Novel sulphonamide-bearing methoxyquinazolinone derivatives as anticancer and apoptosis inducers: synthesis, biological evaluation and in silico studies

Abstract We synthesised a new series of sulphonamide-bearing quinazolinone derivatives 5-18 and evaluated their in vitro cytotoxicity in various cancer cell lines (A549, HepG-2, LoVo and MCF-7) and in normal human cells (HUVEC). Compounds 6 and 10 exhibited the higher activity against all the cancer cell lines compared with 5-flourourcil as positive control. The ability of the most promising compounds 6 and 10 to induce cell cycle arrest and apoptosis in breast cancer (MCF-7) cells was evaluated by flow cytometry. Reverse transcriptase-polymerase chain reaction and western blotting were used to evaluate the expression of apoptosis-related markers. We found that the 2-tolylthioacetamide derivative 6 and the 3-ethyl phenyl thioacetamide derivative 10 exhibited cytotoxic activity comparable to that of 5-fluorouracil as reference drug in MCF-7 and LoVo colon cancer cells. Cell cycle analysis showed a concentration-dependent accumulation of cells in the sub-G1 phase upon treatment with both compounds. The Annexin V-fluorescein isothiocyanate/propidium iodide assay showed that the compounds 6 and 10 increased the early and late apoptosis cell death modes in a dose-dependent manner. These compounds downregulated the expression of B-cell lymphoma-2 (Bcl-2), while increasing that of p53, Bcl-2-like protein 4, and caspase-7, at the mRNA and protein levels. Molecular docking of compounds 6 and 10 with Bcl-2 predicted them to show moderate − high binding affinity (6: −7.5 kcal/mol, 10: −7.9 kcal/mol) and interactions with key central substrate cavity residues. Overall, compounds 6 and 10 were found to be promising anticancer and apoptosis-inducing agents.


Introduction
Cancer is a life-threatening disease that is considered a major medical challenge worldwide 1 . Treatments for cancer include surgery, chemotherapy, hormonal therapy, and biological therapy [2][3][4] . The choice of treatment is influenced by the site and progression of the disease. Chemotherapy is primarily used for the treatment of metastasis and hypoxic tumours. However, its use is limited by the toxicity of the drugs towards healthy cells 5,6 . This toxicity is a result of the low selectivity of existing chemotherapeutic drugs towards cancer cells 7 . In addition, the long-term use of chemotherapeutic agents often gives rise to drug resistance 8 . The continuous search for new anticancer agents that offer both selectivity towards malignant cells and low potential for resistance is required. Interest in quinazolinone derivatives grew after showing numerous activity in medical chemotherapy such as apoptosis induction and antiangiogenic properties. Idelalisib, afatinib, gefitinib, erlotinib, and lapatinib ( Figure 1) are among these compounds that have been reported to exert apoptosis induction and cell cycle arrest in different cancer cells [9][10][11][12][13][14] . The main factors contributing to the interest in these compounds are their good safety profile and potential for oral administration 15,16 . Hybridisation has proven to be beneficial in the preparation of new anticancer agents and in overcoming the drawbacks of conventionally used drugs 17,18 . Therefore, sulphonamides were hybridised with quinazolin-4(3H)-ones to obtain potentially better drug candidates. Sulphonamides mimic the properties of p-aminobenzoic acid and block SH-and NH 2 -containing enzymes and proteins to exhibit antimicrobial activity 19,20 .
We designed a series of novel compounds using the quinazolinone-sulphonamide hybrid scaffold and explored their in vitro cytotoxic effects against a range of cancer cell lines. The hybrid group was fixed and structural modifications focussed on the replacement of the thiol group at the C-2 position of quinazolines with thioacetamide derivatives bearing substituted phenyl rings. The activities of the target compounds were evaluated against MCF-7, HepG-2, LoVo, and A549 cancer cell lines; the most potent compounds were then evaluated for their pro-apoptotic activity, in order to analyse the underlying anticancer mechanisms. To explore the possible pharmacological properties involved in the anticancer activity, the cell cycle and apoptosis were evaluated by flow cytometry. The target compounds were established to initiate apoptosis, which synergistically enhanced the antitumor effects 21,22 . Therefore, the apoptotic effects of the most potent compounds were explored by evaluating caspase-7 activity and by monitoring B-cell lymphoma-2 (Bcl-2) and Bcl-2-like protein 4 (Bax) levels using in vitro and in silico techniques.

