Synthesis of some quinazolinones inspired from the natural alkaloid L-norephedrine as EGFR inhibitors and radiosensitizers

Abstract A set of quinazolinones synthesized by the aid of L-norephedrine was assembled to generate novel analogues as potential anticancer and radiosensitizing agents. The new compounds were evaluated for their cytotoxic activity against MDA-MB-231, MCF-7, HepG-2, HCT-116 cancer cell lines and EGFR inhibitory activity. The most active compounds 5 and 6 were screened against MCF-10A normal cell line and displayed lower toxic effects. They proved their relative safety with high selectivity towards MDA-MB-231 breast cancer cell line. Measurement of the radiosensitizing activity for 5 and 6 revealed that they could sensitize the tumour cells after being exposed to a single dose of 8 Gy gamma radiation. Compound 5 was able to induce apoptosis and arrest the cell cycle at the G2-M phase. Molecular docking of 5 and 6 in the active site of EGFR was performed to gain insight into the binding interactions with the key amino acids.


Introduction
Cancer is characterized by the disturbance of normal cellular processes required for cell growth, division and differentiation [1][2][3] . Surgery, radiotherapy and chemotherapy, including immunotherapy, targeted and combined therapy, are different strategies advocated for cancer treatment [4][5][6] .
Protein kinases (PKs) play a pivotal role in cell proliferation by controlling signal transduction through the phosphorylation of different amino acid residues, namely tyrosine, threonine and serine 7 . Tyrosine kinases (TKs) are divided into receptor tyrosine kinases (RTKs) and non-receptor tyrosine kinases (NRTKs) in human genome. RTKs are vital components of cellular signaling pathways that are active during embryonic development and adult homeostasis. Due to their role as growth factor receptors, many RTKs have been involved in the onset or progression of various cancers, either by mutations or receptor/ligand overexpression; thus, they are considered attractive candidates for therapeutic intervention 7,8 . An example of RTK family members is epidermal growth factor receptor (EGFR). EGFR is a member of the ErbB receptor family and plays an essential role in cell signaling. Signaling is initiated by binding ligands to the extracellular domain of the EGFR, activating kinases and promoting cancer cell survival, invasiveness and drug resistance 9,10 . EGFR has a critical role in regulating several cellular functions such as cell growth, proliferation, differentiation and apoptosis, leading to the development of several types of solid tumors 11 . EGFR (HER-1) and ERB-B2 (HER-2) are characterized in solid tumors as breast, ovary, lung and others. The inhibition of EGFR is classified as targeted therapy as it aims at the differences between cancer and normal cells and is characterized by its high selectivity and lowered side effects.
Quinazolines are fused heterocyclic ring systems known for their variable biological activity [12][13][14][15] . They are well known for their inhibitory activity towards various protein kinase enzymes and their anticancer activity 16 . For example, lapatinib, a dual reversible EGFR and HER2 inhibitor. Also, gefitinib and erlotinib are reversible EGFR inhibitors; they are examples of FDA approved small molecules TK inhibitors 17 . Methaqualone, a potent hypnotic, was considered as an important landmark in synthetic anticonvulsants 18 . The 3-[b-keto-gamma-(3-hydroxy-2-piperidyl)-propyl]-4-quinazolone (A) was the first isolated natural quinazolinone alkaloid known by its antimalarial activity 19 . The quinazolinone derivatives (B) and benzo[g]quinazolinone (C) were reported to possess potent EGFR and HER2 inhibitory activity 20,21 (Figure 1). On the other hand, the Ephedra alkaloid, Norephedrine (NE) is a stereoisomer of phenylpropanolamine that is naturally occurring sympathomimetic 22 . Investigation revealed that long-term use of NE caused severe side effects, including fatality 23 . In addition to medicinal use, the properties of this alkaloid have attracted considerable attention in natural product chemistry field that leads to its use as a starting material in the preparation of chiral ligands for asymmetric catalytic synthesis 24,25 .
In continuation of our studies aiming to find new leads with potential anticancer activities, various substituted quinazolinones have been designed to accommodate different electronic natures as heterocycles representing the primary scaffold in many cytotoxic agents hoping to develop potent and safe anticancer agents and EGFR inhibitors. All the synthesized compounds were screened against MDA-MB-231, MCF-7, HepG-2, HCT-116 cancer cell lines and the most potent compounds were evaluated against MCF-10A normal cells to determine the selectivity of the compounds on the different cell lines. Also, the in vitro EGFR inhibitory activity of the compounds was measured. The effect of the most potent compounds on cell cycle progression and the radiosensitizing activity were evaluated. Docking studies were carried out to confirm the possible mode of action of the promising compounds.

