Elsevier

European Journal of Medicinal Chemistry

Volume 86, 30 October 2014, Pages 257-269
European Journal of Medicinal Chemistry

Original article
Design, synthesis, and structure–activity relationships of novel benzothiazole derivatives bearing the ortho-hydroxy N-carbamoylhydrazone moiety as potent antitumor agents

https://doi.org/10.1016/j.ejmech.2014.08.058Get rights and content

Highlights

  • 36 novel benzothiazole derivatives were designed and synthesized.

  • Target compounds showed excellent antitumor potency in vitro against 5 cancer cell lines.

  • The cytotoxic activities of 15g and 16b were 1.8–8.7 times more active than PAC-1.

  • Enzymatic activities of 15g and 16b were 2.9 times and 16 times better than PAC-1.

Abstract

A series of novel benzothiazole derivatives bearing the ortho-hydroxy N-carbamoylhydrazone moiety were designed and synthesized and their cytotoxic activities against five cancer cell lines (NCI-H226, SK-N-SH, HT29, MKN45, and MDA-MB-231) were screened in vitro. Most of them showed moderate to excellent activity against all the tested cell lines. Among them, compounds 15g (procaspase-3 EC50 = 1.42 μM) and 16b (procaspase-3 EC50 = 0.25 μM) exhibited excellent antitumor activity with IC50 values ranging from 0.14 μM to 0.98 μM against all cancer cell lines, which were 1.8–8.7 times more active than the first procaspase activating compound (PAC-1) (procaspase-3 EC50 = 4.08 μM). The structure–activity relationship (SAR) analyses indicated that the introduction of a lipophilic group (a benzyloxy or heteroaryloxy group) at the 4-position of the 2-hydroxy phenyl ring was beneficial to antitumor activity, and the presence of substituents containing nitrogen that are positively charged at physiological pH could also improve antitumor activity. It was also confirmed that the steric effect of the 4-position substituent of the benzyloxy group had a significant influence on cytotoxic activity.

Graphical abstract

A series of novel benzothiazole derivatives bearing ortho-hydroxy N-carbamoylhydrazone moiety were synthesized and evaluated for their cytotoxic activities. Six potent compounds were further examined for their procaspase-3 kinase activity.

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Introduction

The primary cause of cancer development and progression is the dysregulation of apoptosis [1]. Thus, it would be an effective approach to explore novel apoptosis-inducing compounds for the treatment of cancer. Compounds such as p53 disruptors (tenovin-1) [2] and inhibitors of XIAP (GDC-0152) [3] or Bcl-2 (GDC-0199) [4] act directly on proteins in the apoptotic pathway, to induce apoptosis and lead to the death of cancer cells. However, direct activation of procaspase-3 by small molecules (PAC-1 and S-PAC-1, Fig. 1) could have advantages over the above, because numerous studies have demonstrated that procaspase-3 is overexpressed in a variety of human tumors, including those of colon cancer [5], lung cancer [6], melanoma [7], hepatoma [8], breast cancer [9], lymphoma [10], and neuroblastoma [11].

Structure–activity relationship (SAR) studies revealed that the activity of PAC-1 and S-PAC-1 in vitro and in cell culture is dependent on the presence of the ortho-hydroxy N-acylhydrazone moiety, which is known to participate in metal chelation [12]. In addition, studies have also revealed that the piperazine nitrogens in PAC-1 and S-PAC-1 may play a very important role in their activity [5], because the autoactivation mechanism provides another channel for the activation of procaspase-3 to caspase-3 [13]. It is possible that the piperazine nitrogens in PAC-1 and S-PAC-1 are positively charged at physiological pH, which may directly interact with the triaspartic acid “safety catch” of procaspase-3, as such inducing the autoactivation of procaspase-3 and catalyze the hydrolysis of hundreds of protein substrates, leading to cell death.

