Evaluation of Synthetic 2,4-Disubstituted-benzo[g]quinoxaline Derivatives as Potential Anticancer Agents

A new series of 2,4-disubstituted benzo[g]quinoxaline molecules have been synthesized, using naphthalene-2,3-diamine and 1,4-dibromonaphthalene-2,3-diamine as the key starting materials. The structures of the new compounds were confirmed by spectral data along with elemental microanalyses. The cytotoxic activity of all synthesized benzo[g]quinoxaline derivatives was assessed in vitro against the breast MCF-7 cancer cell line. The tested molecules revealed good cytotoxicity toward the breast MCF-7 cancer cell line, especially compound 3. The results of topoisomerase IIβ inhibition assay revealed that compound 3 exhibits potent inhibitory activity in submicromolar concentration. Additionally, compound 3 was found to cause pre-G1 apoptosis, and slightly increase the cell population at G1 and S phases of the cell cycle profile in MCF-7 cells. Finally, compound 3 induces apoptosis via Bax activation and downregulation of Bcl2, as revealed by ELISA assay.


Introduction
Cancer is one of the most challenging problems worldwide [1]. Breast cancer exceeds lung cancer in terms of mortality rate, with more than two million patients every year (more than 6.9% mortality rate) [2,3]. Targeted therapy has become an important type of cancer treatment, as it could reduce the activity of a specific target or prevent binding to a specific receptor [4,5]. A lot of efforts of pharmaceutical companies in the development of anticancer drugs have focused on targeted therapy for the treatment of different types of cancer [6,7]. In addition, due to the rapid progress in understanding the molecular pathways involved in cell cycle regulation, there is a motivation to target different steps during cell cycle, in order to control cancer cell proliferation and to avoid drawbacks observed with traditional chemotherapeutic agents [8][9][10]. Furthermore, the pro-apoptotic protein Bax and the ant-apoptotic protein Bcl2 are signs of apoptosis induction, which may be useful targets in the mitochondrial-dependent pathway of apoptosis [11,12]. Therefore, apoptosis induction in cancer cells can be used as a new weapon against cancer cell proliferation [13,14].
Benzo [g]quinoxalines are a promising class of potent anticancer agents that display broad-spectrum in vivo and in vitro activity against different types of tumor cell lines [15,16]. They are considered the chromophore-modified analogs of mitoxantrone, a synthetic analogue of Doxorubicin (Dox) I [17]. In addition, compounds having three to four rings give the optimal intercalation with human DNA, leading to reading errors during the replication step in the cell cycle [18,19]. For example, Benzo[g]quinoxaline derivatives II exhibited cytotoxicity comparable to that of Dox against the HCT-15 cancer cell line [20]. Furthermore, benzoquinoxalinedione III exhibited cytotoxicity and was highly active, especially against sarcoma [21]. 1,4-diazaanthraquinone IV was proven to induce apoptosis of breast cancer, which activates p53 and triggers apoptosis of tumor cells, as shown in Figure 1 [22].
broad-spectrum in vivo and in vitro activity against different types of tumor cell lines [15,16]. They are considered the chromophore-modified analogs of mitoxantrone, a synthetic analogue of Doxorubicin (Dox) I [17]. In addition, compounds having three to four rings give the optimal intercalation with human DNA, leading to reading errors during the replication step in the cell cycle [18,19]. For example, Benzo[g]quinoxaline derivatives II exhibited cytotoxicity comparable to that of Dox against the HCT-15 cancer cell line [20]. Furthermore, benzoquinoxalinedione III exhibited cytotoxicity and was highly active, especially against sarcoma [21]. 1,4-diazaanthraquinone IV was proven to induce apoptosis of breast cancer, which activates p53 and triggers apoptosis of tumor cells, as shown in Figure 1 [22].
Based on the above facts, and as a continuation of our search for new anticancer agents [23][24][25][26], new benzo[g]quinoxaline molecules were synthesized. All the synthesized molecules were evaluated for their in vitro cytotoxic activity against the breast MCF-7 cancer cell line. Additionally, the most active molecule was further evaluated for topoisomerase inhibition and for its apoptotic inducing activity.

