Design, synthesis and biological evaluation of 1-Aryl-5-(4-arylpiperazine-1-carbonyl)-1H-tetrazols as novel microtubule destabilizers

Abstract A series of 1-aryl-5-(4-arylpiperazine-1-carbonyl)-1H-tetrazols as microtubule destabilizers were designed, synthesised and evaluated for anticancer activity. Based on bioisosterism, we introduced the tetrazole moiety containing the hydrogen-bond acceptors as B-ring of XRP44X analogues. The key intermediates ethyl 1-aryl-1H-tetrazole-5-carboxylates 10 can be simply and efficiently prepared via a microwave-assisted continuous operation process. Among the compounds synthesised, compound 6–31 showed noteworthy potency against SGC-7901, A549 and HeLa cell lines. In mechanism studies, compound 6–31 inhibited tubulin polymerisation and disorganised microtubule in SGC-7901 cells by binding to tubulin. Moreover, compound 6–31 arrested SGC-7901cells in G2/M phase. This study provided a new perspective for development of antitumor agents that target tubulin.


Introduction
Microtubule, is considered an important target for anticancer drug discovery, playing a crucial role in a wide range of fundamental cell functions including the shape maintenance, intracellular transport and cell division 1 . Microtubule-targeted agents, according to the mechanism of interfering with microtubule dynamics, have been classified into microtubule stabilisers (taxanes and epothilones) and microtubule destabilizers (alkaloids and colchicine (1)) 2 . Given the extensively successful clinical use of vinca alkaloids, the microtubule destabilizers have aroused great interest among medicinal chemists 3 . Over the past decades, a great many outstanding microtubule destabilizers have been reported, such as combretastatin A-4 (CA-4, 2) 4 and XRP44X (3) 5 ( Figure 1).
XRP44X, an arylpyrazole derivative, was developed by Wasylyk et al. as a novel microtubule destabilizer, which prominently inhibited the polymerisation process of tubulin into microtubules by interacting with the colchicine-binding site of tubulin and displayed potent cytotoxic activities against a wide variety of human cancer cell lines at low nanomolar concentrations. A series of aryloxazole derivatives have been synthesised and were found to be potent inhibitors of tubulin polymerisation by Pae and co-workers. Among them, compound 4 showed excellent antiproliferative in vitro and effectively reduced tumour growth in vivo using a tumour xenograft model 6 . Our groups discovered a series of aryltriazole derivatives as a result of structural modifications of the lead compound XRP44X 7 . The most potent compound 5 exhibited excellent antiproliferative activity via disrupting cytoskeleton.
As shown in Figure 1, XRP44X and its analogues molecules can be divided into three major structural elements i.e., A-ring (substituted phenyl), B-ring (five-membered heterocycle, such as pyrazole, oxazole and 1,2,3-triazole), C-ring (piperazine), D-ring (substituted phenyl), and a carbonyl linkage between B-ring and C-ring. Bioisosterism plays a major role in the search for analogues of an active drug molecule. Application of bioisosterism instead of the B-ring on the structure of XRP44X is one of the strategies often used in design of the XRP44X analogues 6 . The modification of B-ring has led to varied cytotoxic activity.
In the last decade, there has been great interest in compounds containing the 1H-tetrazol scaffold because of its unique chemical structure and broad spectrum of biological properties including anticancer activity. For example, the 1-(3,4,5-Trimethoxyphenyl)-5-(4-ethoxyphenyl)-1H-tetrazole was designed as tubulin inhibitor, which showed low IC 50 values at nanomolar level 8 .
As a part of our continuing effort on the development of novel antitumor agents, we designed a series of novel XRP44X analogues by introducing an 1H-tetrazol, hydrogen-bonding acceptors, as B-ring of XRP44X ( Figure 2). Herein, we described the detailed synthetic routes, antiproliferative, tubulin polymerisation, analysis of immunofluorescence staining and cell cycle analysis of these compounds. reacted with ethyl oxalate in dichloromethane to afford corresponding intermediates (8) at room temperature 9,10 . Subsequently, 8 were reacted with triphenylphosphine and carbon tetrachloride to give corresponding (E)-ethyl 2-chloro-2-(arylimino)acetates (9) via Appel reaction under microwave irradiation. Without further purification, compounds 9 reacted with sodium azide in acetonitrile to get the key intermediates ethyl 1-aryl-1Htetrazole-5-carboxylates (10) in a continuous operation process [11][12][13] . Finally, the key intermediates 10 reacted with corresponding arylpiperazines to afford the target compounds (6) in the presence of trimethylaluminium 14 .
Under conventional heating conduction, the reaction of 8 with PPh 3 in CCl 4 to generate (E)-ethyl 2-chloro-2-(arylimino)acetates (9) suffered from long reaction time and low yields ( Table 1, entries 1-5). Microwave irradiation offered many advantages, such as rate enhancements and higher yields, over conventional heating and had become a popular technique that was widely used in organic synthesis today [15][16][17][18] . When microwave irradiation was incorporated into synthesis of 9-8, we found that the reaction rate was greatly improved. We systematically screened the influence of reaction temperature and time on the yield of 9-8 under microwave conditions. As shown in Table 1, the optimised condition was confirmed to be microwave irradiation at 130 C for 20 min (entry 8). Accordingly, all intermediates 9 were obtained smoothly.

