2H-chromene derivatives bearing thiazolidine-2,4-dione, rhodanine or hydantoin moieties as potential anticancer agents

European Journal of Medicinal Chemistry 59 (2013) 15e22

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

Original article

2H-chromene derivatives bearing thiazolidine-2,4-dione, rhodanine or hydantoin moieties as potential anticancer agents Mohammad Azizmohammadi a, Mehdi Khoobi b, Ali Ramazani a, Saeed Emami c, Abdolhossein Zarrin d, Omidreza Firuzi d, Ramin Miri d, Abbas Shafiee b, * a

Chemistry Department, Zanjan University, P.O. Box 45195-313, Zanjan, Iran Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 14176, Iran Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran d Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, 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 26 June 2012 Received in revised form 19 September 2012 Accepted 25 October 2012 Available online 2 November 2012

A variety of (Z)-[(2H-chromen-3-yl)methylene]azolidinones 6aet bearing thiazolidine-2,4-dione, rhodanine or hydantoin scaffolds were designed and synthesized as potential anticancer agents. Inhibitory effect of synthesized compounds 6aet on the viability of cancer and non-cancer cells was assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay. The SAR study revealed that the N-substitution of azolidinone moiety cannot improve the activity but S/NH replacement (thiazolidine-2,4-dione/hydantoin) and S/O alteration (rhodanine/thiazolidine-2,4-dione) enable us to modulate the growth inhibition activity against various cell lines. Moreover, 6-bromo and 2-methyl substituents on chromene ring had positive effects on growth inhibitory activity depending on the tumor cell lines. Among the synthesized compounds, hydantoin derivative 6o with a 6-bromo-2-methyl-2Hchromene substructure showed the best profile of cytotoxicity comparable to that of cisplatin as standard anticancer agent. Ó 2012 Elsevier Masson SAS. All rights reserved.

Keywords: Anti-cancer agents Cytotoxicity 2H-chromene Thiazolidine-2,4-dione Rhodanine Hydantoin

1. Introduction It is known that cancer represents one of the most serious health problems in the world. Thus, in the past several decades researchers have been challenged by the task of finding effective clinical approaches for the treatment of cancer. Apart from the use of surgery and radiotherapy, chemotherapy still remains an important option for the treatment of cancer [1]. Cancer cells differ from their normal counterparts in a number of biochemical processes, particularly during the control of cell growth and division. While major advances have been made in the prevention and treatment of cancer, but chemotherapeutic agents generally act on metabolically active or rapidly proliferating cells, and cannot distinguish between cancer and normal cells [2]. While many chemotherapeutic agents such as 5-fluorouracil, cisplatin, paclitaxel and docetaxel are currently used for treatment of cancer, these diseases still remain tenacious and deadly [3]. Thus, the search for novel anticancer agents and new approaches to cancer

* Corresponding author. Tel.: þ98 21 66406757; fax: þ98 21 66461178. E-mail address: ashafi[email protected] (A. Shafiee). 0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2012.10.044

treatment is a highly active research field, stimulated by the discovery of new biological targets and by the possibility of achieving newer anticancer agents with high efficacy and selectivity, low toxicity, and minimum side effects [4,5]. In the search for potential chemotherapeutic agents, considerable effort has been focused on the development of anticancer agents that contain heterocyclic structures as their main structural motif. The thiazolidine ring has been used as scaffold to develop novel class of anticancer agents with a broad spectrum of cytotoxicity against many human cancer cells [6e11]. Among the thiazolidine derivatives, numerous compounds containing thiazolidine2,4-dione and rhodanine have been recognized as new potential anticancer agents. For example, GSK1059615 (1) is a potent, reversible, ATP-competitive, thiazolidinedione inhibitor of PI3Ka [12]. Recently, Liu et al. identified a thiazolidine-2,4-dione analog 2 with potential anticancer activity mainly through the inhibition of the Raf/MEK/ERK and PI3K/Akt signaling pathways [13]. Also, Yang et al. [14] and Huang et al. [15] have reported that D2CG (3) and D2TG (4), the benzylidene analogs of anti-diabetic drugs troglitazone and ciglitazone act as suppressant of cell proliferation in cancer cells. Moreover, there is a known antiproliferative potential of 5-substituted imidazolidine-2,4-diones (hydantoins) [16] and

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N-Alkylated thiazoline-2,4-dione 11 was prepared form commercially available thiazoline-2,4-dione by alkylating with methyl or ethyl iodide in DMF and in the presence of potassium carbonate as a base. Several bases and solvents were screened for this reaction but the best result obtained in the presence of potassium carbonate or potassium hydroxide in DMF. For preparation of target compounds 6 via Knoevenagel condensation of azolidinones 11 with 2H-chromene-3-carbaldehydes 10aed, several conditions were screened (sodium acetate in acetic acid, sodium acetate in DMF and piperidine in ethanol or methanol in thermal conditions), but the best result was obtained only in refluxing methanol and in the presence of a catalytic amount of piperidine. In the case of Nsubstituted azolidinones, the best result was obtained when the reaction was done in the presence of potassium carbonate as base and DMF as solvent. The structures of target compounds were appropriately characterized by spectral data. For example, compound 6b were fully characterized by IR, 1H and 13C NMR spectra and MS. The mass spectrum of 6b displayed a molecular ion signal at m/z 275. In the 1 H NMR spectrum of compound 6b, in addition to the aromatic protons of chromene ring (d ¼ 6.85e7.27 ppm), a sharp singlet due to vinylic hydrogen was observed at 7.21 ppm. The 1H decoupled 13 C NMR spectrum of 6b showed 13 signals, for example, the CH2 of chromene ring was appeared at 65.6 ppm. In this spectrum, the vinylic methine resonated at 132.0 ppm and the signals for the carbonyl and thiocarbonyl were observed at 169.1 and 194.6, respectively. Meanwhile, in the 1H NMR spectra of target

5-arylidene-2,4-imidazolidinediones [17], which are related to the inhibition of EGFR-kinase epidermal growth factor receptor. Aplysinopsin (5) an indole-derived 2-aminoimidazoline-4-one has been reported as a potent cytotoxic agent against the Kb-cell line [18]. Kondo et al. have reported the anticancer activity of aplysinopsin and methyl-aplysinopsins against L-1210- and Kb-cell lines [19] (Fig. 1). These findings regarding the potential of thiazolidines and imidazolidines with a double bond adjacent to the carbonyl group prompted us to design 2H-chromene-derived arylideneazolidinones 6 as new potential anticancer agents. Thus, we describe herein, the synthesis and cytotoxic activity of (Z)-[(2Hchromen-3-yl)methylene]azolidinones 6 bearing thiazolidine-2,4dione, rhodanine or hydantoin moeities. The 2H-chromene motif is a core structure in many naturally occurring bioactive oxygen heterocycles which found to possess anticancer activity [20]. Therefore, 2H-chromene was considered as aryl part to incorporate in designed structure 6. 2. Chemistry In this work, we describe synthesis of a novel series of chromene derivatives 6aet. As outlined in Scheme 1, the reaction of 2hydroxybenzaldehyde 7aec and acrolein 8a or 3-methylacrolein 8b in the presence of potassium carbonate afforded appropriate chromene-3-carbaldehyde 10aed. This reaction proceeded in 1,4dioxane, at reflux condition via intermediates 9aed [21].