Chemistry
Here, we synthesized 3-(4-chlorophenyl)-1,2-dihydrobenzo[g]quinoxaline (2) using naphthalene-2,3-diamine (1) as the key starting material via the condensation of compound 1 with 4-chlorophenacyl bromide in methanol in the presence of fused sodium acetate. Dehydrogenation of compound 2 in refluxing acetic anhydride led to the formation of 2-(4-Chlorophenyl)benzo[g]quinoxaline (3) [27], (Scheme 1). Based on the above facts, and as a continuation of our search for new anticancer agents [23][24][25][26], new benzo[g]quinoxaline molecules were synthesized. All the synthesized molecules were evaluated for their in vitro cytotoxic activity against the breast MCF-7 cancer cell line. Additionally, the most active molecule was further evaluated for topoisomerase inhibition and for its apoptotic inducing activity.
The structure of the compounds 2 and 3 were confirmed by the spectral data. In the IR spectrum of compound 2 a band in the region 3225 cm −1 characteristic of the NH group was observed, while in the IR spectrum of compound 3, the band of NH group was absent. From study, the 1 H-NMR spectrum of compound 2 showed the structure of this compound in two isomers, as shown in Figure 2.
Proton signals of NH and CH 2 groups in 3,4-dihydrobenzo[g]quinoxaline isomers were observed at δ 8.19 and 4.19 ppm, respectively, while the proton signals of two NH groups in 1,4-dihydrobenzo[g]quinoxaline were observed at δ 9.58 and 9.01 ppm as singlet signals and the proton of olefinic NH-CH= appeared at δ 7.34 ppm as singlet signal.
Additionally, the 13 C-NMR spectrum of compound 2 showed the presence of five characteristic carbon signals at δ 151.02, 150.61, 150.50, 148.01, and 145.33 ppm due to the C=N, =CN groups. In addition, the 13 C-NMR spectrum of compound 2 also showed carbon signals at δ 37.72 and 37.33 ppm assigned to carbon atom of methylene group (CH 2 ).
The 1 H-NMR spectrum of compound 3 showed two sharp singlet signals at δ 9.66 and 8.80 ppm due to the proton of azomethene (CH=N) groups and two protons of H5 and H10 of benzo[g]quinoxaline 3. The residue protons of benzo[g]quinoxaline (3) appeared as multiplet signals in the region at δ 7.68-8.46 ppm due to protons of 4-chlorophenyl and benzene ring. The 13 C-NMR spectrum of compound 3 showed two signals at δ 150.49 and 145.34 ppm due to the carbons of C=N groups. In addition, the 13 C-NMR spectrum of compound 3 displayed 14 signals in the region at δ 138.32-127.53 ppm due to the carbons of naphthalene and 4-chlorophenyl rings. The structure of the compounds 2 and 3 were confirmed by the spectral data. In the IR spectrum of compound 2 a band in the region 3225 cm −1 characteristic of the NH group was observed, while in the IR spectrum of compound 3, the band of NH group was absent. From study, the 1 H-NMR spectrum of compound 2 showed the structure of this compound in two isomers, as shown in Figure 2. Proton signals of NH and CH2 groups in 3,4-dihydrobenzo[g]quinoxaline isomers were observed at δ 8.19 and 4.19 ppm, respectively, while the proton signals of two NH groups in 1,4-dihydrobenzo[g]quinoxaline were observed at δ 9.58 and 9.01 ppm as singlet signals and the proton of olefinic NH-CH= appeared at δ 7.34 ppm as singlet signal. Scheme 1. Synthesis of 2-(4-chlorophenyl)benzo[g]quinoxaline. Reagents and reaction conditions: (i) 4-chlorophenacyl bromide, AcONa/MeOH, (ii) Acetic anhydride/reflux, (iii) Acetic anhydride/CH 3 CN, reflux, (iv) ethyl β-aryl-αcyanoacrylate/reflux. heme 1. Synthesis of 2-(4-chlorophenyl)benzo[g]quinoxaline. Reagents and reaction conditions: (i) 4-chlorophenacyl omide, AcONa/MeOH, (ii) Acetic anhydride/reflux, (iii) Acetic anhydride/CH3CN, reflux, (iv) ethyl aryl-α-cyanoacrylate/reflux. The structure of the compounds 2 and 3 were confirmed by the spectral data. In the IR spectrum of compound 2 a band in the region 3225 cm −1 characteristic of the NH group was observed, while in the IR spectrum of compound 3, the band of NH group was absent. From study, the 1 H-NMR spectrum of compound 2 showed the structure of this compound in two isomers, as shown in Figure 2. Proton signals of NH and CH2 groups in 3,4-dihydrobenzo[g]quinoxaline isomers were observed at δ 8.19 and 4.19 ppm, respectively, while the proton signals of two NH groups in 1,4-dihydrobenzo[g]quinoxaline were observed at δ 9.58 and 9.01 ppm as singlet signals and the proton of olefinic NH-CH= appeared at δ 7.34 ppm as singlet signal. In addition, reaction of 2-(4-chlorophenyl)-3,4-dihydro-benzo[g]quinoxaline (2) with acetic anhydride in the presence of acetonitrile afforded 2-(4-chlorophenyl)-4-acetyl-3,4dihydro-benzo[g]quinoxaline (4). The 1 H-NMR spectrum of compound 4 showed the structure of this compound in two isomers, as shown in Scheme 2. The proton signal of NH group in 4-acetyl-2-(4-chlorophenyl)-1H-benzo [g]quinoxaline (4) appeared at δ 9.32 as singlet signal, while the olefinic proton of this isomer was observed at δ 7.35 with aromatic protons as multiplet signal. The 13 C-NMR spectrum of compound 4 supported the formation of two isomers of these compounds, as it showed that the presented three carbon signals at δ 150.55, 44.48, and 23.79 ppm due to the C=N, -CH 2 N, and methyl groups of the isomer 4-acetyl-2-(4-chlorophenyl)-3,4-dihydro-benzo[g]quinoxaline, respectively, while the 13 C-NMR spectrum displayed carbon signals at δ 145.35, 138.32, and 23.12 ppm assigned to carbon atoms of N-C=C-N and methyl group for the isomer 4-acetyl-2-(4chlorophenyl)-1,2-dihydro-benzo[g]quinoxaline. The carbon signals due to the naphthalene and 4-chlorophenyl rings appeared in the region at δ 138.12-127.56 ppm, while the carbon

