Design, Synthesis, and Antipoliferative Activities of Novel Substituted Imidazole-Thione Linked Benzotriazole Derivatives

A new series of benzotriazole moiety bearing substituted imidazol-2-thiones at N1 has been designed, synthesized and evaluated for in vitro anticancer activity against the different cancer cell lines MCF-7(breast cancer), HL-60 (Human promyelocytic leukemia), and HCT-116 (colon cancer). Most of the benzotriazole analogues exhibited promising antiproliferative activity against tested cancer cell lines. Among all the synthesized compounds, BI9 showed potent activity against the cancer cell lines such as MCF-7, HL-60 and HCT-116 with IC50 3.57, 0.40 and 2.63 µM, respectively. Compound BI9 was taken up for elaborate biological studies and the HL-60 cells in the cell cycle were arrested in G2/M phase. Compound BI9 showed remarkable inhibition of tubulin polymerization with the colchicine binding site of tubulin. In addition, compound BI9 promoted apoptosis by regulating the expression of pro-apoptotic protein BAX and anti-apoptotic proteins Bcl-2. These results provide guidance for further rational development of potent tubulin polymerization inhibitors for the treatment of cancer.


Introduction
Microtubules are chief constituents of the cytoskeleton with a vital role in biological functions of all eukaryotic cells. They are essential for cell proliferation, shape maintenance, intracellular transportation, cell motility, cell division and mitosis. Chemically, they combined the two types of protein subunits, αand β-tubulin heterodimers [1,2]. Targeting microtubules appeared as an effective strategy for anticancer agents, as those agents interfere with microtubule dynamic equilibrium of the reversible assembly-disassembly [3]. This interference is categorized to twin core groups: microtubule-stabilizing agents and microtubule-destabilizing agents that fasten to the tubulin polymer to secure microtubules (taxane) or to the tubulin dimers and destabilize microtubules (vinca alkaloid or colchicine), respectively [4].
Recently, a great effort has been given to picking out innovative microtubule-targeting agents as chemotherapeutic agents and specifically aiming them at the colchicine binding site [5][6][7][8]. Inhibitors binding to the colchicine binding site have numerous benefits to fulfill the criteria required for an ideal chemotherapeutic agent, including uncomplicated structures, enhanced hydrophilicity, scaled down toxicity, powerful anti-vascular activity, and considerable multidrug resistance (MDR) effects [9,10].
Combretastatin A-4 (CA-4, 1, Figure 1) is a descriptive colchicine binding site in the tubulin which acts by inhibition of the microtubule polymerization. This natural phenol originated in the bark of Combretum caffrum, which is known as the African bush willow. [11]. CA-4 is an excellent candidate that has exhibited potent anti-vascular and anticancer activity along with its structural features, which stimulate its eligibility to become a lead molecule for designing new analogs [12]. However, the stilbenoid backbone of CA4 is easily isomerized during stock piling, administration and the breaking down to an E isomer which is thermodynamically way out stable but less potent, which reflects the critical chemical instability feature. Afterwards, an innovative chain of combretastatin spin-offs have been synthesized to bypass this stability drawback, just like isoCA-4 [7,13] and Phenstatin (Figure 1). Structural alterations of the combretastatin stilbene have been explored by joining the 1,2-diarylalkene link to a carbocyclic or heterocyclic ring system to obtain conformationally restricted analogues [14,15]. These hetero-combretastatin derivatives have been synthesized and inspired from several five-membered aromatic heterocyclic rings including tetrazole, pyrazole, imidazole, triazole, isoxazole, thiazole, oxadiazole-2-thione, thiazolidinone, and thiophene rings [16][17][18][19][20][21][22][23][24][25][26].

