Design, eco-friendly synthesis, molecular modeling and anticancer evaluation of thiazol-5(4H)-ones as potential tubulin polymerization inhibitors targeting the colchicine binding site

In recent years, suppressing tubulin polymerization has been developed as a therapeutic approach for cancer treatment. Thus, new derivatives based on thiazol-5(4H)-ones have been designed and synthesized in an eco-friendly manner. The synthesized derivatives have the same essential pharmacophoric features of colchicine binding site inhibitors. The anti-proliferative activity of the new derivatives was evaluated on three human cancer cell lines (HCT-116, HepG-2, and MCF-7) using MTT assay procedure and colchicine was used as a positive control. Compounds 4f, 5a, 8f, 8g, and 8k showed superior antiproliferative activities against the three tested cell lines with IC50 values ranging from 2.89 to 9.29 μM. Further investigation for the most active cytotoxic agents as tubulin polymerization inhibitors was also performed in order to explore the mechanism of their anti-proliferative activity. Tubulin polymerization assay results were found to be comperable with the cytotoxicity results. Compounds 4f and 5a were the most potent tubulin polymerization inhibitors with an IC50 value of 9.33 and 9.52 nM, respectively. Further studies revealed the ability of 5a to induce apoptosis and arrest cell cycle growth at the G2/M phase. Molecular docking studies were also conducted to investigate possible binding interactions between the target compounds and the tubulin heterodimer active site. From these studies, it was concluded that inhibition of tubulin polymerization yields the reported cytotoxic activity.


Introduction
Cancer is a complex, widespread, and lethal disease. It begins when cells start to grow beyond their usual boundaries, then can invade adjoining parts of the body and spread to other organs. 1 Many of the currently available antitumor drugs are unable to differentiate between normal and neoplastic cells, and are also unable to overcome primary or secondary resistance mechanisms evolved in the tumor cells. 2,3 Thus, there is a pressing need for new antitumor agents with high potency, and less toxicity in non-cancerous cells, that are able to act on unique targets.
Microtubules, the key components of the cytoskeleton are essential in all eukaryotic cells. Microtubules are composed of a-tubulin and b-tubulin heterodimers arranged in the form of slender lamentous tubes that can be many micrometres long. 4 They are highly dynamic polymers and their polymerization dynamics are tightly regulated both spatially and temporally. 5 They are crucial in the development and maintenance of cell shape and cell division (mitosis). 6 During mitosis process, the duplicated chromosomes of a cell are separated into two identical sets before cleavage of the cell into two daughter cells. 7 The importance of microtubules in mitosis and cell division makes it an important target for anticancer drugs. 8 Microtubules and their dynamics are considered targets for diverse groups of antimitotic drugs (with various tubulin-binding sites) that have been used with great success in the treatment of cancer. 9 In view of the success of this class of drugs, it has been argued that microtubules represent the best cancer target to be identied so far, and it seems likely that drugs of this class will continue to be important chemotherapeutic agents, even as more selective approaches are developed 10 The tubulin heterodimer contains at least three distinct drug binding sites: the paclitaxel (taxanes alkaloid), vinblastine (vinca alkaloid), and colchicine binding sites. 11 For the rst two of these sites, there are many drugs in current use in clinical oncology. 12,13 All the marketed tubulin inhibitors bind to the paclitaxel and vinblastine binding sites are highly potent but the clinical use is limited for several reasons: (i) they are prone to develop multi-drug resistance, (ii) they are highly lipophilic and have to be solubilized by surfactants which can cause hypersensitivity reactions in patients, (iii) they have to be administered intravenously due to poor water solubility which is not convenient for patients and may lead to poor patient compliance. 