Synthesis and biological evaluation of 3-(trimethoxyphenyl)-2(3H)-thiazole thiones as combretastatin analogs

European Journal of Medicinal Chemistry 70 (2013) 692e702

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Synthesis and biological evaluation of 3-(trimethoxyphenyl)-2(3H)thiazole thiones as combretastatin analogs Mandana Banimustafa a, Asma Kheirollahi b, Maliheh Safavi c, Sussan Kabudanian Ardestani c, Hassan Aryapour d, Alireza Foroumadi e, Saeed Emami f, g, * a

Student Research Committee, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Department of Biochemistry, Institute of Biochemistry and Biophysics, University of Tehran, P.O. Box 13145-1384, Tehran, Iran d Department of Biology, Faculty of Science, Golestan University, Gorgan, Iran e Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran, Iran f Department of Medicinal Chemistry, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran g Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 June 2013 Received in revised form 14 October 2013 Accepted 16 October 2013 Available online 25 October 2013

A series of 3-(trimethoxyphenyl)-2(3H)-thiazole thiones 5 were designed as new heterocyclic analogs of combretastatin A-4 (CA-4). Indeed, the olefinic core structure of CA-4 has been replaced by 2(3H)thiazole thione. The general synthetic strategy to prepare compounds 5 was based on the cyclocondensation reaction between triethylammonium N-(trimethoxyphenyl)dithiocarbamate and appropriate phenacyl halide. The cytotoxic activity evaluation of 3-(trimethoxyphenyl)-2(3H)-thiazole thiones 5 against human cancer cell lines T47D, MCF-7 and MDA-MB-231 demonstrated that 4-methyl analog 5f showed the highest activity against all cell lines. Compound 5f had no significant toxicity towards nontumoral cells MRC-5 and its cytotoxicity was apparently selective for cancer cells. The results of bioassays showed that the representative compound 5f depolymerized tubulin, inhibited cell proliferation, and induced apoptosis in cancer cells. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Anticancer activity Cytotoxicity Combretastatin analogs 2(3H)-Thiazole thiones Thiazolidine-2-thione Tubulin polymerization

1. Introduction Currently, cancer is one of the most serious problem threats human health in the world [1]. Chemotherapy has still been an important fundament for cancer treatment. Drugs that perturb microtubule/tubulin dynamics are used widely in cancer chemotherapy [2]. Several binding sites including taxane, vinca alkaloids and colchicine binding sites have been identified on tubulin. Antimitotic agents with the capability of binding at the colchicine site of tubulin have received much attention and some of them such as combretastatin A-4 (CA-4) and its water-soluble prodrug CA-4P are undergoing clinical trials as antitumor drugs [2,3]. CA-4 is the most biologically significant member of cis-stilbenes isolated from the bark of African willow tree Combretum caffrum * Corresponding author. Department of Medicinal Chemistry, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari 48175-861, Iran. Tel.: þ98 151 3543082; fax: þ98 151 3543084. E-mail address: [email protected] (S. Emami). 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.10.046

[4]. This compound was found to be a potent antiproliferative agent against a broad spectrum of cancer cell lines including multi drugresistant cells [5,6]. Structureeactivity relationship studies on CA-4 derivatives have established that pharmacophore structure for binding to tubulin is the cis-orientation of the two ethenyl-bridged aromatic rings which one of them bearing 3,4,5-trimethoxy substituents [7e9]. The cisstilbene structure of CA-4 is unstable and during storage and administration, the cis-configuration is isomerized to trans-form results in decreasing of both antitubulin activity and cytotoxicity [10,11]. Moreover, the double bond reduction of CA-4 leads compound with moderately reduced activity [8,9,12]. Accordingly, considerable efforts have been focused on the cis-restriction of pharmacophoric backbone (Fig. 1), particularly by the replacement of the double bond with heterocyclic rings [13,14]. For example, a number of five-membered heterocyclic analogs of CA-4 such as imidazoles, pyrazoles, thiazoles, triazoles, oxazolones and furanones, have been reported as cis-restricted biologically active congeners of CA-4 [13e15].

M. Banimustafa et al. / European Journal of Medicinal Chemistry 70 (2013) 692e702

MeO

693

OMe MeO

MeO

MeO MeO

S N

MeO

MeO

S

MeO MeO

OH (A)

Combretastatin A-4 (CA-4)

R

R

(B) Cyclic combretastatin analogues

(C) 2(3H)-Thiazole thiones

Fig. 1. (A) Structure of combretastatin A-4 (CA-4) as unstable cis-stilbenes undergoing to trans-isomerism; (B) cyclic combretastatin analogs locked as cis-oriented stilbenoids; (C) 3(trimethoxyphenyl)-2(3H)-thiazole thiones as new heterocyclic analogs of CA-4.

With the aim of developing new heterocyclic analogs of CA-4, we designed a series of 3-(trimethoxyphenyl)-2(3H)-thiazole thione derivatives in which the olefinic core structure of CA-4 has been replaced by 2(3H)-thiazole thione (Fig. 1). Thus, we report here the convenient synthesis and biological activity of 3-(trimethoxyphenyl)-2(3H)-thiazole thiones and their related compounds. 2. Chemistry The synthesis of target compounds 5aej was carried out according to Scheme 1. Firstly, 3,4,5-trimethoxyaniline (1a) was treated with carbon disulfide in triethylamine, at room temperature to obtain N-(trimethoxyphenyl) dithiocarbamate salt 2. Compound 2 was reacted with appropriate phenacyl halide to produce a gummy adduct, which was dehydrated by refluxing in 0.5% HCl to give 4-aryl-3-(3,4,5-trimethoxyphenyl)-2(3H)-thiazole thione 5. Structurally, the intermediate adduct would be either acyclic Sphenacyl dithiocarbamate 3 or cyclic 4-hydroxythiazolidine-2thione 4. In the case of the reaction of dithiocarbamate 2 and 4methoxylphenacyl bromide, the intermediate was isolated and characterized as acyclic adduct 3a. Moreover, the reaction 4bromophenacyl bromide or 2,4-dichlorophenacyl chloride with the dithiocarbamate salt 2 resulted in corresponding cyclic adducts which were unambiguously purified and characterized as 4hydroxythiazolidine-2-thione 4a,b. It should be noted that compounds 4a and 4b which have a chiral center at the C-4 of their thiazolidine-2-thione scaffold are racemates. Due to the problem of isolation and purification the rather labile intermediates 3 and 4, the rest of final compounds 5 were obtained from refluxing of crude mixture of 3 and 4 in 0.5% hydrochloric acid. For preparation of 3-(4-methoxyphenyl) and 3-(3,4dimethoxyphenyl) analogs of 5 (compounds 6a,b), we have attempted to synthesize corresponding dithiocarbamates by following the general procedure which was used for compound 2. Due to the low solubility of 4-methoxyaniline and 3,4dimethoxyaniline in triethylamine, these attempts were failed. Luckily, compounds 6a,b were prepared by using an one-pot sequential reaction (Scheme 2). Thus, treatment of 4methoxyaniline or 3,4-dimethoxyaniline with carbon disulfide in DMF in the presence of K3PO4 afforded corresponding dithiocarbamate which was subsequently reacted with phenacyl bromide. The crude product was refluxed in 0.5% HCl to give pure compound 6. As described above, the intermediate compounds 3a, 4a and 4b were isolated and unambiguously characterized by spectral data. The IR spectrum of compound 3a showed a strong band at 1681 cm1 due to the carbonyl group. The NH absorption of compound 3a was appeared at 3313 cm1. In the 1H NMR of compound