Design and Chemistry
As illustrated in scheme 1, some substituted thiourea derivatives T1-12 were synthesized by refluxing some selected substituted anilines with ammonium thiocyanate in acidic aqueous solution. Novel benzotriazole bearing 3-substitutedphenylimidazol-2-thiones BI1-12 were prepared in two steps as stated in scheme 2. Initially, preparation of chloroethanone of benzotriazole, as intermediate, was prepared through the reaction between benzotriazole and chloroacetyl chloride. Then, the target structure BI1-12 were synthesized via refluxing chloroethanone of benzotriazole with substituted thiourea derivatives T1-12 in ethanol containing catalytic amount of anhydrous sodium acetate.
The structures of compounds BI1-12 were confirmed using different spectroscopic methods and by elemental analysis. The IR spectra characteristically revealed the existence of peaks for NH groups which are tautomeric with CH2 group (C5) of imidazole ring moiety at range 3270-3340 cm −1 , in addition to the presence of peaks of C=N at 1664-1662 cm −1 . Regarding 1 H NMR spectra, all synthesized benzotriazole imidazole-thione derivatives BI1-12 present in two tautomeric forms I and II as stated in scheme 2. They all exhibited the significant singlet signals corresponding to CH2 group (C5, imidazole) at δ 3.99-4.03 ppm which is tautomeric with NH group. Moreover, NH singlet signal observed at δ 11.04-11.75 ppm D2O exchangeable in compounds BI1-6, BI8 and BI11-12. On the other hand, NH group in compounds BI7, BI9 and BI10 obviously showed singlet signals at δ 4.89-6.11 ppm with D2O exchangeable behavior in their spectra. Through 1 HNMR, all of the benzotriazole analogues show a mixture of tautomer I and II in imidazole ring displaying that tautomer I, where C-5 in the imidazole ring is in the form of (-CH2-), represents the major one with ratio two times of tautomer II, where imidazole ring nitrogen Benzotriazole-based compounds are unique nitrogen-containing heterocycles that have attracted significant attention from medicinal chemists as a promising class of bioactive heterocyclic products that exhibit numerous biological properties, such as anticancer [27][28][29][30][31] (BZI, Figure 1), antibacterial [32,33], antiviral [34,35], and anti-inflammatory activities [36,37]. Furthermore, substitution of the 3,4,5-trimethoxyphenyl ring of isoCA-4 by a quinazoline nucleus [38] or quinoline [39][40][41] led to tubulin inhibitor with potent antiproliferative deeds versus a variety of cancerous cell lines. Recently, BZII with a benzotriazole moiety for the replacement for trimethoxyphenyl moiety of colchicine was reported (Figure 1), and the docking studies of BZII showed that benzotriazole formed polar and hydrophobic interaction with the critical residue amino acids of αand β-tubulin subunits in the colchicine-binding pocket. These data demonstrated that benzotriazole moiety might be a surrogate of the traditional 3,4,5-trimethoylphenyl moiety when binding to the colchicine site [9,30].
Based on these inspiring results, we have dedicated ourselves to designing and introducing novel anticancer agents pursuing a tubulin-microtubule system. We proposed a chain of 3-(benzotriazole)-2(3H)-imidazole-2-thiones as novel heterocyclic analogs of CA-4, in which the olefinic core structure of CA-4 is substituted by 2(3H)-imidzaole-2thione. A new approach towards the development of a new series of novel imidazolthione derivatives was proposed by replacing the 3,4,5-trimethoxyphenyl of CA-4 with the benzotriazole ring as ring A, and the hosted ring B with different substituents. In this paper we report on their synthesis and potent antitumor activities versus human cancer cell lines as well as cytotoxicity toward a representative normal human cell line HUVEC. In addition, the fundamental cytotoxic mechanisms of the typical compound BI9 were also interpreted.

