Anticancer and radiosensitizing evaluation of some ...

European Journal of Medicinal Chemistry 53 (2012) 403e407

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

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Anticancer and radiosensitizing evaluation of some new pyranothiazole-Schiff bases bearing the biologically active sulfonamide moiety Mostafa M. Ghorab a, Mohamed A. Shaaban b, Hanan M. Refaat b, *, Helmy I. Heiba c, Sara S. Ibrahim c a

Medicinal, Aromatic and Poisonous Plants Research Center (MAPPRC), College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia Department of Organic Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini St., Cairo 11562, Egypt c Department of Drug Radiation Research, National Center for Radiation Research and Technology, Nasr City, Cairo, Egypt b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 January 2012 Received in revised form 3 April 2012 Accepted 7 April 2012 Available online 26 April 2012

The present work reports the synthesis of some new Schiff bases, 5-(substituted benzylideneamino)6-cyano-7H-7-(4-methoxyphenyl)-2-(4-sulphamoylphenylamino) pyrano[2,3-d]thiazole (5e15). The design of the structures of these compounds complies with the general pharmacophoric requirements for CA inhibiting anticancer drugs. The newly synthesized compounds were evaluated for their in vitro anticancer activity against human breast cancer cell line (MCF7). Most of the screened compounds showed interesting cytotoxic activities compared to doxorubicin as a reference drug. Compounds 4, 6e8 and 11 (IC50: 27.51, 10.25, 9.55, 9.39 and 9.70 mM, respectively) exhibited higher cytotoxic activities than the reference drug doxorubicin (IC50: 32.00 mM). Additionally, the previously mentioned compounds were evaluated again for their ability to enhance the cell killing effect of g-radiation. Ó 2012 Elsevier Masson SAS. All rights reserved.

Keywords: Pyranothiazole Sulfonamides Carbonic anhydrase inhibitors Cytotoxicity Radiosensitizing activities

1. Introduction Sulfonamides are currently an important group of organic compounds that possess several types of biological activities [1e4] including anticancer activity [5e10]. The anticancer activity of sulfonamides was found to take place through a variety of mechanisms such as cell cycle perturbation in the G1phase, disruption of microtubule assembly, angiogenesis inhibition, and functional suppression of the transcriptional activator NF-Y, and carbonic anhydrase inhibition [11e14] which was reported to be the most prominent mechanism [15]. Carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to give bicarbonate and a proton. CAs are involved in pH regulation, secretion of electrolytes, respiration [16,17], biosynthetic reactions which require CO2/bicarbonate as substrate such as gluconeogenesis, lipogenesis, ureagenesis, and pyrimidines synthesis [18]. The catalytic domain of CAs contains an active site Zn2þ; a strong Lewis acid that binds to and activates a substrate H2O molecule to catalyze the reversible hydration reaction of carbon dioxide. The hydration of CO2 does not proceed at an appreciable rate under physiological conditions in the absence of CA enzymes [19]. CA inhibition has been found to have an important role in cancer

* Corresponding author. Tel.: þ20 02 3639307; fax: þ20 02 3635140. E-mail address: [email protected] (H.M. Refaat). 0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2012.04.009

treatment through reducing the provision of bicarbonate for the synthesis of nucleotides and other cell components such as membrane lipids [20]. E7070 II (Fig. 1) is an example of carbonic anhydrase inhibitors. Furthermore, among the compounds that had shown significant anticancer activity were the thiazole derivatives [21e26]. Also, combination of several pyranothiazole with sulfonamide moiety was reported to have significant anticancer activity [27e29]. In addition, Schiff bases are important class of compounds in medicinal and pharmaceutical field that show vast biological applications including antitumor activity [30,31]. In the light of these facts, and in a hope to obtain some novel compounds with significant anticancer activity, this work reports the synthesis of a novel series of Schiff base derivatives containing pyranothiazole bearing a sulfonamide moiety and the testing of these compounds for their in vitro anticancer activity. We also aimed to evaluate the most potent compounds for their in vitro anticancer activity in combination with g-radiation, to evaluate their ability to enhance the cytotoxic activity of g-radiation. 2. Result and discussion 2.1. Chemistry The newly synthesized compounds were obtained starting with 4-(4-oxo-4,5-dihydrothiazol-2-ylamino)benzenesulfonamide

404

M.M. Ghorab et al. / European Journal of Medicinal Chemistry 53 (2012) 403e407

Fig. 1. Compound E7070, a sulfonamide compound in advanced clinical trials as anticancer agent.