Chemistry
Melting points were determined uncorrected by 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 solvent system of chloroform/methanol (8:2), spots were detected by UV light. IR spectra (KBr discs) were recorded using FT-IR spectrophotometer (Perkin Elmer, USA). 1 H, 13 C NMR and 2D NMR experiments 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 and CDCl 3 as solvents. EIMS were measured using Shimadzu-GC/MS. Elemental analyses were performed on a model 2400 CHNSO analyser (Perkin Elmer, USA). All the values were within ±0.4% of the theoretical values. The X-ray data were collected at T ¼ 298 K on Enraf Nonius 590 Kappa CCD single crystal diffractometer equipped with graphite monochromated Mo Ka (k¼0.71073 Å) radiation using w-x scan technique. All reagents used were of AR grade.

Radiosensitizing evaluation
Irradiation was performed at the National Centre for Radiation Research and Technology (NCRRT), Egyptian Atomic Energy Authority, using Gamma cell-40 ( 137 Cs) source. The promising compounds 5 and 6 were selected to be re-evaluated for in vitro cytotoxic activity after the cells containing the compounds were gamma-irradiated at a dose level of 8 Gy with a dose rate of 0.758 rad/s for 17.59 min. Cytotoxicity was measured two days after irradiation. The IC 50 of the tested compounds is calculated using GraphPad Prism 5.

Cell cycle analysis
The MDA-MB-231 cells (10 5 /well) were incubated with compound 5 at its IC 50 . After 24 h, the cells were washed twice with PBS, then collected and fixed with ice-cold ethanol 70% (v/v). The cells were re-suspended with 0.1 mg/mL RNase, stained with 40 mg/mL Scheme 3. Synthesis of compounds 9-11. PI and examined using flow cytometry (FACScalibur-Becton Dickinson).

Apoptotic assay
Cells were prepared as previously mentioned. Treatment of cells (10 5 ) with Annexin V-FITC and propidium iodide (PI) was by apoptosis detection kit [BD Biosciences, San Jose, CA]. The binding of Annexin V-FITC and PI was examined using flow cytometry FACScalibur (BD Biosciences, San Jose, CA). CellQuest software was used for performing quadrant analysis of co-ordinate dot plots.

Molecular docking
Docking studies were performed using Molecular Operating Environment software (MOE, 2015.10) provided by chemical computing group, Canada. The software was used to carry out the docking of the promising compounds in the receptor's active site. The protein crystal structure was obtained from the Protein Databank, PDB: 1M17 containing the EGFR enzyme co-crystallized with erlotinib. All the water molecules were removed, 3D protonation was performed. The pocket was determined by the alpha triangle matcher technique. The Energy Minimization was performed using MMFF94X force field with RMSD gradient of 0.001 kcal mol À1 Å À1 and the partial charges were calculated. The co-crystallized ligand was self-docked inside the active site. The compounds to be docked were drawn on ChemBioOffice 12 and copied as smiles to MOE followed by docking of 5 and 6.