3a, a signal was observed at 3.79 ppm that belongs to the protons of CH2. The chemical shift value of aromatic H-2 and H-6 signal (8.05 ppm) was in accord to the protons ortho to the carbonyl group. The 13C NMR data showed that the resonance related to the carbonyl carbon of compound 3a was occurred at 197.96 ppm. Cyclization of compound 3a to 5j resulted in disappearance of carbonyl signal from this region of 13C NMR spectrum. These data confirmed the acyclic form of intermediate 3a. In the 1H NMR spectra of compound 4a, two characteristic doublets at 3.62 and 3.80 ppm with geminal coupling constants w12.3 Hz indicated the diastereotopic nature of C-5 methylene protons because of neighboring with C-4 stereocenter in thiazolidine ring. Furthermore, the IR, 1H NMR, 13C NMR and MS spectral data were used for structural characterization of target compounds 5. Representatively, the 1H NMR spectrum of compound 5b showed six protons as a singlet at 3.56 ppm, which was related to the 3- and 5-methoxy groups of trimethoxyphenyl moiety. The protons of 4methoxy group were appeared at 3.69 ppm as a singlet peak. A singlet at 6.57 ppm attributed to the H-5 of 2(3H)-thiazole thione. The H-2 and H-6 aromatic protons of trimethoxyphenyl moiety were appeared at 6.26 ppm upfield respect to the other aromatic protons. The protons of 4-bromophenyl displayed two doublets at 6.86 and 7.27 ppm bearing coupling constant of 8.35 Hz. In the decoupled 13C NMR of compound 5b, 13 signals was detected which confirmed correct carbon skeleton of the compound. The mass spectral data of compound 5b provided further evidence of its correct structure. The molecular ion peak was observed at 437 and 439 as expected for m/z values of Mþ and [M þ 2]. 3. Biology 3.1. Cytotoxicity assay The in vitro cytotoxic activity of synthesized compounds against three human cancer cell lines, including MCF-7, MDA-MB-231 and T47D was determined by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide) colorimetric assay [16]. MTT assay is based on reduction of the tetrazolium salt to blue colored formazan by mitochondrial dehydrogenases in viable cells. The relative cell viability was expressed as the mean percentage of viable cells comparing to control cells, and the inhibition percentages of compounds were assessed by the following formula: [(Abscontrol cells  Abstreated cells)/Abscontrol cells]  100. The IC50 values (the concentration of compound required to decrease 50% of cell viability) were calculated from the concentrationeresponse curves by regression analysis. The IC50 values of the compounds against different cancer cell lines are shown in Table 1.

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MeO

MeO

MeO

a

MeO

OMe

OMe

OMe

S

MeO

NH2

N H 2

1a

S

b N H O

MeO

S HNEt3

S

3

MeO

MeO

S

S

c N

MeO

R

OMe

OMe

S

N

MeO

S

OH R

R

5

_ )- 4 (+

R= H; 4-Br; 4-Cl, 2,4-Cl2; 4-F; 4-CH3; 4-Ph; 4-OH; 4-OCH3; 3,4-(OH)2 Scheme 1. Synthesis of 3-(trimethoxyphenyl)-2(3H)-thiazole thiones 5. Reagents and conditions: (a) CS2, Et3N, rt; (b) appropriate phenacyl halide, acetone; (c) 0.5% HCl, reflux.

3.2. Tubulin polymerization assay To further characterize the effects of selected compound 5f on microtubule polymerization, we examined microtubules assembly incubated with different concentration of compound 5f using UVe Visible spectrometer. The absorbances (l 350 nm) were recorded for a period of 30 min and the results were compared to the DMSOtreated control group to evaluate the relative degree of change in optical density. The percent of inhibition was calculated as follow: % inhibition ¼ (1  A350 sample/A350 control)  100. 3.3. Acridine orange/ethidium bromide staining test The potential of compounds 4b and 5f to induce apoptosis in T47D and MCF-7 cells was determined morphologically by acridine orange/ethidium bromide double staining test. Using fluorescence microscopy, cells can be distinguished as live cells (uniformly stained green) and apoptotic cells that are stained orange because of cell membrane destruction and the intercalation of ethidium bromide between the nucleotide bases of DNA [17]. 3.4. Flow cytometry analyses of the apoptotic cells with Annexin VPE and 7-aminoactinomycin D (7-AAD) double staining The MCF-7 cells were treated with IC50 concentrations of the selected compounds 4b, 5f and etoposide as reference drug. Then,

H3CO NH2

1. CS2, K3PO4, DMF, Phenacyl bromide 2. HCl 0.5%, reflux

R

R S N S

OCH3 1b: R= H 1c: R= MeO

6a: R= H 6b: R= MeO

Scheme 2. One-pot sequential synthesis of 3-(4-methoxyphenyl)- and 3-(3,4dimethoxyphenyl)-2(3H)-thiazole thiones 6.