3, which was prepared according to the reported procedure [32], by refluxing the N-chloroacetyl derivative 2 with ammonium thiocyanate in ethanol, through intramolecular cyclization and elimination of 1 mol of ammonium chloride. Treatment of compound 3 with 2-(4-chlorobenzylidene) malononitrile in ethanol containing a catalytic amount of piperidine, as a base catalyst, resulted in intramolecular cyclization affording the 4-(5-amino-6-cyano-7(2-methoxyphenyl)-7H-pyrano[2,3-d]thiazol-2-ylamino) benzenesulfonamide 4. The structure of compound 4 was confirmed by its microanalytical and spectral data; where the IR spectrum showed bands at 2187 attributed to the presence of (C^N) group and the 1H NMR spectrum showed a peak at d 4.70 ppm attributed to the presence of C-4 pyran H (Scheme 1). Treatment of compound 4 with aromatic aldehydes in acetic acid yielded the corresponding Schiff bases 5e15. The structures of synthesized compounds 5e15 were supported from their microanalytical and spectral data. The 1H NMR spectra of compounds 5e15 displayed the disappearance of NH2 absorption of the precursor 4 and the presence of singlets at the range d 8.23e8.79 ppm for (N] CHe), in addition to the presence of the expected methoxyphenyl and sulphamoylphenyl protons signals together with other signals assigned to N-substituted benzylidene groups (Scheme 2). 2.2. In vitro anticancer screening The newly synthesized compounds were evaluated for their in vitro cytotoxic activity against human breast cancer cell line, MCF7. Doxorubicin, which is one of the most effective anticancer agents, was used as the reference drug in this study. The relationship between surviving fraction and drug concentration was plotted to obtain the survival curve of breast cancer cell line (MCF7). The response parameter calculated was the IC50 value, which corresponds to the concentration required for 50% inhibition of cell viability. Table 1 shows the in vitro cytotoxic activity of the synthesized compounds. Most of the synthesized compounds exhibited significant activity compared to the reference drug. From the analysis of Table 1, it was found that all compounds showed significant antitumor activities. Interestingly, compounds 6e8 and 11 (IC50: 10.25, 9.55, 9.39 and 9.70 mM, respectively) exhibited 3.1e3.4 fold more potent antitumor activity than doxorubicin (IC50: 32.00 mM). Further, the key compound 4 (IC50: 27.5 1 mM) showed better cytotoxicity than doxorubicin. Compounds 9, 10 and 13 (IC50: 28.85, 23.48, 28.85 mM) showed also better activity than doxorubicin. While compound 5, 12, 14 and 15 showed slightly lower activity than that of doxorubicin.