Design and Chemistry
As illustrated in Scheme 1, some substituted thiourea derivatives T1-12 were synthesized by refluxing some selected substituted anilines with ammonium thiocyanate in acidic aqueous solution. Novel benzotriazole bearing 3-substitutedphenylimidazol-2-thiones BI1-12 were prepared in two steps as stated in Scheme 2. Initially, preparation of chloroethanone of benzotriazole, as intermediate, was prepared through the reaction between benzotriazole and chloroacetyl chloride. Then, the target structure BI1-12 were synthesized via refluxing chloroethanone of benzotriazole with substituted thiourea derivatives T1-12 in ethanol containing catalytic amount of anhydrous sodium acetate. The structures of compounds BI1-12 were confirmed using different spectroscopic methods and by elemental analysis. The IR spectra characteristically revealed the exis-tence of peaks for NH groups which are tautomeric with CH 2 group (C5) of imidazole ring moiety at range 3270-3340 cm −1 , in addition to the presence of peaks of C=N at 1664-1662 cm −1 . Regarding 1 H NMR spectra, all synthesized benzotriazole imidazolethione derivatives BI1-12 present in two tautomeric forms I and II as stated in Scheme 2. They all exhibited the significant singlet signals corresponding to CH 2 group (C5, imidazole) at δ 3.99-4.03 ppm which is tautomeric with NH group. Moreover, NH singlet signal observed at δ 11.04-11.75 ppm D 2 O exchangeable in compounds BI1-6, BI8 and BI11-12.
On the other hand, NH group in compounds BI7, BI9 and BI10 obviously showed singlet signals at δ 4.89-6.11 ppm with D 2 O exchangeable behavior in their spectra. Through 1 HNMR, all of the benzotriazole analogues show a mixture of tautomer I and II in imidazole ring displaying that tautomer I, where C-5 in the imidazole ring is in the form of (-CH2-), represents the major one with ratio two times of tautomer II, where imidazole ring nitrogen bears hydrogen atom (NH) and C-5 is in form of (-CH=) [42]. This finding is consistent with the stability sequence of tautomer I, which is stabilized through conjugation (Supplementary Figure S1). Also, 13 C NMR spectra of the prepared compounds BI1-12 confirmed the formation of imidazole core as well as showed the presence of thione carbon at δ 188-188.55 ppm [43][44][45].

In Vitro Cell Growth Inhibitory Activity
Numerous Tumor-derived cell lines have been utilized to assess the influence of the synthesised compounds on cell viability, for example on MCF-7 breast adenocarcinoma, HL-60 leukemia, and HCT-116 colorectal carcinoma, as well as HUVEC human normal cell line was assayed by MTT assay. The outcomes are reviewed in Table 1. The imidazol-2-thiones compounds which are evaluated in this study were considered to enclose the benzotriazole substituent that mimics ring A in CA-4. The majority of compounds which were designed contain different substituents at position C-4 of the phenyl ring of the imidazol-2-thiones. Overall, those compounds reported a modest to potent growth inhibitory activity versus all triplet tumor cell lines used in this study. The substituted phenyl ring of the imidazol-2-thiones compounds was reported to considerably affect the biological activity based on the category and nature of the substituent. The unsubstituted phenyl ring compound BI1 demonstrated low micromolar range in MCF-7 and HL-60 while poor activity against HCT-116. Adding methyl substituent on the phenyl ring as in compound BI2 lessened the activity, with IC 50 values exceeding 10 µM entirely in the three cell lines. Substitution of the substituent in BI2 by a greater electronreleasing ethyl group in BI3 or methoxy group in BI4, resulted in an improvement in the antiproliferative action; BI3 and BI4 were 3.8-fold and 2.5-fold respectively more active than their corresponding methyl-containing derivative BI2. At the same time, trading the methyl group by polar group hydroxy to the phenyl ring of the imidazol-2-thione in BI5 caused a weighty enhancement in the antiproliferative activity in submicromolar range versus MCF-7, HL-60 and HCT-116 with IC 50 values of 4.4, 1.1 and 2.1 µM respectively.
With the exception of compounds BI2, BI5, and BI10, all of the benzotriazole analogues (BI1, BI3, BI4, BI6-BI9, BI11 and BI12) had little effect on the normal human umbilical vein endothelial cell, HUVEC (IC 50 : 17.3-135 µM). Most interestingly, the 2,4chloro-substituted compound BI9 exhibited potent anticancer activity toward the three cancer cell lines but low toxicity against HUVEC cells with an IC 50 of 118 µM and 13.6 µM for the reference CA-4. The results demonstrated that these designed compounds might possess excellent selectivity over normal human cells, indicating a high safety index. Owing to the excellent antiproliferative activity of compound BI9 in HL-60 (0.4 µM), it was considered in further studies as below.