Biological evaluation
2.2.1. In vitro antiproliferative activity All the target compounds have been evaluated for in vitro the antiproliferative activities against different three human cancer cell lines (gastric adenocarcinoma SGC-7901 cells, lung adenocarcinoma A549 cells and cervical carcinoma HeLa cells) using MTT assay with colchicine and CA-4 as references. To examine the more detailed structure-activity relationships, modifications towards A-ring and D-ring were performed.
Careful observation of Table 2 revealed that introduction of substituent into the ortho-position of A-ring could remarkably enhance the antiproliferative effect, such as 6-28 $ 6-30. The overall preference order of substituent at the ortho-position of A-ring is as follows: 2-methyl > 2-fluoro > 2-chloro > H. Furthermore, in the case of D-ring 3,5-dimethoxyphenyl fused  compounds showed significant anticancer activities. Compound 6-31 was found to be the most potent compound among all the target compounds with IC50 value of 0.090-0.650 lM against the three cancer cell lines.

Effect on tubulin polymerisation
In order to examine whether the compounds interact with tubulin, we chose the most active compound 6-31 to evaluate for its inhibition of tubulin polymerisation in vitro. Microtubule polymerisation inhibitor (CA-4) and microtubule stabilising agent (Paclitaxel) were used as the positive and negative controls, respectively. As shown in Figure 3, compound 6-31 inhibited tubulin assembly in a concentration-dependent manner. In contrast, paclitaxel could raise the proportion of tubulin polymerisation in comparison with the untreated cells. The results suggested that compound 6-31 interferes with the microtubule polymerisation in a similar way as CA-4.

Analysis of immunofluorescence staining
To further confirm the influence of inhibition of tubulin polymerisation in cells, immunofluorescence staining was carried out. SGC-7901 cell lines were treated for 24 h with CA-4 and compound 6-31, at their respective 2-fold IC 50 concentrations. As given in Figure 4, the microtubule network without drug treatment displays normal arrangement and organisation in control cells. Whereas SGC-7901 cells were treated with CA-4 and compound 6-31 and the results demonstrated that microtubules were destroyed and wrapped around the nucleus in comparison with the control. These results suggest that compound 6-31 inhibits microtubule assembly and disrupts cytoskeleton similarly to CA-4.