Cytotoxic Activity against Breast MCF-7 Cancer Cell Line
The effect of the prepared benzo[g]quinoxaline derivatives 2-9 on the viability of breast MCF-7 cancer cell line was studied using MTT assay. The cytotoxicity was assessed using Dox as positive reference drug. Treatment of the MCF-7 cell line with different concentration of benzo[g]quinoxaline derivatives revealed that some of the tested compounds displayed good cytotoxic activity against MCF-7 cells as concluded from their IC 50 values presented in Table 1. Benzo[g]quinoxaline molecule 3 exhibited the highest cytotoxic activ- DNA synthesis has been regarded as one of the most effective targets for the development of novel anticancer agents with apoptosis induction [28]. In addition, several reported experimental results showed that compounds having polycyclic molecular skeleton appear to give the optimal base pair intercalation and enzyme binding with human DNA, leading to reading errors during the replication step [19]. To obtain a further insight into the mechanism of cell proliferation inhibition activity, benzo[g]quinoxaline molecule 3 was investigated for topoisomerase IIβ (topo IIβ) inhibitory activity using the Human DNA topo IIβ ELISA Kit # MBS942146. The results revealed that the tested benzo[g]quinoxaline molecule 3 had a good inhibitory activity against topo IIβ at submicromolar level with IC 50 = 32.16 µM compared with value of 3.31 µM for Dox, a known potent topo IIβ inhibitor ( Figure 3).  DNA synthesis has been regarded as one of the most effective targets for the development of novel anticancer agents with apoptosis induction [28]. In addition, several reported experimental results showed that compounds having polycyclic molecular skeleton appear to give the optimal base pair intercalation and enzyme binding with human DNA, leading to reading errors during the replication step [19]. To obtain a further insight into the mechanism of cell proliferation inhibition activity, benzo[g]quinoxaline molecule 3 was investigated for topoisomerase IIβ (topo IIβ) inhibitory activity using the Human DNA topo IIβ ELISA Kit # MBS942146. The results revealed that the tested benzo[g]quinoxaline molecule 3 had a good inhibitory activity against topo IIβ at submicromolar level with IC50 = 32.16 µM compared with value of 3.31 µM for Dox, a known potent topo IIβ inhibitor (Figure 3). Graphical representation for comparison of topo IIβ IC50 of compound 3 and Dox; topo II was performed using four doses protocol for compound 3 and Dox, and the concentration that induced 50% maximum inhibition of topo II was determined from sigmoidal dose-response. Data are expressed as the mean ± SD (n = 3 experiments) and statistical comparisons were carried out using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test (** p < 0.01). **-Significant.

