Novel sulphonamide benzoquinazolinones as dual EGFR/HER2 inhibitors, apoptosis inducers and radiosensitizers

Abstract A series of sulphonamide benzoquinazolinones 5–18 was synthesized and evaluated for cytotoxic activity against MDA-MB-231 cell line. The compounds showed IC50 ranging from 0.26 to 161.49 µM. The promising compounds were evaluated for their inhibitory profile against epidermal growth factor (EGFR) and HER2 enzymes. Compound 10 showed more potent activity on both EGFR and HER2 than erlotinib (IC50 3.90 and 5.40 µM versus 6.21 and 9.42 µM). The pro-apoptotic activity of 10 was evaluated against caspase-3, Bax, B-cell lymphoma protein 2 (Bcl-2) expression levels, and cell cycle analysis. Compound 10 increased the level of caspase-3 by 10 folds, Bax level by 9 folds, decreased the level of the Bcl-2 by 0.14 and arrested the cell cycle in the G2/M phase. The radio-sensitizing activity of 10 was measured using a single dose of 8 Gy gamma radiation (IC50 decreased from 0.31 to 0.22 µM). Molecular docking was performed on EGFR and HER2 receptors.


Introduction
The major challenge in cancer therapy is the induction of apoptosis through anticancer agents [1][2][3] . Apoptosis is a crucial process in maintaining normal tissue homeostasis in the human body, mediated by signal transduction pathways. The two major apoptotic pathways are extrinsic and intrinsic. The extrinsic pathway is induced by the trans-membrane death receptors, while the intrinsic is through mitochondrial stress caused by DNA damage and heat shock 4 . Activated caspases are the executioners of apoptosis 5 . So, more effective therapeutic strategies for better understanding of signaling pathways and molecular targets should be further provided.
Breast cancer is the world's second leading cause of cancerrelated death 6 . The overexpression of the HER2 enzyme in breast cancer is correlated with poor prognosis and drug resistance 7 . HER2 belongs to the epidermal growth factor family (EGFR), also called ErbB. It is a member of receptor tyrosine kinases (TKs) involved in signaling pathways controlling angiogenesis, cell differentiation, and proliferation 8 . The EGFR consists of a subfamily of EGFR (HER1), HER2, HER3, and HER4, that are only expressed at low levels in normal human tissues 9 . Although most patients with EGFR mutant cancers respond to therapies, the patients develop resistance after an average of one year on treatment 10 . The resistance to HER2 targeted therapies is associated with the overexpression of EGFR family enzymes 11 . It is obvious that HER family is interdependent and shows functional redundancy. The blockage of one HER receptor can be compensated by another HER family member 9,12 . The cross-linking and compensatory activities of the HER family members can provide a strong rationale for co-targeting of both EGFR and HER2 enzymes.
Molecular hybridization is a simple and effective tool to combine covalently two drug pharmacophores into a single molecule 13 . Lately, it has been observed that benzo [g]quinazoline and sulphonamides demonstrated profound growth inhibitory activity against different cancer cells and TK enzymes 14,15 . The quinazoline is a privileged scaffold that constitutes an important class of heterocyclic compounds owing to its varies pharmacological properties 16,17 . Afatinib, lapatinib, gefitinib, and erlotinib ( Figure 1) are the representative drugs in this class in clinical use for targeted anticancer therapies [18][19][20][21] . The use of them has paved the way to develop new quinazoline-based molecules acting as EGFR inhibitors. Also, it is well-known that sulphonamides are strongly related to anticancer activity 22,23 . They have several targets, most of which are directly connected to oncogenesis 24 . They proved to exhibit good activity through many mechanisms as carbonic anhydrase 24 , matrix metalloprotienase 25 , NADPH reductase 26 , histone deacetylase 27 , and PI3K inhibition 28 .
In this context, we desire to exploit newer leads with tuneable anticancer activity and low toxicity 14,29 . A series of substituted benzo[g]quinazolinone benzenesulfonamide hybrids were designed, synthesized, and evaluated as dual EGFR/HER2 inhibitors. The apoptotic activity of the most potent compound was evaluated through the activation of the proteolytic caspase-3, Bax and B-cell lymphoma protein 2 (Bcl2) expression levels, cell cycle analysis, and radiosensitizing activity. Molecular docking was carried out inside the binding site of EGFR and HER2 receptors in order to confirm their possible mechanism of action.

