Synthesis and characterization of novel combretastatin analogues of 1,1-diaryl vinyl sulfones, with antiproliferative potential via in-silico and in-vitro studies

Novel 1,1-diaryl vinyl-sulfones analogues of combretastatin CA-4 were synthesized via Suzuki–Miyaura coupling method and screened for in-vitro antiproliferative activity against four human cancer cell lines: MDA-MB 231(breast cancer), HeLa (cervical cancer), A549 (lung cancer), and IMR-32 (neuroblast cancer), along with a normal cell line HEK-293 (human embryonic kidney cell) by employing 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The compounds synthesised had better cytotoxicity against the A549 and IMR-32 cell lines compared to HeLa and MDA-MB-231 cell lines. The synthesized compounds also showed significant activity on MDA-MB-231 cancer cell line with IC50 of 9.85–23.94 µM, and on HeLa cancer cell line with IC50 of 8.39–11.70 µM relative to doxorubicin having IC50 values 0.89 and 1.68 µM respectively for MDA-MB-231 and HeLa cell lines. All the synthesized compounds were not toxic to the growth of normal cells, HEK-293. They appear to have a higher binding affinity for the target protein, tubulin, PDB ID = 5LYJ (beta chain), relative to the reference compounds, CA4 (− 7.1 kcal/mol) and doxorubicin (− 7.2 kcal/mol) except for 4E, 4M, 4N and 4O. The high binding affinity for beta-tubulin did not translate into enhanced cytotoxicity but the compounds (4G, 4I, 4J, 4M, 4N, and 4R, all having halogen substituents) that have a higher cell permeability (as predicted in-silico) demonstrated an optimum cytotoxicity against the tested cell lines in an almost uniform manner for all tested cell lines. The in-silico study provided insight into the role that cell permeability plays in enhancing the cytotoxicity of this class of compounds and as potential antiproliferative agents.


Synthesised compounds. Synthesis of 5-(2,2-dibromovinyl)-1,2,3-trimethoxybenzene
Most of the compounds having very low cell permeability, suggests that the compounds may not be sufficiently bioavailable at the site of action within the cell lines tested to cause a very strong inhibitory effect as observed in the cell line assays. The compounds with the highest binding affinity for beta-tubulin did not turn out to be the best cell line inhibitors, but the compounds 4G, 4I, 4J, 4M, 4N, and 4R all have relatively higher cell permeability ( Table 2; Fig. 3b) and they appear to be the ones that perform better in the cell line assays (across board for all cell lines-having close IC 50 values for the different cell lines tested) 39,40 . There were a few exceptions to the generally observed trend, such as compounds 4A and 4B which did not follow the observed general trend among the test compounds (Table 1; Fig. 3b). These two exceptions are among the compounds with the highest binding affinity for the beta-tubulin, have very low permeability but still have lower IC 50 values for the A549 and IMR-32 cell lines relative to every other test compound while their IC 50 values for MDA-MB 231, HeLa are significantly higher than those observed for A549 and IMR-32 cell lines and for many of the other test compounds (Table 1; Fig. 3b). It is however, notable that the only compounds that have significantly high cell permeability (relative to the other test compounds) as predicted, are those with the halogen substituents, 4G (R3 = F), 4M (R3 = Cl), 4N (R2 & R3 = Cl), 4R (R5 = F). It is worthy to note that the halogen groups confer extra lipophilicity which may facilitate cell-permeability. Compound 4T (R4 = F) also has cell-line inhibition profile similar to those of 4M, 4N, 4R and 4G but its predicted cell permeability is quite low compared to the four compounds-it however, has a halogen group like the others that follow the general trend.
The docking poses of the compounds revealed that the compounds occupy the colchicine binding site ( Fig. 3c(1&2)), assuming different conformations and eleven out of seventeen docked compounds have polar interactions. The polar interactions were majorly with LYS254 and ASN350. Other residues involved in polar Table 2. Binding affinity from molecular docking and some predicted pharmacokinetic properties.
Only two (4I and 4N) of the compounds identified to have optimum cytotoxicity based on their pharmacokinetic profile, have polar contacts with the target protein (Table 3; Fig. 3d) and since these two are not the most potent in the MTT assay, it is believed that the polar contacts did not play a role in enhancing the cytotoxic property of the compounds.
Comparing the sulfonyl bridge analogues prepared in this study with other analogues prepared by other groups of researchers, such as the cyclopropyl amide analogues by Huan et al. 41 , pyridine-bridge analogues by Zheng et al. 42,43 and biaryl aryl stilbenes by Kumar et al. 44 , it was observed that the pyridine-bridge analogues, appear to perform better than the sulfonyl bridge analogues in this study, based on a comparison of IC 50 values on a relative scale-the pyridine-bridge analogues are active at a generally lower threshold of IC 50 values compared to the compounds being reported here. However, the sulfonyl bridge analogues studied in this work appear to have better cytotoxicity compared to the cyclopropyl amide analogues and biaryl aryl stilbenes against the tested cell lines (HeLa and A549) comparing the reported IC 50 values for the compounds synthesized and tested by Huan Table 3. Residues within 4 Å and polar interactions of ligands with target protein.

