Design and green synthesis of novel quinolinone derivatives of potential anti-breast cancer activity against MCF-7 cell line targeting multi-receptor tyrosine kinases

Abstract A new set of 4,6,7,8-tetrahydroquinolin-5(1H)-ones were designed as cytotoxic agents against breast cancer cell line (MCF-7) and synthesised under ultrasonic irradiation using chitosan decorated copper nanoparticles (CS/CuNPs) catalyst. The new compounds 4b, 4j, 4k, and 4e exhibited the most potent cytotoxic activity of IC50 values (0.002 − 0.004 µM) comparing to Staurosporine of IC50; 0.005 μM. The latter derivatives exhibited a promising safety profile against the normal human WI38 cells of IC50 range 0.0149 − 0.048 µM. Furthermore, the most promising cytotoxic compounds 4b, 4j were evaluated as multi-targeting agents against the RTK protein kinases; EGFR, HER-2, PDGFR-β, and VEGFR-2. Compound 4j showed promising inhibitory activity against HER-2 and PDGFR-β of IC50 values 0.17 × 10−3, 0.07 × 10−3 µM in comparison with the reference drug sorafenib of IC50; 0.28 × 10−3, 0.13 × 10−3 µM, respectively. In addition, 4j induced apoptotic effect and cell cycle arrest at G2/M phase preventing the mitotic cycle in MCF-7 cells.


Introduction
Breast cancer is one of the most common causes that threatens women's health impacting $15% of all cancer deaths occurred in women all over the world 1,2 . The incidence of breast cancer is increasing in all the regions of globe. Although chemo and radiotherapies are considered the most important prime option for the treatment of cancer disease, 3 the drug-induced toxicity to the normal cells and drug-resistance constitute problems that still need to be resolved 4 . Therefore, the discovery of new selective and safer anticancer agents is still a great interest in the field of medicinal chemistry 5 . inhibitors [6][7][8][9] . In addition, these suppressors can inhibit the overexpression of tyrosine kinase and maintain its physiological balance 10 . The US FDA approves more than 60 small-molecule protein kinase inhibitors. Most of them are multi-targeting suppressors synergistically functioning on many signalling pathways by interacting with each target simultaneously 11 . Targeting multiple kinases increases the anticancer potency as well as reduces the risk of developing drug resistance [11][12][13] (Figure 1).
Quinoline is naturally present in many alkaloids having potent antitumor activity, for example, camptothecin 14 (Figure 1). Quinoline scaffold and its related derivatives represent a broad spectrum of pharmacological activities, especially in drug discovery of new anticancer agents [15][16][17][18] . Food and Drug Administration (FDA) has approved various quinoline small molecules acting as protein kinases inhibitors for clinical uses in cancer disease [19][20][21][22] (Figure 1). The occurrence of the nitrogen atom in the quinoline nucleus withdraws electrons by resonance and interferes with the equal distribution of p electron density. It has been reported that the quinoline nucleus has a great tendency to bind to the active site of various proteins via the formation of hydrogen bonds with its nitrogen atom and p-p stacking complexes with complementary amino acid residues 15,16 .
Molecular hybridisation currently appears as a promising drug design strategy, specifically in discovering new anticancer drugs 28,29 . It has been reported that conjugating of two or more pharmacophores in the same molecular architecture might decrease the risk of drug-drug interactions, overcome the problem of drug resistance as well as enhance the biological efficacy via the binding with different targets as one single entity 30 .
Based on the above findings, in continuation of our recent work of using green synthetic approaches to develop a variety of heterocyclic systems with great biological importance [31][32][33][34] , and in an attempt to prepare new potent anti-breast cancer leads of potential suppression activity against different RTKs EGFR, HER-2, PDGFR-b, and VEGFR-2, this study deals with the synthesis of new tetrahydroquinolines hybridised with other substituted phenylsulfonyl-phenyl moieties at C-4 position and conjugated with different groups at C-2 and C-3 positions (Figure 3). It has been taken in consideration the effect of molecular orientation, ring size variation and the occurrence of different heteroatoms that could provide hydrogen binding with various RTKs binding pockets ( Figure 3). The cytotoxic activity against human breast cancer cells (MCF-7) was evaluated for all the new prepared analogues. In addition, in vitro multi-targeting inhibition assessment against EGFR, HER-2, PDGFR-b, and VEGFR-2 of the most active cytotoxic candidates was also carried out. Extra investigations of different mechanistic pathways such as cell cycle analysis and apoptosis were evaluated for the most promising compound as a representative example for the new active analogues. Furthermore, molecular modelling studies were performed to explore the modes of interaction between the promising target compounds and the vital amino acids residues of different kinases to ascertain binding stability and the relationship between their physicochemical characteristics and their favourable suppression effects.