Chemistry
In this work, it seemed of interest to search for new heterocyclic compounds with anticancer activity. A novel series of 3,4-dihydroquinazolinone conjugated to a biologically active benzenesulphonamide moiety was synthesised by the introduction of benzenesulphonamide at the 3-position with the incorporation of different types of acetamide terminal at 2-position aimed at exploring the potential anticancer activity. Scheme 1 shows the synthesis of quinazolinone-benzenesulphonamide derivatives 5-18. The starting material, 4-(2-mercapto-4-oxoquinazolin-3(4H)yl) benzenesulphonamide (4) was prepared in quantitative yield by cyclocondensation of 4-isothiocyanatobenzenesulfonamide (2) 23 and 2-amino-3-methoxybenzoic acid (3) in refluxing 1,4dioxan containing a catalytic amount of triethylamine. The structure of compound 4 was characterised from correct analytical data as well as its infra-red (IR) spectrum which showed a characteristic bands at 3321, 3262, 3181 cm À1 (NH 2 ), 3055 cm À1 (CH aromatic), 1691 cm À1 (CO), 1620 cm À1 (CN), 1381, 1156 cm À1 (SO 2 ). 1 H-NMR spectrum exhibited signals at 3.9 ppm attributed to OCH 3 and 12.3 ppm assigned to SH group. 13 C-NMR spectrum revealed signals at 55.7 ppm due to OCH 3 , 160.5 ppm for C¼N and 160.9 ppm attributed to C¼O. The coupling of 4 and 2-chloro-N-substituted acetamides in dry acetone, in the presence of anhydrous K 2 CO 3 at room temperature yielded the corresponding 2-((8-methoxy-4-oxo-3-(4-sulfamoylphenyl)-3,4-dihydroquinazolin-2-yl)thio)-N-substituted phenyl acetamides 5-18. The synthesised compounds 5-18 were characterised on the basis of their spectral data. The IR spectra of compounds 5-18 displayed additional NH, NH 2 , CH aromatic, CH aliphatic, 2CO, CN and SO 2 characteristic bands in the assigned regions. Proton nuclear magnetic resonance ( 1 H-NMR) spectra of compounds 5-18 revealed two singlet signal peaks (3.9 À 4.1 ppm, representing the CH 2 ; 7.9 À 10.3 ppm, representing the NH) and the loss of the SH singlet of 3. The 13 C-NMR spectra of compounds 5-18 showed two signals peculiar to the CH 2 and CO carbons. The 1 H-NMR spectra of compounds 6-8 showed singlet peaks at 2.4, 2.2 and 2.3 ppm, which were attributed to the CH 3 groups at the ortho, meta and para-positions of the phenyl group, respectively. The 1 H-NMR and 13 C-spectra of compounds 9-11 showed triplets (1.0, 1.1, 1.1 ppm, respectively) and quartettes at 2.5 ppm, attributed to the CH 3 and CH 2 , respectively of the ethyl groups, at the ortho, meta and para positions of the phenyl ring. The 13 C-NMR spectra of compounds 9-11 showed signals corresponding to the CH 3 (14.5, 15.9 and 16.1 ppm, respectively) and CH 2 (24.0, 28.7, and 28.0, respectively) of the ethyl groups. The IR spectra of compounds 16-18 showed bands corresponding to the NO 2 groups in the specified region. The 1 H-NMR spectra of 16 and 17 showed singlets at 2.3 and 2.2 ppm, respectively, due to the CH 3 group, while the 13 C-NMR showed signals at 19.3 and 18.0 ppm, respectively.