In Vitro Inhibition of Tubulin Polymerization and Colchicine Binding
Colchicine and CA-4 drag to the colchicine binding site by attachment to tubulin which resulting in hang-up of microtubule polymerization [22,46]. To explore if the antiproliferative activities of the topmost powerful compounds of the of imidazol-2-thione chain derived from a collaboration with tubulin, compounds BI3, BI5-BI9 and BI12 along with the benchmark compound CA-4 were assessed for their anti-tubulin polymerization activities besides the consequences of the binding of [ 3 H] colchicine to tubulin, the results presented in Table 2.
Most compounds strongly inhibited tubulin assembly compared toward of CA-4 with average IC 50 values 0.49-0.92 µM, except for the compound BI8, which was the least with an IC 50 value of 3.05 µM. Compounds BI5 and BI9 were found to be the most active with IC 50 values of 0.49 and 0.52 µM respectively, which are comparable to that of CA-4 (IC 50 = 0.57 µM). Their results are in harmonization alongside the potent cell growth inhibitory action of BI3 and BI9 in cancer cells. The same series of compounds were also evaluated at twin unalike concentrations (1 and 5 µM) on behalf of its capability to compete with colchicine for binding to tubulin by means of [ 3 H] colchicine binding assay. All the compounds effectively hindered colchicine binding to tubulin by average 59-65% at 1 µM and 77-86% at 5 µM. Interestingly, compound BI9 powerfully hindered colchicine binding to tubulin by 49% at 1 µM and 66% at 5 µM plus to a 1.2 fold greater potency compared to CA-4, with 64% and 85% inhibition, respectively. Consequently, that implies the involvement of compound BI9 in tubulin polymerization inhibition over the colchicine-binding site. These outcomes are matched with formerly stated one on behalf of a sequence of linked benzotriazole correspondents, which powerfully evacuated colchicine from its binding site on the tubulin [30,31].

HL-60 Cell Cycle Arrest
It is well known that CA-4 as microtubule-targeting agent modify the tubulin-microtubule equilibrium and thus challenge the development of the mitotic spindles throughout the M phase, causing cell cycle arrest at G 2 /M phase and eventually prompt apoptosis [12,47,48]. The effect of the functioning compound benzotriazole analogue BI9 at concentration 1 µM on the leukemia HL-60 cells was evaluated by flow cytometry for 48 and 72 h. As shown in Figure 2A, Compounds BI9 brought clear G 2 /M arrest at 48 and 72 h with the proportions of HL-60 cells in G 2 /M phase of 32.3% and 35.1%, respectively. This discovery is comparable with CA-4 (50 nM) which produced a substantial escalation in the proportion of cells in G 2 /M arrest at 48 and 72 h with 40.1 and 46.6% of HL-60 cells respectively, connected to a lessening of cells in the cell cycle G0 phase ( Figure 2B).
Furthermore, BI9 initiated an important upturn in apoptosis as the population in the sub-G1 phase was amplified by 23.1 and 27.5% at 48 and 72 h, respectively, compared to 1.5 and 3.5% for untreated cells ( Figure 2B). From the aforementioned results we can conclude that BI9 demonstrated a significant upturn in the cell population of the HL-60 cancer cells in the G 2 /M phase, and induced cellular apoptosis in pre-G1 phase. These outcomes are in harmony with the formerly detected results for antimitotic spin-offs in the sequences of linked analogues which considerably encourage G 2 /M cycle arrest and apoptosis in MCF-7 cells [30,31].   Figure 3A, after HL-60 cells were exposed to CA-4 and BI9 for 48 h time point, the total fractions of early apoptotic cells (annexin V + /PI − ) and late apoptotic cells (annexin V + /PI − ) considerably amplified from 1.6% in control cells to 18.7% and 21.5%, respectively. The average fraction of Annexin V-staining positive cells (total apoptotic cells) for CA-4 and BI9 were 23.7% and 19.2% after 72 h, respectively, when compared to the control cells (1.6%) ( Figure 3B). As stated from the cell cycle arrest and apoptosis outcomes mentioned earlier (Figure 2A,B), these outcomes confirmed that CA-4 analouge BI9 could induce efficient apoptosis in HL-60 cancer cells.  For additional examination of the effect of BI9 on the apoptosis-related proteins expression in HL-60 cells, the consequences of compound BI9 dosing on the expression of the pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 in HL60 cells was assessed. As shown in Figure 4, Western blotting of HL-60 cell extracts dosed by compound BI9 at 0.5 and 1 µM for 84 h, the expression level of pro-apoptotic Bax protein was significantly up-regulated, and correspondingly the pro-survival protein of Bcl-2 was down-regulated compared to the untreated group. Furthermore, additional indicator of apoptosis was confirmed by poly ADP-reibose polymerase (PARP) cleavage of compound BI9 after a 48 h treatment, confirming its proapototic activity. Taken together, the pattern in cell cycle analysis and apoptosis reveal that benzotriazole BI9 was the most effective at regulating apoptosis-related protein expression in HL-60 cancer cells.