Cell Cycle Analysis
Cell cycle analysis was conducted to determine the mechanism of cytotoxic activity of the most active molecule against MCF-7 cells by using DNA flow cytometric analysis [29]. Compound 3 was tested at its IC50 concentration dose value. The results showed that the tested compound 3 increased the percentage of cell population at G1 and S phases compared to the untreated control. Moreover, the results showed that compound 3 increased the percentage of cells at the pre-G1 phase from 2.41% to 38.24%, compared to the untreated control ( Figure 4). Graphical representation for comparison of topo IIβ IC 50 of compound 3 and Dox; topo II was performed using four doses protocol for compound 3 and Dox, and the concentration that induced 50% maximum inhibition of topo II was determined from sigmoidal dose-response. Data are expressed as the mean ± SD (n = 3 experiments) and statistical comparisons were carried out using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test (** p < 0.01). **-Significant.

Cell Cycle Analysis
Cell cycle analysis was conducted to determine the mechanism of cytotoxic activity of the most active molecule against MCF-7 cells by using DNA flow cytometric analysis [29]. Compound 3 was tested at its IC 50 concentration dose value. The results showed that the tested compound 3 increased the percentage of cell population at G1 and S phases compared to the untreated control. Moreover, the results showed that compound 3 increased the percentage of cells at the pre-G1 phase from 2.41% to 38.24%, compared to the untreated control ( Figure 4).

Annexin V/FITC Apoptosis Staining Assay
Apoptosis staining assay was performed to detect the effect of compound 3 on the percentage of early and late apoptosis in the breast MCF-7 cancer cell line [30]. As shown in Figure 5, compound 3 increases the apoptosis percentage at the early stage from 0.77% to 1.59% compared with the untreated control. Additionally, the apoptosis percentage at late stage was increased from 0.13% to 21.38% compared with the untreated control. Therefore, compound 3 can be considered an apoptosis inducer in the breast MCF-7 cell line.

Annexin V/FITC Apoptosis Staining Assay
Apoptosis staining assay was performed to detect the effect of compound 3 on the percentage of early and late apoptosis in the breast MCF-7 cancer cell line [30]. As shown in Figure 5, compound 3 increases the apoptosis percentage at the early stage from 0.77% to 1.59% compared with the untreated control. Additionally, the apoptosis percentage at late stage was increased from 0.13% to 21.38% compared with the untreated control. Therefore, compound 3 can be considered an apoptosis inducer in the breast MCF-7 cell line.