14 Tubulin inhibitors that bind to the colchicine binding site can largely overcome the above drawbacks and have therapeutic advantages over taxanes and vinca alkaloids. For example, they can be administered orally owing to the higher water solubility, they do not require surfactants for solubilization, thus are devoid of surfactant-induced hypersensitivity reaction. More importantly they are less prone to develop multi-drug resistance. Therefore, tubulin inhibitors that bind to colchicine binding site have received extraordinary attention in the last ten years. 15 Colchicine binding site inhibitors (CBSIs) exert their biological effects by inhibiting tubulin assembly and suppressing microtubule formation. 14 Colchicine I itself binds to tubulin very tightly, but neither colchicine nor compounds that bind to the colchicine binding site on tubulin have yet found signicant use in cancer treatment. 16 Combretastatin A-1 (CA-1) II and combretastatin A-4 (CA-4) III are two combretastatin analogs, both showed similar microtubule inhibitory activity but have limited water solubility. 17 In order to improve the water solubility, both compounds were prepared as prodrugs of monosodium phosphate salt, and they can be transformed into the active components CA-1 and CA-4 in vivo. 18,19 In phase II clinical trial, CA-4P showed no bone marrow toxicity, stomatitis, and hair loss. 20 Ombrabulin IV is another CA-4 analog which has better solubility, oral bioavailability, improved anti-cancer activity and decreased toxicity. 14   This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 2791-2811 | 2793 ZD6126 V is a NAC (N-acetylcolchicinenol) phosphate prodrug which showed microtubule inhibitory activity in vivo. Moreover, it showed no obvious neurotoxicity and displayed good antitumor activity. 21,22 E7010 VI is an orally bioavailable sulfonamide that inhibits tubulin polymerization by binding to the colchicine binding site. It exhibited a broad spectrum of antitumor activity in vitro and in vivo. 23 Plinabulin VII is in a world-wide Phase III clinical trial for non-small cell lung cancer. 24 Plinabulin blocks the polymerization of tubulin in a unique manner, resulting in multifactorial effects including an enhanced immune-oncology response, 25 activation of the JNK pathway and disruption of the tumor blood supply. 26 Indibulin VIII has shown promising anticancer activity with a minimal neurotoxicity in preclinical animal studies and in Phase I clinical trials for cancer chemotherapy. 27 The antitumor activity of indibulin is believed to be primarily related to its effects on microtubules. 28 Recently, many molecules (e.g. compounds IX, X, XI & XII) interacting with the colchicine binding site have been designed and synthesized with signicant structural diversity. These  This journal is © The Royal Society of Chemistry 2020 compounds were modied and tested in order to nd a highly potent, low toxicity agent for treatment of cancers 29,30 (Fig. 1).
In the present work, our research group synthesized a series of thiazol-5(4H)-ones having the same pharmacophoric features of CBSIs and targeting the colchicine binding site, to examine their effect as anticancer agents with potential inhibitory effect on tubulin assembly.

Rational drug design
The colchicine binding site is positioned at the interface between the a and b subunits of the tubulin protein, with the major part of it buried in the b subunit and lined by the helices 7 and 8. The cavity, which is funnel shaped, has a volume of about 600 A and opens up towards the a subunit of the interface surrounded by residues Asn101a, Thr179a, Ala180a, Val181a, Thr314b, Asn349b, Asn350b, and Lys352b. The other, b subunit, end of the cavity is surrounded by residues Tyr202b, Val238b, Thr239b, Cys241b, Leu242b, Leu248b, Leu252b, Leu255b, Ile378b, and Val318b and forms the narrow funnel end-like part of the cavity. The predominance of hydrophobic residues confer a strong hydrophobic character to this part of the cavity. At the wider portion, the cavity is surrounded by Ala250b, Asp251b, Lys254b, Asn258b, Met259b, Ala316b, Ala317b, Thr353b and Ala354b making it moderately polar/moderately hydrophobic. 31 As shown in Fig. 2, colchicine binding site inihbitors have the following seven pharmacophoric points: three hydrogen This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 2791-2811 | 2795 bond acceptors (A1, A2, and A3), one hydrogen bond donor (D1), two hydrophobic centers (H1 and H2) and one planar group (R1). 14,32 Depending on these previously reported facts we can say that the molecules that will have these seven pharmacophoric features will be considered as promising tubulin inhibitors.