O

N

NH S

O

O

N

S

NH2

O

N 2

1, GSK1059615

O

O S

O 3,

NH O

O

S

O

NH O

HO

2CG

2TG

4,

H3C NH

N N N H

O

5, Aplysinopsin

CH3

O R1 O

R2

Y

3 N R

X

6 X = O, S; Y = S, NH, NMe R1 = H, Br, OMe; R2 = H, Me R3 = H, Me, Et, CH2CO2H

Fig. 1. Structure of some previously described azolidinones 1e5 as anticancer agents and structure of designed compounds (Z)-[(2H-chromen-3-yl)methylene]azolidinones 6 as potential anticancer agents.

M. Azizmohammadi et al. / European Journal of Medicinal Chemistry 59 (2013) 15e22

O

O H

R1

OH

K2CO3

R2

7a-c

O

O H

+

17

H

1,4-dioxane 8a,b

R2

O

R1

9a-d

R1= H, 6-Br, 8-OMe R2= H, Me O

O

Y

H

R1 O 10a-d

R2

N R3

O

11 X

R1

Piperidine, MeOH or K2CO3, DMF

O

R2

Y

3 N R

X

6a-t X = O, S; Y = S, NH, NMe R1 = H, Br, OMe; R2 = H, Me R3 = H, Me, Et, CH2CO2H

Scheme 1. Synthesis of (Z)-[(2H-chromen-3-yl)methylene]azolidinones 6.

compounds only a set of signals was appeared which confirmed preparation of one stereoisomer during the condensation. The aldol products 6 were exclusively obtained in (Z)-configuration as thermodynamically favored structures. The (Z)- or (E)-geometry was readily identified by 1H NMR, as the vinylic proton is more deshielded in the (Z)-isomer than the (E)-isomer. In (Z)-form, as discussed above, vinylic proton appeared at 7.21 ppm due to the magnetic anisotropy effects of carbonyl group on the vinylic proton, while in (E)-form the resonance should be around 6.50 ppm. Since in the resulting product, no signal was detected around 6.50 ppm, the (E)-configuration was consequently ruled out which were in accord with the previously reported data [22,23]. All described 2-methylchromene derivatives 6ler, which possess a chiral center on their chromene ring on C-2 position, are racemates. 3. Pharmacology The effect of synthesized compounds 6aet on cell viability was tested using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) reduction assay [24,25] against A549 (human alveolar basal epithelial adenocarcinoma), K562 (human chronic myelogenous leukemia), MCF-7 (human breast adenocarcinoma), and MOLT-4 (human acute lymphoblastic leukemia) cancer cell lines, as well as NIH/3T3 (mouse embryo fibroblasts) non-cancer cells. The percent inhibition of viability for each concentration of the compounds was calculated with respect to the control and IC50 values were estimated with the software CurveExpert version 1.34 for Windows. Each experiment was repeated 3e5 times and the results are summarized in Table 1 as mean  SD (in mM). Blank wells of all agents, which contained the same concentrations of test compounds, but did not contain MTT, had very low absorbance (data not shown). Therefore, interference of the color of compounds in the assay seems unlikely. 4. Results and discussion The results of the cell viability assay of compounds 6aet against four cancer cell lines (A549, K562, MCF-7 and MOLT-4) and one

non-malignant cell line (NIH/3T3) in comparison with cisplatin were presented in Table 1. Compound 6o was the most potent compound against A549 and K562 cell lines with IC50 values of 17.5 and 10.6 mM. Moreover, compound 6j showed good cytotoxic activity against A549 cells (IC50 < 20 mM). In case of MCF-7 cell line, compound 6c followed by 6o exhibited the highest growth inhibitory activity at concentrations of 8.1 and 15.3 mM, respectively. Also, compound 6m showed significant activity against MCF-7 cells. The results in human acute lymphoblastic leukemia cells (MOLT-4) demonstrated that 6s with an IC50 value of 13.2 mM was the most potent compound. Notably, this compound showed poor or no activity (IC50  89.1 mM) against other cell lines. Also, compounds 6c, 6m and 6o had an inhibitory effect on viability of MOLT-4 cells at concentrations less than 25 mM. As it could be seen in Table 1, the compounds 6eeg, 6k, 6per and 6t have not shown cancer cell growth inhibition within the tested concentrations. It should be mentioned that due to low solubility, compounds 6p, 6r and 6k were tested at the maximum concentrations of 50, 50, and 25 mM, respectively, instead of the usual maximum concentrations of 100 or 200 mM used for most of the compounds. Generally, among all tested compounds, hydantoin derivative 6o showed the best activity with IC50s comparable to those of cisplatin as a reference drug. The structureeactivity relationships (SAR) analyses indicate that the N-substitution with methyl, ethyl and carboxymethyl moieties produced inactive compounds (6eeg, 6per and 6t). Thus, in this series of compounds, the presence of free imidic NH is required for cytotoxic activity. Exceptionally, N-ethyl derivative 6s bearing 8methoxy group on chromene ring showed significant activity against MOLT-4 cells. By comparing the activities of 8-methoxy analog 6s with its unsubstituted counterpart 6f, it revealed that the presence of methoxy group on the C-8 position of chromene ring confers the highest cytotoxic activity of this compound against hematopoietic cell line MOLT-4. The comparison of thiazolidine2,4-dione derivatives 6a, 6h and 6l with their rhodanine counterparts 6b, 6i and 6m, demonstrated that the replacement of O with S increases the activity.

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Table 1 Inhibition of cell viability (IC50, mM)a of (Z)-5-[(2H-chromen-3-yl)methylidene]azolidinones 6aet against different cell lines in comparison with standard drug cisplatin.

