 family of proteins plays a significant role in apoptosis [39]. The caspase family comprises cysteine proteases that are classified as either executioner-caspase-3, or as initiators-caspase-9 and caspase-8 [40]. The results in Table 4 showed the strong stimulated expression of the pro-apoptotic Bax gene (5.34 folds) and apoptotic genes, along with Caspase-3 (5.02 folds) and Caspase-9 (3.5 folds), compared to the negative controls. Nevertheless, considerable downregulation of the anti-apoptotic gene, Bcl-2 (0.26 folds), was described, leading to an increased expression ratio of Bax/Bcl-2 (1→20.53). The Bax/Bcl-2 gene expression ratio can serve as an early predictor for cancer in patients and is a sensitive monitor of cancer progression [41]. Collating all the provided evidence, the upregulation of caspase-3, caspase-9, and Bax is clear, while the downregulation of Bcl-2 genes makes it evident that compound 6b induces apoptosis within the liver cancer cell line (HepG2).  Investigation of the mechanistic growth-inhibitory action of compound 6b on liver cancer cells (HepG2), a cell-cycle and apoptosis rate analysis was conducted at the compound's approximated IC 50 value (6.83 µM). Programmed cell death is a fundamental cellular program that is inherent in every cell of the human body. One of the most extensively studied forms of programmed cell death is apoptosis, which plays an important role during the various physiological processes and is involved in a variety of pathological conditions [42]. The HepG2 cells were exposed to compound 6b for 24 h and the cell cycle was monitored via flow cytometry; the results are reported in Table 5. The impact of compound 6b on the cell cycle distribution revealed that the predominant cell population at the G2/M stage (31.26%) was significantly higher than that of the untreated cell line (7.12%). Furthermore, a significant reduction at the S stage complemented the elevated cell population at the G2/M stage of the treated cells compared to the negative controls (28.37% vs. 43.16%) (see Figure 5).
To identify the mode of cell death promoted by compound 6b within the liver cancer cells (HepG2), apoptosis rate analysis was performed, following 24 h of exposure. Compound 6b, at a concentration of 6.83 µM, induced both early-and late-stage apoptosis in the HepG2 cell line, with significantly elevated percentage apoptotic cell levels compared to the controls (1.64% and 10.91%, respectively). Moreover, the average proportion of Annexin-V-stained positive cells (total apoptotic cells) was elevated from 2.02% within the untreated cells to 16.84% within the treated ones. Interestingly, compound 6b showed little influence on the necrosis of liver cancer cells. The provided findings are supported by the previous cell cycle analysis, confirming the potentiality of 6b as a promising anticancer agent (see Table 6 and Figure 6).  To identify the mode of cell death promoted by compound 6b within the liver cancer cells (HepG2), apoptosis rate analysis was performed, following 24 h of exposure. Compound 6b, at a concentration of 6.83 μM, induced both early-and late-stage apoptosis in the HepG2 cell line, with significantly elevated percentage apoptotic cell levels compared to the controls (1.64% and 10.91%, respectively). Moreover, the average proportion of Annexin-V-stained positive cells (total apoptotic cells) was elevated from 2.02% within the untreated cells to 16.84% within the treated ones. Interestingly, compound 6b showed little influence on the necrosis of liver cancer cells. The provided findings are supported by the previous cell cycle analysis, confirming the potentiality of 6b as a promising anticancer agent (see Table 6 and Figure 6).

Chemistry and Analysis
Melting points were recorded in open capillaries, using an electrothermal 9200 melting point apparatus (Cole-Parmer GmbH, Wertheim, Germany). Nuclear magnetic resonance (NMR) spectra were recorded (700 MHz for proton 1 H and 176 MHz for carbon 13 C) in deuterated dimethyl sulfoxide (DMSO-d6) as a solvent and tetramethylsilane (TMS) as an internal standard, using Bruker spectrometers (Bruker, Coventry, Germany) in the College of Pharmacy, King Saud University, Saudi Arabia. All chemical shifts were expressed per the δ scale (ppm), while coupling constants (J) for 1 H were given in Hz and expressed as (s) for a singlet, (bs) for a broad singlet, (d) for a doublet, (t) for a triplet, (dd)

Chemistry and Analysis
Melting points were recorded in open capillaries, using an electrothermal 9200 melting point apparatus (Cole-Parmer GmbH, Wertheim, Germany). Nuclear magnetic resonance (NMR) spectra were recorded (700 MHz for proton 1 H and 176 MHz for carbon 13 C) in deuterated dimethyl sulfoxide (DMSO-d6) as a solvent and tetramethylsilane (TMS) as an internal standard, using Bruker spectrometers (Bruker, Coventry, Germany) in the College of Pharmacy, King Saud University, Saudi Arabia. All chemical shifts were expressed per the δ scale (ppm), while coupling constants (J) for 1 H were given in Hz and expressed as (s) for a singlet, (bs) for a broad singlet, (d) for a doublet, (t) for a triplet, (dd) for a doublet of doublets, (td) for a triplet of doublets, and (m) for a multiplet. Mass spectra were recorded using an Agilent Single Quad mass spectrometer (Bruker, Billerica, MA, USA). Elemental analyses were performed on a Perkin Elmer 2400 CHN elemental analyzer (PerkinElmer, Inc., Waltham, MA, USA). Reactions were monitored via thin-layer chromatography (TLC) using systems (ethylacetate 20%, hexane 80%) and (methanol 5%, chloroform 95%), while spots were visualized using an ultraviolet lamp.