Chemistry
All organic solvents were purchased from commercial sources and used as received unless otherwise stated. All other chemicals were purchased from Merck, Aldrich, or Acros and used without further purification. Thin-layer chromatography (TLC) was performed on precoated Merck 60 GF254 silica gel plates with a fluorescent indicator, and detection by means of UV light at 254 and 360 nm. The melting points were measured on a Stuart melting point apparatus and are uncorrected. IR spectra were recorded on a Smart iTR, which is an ultrahigh-performance, versatile Attenuated Total Reflectance (ATR) sampling accessory on the Nicolet iS10 FT-IR spectrometer. The NMR spectra were recorded on a Bruker Avance III 400 (9.4 T, 400.13 MHz for 1 H, 100.62 MHz for 13 C) spectrometer with a 5-mm BBFO probe, at 298 K and a Bruker High Performance Digital FT-NMR Spectrometer Avance III 850 MHz. Chemical shifts (d in ppm) are given relative to internal solvent, DMSO-d 6 2.50 for 1 H and 39.50 for 13 C, CDCl 3 7.25 for 1 H and 77.7 was used as an external standard. Mass spectra were recorded on a Thermo ISQ Single Quadrupole GC-MS. Elemental analyses were carried out on a Euro Vector instrument C, H, N, S analyser EA3000 Series. Sonication was performed by Techno-gaz sonicator (with a frequency of 37 kHz and ultrasonic peak max. 320 W).
The catalyst (CS/CuNPs) 35 , 4-(phenylsulfonyl)benzaldehyde (2a) 36 and 4-tosylbenzaldehyde (2b) 37 were prepared according to reported literature. (1 mmol), active methylene compounds 3a-f (1 mmol) and ammonium acetate (9 mmol) in ethanol (25 ml) containing a catalytic amount of Cu-chitosan NPs (0.1 g) was refluxed at 60 C for the appropriate time (cf. Table 1) until completion of the reaction (monitored by TLC). The reaction mixture was filtered to separate the catalyst; then, the filtrate was cooled at room temperature, and the reliable product obtained was filtered, dried, and purified by recrystallisation from ethanol.

Sonicated reactions
These processes were performed on the same scale described above for silent reactions. All The reactions were kept at 60 C, which was attained by adding or removing water in an ultrasonic bath (the temperature inside the reaction vessel was 58-63 C). The sonochemical reactions were continued for a suitable time (cf. Table 1) until the starting materials were no longer detectable by TLC. Then, the catalyst was separated, and the products obtained were purified as described above in silent reaction procedures. The synthesised compounds with their physical data are listed below.

Kinase assays
The activities of the examined compounds against EGFR, HER2, PDGFR-b, and VEGFR2 were in vitro tested using Abcam's Human In cell ELISA Kit (ab 126419) for EGFR, ADP-Glo TM Kinase Assay for Her2, PDGFR-b, Active, Recombinant protein expressed in Sf9 cells for VEGFR2 (KDR) Kinase Assay Kit Catalog #40325, respectively. The procedure of the used kits was done according to the manufacturer, s instructions.

Cell cycle analysis and apoptosis detection
Cell cycle analysis and apoptosis investigation were carried out by flow cytometry. 39,40 MCF-7 cells were seeded at 8 Â 104 and incubated at 37 C, 5% CO 2 overnight. After treatment with the tested compound, for 24 h, cell pellets were collected and centrifuged (300 g, 5 min).

Molecular docking method
The docking studies had been calculated and studies using Molecular Operating Environment (MOE, 2019.0102) software [41][42][43] . All minimisations were performed with MOE until an RMSD gradient of 0.1 kcalÁmol À1 Å À1 with MMFF94x force field and the partial charges were automatically calculated.