It worth mentioning that, the seven pharmacophoric features can be partitioned among two planes. Features A1, D1, H1, and R1 lie in plane A, and features A2, A3, and H2 lie in plane B. Relative to one another, the two planes have a tilt of about 45 and match the shape of the colchicine site 32 (Fig. 3).
Taking colchicine, as a lead compound for synthesis of the new derivatives, it is formed of three parts: ring A, ring B (linker), and ring C. Structure-activity study reveals that the A and C ring of colchicine comprise the minimal structural feature of the molecule needed for its high affinity binding to tubulin. 33 The changes to the linker region affect the cytotoxic activity of the most reported colchicine binding site inihbitors. 34  In continuation for our previous work of design and synthesis of new anticancer agents, [35][36][37][38][39][40][41][42][43][44][45] the main target of this work was the synthesis of new thiazol-5(4H)-ones having the same essential pharmacophoric features of the reported CBSIs (Fig. 5). The core of our molecular design rational comprised bioisosteric modication strategies of CBSIs at three different positions.
The wide variety of modications enabled us to study the SAR of these compounds as effective anti-cancer agents with potential tubulin polymerization inhibitory activity which is considered as a crucial objective of our work. All modication pathways and molecular design rationale were illustrated and summarized in Fig. 7.

Chemistry
On the bases of green considerations and in continuation of our earlier endeavors 46-48 toward the development of eco-friendly synthetic routes for heterocyclic systems, we report herein Scheme 1 Synthesis of target compounds 4a-k and 5a,b. Reagents and conditions: (i), (ii) and (iii): absolute ethanol and gl. acetic acid/reflux. facile routes to various thiazol-5(4H)-one derivatives. In addition to conventional method, ultra-sound irradiation and microwave irradiation (solvent less) techniques were used in the synthesis of the new members. The reactions have been worked well in a one-pot fashion and were completed in a few minutes, with the desired products obtained in good yields.
Finally, we repeated the two consequence steps of the reaction under both ultrasonic (in ethanol and catalytic amount of acetic acid) and microwave irradiation solvent-free conditions without isolation of the intermediates 3a,b and 7a,b. The reaction mixture afforded the same products with increased yield and shortage in the reaction time under ultrasonic method. Surprisingly, the yield was increased dramatically to 92%.
The structures of the synthesized compounds were established based on spectral data. The IR spectra of compound 3a,b showed the presence of NH 2 and NH absorptions at a range of 3376-3151 cm À1 . The 1 H NMR spectra of compounds 3a,b exhibited singlet signals of methyl group at 2.32 ppm, singlet For compounds 4a-k, 5a,b, 8a-l, and 9a,b the IR spectra showed absorption bands at ranges of 3115-3441, 1688-1722 and 1605-1648 corresponding to NH, C]O, C]N groups, respectively. The 1 H NMR spectrum of 4a-k, 5a,b, 8a-l, and 9a,b showed characteristic peaks attributed to imine methyl protons between 2.47 ppm and 3.92 ppm. The proton present in the Schiff base appeared at a range of 8.25-8.66 ppm. The characteristic peaks due to NH proton appeared between 10.07 and 12.87 ppm as singlet peaks. The 13 C NMR spectra of compounds 4a-k, 5a,b, 8a-l, and 9a,b provided additional evidence in support of the proposed structures. All spectra that supports elucidating the chemical structures of the new derivatives are supplied with this research work as ESI le. † 2.2. Biological evaluation 2.2.1. In vitro anti-proliferative activity. The synthesized compounds were tested for their in vitro cytotoxic activities using standard MTT method, 49-51 against a group of human cancer cell lines namely; colorectal carcinoma (HCT-116), hepatocellular carcinoma (HepG-2), and breast cancer (MCF-7). Colchicine was used as a positive control. The results of cytotoxicity test were reported as growth inhibitory concentration (IC 50 ) values and summarized in Table 1.