the apoptosis induction was evaluated by Annexin-V binding and 7AAD uptake test. Annexin V-PE was used to quantitatively determine the percentage of cells within a population that are undergoing apoptosis [18]. Using this technique, cells that are viable are Annexin V-PE and 7-AAD negative; cells that are in early apoptosis are Annexin V-PE positive and 7-AAD negative; and cells that are in late apoptosis or necrosis are both Annexin V-PE and 7-AAD positive. The double stained cells were analyzed by flow cytometry. 4. Results and discussion The IC50 values of 3-(trimethoxyphenyl)-2(3H)-thiazole thiones 5aej in comparison with etoposide as standard drug are presented in Table 1. Among the compounds 5aej, 4-methyl analog 5f showed the highest activity against all cell lines with IC50 values 11.8e 19.7 mg/mL. Moreover, compounds 5c, 5e and 5h exhibited significant growth inhibitory activity against all tested cell lines (IC50 values < 50 mg/mL). The comparison of IC50 values of 5f and 5h with those of unsubstituted analog 5a revealed that the introduction of methyl or hydroxyl group on 4-phenyl ring increases the cytotoxic activity. The compounds 5bee containing halogen substituent showed lower activities against T47D cells compared to unsubstituted counterpart 5a. Thus, halogen substitution could not improve the inhibitory activity against T47D cell line. While, the introduction of 4-fluoro- or 2,4-dichloro- substituents improved the cytotoxic activity toward MCF-7 and MDA-MB-231 (compounds 5c and 5e vs. 5a). The O-methylation of 4-hydroxy derivative (compound 5j compared to 5h) reduced the cytotoxic potential against all cell lines. The observed activity with 4-hydroxy derivative 5h and 3,4dihydroxy analog 5i demonstrated that the insertion of second hydroxy group could not improve the cytotoxic activity. To investigate the impact of 3,4,5-trimethoxyphenyl functionality on cytotoxic activity, we synthesized and evaluated the monomethoxy and dimethoxy congeners of compound 5a (compounds 6a and 6b, respectively). Interestingly, these compounds showed comparable or superior activity respect to the corresponding 3,4,5trimethoxyphenyl derivative 5a. These findings revealed that the intrinsic cytotoxic activity of compounds significantly depends on 3,4-diaryl-2(3H)-thiazole thione backbone.

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695

Table 1 Cytotoxic activity (IC50, mg/mL) of compounds 3e6. Compound

Structure

T47D

MCF-7

MDA-MB-231

29.9  5.2

18.6  4.6

16.4  3.6

16.3  3.3

16.5  1.0

15.1  2.7

16.9  4.0

9.6  2.5

14.5  2.1

S

31.6  3.8

>100

52.0  14.1

S

>100

>100

58.0  1.4

S

38.9  6.7

38.5  2.1

19.5  1.9

S

>100

>100

>100

S

43.4  8.8

23.6  3.2

49.0  5.2

OCH3 OCH3

H3CO

S

3a

N H

H3CO

S O

OMe MeO 4a

S N

MeO

S OH

Br OMe MeO 4b

S N

MeO

S OH Cl

Cl

OMe MeO 5a

S N

MeO

OMe MeO 5b

S N

MeO

Br OMe MeO 5c

S N

MeO

F OMe MeO 5d

S N

MeO

Cl

OMe MeO 5e

MeO

S N

(continued on next page)

Cl Cl

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M. Banimustafa et al. / European Journal of Medicinal Chemistry 70 (2013) 692e702