Chemistry
All reagents and solvents were used without further purification. All the recorded melting points were taken in an open glass capillary on a Griffin apparatus and the values given were uncorrected. Microanalyses for C, H, N, and S were carried out at the Regional Center for Mycology and Biotechnology, Faculty of Pharmacy, Al-Azhar University. C, H, N, S analysis values were accepted within a range of ± 0.4% of theoretical calculated percentages. Also, Mass spectra were recorded on Jeol-Gas chromatography-mass spectrometry, JMS-Q1500GC (Tokyo, Japan) controlled with Escrime software. A direct insertion probe was used for sample infusion, source (EI + ): source temperature was 210 °C. Electron energy was 70 eV, and trap-emission was 100 V. Data was acquired by applying a total MS scan from 40 to 1000 m/z (500 scan/s). IR spectra were determined using potassium bromide discs and values were represented in cm −1 . IR spectra were recorded on Shimadzu IR 435 spectrophotometer (Shimadzu Corp., Kyoto, Japan) Faculty of Pharmacy, Cairo University. 1H NMR spectra were carried out on Bruker 400 MHz (Bruker Corp., Billerica, MA, USA) spectrophotometer, Faculty of Pharmacy, Cairo University. Chemical shifts were recorded in ppm on δ scale, coupling constants (J) were given in Hz and peak multiplicities are designed as follows: s, singlet; d, doublet; dd, doublet of doublet; t, tri- Quantification of the tested proteins in HL-60 leukemia cell lysates, both normalized the β-actin, as a protein internal control. The control group was set to '1', and all data from three separate experiments are shown as mean ± SEM. a and b indicate statistically significant differences from the control and BI9 (0.5 µM) group, respectively at p < 0.05, in one-way ANOVA with Tukey's multiple comparisons test.

Chemistry
All reagents and solvents were used without further purification. All the recorded melting points were taken in an open glass capillary on a Griffin apparatus and the values given were uncorrected. Microanalyses for C, H, N, and S were carried out at the Regional Center for Mycology and Biotechnology, Faculty of Pharmacy, Al-Azhar University. C, H, N, S analysis values were accepted within a range of ±0.4% of theoretical calculated percentages. Also, Mass spectra were recorded on Jeol-Gas chromatography-mass spectrometry, JMS-Q1500GC (Tokyo, Japan) controlled with Escrime software. A direct insertion probe was used for sample infusion, source (EI + ): source temperature was 210 • C. Electron energy was 70 eV, and trap-emission was 100 V. Data was acquired by applying a total MS scan from 40 to 1000 m/z (500 scan/s). IR spectra were determined using potassium bromide discs and values were represented in cm −1 . IR spectra were recorded on Shimadzu IR 435 spectrophotometer (Shimadzu Corp., Kyoto, Japan) Faculty of Pharmacy, Cairo University. 1H NMR spectra were carried out on Bruker 400 MHz (Bruker Corp., Billerica, MA, USA) spectrophotometer, Faculty of Pharmacy, Cairo University. Chemical shifts were recorded in ppm on δ scale, coupling constants (J) were given in Hz and peak multiplicities are designed as follows: s, singlet; d, doublet; dd, doublet of doublet; t, triplet; q, quartet; qui, quintet; m, multiplet. 13C NMR spectra were carried out on Bruker 100 MHz spectrophotometer, Faculty of Pharmacy, Cairo University. The reaction progress was monitored by TLC using aluminum sheets precoated with UV fluorescent silica gel (MERCK 60F 254), spots were visualized using UV Lamp. The solvent system used was methanol, ethyl acetate, and toluene with a ratio of 1:2:3. The synthesis of some substituted thiourea T1-12 as shown in Scheme 1 was achieved through refluxing a mixture of substituted aniline and ammonium thiocyanate in acidic aqueous solution according to the reported method [51].

Biochemical Evaluation of Activity
All biochemical assays were accomplished in triplicate on a minimum of three independent times for the calculation of the mean values and reporting.