Our group have reported a series of derivatives bearing an ortho-hydroxy N-acylhydrazone moiety (3, 4, Fig. 2), the SAR of which exhibited that the introduction of either a benzyloxy or heteroaryloxy group at the 2-hydroxy phenyl ring could enhance activity in vitro [14], [15], [16]. Benzothiazoles have attracted much attention over many years due to their diverse biological properties, including antifungal [17], anticancer [18], [19], antiamyloid [20], and antirheumatic [21] utility, and have been widely used as important structural components in modern drug design for the treatment of cancer [22], [23], [24].

In our continued exploration for potent and novel procaspase-3 activators as potential anticancer agents with multi-targeted molecular mechanisms, we combined benzothiazole with the ortho-hydroxy carbamoylhydrazone moiety in target compounds based on a hybrid pharmacophore design. Some groups (R1) that are positively charged at physiological pH (e.g., 4-methylpiperidine, morpholine, dimethylamine, and diethylamine), were introduced at the 6-position of benzothiazole to avoid the metabolic pathway, and the effect of positively charged groups on the activity was examined by changing the number of carbon atoms (n). Substituted benzyloxy and heteroaryloxy groups (R), which were beneficial to the antitumor activity in our previous study, were also introduced into the 4-position of the 2-hydroxy phenyl ring to heighten the activity (Fig. 3).

Herein, we describe a series of novel target compounds and their in vitro antitumor activities against two procaspase-3 over-expression cancer cell lines (NCI-H226, Lung cancer; and SK-N-SH, neuroblastoma) and one cancer cell line moderately sensitive to procaspase-3 (MDA-MB-231, breast cancer). Furthermore, two cancer cell lines with low-sensitivity to procaspase-3 (MKN45, human gastric cancer cell; and HT29, human colorectal cancer cell) were evaluated to rule out off-target effects [25]. Several compounds with potent activity were selected for the enzymatic assays to determine procaspase-3 kinase activation.

Section snippets

Chemistry

The preparation of key intermediates 9ae is described in Scheme 1. The commercially available starting materials 4-nitrobenzyl bromide or 4-chloronitrobenzene combined with excessive secondary amines (4-methylpiperidine, morpholine, dimethylamine and diethylamine) in acetonitrile at room temperature for 3 h underwent a nucleophilic substitution reaction to provide intermediates 5ae. These were then reduced in the presence of 80% hydrazine, FeCl6H2O, and activated carbon to obtain the aryl

In vitro cytotoxicity and structure–activity relationships

All target compounds were evaluated for their cytotoxicity in vitro against two procaspase-3 over-expression cancer cell lines (NCI-H226), Lung cancer cells and SK-N-SH, neuroblastoma cells and one cancer cell line moderately sensitive to procaspase-3 (MDA-MB-231), breast cancer cells by using an MTT assay. In addition, two cancer cell lines with low-sensitivity to procaspase-3 (MKN45, human gastric cancer cells and HT29, human colorectal cancer cells) were further evaluated to rule out

Conclusions

In summary, we designed and synthesized a series of novel benzothiazole derivatives bearing an ortho-hydroxy N-carbamoylhydrazone. The prepared compounds were evaluated for in vitro cytotoxic activity in five human cancer cell lines (NCI-H226, SK-N-SH, MKN45, HT29, and MDA-MB-231). Most of them had moderate to excellent activity against all tested cancer cell lines and were sensitive to the level of cellular concentration of procaspase-3. Compounds 15g and 16b exhibited more potent antitumor

Chemistry

All the mass spectra (MS) were taken in ESI mode on Agilent 1100 LC-MS (Agilent, Palo Alto, CA, USA). Proton (1H) nuclear magnetic resonance spectroscopy was performed using Bruker ARX-300, 400 MHz spectrometers (Bruker Bioscience, Billerica, MA, USA) with TMS as an internal standard. Column chromatography was run on silica gel (200–300 mesh) from Qingdao Ocean Chemicals (Qingdao, Shandong, China). The IR spectra were recorded by means of the KBr pellet technique on a Bruker FTS 135

Acknowledgments

The work was supported by Program for Innovative Research Team of the Ministry of Education of the People's Republic of China (IRT1073), National High Technology Research and Development Program of China (863 Program) (No. 2012AA020305) and National Natural Science Foundation of China (No. 81102470).

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