Table 1 (continued ) Compound

Structure

T47D

MCF-7

MDA-MB-231

S

16.3  3.7

19.7  0.8

11.8  1.3

S

>100

45.3  10.6

>100

S

26.4  6.2

35.1  3.1

22.9  7.2

S

24.9  3.7

>100

29.8  0.3

S

>100

>100

53.8  8.4

S

23.2  2.9

88.5  12.9

36.8  5.6

S

33.1  8.2

17.6  1.5

19.1  1.4

8.2  1.2

9.4  3.2

7.2  1.6

OMe MeO 5f

S N

MeO

Me

OMe MeO 5g

S N

MeO

Ph OMe MeO 5h

S N

MeO

HO

OMe MeO

5i

S N

MeO

HO

OH OMe

MeO 5j

S N

MeO

MeO

MeO

S N

6a

OMe MeO 6b

Etoposide

S N

M. Banimustafa et al. / European Journal of Medicinal Chemistry 70 (2013) 692e702

Furthermore, the cytotoxic activity of acyclic and alcoholic intermediates (compounds 3a and 4a,b, respectively) were also evaluated against test cell lines (Table 1). Surprisingly, by comparing the IC50 values of acyclic analog 3a with those of heterocyclic congener 5j, it was demonstrated that acyclic form had more cytotoxicity than compound 5j. It was worthy of note that the alcoholic derivatives 4a and 4b showed more potent activity respect to the corresponding dehydrated analogs 5b and 5e, respectively. Alcoholic compound 4b was as potent as standard drug etoposide against MCF-7 cells. Its activity against the latter cell line was superior than all tested compounds. The cytotoxic activity of promising compounds 4b and 5f were also evaluated on non-tumoral cell line MRC-5. Results of MTT assay showed that compounds 4b and 5f have less cytotoxic activity against non-tumoral cells. The IC50 values of compounds 4b and 5f on MRC-5 cells were 47.3  4.2 mg/mL and >100 mg/mL, respectively. According to the results, the cytotoxicity of these compounds was apparently selective for cancer cell lines T47D, MCF-7 and MDA-MB-231. Particularly, compound 5f had no significant toxicity towards non-tumoral cell line MRC-5 (Selectivity index > 5). To verify whether the cytotoxic activity of the designed compounds was correlated to tubulin inhibition, the inhibitory effect of the most active compound 5f on the polymerization of purified tubulin was evaluated. The effect of compound 5f on the microtubules polymerization is shown in Fig. 2. The results showed that compound 5f evidently inhibited the assembly of purified sheep brain MTP in a concentration dependent manner (Fig. 2). It is well documented that exposure to microtubule-targeting agents leads to malformed mitotic spindles, mitotic arrest, and apoptosis. Thus, we next examined the effects of selected compounds 4b and 5f on the apoptosis of T47D and MCF-7 cells by acridine orange/ethidium bromide double staining technique. T47D and MCF-7 cells were treated with and without IC50 concentration of compounds 4b and 5f for 24 h and stained with a mixture of acridine orange and ethidium bromide. Analysis of the acridine orange/ethidium bromide staining results revealed that the test compounds 4b and 5f induced apoptosis in T47D and MCF-7 cell lines. As shown in Fig. 3, the non-apoptotic control cells were stained green and the apoptotic cells had orange particles in their nuclei due to nuclear DNA fragmentation. The appearance of chromatin condensation and nuclear fragmentation are evident in this figure. To further confirm of apoptosis induction by compounds 4b and 5f, the treated MCF-7 cells were subjected to Annexin V/7-AAD double staining followed by flow cytometry analyses (Figs. 4 and 5). Annexin V/7-AAD flow cytometric analyses revealed that cells undergo apoptosis after treatment with IC50 concentrations of compounds 4b and 5f. As shown in Fig. 5, compounds 4b and 5f induced 51.67% and 66.47% apoptosis in the cancer cells, respectively. Therefore, it is evident that these compounds have strong anti-proliferative activity. 5. Conclusion In summary, we designed a series of 3-(trimethoxyphenyl)2(3H)-thiazole thiones 5 as new heterocyclic analogs of CA-4, in which the olefinic core structure of CA-4 replaced by 2(3H)-thiazole thione. The general synthetic strategy employed to prepare the 3(trimethoxyphenyl)-2(3H)-thiazole thiones 5 was based on the cyclocondensation reaction between N-(trimethoxyphenyl)dithiocarbamate and appropriate phenacyl halide. In some cases, the intermediate adducts including acyclic S-phenacyl dithiocarbamate 3 or cyclic 4-hydroxythiazolidine-2-thione 4 were purified and characterized as individual compounds. The cytotoxic activity evaluation of 3-(trimethoxyphenyl)-2(3H)-thiazole thiones 5

697

Fig. 2. Inhibition of tubulin polymerization by compound 5f. (A) Kinetics of the inhibition of microtubule assembly in different concentrations of compound 5f. (B) Plot of percent tubulin polymerization against log concentration (M) of compound 5f at 37  C.

against human cancer cell lines, including T47D, MCF-7 and MDA-MB-231 demonstrated that 4-methyl analog 5f showed the highest activity against all cell lines. The cytotoxicity of compound 5f was apparently selective for tested cancer cell lines and compound 5f had no significant toxicity towards non-tumoral cell line MRC-5. Besides 2(3H)-thiazole thiones 5, 4-hydroxy-3-(3,4,5trimethoxyphenyl)-4-(2,4-dichlorophenyl)thiazolidine-2-thione (4b) which was separated and characterized as a stable intermediate showed better profile of antiproliferative activity against tested cell lines. The representative compound 5f depolymerized tubulin, inhibited cell proliferation, and induced apoptosis in cancer cells. These compounds can be considered as more stable analogs of combretastatin A-4 for further biological evaluations. 6. Experimental protocols 6.1. Chemistry 6.1.1. General methods Triethylammonium N-(3,4,5-trimethoxyphenyl)dithiocarbamate was prepared according to the literature method [19]. Required phenacyl halides including 2-bromo-40 -hydroxyacetophenone and 2-bromo-40 -chloroacetophenone were synthesized by bromination of corresponding acetophenones using CuBr2 in CHCl3eEtOAc [20]. Other phenacyl halides were

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Fig. 3. Acridine orange/ethidium bromide double staining of T47D (A) and MCF-7 (B) cells with characteristic symptoms of apoptosis: (a) DMSO 1% as control; (b) etoposide as positive control; (c) cells treated with IC50 concentration of compound 4b for 24 h; (d) cells treated with IC50 concentration of compound 5f for 24 h. White arrow indicates live cells and dashed arrow indicates apoptotic cells. The images of cells were taken with a fluorescence microscope at 400.

commercially available materials from Merck or Fluka companies. The progress of reactions was checked by thin-layer chromatography (TLC) using silica gel 60 F254 plastic sheets (Merck). The ultra-violet light (254 nm) was used for TLC visualization. Yields are based on isolated product and were not optimized. Melting points were determined in open glass capillaries using Bibby Stuart Scientific SMP3 apparatus (Stuart Scientific, Stone, UK) and are uncorrected. The IR spectra were obtained on a PerkinElmer FT-IR spectrophotometer using KBr disks. The NMR spectra were recorded using a Bruker 500 spectrometer and chemical shifts are expressed as d (ppm) with tetramethylsilane (TMS) as internal standard. The mass spectra were obtained using a HP 5937 Mass Selective Detector (Agilent technologies). 6.1.2. General procedure for the synthesis of intermediates Sphenacyl dithiocarbamate 3 or 4-hydroxythiazolidine-2-thione 4 N-(Trimethoxyphenyl) dithiocarbamate salt 2 (1.5 mmol) was dissolved in acetone (10 mL) and the solution was stirred in an ice bath. An appropriate phenacyl halide (1.5 mmol) was added portionwise over a 30 min period. After completion of the reaction (consumption of phenacyl halide, 0.5e2 h), the minimum volume of water to cause solution was added and the cold solution was stirred for 15 min longer. After evaporation of acetone under reduced pressure, a viscose oily residue was separated. Water (5 mL) was added to the oily residue and the aqueous phase was decanted. The crude product was crystallized from methanol or diethyl ether. 6.1.3. General procedure for the synthesis of 3-(trimethoxyphenyl)2(3H)-thiazole thiones 5 N-(Trimethoxyphenyl) dithiocarbamate salt 2 (1.5 mmol) was dissolved in acetone (10 mL) and the solution was stirred in an ice bath. An appropriate phenacyl halide (1.5 mmol) was added portionwise over a 30 min period. After completion of the reaction (consumption of phenacyl halide, 0.5e2 h), the minimum volume of water to cause solution was added and the cold solution was stirred for 15 min longer. After evaporation of acetone under reduced pressure, a viscose oily residue was separated. Water (5 mL) was added to the oily residue and the aqueous phase was decanted. The oily crude product was mixed with 0.5% HCl (20 mL) and methanol (4 mL), and the mixture was refluxed for 45 min. After cooling up to room temperature, the reaction mixture was left