O R1 O

R2

Y

3 N R

X

Compound

R1

R2

R3

X

Y

MOLT-4

MCF-7

K562

A549

NIH/3T3

6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 6l 6m 6n 6o 6p 6q 6r 6s 6t Cisplatin

H H H H H H H 6-Br 6-Br 6-Br 6-Br 6-Br 6-Br 6-Br 6-Br 6-Br 6-Br 6-Br 8-OMe 8-OMe

H H H H H H H H H H H Me Me Me Me Me Me Me H H

H H H H Me Et CH2CO2H H H H CH2CO2H H H H H Me Et CH2CO2H Et CH2CO2H

O S O NH O O S O S O S O S NH O O O S O S

S S NH NMe S S S S S NH S S S NMe NH S S S S S

100.7  20.3 52.0  9.6 21.3  7.8 72.0  14.9 > 100 > 200 > 100 54.7  9.3 38.2  12.6 77.5  30.8 > 25 64.5  10.2 20.5  3.4 56.6  10.2 24.8  5.8 > 50 > 100 > 50 13.2  3.6 > 200 4.1  0.90

99.4  20.9 68.7  18.8 8.1  2.4 148.9  22.4 > 100 > 200 > 100 57.9  8.5 40.4  13.6 51.0  18.7 > 25 67.6  4.8 19.9  7.2 84.8  26.7 15.3  4.3 > 50 > 100 > 50 89.1  26.0 > 200 11.9  3.8

98.9  14.7 55.3  21.2 55.7  10.7 96.5  18.3 > 100 > 200 > 100 49.8  15.1 33.7  8.9 79.3  22.5 > 25 77.0  29.8 31.6  12.6 51.8  8.6 10.6  2.6 > 50 > 100 > 50 90.4  34.9 > 200 13.0  1.7

192.2  17.6 73.1  18.8 29.7  10.2 89.4  4.3 > 100 > 200 >100 79.5  9.9 51.0  7.9 19.3  5.5 > 25 65.5  19.1 47.5  12.3 120.9  13.0 17.5  3.4 > 50 > 100 > 50 > 200 > 200 14.1  0.80

> 200 72.4  17.5 180.1  43.5 105.8  9.6 ND b ND ND 56.5  13.0 27.8  6.4 35.9  11.3 ND 56.2  13.7 28.7  8.1 87.7  12.6 25.2  10.0 ND ND ND 75.0  17.2 ND 2.8  1.9

a b

Values represent mean  S.D. of 3e5 experiments. Not determined.

The IC50s of unsubstituted chromene derivatives 6aeg revealed that the hydantoin derivative 6c had a better profile of activity, thus it seems that the hydantoin is more favorable than thiazolidine-2,4dione and rhodanine. By comparing the activity of hydantoins 6j and 6o, it can be suggested that the introduction of 2-methyl group on chromene ring has led to a higher inhibitory activity. A similar result could be found by comparing the IC50 values of compounds 6i and 6m with a rhodanine structure. Aside from the clear effect of 2-methyl substitution, the results were not unequivocal in the case of 6-bromo substitution on the chromene ring. Although 6-bromo substitution led to increased cytotoxic activities of 6h and 6i compared to 6a and 6b, respectively, but the same substitution decreased the potency of 6j compared to 6c against all cell lines with the exception of A549 cells. In order to assess the effect of synthesized compounds on noncancer cells, we selected active compounds against cancer cells and tested them on NIH/3T3 fibroblast cell line (Table 1). Two of the most active compounds (6c and 6o) had higher IC50 values against NIH/3T3 cells compared to the cancer cell lines. Furthermore, compounds 6a, 6d and 6n were generally less potent on normal cells compared to cancer cells. However, the rest of the compounds could also inhibit NIH/3T3 cell growth in a similar fashion to cancer cells. It should be noted that cisplatin, which is a standard reference anti-cancer drug, was very effective on normal cells, a phenomenon that has been observed also in other reports [26,27]. Inhibition of viability was also tested on MOLT-4 cells by using trypan blue dye exclusion assay (Table 2). MOLT-4 cells were chosen for this assay, because synthesized compounds showed overall a better inhibition against these cells in MTT assay. As shown in Table 2, all compounds were able to inhibit cell viability by around 50% at the concentrations equal to their IC50 values obtained in the MTT assay. These findings confirm the results of MTT assay.

The results of our study revealed that chromene-containing hydantoin namely (Z)-5-[(2H-chromen-3-yl)methylene]imidazolidine-2,4-dione is an excellent scaffold for further biomolecular study in the field of anti-cancer chemotherapy. From the molecular mechanism point of view, hydantoins were shown to inhibit MMPs [28] and were patented as tyrosine kinase inhibitors [29]. A series of substituted hydantoins have also been patented as MEK1 and MEK2 inhibitors [30]. Zuliani et al. reported that 5-benzylidene hydantoins inhibite A549 lung cancer cell line proliferation via dual mechanism; inhibiting EGFR autophosphorylation and increasing p53 level [31]. As patented by Shapiro et al., 2-[4-[(5oxo-2-thioxo-4-imidazolidinylidene)-methyl]phenoxy] propionic acid derivatives bind the ERK1 or ERK2 kinase, and inhibit phosphorylation [32]. Based on these data, it seems that our target compounds derived from (Z)-5-[(2H-chromen-3-yl)methylene] imidazolidine-2,4-dione could have diverse mechanisms of action

Table 2 Inhibition of cell viability in MOLT-4 cells assessed by trypan blue exclusion assay. Compound

Concentration (mM)

% Viabilitya

6a 6b 6c 6d 6h 6i 6j 6l 6m 6n 6o 6s Cisplatin

100 50 20 75 50 40 75 65 20 50 25 10 4

53.4  6.1 49.3  4.7 53.3  23.9 49.5  6.0 52.7  4.2 46.7  6.1 60.1  8.4 53.3  5.6 53.1  3.1 54.3  5.1 41.3  8.9 53.2  4.4 26.6  4.8

a

Values represent mean  S.D.

M. Azizmohammadi et al. / European Journal of Medicinal Chemistry 59 (2013) 15e22

especially kinases inhibitory activity. Therefore, the results of our study on compound 6o prototype prompt further investigation of such bimolecular studies to help us understand their properties and mechanism of action.

19

1690 (C]O). 1H NMR (400 MHz, DMSO-d6) d: 9.59 (s, 1H, H-aldehyde), 7.37 (dd, J ¼ 2.5 and 8.0 Hz, 1H, H-7), 7.31 (d, J ¼ 2.5 Hz, 1H, H-5), 7.10 (s, 1H, H-4), 6.76 (d, J ¼ 8.0 Hz, H-8), 5.03 (2H, s, OeCH2). Anal. Calcd for C10H7BrO2 (239.07): C, 50.24; H, 2.95. Found: C, 50.44; H, 2.71.