Annexin V/FITC Apoptosis Staining Assay
Apoptosis staining assay was performed to detect the effect of compound 3 on the percentage of early and late apoptosis in the breast MCF-7 cancer cell line [30]. As shown in Figure 5, compound 3 increases the apoptosis percentage at the early stage from 0.77% to 1.59% compared with the untreated control. Additionally, the apoptosis percentage at late stage was increased from 0.13% to 21.38% compared with the untreated control. Therefore, compound 3 can be considered an apoptosis inducer in the breast MCF-7 cell line.

Bax and Bcl2 Enzyme Assay
In order to investigate the molecular pathway involved in apoptosis, compound 3 was screened for its ability to activate pro-apoptotic protein; Bax and downregulate antiapoptotic protein; Bcl2 [31]. The MCF-7 cancer cell line was treated with compound 3 at its Pharmaceuticals 2021, 14, 853 7 of 13 IC 50 value (µM) and the level of expression of proteins involved in apoptosis was quantized using ELISA assay. From the results presented in Figure 6, compound 3 increased the expression of Bax in MCF-7 cancer cells by 3.89-fold compared with the untreated control. In addition, compound 3 resulted in downregulation of the anti-apoptotic protein Bcl2 by 4.35-fold compared with the untreated control. Therefore, it can be concluded that compound 3 can induce the intrinsic pathway of apoptosis by activation of Bax and downregulation of Bcl2. In order to investigate the molecular pathway involved in apoptosis, compound 3 was screened for its ability to activate pro-apoptotic protein; Bax and downregulate anti-apoptotic protein; Bcl2 [31]. The MCF-7 cancer cell line was treated with compound 3 at its IC50 value (µM) and the level of expression of proteins involved in apoptosis was quantized using ELISA assay. From the results presented in Figure 6, compound 3 increased the expression of Bax in MCF-7 cancer cells by 3.89-fold compared with the untreated control. In addition, compound 3 resulted in downregulation of the anti-apoptotic protein Bcl2 by 4.35-fold compared with the untreated control. Therefore, it can be concluded that compound 3 can induce the intrinsic pathway of apoptosis by activation of Bax and downregulation of Bcl2. Figure 6. Effect of compound 3 on apoptosis related proteins, Bax, Bcl2, and Fas/Fas-L in MCF-7 cells. The graph represents the increased expression of Bax and decreased expression of Bcl2, during apoptotic event that occurred after the treatment of compound 3 (2.89 µM) in MCF-7 cells and was determined by ELISA assay. Data was expressed as mean ± SD (n = 3 experiments) and statistical comparisons were carried out using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test (*** p < 0.001).

Molecular Docking Study
Molecular docking study was performed to investigate its plausible binding interaction with the key amino acid in the active site of topoisomerase II. Benzo[g]quinoxaline molecule 3 was docked with the crystal structure of topoisomerase II (PDB code 1ZXM). As can be seen, the molecular interaction of compound 3 in Figure  7 revealed π-π interaction with the target protein. In addition, the hydrophobicity induced by compound 3 resulted in docking score of −8.28 kcal/mol. Figure 6. Effect of compound 3 on apoptosis related proteins, Bax, Bcl2, and Fas/Fas-L in MCF-7 cells. The graph represents the increased expression of Bax and decreased expression of Bcl2, during apoptotic event that occurred after the treatment of compound 3 (2.89 µM) in MCF-7 cells and was determined by ELISA assay. Data was expressed as mean ± SD (n = 3 experiments) and statistical comparisons were carried out using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test (*** p < 0.001).