in refrigerator overnight. The precipitated solid was collected by filtration and washed with water. The product was recrystallized from methanol or diethyl ether to give pure 3-(trimethoxyphenyl)2(3H)-thiazole thione 5. 6.1.4. General procedure for the synthesis of 3-(4-methoxyphenyl)and 3-(3,4-dimethoxyphenyl)-2(3H)-thiazole thiones 6 A mixture of 4-methoxyaniline or 3,4-dimethoxyaniline (1 mmol) and K3PO4 (215 mg, 1 mmol) in DMF (7.5 mL) was cooled in ice bath, and then CS2 (380 mg, 5 mmol) was added. After stirring for 20 min, 2-bromoacetophenone (199 mg, 1 mmol) was added and the mixture was allowed to warm to room temperature gradually. Water (20 mL) was added to the mixture and left in refrigerator overnight. The precipitated solid was collected by filtration and washed with water. The obtained crude product was mixed with 0.5% HCl (20 mL) and methanol (4 mL), and the mixture was refluxed for 45 min. After cooling up to room temperature, the reaction mixture was left in refrigerator overnight. The precipitated solid was collected by filtration and washed with water. The product was recrystallized from methanol to give compound 6. 6.1.5. Physicochemical and spectral data of synthesized compounds 6.1.5.1. 4-Methoxyphenacyl N-(3,4,5-trimethoxyphenyl)dithiocarbamate (3a). Yield 60%; mp 142e143  C; IR (KBr, cm1) nmax: 3313, 3076, 2938, 2838, 1681, 1599, 1508, 1454, 1312, 1261, 1177, 1127, 1015, 987, 835. 1H NMR (500 MHz, CDCl3) d: 3.79 (s, 2H, CH2), 3.85 (s, 6H, 3-OCH3 and 5-OCH3), 3.87 (s, 3H, 4-OCH3), 3.91 (s, 3H, 40 OCH3), 4.75 (br s, 1H, NH), 6.35 (s, 2H, H-2 and H-6), 7.00 (d, 2H, J ¼ 8.82 Hz, H-30 and H-50 ), 8.05 (d, 2H, J ¼ 8.82 Hz, H-20 and H-60 ). 13 C NMR (125 MHz, CDCl3) d: 56.02, 56.49, 56.68, 61.36, 101.19, 107.19, 114.06, 114.51, 127.83, 131.59, 153.29, 153.71, 160.45, 164.74, 197.96. MS (m/z, %): 406 (Mþ, 5), 389 (100), 343 (30), 288 (16), 164 (18), 135 (43). Anal. Calcd for C19H21NO5S2: C, 56.00; H, 5.19; N, 3.44. Found: C, 56.23; H, 5.08; N, 3.50. 6 .1. 5 . 2 . ( ) - 4 - H y d ro x y- 3 - ( 3 , 4 , 5 - t r i m e t h o x y p h e nyl ) - 4 - ( 4 bromophenyl)thiazolidine-2-thione (4a). Yield 92%; mp 134e 135  C; IR (KBr, cm1) nmax: 3265, 2933, 1593, 1504, 1403, 1198, 1072, 990, 833, 820, 781, 631, 567. 1H NMR (500 MHz, CDCl3) d: 3.57 (s, 6H, 3-OCH3 and 5-OCH3), 3.62 (d, 1H, J ¼ 12.25 Hz, H-5a Thiazolidine), 3.68 (s, 3H, 4-OCH3), 3.80 (d, 1H, J ¼ 12.28 Hz, H-5b Thiazolidine), 6.23 (s, 2H, H-2 and H-6), 7.33 (s, 4H, BrPh), 7.53 (s,

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Fig. 4. Histogram of flow cytometric analysis of MCF-7 cells treated with compounds 4b and 5f. Cells were stained with Annexin V/7-AAD and quantitated by flow cytometry. MCF-7 cells were treated with DMSO 1% (negative control) or with IC50 concentrations of etoposide (positive control) and compounds 4b and 5f. Fluorescence intensity of Annexin V-PE was shifted to the right.

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Fig. 5. Flow cytometric analysis of MCF-7 cells treated with compounds 4b and 5f. Cells were stained with Annexin V/7-AAD and quantitated by flow cytometry. Percentage of PEpositive events (% apoptotic cells) was calculated.

1H, OH). 13C NMR (125 MHz, CDCl3/DMSO-d6) d: 44.99, 56.37, 56.53, 61.04, 100.77, 107.32, 123.16, 128.38, 130.44, 131.58, 132.37, 133.85, 137.89, 140.51, 153.00, 197.77. MS (m/z, %): 455 (Mþ, 5), 423 (45), 408 (42), 395 (35), 243 (38), 194 (64), 183 (100), 155 (34), 89 (35), 76 (26), 50 (37). Anal. Calcd for C18H18BrNO4S2: C, 47.37; H, 3.98; N, 3.07. Found: C, 47.33; H, 4.22; N, 3.05. 6.1.5.3. ()-4-Hydroxy-3-(3,4,5-trimethoxyphenyl)-4-(2,4dichlorophenyl)thiazolidine-2-thione (4b). Yield 47%; mp 151e 152  C; IR (KBr, cm1) nmax: 3431, 2939, 1592, 1503, 1318, 1231, 1121, 1071, 947, 820. 1H NMR (500 MHz, CDCl3) d: 3.43 (d, 1H, J ¼ 12.37 Hz, H-5a Thiazolidine), 3.69 (s, 6H, 3-OCH3 and 5-OCH3), 3.75 (s, 3H, 4-OCH3), 4.24 (d, 1H, J ¼ 12.36 Hz, H-5b Thiazolidine), 6.61 (s, 2H, H-2 and H-6), 7.13 (dd, 1H, J ¼ 8.62 and 2.08 Hz, H-50 ), 7.31 (d, 1H, J ¼ 2.08 Hz, H-30 ), 7.78 (d, 1H, J ¼ 8.61 Hz, H-60 ). 13C NMR (125 MHz, CDCl3/DMSO-d6) d: 42.52, 56.46, 56.70, 61.13, 61.82, 99.37, 106.51, 198.62, 127.50, 127.77, 130.80, 133.43, 136.00, 138.03, 152.88. MS (m/z, %): 427 (32), 253 (10), 225 (93), 210 (73), 182 (61), 173 (100), 152 (65), 140 (19), 124 (18), 109 (43), 96 (29), 86 (22), 75 (22), 57 (14). Anal. Calcd for C18H17Cl2NO4S2: C, 48.43; H, 3.84; N, 3.14. Found: C, 48.71; H, 3.90; N, 2.95. 6.1.5.4. 3-(3,4,5-Trimethoxyphenyl)-4-phenylthiazole-2(3H)-thione (5a). Yield 66%; mp 184e185  C; IR (KBr, cm1) nmax: 2934, 2827, 1600, 1505, 1450, 1228, 1126. 1H NMR (500 MHz, CDCl3) d: 3.71 (s, 6H, 3-OCH3 and 5-OCH3), 3.85 (s, 3H, 4-OCH3), 6.42 (s, 2H, H-2 and H-6), 6.64 (s, 1H, Thiazole-H), 7.11 (d, 2H, J ¼ 7.0 Hz, H-20 and H-60 ), 7.25 (t, 2H, J ¼ 7.5 Hz, H-30 and H-50 ), 7.31 (t, 1H, J ¼ 7.5 Hz, H-40 ). MS (m/z, %): 359 (Mþ, 100), 344 (43), 312 (18), 258 (18), 135 (35), 105 (31), 77 (37), 43 (20). Anal. Calcd for C18H17NO3S2: C, 60.14; H, 4.77; N, 3.90. Found: C, 60.32; H, 5.03; N, 3.77.

