Synthesis, potential antitumor activity, cell cycle analysis, and multitarget mechanisms of novel hydrazones incorporating a 4-methylsulfonylbenzene scaffold: a molecular docking study

Abstract Hydrazone is a bioactive pharmacophore that can be used to design antitumor agents. We synthesised a series of hydrazones (compounds 4–24) incorporating a 4-methylsulfonylbenzene scaffold and analysed their potential antitumor activity. Compounds 6, 9, 16, and 20 had the most antitumor activity with a positive cytotoxic effect (PCE) of 52/59, 27/59, 59/59, and 59/59, respectively, while compounds 5, 10, 14, 15, 18, and 19 had a moderate antitumor activity with a PCE of 11/59–14/59. Compound 20 was the most active and had a mean 50% cell growth inhibition (GI50) of 0.26 µM. Compounds 9 and 20 showed the highest inhibitory activity against COX-2, with a half-maximal inhibitory concentration (IC50) of 2.97 and 6.94 μM, respectively. Compounds 16 and 20 significantly inhibited EGFR (IC50 = 0.2 and 0.19 μM, respectively) and HER2 (IC50 = 0.13 and 0.07 μM, respectively). Molecular docking studies of derivatives 9, 16, and 20 into the binding sites of COX-2, EGFR, and HER2 were carried out to explore the interaction mode and the structural requirements for antitumor activity.

In this study, we synthesised a series of hydrazones (compounds 4-24) incorporating a 4-methylsulfonylbenzene scaffold (  Figure 2. The reported anticancer agents bearing hydrazone, sulphonyl, and benzamide fragments. and 2. A 4-methylsulfonylbenzene core was linked with a hydrazone moiety and connected with various arylidenes with or without hydroxyl and N,N-diethylamine fragments ( Figure 3). We also evaluated the in vitro antitumor activities of the synthesised compounds using 59 human cancer cell lines and investigated the structure-activity relationship (SAR) of the compounds with various substituents, depending on their antitumor activities. Next, we performed a cell cycle analysis and apoptotic induction assay of the most active compounds using the HL-60 cell line. We performed an enzymatic assay of the EGFR and HER2 inhibitory activity of the most promising compounds and evaluated the COX-2 inhibitory activity of the most active derivatives. Finally, we used molecular docking to predict the interaction mode of the biologically active compounds in the binding pockets of COX-2 isozyme, and EGFR, and HER2 tyrosine kinases.

Annexin V-FITC apoptosis assay
Apoptosis induction is the most important mechanism by which major chemotherapeutics kill cancer cells 71 . Apoptosis causes cellular changes whereby the translocation of phosphatidylserine (PS) occurs through the plasma membrane from the inside to the outside 71 . Annexin-V can bind to PS, which can be used as a sensitive probe for PS on the outer side of the plasma membrane 72 . We performed cytometric analysis to distinguish apoptosis from the necrosis mode of HL60 cell death induced by the most active compounds 6, 9, 16, and 20 using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (AV/PI) dual-staining assay with the BD FACSCalibur (BD Biosciences, San Jose, CA, USA) ( Table 5). The AV/PI staining of HL60 cells was performed at a mixed molar concentration of 10 mM with compounds 6, 9, 16, and 20 for 24 h. Figure 4 and Table 5 show the results of treating HL60 cells with compounds 6, 9, 16, and 20 for 24 h. We found an increase in the early apoptosis ratio (Figure 4, lower-right quadrant of the cytogram) from 0.57% in the control sample (DMSO) to 3.52%-8.42% and a sharp increase in the late apoptosis ratio (Figure 4, upperright quadrant of the cytogram) from 0.22% to 7.64%-13.41%. These data support the apoptotic mechanism underlying programmed cell death induced by compounds 6, 9, 16, and 20 rather than the necrotic pathway.

In vitro cell cycle analysis
Antitumor agents can induce apoptosis by activating signalling pathways, leading to G2/M phase arrest 73,74 . Flow cytometry is used to measure cell growth in different cell cycle phases (pre-G1, G1, S, and G2/M) 73,74 . We selected the most active compounds 6, 9, 16, and 20 for further analysis of their effects on cell cycle progression in the HL60 cell line ( Figure 5 and Table 6). We used the solvent DMSO as a negative control. Briefly, we incubated HL60 cells with 10 mM compounds 6, 9, 16, and 20 for 24 h. Compounds 6,9,16, and 20 interfered with the normal cell cycle of HL60 cells. There was a significant effect on the percentage of apoptotic cells, as indicated by an increase in cells in the pre-G1 phase (12.57%-24.31%) and the G2/M phase (23.27%-38.09%) compared to the control (1.71% and 12.03% cells, respectively). In contrast, the percentage of cells in S and G0/G1 phases significantly decreased (22.49%-29.43% and 36.28%-48.31%, respectively) compared to the control (35.3% and 52.67%, respectively), causing cell cycle arrest. These results clearly indicated that compounds 6,9,16, and 20 arrests the G2/M phase of the cell cycle ( Figure 5 and Table 6).