Materials and methods
Melting points were uncorrected and measured 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 TLC with a developing solvent system of chloroform/methanol (7:3) and detected by the UV lamp. IR spectra were recorded using FT-IR spectrophotometer (Perkin Elmer, USA). NMR spectra were scanned on an NMR spectrophotometer (Bruker AXS Inc., Switzerland) operating at 500 MHz for 1 H and 125.76 MHz for 13 C. Chemical shifts are expressed in d-values (ppm) relative to TMS as an internal standard, using DMSO-d 6 as a solvent. Mass spectra were recorded on ISQ LT Thermo Scientific GCMS model (Massachusetts, USA). Elemental analyses were performed on a model 2400 CHNSO analyser (Perkin Elmer, USA). All the values were within ±0.4% of the theoretical values. All reagents were obtained from Sigma-Aldrich of AR grade. General procedure A mixture of 4 (0.383 g, 0.001 mol) and 2-chloro-N-substituted acetamide derivatives (0.001 mol) in dry acetone (30 ml) and anhydrous K 2 CO 3 (0.138 g, 0.001 mol) was stirred at room temperature for 10 h. The mixture was filtered and the product formed was crystallized from ethanol to give 5-18.    In vitro enzymatic activity assay EGFR and HER2 kinase kits were purchased from Invitrogen. EGFR (PV3872), 0.200 mg/mL and HER2 (PV3366), 0.192 mg/mL were used. ATP solution and a kinase/peptide mixture were prepared. The plate was incubated for 1 h at room temperature. About 5 mL of the developing solution was added to each well. The plate was incubated for 1 h and then read by ELISA Reader (PerkinElmer, USA). Every experiment was repeated three times. Data represented as means ± SE from three independent experiments. Curve fitting was performed using Graph Pad Prism 5.
The effect on caspase-3 The Quantikine-Human active caspase-3 immunoassay (R&D Systems Inc., USA) is used to measure the active caspase-3 level, by adding 100 mL of the standard diluent to the zero standard wells. Cover and incubate for 2 h at room temperature. Add 100 lL of caspase-3 (active) detection antibody solution into each well except the chromogen blank. Incubate for 1 h then add 100 mL anti-rabbit IgG HRP working solution to each well and incubate for 30 min. The absorbance of each well was measured at 450 nm.
The effect on BAX and Bcl-2 levels Cells were grown in RPMI 1640 containing 10% foetal bovine serum at 37 C, stimulated with the compounds to be tested for Bax, and lysed with cell extraction buffer. This lysate was diluted in the standard diluent buffer over the range of the assay and measured for human active Bax and Bcl2 content according to the reported method 31 .

Analysis of the cell cycle distribution
To determine the effect of compound 10 and erlotinib on the cell cycle distribution of MDA-MB-231 cell line; cell cycle analysis was performed using the CycleTEST TM PLUS DNA Reagent Kit (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). Control cells with known DNA content (peripheral blood mononuclear cells) were used as a reference point for determining the DNA index for the test samples. The cells were stained with propidium iodide stain following the procedure provided by the kit then incubated at room temperature for 5 min in the dark and run on the DNA cytometer. Cell cycle distribution was calculated using CELLQUEST software (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA).

Radiosensitizing activity
Irradiation was performed at the National Center for Radiation Research and Technology (NCRRT), Egyptian Atomic Energy Authority (EAEA), using gamma cell-40 ( 137 Cs) source. Compound 10 was selected to be re-evaluated for the in vitro antiproliferative activity in combination with c-irradiation using MTT assay. Cells were incubated with compound 10 in molar concentrations of 0.01, 0.1, 1.0, and 10 mM. After 2 h, cells were subjected to a single dose of 8 Gy of c-radiation at a dose rate of 0.758 rad/s for 17.73 min, and then the anti-proliferative activity was measured 48 h after irradiation.
The IC 50 of the tested compounds was calculated after irradiation.