5. Conclusion In conclusion, we describe the synthesis and cytotoxic activity of (Z)-[(2H-chromen-3-yl)methylene]azolidinones bearing thiazolidine-2,4-dione, rhodanine or hydantoin scaffolds. The effect of substitution on chromene ring and azolidinone part was explored by preparing twenty compounds. Among the synthesized compounds, hydantoin derivative 6o with a 6-bromo-2-methyl2H-chromene substructure showed the best anti-cancer activity comparable to that of cisplatin as a standard antitumoral agent. The SAR study revealed that the N-substitution of azolidinone moiety cannot improve the activity but S/NH replacement (thiazolidine2,4-dione /hydantoin) and S/O alteration (rhodanine / thiazolidine2,4-dione) enable us to modulate the cytotoxic activity against various cancer cell lines. Moreover, 6-bromo and 2-methyl substituents on chromene ring had positive effects on growth inhibitory activity depending on the tumor cell lines. Therefore, it was concluded that (Z)-5-[(2H-chromen-3-yl)methylene]imidazolidine-2,4-dione is an excellent scaffold for further study in the field of cancer chemotherapy. 6. Experimental protocols 6.1. Chemistry All commercially available reagents were used without further purification. Column chromatography was carried out on silica gel (70e230 mesh). TLC was conducted on silica gel 250 micron, F254 plates. Melting points were measured on a Kofler hot stage apparatus and are uncorrected. The IR spectra were taken using Nicolet FT-IR Magna 550 spectrographs (KBr disks). 1H NMR spectra were recorded on a Bruker 400 or 500 MHz NMR instruments. The chemical shifts (d) and coupling constants (J) are expressed in parts per million and hertz, respectively. Mass spectra of the products were obtained with an HP (Agilent technologies) 5937 Mass Selective Detector. Elemental analyses were carried out by a CHNRapid Heraeus elemental analyzer. The results of elemental analyses (C, H, N) were within 0.4% of the calculated values. 6.1.1. General procedure for the synthesis of 2H-chromene-3carbaldehyde 10aed A mixture of appropriate 2-hydroxybenzaldehyde (7 mmol) and potassium carbonate (7 mmol) in 1,4-dioxane (12.5 mL) was treated with acrolein or 3-methylacrolein (0.5 mL). The mixture was heated at 100  C for 8 h and allowed to cool. The mixture was diluted with water and extracted several times with ether. The combined ether extracts were dried (Na2SO4) and evaporated to give compound 10 as a yellow solid which was crystallized from ethyl acetateehexane. 6.1.1.1. 2H-chromene-3-carbaldehyde (10a, R1 ¼ R2 ¼ H). Light yellow solid, yield: 62%, mp.: 34e36  C. IR (KBr, cm1): 1712 (C]O). 1 H NMR (400 MHz, DMSO-d6) d: 9.59 (s, 1H, H-aldehyde), 7.30 (t, J ¼ 7.8 Hz, 1H, H-7), 7.26 (s, 1H, H-4), 7.20 (d, J ¼ 7.8 Hz, 1H, H-5), 6.96 (t, J ¼ 7.8 Hz, 1H, H-6), 6.87 (d, J ¼ 7.8 Hz, 1H, H-8), 5.04 (2H, s, OeCH2). Anal. Calcd for C10H8O2 (160.17): C, 74.99; H, 5.03. Found: C, 74.71; H, 5.32. 6.1.1.2. 6-Bromo-2H-chromene-3-carbaldehyde (10b, R1 ¼ 6-Br, R2 ¼ H). Yellow solid, yield: 56%, mp.: 36e38  C. IR (KBr, cm1):

6.1.1.3. 6-Bromo-2-methyl-2H-chromene-3-carbaldehyde (10c, R1 ¼ 6Br, R2 ¼ CH3). Yellow solid, yield: 52%, mp.: 47e49  C. IR (KBr, cm1): 1697 (C]O). 1H NMR (400 MHz, DMSO-d6) d: 9.59 (s, 1H, H-aldehyde), 7.36 (d, J ¼ 8.0 Hz, 1H, H-7), 7.33 (s, 1H, H-5), 7.11 (s, 1H, H-4), 6.77 (d, J ¼ 8.0 Hz, H-8), 5.42 (q, J ¼ 6.4 Hz, 1H, OeCH), 1.35 (d, J ¼ 6.4 Hz, 3H, CH3). Anal. Calcd for C11H9BrO2 (253.09): C, 52.20; H, 3.58. Found: C, 52.53; H, 3.39. 6.1.1.4. 8-Methoxy-2H-chromene-3-carbaldehyde (10d, R1 ¼ 8-OMe, R2 ¼ H). Light yellow solid, yield: 50%, mp.: 65e67  C. IR (KBr, cm1): 1690 (C]O). 1H NMR (400 MHz, DMSO-d6) d: 9.58 (s, 1H, Haldehyde), 7.27 (s, 1H, H-4), 6.94e6.85 (m, 3H, H-5, H-6 and H-7), 5.10 (2H, s, OeCH2), 3.89 (s, 3H, OeCH3). Anal. Calcd for C11H10O3 (190.20): C, 69.46; H, 5.30. Found: C, 69.61; H, 5.04. 6.1.2. General procedure for N-alkylation of thiazolidine-2,4-dione 11 Thiazolidine-2,4-dione (1 mmol) and potassium carbonate (1 mmol) were dissolved in DMF (5 mL). The solution was stirred at room temperature for several minutes and then methyl or ethyl iodide (1.2 mmol) was added dropwise to the mixture. The solution was heated to 80  C for 6 h. After completion of the reaction (monitored by TLC), the mixture was cooled to room temperature and diluted with water. The precipitate was filtered and washed with water [33]. 6.1.3. General procedure for the synthesis of compounds 6aet Azolidinones 11 (1 mmol) and appropriate 2H-chromene-3carbaldehyde 10 (1 mmol) were dissolved in methanol (5 mL). The solution was refluxed for 2e18 h in the presence of a small amount of piperidine as a catalyst. After completion of the reaction (monitored by TLC), the mixture was cooled, the precipitate was filtered and crystallized from ethanol to give corresponding products. In the case of N-substituted azolidinones the best result was obtained when the reaction was done in the presence of potassium carbonate (1.5 mmol) and DMF (5 mL) as solvent. 6.1.3.1. (Z)-5-((2H-chromen-3-yl)methylene)thiazolidine-2,4-dione (6a). From compound 10a (1 mmol, 0.16 g) and thiazolidine-2,4dione (1 mmol, 0.12 g), for 6 h, product 6a was obtained, yellow solid, yield: 50%, mp.: >260  C. IR (KBr, cm1): 3446 (NH), 1739 (C] O), 1690 (C]O). 1H NMR (500 MHz, DMSO-d6) d: 7.24e7.28 (m, 3H, H-vinylic, H-5 and H-7), 6.99 (s, 1H, H-4), 6.94 (t, J ¼ 7.4 Hz, 1H, H6), 6.82 (d, J ¼ 7.4 Hz, 1H, H-8), 5.02 (s, 2H, OeCH2). 13C NMR (125 MHz, DMSO-d6) d: 170.1, 168.7, 153.3, 132.9, 132.6, 130.9, 129.2, 128.0, 126.6, 125.9, 121.8, 115.4, 65.8. Anal. Calcd for C13H9NO3S (259.28): C, 60.22; H, 3.50; N, 5.40. Found: C, 60.47; H, 3.27; N, 5.19. 6.1.3.2. (Z)-5-((2H-chromen-3-yl)methylene)-2-thioxothiazolidin-4one (6b). From compound 10a (1 mmol, 0.16 g) and 2-thioxothiazolidin-4-one (1 mmol, 0.13 g), for 6 h, product 6b was obtained, yellow solid, yield: 44%, mp.: 258e260  C. IR (KBr, cm1): 3380 (NH), 1714 (C]O). 1H NMR (500 MHz, DMSO-d6) d: 7.27e7.23 (m, 2H, H-5 and H-7), 7.21 (s, 1H, H-vinylic), 7.14 (s, 1H, H-4), 6.96 (t, J ¼ 7.8 Hz, 1H, H-6), 6.85 (d, J ¼ 7.8 Hz, 1H, H-8), 5.05 (s, 2H, OeCH2). 13 C NMR (125 MHz, DMSO-d6) d: 194.6, 169.1, 153.4, 132.0, 131.7, 128.7, 128.5, 128.0, 124.8, 122.0, 121.9, 115.5, 65.6. MS (m/z, %): 275 (Mþ, 72), 187 (100), 149 (43), 115 (30), 71 (34), 57 (65). Anal. Calcd for C13H9NO2S2 (275.35): C, 56.71; H, 3.29; N, 5.09. Found: C, 56.51; H, 3.07; N, 5.33.

