Synthesis of polybrominated benzimidazole and benzotriazole derivatives containing a tetrazole ring and their cytotoxic activity

Abstract A series of new benzimidazole and benzotriazole derivatives containing a tetrazole moiety was synthesized by N-alkylation of 5-aryltetrazole with 4,5,6,7-tetrabromo-1-(3-chloropropyl)-1H-benzimidazole and 4,5,6,7-tetrabromo-2-(3-chloropropyl)-2H-benzotriazole. The reaction was regioselective and mostly 2,5-disubstituted tetrazole derivatives were obtained. The effect of all synthesized compounds on human recombinant casein kinase 2alpha subunit (rhCK2α) and cytotoxicity against human T-cell lymphoblast (CCRF-CEM) and breast adenocarcinoma (MCF-7) cell lines were evaluated. The results have shown that many of the synthesized compounds exhibit significant cytotoxicity at micromolar concentration. Graphical abstract


Introduction
Azole derivatives have been reported to possess diverse biological activity and are widely used in a medicinal chemistry [1][2][3]. Nowadays, many research groups focus on designing and synthesis of compounds containing two or more azoles, in search for obtaining more active compounds. Many structures containing indole, benzotriazole, or benzimidazole linked with tetrazolyl substituent have been obtained and characterized. It was shown that the attachment of a tetrazolyl substituent to indole derivatives resulted in an increase of both; antibacterial activity against Gram-positive bacteria (Bacillus subtilis, Streptococcus lactis) and Gramnegative bacteria (Escherichia coli, Pseudomonas aeruginosa), and antifungal activity against Candida albicans, Aspergillus niger, and Penicillium sp. [4]. Furthermore, it is known, that certain indolyltetrazole derivatives exhibit significant anticancer activity against human liver carcinoma cell line (HepG2) [5]. The incorporation of the tetrazole moiety into benzotriazole and benzimidazole derivatives resulted in the improvement of biological action of the compound. Structures containing both benzotriazole and tetrazole moiety in addition to antibacterial and antifungal properties exhibit significant, anticonvulsant, anti-inflammatory, and anti-nociceptive activity [6,7]. Compound 2-[2-(5-phenyl-1H-tetrazol-1-yl)phenyl]-1H-benzimidazole exhibits significant antibacterial (against Staphylococcus aureus and Escherichia coli) and antifungal (against C. albicans and A. niger) activity comparable with ciprofloxacin and fluconazole, respectively [8]. Similar properties are also described for 2-(a-p-substituted phenyl-a-benzimidazolo)methyl-1,2,3,4-tetrazole [9] and other derivatives [10,11]. Furthermore, there are known benzimidazole-tetrazole derivatives, which show anti-hypertensive activity compared with losartan and telmisartan [12][13][14]. There are also obtained benzimidazole derivatives bearing a tetrazole ring, that reveal moderate anti-asthmatic, and anti-diabetic activity as well as antioxidant and antitumor activity against mice Dalton's lymphoma ascites (DLA), human breast adenocarcinoma (MCF-7), and human colon adenocarcinoma (HCT) cell lines [11,[15][16][17][18].
Taking into consideration the biological importance of benzimidazole, benzotriazole, and tetrazole derivatives and fact that the fusion of various tetrazoles to azoles generally leads to an increase of the biological activity, we decided to synthesize a new group of compounds containing benzimidazole/benzotriazole and tetrazole moieties and evaluate their cytotoxic effect on two human cancer cell lines: T-cell lymphoblast (CCRF-CEM) and breast adenocarcinoma (MCF-7). Knowing that 4,5,6,7-tetrabromo-1Hbenzotriazole (TBBt) and 4,5,6,7-tetrabromo-1H-benzimidazole (TBBi) derivatives are inhibitors of casein kinase CK2 [19,20], which is significantly increased in many tumor cells [21,22], these derivatives were chosen for further modifications.
First attempt of synthesis of compounds 6a and 7a was performed in n-butanol in the presence of K 2 CO 3 and KI at 90°C, but in these conditions products were not formed. In other approach only K 2 CO 3 and CH 3 CN were used. The reaction was performed at reflux and the progress was monitored by thin-layer chromatography (Scheme 1). Products 6a and 7a were obtained with total yield 86 %. The ratio of 2,5-disubstituted isomer 6a to 1,5-disubstituted isomer 7a equaled 7.42 to 1, and showed the regioselectivity of the reaction. In this manner N-alkylation of other aryltetrazole derivatives 5b-5d was performed. In all cases reactions were regioselective and formation of 2,5-disubstituted tetrazoles was preferred. Additionally, the weight ratio of 2,5-isomer 6a-6d to 1,5-isomer 7a-7d depended on aryl substituent in the tetrazole ring and range from 13.5/1 to 2.8/1. The times, yields, and isomers ratios are summarized in Table 1. All isomeric products 6a-6d and 7a-7d were separated and purified by column chromatography on silica gel. Their structures were elucidated on the basis of 1 H NMR, 13 C NMR, IR, and HRMS spectral data.
The same procedure was applied for the synthesis of 4,5,6,7-tetrabromo-2H-benzotriazole derivatives 8a-8d. The progress of the reaction was monitored by TLC using chloroform:cyclohexane (9:1 v/v) as an eluent. Products were purified three times by column chromatography. There is no doubt, that repeated purification contributed to lower yields of the reactions (Table 1). In this case only 2,5-disubstituted tetrazoles were obtained, what is consistent with our previous studies [27].
In the case of the leukemia cell line (CCRF-CEM), the highest cytotoxicity exhibited compounds 7a and 7d, that at the 25 lM concentration caused a complete (7a) or almost complete (7d) loss of cell viability after 48 h treatment. Worth attention are also compounds 7b, 7c, that effectively inhibited cell growth at the 50 lM concentration. As can be seen from the results presented in the Table 2 the cytotoxic activity of investigated compounds depended on their structure. All 1,5-disubsituted tetrazoles 7a-7d appeared more cytotoxic than their 2,5-disubstituted isomers 6a-6d. It has to be pointed out, that compounds 7a-7d exhibited higher cytotoxicity than TBBi (1) and its 3-chloropropyl derivative 3.
Human breast adenocarcinoma cell line (MCF-7) reveals higher resistance for all tested compounds as compared with leukemia cell line ( Table 3). All benzimidazole derivatives exhibited lower cytotoxicity against MCF-7 than it was observed for CCRF-CEM cell line, and higher compound concentration and/or prolongation of treatment time to 48 h were necessary to observe significant decrease of cell viability. The most active compounds were 7a-7d (25 lM) and 6b-6d (50 lM). Moreover, comparing the cytotoxicity of TBBI (1) and 4,5,6,7-tetrabromo-1-(3-chloropropyl)-1H-benzimidazole (2), with the effect of these compounds, it appeared that synthesized derivatives 7a-7d and 6b-6d reduced the viability of MCF-7 cells more effectively.
It can be concluded that the viability of tested human cancer cell lines was reduced by all synthesized compounds in the concentration-and time-dependent manner. It is also evident, that these close structurally related molecules displayed remarkable differences in cytotoxicity. Benzimidazole derivatives containing tetrazole ring appeared more cytotoxic against both cell lines then their benzotriazole analogs. It has to be pointed out, that many of the synthesized azoles (7a-7d, 6a-6d, 8a-8c) showed higher cytotoxicity than basic compounds TBBi 1 or TBBt 2 and their corresponding chloropropyl derivatives 3 and 4, what confirms, that the aryltetrazolyl substituent can increase the biological activity.
To explain the molecular mechanism of action of the synthesized tetrazole derivatives, their effect on the activity of human recombinant casein kinase 2a subunit (rhCK2a) was estimated. Unexpectedly, all obtained derivatives did not inhibit kinase activity at 1-20 micromolar concentrations, what indicates that this enzyme is not a target for these compounds. It is known that TBBt and TBBi and their derivatives affect other kinases and proteins not dependent on ATP, i.e. quinone reductase [29], or