6.1.5.5. 3-(3,4,5-Trimethoxyphenyl)-4-(4-bromophenyl)thiazole2(3H)-thione (5b). Yield 73%; mp 239e240  C; IR (KBr, cm1) nmax: 2940, 1598, 1505, 1418, 1275, 1263, 1231, 1131, 955, 750. 1H NMR (500 MHz, CDCl3) d: 3.56 (s, 6H, 3-OCH3 and 5-OCH3), 3.69 (s, 3H, 4OCH3), 6.26 (s, 2H, H-2 and H-6), 6.57 (s, 1H, Thiazole-H), 6.86 (d, 2H, J ¼ 8.35 Hz, H-30 and H-50 ), 7.24 (d, 2H, J ¼ 8.35 Hz, H-20 and H60 ). 13C NMR (125 MHz, CDCl3/DMSO-d6) d: 56.62, 61.19, 106.68, 109.87, 123.90, 129.86, 130.34, 131.99, 132.34, 132.96, 138.62, 143.98, 153.71. MS (m/z, %): 439 (M þ 2, 100), 437 (Mþ, 89), 424 (50), 392 (23), 194 (27), 183 (43), 89 (34). Anal. Calcd for C18H16BrNO3S2: C, 49.32; H, 3.68; N, 3.20. Found: C, 49.34; H, 3.55; N, 3.14. 6.1.5.6. 3-(3,4,5-Trimethoxyphenyl)-4-(4-fluorophenyl)thiazole2(3H)-thione (5c). Yield 47%; mp 190e191  C; IR (KBr, cm1) nmax: 3060, 2932, 1601, 1505, 1459, 1415, 1271, 1226, 1136, 1004, 852, 729. 1 H NMR (500 MHz, CDCl3) d: 3.73 (s, 6H, 3-OCH3 and 5-OCH3), 3.87 (s, 3H, 4-OCH3), 6.42 (s, 2H, H-2 and H-6), 6.64 (s, 1H, Thiazole-H), 6.95e6.99 (m, 2H, H-30 and H-50 ), 7.09e7.14 (m, 2H, H-20 and H-60 ). 13 C NMR (125 MHz, CDCl3) d: 56.73, 61.39, 106.80, 109.44, 116.14 (d, JC,F ¼ 21.75 Hz), 127.29, 130.96 (d, JC,F ¼ 8.5 Hz), 133.12, 138.81, 144.31, 153.88, 163.39 (d, JC,F ¼ 249.38 Hz), 190.32. Anal. Calcd for C18H16FNO3S2: C, 57.28; H, 4.27; N, 3.71. Found: C, 57.25; H, 4.32; N, 3.70. 6.1.5.7. 3-(3,4,5-Trimethoxyphenyl)-4-(4-chlorophenyl)thiazole2(3H)-thione (5d). Yield 70%; mp 214e215  C; IR (KBr, cm1) nmax: 2928, 1597, 1505, 1274, 1262, 1231, 1133, 1092, 1050, 821. 1H NMR (500 MHz, CDCl3) d: 3.76 (s, 6H, 3-OCH3 and 5-OCH3), 3.90 (s, 3H, 4OCH3), 6.43 (s, 2H, H-2 and H-6), 6.66 (s, 1H, Thiazole-H), 7.08 (d, 2H, J ¼ 8.22 Hz, H-30 and H-50 ), 7.27 (d, 2H, J ¼ 8.03 Hz, H-20 and H60 ). MS (m/z, %): 395 (M þ 2, 45), 393 (Mþ, 100), 378 (40), 346 (14),

