Antiproliferative benzothiazoles incorporating a trimethoxyphenyl scaffold as novel colchicine site tubulin polymerisation inhibitors

Abstract Tubulin polymerisation inhibitors exhibited an important role in the treatment of patients with prostate cancer. Herein, we reported the medicinal chemistry efforts leading to a new series of benzothiazoles by a bioisosterism approach. Biological testing revealed that compound 12a could significantly inhibit in vitro tubulin polymerisation of a concentration dependent manner, with an IC50 value of 2.87 μM. Immunofluorescence and EBI competition assay investigated that compound 12a effectively inhibited tubulin polymerisation and directly bound to the colchicine-binding site of β-tubulin in PC3 cells. Docking analysis showed that 12a formed hydrogen bonds with residues Tyr357, Ala247 and Val353 of tubulin. Importantly, it displayed the promising antiproliferative ability against C42B, LNCAP, 22RV1 and PC3 cells with IC50 values of 2.81 μM, 4.31 μM, 2.13 μM and 2.04 μM, respectively. In summary, compound 12a was a novel colchicine site tubulin polymerisation inhibitor with potential to treat prostate cancer.


Introduction
Prostate cancer is the second most commonly diagnosed cancer and the sixth leading cause of cancer death among men worldwide, with an estimated 1276000 new cancer cases and 359000 deaths in 2018 [1][2][3] . Microtubules play an important role in various cellular processes, including spindle formation, cellular shape maintenance, and intracellular transportation [4][5][6] . Microtubule has been an attractive target for chemotherapeutic agents to treat prostate cancer 7 . Anticancer agents targeting tubulin bind at four binding sites: taxanes, vinca alkaloids, colchicine and laulimalide sites 8,9 . Although some tubulin inhibitors that bind to vinca alkaloids and taxanes sites have been approved by Food and Drug Administration (FDA), there are no FDA approved colchicine site tubulin inhibitors currently. Therefore, it is necessary to develop novel tubulin polymerisation inhibitors targeting the colchicine binding site to treat prostate cancer.
Recently, benzothiazole scaffold has been an attractive subject due to its potently anticancer activity in medicinal chemistry 10-12 . 2-(4-Aminophenyl) benzothiazole analogue 1 (Figure 1) exhibited the antiproliferative activity against MCF-7 and MDA-468 cells 13,14 . Benzothiazole 2 induced apoptosis and inhibited the growth of HL60 and U937 cells 15 . Benzothiazole linked a phenylpyridopyrimidinone 3 showed the significant cytotoxicity against human cervical cancer cell line ME-180 with an IC 50 value of 4.01 lM 16 . Based on these findings, benzothiazole was a promising scaffold to design anticancer agents. On the other hand, our group also reported three series of colchicine site tubulin inhibitors: (1) compound 4 could change the membrane potential of the mitochondria against MGC-803 cells 17 ; (2) b-lactam containing a 3,4,5trimethoxyphenyl unit 5 exhibited the well antiproliferative activity against MGC-803 cells in vitro and in vivo 18 ; (3) compound 6 could inhibit MGC803 cell growth and colony formation, induce G2/M phase arrest and promote apoptosis 19 .
Bioisosterism is a useful strategy in rational drug design to improve pharmacodynamic or pharmacokinetic properties of lead compounds 20 . In order to optimise the reported colchicine site tubulin inhibitor 4 and discovery more potently anticancer agents [21][22][23][24][25] , benzothiazole was used as a bioisostere of the benzoxazole in the lead structure. Herein, we synthesised a variety of benzothiazole derivatives based on the lead compound 4 by the bioisosterism approach. In addition, the preliminary structure activity relationship (SAR) was explored. The rational design of benzothiazole analogues was described in Figure 2. Compared with the reported compound 4, compound 12a showed the better antiproliferative activity against prostate cancer cells.

Chemistry
General procedure for preparation of compounds 9a$9k and 12a$12d Imine intermediates 7a~7k and 10a~10d were synthesised according to our reported references17, 19. A solution of intermediates 7a~7k and 10a~10d (3 mmol) in dichloromethane (20 ml) was reacted with 2-chloroacetyl chloride (6 mmol) at room temperature. After the mixture was stirred for 3 h, the reaction mixture was diluted with water. The organic layer was washed by aqueous NaHCO 3 , water, and brine, dried over anhydrous Na 2 SO 4 . The system was concentrated to provide the crude products 8a~8k and 11a~11d without purification.