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6.1.3.3. (Z)-5-((2H-chromen-3-yl)methylene)imidazolidine-2,4-dione (6c). From compound 10a (1 mmol, 0.16 g) and imidazolidine-2,4dione (1 mmol, 0.10 g), for 12 h, product 6c was obtained, yellow solid, yield: 40%, mp.: 240e242  C. IR (KBr, cm1): 3269 and 3221 (NH), 1780 (C]O), 1768 (C]O). 1H NMR (400 MHz, DMSO-d6) d: 7.09e7.13 (m, 3H, H-vinylic, H-5, H-7), 6.94 (s, 1H, H-4), 6.93 (t, J ¼ 7.5 Hz, 1H, H-6), 6.79 (d, J ¼ 7.5 Hz, 1H, H-8), 5.96 (s, 1H, NH), 4.94 (s, 2H, OeCH2). Anal. Calcd for C13H10N2O3 (242.23): C, 64.46; H, 4.16; N, 11.56. Found: C, 64.18; H, 4.33; N, 11.29. 6.1.3.4. (Z)-5-((2H-chromen-3-yl)methylene)-2-amino-1-methyl-1Himidazol-4(5H)-one (6d). From compound 10a (1 mmol, 0.16 g) and 2-amino-1-methyl-1H-imidazol-4(5H)-one (1 mmol, 0.11 g), for 8 h, product 6d was obtained, yellow solid, yield: 36%, mp.: 253e255  C. IR (KBr, cm1): 3380 and 3278 (NH2), 1724 (C]O). 1H NMR (500 MHz, DMSO-d6) d: 7.13e7.09 (m, 3H, H-vinylic, H-5 and H-7), 6.96 (s, 1H, H-4), 6.89 (t, J ¼ 7.8 Hz, 1H, H-6), 6.80 (d, J ¼ 7.8 Hz, 1H, H-8), 5.91 (s, 2H, NH2), 5.19 (s, 2H, OeCH2), 3.13 (s, 3H, NeCH3). 13C NMR (125 MHz, DMSO-d6) d: 174.2, 166.2, 153.5, 135.1 129.5, 129.3, 126.9, 126.5, 125.4, 121.5, 115.2, 110.0, 65.2, 28.6. Anal. Calcd for C14H13N3O2 (255.27): C, 65.87; H, 5.13; N, 16.46. Found: C, 65.62; H, 5.33; N, 16.19. 6.1.3.5. (Z)-5-((2H-chromen-3-yl)methylene)-3-methylthiazolidine2,4-dione (6e). From compound 10a (1 mmol, 0.16 g) and 3methylthiazolidine-2,4-dione (1 mmol, 0.13 g), for 3 h, product 6e was obtained, yellow solid, yield: 74%, mp.: 207e209  C. IR (KBr, cm1): 1731 (C]O), 1686 (C]O). 1H NMR (400 MHz, DMSO-d6) d: 7.44 (s, 1H, H-vinylic), 7.24 (t, J ¼ 7.2 Hz, 1H, H-7), 7.10 (d, J ¼ 7.2 Hz, 1H, H-5), 6.94 (t, J ¼ 7.2 Hz, 1H, H-6), 6.85 (s, 1H, H-4), 6.84 (d, J ¼ 7.2 Hz, 1H, H-8), 5.07 (s, 2H, OeCH2), 3.24 (s, 3H, NeCH3). MS (m/ z, %): 273 (Mþ, 8), 187 (30), 167 (18), 149 (84), 115 (17), 107 (86), 91 (72), 57 (100). Anal. Calcd for C14H11NO3S (273.30): C, 61.52; H, 4.06; N, 5.12. Found: C, 61.71; H, 4.32; N, 5.39. 6.1.3.6. (Z)-5-((2H-chromen-3-yl)methylene)-3-ethylthiazolidine-2,4dione (6f). From compound 10a (1 mmol, 0.16 g) and 3ethylthiazolidine-2,4-dione (1 mmol, 0.14 g), for 1 h, product 6f was obtained, yellow solid, yield: 55%, mp.: 148e150  C. IR (KBr, cm1): 1729 (C]O), 1682 (C]O). 1H NMR (500 MHz, CDCl3) d: 7.45 (s, 1H, H-vinylic), 7.22 (t, J ¼ 7.5 Hz, 1H, H-7), 7.11 (d, J ¼ 7.5 Hz, 1H, H5), 6.94 (t, J ¼ 7.5 Hz, 1H, H-6), 6.83 (s, 1H, H-4), 6.82 (d, J ¼ 7.5 Hz, 1H, H-8), 5.06 (s, 2H, OeCH2), 3.80 (q, J ¼ 7.1 Hz, 2H, OeCH2CH3), 1.27 (t, J ¼ 7.1 Hz, 3H, OeCH2CH3). 13C NMR (125 MHz, CDCl3) d: 166.9, 165.8, 154.0, 133.0, 131.6, 130.4, 128.2, 127.6, 122.1, 121.9, 120.8, 116.0, 66.1, 37.2, 13.0. Anal. Calcd for C15H13NO3S (287.33): C, 62.70; H, 4.56; N, 4.87. Found: C, 62.51; H, 4.81; N, 4.60. 6.1.3.7. (Z)-2-(5-((2H-chromen-3-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (6g). From compound 10a (1 mmol, 0.16 g) and 2-(4-oxo-2-thioxothiazolidin-3-yl)acetic acid (1 mmol, 0.19 g), for 1 h, product 6g was obtained, yellow solid, yield: 50%, mp.: 233e235  C. IR (KBr, cm1): 3433 (OH), 1704 (C]O), 1642 (C]O). 1 H NMR (400 MHz, CDCl3) d: 9.23 (br s, 1H, CO2H), 7.36 (s, 1H, Hvinylic), 7.29e7.25 (m, 2H, H-5 and H-7), 7.20 (s, 1H, H-4), 6.97 (t, J ¼ 7.8 Hz, 1H, H-6), 6.85 (d, J ¼ 7.8 Hz, 1H, H-8), 5.11 (s, 2H, OeCH2), 4.36 (s, 2H, NeCH2). MS (m/z, %): 333 (Mþ, 3), 318 (11), 273 (3), 167 (71), 151 (49), 136 (6), 122 (6), 103 (100). Anal. Calcd for C15H11NO4S2 (333.38): C, 54.04; H, 3.33; N, 4.20. Found: C, 54.37; H, 3.58; N, 4.01. 6.1.3.8. (Z)-5-((6-bromo-2H-chromen-3-yl)methylene)thiazolidine2,4-dione (6h). From compound 10b (1 mmol, 0.24 g) and thiazolidine-2,4-dione (1 mmol, 0.12 g), for 8 h, product 6h was obtained, yellow solid, yield: 53%, mp.: 248e250  C. IR (KBr, cm1): 3443 (NH), 1728 (C]O), 1689 (C]O). 1H NMR (500 MHz, CDCl3) d: 7.48 (s,