subclinical metastases and if the primary local tumor is effectively treated by radiotherapy. In this regard, no interaction between radiotherapy and chemotherapy is required. The other idea is the enhancement of radiation effects. Cytotoxic agents can enhance radiation effects by direct enhancement of the initial radiation damage by incorporating drugs into DNA, inhibiting cellular repair, accumulating cells in a radiosensitive phase or eliminating radioresistant phase cells, eliminating hypoxic cells or inhibiting the accelerated repopulation of tumor cells. Virtually, all chemotherapeutic agents have the ability to sensitize cancer cells to the lethal effects of ionizing radiation [33]. The ability of the most five active compounds 4, 6e8 and 11 to enhance the cell killing effect of g-irradiation was studied. Compounds 4, 6e8 and 11 exhibited (IC50: 27.51, 10.25, 9.55, 9.39 and 9.70 mM, respectively) when used alone (Table 1). The IC50 values were decreased to 14.76, 9.11, 8.72, 8.72 and 7.55 mM when the cells were treated with compound 4, 6e8 and 11 in combination with a single dose of g-radiation at a dose level of 8 Gy (Table 2). The survival curve for MCF7 cell line for compound 11 alone and in combination with g-irradiation is illustrated in (Fig. 2). 3. Conclusion The objective of the present study was to synthesize and investigate the anticancer activity of new Schiff bases of pyrano[2,3-d] thiazole containing a free sulfonamide moiety. Some of the new synthesized compounds 4, 6e8 and 11 showed promising anticancer activity against human breast cancer cell line [MCF7]. Also, combination of these active compounds with radiation resulted in a remarkable increase of their potency toward cancer cells. 4. Experimental 4.1. Chemistry Melting points are uncorrected and were determined on a Stuart melting point apparatus (Stuart Scientific, Redhill, UK). Elemental analysis (C, H, N) were performed on PerkineElmer 2400 analyser (PerkineElmer, Norwalk, CT, USA) at the microanalytical laboratories of the Faculty of Science, Cairo University. All compounds were within 0.4% of the theoretical values. The IR spectra (KBr) were measured on Shimadzu IR 110 spectrophotometer (Shimadzu, Koyoto, Japan), 1 H NMR spectra were obtained on a Bruker proton NMR-Avance 300 (300 MHz) (Bruker, Munich, Germany), in DMSO-d6 as a solvent, using tetramethylsilane (TMS) as internal standard. Mass spectra were run on JEOL JMS AX-500 mass spectrometer (JEOL JMS, Tokyo, Japan). All reactions were monitored by thin layer chromatograph (TLC) using precoated Aluminum sheets Silica gel Merck 60 F254 and were visualized by UV lamp (Merck, Darmstadt, Germany). 4.1.1. 2-Chloro-N-(4-sulfamoylphenyl)acetamide (2) M.p.: 215e217 C as reported [31]. 4.1.2. 4-(4-Oxo-4,5-dihydrothiazol-2-ylamino)benzenesulfonamide (3) Compound 3 was prepared by another method. A mixture of compound 2 (2.48 g, 0.01 mol) and ammonium thiocyanate (0.76 g, 0.01 mol) in ethanol (50 mL) was refluxed for 1 h. The solid obtained was crystallized from dioxane. M.p.: 244e246 C as reported [11].

2.3. Radiosensitizing activity The rationale for combining chemotherapy and radiotherapy is based mainly on two ideas, one being spatial cooperation, which is effective if chemotherapy is sufficiently active to eradicate

4.1.3. 5-Amino-6-cyano-7-(4-methoxyphenyl)-7H-2(4-sulphamoylphenylamino) pyrano[2,3-d]thiazole (4) A mixture of compound 3 (2.71 g, 0.01 mol) with 2-(4-methoxy benzylidene) malononitrile (0.01 mol) in ethanol (30 mL) containing