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292 (14), 181 (11), 168 (17), 152 (16), 136 (20), 123 (13), 109 (13), 89 (11), 81 (10), 66 (13), 57 (10). Anal. Calcd for C18H16ClNO3S2: C, 54.88; H, 4.09; N, 3.56. Found: C, 54.76; H, 3.88; N, 3.86. 6.1.5.8. 3-(3,4,5-Trimethoxyphenyl)-4-(2,4-dichlorophenyl)thiazole2(3H)-thione (5e). Yield 39%; mp 173e174  C; IR (KBr, cm1) nmax: 3107, 2935, 1600, 1504, 1465, 1308, 1292, 1232, 1123, 1096. 1H NMR (500 MHz, CDCl3) d: 3.76 (s, 6H, 3-OCH3 and 5-OCH3), 3.85 (s, 3H, 4OCH3), 6.47 (s, 2H, H-2 and H-6), 6.67 (s, 1H, Thiazole-H), 7.11 (d, 1H, J ¼ 8.24 Hz, H-60 ), 7.19 (dd, 1H, J ¼ 8.24 and 1.92 Hz, H-50 ), 7.38 (d, 1H, J ¼ 1.9 Hz, H-30 ). 13C NMR (125 MHz, CDCl3) d: 56.66, 61.32, 106.16, 111.62, 127.58, 128.74, 130.12, 132.69, 133.30, 135.72, 137.21, 138.79, 140.57, 153.7. MS (m/z, %): 427 (Mþ, 88), 412 (29), 382 (12), 326 (13), 167 (18), 149 (31), 137 (30), 123 (44), 109 (54), 97 (85), 83 (86), 69 (93), 57 (100), 43 (85). Anal. Calcd for C18H15Cl2NO3S2: C, 50.47; H, 3.53; N, 3.27. Found: C, 50.61; H, 3.52; N, 3.44. 6.1.5.9. 3-(3,4,5-Trimethoxyphenyl)-4-(p-tolyl)thiazole-2(3H)-thione (5f). Yield 75%; mp 164e165  C; IR (KBr, cm1) nmax: 1598, 1505, 1463, 1417, 1267, 1231, 1128, 717. 1H NMR (500 MHz, CDCl3) d: 2.33 (s, 3H, CH3), 3.74 (s, 6H, 3-OCH3 and 5-OCH3), 3.88 (s, 3H, 4-OCH3), 6.43 (s, 2H, H-2 and H-6), 6.61 (s, 1H, Thiazole-H), 7.01 (d, 2H, J ¼ 8.05 Hz, H-30 and H-50 ), 7.07 (d, 2H, J ¼ 7.94 Hz, H-20 and H-60 ). 13 C NMR (125 MHz, CDCl3) d: 21.65, 56.69, 61.36, 106.88, 108.87, 128.29, 128.85, 129.19, 129.58, 133.39, 139.80, 145.66, 153.74, 190.25. Anal. Calcd for C19H19NO3S2: C, 61.10; H, 5.13; N, 3.75. Found: C, 61.11; H, 5.01; N, 3.64. 6.1.5.10. 3-(3,4,5-Trimethoxyphenyl)-4-(4-biphenyl)thiazole-2(3H)thione (5g). Yield 33%; mp 209e210  C; IR (KBr, cm1) nmax: 1584, 1504, 1417, 1231, 1127, 1004, 753. 1H NMR (500 MHz, CDCl3) d: 3.65 (s, 6H, 3-OCH3 and 5-OCH3), 3.79 (s, 3H, 4-OCH3), 6.69 (s, 2H, H-2 and H-6), 7.23 (s, 1H, Thiazole-H), 7.34e7.71 (m, 9H, Biphenyl). Anal. Calcd for C24H21NO3S2: C, 66.18; H, 4.86; N, 3.22. Found: C, 66.20; H, 4.99; N, 3.07. 6.1.5.11. 3-(3,4,5-Trimethoxyphenyl)-4-(4-hydroxyphenyl)thiazole2(3H)-thione (5h). Yield 53%; mp 254e255  C; IR (KBr, cm1) nmax: 3400, 1602, 1505, 1464, 1278, 1233, 1128, 834. 1H NMR (500 MHz, DMSO-d6) d: 3.64 (s, 6H, 3-OCH3 and 5-OCH3), 3.66 (s, 3H, 4-OCH3), 6.58 (s, 2H, H-2 and H-6), 6.64 (d, 2H, J ¼ 8.55 Hz, H-30 and H-50 ), 7.03 (s, 1H, Thiazole-H), 7.05 (d, 2H, J ¼ 8.52 Hz, H-20 and H-60 ), 9.70 (s, 1H, OH). MS (m/z, %): 375 (Mþ, 100), 360 (76), 329 (92), 293 (26), 274 (21), 205 (20), 180 (29), 172 (28), 150 (68), 137 (31), 118 (65), 109 (36), 97 (36), 89 (48), 81 (34), 69 (40), 57 (40), 43 (36). Anal. Calcd for C18H17NO4S2: C, 57.58; H, 4.56; N, 3.73. Found: C, 57.31; H, 4.48; N, 3.85. 6.1.5.12. 3-(3,4,5-Trimethoxyphenyl)-4-(3,4-dihydroxyphenyl)thiazole-2(3H)-thione (5i). Yield 52%; mp 237e238  C; IR (KBr, cm1) nmax: 3390, 3111, 1599, 1505, 1306, 1233, 1126, 1025. 1H NMR (500 MHz, DMSO-d6) d: 3.65 (s, 6H, 3-OCH3 and 5-OCH3), 3.66 (s, 3H, 4-OCH3), 6.50 (dd, 1H, J ¼ 8.18 and 2.01 Hz, H-60 ), 6.57 (s, 2H, H2 and H-6), 6.59 (d, 1H, J ¼ 8.18 Hz, H-50 ), 6.60 (d, 1H, J ¼ 2.01 Hz, H20 ), 6.98 (s, 1H, Thiazole-H), 9.00 (s, 1H, OH), 9.22 (s, 1H, OH). MS (m/ z, %): 391 (Mþ, 100), 376 (51), 344 (23), 290 (17), 166 (34), 149 (35), 134 (68), 120 (35), 109 (46), 97 (45), 83 (40), 69 (48), 57 (54), 43 (43). Anal. Calcd for C18H17NO5S2: C, 55.23; H, 4.38; N, 3.58. Found: C, 55.20; H, 4.46; N, 3.56. 6.1.5.13. 3-(3,4,5-Trimethoxyphenyl)-4-(4-methoxyphenyl)thiazole2(3H)-thione (5j). Yield 80%; mp 174e175  C; IR (KBr, cm1) nmax: 1599, 1505, 1465, 1417, 1275, 1249, 1052, 833. 1H NMR (500 MHz, CDCl3) d: 3.74 (s, 6H, 3-OCH3 and 5-OCH3), 3.79 (s, 3H, 4-OCH3), 3.88 (s, 3H, 40 -OCH3), 6.43 (s, 2H, H-2 and H-6), 6.58 (s, 1H,