Conclusion
A series of 20 compounds of the analogues of CA-4 using Suzuki Miyaura coupling method were successfully synthesized and fully characterized using different spectroscopic techniques. The compounds possessing electron donating substituents gave higher percentage yields than those possessing electron withdrawing substituent. This is due to their positive electronic induction effect. All the synthesized compounds evaluated for their anticancer activity against four cancer cell lines, MDA-MB 231 (breast cancer), HeLa (cervical cancer), A549 (lung cancer), and IMR-32 (neuroblast cancer) showed moderate to good activity and non were cytotoxic towards normal healthy cell lines (HEK 293) except compound 4V which showed some level of cytotoxicity. The observed results from both in-silico and in-vitro study shows that the compounds are a source of potential lead compounds that can be optimized further especially by improving their cell permeability. Based on the observation of improved cell-lines inhibition profile of test compounds by halogen substitution, the compounds may be optimized further by incorporating more halogen substituents at different positions (and perhaps on the other aromatic ring) and investigate if there would be an improvement in the anticancer profile of the optimized compounds.

Methodology
Experimental section. General information [3][4][5] . Melting points were obtained in open capillary tubes with a 1101D Mel-Temp ® Digital Melting Point Apparatus from Cole-Palmer Limited and are unuttered. The infrared spectra were documented on Thermo Nicolet Nexus 670 Spectrometer with 4 cm −1 resolution using KBr beam splitter and wave numbers of maximal absorption peaks were presented n cm −1 . NMR spectra were recorded on Bruker 300, 400, 500 and 600 MHz NMR Spectrometers, 13 C-NMR spectra were documented at 75, 100, 125, 150 MHz. The proton resonances are annotated as chemical shifts δ parts per million (ppm) relative to tetramethylsilane (δ 0.0) using the residual solvent signal as an internal standard or tetramethylsilane itself: chloroform-d (δ 7.26, singlet), multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad), coupling constant J, in hertz (Hz), and the number of protons for a given resonance indicated by nH. The chemical shifts of 13 C NMR are reported in ppm, relative to the central line of the triplet at ẟ 77.0 ppm for CDCl 3 . ESI spectra were recorded on Micro mass Quattro LC using ESI+ software with a capillary voltage of 3.98 kV and an ESI mode positive ion trap detector. High-resolution mass spectra (HRMS) were recorded on a QSTAR XL hybrid MS-MS mass spectrometer.
A The organic layer was dried over anhydrous Na 2 SO 4 and evaporated to dryness. The crude was purified on SiO 2 (60-120 mesh) column using Hexanes/EtOAc (95:5) as eluents to afford the analytically pure 1A (Fig. 4a).

Synthesis of 5-ethynyl-1,2,3-trimethoxybenzene 46 .
To a stirred solution of dibromoalkene 2A (3.786 g, 10.76 mmol) in anhydrous CH 3 CN was added DBU (6.552 g, 43.10 mmol) drop wise over a period of 10 min at ambient temperature (25-30 °C). The reaction mixture was allowed to stir at ambient temperature for 16 h. After completion of reaction (monitored by TLC), reaction mixture was cooled at 15 °C and quenched by drop wise addition of 5 N aqueous HCl (10 mL) over a period of 15 min then continued stirring for 5 min.
The reaction mixture was extracted with EtOAc/hexane (1:1, 2 × 10 mL); organic layers were washed with water (10 mL). The organic layers were dried over anhydrous Na 2 SO 4 , solvent was evaporated under reduced pressure, and resulting residues were dried in high vacuum to afford the analytically pure 2A (Fig. 4b) as yellow oil (2.560 g, 92% yield). 47  , filtered, and concentrated. The residue was purified by column chromatography to afford 3A (Fig. 4c).