O

NH2 Cl

O HN C

Cl

Cl

DMF/ r.t

NH4SCN

O S O NH 2

reflux ,ethanol

SO2NH2

SO2NH2

1

405

NH

O

N S

3

2

CN CN

Ar

Ar= C6H4OCH3 -4

Ar O S O NH 2

S

H N

N

H

O

CN NH2

4 Scheme 1. Preparation of compound 4.

piperidine (1 mL) was refluxed for 8 h. The reaction mixture was filtered and the filtrate was poured onto ice cold water. The solid obtained was crystallized from dioxane to give compound 4: yield, 70%; m.p.: 150e152 C. IR (KBr, cm1): 3333, 3217 (NH, NH2), 3075 (CH arom.), 2936, 2850 (CH aliph.), 2187 (C^N), 1593 (C]N), 1330, 1159 (SO2). 1H NMR (DMSO-d6) d: 3.78 [s, 3H, OCH3], 4.70 [s, 1H, CH pyran], 7.05e7.90 [m, 10H, AreH þ SO2NH2], 8.20 [s, 1H, NH, exchangeable with D2O], 10.00 [s, 2H, NH2 exchangeable with D2O]. MS (m/z): 456 [M þ 1] (28.41), 277 (100). 4.1.4. 5-(Substituted benzylideneamino)-6-cyano-7H-7-(4methoxyphenyl)-2-(4-sulphamoylphenylamino)pyrano[2,3-d] thiazole (5e15) A mixture of compound 4 (0.45 g, 0.001 mol) and the appropriate aromatic aldehyde (0.001 mol) in acetic acid (20 mL) was refluxed for 5 h. The reaction mixture was then poured onto cold water; the obtained solid was recrystallized from dioxane. 4.1.4.1. 5-(Benzylideneamino)-6-cyano-7H-7-(4-methoxyphenyl)2-(4-sulphamoylphenylamino)pyrano[2,3-d]thiazole (5). Yield, 51%; m.p.: 188e190 C. IR (KBr, cm1): 3250, 3203, 3150 (NH, NH2), 3065 (CH arom.), 2938, 2848 (CH aliph.), 2201 (C^N), 1598 (C]N), 1320, 1162 (SO2). MS (m/z): 543 [Mþ] (1.91), 169 (100). 4.1.4.2. 5-(2-Chlorobenzylideneamino)-6-cyano-7H-7-(4methoxyphenyl)-2-(4-sulphamoylphenylamino)pyrano[2,3-d]thiazole (6). Yield, 47%; m.p.: 180e182 C. IR (KBr, cm1): 3329, 3251, 3211 (NH, NH2), 3071 (CH arom.), 2932, 2843 (CH aliph.), 2203

(C^N), 1595 (C]N), 1329, 1165 (SO2). 1H NMR (DMSO-d6) d: 3.78 [s, 3H, OCH3], 4.70 [s, 1H, CH pyran], 6.60e7.80 [m, 14 H, AreH þ SO2NH2], 8.00 [s, 1H, NH, D2O exchangeable], 8.54 [s, 1H, CH]N]. MS (m/z): 580 [M þ 3] (0.26), 578 [M þ 1] (0.78), 149 (100). 4.1.4.3. 5-(4-Chlorobenzylideneamino)-6-cyano-7H-7-(4methoxyphenyl)-2-(4-sulphamoylphenylamino)pyrano[2,3-d]thiazole (7). Yield, 67%; m.p.: 206e208 C. IR (KBr, cm1): 3331, 3273, 3226 (NH, NH2), 3039 (CH arom.), 2934, 2851 (CH aliph.), 2201 (C^N), 1603 (C]N), 1331, 1170 (SO2). 1H NMR (DMSO-d6) d: 3.78 [s, 3H, OCH3], 4.10 [s, 1H, CH pyran], 7.06e7.07 [m, 2 H, AreH], 7.15 [d, 2 H, J ¼ 7.8 Hz, AreH], 7.34 [s, 2 H, SO2NH2, D2O exchangeable], 7.49 [d, 2 H, AreH], 7.62 [d, 2 H, AreH],7.77 [d, 2 H, J ¼ 7.8 Hz, AreH], 7.80 [m, 2 H, AreH], 8.00 [s, 1H, NH, D2O exchangeable], 8.23 [s, 1H, CH]N]. 4.1.4.4. 5-(3-Bromobenzylideneamino)-6-cyano-7H-7-(4methoxyphenyl)-2-(4-sulphamoylphenylamino)pyrano[2,3-d]thiazole (8). Yield, 54%; m.p.: 228e230 C. IR (KBr, cm1): 3320, 3260, 3210 (NH, NH2), 3057 (CH arom.), 2933, 2846 (CH aliph.), 2197 (C^N), 1603 (C]N), 1328, 1144 (SO2). 1H NMR (DMSO-d6) d: 3.85 [s, 3H, OCH3], 4.45 [s, 1H, CH pyran], 7.05e7.90 [m, 14 H, AreH þ SO2NH2 at 7.32 D2O exchangeable], 8.2 [s, 1H, NH, D2O exchangeable], 8.50 [s, 1H, CH]N]. 4.1.4.5. 6-Cyano-5-(4-fluorobenzylideneamino)-7H-7-(4methoxyphenyl)-2-(4-sulphamoylphenylamino)pyrano[2,3-d]thiazole (9). Yield, 63%; m.p.: 108e110 C. IR (KBr, cm1): 3334, 3208,