Molecular Docking Study
Molecular docking study was performed to investigate its plausible binding interaction with the key amino acid in the active site of topoisomerase II. Benzo[g]quinoxaline molecule 3 was docked with the crystal structure of topoisomerase II (PDB code 1ZXM). As can be seen, the molecular interaction of compound 3 in Figure 7 revealed π-π interaction with the target protein. In addition, the hydrophobicity induced by compound 3 resulted in docking score of −8.28 kcal/mol.

Chemistry
The melting point of the synthesized derivatives was determined with electro thermal melting point apparatus and has not been corrected. The IR data were obtained

Chemistry
The melting point of the synthesized derivatives was determined with electro thermal melting point apparatus and has not been corrected. The IR data were obtained on a Jasco FTLIR 6100 infrared spectrometer using KBr disc technique. 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) spectra were run with a Bruker 400 DRX-Avance NMR spectrometer, and measured using tetramethylsilane (TMS) as the internal standard. The mass spectra were measured with a Finnigan MATSSQ-7000 mass spectrometer. Elemental micro-analyses were carried out at the micro analytical unit, Central Services laboratory, National Research Centre, Dokki, Cairo, Egypt, using Vario Elemental and were found to be within ± 0.5% of the theoretical values. All reagents were obtained from Aldrich Chemical Company and used as supplied.

Cell Cycle Analysis Compound 3
MCF-7 cells (2 × 10 5 /well) were harvested and washed twice in PBS. After that, cells were incubated at 37 • C and 5% CO 2 . The medium was replaced with (DMSO 1% v/v) containing the tested compound, then incubated for 48 h, washed twice in PBS, fixed with 70% ethanol, rinsed again with PBS, and then stained with DNA fluorochrome PI for 15 min at 37 • C. DNA content was analyzed by flow cytometry on a FACS Calibur flow cytometer (Becton and Dickinson, Heidelberg, Germany).

Annexin V FITC/PI Apoptosis Detection Staining Assay
Apoptosis in MCF-7 cells was investigated using fluorescent Annexin V-FITC/PI detection kit by flow cytometry assay. Briefly, MCF-7 cells (2 × 10 5 ) after incubation for 12 h. Cells were treated with compound 3 at its IC 50 concentration for 48 h, then the cells were harvested and stained with Annexin V-FITC/PI dye for 15 min in the dark at 37 • C. Flow cytometry analyses were performed using FACS Calibur flow cytometer (Becton and Dickinson, Heidelberg, Germany).

In Vitro ELISA Measurement for the Concentration of Bax, Bcl2, and Fas/Fas-l Proteins
The levels of the apoptotic marker Bax, Bcl-2, and Fas/Fas-l were assessed using ELISA kit. The procedure of the used kits was completed according to the manufacturer's instructions. Briefly, cell lysates were prepared from control and MCF-7 cells (2.5 × 10 5 ) treated with IC 50 concentration of compound 3. Then, equal amounts of cell lysates were loaded then probed with specific antibodies. The samples were measured at 450 nm in ROBONEK P2000 ELISA reader. All experiments were performed in duplicate.

Molecular Docking Study
Molecular docking study was performed using MOE software program (MOE 2009.10). The topoisomerase II crystal structure (PDB entry: 1ZXM) was obtained from protein data bank (Supplementary Materials).

Conclusions
A series of 2,4-disubstituted benzo[g]quinoxaline molecules were synthesized, and their in vitro cytotoxic activity were evaluated against the breast MCF-7 cancer cell line using MTT colorimetric assay. The tested molecules revealed good activity against MCF-7 cells. The results of DNA topoisomerase IIβ inhibition activity showed that compound 3 exhibited good inhibition activity against topo IIβ at submicromolar level compared with Dox, a known potent topo IIβ inhibitor. Moreover, these compounds were found to be apoptotic inducers via activation of Bax and downregulation of Bcl2. Therefore, the prepared benzo[g]quinoxaline molecules can be considered a scaffold for further optimization to find a more potent cytotoxic agent with better apoptotic inducing activity.