Molecular docking
Molecular modeling was performed using the Molecular Operating Environment (MOE, 10.2008) software. The protein data bank files (PDB: 1M17 and 3RCD) were selected for this purpose. Water molecules were ignored and hydrogen atoms were added. The cocrystallized ligands in both receptors were re-docked into the active site for method standardization. The structure of compound 10 was drawn on ChemDraw and copied as smiles to MOE. Energy minimizations were performed for compound 10 using MMFF94X force field and the partial charges were calculated. Docking of 10 inside the active site of the enzyme to generate one hundred conformations. Top-scored conformation was captured by 2D and 3D images.

Chemistry
The synthesis of the target compounds 5-18 was described in        (MDA-MB-231), and erlotinib was used as the reference drug.

Activation of caspase-3
Caspase-3 is a member of the cysteine-aspartic acid protease family that plays a crucial role in apoptosis 32 . It is an inactive proenzyme converted to the active form through caspases 8, 9, and 10 33 . Caspase-3 is activated in the apoptotic cell by both extrinsic (death ligand) and intrinsic (mitochondrial) pathways 34 by cleaving multiple proteins in the cells leading to cell death 35 .
The effect of compound 10 on caspase-3 was evaluated in reference to erlotinib. Compound 10 showed an increase in the level of the active caspase 3 by 10 folds compared to the control cells. While erlotinib increases the level of caspase 3 by 9 folds (Table 3).

Effects on Bcl-2 family proteins
The Bcl-2 family plays a central role in tumour progression or inhibition of mitochondrial intrinsic apoptotic pathway 36 . The pro-apoptotic Bax is essential for cell apoptosis. However, the anti-apoptotic Bcl-2 overexpression enhances cell survival by suppressing apoptosis 37 . Thus, the balance between these two different proteins determines the cell fate 38,39 . Increments in the Bax/Bcl2 ratio trigger a cascade of caspases that leads to the activation of caspase 3; the apoptosis executioner 40 . In this study, MDA-MB-231 breast cells were treated with compound 10 and their effect on the expression levels of Bcl2, and Bax were illustrated in Table 4. Compound 10 and erlotinib boosted the level of the pro-apoptotic protein Bax by 9 and 11 folds, respectively, compared to the control cells. On the other hand, they markedly reduced the levels of the anti-apoptotic proteins Bcl2 by 0.14 and 0.07 folds, respectively. The results showed that both compound 10 and erlotinib markedly boosted the Bax level and down-regulated Bcl2 level proving their pro-apoptotic effect.

Cell cycle analysis
Cell cycle progression is responsible for normal cell growth and proliferation. DNA damage can lead to either DNA repair or cell  Table 7. IC 50 of compound 10 on MDA-MB-231 cells before and after being subjected to a single dose of 8 Gy c-radiation.
Compound no. IC 50 before Irradiation (mM) IC 50 after Irradiation (mM) 10 0.31 ± 0.01 0.22 ± 0.03  Table 5 indicate that compound 10 arrested the cell cycle at the G2/M phase when compared to the untreated control (17.52% and 6.44%, respectively; Figure 2(A,C)). While erlotinib arrested the cell cycle at the G2/M phase by 24.81% (Figure 2(B)). Also, the cell population in G1 and S phases decreases after treatment (49.36% and 18.28% versus 69.55% and 23.04%, respectively) in case of  compound 10 compared to control. While in the case of erlotinib, the cell population in G1 and S phases markedly decreases after treatment to (41.55% and 16.31%, respectively). These results reveal that in MDA-MB-231 cells, cell cycle arrest occurs in the G2/ M phase in the case of compound 10 and erlotinib.

Cytotoxicity against normal breast cells
The cytotoxicity of compound 10 compared to erlotinib was measured against 184A1 normal breast cells using MTT assay in order to determine the relative safety of compound 10 on normal tissues. Compound 10 and erlotinib showed mild cytotoxic effect with an IC 50 of 84.5 and 101.9 mM, respectively ( Table 6).

Radiosensitizing evaluation
Most cancer patients receive radiation therapy during the course of treatment. Gamma rays are high energy radiation used in therapy to shrink tumours and kill malignant cells by damaging their DNA either directly or indirectly through free radicals formation. The major drawback of radiation therapy is that they cannot differentiate between normal and cancerous cells. So, the use of radiotherapy and selective chemotherapy are required in order to eliminate normal tissue damage 42 . The cytotoxicity of compound 10 was measured on MDA-MB-231 cell line before and after being subjected to a single dose of 8 Gy c-irradiation. The ability of compound 10 to enhance the cell-killing effect of c-irradiation was examined. The results showed that compound 10 is able to sensitize the cancer cells to the lethal effects of gamma radiation (Table 7).