OCH3

O S O NH 2

H N

H

S N

OCH3

AcOH

CN

+ O

NH2

ArCHO

O S O NH 2

4

H N

H

S N

O

CN N

5-15 5:Ar= C6H4

11:Ar= 4-(C6H4)NO2

6:Ar= 2-(C6H4)Cl

12:Ar= 4-(C6H4)N(CH3)2 13:Ar= CH=CH-C6H5

7:Ar= 4-(C6H4)Cl 8:Ar= 3-(C6H4)Br

14:Ar= 4-(C6H4)OH

9:Ar= 4-(C6H4)F

15:Ar= piperonyl

10:Ar= 2,4-(C6H3)Cl2 Scheme 2. Preparation of Schiff bases 5e15.

Ar

406


Table 1 In vitro anticancer screening of the synthesized compounds against human breast cell line (MCF7). Cpd. no.

Compound concentration (mM) 10 (mM)

25 (mM)

50 (mM)

IC50 (mM)

100 (mM)

Surviving fraction (mean SE)a DOX 4 5 6 7 8 9 10 11 12 13 14 15 a

0.551 0.810 0.825 0.541 0.443 0.435 0.845 0.693 0.480 0.792 0.828 0.872 0.814

0.026 0.022 0.013 0.003 0.017 0.009 0.013 0.023 0.010 0.021 0.020 0.025 0.008

0.480 0.550 0.668 0.323 0.251 0.233 0.551 0.503 0.327 0.701 0.601 0.638 0.660

0.003 0.019 0.021 0.020 0.012 0.006 0.021 0.033 0.016 0.031 0.019 0.016 0.025

0.139 0.331 0.307 0.360 0.355 0.371 0.380 0.377 0.313 0.347 0.242 0.370 0.419

0.005 0.013 0.007 0.018 0.020 0.018 0.003 0.010 0.005 0.017 0.013 0.030 0.023

0.130 0.350 0.271 0.460 0.290 0.309 0.443 0.391 0.381 0.378 0.370 0.307 0.393

0.016 0.015 0.017 0.015 0.009 0.011 0.017 0.015 0.007 0.018 0.026 0.005 0.006

32.00 27.51 33.55 10.25 9.55 9.39 28.85 23.48 9.70 36.24 28.85 34.50 38.25

Fig. 2. Interaction map of E7070 with the active site of hCA II showing similar interactions as those previously reported.

Each value is the mean of three experiments standard error.