Platelet-derived growth factor receptor a (PDGFRA)
The X-ray crystallographic structure of human platelet-derived growth factor receptor a (PDGFRA) co-crystalised with Imatinib (PDB ID: 6JOL) was downloaded from the protein data bank (https://www.rcsb.org/structure/6JOL). For each co-crystallised enzyme, water molecules and ligands which are not involved in the binding were removed. The protein was prepared for the docking study using Protonate 3D protocol in MOE with default options. The co-crystalised ligand (imatinib) was used to define the binding site for docking. Triangle Matcher placement method and London dG scoring function were used for docking.
The progress of reactions was monitored by TLC till the disappearance of reactants. The beneficial effect of ultrasound is noticeable over conventional conditions on this reaction (Table 1), reducing the reaction time from 5 to 6 h into 20-30 min. and the yield of reaction increased to 90-95% under ultrasound irradiation compared to 78-84% under conventional conditions ( Table 1).
The reaction products were identified as the polysubstituted 4,6,7,8-tetrahydroquinolin-5(1H)-ones 4a-l in all cases based on its 1 -H NMR spectrum. For example, the 1 H NMR spectrum of the isolated reaction product 4a revealed five singlet signals at d 0.80, 0.98, 2.28, 3.49, and 4.91 ppm due to the four methyl groups and CH-4, respectively, two shielded doublets (dd) at d 1.97, 2.15, ppm with coupling constants of approximately 16 Hz due to the H8a, H8b, another two shielded doublets (dd) at d 2.33, and 2.41 ppm with coupling constant of 16.4 Hz due to H6a, H6b and two doublets and aromatic multiplet in the region 7.76-7.80 ppm due to 9 aromatic protons. In addition to a D 2 O exchangeable signal at 9.21 indicates the presence of NH group. Also, the 13 C NMR of the isolated product 4a adds strong evidence for the proposed structure, which agrees with the structure formed as shown in Scheme 1 (cf. experimental part).
The apparent effect of ultrasound on the reaction mentioned above may be attributed to the fact that ultrasonic irradiation gives the reactants sufficient energy to exceed the energy barrier of the reaction, thus, 4,6,7,8-tetrahydroquinolin-5(1H)-ones 4a-l formed. This sufficient energy can be reasonably interpreted in terms of the physical phenomenon called acoustic cavitation (in our case, at solid-liquid interfaces). The most accepted proposed mechanism for the effect of cavitation near surfaces 44,45 is microjet impact and shockwave damage. Along with the shock wave associated with the cavitation collapse, the jet causes localised deformation and surface erosion, which increases the possible reaction area. Therefore, the treated surfaces contain an increased number of dislocations that are widely considered to be the active sites in catalysis.

In vitro anticancer activity
This work represents the construction of a new series of compounds bearing 7,7-dimethyl-4-(4-(phenylsulfonyl)phenyl)-4,6,7,8tetrahydroquinolin-5(1H)-one scaffold 4a-l of potential anti-breast cancer activity. To evaluate the cytotoxic activity of the new derivatives, they were subjected to MTT cell viability assay against human breast cancer MCF-7 cell line utilising staurosporine as a reference drug. [46][47][48] The obtained data were represented as IC 50 (mM) values which are the average of at least three independent experiments and tabulated in Table 2.
The new derivatives showed variable degrees of cytotoxic activities. Interestingly, the 2-trifluoromethyl derivative 4b exhibited about 2.5-fold more potent activity than the standard drug Scheme 1. Synthesis of novel tetrahydroquinolin-5(1H)-ones 4a-l. staurosporine (IC 50 ; 0.002 mM, IC 50staurosporine ; 0.005 mM). The potent cytotoxic effect of 4b could be explained due to the electronwithdrawing power of fluorine atom, alongside the increased carbon-fluorine bond energy, that significantly potentiates the metabolic stability of the host molecule and enhances its lipophilicity facilitating the cell membrane permeation and its pronounced cytotoxic effect 38 . A slight decrease in the potency was detected upon replacement of -CF 3 group by NH 2 as compounds 4j, 4e, 4k exhibiting about 1-1.6-fold more potent activity than that resulted by staurosporine of IC 50 values of 0.003, 0.004, and 0.004 mM, respectively. Further decrease in the inhibitory activity to be 1.4-fold less potent than the standard drug was obtained upon replacement of the 3-methoxy/ethoxy groups by phenyl or p-bromophenyl groups as compounds 4d, 4 l, of IC 50 ; 0.007 mM. The rest of the compounds produced a detectable decrease in the cytotoxic potency of IC 50, ranging from 0.023 to 0.103 mM. It could be noted that the significant increase in the hydrophobicity of the compounds negatively affects the cytotoxic activity of the new tetrahydroquinolinone derivatives.

