Synthesis and Antitumor Evaluation of New Heterocycles Derived from 3-Methyl-2-benzothiazolinone Hydrazone

3-Methyl-2-benzothiazolinone hydrazone condensed with some selected aldehydes to give the corresponding Schiff bases. Cyclizing the obtained Schiff base derivatives with thioglycolic acid afforded the respective thiazolylideneaminothiazolidenones. Condensation of 3-methyl-2benzothiazolinone hydrazone with ethyl cyanoacetate yielded a mixture of the bishydrazine and the cyanoacetohydrazide derivatives. Meanwhile, its condensation with ethyl acetoacetate produced a mixture of the monoketone and the β-diketone derivatives. Besides, the reaction of the starting hydrazone with malononitrile afforded a mixture of the cyanomethylylidene and cyanomethyl hydrazone derivatives beside the bishydrazine derivative. Elementary and spectroscopic measurements were in good accord with the structures postulated for the new compounds. The antitumor activities of certain selected new compounds were screened, in vitro, against a panel of four (liver, HepG2; breast, MCF-7; lung, A549; and colon; HCT116) human solid tumor cell lines. The cells that showed better activity were HepG2 and MCF-7. Structure-activity relationship (SAR) was also discussed.


Introduction
Benzothiazoles are interesting heterocycles which are ubiquitous in nature and show antiviral, 1 antibacterial, 2 antimicrobial 3 and antifungal activities. 4They are also useful as anti-allergic, 5 antidiabetic, 6 antitumor, 7 antiinflamatory, 8 anthelemintic, 9 and anti-HIV agents. 103][14] Many benzothiazole derivatives have been synthetically and biologically evaluated through reacting 2-aminobenzothiazoles with various reagents. 15,16Limited attention, on the other hand, has been paid to making use of 2-hydrazonobenzothiazoles for similar purposes. 17,18In context to our interest in this realm, 19 the present work has been endeavored aiming at designing and synthesizing new compounds bearing the benzothiazole scaffold in their molecules to be evaluated for their anticancer activities.This has been achieved through reacting 3-methyl-2-benzothiazolinone hydrazone 1 with certain carbonyl reagents and active methylenes.

Chemistry
It has been found that hydrazone 1 condenses with aldehydes 2a-h in absolute ethanol at the reflux temperature in the presence of a few drops of piperidine to give the respective Schiff bases 3a-h in high percentage yields (Scheme1).The assigned structures were attested by compatible analytical and spectroscopic measurements.
The first product was proved to be 1,2-bis(3methylbenzo[d]thiazol-2(3H)-ylidene)hydrazine 5 by comparing its melting point (mp) and IR spectra with a reference sample. 19An ORTEP overview of compound 5 is represented in Figure 1. 19he second product was formulated as 2-cyano-N'-   Condensation of hydrazone 1 with malononitrile proceeded in absolute ethanol at the reflux temperature to give a mixture of three products which were separated by column chromatography (Scheme 6).The first (30%) was proved to be compound 5 by comparing its mp and IR spectra with those of a reference sample. 19he second product (20%, pale brown crystals) was formulated as 2-(2-(3-methylbenzo[d]thiazol-2(3H)ylidene)hydrazinyl)acetonitrile 9 for the following reasons: (i) its elementary analyses and molecular weight determination (MS) corresponded to C 10 H 10 N 4 S (m/z 218, M + , < 5%); (ii) the IR spectrum of 9 revealed strong absorption bands at ν/cm −