Biology
Cell culture and MTT assay PC3, C42B, 22RV1, and LNCAP cell lines were cultured in an atmosphere containing 5% CO 2 at 37 C, with RPMI-1640 medium with 10% foetal bovine serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin. Cells were seeded at a density of 1500 per well in 96-well plates for 72 h. Then, 20 lL MTT (thiazolyl blue tetrazolium bromide) solution was added to each well, and incubated for 4 h at 37 C. 150 lL DMSO was added to each well to dissolve the formazan after removing the liquid, the absorbance was determined at 570 nm.
In vitro tubulin polymerisation assay Tubulin (5.6 mg/ml) was resuspended in PEM buffer (containing 80 mM PIPES, 1 mM ATP, 1 mM EGTA, 10.2% glycerol, 0.5 mM MgCl 2 ) and then was preincubated with compound 12a, colchicine or vehicle DMSO on ice. The reaction was monitored by a spectrophotometer in absorbance at 420 nm (excitation wavelength is 340 nm).

Immunostaining and microscopy
PC3 cells were seeded on the slices and incubated overnight. Then, cells were treated with different concentrations of 12a. After 48 h, slices were fixed by 4% paraformaldehyde for 15 min after washed by PBS for 3 times. 0.5% Triton-X-100 was added and shaked for 20 min. 0.1% BSA was used to block for 30 min and then removed. The slices were added b-tubulin antibody (1:100) and incubated overnight. Then slices were washed by PBST 3 times, bind with secondary antibody with FITC signal (1:500) in a dark. DAPI was used to stain for 3 min and then removed. After that, images were captured by Laser scanning confocal microscope (Nikon, Japan).

EBI competition assay
6-Well plates were seeded with PC3 cells for 24 h. Then, cells were incubated with compound 12a, colchicine or DMSO for 2 h and afterward treated with EBI (100 mM) for 2 h. Then, the cells were harvested and lysed. Cell extracts were used for Western blotting analysis.

Western blot analysis
Cellular protein lysates were denatured in loading buffer at 100 C prior to adding into SDS-PAGE. Proteins were transferred to nitrocellulose membranes, probed with indicated antibodies, and visualised by an enhanced chemiluminescence detection system. The western blotting bands were semi-quantified using Image J and adjusted for loading control.

DAPI staining
Cells were seeded on a 6-well plate (2 Â 105/well) and treated with the test compound after the incubation for 24 h. Then, cells were stained by DAPI (4 0 ,6-diamidino-2-phenylindole) in the dark for 30 min. The images of cells were observed in a fluorescence microscope (Nikon, Japan).

Molecular modelling
Molecular modelling was performed using the autodock 4 software from the Scripps Research Institute. Molecules were first generated using Pymol software. For the receptor preparation, the PDB code (1SA0) was downloaded from the Protein Data Bank. Dockings were initially performed using a blind docking method on a lower resolution docking. This was followed by a series of resolving steps to attempt to fine tune the compound. The final results were analysed by Pymol software.

Results and discussion
Synthesis of the target analogues 9a$9k and 12a$12d The synthetic route of benzothiazoles containing the 3, 4, 5-trimethoxyphenyl fragment was shown in Scheme 1. Aniline derivatives and various benzyl chlorides were subjected to nucleophilic substitution reaction to afford imines 7a~7k and 10a~10d in the presence of potassium carbonate. The imine intermediates were reacted with chloroacetyl chloride to obtain the amide analogues 8a~8k and 11a~11d in the dichloromethane system. The target analogues 9a~9k and 12a~12d were easily obtained at the reflux condition with benzo[d]thiazole-2-thiol in the presence of sodium hydroxide. The chemical structures of all benzothiazole derivatives 9a~9k and 12a~12d were characterised using spectral methods, and all spectral data corroborated the structures.