3188 (NH, NH2), 3032 (CH arom.), 2934, 2849 (CH aliph.), 2198 (C^N), 1603 (C]N), 1331, 1171 (SO2). 1H NMR (DMSO-d6) d: 3.78 [s, 3H, OCH3], 4.80 [s, 1H, CH pyran], 6.81 [d, 2H, J ¼ 9.6 Hz, AreH], 7.06 [d, 2H, J ¼ 9.6 Hz, AreH], 7.13 [d, 2H, J ¼ 9.6 Hz, AreH], 7.32 [s, 2H, SO2NH2, D2O exchangeable], 7.49 [d, 2H, J ¼ 9.6 Hz, AreH], 7.55e7.80 [m, 4H, AreH], 8.10e8.20 [s, 1H, NH, D2O exchangeable], 8.79 [s, 1H, CH]N]. 13C NMR (DMSO-d6) d: 26.25, 55.91, 73.09, 114.46, 115.01, 115.38, 120.72, 126.25, 126.75, 127.13, 127.52, 130.09, 130.65, 131.92, 132.36, 140.56, 158.95, 160.92, 161.48, 167.97, 173.37. MS (m/z): 560 [M 1] (0.33), 63 (100). 4.1.4.6. 6-Cyano-5-(2,4-dichlorobenzylideneamino)-7H-7-(4methoxyphenyl)-2-(4-sulphamoylphenylamino)pyrano[2,3-d]thiazole (10). Yield, 77%; m.p.: 84e86 C. IR (KBr, cm1): 3330, 3266, 3189 (NH, NH2), 3079 (CH arom.), 2928, 2858 (CH aliph.), 2207 (C^N), 1585 (C]N), 1377, 1168 (SO2). 1H NMR (DMSO-d6) d: 3.78 [s, 3H, OCH3], 4.45 [s, 1H, CH pyran], 7.03 [d, 2H, J ¼ 6.9 Hz, AreH], 7.06 [d, 2H, J ¼ 6.9 Hz, AreH], 7.33 [s, 2H, SO2NH2, D2O exchangeable], 7.55e7.59 [m, 4H, AreH], 7.71 [s, 1H, AreH], 7.80 [d, 1H, J ¼ 6.4 Hz, AreH], 7.84 [d, 1H, J ¼ 6.4 Hz, AreH], 8.20 [s, 1H, NH, D2O exchangeable], 8.50 [s, 1H, CH]N]. 13C NMR (DMSO-d6) d: 26.25, 55.91, 73.09, 114.46, 115.01, 115.38, 120.72, 126.25, 126.75, 127.13, 127.52, 130.09, 130.65, 131.92, 132.36, 140.56, 158.95, 160.92, 161.48, 167.97, 173.37. 4.1.4.7. 6-Cyano-7H-7-(4-methoxyphenyl)-5-(4nitrobenzylideneamino)-2-(4-sulphamoylphenylamino)pyrano[2,3-d] thiazole (11). Yield, 75%; m.p.: 198e200 C. IR (KBr, cm1): 3339, 3224, 3150 (NH, NH2), 3068 (CH arom.), 2937, 2842 (CH aliph.), 2201 (C^N), 1595 (C]N), 1336, 1165 (SO2). 1H NMR (DMSO-d6) d: 3.80 [s, 3H, OCH3], 4.25 [s, 1H, CH pyran], 6.96e7.22 [m, 4H, AreH],