Synthesis of (E)-5-(1-iodo-2-tosylvinyl)-1,2,3-trimethoxybenzene
General method for the synthesis of 1,1-diarylvinyl sulfones CA4 analogues (4a-u) [48][49][50] . To a stirred mixture of (E)-5-(1-iodo-2-tosylvinyl)-1,2,3-trimethoxybenzene 4A (100 mg, 0.211 mmol, 1 eq), different aryl boronic acids (30.64 mg, 0.253 mmol, 1.2 eq) and Na 2 CO 3 (44.73 mg, 0.422 mmol, 2 eq) in water and dimethylformalmide (1:2), with palladium acetate (Pd(OAc) 2 ) catalyst (0.0006 mmol, 0.005 eq) was added and reaction was stirred at room temperature under nitrogen for 3 h. The residue was purified by chromatography (EtOAc/hexanes) to afford the corresponding coupled product 4A-V (Fig. 4d). www.nature.com/scientificreports/ Antiproliferative assay. The cytotoxicity of synthesized compounds 4A-V were determined in triplicates by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay 51,52 . The pale yellow coloured tetrazolium salt (MTT) reduced to a dark blue water-insoluble formazan by metabolically active cells and the product was measured quantitatively after solubility in DMSO. The absorbance of the soluble formazan is directly proportional to the number of viable cells. A panel of four cancer cell lines were used for testing the in-vitro cytotoxicity. These cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) containing non-essential amino acids and 10% FBS. All the cells were maintained under humidified conditions of 5% CO 2 atmosphere at 37 °C in a CO 2 incubator (Model Galaxy 170S, Eppendorf, USA). The 96-well micro titre plates were incubated for 24 h prior to addition of the experimental compounds. Cells were treated with vehicle alone (DMSO) or compounds (drugs (1 µg/mL) were dissolved in 100 µL DMSO previously) at different concentrations (0.1, 1, 10 and 25 µM) of test compounds for 48 h. The assay was completed with the addition of MTT (5%, 10µL) and incubated for 60 min at 37 °C. The supernatant was aspirated and plates were air dried and the MTT-formazan crystals were dissolved in 100 µL of DMSO. The optical density (O.D) was measured at 570 nm using TECAN multimode reader (Infinite ® M200Pro, Tecan, Switzerland). The percent cell viability of each treated well of 96 well plate was calculated based on test wells relative to control wells. Doxorubicin was used as positive controls for comparison purpose and 1% DMSO as a vehicle control. The cytotoxicity of test compounds was expressed in terms of IC 50 value, which is defined as a concentration of compound that produced 50% reduction absorbance relative to control, and the absorbance at 570 nm wavelength was recorded 51 . Molecular docking and in-silico pharmacokinetics prediction. The coordinates of the tubulin (protein) were downloaded from the protein databank (PDB code = 5LYJ). The beta tubulin portion (B chain) that consists of the colchicine site ( Fig. 3c(1&2)), that is the target for combretastatins, was extracted from the whole protein and stripped of other associated ligands, water of crystallization and ions (Ca 2+ and Mg 2+ ) and saved in a pdb format. This extracted chain B was processed further using the AutoDock program (MGL Tools 1.5.6). Gasteiger charges were added and the search space area was set by centering the grid box at the colchicine site, capturing all the residues within 4 Å of the native combretastatin within the protein. The grid box parameters (x, y, z) in Angstrom units are (22,20,24) for size and (16.061, 66.352, 38.609) for centre and the processed protein chain was saved as a pdbqt file. The native doxorubicin ligand was redocked with the protein and the rmsd of the native ligand and redocked native ligand was estimated (0.7029 Å) in order to validate the docking protocol. The 3D structures of the synthesized compounds were prepared using chem3D and their energies were minimized with MM2 Force Field of Chem3D application interface and the ligands were saved as pdb files. The command line version of the obabel program, installed on Ubuntu LTS operating system, was used to convert the pdb files into pdbqt files. The pdbqt files for the protein and prepared ligands were used for the docking calculation with AutoDock Vina. The resulting binding poses and interactions were visualized and processed using the Pymol program. The binding energy of the compounds as well as interacting residues are presented in Table 2. The compounds were submitted to the online server, preADMET, for in-silico pharmacokinetic property prediction to obtain estimates for cell permeability, plasma protein binding, blood brain barrier penetration and Intestinal absorption.