Chemistry
The behaviour of the methyl 2-isothiocyanatobenzoate 1 towards the natural alkaloid L-norephedrine 2 was studied. When the isothiocyanate 1 reacted with 2 in chloroform containing a catalytic amount of triethylamine (TEA) at room temperature the unexpected quinazolinone 4 was formed despite the prospective thiourea derivative 3. In a trial to obtain the open-chain thiourea derivative 3, NMR tube reaction was carried out between 1 and 2.  MS spectra of 6 and 7 at 193 and 207 m/z, respectively were in full support of the proposed structures (Scheme 1).
When 1 reacted with 2 in DMF in the presence of a few drops of TEA yielded the thiazoloquinazolinone 8 rather than the expected 4 (Scheme 2, see Supplementary data 1). The structure of 8 was confirmed by different spectroscopic data and X-ray crystallographic analysis 29 is displayed in Figure 2.
In Scheme 3, the interaction of 3 with hydrazine hydrate in absolute EtOH gives the expected product 11 in addition to two other derivatives 9 and 10 that were apparent in TLC. The mechanism of formation of 9 and 10 is explained in Figure 3. d C 73.43 ppm indicated a non-substituted OH group. All these data proved that 11 lack the ring structure present in 9 and 10. The M þ at 310 m/z further confirmed the formation of 11. Treatment of 7 with 2 in EtOH 95%, in the presence of K 2 CO 3 afforded a mixture of 12 and 13. While when the same reaction was repeated in DMF instead of EtOH the unexpected dimer 14 was formed. However, both reactions were expected to give 11 (Scheme 4). The formation of 12 and 13 was assumed to proceed via addition-elimination mechanisms, as depicted in Figure  4. The NMR data structure of 12 and 13 indicated the disappearance of the S-CH 3 signals present in 7. 1 H NMR spectrum of 12 showed NH 2 signal at d H 5.50 and OH signal at d H 11.62 ppm. The mass spectrum of 12 showed an M þ at 177 m/z provided further evidence for replacing the S-CH 3 with OH group. In 13 signals for an ethoxy group at d  The signals of two moieties of L-norephedrine were overlapped in both 1 H and 13 C NMR spectra. HR ESI showed a quasi-molecular ion at 430.2127 m/z (calc. 430.2131) for M þ þ 1 ion certainly supporting the structure of 18. The NH proton appeared as two broad singlets at 4.15, 4.40 each integrated for half proton diagnostic for the suggested tautomerisation in the structure. All the assignments of 1 H and 13 C NMR signals were performed based on DEPT 135 as well as 2D NMR experiments including COSY, HSQC and HMBC (see Supplementary data 2).

In vitro cytotoxic activity evaluation
The in vitro cell viability activity of the targeted compounds 4-18 was measured through MTT assay against a panel of cell lines MDA-MB-231, MCF-7, HepG-2 and HCT-116 human cancer cell lines derived from breast, liver and colon tumors. A closer look at Table 1 indicates that compounds 4-18 showed variable IC 50 values against the tested cell lines and was compared to erlotinib and staurosporine, as standards.   (Table 2).

EGFR kinase assay
All the newly synthesized compounds, 4-18, were subjected to EGFR-TK inhibitory assay. Furthermore, a representative compound eliciting superior EGFR inhibition was subjected to cell cycle analysis and apoptotic assay to investigate its effect on cell cycle progression and apoptosis. Table 1 shows the inhibition data of EGFR (IC 50 values) for the examined compounds, erlotinib and        staurosporine, as reference standards. All analogues showed excellent EGFR inhibition potential ranging from 0.76 to 9.78 mM.  replacement of the thione group in 6 with methyl mercaptan 7, 1-hydroxy-1-phenylpropan-2-ylamino 11, hydroxy 12 or ethoxy group 13 reduces the activity. Regarding the two homologues 8 and 15, the replacement of sulphur with oxygen greatly enhances the EGFR activity, while the opposite occurs in 9 and 10. The two dimers 14 and 17 show very potent activity that demonstrates that a bulky rigid structure is favourable for binding with the receptor.

Radiosensitizing activity
Radiotherapy is second to surgery in cancer treatment. The major drawback of radiotherapy is its inability to differentiate between cancerous and normal tissues. Radiation causes ionization and excitation of atoms that result in the generation of short-lived free radicals. These free radicals can damage proteins and membranes, leading to single or double DNA strand breaks 31,32 . A radiosensitizing agent can induce tumor sensitization to ionizing radiation, thus lowering the required dose for treatment. This enhancement of radiation effects not only control the local tumors but also limit the metastatic spread. EGFR inhibitors can adopt another mechanism of action by inhibiting accelerated repopulation of tumor cells during fractionated radiotherapy as they block the membrane receptors of growth factors or interfere with the signaling pathways involved in cell proliferation 33,34 .
The ability of the most active compounds 5 and 6 to enhance gamma radiation-induced tumor cell death was examined. The results proved the ability of the two compounds to sensitize the cancerous cells to the lethal effects of ionizing radiation (Table 3). Compounds 5 and 6 showed enhanced cytotoxicity on all cell lines after irradiation with a single dose of 8 Gy gamma radiation. Compound 5 was more potent on all the tested cell lines with IC 50 < 5 mM.