Cell cycle analysis
It is well known that most tubulin inhibitors induce cell cycle arrest in the G2/M phase 7 . Thus, the effect of compound 6-31on the cell cycle of SGC-7901 cells was analysed by flow cytometry. SGC-7901 cells were incubated with CA-4 or compound 6-31, at their respective two-fold IC 50 concentrations, and the proportion of tested cells at different cell cycle phases was analysed by flow cytometry after 0, 12, 24, 36, 48, and 72 h of treatment, respectively.     6-31, respectively, indicating the induction of apoptosis. The cell cycle analysis suggests that compound 6-31 arrests SGC-7901 cell in G2/M phase followed by cellular apoptosis.

Molecular modelling
To further understand the binding interactions, molecular docking of the most active compound 6-31 was carried out with tubulin crystal structure (PDB code: 3HKC) using the CDOCKER programme of Discovery Studio 3.0 software. As shown in Figure 6, XRP44X and compound 6-31 are located at the same position with a similar conformation in the binding pocket. Meanwhile, the hydrogen bond exists between the carbonyl group of XRP44X or compound 6-31 with amino acid residue Alab317. Moreover, it is worth noting that the residue of Asnb258 forms a hydrogen bond with the 2 N of 1Htetrazole of compound 6-31 and the residue of Lysb352 forms a hydrogen bond with the 4 N of 1H-tetrazole of compound 6-31. It suggests that the 1H-tetrazole derivatives can not only maintain the right conformation, but also nicely nestle in the active site. The 1Htetrazole moiety and unique active site interactions set the stage for structure-based design of more potent derivatives.

Prediction of drug-like properties
Furthermore, to explore the drug-like properties of the target compounds, some physicochemical properties of XRP44X and compound 6-31 were predicted using free online website (http:// www.swissadme.ch/index.php) for their adaptability with Lipinski's rule of five 19 . As shown in Table 3, XRP44X and compound 6-31 conform well to the Lipinski's rule of five. Compared with XRP44X, compound 6-31 has a lower value of lipid-water partition coefficient. This data indicates that compound 6-31 may have better water solubility than XRP44X. In addition, compound 6-31 exists six hydrogen bond receptors, much more than XRP44X, which helps to reduce the binding energy between the compound and the action site.

Conclusion
In summary, we had designed and synthesised a series of XRP44X derivatives having a 1H-tetrazole B-ring as the hydrogen-bonding acceptors and found that these compounds showed good growth inhibition activities against a range of human cancer cells. Among them, compound 6-31, represented the most active compound with IC 50 values of 0.090-0.650 lM against three cancer cell lines. Moreover, compound 6-31 could disrupt microtubule network in living cancer cells, arrest cell cycle at G2/M phase and induce apoptosis in a dose-and time-dependent manner. Docking studies suggest that compound 6-31 may be a potential tubulin inhibitor. A hydrogen bond was present between Alab317 with the carbonyl group of compound 6-31. Another two hydrogen bonds were also observed between Asnb258 and Lysb352 with the 1H-tetrazole (B-ring). In addition, the prediction of drug-like properties studies shows that compound 6-31 has better pharmacokinetic properties than XRP44X. All of these results indicate compound 6-31 as a promising microtubule destabilizer for further investigation in anticancer drug development.

Materials and methods
All of reagents and solvents were purchased from chemical company. 1 H NMR and 13 C NMR spectra were tested in CDCl 3 with TMS as the internal reference on a Bruker AVANCE 400 or 600 ( 1 H at 400 or 600 MHz, 13 C at 150 MHz). Mass spectra (MS) were measured on an Agilent 1100-sl mass spectrometer with an electrospray ionisation source from Agilent Co. Ltd. High resolution accurate mass determinations (HRMS) for all of the final target compounds were obtained on a Bruker Micromass Time of Flight mass spectrometer equipped with electrospray ionisation (ESI). TLC analysis was used for determining the extent of reactions under UV light (wavelength: 365 nm and 254 nm). Melting point was measured (uncorrected) on hot-stage microscope (Beijing Taike, X-4). The microwave reactions were carried out in a single mode cavity microwave synthesiser (CEM Corporation, NC, USA).