Cell cycle analysis
The capacity of anticancer drugs to influence cell cycle distribution can provide an insight into the mechanism of their cytotoxic activity 24 . In fact, several cell cycle inhibitors have emerged as prospective therapeutic medications for the treatment of several tumours 25 . Following the cytotoxicity screening, the effect of the most active compounds 6 and 10 on cell cycle progression in the MCF-7 cell line was evaluated. Compared with untreated cells, MCF-7 cells treated with compounds 6 and 10 had a significantly higher percentage of cells in the sub-G1. This increase in sub-G1 phase cells exhibited dose-dependence. Treatment with 10, 20, and 30 mM of the promising compound 6 increased the proportion of sub-G1 phase cells to 3.35 ± 0.07%, 7.8 ± 0.14%, and 22.85 ± 1.2%, respectively, versus the control (0.75 ± 0.07%) ( Figure 2). Similarly, treatment of MCF-7 cells with the active compound 10 (10, 20, and 30 mM) also caused an accumulation of cells in the sub-G1 phase (4.3 ± 0.28%, 7.25 ± 2. 89% and 30.6 ± 0.07%, respectively), compared to the control (0.75 ± 0.07%) ( Figure 3). This increase in the proportion of sub-G1 phase cells was accompanied by a significant decrease in the percentage of cells in the G1 and G2-M phases. It has been proposed that the increment in the sub-G1 cell fraction is indicative of apoptotic cell death 26 , suggesting that both the biologically active compounds 6 and 10 induced apoptosis in MCF-7 cells.

Quantification of apoptosis
Apoptosis evasion is also a hallmark of the transformation of normal cells into tumour cells 27 . Common anticancer drugs aim to induce cell death through apoptosis; this is viewed as a requirement for blocking malignant cell growth 28 . To verify that apoptotic cell death was caused by the promising compounds 6 and 10, an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) assay was used to quantify the cells undergoing apoptosis. In addition to the accumulation of cells in the sub-G1 phase, the Annexin V/PI assay has been widely utilised for the detection of apoptotic cells 29 . As shown in Figure 4, the treatment of MCF-7 cells with compound 6 (10, 20, and 30 mM) for 24 h cells increased the proportion of cells in early apoptosis (7.6 ± 1.4%, 11.4 ± 1.97%, and 18.35 ± 2.4%, respectively), compared to the control group (2.9 ± 0.84%). Similarly, the number of early apoptotic MCF-7 cells increased to 9.55 ± 0.2%, 11.4 ± 0. 7%, and 16 ± 0.84% on 24 htreatment with 10, 20, and 30 mM compound 10, respectively, compared to the control group (3.3 ± 0.28%). An increase in the number of late apoptotic cells was also observed ( Figure 5).
Overall, the flow cytometry data suggested that both compounds induced cell death through the induction of apoptosis, in a dosedependent manner.

Expression of p53, bax, caspase-7 and bcl-2
Next, we explored whether the induction of apoptosis caused by the active compounds 6 and 10 was associated with the activation of apoptosis-related genes. Members of the Bcl-2 family, especially the pro-apoptotic Bax and anti-apoptotic Bcl-2 genes, are known to play a crucial role in the regulation of the apoptotic pathway. The Bax and Bcl-2 genes participate in the downstream initialising of caspase proteins 30 . Therefore, the expression of key genes and the levels of proteins that control the apoptosis pathway were investigated to evaluate the pro-apoptotic effect of the compounds. The expression of p53, Bax, caspase-7, and Bcl-2 was evaluated using specific primers and antibodies. b-actin was used as an internal control. A remarkable change in the expression of apoptotic genes was reported after 24 h of treatment with increasing concentrations of the two compounds. The expression of p53, Bax, and caspase-7 mRNA increased as compared to the control, when the concentrations of compounds 6 and 10 were increased ( Figure 6(A,B)). Meanwhile, the expression of Bcl-2 was downregulated with increasing doses of compounds 6 and 10, as compared to that of the control.
To assess the changes in expression of p53, Bax, Bcl-2, and caspase-7 proteins upon treatment with the compounds, measurements of the protein levels in MCF-7 cells treated with different dosed of compounds 6 and 10 were carried out. Western blot analysis revealed that treatment with compounds 6 and 10 markedly increased the levels of p53, Bax, and caspase-7 ( Figure  7(A,B)). In contrast, the expression of Bcl-2 was down-regulated; this shift in the Bax/Bcl-2 ratio corresponds with the onset of cell apoptosis 31 . The activation of caspase-7 was also evident (Figure 7), indicating the initiation of cellular apoptosis by both compounds.

Structure-activity relationship
By comparing the experimental cytotoxicity of the novel synthesised compounds reported in this study to their structures, the following structure-activity relationships (SAR) were postulated.