7.33 [s, 2H, SO2NH2, D2O exchangeable], 7.41e7.64 [m, 4H, AreH], 7.71e7.95 [m, 4H, AreH], 8.20 [s, 1H, NH D2O exchangeable], 8.50 [s, 1H, CH]N]. 13C NMR (DMSO-d6) d: 29.32, 55.86, 77.61, 114.75, 116.37, 117.22, 127.16, 127.49, 128.82, 129.73, 130.12, 130.19, 132.32, 146.84, 158.13, 160.92, 161.13, 163.56, 169.27. MS (m/z): 587 [M 1] (0.34), 76 (100). 4.1.4.8. 6-Cyano-5-(4-(dimethylamino)benzylideneamino)-7H-7-(4methoxyphenyl)-2-(4-sulphamoylphenylamino)pyrano[2,3-d]thiazole (12). Yield, 43%; m.p.: 162e164 C. IR (KBr, cm1): 3333, 3267, 3213 (NH, NH2), 3071 (CH arom.), 2935, 2846 (CH aliph.), 2205 (C^N), 1597 (C]N), 1378, 1169 (SO2). 1H NMR (DMSO-d6) d: 3.10 [s, 6H, 2CH3], 3.80 [s, 3H, OCH3], 4.25 [s, 1H, CH pyran], 6.80 [d, 2H, J ¼ 9.0 Hz, AreH], 7.06 [d, 2H, J ¼ 7.2 Hz, AreH], 7.12 [d, 2H, J ¼ 7.2 Hz, AreH], 7.36 [s, 2H, SO2NH2, D2O exchangeable], 7.56 [d, 2H, J ¼ 8.1 Hz, AreH], 7.60 [d, 2H, J ¼ 8.1 Hz, AreH], 7.69 [d, 2H, J ¼ 9.0 Hz, AreH], 7.80 [s, 1H, NH, D2O exchangeable], 8.30 [s, 1H, CH]N]. 4.1.4.9. 6-Cyano-7H-(7-(4-methoxyphenyl)-5-(3-phenylallylidene) amino)-2-(4-sulphamoylphenylamino)pyrano[2,3-d]thiazole (13). Yield, 39%; m.p.: 92e94 C. IR (KBr, cm1): 3311, 3190, 3152 (NH, NH2), 3035 (CH arom.), 2931, 2846 (CH aliph.), 2202 (C^N), 1593 (C]N), 1331, 1165 (SO2). 1H NMR (DMSO-d6) d: 3.83 [s, 3H, OCH3], 4.30 [s, 1H, CH pyran], 6.82, 6.90 [2d, 2H, J ¼ 8.5 Hz, CH ¼ CH], 7.08e7.16 [m, 4H, AreH], 7.37 [s, 2H, SO2NH2, D2O exchangeable], 7.51 [m, 1H, AreH], 7.59 [d, 2H, J ¼ 6.8 Hz, AreH], 7.61 [d, 2H, J ¼ 6.8 Hz, AreH], 7.72e7.78 [m, 4H, AreH], 8.15 [s, 1H, NH, D2O exchangeable], 8.30 [s, 1H, CH]N]. 4.1.4.10. 6-Cyano-5-(4-hydroxybenzylideneamino)-7H-7-(4methoxyphenyl)-2-(4-sulphamoylphenylamino)pyrano[2,3-d]thiazole (14). Yield, 46%; m.p.: 170e172 C. IR (KBr, cm1): 3333, 3267,

Table 2 In vitro anticancer screening of compounds 4, 5bed and 5g against human breast cell line (MCF7) in combination with radiation. Cpd. no.

Control

Irradiated (8 Gy)

4 6 7 8 11

1.000 1.000 1.000 1.000 1.000

0.927 0.927 0.927 0.927 0.927

Compound concentration (mM) þ irradiation (8 Gy) Surviving fraction (mean SE)a 10

0.02 0.02 0.02 0.02 0.02

*Significant difference from control group at p < 0.001. a Each value is the mean of three values Standard Error.

0.618 0.453 0.311 0.319 0.403

25

0.01* 0.017* 0.013* 0.02* 0.01*

0.346 0.237 0.217 0.211 0.201

IC50 (mM)

