Synthesis and anticancer activity of novel hydrazone linkage-based aryl sulfonate derivatives as apoptosis inducers

In the present study, the various 28 hybrid molecules containing hydrazone and sulfonate moieties were synthesized and characterized by FTIR, 1H-NMR, 13C-NMR spectroscopy and LC-MS spectrometry, besides elemental analysis. The compounds were evaluated for their antiproliferative effects against six cancer cell lines, namely A549 (non-small cell lung cancer), MCF-7 (breast cancer), HT-29 (colorectal adenocarcinoma cancer), PC-3 (androgen-independent prostate adenocarcinoma), Hep3B (hepatocellular carcinoma cancer), and HeLa (epitheloid cervix carcinoma cancer). Among all the target compounds, compounds 4g and 4h exhibited more promising effects on MCF-7 cell lines (IC50 = 17.8 μM and 21.2 μM, respectively) with high selectivity. Further mechanistic studies proposed that compounds 4g and 4h induced apoptosis is mediated through the intrinsic apoptotic pathway with changes in mitochondrial membrane potential by finally activating caspase-9 and caspase-3. The results have been encouraging enough to merit further investigation.


Introduction
Hydrazone derivatives are known for their broad spectrum of biological activities [1]. A variety of hydrazones have been utilized as anticancer drugs. Representative members of this class are zorubicin and bisantrene which are widely used for treatment of different cancer types (Fig. 1).
Sulfonates are widely used in medicine researches and have significant pharmacological applications such as antifungal [6], acaricidal [7] and antioxidant [8]. In particular, there have been many reports on the anticancer properties of compounds bearing aryl sulfonate moiety [9][10][11] (Fig. 2). Sulfonate-based compounds have a great affinity for lipid phases and can easily pass the membrane to attach to target sites due to their physicochemical features [7]. Also, the irreversible enzyme inhibitors usually possess reactive functional groups such as nitrogen mustards, aldehydes, alkenes and phenyl sulfonates [12]. These irreversible enzyme inhibitors may block certain enzymes that cancer cells.
On a pharmaceutical target, combining two or more effective pharmacophores of various potent bioactive agents can lead to potent new compounds. Based on the aforementioned compounds and in continuation of our work on hydrazones we were interested in studying the reaction of hydrazides with sulfonate aldehydes. Thus, we report here on the synthesis, characterization and anticancer evaluation of hydrazone derivatives with tosyl sulfonate moieties.
Treatment of vanillin/p-hydroxybenzaldehyde with tosyl chloride and triethylamine in dimethylformamide gave the aldehyde derivatives (2a-b) [21]. In the final step of this study, hydrazone linkage-based aryl sulfonate derivatives were obtained by the reaction of compounds 1a-n with related aldehydes (2a-b) in ethanol, as shown in Scheme 1.
The title compounds were characterized by FT-IR, 1 H-NMR, 13 C-NMR and LC-MS spectroscopy, besides elemental analysis and melting point. All of the spectral data are listed in Supplementary Material.
The C=N and C=O groups of the synthesized compounds were confirmed by IR spectra. The IR spectral peaks of compounds were recognized for C=O of CONH from 1637 to 1703 cm −1 ; for C=N at 1583-1627 cm −1 .
In the 1 H-NMR spectra of compounds, characteristic singlet signals in the range of 2.38-2.44 ppm are attributable to the tosyl groups. In agreement with the literature data, CH=N and CONH groups exhibited two separate singlet signals in the 1 H-NMR and 13 C-NMR spectra of compounds 3a, 3c-f, 3h, 4a, 4c-f, and 4h due to the restricted rotation around the C-N bond [22]. In the cases of aromatic aldehydes-condensed N-acylhydrazones, 1 H-NMR spectra in DMSO-d 6 showed two peaks of the -CH 2 CO and CH-CH 3 groups that be attributed to the presence of two possible syn-anti isomers of the amide bond or E-Z isomer. For all the final compounds, the protons of azomethine and amide resonated at the expected regions in the literature [13][14][15]23].
The 13 C-NMR signals corresponding to the carbons from the azomethine and amide groups appear in the range 145.5-149.3 ppm and 160. 6