Enzymatic inhibition assay
2.4.1. Cox-2 inhibition activity COX-2 is overexpressed in several cancer cell lines during cell proliferation. Its inhibition is used as a target for cancer treatment and prevention 51,52,75 . Therefore, we performed COX-2 inhibition assays (kit catalogue no. 560101; Cayman Chemicals Inc., Ann Arbour, MI, USA) using compounds 6,9,16, and 20, which showed the highest antitumor activity, in addition to the reference drug celecoxib 15,76 . The results were expressed as IC 50 (mM) as the mean of three acquired determinations ( Table 7). The IC 50 of celecoxib as a COX-2 inhibitor was 2.79 mM. Compounds 9 and 20 were the most active COX-2 inhibitors (IC 50    alkylphenyl moiety, such as the 4-tolyl fragment in compound 9 and the N,N-diethylaminophenyl fragment in compound 20, have high COX-2 inhibition compared to compounds devoid of the 4-alkylphenyl moiety, such as compounds 6 and 16.

Molecular docking study
Molecular modelling is an important tool for studying the biological activity and SARs of bioactive compounds and exploring the binding mode of ligand molecules within the receptor-or putative enzyme-binding sites [77][78][79][80] . We performed molecular docking using the MOE 2008.10 program protocol obtained from Chemical Computing Group Inc. (Montreal, QC, Canada) 81 . We subjected the selected compounds and the co-crystallized bound inhibitors to molecular docking into the putative active site of the protein to ensure docking accuracy and generate an appropriate binding orientation 27,40,41,82,83 .
2.5.1. Molecular docking of compound 9 with COX-2 Molecular docking was performed to study the mode of interaction between the most active compound 9 and the COX-2 pocket-binding site (Figure 6). We derived the crystallographic binding site on the COX-2 isozyme in a complex with the SC-558 ligand, a celecoxib analogue, from the Protein Data Bank (PDB code: 1CX2) ( Figure 6, left panel). We used the interaction energy and hydrogen bond formation among compound 9 and the amino acids Table 4. Average antitumor activity of compound 20, and reference drugs against tumour cell lines from nine different organs at 10-fold dilution of five concentrations; median growth inhibitory (GI 50 , mM), total growth inhibitory (TGI, mM) and median lethal (LC 50 , mM) a .  within the putative active pocket of the COX-2 isozyme to predict the mode of interaction. Figure 6 shows the molecular docking results for compound 9.    phenyl part of the benzylidenehydrazine moiety interacted with the amino acid residue Arg-120 by forming a nonclassical H-bond (2.99 Å) and CH-p interaction (3.98 Å) ( Figure 6, right panel).

Molecular docking of compound 20 with EGFR
The results of the antitumor activity and enzymatic assay of compound 20 against EGFR prompted us to perform molecular docking studies of the ATP-binding site of EGFR, along with the reference drug erlotinib to predict the binding interactions of the target compound (Figure 7). We retrieved the ligand erlotinib from the PDB as a co-crystallized ligand in a complex with EGFR (PDB code: 1M17) (Figure 7, left panel). Both phenyl rings of compound 20 surrounded and interacted with amino acid residues lining the hydrophobic pocket in EGFR-TK, such as Gly-772, Leu-768, Pro-770, Leu-694, Leu-820, and Val-702 ( Figure 7, right panel). Also, the -OH group of compound 20 formed triple hydrogen bonds with amino acid residues Met-769 (3.19 Å), Pro-770 (3.49 Å), and Gly-772 (3.73 Å). The methylsulfonyl (CH 3 -SO 2 -) moiety showed significant interactions, where the oxygen atom of the CH 3 -SO 2group directly formed hydrogen bonds with the amino acid residue Thr-766 (3.26 Å) and the Thr-830 side chain (3.00 Å) and showed additional binding with a water molecule (HOH-10)mediated hydrogen bonding with Thr-766 (2.78, and 3.10 Å). Also, the methyl moiety of the CH 3 -SO 2group interacted with amino acid residue Met-742 by a nonclassical hydrogen bond of 3.91 Å with the sulphur (S-) part of Met-742, while the phenyl part attached to the methylsulfonyl moiety interacted with amino acid residue Leu-820 through CH-p interaction (4.34 Å). These binding interactions indicated that both 2-hydroxyphenyl and 4-methylsulfonylbenzene fragments are important for binding and subsequent inhibitory effects (Figure 7).