Cell Viability Assay
The Benzotriazole compounds were evaluated for antiproliferative effect using the MTT viability assay of various cancer cell lines (MCF-7, HCT-116 and HL-60) and the normal human umbilical vein endothelial cell line (HUVEC) to calculate the relative IC 50 values for each compound. Cells were seeded in triplicate in 96-well plates at a density of 10 × 10 3 cells/mL in a total volume of 200 µL per well. 0.1% of DMSO was used as a vehicle control. After this time, they were treated with 2 µL test compound which had been pre-prepared as stock solutions in ethanol to furnish the concentration range of study, 10 nM to 100 µM, and re-incubated for a further 72 h. The culture medium was then removed, and the cells washed with 100 µL phosphate buffered saline (PBS) and 50 µL MTT added, to reach a final concentration of 1 mg/mL MTT added. Cells were incubated for 2 h in darkness at 37 • C. At this point solubilisation was begun through the addition of 200 mL DMSO and the cells maintained at room temperature in darkness for 20 min to ensure thorough colour diffusion before reading the absorbance. Plates were incubated for 72 h at 37 • C + 5% CO 2 . The MTT (5 mg/mL in PBS) was added and incubated for another 4 h, and the optical density was detected with a microplate reader at 570 nm. Results were expressed as percentage viability relative to vehicle control (100%). Dose response curves were plotted and IC 50 values (concentration of drug resulting in 50% reduction in cell survival) were obtained using the commercial software package Prism (GraphPad Software, Inc., La Jolla, CA, USA). All the experiments were repeated in at least three independent experiments.

Tubulin Polymerization Assay
The assembly of purified bovine tubulin was monitored using a kit, BK006, purchased from Cytoskeleton Inc., (Denver, CO, USA). The assay was carried out in accordance with the manufacturer's instructions using the standard assay conditions [54]. Briefly, purified (>99%) bovine brain tubulin (3 mg/mL) in a buffer consisting of 80 mM PIPES (pH 6.9), 0.5 mM EGTA, 2 mM MgCl 2 , 1 mM GTP and 10% glycerol was incubated at 37 • C in the presence of either vehicle (2% (v/v) ddH 2 O), CA-4, benzotriazole compounds. Light was scattered proportionally to the concentration of polymerized microtubules in the assay. Therefore, the tubulin assembly was monitored turbidimetrically at 340 nm in a Spectramax 340 PC spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). The concentration that inhibits tubulin polymerization by 50% (IC 50 ) was determined using area under the curve (AUC). The AUC of the untreated controls were considered as 100% polymerization. The IC 50 value for each compound was computed using GraphPad Prism Software.

Colchicine Site Competitive Binding Assay
The affinity of Benzotriazole compounds to colchicine binding site was determined using a Colchicine Site Competitive Assay kit CytoDYNAMIX Screen15 (Cytoskeleton, Inc., Denver, CO, USA) using the standard protocol of the manufacturer to determine Ki values (µM). Biotin-labelled tubulin (0.5 µg) in 10 µL of reaction buffer was mixed with [3H]colchicine (0.08 µM, PerkinElmer, Waltham, MA) and the test compounds (positive control colchicine, negative control vinblastine, G-1, fluorescent G-1, or 2-ME) in a 96well plate (final volume: 100 µL). After incubating for 2 h at 37 • C with gentle shaking, streptavidin-labelled yttrium SPA beads (80 µg in 20 µL reaction buffer, PerkinElmer, Waltham, MA) were added to each well and incubated for 30 min at 4 • C. The plates were then read on a scintillation counter (Packard Instrument, Topcount Microplate Reader) and the percentage of inhibition was calculated [55,56].

Cell Cycle Analysis
HL-60 cells were seeded at a density of 1 × 10 5 cells/well in 6-well plates and treated with CA-4 and compound BI9 (1 µM) for 48 and 72 h. The cells were collected by trypsinization and centrifuged at 800× g for 15 min. Cells were washed twice with ice-cold PBS and fixed in ice-cold 70% ethanol overnight at −20 • C. Fixed cells were centrifuged at 800× g for 15 min and stained with 50 µg/mL of PI, containing 50 µg/mL of DNase-free RNase A, at 37 • C for 30 min. The DNA content of cells (10,000 cells/experimental group) was analysed by flow cytometer at 488 nm using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) and all data were recorded and analysed using the CellQuest Software (Becton-Dickinson).