The tested compounds exhibited different degrees of antiproliferative activities against the three tested cell lines. Their activities range from excellent, good, moderate to weak.
In general, compounds 4f, 5a, 8f, 8g, and 8k showed superior antiproliferative activities against the three cell lines with IC 50 Table 1 In vitro anti-proliferative activities of the tested compounds and in vitro tubulin polymerization inhibition Comp.
Moreover, several compounds such as 4e, 4g, 4k, 8b, 8c, and 8h demonstrated strong anti-proliferative activities over all examined cell lines with IC 50 values ranging from 10.32 to 20.00 mM. Also, compounds 3b, 4b, 4d, 5b, and 8i showed strong antiproliferative activities against only two cell lines with IC 50 values ranging from 10.64 to 18.40 mM.
On the other hand, compounds 4a, 4c, 4h, 4i, 4j, 8a, and 8d displayed weak anti-proliferative activities against at least two cell lines with IC 50 values ranging from 31.86 to 48.83 mM.
Finally, compounds 3a, 7a, 7b, and 8j showed no activity against any of the tested cancer cell lines. In addition,   To investigate whether the cytotoxic activity of the synthesized compounds was related to an interaction with the tubulin system, an in vitro tubulin polymerization assay was performed for the most cytotoxic members. The inhibition assay on microtubule polymerization was evaluated turbidimetrically using a uorescent plate reader. 52 Colchicine was used as a positive control (Table  1).
Compounds 4f and 5a were the most potent tubulin polymerization inhibitors with an IC 50 values of 9.33 and 9.52 nM, respectively. These compounds had activities higher than that of colchicine (IC 50 ¼ 10.65 nM). Additionally, compounds 8f, 8k, and 8l showed promising activities nearly equal to colchicine with IC 50 values of 11.59, 13.50, and 13.16 nM, respectively. Also, compounds 4d and 8g showed strong tubulin polymerization inhibitory activities with IC 50 values of 19.68 and 14.17 nM, respectively. Finally, compounds 3b, 4e, 4k, 8h, 8i and 9a exhibited moderate tubulin polymerization inhibitory activities with IC 50 values ranging from 38.37 to 50.01 nM. The results strongly implicated a direct interaction between the examined compounds and tubulin. It can be concluded that the cytotoxic activity of the synthesized compounds may derive from an interaction with tubulin and an interference with microtubule assembly.
2.2.3. Cell cycle analysis. To gain a better insight into the impact of compound 5a on cancer cell growth inhibition, its impact on cell cycle distribution and apoptosis induction was assessed using HepG-2 cells according to the method outlined by Wang et al. 53 In fact, anticancer agents hinder cancer cell growth and multiplication by arresting cell division at distinct checkpoints, and that cells resist apoptosis are highly resistant to cancer treatment. 54 In the present work, HepG-2 cell line was  treated with compound 5a at a concentration equals its IC 50 value on tubulin (9.52 nM) for 24 h.
As shown in Table 2, Fig. 8 and 9, the percentage of HepG-2 cells at S phase was increased from 38.15% to 39.28% aer incubation with compound 5a. Additionally, cells in G2/M phase markedly increased from 5.09% to 20.55% and the G1 phase decreased from 56.76% in control to 40.17%, indicating that compound 5a caused cell arrest at G2/M phase. Also, it was found that the cells increased from 2.14% to 15.33% at pre-G1 phase, indicating that compound 5a caused apoptosis at pre-G1 phase.
2.2.4. Annexin V-FITC apoptosis assay. To further conrm Apoptotic effect of compound 5a in HepG-2 cells, Annexin V and PI double staining assay was performed. 55 In this test, HepG-2 cells were incubated with compound 5a at concentration of 2.5 mM for 24 h. The results were reported in Table 3, Fig. 10 and 11.