Molecular docking analysis
Molecular docking simulations for promising compounds were also performed to obtain further understanding into differential cytotoxic action of synthesised compounds. The molecular docking procedure implemented in this study was validated as described by Al Ajmi and co-workers 32   phenyl)-4-chloro-5-methyl-N,N-diphenyl-1H-pyrazole-3-carboxamide (DRO), was extracted from the ligand-bound X-ray co-crystal structure of Bcl-2, and re-docked. The poses of the bound and redocked ligand were compared, and the root mean square deviation (RMSD) was calculated. The RMSD of the re-docked DRO was found to be 0.3842 Å. Since the calculated RMSD was within the acceptable limit (2.0 Å), we were confident in adopting the docking protocol to predict the binding of compounds 6 and 10 with Bcl-2.

Chemistry
The melting points (MP; uncorrected) of the compounds were determined in an open capillary on a Gallen Kamp melting point apparatus (Sanyo Gallen Kamp, UK). Precoated silica gel plates (Kieselgel 0.25 mm, 60 F254, Merck, Germany) were used for thinlayer chromatography. A developing solvent system of chloroform/methanol (8:2) was used, and the spots were visualised using ultraviolet light. The IR spectra (KBr disc) were recorded using a Fourier transform-IR spectrophotometer (Perkin Elmer, USA). A nuclear magnetic resonance spectrophotometer (Bruker AXS Inc., Switzerland) was used for the 1 H-and 13 C-NMR experiments, operating at 500 MHz and 125.76 MHz, respectively. Chemical shifts are reported as d-values (ppm) relative to tetramethylsilane (internal standard), using deuterated dimethyl sulfoxide (DMSO-d 6 ) as the solvent. Elemental analyses were conducted using a model 2400 CHNSO analyser (Perkin Elmer, USA). All results were within ± 0.4% of the theoretical values. All reagents used were of analytical grade.

Cytotoxicity assay
The MTT assay was performed to test the antiproliferative activity of the synthesised compounds, as per the methodology described by Alqahtani and co-workers 35 . Briefly, A549, HepG2, LoVo, MCF-7 and HUVEC cell lines were seeded in 96-cell culture plates (5 Â 10 4 per well) and allowed 24 h for adherence. The cells were then treated with different concentrations of each compound, and 5fluorouracil was used as a positive control. Following the treatment period (48 h), 10 mL of MTT solution (5 mg/mL) was added to each well and the plates were incubated at 37 C for 2-4 h. Isopropanol (100 mL) acidified with 0.1 N HCl was added to solubilise the formazan products and the plate was kept on a shaker for 10 min. The optical density of each mixture was measured at 570 nm using an enzyme-linked immunosorbent assay plate reader (ELISA plate reader, Bio-Tek, USA). The concentrations of tested compounds required to inhibit cell growth by 50% (IC 50 ) were calculated using a dose-response curve. Cell survival was calculated using the following equation: Cell survival ð%Þ ¼ ðOD of treated sampleÞ= ðOD of untreated sampleÞ Â 100

Cell cycle analysis
Cell cycle analysis was conducted as previously described by Alqahtani and co-workers 35 . Briefly, MCF-7 cells were seeded in 6well plates and incubated for 24 h before the addition of various concentrations (10, 20 and 30 mM) of compounds 6 and 10. After incubating for 24 h, the cells were harvested, washed, and resuspended in PBS. The cells were fixed with 70% ethanol at 4 C for 4 h. The cells were then incubated with RNase (100 mg/mL) and PI (50 mg/mL) for 30 min in the dark. Flow cytometry analysis was performed using Cytomics FC 500 (Beckman Coulter, Brea, CA, USA).  Table 3. The program was run as follows: initial denaturation at 95 C for 5 min, denaturation at 95 C for 30 s, annealing at 55 C for 45 s, elongation at 72 C for 45 s (30 cycles), and final extension at 72 C for 10 min. The RT-PCR products were electrophoresed on a 1.2% agarose gel containing ethidium bromide, and the gel was imaged on a Licor machine.