Molecular docking of compound 20 with HER2
We retrieved the crystal 3 D structure of HER2 co-crystallized with its bound inhibitor 03Q from the PDB (PDB code: 3PP0) (Figure 8

Physicochemical and pharmacokinetic predictions
We predicted the pharmacokinetic and physicochemical properties of the most active compounds 6,9,16,20, and reference drugs celecoxib, erlotinib, gefitinib, and vismodegib using the automated SwissADME online calculation system (Table 8) 84 . Compounds 6,9,16,20 showed high gastrointestinal absorption, while compound 20 was predicted as an inhibitor of CYP2C19, CYP2C9, and CYP3A4 isoforms and a non-inhibitor of CYP1A2 and CYP2D6 isoforms (Table 8). In contrast, compound 16 was predicted as a non-inhibitor of all CYP isoforms. In addition, compounds 6 and 9 were predicted as inhibitors of CYP2C19 and CYP1A2 isoforms and non-inhibitors of CYP2C9, CYP3A4, and CYP2D6 isoforms. In addition, we calculated the drug-likeness properties, as indicated by major Lipinski's (Pfizer), Ghose's (Amgen), Veber's (GSK), and Egan's (Pharmacia) pharmaceutical rules [85][86][87] . Compounds 6,9,16,20 successfully passed all filters ( Table 8). The BOILED-Egg graph 88 of the WlogP/tPSA (topological polar surface area) showed that compounds 6, 9, 16, 20, together with celecoxib and vismodegib, are located in the human intestinal absorption (HIA) region with no BBB permeation, indicating few CNS side effects ( Figure 9). Indeed, compounds 6, 9, 16, 20 are not P-glycoprotein (P-gpÀ) substrates, suggesting that they are not susceptible to the efflux mechanism carried out by P-gp that is used by many cancer cell lines as a drug resistance mechanism (Figure 9) [89][90][91] . In addition, the bioavailability radar chart of compounds 6, 9, 16, 20, and reference drugs are shown in   [89][90][91] . The optimal property ranges are shown as a pink area, while the red line represents predicted properties for the examined molecule. The SwissADME tool calculation of compounds 6, 9, 16, and 20 predicts that they possess appropriate physicochemical and pharmacokinetic properties.

Chemistry
Melting points (uncorrected) were recorded on a Barnstead 9100 Electrothermal melting apparatus (APS Water Services Corporation, Van Nuys, CA, USA), while the IR spectra were recorded on a FT-IR Perkin-Elmer spectrometer (PerkinElmer Inc., Waltham, MA, USA). The 1 H NMR and 13 C NMR were measured in DMSO-d 6 or CDCl 3 , on Bruker 700 or 500 and 176 or 125 MHz instruments, respectively (Bruker, Billerica, MA, USA). Chemical shifts are reported in d ppm. Mass spectra were recorded on an Agilent 6320 Ion Trap mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). C, H, and N were analysed at the Research Centre, College of Pharmacy, King Saud University, Saudi Arabia. The results were within ± 0.4% of the theoretical values. Compounds 3,11,16,19, and 20 were prepared according to a previous report 65 .
4.1.1. General procedure for the synthesis of hydrazones 4-24 (Scheme 1) A mixture of 4-(methylsulfonyl)benzohydrazide 3 (10 mmol) and an appropriate aromatic aldehyde (10 mmol) was stirred in methanol (10 ml) containing a catalytic amount of acetic acid (0.5 ml) at room temperature for 24 h. The obtained solid was filtered, dried, and recrystallized from absolute ethanol.

Apoptosis assay
According to our previous report, apoptosis induction was performed using the Leukaemia HL-60 cell line and well-established Annexin 5-FITC/PI detection kit. The cell line samples were analysed using FACSCalibur flow cytometer 40,72 .

Cell cycle analysis
Cell cycle analysis was carried out similar to our previous report using the Leukaemia HL-60 cell line stained with the DNA fluorochrome PI and analysed by FACSCalibur flow cytometer 40,74 .

In vitro cyclooxygenase (COX) inhibition assay
The colorimetric COX-2 inhibition assay (kit catalogue number 560101, Cayman Chemical, Ann Arbour, MI) was used to measure the ability of the tested derivatives and celecoxib to inhibit COX-2 isozyme under the manufacturer's instructions 15,76 .

Egfr and HER2 tyrosine kinases assay
In vitro luminescent EGFR tyrosine kinase assay using Kinase-GloV R MAX as a detection reagent, and In vitro HER2 tyrosine kinase assay using DP-Glo TM reagent that measures ADP formed from a kinase reaction, this luminescent signal positively correlates with ADP amount and kinase activity 40 .

Molecular docking and ADME methodology
Molecular docking protocols were carried out using the MOE 2008.10 software from Chemical Computing Group Inc. (Montreal, QC, Canada) following established methods 40,81 . The crystal structures of COX-2 (PDB code: 1CX2), EGFR (PDB Code: 1M17), and HER2 (PDB Code: 3PP0) were retrieved from the protein data bank. The Swiss Target Prediction and the Swiss ADME online tools were used to predict the physicochemical, pharmacokinetic, and drug-likeness properties of the test compounds and used reference drugs 84 .