General Protocol for the Synthesis of 2-[(1,3-benzoxazol-2-yl) sulfanyl]
Acetohydrazide (3) Compound 2 (0.01 mol) and hydrazine hydrate (0.05 mol) were mixed well and heated over a water bath for 10 min before adding 30 mL of ethanol. The reaction mixture was then heated with reflux for 6-10 h. When the reaction was completed, the mixture was cooled to room temperature, added to iced water, filtered, and, finally, recrystallized from the absolute ethanol [43][44][45].

In Vitro CDK2, EGFR, HER2, and VEGFR-2 Enzyme Assays
Compound 6b was biologically evaluated for its inhibitory effect against CDK2, EGFR, HER2, and VEGFR-2. Human CDK2, EGFR, HER2, and VEGFR-2 ELISA kits (Enzyme-Linked-Immunosorbent Serologic Assay) were used during this evaluation. At first, specified antibody proteins were added separately to a 96-well plate, then 0.1 mL of the standard solution or the evaluated molecule was added; these were then incubated for 2.5 h at RT. After rinsing, 100 µL of the ready-made biotin antibody protein was added, then incubated for one extra hour at room temperature and, finally, washed. Next, 100 µL of streptavidin solution was added, incubated at room temperature for 45 min, and then washed. Next, 100 µL of the TMB substrate reagent was added and incubated for 0.5 h at RT, followed by adding 50 µL of the stop solution. The absorbance of the ELISA plate was promptly measured at 450 nm using the ELISA plate reader. The standard curve that was generated had absorbances on the Y-axis and concentrations on the X-axis.

Determination of Apoptosis-Related Proteins
The HepG2 cells were incubated in 96-well plates in triplicates and left for 24 h. After 24 h of incubation, the cells were treated with compound 6b at different concentrations, while control cells were only treated with 0.1% DMSO (v/v). The treated cells, along with control cells, were then incubated for another 24 h; then, caspases-3 and 9, BAX, and Bcl-2 levels were determined using ELISA assay kits; KHO1091 (Invitrogen TM , Grand Island, NY, USA), EIA-4860 (DRU International, Inc., Mountainside, NJ, USA), EIA-4487 (DRU International INC., Mountainside, NJ, USA) and 99-0042 (Invitrogen TM , Grand Island, NY, USA), respectively, according to their manufacturers' procedures.

Cell-Cycle Flow Cytometry Analysis
To detect the effect of the synthesized compound, 6b, on cell cycle distribution, flow cytometry analysis was carried out, utilizing propidium iodide (PI) staining. The kit used for the cell cycle flow cytometry analysis was the K101-100 Annexin V-FITC Apoptosis Detection Kit (BioVision, Mountain View, CA, USA). First, HepG2 cells were manipulated with compound 6b (6.4 µM) for nearly 24 h. Then, the treated cells were fixed in 70% ethanol for 12 h at 4 • C. Following that, the cells were rinsed with cold PBS, incubated with 100 µL RNase A for 0.5 h at 37 • C, and stained with propidium iodide (400 µL) in the dark at RT for an extra 0.5 h. The stained cells were determined, utilizing Epics XLMCL™ flow cytometer equipment (Beckman Coulter, Apeldoorn, Netherlands), then the results were collected and analyzed with the Flowing software (version 2.5.1, Turku Center for Biotechnology, Turku, Finland).

Annexin V-FITC Dual-Staining Apoptosis Analysis
Apoptosis analysis was employed to evaluate the apoptotic effect of compound 6b. HepG2 cells (2.0 × 10 5 cells) were treated with compound 6b (6.4 µM) for 24 h, collected by trypsin, centrifuged, rinsed twice successively with PBS, suspended in 0.5 mL of binding buffer, then dual-stained with Annexin V-FITC (5 µL) and propidium iodide (5 µL) in the dark for 15 min at RT. These stained cells were evaluated using flow cytometry equipment Epics XL-MCL™ (Beckman Coulter, Apeldoorn, Netherlands) with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. The results were then analyzed with the Flowing software.

Conclusions
A series of 2-mercaptobenzoxazoles, hybridized with either substituted benzenes, substituted isatins, or heterocycles were synthesized and obtained as E/Z-diastereomers. The compounds have been assigned Z or E configurations by 1HNMR analysis. The biological investigations of the synthesized compounds revealed good cytotoxic activities for four compounds against four cancer cell lines: hepatocellular carcinoma (HepG2), mammary gland (MCF-7), breast cancer (MDA-MB-231), and epithelioid cervix carcinoma (HeLa). Furthermore, biological screening for the most active compound, 6b, showed that it was found to work via more than one mechanism of action, in a "multitarget phenomenon". Compound 6b induced apoptosis by increasing the gene expression of Bax, caspase-3, and caspase-9, and the suppression of Bcl-2. Likewise, it caused cell-cycle arrest at the G2/M phase. In addition to its effect on apoptosis and the cell cycle, compound 6b appeared to inhibit various kinases, such as CDK2, EGFR, HER2, and VEGFR2, which are overexpressed in cancer cells. Thus, it is worthwhile to conclude that the synthesized compounds have a promising anticancer effect via multiple mechanisms, which give these compounds the privilege of avoiding cancer resistance and increasing efficacy.