The results revealed that compound 5a induced total apoptotic effect equal 13.79% which was eight time more than  the control (1.66%). In details, compound 5a obviously induced early apoptosis by 8.25% and enhanced late apoptosis by 5.54% when compared with the untreated control HepG-2 cells (1.02% and 0.64%, respectively).

Docking studies
Molecular docking studies were conducted to give a guidance of molecular binding modes of the tested molecules inside the pocket of tubulin heterodimers. The selected compounds have been docked against tubulin heterodimers using MOE2014 to determine the free energy and binding mode. The selection of the most promising molecules depended on the rightbinding mode and the binding free energy (DG). 56 The binding free energies of the synthesized compounds and the reference ligand were summarized in Table 4. The binding mode of the co-crystallized ligand, DAMA-colchicine, exhibited  an energy binding of À13.08 kcal mol À1 . The ring A (trimethoxy phenyl moiety) formed a hydrogen bond with Cys241. Also, it formed ve hydrophobic interactions with Ala250, Leu255 and Cys241. The 2-mercaptoacetamide moiety formed two hydrogen bonds with Leu248 and Ser178. The ring C formed one hydrogen bonding and one hydrophobic interaction with Lys352 (Fig. 12).
Compound 4d as a representative example showed a binding mode like that of DAMA-colchicine, with affinity value of À11.44 kcal mol À1 . The 2,4-dichlorophenyl moiety formed ve hydrophobic interactions with Ala316, Lys352, Leu248 and Ala250. The thiazol moiety formed one hydrogen bond with Ser178 and one hydrophobic interaction with Lys254. The ptolyl moiety formed three hydrophobic interactions with Ile171 and Ala12 (Fig. 13).
The binding mode of compound 4f exhibited an affinity value of À11.80 kcal mol À1 . The 2,4-dihydroxybenzyl moiety formed two hydrogen bonds with Gly246 and Leu248. Also, it formed two hydrophobic interactions with Gln247 and Gln11. The hydrazinyl moiety formed two hydrogen bonds with Asn258 and Asn101. The p-tolyl moiety formed ve hydrophobic interactions with Leu255, Met259, Ala316, and Lys352 (Fig. 14).
The binding mode of compound 5a exhibited an affinity value of À10.41 kcal mol À1 . The thiophene moiety formed two hydrophobic interactions with Ala316 and Lys352. The hydrazinylthiazol moiety formed two hydrogen bonds with Lys352 and Lys254. Also, it formed one hydrophobic interaction with Lys352. The p-tolyl moiety formed one hydrophobic interaction with Ala12 (Fig. 15).
The binding mode of compound 8f exhibited an affinity value of À13.19 kcal mol À1 . The 2,4-dihydroxyphenyl moiety formed two hydrogen bonds with Gln11 and Gly246. In addition, it formed one hydrophobic interaction with Leu248. The hydrazinylthiazol moiety formed three hydrogen bonds with Ser178, Asn101 and Asn258. The thiophene moiety formed one hydrophobic interaction and one hydrogen bond with Lys352 (Fig. 16).

Structure-activity relationship (SAR)
As outlined in the rationale molecular design, it was aimed at studying the SAR of the newly synthesized thiazol-5(4H)-one derivatives as potential tubulin polymerization inhibitors.

Conclusion
To sum up, thirty-one new derivatives based on thiazol-5(4H)one were designed and eco-friendly synthesized using conventional, ultrasound irradiation and microwave-assisted methods. The synthesized derivatives were evaluated for their anti-proliferative activities against a group of three human cancer cell lines including; colorectal carcinoma (HCT-116), hepatocellular carcinoma (HepG-2), and breast cancer (MCF-7) using MTT assay. Compound 4f has appeared as the most active member against all examined cells with IC 50 0.3 mM, respectively). In addition, compounds 5a, 8f, 8g and 8k showed excellent antiproliferative activity against the three tested cell lines with IC 50 values ranging from 3.23 to 9.29 mM. Moreover, the most active compounds have been studied for their inhibitory activities of tubulin polymerization. Tubulin polymerization assay ndings were consistent with cytotoxicity data results. Moreover, compound 5a arrested the cell cycle in the G2/M phase and induced apoptosis in HepG-2 cells. Docking experiments assisted these ndings by anticipating potential binding interactions between the target compounds and the active sites of tubulin heterodimers. The most effective candidates in the quest for strong and selective antineoplastic agents will serve as valuable lead compounds and merit further investigations.