1H, H-5), 7.32 (d, J ¼ 7.9 Hz, 1H, H-7) 7.28 (s, 1H, H-vinylic), 7.00 (s, 1H, H-4), 6.02 (d, J ¼ 7.9 Hz, 1H, H-8), 5.04 (2H, s, OeCH2). 13C NMR (125 MHz, CDCl3) d: 167.3, 167.0, 152.5, 133.3, 130.2 129.5, 129.0, 128.0, 124.2, 124.1, 117.7, 113.1, 65.9. MS (m/z, %): 339 ([M þ 2]þ, 54), 337 (Mþ, 54), 267 (100), 224 (30), 211 (22), 209 (20), 187 (22), 158 (21), 115 (66), 102 (17). Anal. Calcd for C13H8BrNO3S (338.18): C, 46.17; H, 2.38; N, 4.14. Found: C, 46.42; H, 2.14; N, 4.39. 6.1.3.9. (Z)-5-((6-bromo-2H-chromen-3-yl)methylene)-2-thioxothiazolidin-4-one (6i). From compound 10b (1 mmol, 0.24 g) and 2thioxothiazolidin-4-one (1 mmol, 0.13 g), for 10 h, product 6i was obtained, yellow solid, yield: 44%, mp.: 250e252  C. IR (KBr, cm1): 3447 (NH), 1690 (C]O). 1H NMR (400 MHz, DMSO-d6) d: 7.49 (s, 1H, H-5), 7.37 (d, J ¼ 7.2 Hz, 1H, H-7), 7.12 (s, 1H, H-vinylic), 7.04 (s, 1H, H-4), 6.80 (d, J ¼ 7.2 Hz, 1H, H-8), 5.06 (2H, s, OeCH2). Anal. Calcd for C13H8BrNO2S2 (354.24): C, 44.08; H, 2.28; N, 3.95. Found: C, 44.27; H, 2.02; N, 3.74. 6.1.3.10. (Z)-5-((6-bromo-2H-chromen-3-yl)methylene)imidazolidine-2,4-dione (6j). From compound 10b (1 mmol, 0.24 g) and imidazolidine-2,4-dione (1 mmol, 0.10 g), for 12 h, product 6j was obtained, yellow solid, yield: 74%, mp.: >260  C. IR (KBr, cm1): 3167 and 3426 (NH), 1769 (C]O), 1709 (C]O). 1H NMR (400 MHz, DMSO-d6) d: 7.31e7.26 (m, 3H, H-vinylic, H-5 and H-7), 6.91 (s, 1H, H-4), 6.76 (d, J ¼ 8.0 Hz, 1H, H-8), 5.93 (s, 1H, NH), 4.97 (s, 2H, Oe CH2). Anal. Calcd for C13H9BrN2O3 (321.13): C, 48.62; H, 2.82; N, 8.72. Found: C, 48.44; H, 2.53; N, 8.99. 6.1.3.11. (Z)-2-(5-((6-bromo-2H-chromen-3-yl)methylene)-4-oxo-2thioxothiazolidin-3-yl)acetic acid (6k). From compound 10b (1 mmol, 0.24 g) and 2-(4-oxo-2-thioxothiazolidin-3-yl)acetic acid (1 mmol, 0.19 g), for 1 h, product 6k was obtained, yellow solid, yield: 57%, mp.: 240e242  C. IR (KBr, cm1): 3444 (OH), 1727 (C]O), 1674 (C]O). 1H NMR (400 MHz, DMSO-d6) d: 9.31 (br s, 1H, CO2OH), 7.52 (s, 1H, H-5), 7.39 (d, J ¼ 7.9 Hz, 1H, H-7), 7.3 (s, 1H, H-vinylic), 7.12 (s, 1H, H-4), 6.82 (d, J ¼ 7.9 Hz, 1H, H-8), 5.12 (s, 2H, OeCH2), 4.33 (s, 2H, NeCH2). MS (m/z, %): 413 ([M þ 2]þ, 31), 411 (Mþ, 30), 267 (82), 222 (18), 187 (45), 115 (68), 72 (100). Anal. Calcd for C15H10BrNO4S2 (412.28): C, 43.70; H, 2.44; N, 3.40. Found: C, 43.49; H, 2.23; N, 3.19. 6.1.3.12. (Z)-5-((6-bromo-2-methyl-2H-chromen-3-yl)methylene) thiazolidine-2,4-dione (6l). From compound 10c (1 mmol, 0.25 g) and thiazolidine-2,4-dione (1 mmol, 0.12 g), for 12 h, product 6l was obtained, yellow solid, yield: 41%, mp.: 221e223  C. IR (KBr, cm1): 3439 (NH), 1741 and 1689 (C]O). 1H NMR (500 MHz, CDCl3) d: 7.29 (s, 1H, H-5), 7.23 (d, J ¼ 8.5 Hz, 1H, H-7), 7.22 (s, 1H, H-vinylic), 6.74 (d, J ¼ 8.5 Hz, 1H, H-8), 6.68 (s, 1H, H-4), 5.26 (q, J ¼ 6.4 Hz, 1H, Oe CH), 1.41 (d, J ¼ 6.4 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3) d: 173.5, 172.1, 151.1, 133.5, 130.0, 128.5, 127.6, 126.7, 123.4, 118.5, 113.6, 72.4, 44.4. Anal. Calcd for C14H10BrNO3S (352.20): C, 47.74; H, 2.86; N, 3.98. Found: C, 47.59; H, 2.61; N, 4.23. 6.1.3.13. (Z)-5-((6-bromo-2-methyl-2H-chromen-3-yl)methylene)-2thioxothiazolidin-4-one (6m). From compound 10c (1 mmol, 0.25 g) and 2-thioxothiazolidin-4-one (1 mmol, 0.13 g), for 12 h, product 6m was obtained, yellow solid, yield: 43%, mp.: >260  C. IR (KBr, cm1): 3415 (NH), 1711 (C]O). 1H NMR (400 MHz, CDCl3) d: 7.33 (d, J ¼ 8.3 Hz, 1H, H-7), 7.25 (s, 1H, H-5), 7.17 (s, 1H, H-vinylic), 6.74 (d, J ¼ 8.3 Hz, 1H, H-8), 6.67 (s, 1H, H-4), 5.24 (q, J ¼ 6.4 Hz, 1H, OeCH), 1.76 (d, J ¼ 6.4 Hz, 3H, CH3). Anal. Calcd for C14H10BrNO2S2 (368.27): C, 45.66; H, 2.74; N, 3.80. Found: C, 45.91; H, 2.52; N, 3.59. 6.1.3.14. (Z)-2-amino-5-((6-bromo-2H-chromen-3-yl)methylene)-1methyl-1H-imidazol-4(5H)-one (6n). From compound 10c (1 mmol,