Biological evaluation
Conventional chemotherapy, although directed toward certain macromolecules or enzymes, typically does not discriminate effectively between rapidly dividing normal cells (e.g., bone marrow and gastrointestinal tract) and tumor cells, thus leading to several toxic side effects.Tumor responses from cytotoxic chemotherapy are usually partial, brief and unpredictable.In contrast, targeted therapies interfere with molecular targets that have a role in tumor growth or progression.These targets are usually located in tumor cells, although some like the antiangiogenic agents may target other cells such as endothelial cells. 20  targeted therapies have a high specificity toward tumor cells, providing a broader therapeutic window with less toxicity.They are also often useful in combination with cytotoxic chemotherapy or radiation to produce additive or synergistic anticancer activity since their toxicity profiles often do not overlap with traditional cytotoxic chemotherapy.Thus, targeted therapies represent a new and promising approach to cancer therapy, leading to beneficial clinical effects.There are multiple types of available targeted therapies, including monoclonal antibodies, inhibitors of tyrosine kinases and antisense inhibitors of growth factor receptors. 20oly(ADP-ribose)polymerase-1 (PARP-1) is a chromatin-bound nuclear enzyme engaged in the detection and repair of DNA damage. 21During poly(ADP) ribosylation, PARP-1 consumes nicotinamide adenine dinucleotide to synthesize poly(ADP)-ribose either on itself or on an array of nuclear target proteins such as histones, topoisomerases, DNA polymerases and DNA ligases. 22his results in high negatively charged nuclear proteins that subsequently unwind and repair the damaged DNA through the base excision repair (BER) pathway.Therefore, inhibition of PARP sensitizes tumor cells to cytotoxic drugs that induce DNA damage, which would normally be repaired through the BER pathway.Undeniably PARP inhibition has been shown to sensitize tumors to DNA alkylating agents (e.g., temozolomide) and topoisomerase I poisons (e.g., irinotecan). 23Moreover, some PARP inhibitors have been shown to display single agent activity for tumors lacking BRCA1-or BRCA2-dependent DNA double-stranded repair mechanisms. 24Thus, development of PARP inhibitors is an appealing research area offering innovative cancer treatment opportunities.
In the same direction and in continuing effort to find more potent and selective anticancer compounds, herein we designed and synthesized a series of benzothiazole compounds for studying their antiproliferative activities against 4 different human cancer cell lines including liver HepG2, breast MCF-7, lung A549 and colon HCT116 cancer cell lines as well as investigating their mechanism of action on poly(ADP-ribose)polymerase-1 inhibition.

Cell lines and culturing
Anticancer activity screening for the tested compounds utilizing 4 different human tumor cell lines including liver HepG2, breast MCF-7, lung A549 and colon HCT116 cancer cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA).The tumor cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat inactivated fetal calf serum (GIBCO), penicillin (100 U mL −1 ) and streptomycin (100 µg mL −1 ) at 37 °C in humidified atmosphere containing 5% CO 2 .Cells at a concentration of 0.50 × 10 6 were grown in a 25 cm 2 flask in 5 mL of complete culture medium.

In vitro cytotoxicity assay
The antiproliferative activity was measured in vitro using the sulfo-rhodamine-B stain (SRB) assay according to the previously reported standard procedure. 25Cells were inoculated in 96-well microtiter plate (10 4 cells well −1 ) for 24 h before treatment with the tested compounds to allow attachment of cell to the wall of the plate.Test compounds were dissolved in dimethyl sulfoxide (DMSO) at 1 mg mL −1 immediately before use and diluted to the appropriate volume just before addition to the cell culture.Different concentrations of tested compounds (0-50 µg mL −1 ) and doxorubicin (DOX) were added to the cells.Triplicate wells were prepared for each individual dose.Monolayer cells were incubated with the compounds for 48 h at 37 °C and in atmosphere of 5% CO 2 .After 48 h, cells were fixed, washed and stained for 30 min with 0.4% (m/v) SRB dissolved in 1% acetic acid.Unbound dye was removed by four washes with 1% acetic acid and attached stain was recovered with Tris-EDTA buffer.Color intensity was measured in an enzyme-linked immunosorbent assay (ELISA) reader.The relation between surviving fraction and drug concentration is plotted to get the survival curve for each cell line after the specified time.The concentration required for 50% inhibition of cell viability (IC 50 ) was calculated and the results are given in Table 1.The results were compared to the antiproliferative effects of the reference control DOX. 26ARP-1 inhibitors comprise a promising new class of compounds for the treatment of cancer.The activity of PARP-1 in the lysate of hepatic HepG2 and breast MCF-7 cancer cells treated with the prepared compounds was measured using ELISA kit purchased from Glory Science Co., Ltd (Del Rio, TX78840, USA).

Statistical analysis
The results are reported as mean ± standard error (S.E.) for at least three time experiments.Statistical differences were analyzed according to one way ANOVA test followed by student's t-test wherein the differences were considered to be significant at p < 0.05.