Chemistry
All melting points were measured on a Gallen Kamp melting point apparatus (Sanyo Gallen Kamp, UK) and were uncorrected. The Microwave reactions were done by Microsynth instrument type MA143 (Micro wave ux). The ultrasoundassisted reactions were performed in Digital Ultrasonic Cleaner CD-4830 (35 KHz, 310 W). The IR spectra were recorded on a Pye-Unicam SP-3-300 infrared spectrophotometer (KBr dicks) and expressed in wave number (cm À1 ). 1 H NMR spectra were run at 300 and 400 MHz, on a Varian Mercury VX-300 and Bruker Avance III NMR spectrometer, respectively, while 13 C NMR spectra were run at 100 MHz. TMS was used as an internal standard in deuterated dimethylsulphoxide (DMSO-d 6 ). The mass spectra were recorded on Shimadzu GCMS-QP-1000EX mass spectrometer at 70 eV. Elemental analyses were performed on CHN analyzer and all compounds were within AE 0.4 of the theoretical values. The reactions were monitored by thinlayer chromatography (TLC) using TLC sheets coated with UV uorescent silica gel Merck 60 F254 plates and were visualized using UV lamp and different solvents as mobile phases. All reagents and solvents were puried and dried by standard techniques.
4.1.2.c. Under sonication method. A mixture of thiosemicarbazide (10 mmol) and appropriate aryl ketones (10 mmol) namely, 4-methyl acetophenone 1a, 3,4-methoxy acetophenone 1b, 2-acetyl thiophene 6a and 2-acetyl furan 6b, in anhydrous ethanol (20 mL) with catalytic amount of glacial acetic acid was placed in Erlenmyer ask (50 mL) and subjected to ultrasound waves at room temperature for 10 min. A mixture of chloroacetic acid (10 mmol) and (1 mmol) of appropriate aromatic aldehydes namely, 4-methoxy benzaldehyde, 4-chloro benzaldehyde, 4-hydroxy benzaldehyde, 2,4-dichloro benzaldehyde, 3,4-dimethoxy benzaldehyde, and 2,4-dihydroxy benzaldehyde was added to the reaction vessel, and subjected to ultrasound waves at room temperature for 15 min. The formed precipitate was ltered, dried, and crystallized from the appropriate solvent to afford the target compounds 4a-k, 5a,b, 8a-l & 9a,b. Reaction time and yield of the conventional, ultrasonic and microwave procedures were summarized in Table 5.  In vitro cytotoxic activity. Evalation of cytotoxic activity of the synthesized compounds was carried out using MTT assay protocol 49,50,57 against a group of cancer cell lines namely; colorectal carcinoma (HCT-116), Hepatocellular carcinoma (HepG-2) and breast cancer (MCF-7) and colchicine was used as a standard drug. The cells were obtained from ATCC (American Type Culture Collection) via the Holding company for biological products and vaccines (VACSERA) (Cairo, Egypt). The anti-cancer activity was measured quantitatively as follows: Into a medium of RPMI-1640 with 10% fetal bovine serum, the cell lines were cultured. Then, penicillin (100 units per mL) and streptomycin (100 mg mL À1 ) were added at 37 C in a 5% CO 2 incubator. Next, seeding the cell lines in a 96-well plate was achieved by a density of 1.0 Â 10 4 cells per well at 37 C for 48 h under 5% CO 2 . Aer incubation period, the cell lines were treated with different concentration of the synthesized compounds and incubated for 24 h. Aer treatment by 24 h, 20 mL of MTT solution (5 mg mL À1 ) was added and incubated for 4 h. Dimethyl sulfoxide (100 mL) was added into each well to dissolve the formed purple formazan. The colorimetric assay was measured and recorded at absorbance of 570 nm using a plate reader (EXL 800, USA). The relative cell viability in percentage was calculated as (A570 of treated samples/A570 of untreated sample) X 100. Results for IC 50 values of the active compounds were summarized in Table 1.