Biological evaluation
The in vitro antiproliferative activity of the title compounds was evaluated against A549 (non-small cell lung adenocarcinoma), MCF-7 (breast adenocarcinoma), HT-29 (colorectal adenocarcinoma), PC-3 (androgen-independent prostate adenocarcinoma), Hep3B (hepatocellular carcinoma), and HeLa (cervical carcinoma) cells by MTT method. The anticancer screening results at 10 μM concentration are summarized in Table 1. Also, whether or not compounds caused cytotoxic effects in noncancer mouse embryonic fibroblast cell line (NIH3T3) was tested. Compounds showed no cytotoxicity effect on NIH3T3 cells. The structures of the compounds 3a-n and 4a-n differ from each other due to substituent on phenyl ring (-2OCH 3 or -H).
Compounds showed strong antiproliferative activity against cancer cells. Compound 3b exerted cytotoxic activity with GI more than 40% against prostate (PC-3) cell line. The compounds 4g and 4h were found to be the best anticancer activity against the MCF-7 cell line (50.37 and 53.98%, respectively) with no effect on normal cell lines. Compound 3b was less potent on normal cells compared to cancer cells. So, two compounds (4g and 4h) were selected for further studies because of their high anticancer activity and low toxicity to normal cells. The cell growth-inhibitory potencies, expressed as IC 50 values of these compounds, are listed in Table 1. Compounds 4g and 4h showed cytotoxicity with IC 50 values of 17.8 and 21.2 μM against MCF-7 cell line, respectively.
In this study, mitochondrial disfunction was determined based on the mitochondria membrane potential (MMP) NA GI% ≤ zero index that was measured using JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) dye as a fluorescent probe. A loss of mitochondrial mass was characterized by a decrease in red fluorescence and an increase in the green fluorescent intensity [24]. As shown in Fig. 3, before the treatment of compounds, the untreated MCF-7 cancer cells exhibited red fluorescence intensity because of the JC-1 dye aggregation into the mitochondria. Compounds 4g and 4h-treated cells displayed a reduction in red emission intensity and an increase in green emission intensity when compared to control cells. The red/green fluorescence intensity ratio in the MCF-7 cells treated with different concentrations of compounds (10, 20, and 50 µM) was displayed in Fig. 4. A clear concentration-dependent decrease in the ratio can be observed for both compounds. Therefore, one of the first steps by which the studied compounds trigger apoptosis is likely to be through changes in mitochondrial membrane potential.
Annexin V is a fluorescent probe that binds to translocated phosphatidylserine (PS) in apoptotic cells and is used to indicate early apoptotic cells [25]. To determine whether selected compounds undergo apoptosis, an annexin V assay was also performed. As shown in Fig. 5, compounds initiated early apoptotic processes in MCF-7 cells and were dependent on dose, especially for the compound 4h.
Apoptosis can be an extrinsic pathway mediated via activation of death receptors or by an intrinsic mitochondriamediated pathway. In the intrinsic pathway, proapoptotic and antiapoptotic proteins stabilize the outer membrane of the mitochondria and activate caspase-9 and caspase-3. Caspase-3 is activated in both extrinsic and intrinsic apoptosis pathways, the initiator caspase-9 protein is only activated via the intrinsic pathway [26]. MMP depolarization results in the release of cytochrome into the cytosol, which activates caspase 9 via the formation of an apoptosome and the subsequent cleavage of caspase 3/7 effectors [27].
To determine whether caspases were involved in the apoptotic cell death induced by hybrid derivatives 4g and 4h in MCF-7 cells, we examined the effect of these compounds on caspase-3 and caspase-9 activation. It is important to underline that compounds significantly suppressed caspase-3 and caspase-9 activation (Fig. 6). These caspases were highly activated (∼3.5-4-fold) with 50 μM concentrations of 4g and 4h in MCF-7 cells, showing that apoptotic cell death takes place in a caspase-dependent pathway.

Conclusions
In conclusion, we designed two series of hybrid molecules (3a-n and 4a-n) based on the biological significance of hydrazone and sulfonate moieties. All the derivatives were characterized and evaluated for in vitro antiproliferative activity. The numerous compounds displayed a wide variety of cytotoxicity against multiple cancer cell lines. The activity results showed that compounds 4g and 4h were found to be most effective against MCF-7 cell assayed with IC 50 values of 17.8 and 21.2 µM, respectively, besides their selectivity. Annexin V staining assay results showed that the selected compounds were induced apoptosis. This result is associated with depolarization of MMP and activation of caspase-3 and caspase-9. These compounds look like promising leads for further modification for the design of the new anticancer agents with high selectivity.

Experimental
All chemical compounds were purchased from Merck and Sigma Aldrich. Melting points were measured with a Thermoscientific 9300 melting point apparatus and were uncorrected. Infrared (IR) Spectra were recorded using Shimadzu FTIR 8400S spectrophotometer. 1 H-NMR and 13 C-NMR experiments were carried out using BRUKER NMR spectrometer, and chemical shifts (δH) are reported relative to TMS as the internal standard. Mass spectra were recorded using Agilent LC/MSD and Xevo G2-XS QTof spectrometer. All reagents and solvents were dried and purified by the standard techniques. Chemical shift (δ) values of rotameric hydrogens whenever identified are presented within parentheses by assigning an asterisk (*) along with that of other forms [28]. Compounds 1a-n and 2a-b are already recorded in the literature [4,[13][14][15][16][17][18][19][20][21]. Compounds 1i, 3e, 3j and methyl 4-[(thiophene-2-carbonyl) amino]benzoate have CAS Registry Numbers but no reference, analytical, or spectral data. General procedure for the synthesis of target compounds
Synthetic route and chemical structures of compounds 3a-n and 4a-n are presented in Scheme 1.