Antiproliferative activity of the tested compounds
The antiproliferative activities were expressed by median growth inhibitory concentration IC 50 (µg mL −1 ).
As shown in Table 1 and Figure 2, the antiproliferative activity of the synthetic compounds was evaluated against human hepatocellular carcinoma HepG2, breast adenocarcinoma MCF-7, human lung cancer A549 and colon cancer HCT116 cell lines using SRB assay, in comparison with DOX as a reference drug.The results revealed that all compounds did not exert any activity against human lung A549 and colon HCT116 cancer cell lines.Many of the tested compounds showed remarkable anticancer activity against liver HepG2 cancer cell lines.While compounds 3b, 3c and 3f had no influence on the cancer cells, compounds 3d, 3e, 3g, 4d, and 4e were found to be potent anticancer agents having IC 50 values near to the standard drug (IC 50 7.00 ± 0.80, 6.20 ± 0.73, 4.30 ± 0.40, 7.00 ± 0.80 and 5.80 ± 0.66 µg mL −1 , respectively versus 4.20 ± 0.44 µg mL −1 for DOX).The rest of the tested compounds revealed slight to moderate activity.In the same sense, evaluation of the anticancer effect of the tested compounds against human breast MCF-7 cancer cell lines revealed that although compounds 3a, 4a and 4b had no effect on the MCF-7 cancer cell, compounds 3d, 3e, 3g, 4d, and 4e showed anticancer activity close to that of the standard drug (IC 50 values 8.10 ± 0.90, 5.30 ± 0.60, 7.90 ± 0.86, 4.90 ± 0.60 and 5.00 ± 0.55 µg mL −1 , respectively, versus 4.80 ± 0.50 µg mL −1 for DOX).The rest of the tested compounds revealed slight activity.From the forgoing result, the synthesized compounds especially 3d, 3e, 3g, 4d, and 4e showed cytotoxicity and growth inhibitory activity on both breast and liver cancer cell lines.

Poly(ADP-ribose)polymerase-1 (PARP-1) activity
To identify the mechanism of action responsible for the cytotoxicity of the prepared compounds 3d, 3e, 3g, 4d, and 4e, the activity of PARP-1 expressed in the two cell lines (hepatic HepG2 and breast MCF-7 cancer cell lines) were estimated quantitatively and the results were calculated as percentage of the control cancer cells as shown in Figure 3.In this work, the activity of PARP-1 in the lysate of hepatic HepG2 and breast MCF-7 cancer cells treated with the prepared compounds and DOX as a known inhibitor was measured and the data were calculated as percentage of inhibition as compared to the control untreated cancer cells.Meanwhile, treatment of hepatic HepG2 and breast MCF-7 cancer cells with DOX resulted in 82% and 76% inhibition, respectively as compared with control cancer cells.The treatment with compounds 3d, 3e, 3g, 4d and 4e resulted in 53%, 61%, 44%, 45% and 60% inhibition, respectively in hepatic HepG2 cells.Similarly, the treatment of breast MCF-7 cells with the compounds resulted in inhibition of the activity of PARP-1 by 47%, 55%, 37%, 40% and 48%, respectively (Figure 3).
In summation, these findings suggest that there are correlation between the cytotoxicity of the tested compounds and inhibition of the PARP-1 activity.The tested compounds exert anti-carcinogenic activity in hepatic HepG2 and breast MCF-7 cancer cell lines through down regulation of the activity of PARP-1 enzyme, which may reduce the cell proliferation and result in significant growth inhibition.

Conclusions
The present study reports on simple and efficient approaches for the synthesis of new benzothiazolinone derivatives.3-Methyl-2-benzothiazolinone hydrazone 1 was successfully utilized as a starting material for implementing this goal.Some of the new products recorded pronounced in vitro antitumor activities when tested against liver (HepG2) and breast (MCF-7) human solid tumor cell lines.The most promising result against liver carcinoma was recorded by compound 3e (IC 50 value of 4.30 ± 0.40 µg mL −1 ) which is the closest in value to that recorded by the reference drug (DOX, IC 50 value of 4.20 ± 0.44 µg mL −1 ).Meanwhile, compounds 4d (IC 50 4.90 ± 0.60 µg mL −1 ), 4e (IC 50 5.00 ± 0.55 µg mL −1 ) and 3e (IC 50 5.30 ± 0.60 µg mL −1 ) showed the highest activity compared to the reference drug (DOX, IC 50 4.80 ± 0.50 µg mL −1 ) when tested against breast carcinoma.The marked cytotoxic and growth inhibitory activity of compound 3e against both liver and breast carcinoma may be correlated with the presence of the thienyl moiety in its molecule, which is known to promote the biological activity. 27On the other hand, hydrazone 1 showed the least appreciable cytotoxic activity amongst the tested compounds.However, it was successfully used to prepare new products (e.g., 3e, 4d, and 4e) of promise as antitumor agents; particularly against liver and breast carcinoma.These results, supplement to the well known activity of many benzothiazole derivatives in reducing the incidence of various cancer types. 7