4.2.2.
In vitro tubulin polymerization assay. The effect of the synthesized compounds on tubulin polymerization was assessed turbidimetrically using a uorescent plate reader method. 52 At rst, the synthesized compounds and reference drug (colchicine) were incubated in mixture of puried bovine tubulin (10 mM) and buffer system containing 20% glycerol and 1 mM ATP at 37 C. Then, the mixture cooled to 0 C. The IC 50 value was dened as the compound concentration that inhibited the extent of tubulin assembly by 50%.
4.2.3. Cell cycle analysis. HepG-2 cells were seeded at density of 2 Â 10 5 cells per well and incubated for 24 h in sixwell plates. Fetal bovine serum (FBS, 10%) was added, aer that cells were incubated at 37 C and 5% CO 2 . The medium was replaced with (DMSO 1% v/v) containing the 2.5 mM of compound 5a, then incubated for 48 h, washed with cold phosphate buffered saline (PBS), xed with 70% ethanol, rinsed with PBS then stained with the DNA uorochrome PI, kept for 15 min at 37 C. Then samples were analyzed with a FACS Caliber ow cytometer. 53 4.2.4. Annexin V-FITC apoptosis assay. The effect of the most cytotoxic compound 5a on apoptosis induction was analyzed using Annexin V-FITC/PI apoptosis detection kit. In this test, HepG-2 cells were stained with Annexin V uorescein isothiocyanate (FITC) and counterstained with propidium iodide (PI). Then, HepG-2 cells in a density of 2 Â 10 5 per well were incubated with compound 5a for 48 h. Next, the cells were trypsinized, washed with phosphate-buffered saline (PBS), and stained for 15 min at 37 C in the dark. Finally, FACS Caliber ow cytometer was used in analysis process. 55

Docking studies
Crystallographic structure of tubulin [PDB ID: 1SA0, resolution 3.00Å] was retrieved from Protein Data Bank (http:// www.pdb.org) and considered as a target for docking simulation. The docking analysis was performed using MOE2014 soware to evaluate the free energy and binding modes of the synthesized compounds against tubulin. At rst, the crystal structure of the target was prepared by removing water molecules and retaining the two essential chains and the cocrystallized ligand, N-deacetyl-N-(2-mercaptoacetyl)-colchicine (DAMA-colchicine). Then, the protein structure was protonated, and the hydrogen atoms were hided. Next, the energy was minimized, and the binding pocket of the protein was dened.
The 2D structures of the synthesized compounds and reference ligand (DAMA-colchicine) were sketched using ChemBio-Draw Ultra 14.0 and saved as MDL-SD format. Then, the saved les were opened using MOE and 3D structures were protonated. Next, energy minimization was applied. Before docking process, validation of the docking protocol was carried out by running the simulation only using the co-crystallized ligand (DAMA-colchicine) which showed low RMSD value. The molecular docking of the synthesized was performed using a default protocol against the target receptor. In each case, 30 docked structures were generated using genetic algorithm searches, London dG was used for scoring and forceeld (MMFF94) for renement. The London dG scoring function estimates the free energy of binding of the ligand from a given pose. The functional form is a sum of terms: where C represents the average gain/loss of rotational and translational entropy; E ex is the energy due to the loss of ex-

Conflicts of interest
This work was funded by the authors and there is no any conict of interest.