701

Thiazole-H), 6.78 (d, 2H, J ¼ 8.81 Hz, H-30 and H-50 ), 7.04 (d, 2H, J ¼ 8.81 Hz, H-20 and H-60 ). 13C NMR (125 MHz, CDCl3) d: 55.71, 56.70, 61.38, 106.86, 108.49, 114.32, 123.49, 130.36, 133.44, 145.50, 153.79, 160.59. Anal. Calcd for C19H19NO4S2: C, 58.59; H, 4.92; N, 3.60. Found: C, 58.75; H, 5.11; N, 3.36. 6.1.5.14. 3-(4-Methoxyphenyl)-4-phenylthiazole-2(3H)-thione (6a). Yield 15%; mp 141e142  C; IR (KBr, cm1) nmax: 3029, 2837, 1609, 1514, 1284, 1257, 1171, 1052, 764, 728. 1H NMR (500 MHz, CDCl3) d: 3.85 (s, 3H, OCH3), 6.66 (s, 1H, Thiazole-H), 6.93 (d, 2H, J ¼ 8.89 Hz, H-3 and H-5), 7.12e7.15 (m, 4H, H-2, H-6, H-20 and H-60 ), 7.27 (t, 2H, J ¼ 7.61 Hz, H-30 and H-50 ), 7.32 (t, 1H, J ¼ 7.35 Hz, H-40 ). Anal. Calcd for C16H13NOS2: C, 64.18; H, 4.38; N, 4.68. Found: C, 64.10; H, 4.49; N, 4.81. 6.1.5.15. 3-(3,4-Dimethoxyphenyl)-4-phenylthiazole-2(3H)-thione (6b). Yield 24%; mp 168e169  C; IR (KBr, cm1) nmax: 1598, 1516, 1442, 1295, 1244, 1220, 1079, 1052, 1023, 766, 732. 1H NMR (500 MHz, CDCl3) d 3.78 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 6.65 (s, 1H, Thiazole-H), 6.71 (d, 1H, J ¼ 2.01 Hz, H-2), 6.80 (dd, 1H, J ¼ 8.52 and 2.19 Hz, H-6), 6.87 (d, 1H, J ¼ 8.54 Hz, H-5), 7.13 (d, 2H, J ¼ 7.61 Hz, H-20 and H-60 ), 7.25e7.35 (m, 3H, H-30 and H-40 and H50 ). Anal. Calcd for C17H15NO2S2: C, 61.98; H, 4.59; N, 4.25. Found: C, 61.92; H, 4.57; N, 4.00. 6.2. Biological activity 6.2.1. Cell lines and cell culture The human cancer cell lines MCF-7, MDA-MB-231 and T47D were obtained from Pasture Institute, Tehran (Iran). The cells were cultured in RPMI 1640 containing 10% FBS, 1% L-Glutamine, and PenicillineStreptomycin, and then cells were incubated at 37  C in a humidified atmosphere with 5% CO2. 6.2.2. Cytotoxicity assay The in vitro cytotoxic activity of test compounds was determined by MTT assay [16]. Briefly, cells in the log-phase of growth were harvested by trypsinization, seeded in 96 well plates (Nunc, Denmark) for 24 h. Then, the cells were treated with various concentrations of the compounds for 48 h. Etoposide and DMSO were used as positive and negative controls, respectively. The final concentration of DMSO was less than 2%. After 48 h, the culture medium was removed, and cells were incubated with 200 mL of MTT solution (0.5 mg/mL) for 4 h. Then, the supernatant was removed and the formazan crystals were dissolved using DMSO. The absorbance was read at 492 nm with an ELISA plate reader (Exert 96, Asys Hitch, Ec Austria) after 30 min. 6.2.3. Tubulin polymerization assay Sheep brain microtubule protein was isolated by two cycles of polymerization-depolymerization in the PEM buffer [100 mM PIPES, pH 6.9, 1 mM MgSO4 and 1 mM ethylene glycol tetraacetic acid (EGTA)], according to the method described by Sengupta et al. [21]. Tubulin was purified from the microtubule protein by phosphocellulose chromatography [22]. The tubulin solution was rapidly frozen as drops in liquid nitrogen and stored at 70  C until used. Protein concentration was determined by the method of Bradford with bovine serum albumin as the standard [23]. The purity of tubulin was determined using polyacrylamide gel electrophoresis, which was performed by the Laemmli method [24]. The tubulin polymerization assay was carried out based on reported method with some modifications [25]. Tubulin pellets were thawed and centrifuged at 0  C to remove any aggregated or denatured tubulin. A drug/DMSO-tubulin pre-incubation, without

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GTP, was performed for 15 min at 0  C in ice. After adding three mL GTP (final concentration, 1 mM), the drug/DMSO-tubulin samples were immediately placed in a Varian Cary 100 UV/visible spectrophotometer, initiated polymerization by setting the temperature controller at 37  C. The absorbance (l 350 nm) for a period of 30 min was recorded and the results were compared to the DMSOtreated control cells to evaluate the relative degree of change in optical density. DMSO was used at the final concentrations of <4% (v/v). 6.2.4. Acridine orange/ethidium bromide staining test T47D cells grown in 12-well plates (50,000 cells/well) were treated with and without IC50 concentration of compounds 4b and 5f for 24 h. After treatment, cells were harvested and washed three times with phosphate buffer saline (PBS). Then, the cells were stained with 100 mL of a mixture of acridine orange and ethidium bromide (1:1, 100 mg/mL) solutions. Stained cell suspension (10 mL) were placed on a clean microscope slide and covered with a coverslip. The cells were immediately analyzed by Axoscope 2 plus fluorescence microscope (Zeiss, Germany). 6.2.5. Annexin V-PE and 7-AAD double staining test and flow cytometric analysis Annexin V-PE/7-AAD double staining test was performed using an Annexin-V-PE kit (BD Pharmingen product) as described in protocol. The MCF-7 cells were treated with IC50 concentrations of the compounds 4b, 5f and etoposide. After incubation, the cells were collected and washed twice with cold PBS and resuspended in the binding buffer (100 mL of calcium buffer containing 10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). Then, the cells were double stained with 5 mL of Annexin V-PE and 5 mL of 7-AAD solution. Finally, the samples were incubated for 15 min at room temperature and then analyzed by flow cytometry [18]. Acknowledgments This work was supported by a grant from the Research Council of Mazandaran University of Medical Sciences, Sari, Iran. A part of this work was related to the Pharm. D thesis of Mandana Banimustafa (Faculty of Pharmacy, Mazandaran University of Medical Sciences).

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