General
Solvents were purified and dried according to the usual procedures.2-Hydrazono-3-methyl-2,3-dihydrobenzo[d] thiazole 1 was prepared according to a known procedure. 28he reacting aldehydes were purified directly before use by distillation and/or recrystallization.They are commercially available except for 4-piperdine-1-ylbenzaldehyde, 2c, which was prepared by reacting 4-fluorobenzaldehyde with piperidine in DMSO in the presence of K 2 CO 3 . 29The reactions were monitored (thin layer chromatography, TLC) and the purity of the isolated products were controlled by using silica gel with fluorescent indicator F 254 coated on aluminum sheets of layer thickness 0.2 mm [Fluka].Column chromatography was performed on silica gel, grain size 0.063-0.2mm (Merck).Melting points were recorded on Electrothermal melting point apparatus and were uncorrected.The IR spectra were measured in KBr pellets using a JASCO FT/IR-300E Fourier Transform Infrared Spectrophotometer and reported in cm −1 .NMR spectra were recorded on a Joel-500 MHz spectrometer ( 1 H NMR at 500 MHz and 13 C NMR at 125 MHz) in deuterated dimethylsulphoxide (DMSO-d 6 ).The proton chemical shifts are reported in d (ppm) downfield from tetramethylsilane (TMS) as an internal standard.Mass spectra were recorded on a Finnigan SSQ 7000 Spectrometer at 70 eV (Electron Impact).Analytical data were obtained at the analytical laboratory of the National Research Centre (or in the Microanalytical unit, Cairo University, Giza, Egypt).Satisfactory elemental analyses were gained for the new products.The antitumor activity was carried out at the Department of Biochemistry at the National Research Centre (NRC).Fetal bovine serum (FBS) and L-glutamine were obtained from Gibco Invitrogen Company (Scotland, UK).DMEM medium was provided from Cambrex (New Jersey, USA).DMSO, DOX, penicillin, streptomycin and sulfo-rhodamine-B stain (SRB) (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) were obtained from Sigma Chemical Company (St. Louis, MO, USA).Human poly(ADP-ribose) polymerase 1 (PARP-1) activity ELISA kit was purchased from Glory Science Co., Ltd (Del Rio, TX78840, USA).

Chemistry
General procedure for the synthesis of Schiff bases 3a-h A mixture of 3-methyl-2-benzothiazolinone hydrazone 1 (1.79 g, 0.01 mol) and the appropriate aldehyde 2a-h (0.01 mol) was refluxed in absolute ethanol (20  General procedure for the reaction of Schiff base derivatives 3a-e with thioglycolic acid Thioglycolic acid (1.4 g, 1.1 mL, 0.015 mol) was added dropwise to a solution of the appropriate Schiff base derivative 3a-e (0.01 mol) in dry benzene (50 mL) at room temperature.After refluxing the reaction mixture for 6-8 h, the volatile materials were removed under reduced pressure.The residual substance was collected and chromatographed on silica gel to give the corresponding 4-thiazolidinone derivatives 4a-e as colorless crystals.

Reaction of 2-hydrazono-3-methyl-2,3-dihydrobenzo[d] thiazole 1 with ethyl cyanoacetate
To a solution of compound 1 (1.79 g, 0.01 mol) in ethanol (50 mL), ethyl cyanoacetate (1.69 g, 1.6 mL, 0.015 mol) was added at room temperature and the reaction mixture was refluxed for 5 h.The volatile materials were removed under reduced pressure and the residual substance was collected and chromatographed on silica gel to give compounds 5 and 6.

Reaction of hydrazone 1 with ethyl acetoacetate
A mixture of compound 1 (1.79 g, 0.01 mol) and ethyl acetoacetate (1.95 g, 1.9 mL, 0.015 mol) was refluxed in absolute ethanol (20 mL) for 6 h.The volatile materials were removed from the reaction mixture under reduced pressure and the residual substance was chromatographed on silica gel to give compounds 7 and 8, respectively.

Figure 2 .Figure 3 .
Figure 2. The in vitro cytotoxicity activity of the tested compounds expressed as IC 50 values against HepG2 and MCF-7 human cancer cell lines.
Protons of the methylene group (2H) appeared as two doublets (each with J 16.3 Hz) at d 3.71 and d 3.87 ppm, indicating that they are magnetically not equivalent.The four protons of the benzothiazole ring gave signals at 7.08 (t, J 7.6 Hz), 7.18 (d, J 7.6 Hz), 7.23 (t, J 7.6 Hz) and at 7.55 (d, J 7.6 Hz), while the two doublets due to the 1,4-disubstituted benzene ring appeared at d 7.43 (2H, J 8.6 Hz) and d 7.51 (2H, J 8.6 Hz).

Table 1 .
The in vitro cytotoxicity activity of the tested compounds expressed as IC 50 values in 4 different human cancer cell lines a,b a IC 50 dose of the compounds which reduces survival to 50%; b data were expressed as average of three independent experiments; N.A.: no activity.