3.2.2.
The effect of compounds 4b, 4j, 4k, and 4e against the normal WI38 cells One of the characteristics differentiating different anticancer agents from each other is the recurrence and severity of their side effects to the normal cells at their therapeutic doses. Accordingly, the safety profile of the most promising candidates (4b, 4j, 4k, and 4e) was evaluated against the normal WI38 cells derived from lung tissues in comparison with staurosporine as a reference drug-using MTT assay [46][47][48] (Table 3). It is worthy of mentioning that the IC 50 values of the target compounds 4j, 4b against the normal WI38 cells were 20 and 24-fold higher than their IC 50 doses against the cancer cells and about 3-fold higher than the IC 50 value of reference drug staurosporine confirming the promising safety profile of both compounds. Whereas, less safety profile was investigated by compounds 4e and 4k producing IC 50 values against WI38 cells that were only 4-fold higher than their IC 50 doses against MCF-7 cells and approximately equal to that obtained by staurosporine (Table 2).

In vitro kinase inhibition assay
To explore the mechanistic insight into the cytotoxic potentials of the new quinolone compounds, in vitro kinase assay was performed to evaluate the kinase suppression activity of the most promising cytotoxic candidates 4b, 4j against four different RTK. Which are: EGFR, human epidermal growth factor receptor (HER2), PDGFR-a, and VEGFR-2. It has been detected that compound 4j was more potent than 4b as EGFR inhibitor with IC 50 values of 0.07 Â 10 À3 and 0.11 Â 10 À3 mM, respectively, but both were less potent than the Sorafenib of IC 50 0.04 Â 10 À3 mM (Table 4). On the Table 2. In vitro cell cytotoxic activity of the new compounds against MCF-7 cancer cells.

Apoptosis assay
The derivatives 4b and 4j were selected to study their apoptotic effects on MCF-7 cancer cells using Annexin-V/PI binding assay based on their promising cytotoxic potency and various kinase inhibitory effects. Staining MCF-7 cells was carried out with the two dyes; Annexin V/propidium iodide (PI) after treating them with compounds 4b and 4j at their IC 50 concentrations of 0.002 and 0.003 mM for 24 h. Flow cytometry method 39,49 has been used to detect the corresponding red (PI) and green (FITC) fluorescence. It has been noted that there was an increment in the percentages of the late apoptosis produced by the evaluated compounds 4b and 4j from 0.13% (control DMSO/MCF-7 cells) to 13.81% and 22.03%, respectively. Also, the tested compounds produced early apoptotic effects of 6.15% and 3.27% compared to 0.58% of the untreated MCF-7 cell with necrosis percent of 4.37% and 9.22%, respectively, vs. 0.66% produced by the DMSO control (Figures 4  and 5). The proportion of the late apoptosis produced by both 4b and 4j was higher than the proportion of the early phase, which makes recovering the dead cells to safe ones is more challenging.

Cell cycle analysis
The induction of apoptosis is one of the most crucial tools that confirm the effectiveness of cancer therapy 50 . Cell cycle checkpoints are G1 (restriction or start), S (metaphase), and G2/M. One of the main functions of the anticancer therapeutics is stoppage of the cell division at these checkpoints. 51 Thus, MCF-7 cells were incubated with compounds 4b and 4j at their IC 50 concentrations (0.002, 0.003 mM) for 24 h. The cells were stained with Annexin V/ PI and examined using flow cytometry procedure. The resultant data revealed that there was cell accumulation of percentages 24.33% and 18.03% at pre G1 and G2/M phases in MCF-7 cells treated with compound 4b and cell accumulation percentages of 34.52% and 31.17% at pre G1 and G2/M phases in MCF-7 cells treated with compound 4j comparing to 1.43% and 5.03% of the untreated MCF-7 cells. This result represents that there was cell cycle arrest at G2/M phase with mitotic cycle cessation (Table 5, Figure 6).