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0.25 g) and 2-amino-1-methyl-1H-imidazol-4(5H)-one (1 mmol, 0.11 g), for 17 h, product 6n was obtained, yellow solid, yield: 78%, mp.: 185e187  C. IR (KBr, cm1): 3362 and 3255 (NH2), 1666 (C]O). 1H NMR (500 MHz, DMSO-d6) d: 7.47 (s, 1H, H-5), 7.36 (d, J ¼ 8.3 Hz, 1H, H-7), 7.27 (s, 1H, H-vinylic), 7.00 (s, 1H, H-4), 6.79 (d, J ¼ 8.3 Hz, 1H, H-8), 5.04 (q, J ¼ 6.4 Hz, 1H, OeCH), 3.35 (s, 3H, NeCH3), 2.09 (d, J ¼ 6.4 Hz, 3H, CH3). 13C NMR (125 MHz, DMSO-d6) d: 167.3, 167.0, 152.5, 133.3, 130.2, 129.5, 129.0, 128.0, 124.3, 124.1, 117.7, 113.1, 65.9, 37.3, 21.0. MS (m/z, %): 349 ([M þ 2]þ, 3), 347 (Mþ, 3), 167 (9), 103 (16), 95 (7), 84 (52), 77 (12), 69 (27), 57 (73), 43 (100). Anal. Calcd for C15H14BrN3O2 (348.19): C, 51.74; H, 4.05; N, 12.07. Found: C, 51.92; H, 4.33; N, 12.29. 6.1.3.15. (Z)-5-((6-bromo-2-methyl-2H-chromen-3-yl)methylene)imidazolidine-2,4-dione (6o). From compound 10c (1 mmol, 0.25 g) and imidazolidine-2,4-dione (1 mmol, 0.10 g), for 17 h, product 6o was obtained, yellow solid, yield: 42%, mp.: >260  C. IR (KBr, cm1): 3250 and 3220 (NH), 1695 (C]O). 1H NMR (500 MHz, DMSO-d6) d: 7.29 (s, 1H, H-vinylic), 7.28 (d, J ¼ 8.3 Hz, 1H, H-7), 6.90 (s, 1H, H-4), 6.78 (d, J ¼ 8.3 Hz, 1H, H-8), 5.86 (s, 1H, NH), 5.30 (q, J ¼ 6.1 Hz, 1H, OeCH), 1.26 (d, J ¼ 6.1 Hz, 3H, CH3). 13C NMR (125 MHz, DMSO-d6) d: 164.9, 155.2, 150.0, 132.9, 131.9, 129.0, 128.9, 124.2, 123.1, 118.2, 112.5, 104.1, 73.3, 44.3. Anal. Calcd for C14H11BrN2O3 (335.15): C, 50.17; H, 3.31; N, 8.36. Found: C, 50.36; H, 3.09; N, 8.58. 6.1.3.16. (Z)-5-((6-bromo-2-methyl-2H-chromen-3-yl)methylene)-3methylthiazolidine-2,4-dione (6p). From compound 10c (1 mmol, 0.25 g) and 3-methylthiazolidine-2,4-dione (1 mmol, 0.13 g), for 3 h, product 6p was obtained, yellow solid, yield: 51%, mp.: 213e 215  C. IR (KBr, cm1): 1737 (C]O), 1681 (C]O). 1H NMR (500 MHz, CDCl3) d: 7.40 (s, 1H, H-5), 7.31 (d, J ¼ 8.5 Hz, 1H, H-7), 7.25 (s, 1H, H-vinylic), 6.75 (d, J ¼ 8.5 Hz, 1H, H-8), 6.72 (s, 1H, H-4), 5.25 (q, J ¼ 6.4 Hz, 1H, OeCH), 3.24 (s, 3H, NeCH3), 1.42 (d, J ¼ 6.4 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3) d: 166.9, 166.0, 151.2, 135.6, 134.0, 132.9, 131.2, 130.2, 121.2, 119.1, 118.6, 113.7, 72.2, 28.1, 20.1. Anal. Calcd for C15H12BrNO3S (366.23): C, 49.19; H, 3.30; N, 3.82. Found: C, 49.33; H, 3.07; N, 3.60. 6.1.3.17. (Z)-5-((6-bromo-2-methyl-2H-chromen-3-yl)methylene)-3ethylthiazolidine-2,4-dione (6q). From compound 10c (1 mmol, 0.25 g) and 3-ethylthiazolidine-2,4-dione (1 mmol, 0.14 g), for 1 h, product 6q was obtained, yellow solid, yield: 50%, mp.: 153e155  C. IR (KBr, cm1): 1737 (C]O), 1680 (C]O). 1H NMR (500 MHz, CDCl3) d: 7.37 (s, 1H, H-5), 7.30 (d, J ¼ 8.5 Hz, 1H, H-7), 7.25 (s, 1H, Hvinylic), 6.75 (d, J ¼ 8.5 Hz, 1H, H-8), 6.72 (s, 1H, H-4), 5.24 (q, J ¼ 6.5 Hz. 1H, OeCH), 3.82 (q, J ¼ 7.0 Hz, 2H, NeCH2CH3), 1.42 (d, J ¼ 6.5 Hz, 3H, CH3), 1.27 (t, J ¼ 7.0 Hz, 3H, NeCH2CH3). 13C NMR (125 MHz, CDCl3) d: 166.7, 165.8, 151.2, 134.0, 133.0, 130.2, 129.8, 123.1, 121.4, 118.6, 113.7, 72.3, 37.3, 20.1, 13.0. Anal. Calcd for C16H14BrNO3S (380.26): C, 50.54; H, 3.71; N, 3.68. Found: C, 50.81; H, 3.41; N, 3.32. 6.1.3.18. (Z)-2-(5-((6-bromo-2-methyl-2H-chromen-3-yl)methylene)4-oxo-2-thioxothiazolidin-3-yl)acetic acid (6r). From compound 10c (1 mmol, 0.25 g) and 2-(4-oxo-2-thioxothiazolidin-3-yl)acetic acid (1 mmol, 0.19 g), for 12 h, product 6r was obtained, yellow solid, yield: 50%, mp.: 215e217  C. IR (KBr, cm1): 3446 (OH), 1739 (C]O), 1690 (C]O). 1H NMR (400 MHz, CDCl3) d: 9.11 (bs, 1H, CO2H), 7.27e 7.35 (m, 3H, H-vinylic, H-5 and H-7), 6.76e6.78 (m, 2H, H-4 and H8), 5.27 (q, J ¼ 6.2 Hz, 1H, OeCH), 4.88 (s, 2H, NeCH2), 1.43 (d, J ¼ 6.2 Hz, 3H, CH3). Anal. Calcd for C16H12BrNO4S2 (426.30): C, 45.08; H, 2.84; N, 3.29. Found: C, 45.31; H, 2.66; N, 3.01. 6.1.3.19. (Z)-3-ethyl-5-((8-methoxy-2H-chromen-3-yl)methylene) thiazolidine-2,4-dione (6s). From compound 10d (1 mmol, 0.19 g)