General synthetic procedures for arylpiperazines
A solution of arylamine (1 mmol), bis(2-chloroethyl)amine hydrochloride (1.1 mmol) and K 2 CO 3 (3 mmol) in n-BuOH were stirred at irradiated in a microwave reactor for 30 min at 150 C. The   reaction mixture was cooled to room temperature and dissolved in methanol (4 ml), followed by the addition of diethyl ether (150 ml). The precipitate formed was recovered by filtration and washed with diethyl ether to obtain arylpiperazine as HCl salt. The HCl salt was used for the next reaction without further purification 20,21 .

General synthetic procedures for ethyl 2-oxo-2-(arylamino)acetates (8)
To a solution of substituted aniline (10 mmol) and triethylamine (1 ml, 10 mmol) in DCM was added ethyl chlorooxoacetate (10 mmol) in DCM. The reaction mixture was stirred for 1 h at room temperature. The reaction mixture was poured into water and extracted with DCM (50 ml Â 3). The combined organic layer was washed with brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated to yield the crude product. The crude product was purified by column chromatography (n-hexane/EtOAc ¼ 1:2) on silica gel to afford pure products. For example:

General synthetic procedures for (E)-ethyl 2-chloro-2-(arylimino)acetates (9)
A solution of triphenylphosphine (1.5 mmol) and ethyl 2-oxo-2-(arylamino)acetate (1 mmol) in CCl 4 (5 ml) were stirred at irradiated in a microwave reactor for 20 min at 130 C. The reaction mixtures were cooled to room temperature and the precipitate was filtered off. The filtrate was concentrated to yield the crude product. The crude product was purified by column chromatography (n-hexane/EtOAc ¼ 4:1) on silica gel to afford pure products.

General synthetic procedures for ethyl 1-aryl-1H-tetrazole-5carboxylates (10)
Because of the instability of intermediates 9, we described a microwave-assisted continuous operation process method rather than stepwise operation process for the conversion of 8-10. A solution of triphenylphosphine (1.5 mmol) and ethyl 2-oxo-2-(arylamino)acetate 8 (1 mmol) in CCl 4 (5 ml) were stirred at irradiated in a microwave reactor for 20 min at 130 C. The reaction mixtures were cooled to room temperature and the precipitate was filtered off. The filtrate was evaporated and dissolved in CH 3 CN. Sodium azide (1.2 mmol) was added at room temperature under N 2 for 16 h. The solvent was then removed under reduced pressure and extracted with ethyl acetate (50 ml Â 3). The combined organic layer was washed with water and brine and then dried over Na 2 SO 4 , filtered and concentrated to yield the crude product. The crude product was purified by column chromatography (n-hexane/EtOAc ¼ 3:1) on silica gel to afford pure products. 4.1.6. General synthetic procedures for 1-aryl-5-(4-arylpiperazine-1-carbonyl)-1H-tetrazols (6) To a solution of arylpiperazine (0.1 mmol) in anhydrous DCM was added trimethylaluminum (0.5 ml, 1 M in heptane). The reaction was stirred at room temperature under N 2 for 15 min. A solution of an appropriate ethyl 1-aryl-1H-tetrazole-5-carboxylate (0.1 mmol) in anhydrous DCM was added and the reaction was stirred at room temperature under N 2 for 16 h. The reaction was quenched with 5 ml of 1 M HCl and diluted with DCM. The combined organic layer was washed with water and brine and then dried over Na 2 SO 4 , filtered and concentrated to yield the crude product. The crude product was purified by column chromatography (n-hexane/EtOAc ¼ 1:1) on silica gel to afford pure products.  13 13

Cell cycle analysis
Cell cycle analysis assay was followed the procedure of relevant report 26 .

Molecular modelling
Molecular modelling studies. Molecular docking was carried out by CDOCKER programme of Discovery Studio 3.0 software (PDB: 3HKC). The 3 D structure of 3HKC in docking study was downloaded from Protein Data Bank. The docking poses were selected according to previous studies 7 .

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
We gratefully acknowledge the National Natural Science