Molecular docking
Molecular docking was performed using MOE 10.2008 inside the active site of EGFR (PDB ID: 1M17) 43 and HER2 receptors (PDB ID: 3RCD) 44 . In order to rationalize the biological results and to gain insight into the SAR of the target compounds, an attempt to interpret the observed enzymatic activities of the tested compounds on the basis of the ligand-protein interactions was done. The enzymatic activity of EGFR and HER2 inhibitors depends on the ability of the compound to properly dock into the binding site  and to establish interactions with the key amino acids. Accordingly, the active compound in this study should attain the same binding mode observed for the ligand.

Docking on EGFR
The EGFR catalytic domain consists of an N-terminal lobe, which consists mainly of one a-helix and C-helix. The C-terminal lobe is mainly a-helical, and a short strand termed the hinge region connects the two lobes 45 . The N-(3-ethynylphenyl)-6,7-bis(2methoxyethoxy)quinazolin-4-amine (erlotinib) is the co-crystallized ligand inside the EGFR receptor 46 . Erlotinib was located well in the ATP pocket and interacts with Met 769 by a hydrogen bond of 2.70 A length, and hydrophobic interactions with Leu 694, Leu 820, Lys 721, and Thr 766 (hinge region; Figure 3). Compound 10 was docked in the active site of the enzyme and bound in the same manner as the ligand. Compound 10 binds with energy score (S ¼ À9.88 Kcal/mol) and interact with the active site through Met 769 by a hydrogen bond of 0.85 A , Cys 773 with the CO of quinazolinone and Phe 699 with the phenyl ring of the acetamide through a p-p interaction ( Figure 4, Table 8).

Docking on HER2
The crystal structure of HER2 complexed with TAK-285 (PDB ID: 3RCD) showed that Ala 751, Leu 800, Met 801, Leu 852, and Asp 863 are the key amino acids. The X-ray co-crystallized structure of TAK-285 with HER2 demonstrated that it binds to the ATP pocket through an H-bond with Met 801 and to the hinge region by a series of hydrophobic interactions with Leu 852, Leu 726, Phe 1004, Thr 798, Thr 862, and Leu 785 47 ( Figure 5). Compound 10 pursued the similar binding pattern in HER2 with Met 801 by the SO 2 of the sulphonamide group, Thr 862 and Asp 863 by the CO of the acetamide and Lys 753 with the N-1 of quinazolinone ( Figure 6, Table 8).

Conclusion
An array of new 3,4-dihydrobenzo[g]quinazolinone derivatives containing sulphonamide moiety was designed, synthesized, and evaluated for their cytotoxic effect on MDA-MB-231 breast cancer cell line. The tested compounds showed IC 50 values ranging from 0.26 to 161.49 mM on MDA-MB-231. The new compounds were tested for their inhibitory profile against EGFR and HER2 enzymes. The 3,4-dimethyl phenyl derivative 10 was more potent than erlotinib on both EGFR and HER2 (IC 50 3.90 and 5.40 mM versus 6.21 and 9.42 mM, respectively). The 2,5-dimethyl phenyl derivative 11 was the most potent towards EGFR, while the anthraquinone derivative 18 was the most potent towards HER2. Compound 10 was evaluated as an apoptosis inducer through the activation of the proteolytic caspase-3, Bax and Bcl-2 expression levels, and cell cycle analysis. It was found that compound 10 increases the level of caspase-3 by 10 folds, Bax level by 9 folds, decreases the level of Bcl-2 by 0.14 folds and arrested the cell cycle in the G2/M phase. The radiosensitizing activity of 10 was measured on MDA-MB-231 cell line after being irradiated by a single dose of 8 Gy. IC 50 decreased from 0.31 to 0.22 mM after being irradiated. Docking of 10 inside the active site of EGFR and HER2 receptors revealed that it binds in the same manner as that of the co-crystallized ligand.