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and 3-ethylthiazolidine-2,4-dione (1 mmol, 0.14 g), for 1 h, product 6s was obtained, yellow solid, yield: 48%, mp.: 104e106  C. IR (KBr, cm1): 1742 (C]O), 1683 (C]O). 1H NMR (500 MHz, CDCl3) d: 7.45 (s, 1H, H-vinylic), 6.92e6.89 (m, 2H, H-5 and H-6), 6.84 (s, 1H, H-4), 6.75 (d, J ¼ 6.6 Hz, 1H, H-7), 5.13 (s, 2H, OeCH2), 3.90 (s, 3H, OeCH3), 3.81 (q, J ¼ 7.0 Hz, 2H, NeCH2CH3), 1.26 (t, J ¼ 7.0 Hz, 3H, NeCH2CH3). 13C NMR (125 MHz, CDCl3) d: 166.8, 165.8, 147.8, 142.8, 132.8, 130.2, 127.6, 122.6, 121.8, 121.1, 120.2, 114.1, 66.4, 56.0, 37.2, 13.0. Anal. Calcd for C16H15NO4S (317.16): C, 60.55; H, 4.76; N, 4.41. Found: C, 60.82; H, 4.51; N, 4.26. 6.1.3.20. (Z)-2-(5-((8-methoxy-2H-chromen-3-yl)methylene)-4-oxo2-thioxothiazolidin-3-yl)acetic acid (6t). From compound 10d (1 mmol, 0.19 g) and 2-(4-oxo-2-thioxothiazolidin-3-yl)acetic acid (1 mmol, 0.19 g), for 12 h, product 6t was obtained, Yellow solid, yield: 50%, mp.: 186e188  C. IR (KBr, cm1): 3386 (OH), 1724 (C]O), 1695 (C]O). 1H NMR (400 MHz, DMSO-d6) d: 9.23 (br s, 1H, CO2H), 8.26 (s, 1H, H-vinylic), 6.86e6.95 (m, 3H, H-4, H-5 and H-6), 6.55 (d, J ¼ 6.7 Hz, 1H, H-7), 5.07 (s, 2H, OeCH2), 4.63 (s, 2H, NeCH2), 3.85 (s, 3H, OeCH3). Anal. Calcd for C16H13NO5S2 (363.41): C, 52.88; H, 3.61; N, 3.85. Found: C, 52.61; H, 3.33; N, 3.69. 6.2. Biological assays 6.2.1. Reagents and chemicals 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma and penicillin/streptomycin was purchased from Invitrogen. Fetal bovine serum (FBS), phosphate buffered saline (PBS), RPMI 1640, trypan blue and trypsin were purchased from Biosera. Cisplatin and dimethyl sulfoxide were obtained from EBEWE Pharma and Merck, respectively. 6.2.2. Cell lines A549 (human alveolar basal epithelial adenocarcinoma), K562 (human chronic myelogenous leukemia), MCF-7 (human breast adenocarcinoma), MOLT-4 (human acute lymphoblastic leukemia) and NIH/3T3 (mouse embryo fibroblast) cells were obtained from the National Cell Bank of Iran, Pasteur Institute, Tehran, Iran. Cells were maintained at 37  C in humidified air containing 5% CO2. All cell lines were maintained in RPMI 1640 supplemented with 10% FBS, and 100 units/ml penicillin-G and 100 mg/ml streptomycin. A549, MCF-7 and NIH/3T3 cells were grown in monolayer cultures, while K562 and MOLT-4 cells were grown in suspension. 6.2.3. Cell viability assay Cell viability following exposure to synthetic compounds was estimated by using the MTT reduction assay [24,25]. A549, K562, MCF-7, MOLT-4 and NIH/3T3 cells were plated in 96-well microplates at densities of 40,000, 40,000, 30,000, 40,000 and 50,000 cells/ml, respectively (100 ml per well). After overnight incubation at 37  C, 3e4 different concentrations of test compounds were added to the wells. Compounds were all first dissolved in DMSO and then diluted in the growth medium. The concentration of DMSO in the wells did not exceed 0.5%. Cells were further incubated for 72 h and at the end of the incubation time the medium was replaced with fresh medium containing 0.5 mg/ml of MTT. Plates were incubated for another 4 h at 37  C, the media was removed and formazan crystals formed in the cells were dissolved in 200 ml of DMSO. Optical density was measured at 570 nm with background correction at 655 nm using a Bio-Rad microplate reader (Model 680). Blank wells of all agents, which contained the same concentrations of test compounds but did not contain MTT, were run in parallel.

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