50

0.02* 0.012* 0.02* 0.016* 0.02*

0.171 0.135 0.170 0.119 0.100

100

0.01* 0.015* 0.01* 0.015* 0.012*

0.201 0.119 0.201 0.235 0. 130

0.01* 0.007* 0.008* 0.01* 0.009*

14.76 9.11 8.72 8.72 7.55


3221 (NH, NH2), 3079 (CH arom.), 2934, 2846 (CH aliph.), 2202 (C^N), 1598 (C]N), 1336, 1169 (SO2). 1H NMR (DMSO-d6) d: 3.80 [s, 3H, OCH3], 4.50 [s, 1H, CH pyran], 6.82e7.81 [m, 14 H, AreH þ SO2NH2], 8.09 [s, 1H, NH, D2O exchangeable], 8.30 [s, 1H, CH]N], 9.90 [s, 1H, OH, D2O exchangeable]. 4.1.4.11. 5-((Benzo [1,3-b] dioxol-5-ylmethylene)amino)-6-cyano7H-7-(4-methoxyphenyl)-2-(4-sulphamoylphenylamino)pyrano[2,3d]thiazole (15). Yield, 71%; m.p.: 122e124 C. IR (KBr, cm1): 3333, 3227, 3171 (NH, NH2), 3073 (CH arom.), 2935, 2849 (CH aliph.), 2206 (C^N), 1597 (C]N), 1333, 1169 (SO2). 1H NMR (DMSO-d6) d: 3.80 [s, 3H, OCH3], 4.60 [s, 1H, CH pyran], 6.28 [s, 2H, CH2], 6.61 [d, 2H, J ¼ 6.8 Hz, AreH], 6.92 [d, 2H, J ¼ 6.8 Hz, AreH], 7.08 [d, 2H, J ¼ 8.5 Hz, AreH at C-6 & C-7], 7.38 [d, 1H, J ¼ 5.0 Hz, AreH at C-4], 7.28 [s, 2H, SO2NH2, D2O exchangeable], 7.58 [d, 2H, J ¼ 7.2 Hz, AreH], 7.62 [d, 2H, J ¼ 7.2 Hz, AreH], 8.10 [s, 1H, NH, D2O exchangeable], 8.30 [s, 1H, CH]N]. 4.2. Biological testing 4.2.1. Materials and methods The human tumor cell lines [MCF7] were available at the National Cancer Institute, Cairo, Egypt. Irradiation was performed in the National Center for Radiation Research and Technology [NCRRT], Atomic Energy Authority using Gamma cell (137 Cs) source, Graphpad Prism 5 program was used for the calculation of the IC50 value and curve fitting (non linear regression). 4.2.2. In vitro anticancer screening The cytotoxic activity was measured in vitro for the newly synthesized compounds using the Sulfo-Rhodamine-B stain (SRB) assay using the method of Skehan et al. [34]. The in vitro anticancer screening was done at the pharmacology unit, the National Cancer Institute, Cairo University. Cells were plated in 96-multiwell microtiter plate (104 cells/well) for 24 h before treatment with the compound(s) to allow attachment of cell to the wall of the plate. Test compounds were dissolved in DMSO and diluted with saline to the appropriate volume. Different concentrations of the compound under test (10, 25, 50 and 100 mM) were added to the cell monolayer. Triplicate wells were prepared for each individual dose. Monolayer cells were incubated with the compound(s) for 48 h at 37 C and in atmosphere of 5% CO2. After 48 h, cells were fixed, washed, and stained for 30 min with 0.4% (wt/vol) with SRB dissolved in 1% acetic acid. Excess unbound dye was removed by four washes with 1% acetic acid and attached stain was recovered with TriseEDTA 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 breast tumor cell line after the specified time [34]. The molar concentration required for 50% inhibition of cell viability (IC50) was calculated and the results are given in Table 1. The relationship between surviving fraction and drug concentration was plotted to obtain the survival curve of breast cancer cell line [MCF7]. The response parameter calculated was IC50 value, which corresponds to the concentration required for 50% inhibition of cell viability. 4.2.3. Radiosensitizing activity The most active compounds 4, 6e8 and 11 resulted from the in vitro anticancer screening, were selected for further screening in combination with g-radiation. This study was conducted to evaluate the ability of the most biologically active compounds to enhance the cell killing effect of gradiation. Cells were subjected to a single dose of g-radiation at

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a dose level of 8 Gy with a dose rate of 1 Gy/min. The surviving fraction was measured 1 h after subjection to radiation [35]. The surviving fractions were expressed as means standard error. The results were analyzed using 1-way ANOVA test and the percentage of change in the surviving fraction from control for each compound was calculated considering the control as 100% in the surviving fraction. The results are given in Table 2. Appendix A. Supplementary material Supplementary data related to this article can be found online at doi:10.1016/j.ejmech.2012.04.009. References [1] [2] [3] [4] [5] [6] [7] [8]

[9] [10] [11] [12] [13] [14] [15] [16] [17]

[18]

[19] [20] [21] [22] [23]

[24] [25] [26] [27] [28] [29] [30] [31] [32] [33]

[34] [35]

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