Antiproliferative activity of novel benzothiazole derivatives
To discovery novel anti-prostate cancer agents and tubulin polymerisation inhibitors, we evaluated the antiproliferative activity of all benzothiazole derivatives 9a~9k and 12a~12d against a prostate cancer cell line 22RV1 using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Based on previous references, 5-fluorouracil (5-Fu) and was usually selected as a reference drug in the MTT assay to evaluate the antiproliferative activity of synthetic benzothiazole derivatives 26,27 . Therefore, 5-Fu was used as the control drug in this work. In addition, compound 4 was also the control drug to compare the antiproliferative activity.
From the antiproliferative results of benzothiazole derivatives in Table 1, benzothiazoles without the 3, 4, 5-trimethoxyphenyl fragment (9i~9k) displayed very weak activity against 22RV1 cells with IC 50 values of >20 lM. However, benzothiazole-trimethoxyphenyl hybrids 9a~9h showed moderate to potent activity with IC 50 values from 5.24 lM to 19.07 lM against 22RV1 cells. Based on this findings, it revealed that 3, 4, 5-trimethoxyphenyl fragment exhibited an important role for the antiproliferative activity of these benzothiazole derivatives.
To obtain potently antiproliferative compounds, trimethoxyphenyl fragment as B ring was reserved and the molecular deversity of different substituent groups attaching on the A ring was explored. Among them, compound 9 h showed the most potent activity with an IC 50 value of 5.24 lM against 22RV1 cells. We found that the substitution on the A ring was important for the activity showing an over 3-fold activity loss, when the p-Br group was replaced with the m-Cl group (compounds 9d vs. 9 b). In addition, compounds 9 g~9h with an electron-donating methoxy group on the A ring had a more potent inhibitory effect (7.37 and 5.24 lM, respectively) against 22RV1 cells than compounds 9 b~9c (IC 50 >12 lM) with an electron-withdrawing group. From the biological data of compounds 9a~9h, we can conclude that the substituents on the A ring had a remarkable effect on their cytotoxic activity.
To determine whether the aromatic ring or heterocycles have an effect on the activity, compounds with a 3, 5-dimethylisoxazole ring (12a), a pyridine ring (12 b), a 2-chloropyridine ring (12c), and a naphthalene ring (12d) were synthesised and their antiproliferative activity results were shown in Table 2. Replacement of the pyridine scaffold of compound 12 b with a 2-chloropyridine fragment (12c) or a naphthalene unit (12d) led to a loss of the activity. However, changing the pyridine ring of compound 12 b to a 3, 5-dimethylisoxazole ring of compound 12a led to an improvement of the activity against 22RV1 cells. All these results indicated that 3, 5-dimethylisoxazole displayed the better inhibitory activity than other selected heterocycles and phenyl ring.

Compound 12a inhibited cell proliferation against prostate cancer
Among all benzothiazole derivatives 9a~9k and 12a~12d, 12a displayed the most potently antiproliferative activity with an IC 50 value of 2.13 lM against 22RV1 cells. Compound 12a was further examined for possible cytotoxicity against RWPE-1 (normal human prostatic cell line). We found that compound 12a exhibited no cytotoxicity against RWPE-1 (>20 lM). The results indicated that compound 12a had good selectivity between the selected cancer cell line (22RV1) and a normal cell line (RWPE-1). To evaluate its antiproliferative activity against other prostate cancer cell lines, C42B, LNCAP and PC3 cells were selected to perform MTT assay. As shown in the Figure 3, benzothiazole derivative 12a exhibited the well antiproliferative activity against C42B, LNCAP and PC3 cell lines with IC 50 values of 2.81 ± 0.43 lM, 4.31 ± 0.27 lM and 2.04 ± 0.51 lM, respectively.
Compound 12a inhibited tubulin polymerisation by immunostaining assay As the microtubule system played an important role in the maintenance of cell shape and basic cellular functions, an immunofluorescence staining assay was performed to study whether benzothiazole hybrid 12a could disrupt the microtubule dynamics in living cells 28 . As shown in Figure 4, in the control group, the microtubule network in PC3 cells exhibited normal arrangement and organisation, characterised by regularly assembled, normal microtubules wrapped around the cell nucleus. Benzothiazole hybrid 12a at the concentration of 1 lM moderately depolymerised interphase microtubules whereas 12a at the higher concentration of 4 lM induced much stronger depolymerisation effects. These immunofluorescence experiments indicated that compound 12a inhibited tubulin polymerisation in a concentration dependent manner against PC3 cells.