Annexin V/PI Apoptotic Assay
Apoptotic cell death was detected by flow cytometry using Annexin V and propidium iodide (PI). HL-60 Cells were seeded in 6 well plated at density of 1 × 10 5 cells/mL and treated with vehicle (0.1% (v/v) EtOH), positive control (CA-4) or compound BI9 (1 µM) for 48 and 72 h. Cells were then harvested and prepared for flow cytometric analysis. Cells were washed in 1X binding buffer (20X binding buffer: 0.1M HEPES, pH 7.4; 1.4 M NaCl; 25 mM CaCl 2 diluted in dH 2 O) and incubated in the dark for 30 min on ice in Annexin V-containing binding buffer [1:100]. Cells were then washed once in binding buffer and then re-suspended in PI-containing binding buffer [1:1000]. Samples were analysed immediately using the BD accuri flow cytometer and prism software for analysis the data. Four populations are produced during the assay Annexin V and PI negative (Q4, healthy cells), Annexin V positive and PI negative (Q3, early apoptosis), Annexin V and PI positive (Q2, late apoptosis) and Annexin V negative and PI positive (Q1, necrosis).
3.2.7. Evaluation of Expression Levels of Anti-Apoptotic Proteins Bcl-2, Pro-Apoptotic Proteins Bcl-2, Bax and PARP Cleavage HL-60 cells were seeded at a density of 1 × 10 5 cells/flask in T25 flasks. After 48 h, whole cell lysates were prepared from untreated cells or cells treated with vehicle control (EtOH, 0.1% v/v) or compound BI9 (0.5 and 1 µM). HL-60 cells were harvested in RIPA buffer supplemented with protease inhibitors (Roche Diagnostics), phosphatase inhibitor cocktail 2 (Sigma-Aldrich), and phosphatase inhibitor cocktail 3 (Sigma-Aldrich). Equal quantities of protein (as determined by a BCA assay) were resolved by SDS-PAGE (12%) followed by transfer to PVDF membranes. Membranes were blocked in 5% bovine serum albumin/TBST for 1 h. Membranes were incubated in the relevant primary antibodies at 4 • C overnight, washed with TBST, and incubated in horseradish peroxidase conjugated secondary antibody for 1 h at rt and washed again. Western blot analysis was performed as described above using antibodies directed against BAX [1:1000] (Cell Signaling Technology), PARP [1:500] (Cell Signaling Technology) and Bcl-2 [1:500] (Cell Signaling Technology) followed by incubation with a horseradish peroxidase-conjugated anti-mouse antibody [1:2000] (Promega, Madison, WI, USA). All blots were probed with β-actin antibody [1:5000] (Sigma) to confirm equal loading. Proteins were detected using enhanced chemiluminescent Western blot detection (Clarity Western ECL substrate) (Bio Rad) on the ChemiDoc MP System (Bio Rad). Experiments were performed on three independent occasions.

Conclusions
This paper described the synthesis of a series of innovative combretastatin-A4 analogues in which the cis-olefinic bridge is exchanged with an imidazol-2-thiones and benzotriazole substituent mimics ring A in CA-4. These compounds displayed encouraging antiproliferative activity versus different cancer cell lines. Between them, compound BI9, bearing 2,4-chloro-substituted at phenyl attached imidazol-2-thione, showed strong antiproliferative activity versus numerous cancer cell lines such as MCF-7, HL-60 and HCT-116 with IC 50 3.57, 0.40 and 2.63 µM, respectively. Importantly, compound BI9 reported low cytotoxicity in HUVEC cell lines, demonstrating its superior toxicity to proliferating cancer cells. The mechanism ofaction studies suggested that compound BI9 induced G 2 /M arrest, apoptosis through PARP cleavage and regulated the expression of pro-apoptotic protein BAX and anti-apoptotic proteins Bcl-2. It exerted anticancer activity by hang-up tubulin polymerization in the colchicine binding site. These results strongly suggest that novel benzotriazole moiety bearing imidazol-2-thiones as CA-4 analogues can be further explored to develop promising anti-cell proliferative agents for the more effective tubulin polymerization inhibitors dealing with cancer.