Western blot analysis
MCF-7 cells were treated with compounds 6 and 10 for 24 h. The cells were then harvested and washed twice with 1x PBS, by adding lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM sodium ethylenediaminetetraacetic acid, 1 mM ethylene glycol-bis (b-aminoethyl ether)-N,N,N 0 ,N 0 -tetraacetic acid, 1% Triton X100, 1 mg/mL leupeptin, and 100 mM phenylmethylsulphonyl fluoride, to prepare the total cell extract. The lysate was cooled on ice for 1 h and clarified by centrifugation at 13000 rpm at 4 C for 15 min. The supernatants were collected. The protein concentrations of all the samples were determined using Bradford reagent (BioRad, Hercules, CA, USA). For western blot analysis, 30 À 35 mg of the total protein was loaded onto 10% sodium dodecyl sulphate-polyacrylamide gel and then transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% bovine serum albumin in 0.1% Tween-tris-buffered saline (TBST) buffer for 2 h at room temperature, and then washed thrice with TBST. The membrane was incubated with primary antibodies for Bcl-2, p53, caspase7, Bax, and b-actin (1:200, Santa Cruz Biotechnology). To ensure equal loading, an anti-actin antibody was also used. The membrane was incubated with horseradish peroxidase-conjugated anti-mouse secondary antibody diluted 1: 1000 for 1 h at room temperature. After incubation, the membrane was washed thrice with TBST. It was then ready for immunodetection; the membrane was incubated with enhanced chemiluminescence western blotting detection reagents (Amersham, Pharmacia Biotech Inc., Piscataway, NJ, USA) and bands were obtained on exposing to Xray films (Amersham).

Molecular docking
The potential of the two most promising compounds 6 and 10 to inhibit Bcl-2 was evaluated in molecular docking experiments, conducted as described by Al-Shabib and co-workers 32 . The 3 D coordinates of Bcl-2 were retrieved from the PDB-RCSB databank (PDB ID: 2W3L). The X-ray crystal structure of DRO-bound Bcl-2 has previously been solved at a resolution of 2.10 Å 36 . Prior to molecular docking, the protein was pre-processed to remove crystallographic water molecules and any other heteroatoms, add hydrogen atoms, assign proper bond order, and define rotatable bonds, as previously described by Rehman and co-workers 37 . A network of H-bonds was created, and the energy of the protein was minimised using the Merck Molecular Force Field. A grid box of 27 Â 30 Â 25 Å, centred at 39 Â 28 Â À12 Å, with 0.375 Å spacing was defined as a conformation search space for the binding of ligands to Bcl-2. Finally, molecular docking between ligands and proteins was performed using Autodock 4.2 (Scripps Research, San Diego, CA, USA), as previously described by Rabbani and co-workers 38 . Molecular docking was performed using the Lamarckian Genetic Algorithm and Solis and Wets local search methods. The initial torsions, positions, and orientations of the ligands were set randomly. For each docking run, a maximum of 2.5 Â 10 6 calculations was enumerated after setting a population size of 150 and a translational step of 0.2 Å. The quaternion and torsion steps were set to 5. Discovery Studio (BIOVIA, San Diego, CA, USA) was used to analyse the docking results and prepare the figures. Binding affinities of compounds 6 and 10 for Bcl-2 were determined from their respective binding energies (DG), using the following relationship 39 .
Here, R is the Boltzmann gas constant (1.987 cal mol 21 K 21 ) and T is the temperature (298 K).

Conclusion
In conclusion, a new series of quinazoline-sulphonamide derivatives 5-18 were synthesised and evaluated in vitro for their antiproliferative activity. Most of the prepared compounds were found to exhibit remarkable cytotoxicity in the MCF-7 breast cancer cell line. Compounds 6 and 10 were found to be the most promising. Flow cytometry data revealed that compounds 6 and 10 arrested the cell cycle of MCF-7 cells in the sub-G1 and induced apoptosis in cell death mode. Furthermore, changes in the expression of apoptosis-related markers at the gene and protein level were also indicative of apoptotic activity. The 2-tolyl derivative 6 and the 3-ethylphenyl derivative 10 downregulated the expression of B-cell lymphoma-2 (Bcl-2), while increasing that of p53, Bcl-2-like protein 4, and caspase-7, at the mRNA and protein levels. Molecular docking of compounds 6 and 10 also suggested that they possess good binding affinity for Bcl-2. Overall, the findings suggest that compounds 6 and 10 both possess promising anti-proliferative activity. These molecules may be further modified to develop more selective, clinically useful analogues.

Disclosure statement
The authors declare no conflicts of interest.