Compound 12a inhibited tubulin polymerisation targeting the colchicine binding site
The in vitro tubulin polymerisation inhibition activity of compound 12a was evaluated because of its best antiproliferative activity results among all benzothiazole hybrids. When tubulin was incubated with the tested compound 12a, the increased tendency of the fluorescence intensity was obviously slowed down compared with the control. The IC 50 value of compound 12a was 2.87 lM against tubulin (Figure 5(A)). It revealed thatbenzothiazole hybrid 12a was a novel tubulin polymerisation inhibitor.  In order to evaluate whether benzothiazole hybrid 12a directly binds to tubulin at the colchicine binding site, we carried out N,N 0 -ethylenebis(iodoacetamide) (EBI) competition assay in PC3 cells according to previously published references 29,30 . EBI is an alkylating agent that has the property to specifically cross-link the Cys239 and the Cys354 residues of b-tubulin involved in the colchicine-binding site 31 . The occupancy of colchicine-binding site by tubulin polymerisation inhibitors could inhibit the formation of the EBI: b-tubulin adduct. Preincubation of compound 12a at 10 lM prevented the formation of the EBI: b-tubulin adduct,  resulting in the decrease of the adduct band comparing with DMSO and EBI treatment. Thus, the assay ( Figure 5(B)) indicated that compound 12a directly bind to the colchicine-binding site of b-tubulin.
Compound 12a induced morphological changes and affected the expression of apoptosis-related proteins As most tubulin destabilising agents displayed the ability to induce morphological changes and affect the expression of apoptosis-related proteins 32 , DAPI staining and western blotting experiments were performed to determine the effects of benzothiazole hybrid 12a. After 48 h incubation with compound 12a (0, 1 lM, 2 lM and 4 lM), characteristic apoptotic morphological changes were observed by fluorescence microscopy, including chromatin shrinkage and formation of apoptotic bodies (Figure 6(A)). The studies revealed that compound 12a induced morphological changes of prostate cancer (22RV1 cells and PC3 cells) possibly by inducing apoptosis. Bcl-2 family proteins are key regulators of apoptosis and overexpression of the prominent pro-survival Bcl-2 family members like Bcl-2 is a common feature responsible for deregulation of apoptosis in prostate cancer cells 33 . In order to investigate the apoptosis effects of compound 12a, the expression of apoptosisrelated proteins (Bcl-2 and Cleaved-parp) were tested. As shown in the Figure 6(B), the expression level of Bcl-2 was decreased and the expression level of Cleaved-parp was increased in a concentration-dependent manner against prostate cancer (22RV1 cells and PC3 cells).

Molecular modelling analysis of compound 12a
In the current work, trimethoxyphenyl-benzothiazole hybrid 12a has been identified as a novel colchicine site tubulin polymerisation inhibitor by the immunostaining, in vitro tubulin polymerisation assay and EBI competition assay. Furthermore, molecular docking methodologies were also used to explore any molecular interaction exist between 3,4,5-trimethoxyphenyl-benzothiazole hybrid 12a and residues lies in the active site cativity of tubulin. We have used Autodock as an automated tool to perform docking and selected PDB code 1SA0. As shown in Figure 7, benzo[d]thiazole, 3,4,5-trimethoxyphenyl ring, and isoxazole unit formed three hydrogen bonds with residues Tyr357, Ala247, and Val353, respectively. This finding illustrated the importance of 3,4,5-trimethoxyphenyl scaffold for tubulin polymerisation inhibitors. 3,4,5-Trimethoxyphenyl-benzothiazole hybrid 12a formed hydrophilic interactions with the residues of Ser178, Asn329 and Tyr224. In addition, 12a also formed hydrophobic interactions with the residues of Pro325, Val177 and Ile355. All these results suggested that hybrid 12a as a novel tubulin polymerisation inhibitor could display the potently antiproliferative activity.

Conclusions
A series of benzothiazole derivatives were designed, synthesised and evaluated for their antiproliferative activity against prostate cancer. Among them, compound 12a possessed the potently antiproliferative ability against C42B, LNCAP, 22RV1 and PC3 cell lines with IC 50 values of 2.81 lM, 4.31 lM, 2.13 lM and 2.04 lM, respectively. Molecular mechanisms illustrated that compound 12a could induce morphological changes and affect the expression levels of apoptosis-related proteins (Bcl-2 and Cleaved-parp) against prostate cancer cells. From the in vitro tubulin polymerisation inhibition assay and EBI competition assay, compound 12a was a novel colchicine site tubulin polymerisation inhibitor. Taken together, compound 12a might be a lead candidate for its further development in treatment of prostate cancer.