Conclusion
A series of twelve new azoles was synthesized by the reaction of 4,5,6,7-tetrabromo-1-(3-chloropropyl)-1H-benzimidazole (3) and 4,5,6,7-tetrabromo-2-(3-chloropropyl)-2H-benzotriazole (4) with various 5-aryltetrazoles 5a-5d. Products were obtained in relatively short time and with satisfactory yields. In all reactions, formation of 2,5-disubstituted tetrazoles was preferred. For all compounds, the influence on human casein kinase CK2a subunit and cytotoxic activity against human CCRF-CEM and MCF-7 cancer cell lines were evaluated. All synthesized benzimidazole derivatives exhibited higher cytotoxicity against CCRF-CEM cells than against MCF-7 cells, and in the case of benzotriazole derivatives opposite trend was observed. According to our expectations, the introduction of aryltetrazole moiety into the alkyl side chain of the benzimidazole or benzotriazole resulted in higher cytotoxicity as compared to the nonsubstituted TBBi (1) and TBBt (2), and its analogs 3 and 4. It has been proved that the cytotoxic effect of synthesized compounds is not caused by the interaction with rhCK2a subunit.

CK2a assay
CK2a was expressed and purified according to an earlier published procedure [28]. Isotopic assay was used to determine CK2a activity with synthetic peptide substrate (RRRDDDSDDD) from Biaffin GmbH & Co KG, Germany. Experiments were carried out at 30°C for 20 min in the presence of the increasing concentrations of inhibitors.