Molecular docking study
The molecular modelling studies were carried out using MOE (2019.0102) software. All minimisations were performed with MOE until an RMSD gradient of 0.1 kcalÁmol À1 Å À1 with MMFF94x force field, and the partial charges were automatically calculated.

Human epidermal growth factor receptor 2 (HER2)
The X-ray crystallographic structure of human HER2 (HER2) co-     (03Q) (PDB ID: 3PP0) was downloaded from the protein data bank (https://www.rcsb.org/structure/3PP0). For each co-crystallised enzyme, water molecules and ligands that are not involved in the binding were removed. The protein was prepared for the docking study using Protonate 3D protocol in MOE with default options. The co-crystalised ligand (03Q) was used to define the binding site for docking. Triangle Matcher placement method and London dG scoring function were used for docking.
Through examination of the binding interactions of 03Q to the active site of the enzyme, it shows strong bond interactions with Gln799, Leu800, Met801, Arg849, and Asp863 (Figure 7).   The docking setup was first validated by self-docking the cocrystallised ligand (30Q) in the vicinity of the enzyme's binding site. The docking score (S) was À17.1413 kcal/mol, and the root means square deviation (RMSD) was 0.14339 Å (Figure 8).
The 4j compound showed high energy binding score (À17.3597 kcal/mol.) similar to that of the co-crystallised ligand and higher than Sorafenib. Moreover, it showed good binding interactions with the amino acids in the active site of the receptor. The results are summarised in Table 6 and Figures 9 and 10.

Platelet-derived growth factor receptor a (PDGFr-a)
The X-ray crystallographic structure of human PDGFRA co-crystalised with Imatinib (PDB ID: 6JOL) was downloaded from the protein data bank (https://www.rcsb.org/structure/6JOL). For each co-crystallised enzyme, water molecules and ligands that are not involved in the binding were removed. The protein was prepared for the docking study using Protonate 3D protocol in MOE with default options. The co-crystalised ligand (imatinib) was used to define the binding site for docking. Triangle Matcher placement method and London dG scoring function were used for docking.
By examining the binding interactions of imatinib to the enzyme's active site, it shows strong bond interactions with Val607, Glu644, Thr674, Cys677, His816, and Asp836 ( Figure 11).    The docking setup was first validated by self-docking the cocrystallised ligand (imatinib) in the vicinity of the enzyme's binding site. The docking score (S) was À18.0520 kcal/mol, and RMSD was 0.6983 Å (Figure 12).
The 4j compound showed a similar energy binding score to that of Sorafenib. It exhibited good binding interactions with the amino acid in PDGFR-a active site. The results are summarised in Table 7 and Figures 13 and 14.

Conclusion
A new class of 4,6,7,8-tetrahydroquinolin-5(1H)-one-based derivatives has been green synthesised as anti-breast cancer (MCF-7) agents of potential multi-targeting RTKs. The compounds 4a-l were examined as cytotoxic agents against MCF-7 cancer cells using MTT assay utilising staurosporine as a standard drug. The compounds 4b, 4e, 4j, and 4k appeared as the most promising cytotoxic candidates. That revealing a more potent inhibiting effect than staurosporine, displaying IC 50 values ranging from 0.002 to 0.004 mM vs. IC 50 value of staurosporine, 0.007 mM. The safety profile of the latter derivatives was evaluated against the normal WI38 cells. The compounds 4b and 4j appeared as the safest agents on the normal cells. Furthermore, the compound 4b, 4j were selected as representative examples to evaluate their suppression activity against EGFR, HER-2, PDGFR-a, and VEGFR-2 protein kinases. The compound 4j investigated potent multi-targeting inhibitory activity in comparison with Sorafenib. In addition, the biological evidence revealed that the compound 4j caused a marked apoptotic degree with a necrosis percentage 4.2%, leading to cell cycle disruption at G2/M phase in MCF-7 cancer cells. Accordingly, the new derivatives bearing 4,6,7,8-tetrahydroquinolin-5(1H)-one scaffold could be considered primary nuclei for further structural optimisation to get more potent, selective, and safer anticancer candidates. A molecular docking study was found in complete agreement with the obtained experimental results.