Effect on cell cycle progression
The therapeutic effect of the anticancer agent depends upon its ability to stop cell cycle progression by arresting cell division at certain checkpoints promoting apoptosis. These checkpoints exist at G1-S, S and G2-M phases 35,36 . The most potent and selective compound 5 was chosen to determine its ability to induce apoptosis using MDA-MB-231 cells according to the reported method 37 . The cells were treated with compound 5 at a concentration equals to its IC 50 value on EGFR (0.76 lM) for 24 h. It is clear from Figures  8 and 9 that compound 5 interfered with the cell cycle in the G2-     M phase. At that phase, accumulating cells reached 40.39% after treatment of control MDA-MB-231 cells (6.82%) with compound 5. Furthermore, compound 5 raised the percentage of cells at pri-G1 phase by 10 folds to reach 19.23% after being 1.91% in control cells. On the contrary, the cell population in G1 and S phases decrease after treatment with compound 5. So, compound 5 induces apoptosis through cell cycle arrest in the G2-M phase.

Apoptotic assay
Phosphatidylserine (PS) exposure on the outer plasma membrane was detected during apoptosis and forms the basis for Annexin V/ PI (propidium iodide) double staining assay to detect apoptotic cell death. At early apoptosis, the cell membrane excludes viability dyes such as PI and permits the determination of apoptotic cell kinetics according to the cell cycle 38,39 . To investigate the mode of induced cell death, MDA-MB-231 cells were incubated with compound 5 at 0.76 lM for 24 h. Compound 5 induced apoptosis (19.1%) by more than 12 folds over the control (1.54%). Compound 5 induced early apoptosis by 7.36% and enhanced late apoptosis by 11.74% compared with the untreated control cells (Figures 10 and 11).

Molecular docking
Molecular docking of compounds 5 and 6 was performed on the active site of EGFR co-crystallized with erlotinib (PDB: 1M17) (Figure 12) 40 . The active site of 1M17 consists mainly of these key amino acids; Met 769, Leu 694, Thr 766, Ala 719, Leu 764, Gln 767, Leu 768, Pro 770, Phe 771, Gly 772, Leu 820, Thr 830 and Asp 831. The ligand compounds 5 and 6 were docked into the active site of the target protein 1M17 and the binding affinities, energy scores and RMSD values for compounds were recorded. Validation of molecular docking showed that the RMSD values are within acceptable limits (less than 2 Å) 41 . The best binding affinity with the lowest energy score for the compounds was computed as À10.14 kcal mol À1 (compound 5) and À10.03 kcal mol À1 (compound 6). According to these findings, together with the abovementioned biological evaluation, compounds 5 and 6 may act as effective docking material for EGFR tyrosine kinase. The 2D and 3D visuals of the interaction map for compound 5 can be seen in Figure 13. The hydrogen bond formation connected the CO of quinazolinone with Met 769 of the target protein with a length of 2.39 Å and Thr 766 by OH with 2.63 Å. Superimposing compound 5 with erlotinib showed that they adopt the same orientation inside the active site with RMSD ¼ 1.243 Å (Figure 14). On the other hand, four conventional hydrogen bond interactions were observed between compound 6 and the macromolecule 1M17 as follows; the CO of the quinazolinone with Met 769 and Leu 768 with a recorded distance of 2.54 and 2.79 Å, respectively. In addition to Thr 766 that forms two hydrogen bonds with NH and CS at a distance of 2.88 and 3.01 Å (Figure 15). Overlaying of erlotinib and compound 6 can be observed in Figure 16 with RMSD ¼ 1.236 Å.

Conclusion
In this study, a novel series of quinazolinone and fused quinazolinone derivatives synthesized by the aid of L-norephedrine were obtained. All these compounds showed variable anticancer activity against MDA-MB-231, MCF-7, HepG-2 and HCT-116 cancer cell lines and EGFR inhibitory activity comparable to erlotinib. The 3-(1-hydroxy-1-phenylpropan-2-yl)-2-(methylthio)quinazolin-4(3H)one 5 and 3-amino-2-thioxo-2,3-dihydroquinazolin-4(1H)-one 6 were the most promising in this series towards the cancer cell lines and EGFR. Compounds 5 and 6 were further selected to measure their relative safety and selectivity towards normal cells. They showed mild cytotoxic activity towards MCF-10A normal cell line and high selectivity towards MDA-MB-231 cell line. Besides, they displayed radiosensitizing activity through their ability to sensitize the cancer cells to the lethal effect of gamma irradiation. The most potent compound in this series, 5, undergoes cell cycle analysis and annexin V/PI assay to detect apoptotic cell death. Compound 5 proved to arrest the cell cycle progression at the G2-M phase, induce early apoptosis and enhance late apoptosis. Moreover, molecular docking of compounds 5 and 6 showed the key interactions required for EGFR inhibition. Finally, compounds 5 and 6 could be considered as promising leads for the development of new anticancer and radiosensitizing agents.