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1,3,4-oxadiazole-2-thione Derivatives; Novel Approach for Anticancer and Tubulin Polymerization Inhibitory Activities

Mohamed Abdel-Aziz1, Kamel A. Metwally2, Amira M. Gamal-Eldeen3 and Omar M. Aly1,*

1Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, 61519- Minia, Egypt; 2Department of Medicinal Chemistry, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt; 3Cancer Biology Laboratory, Center of Excellence for Advanced Sciences, National Research Center, Cairo, Egypt

Abstract: A series of novel 5-(substituted phenyl)-3-[(substituted phenylamino)methyl]-3H-[1,3,4]oxadiazole-2-thione derivatives were prepared and their in vitro cytotoxicity was evaluated against a panel of three cancer cell lines, namely, hepatocarcinoma HepG2, breast adenocarcinoma MCF-7, and leukemia HL-60 cells, using the widely accepted MTT assay. In general, the synthesized compounds displayed weak to moderate cytotoxic activity against the three tested cell lines. Compound 5a, which has trimethoxy substituents on both phenyl rings, exhibited the highest cytotoxic effect against all cell lines tested with IC50 values of 12.01, 7.52 and 9.7 µM against HepG2, MCF-7 and HL-60 cells, respectively. Mechanistic studies revealed that the test compounds showed a good inhibitory effect on cellular tubulin of hepatocellular carcinoma. Compound 5h was the most potent tubulin inhibitor in HepG2 cells, with 81.1 % inhibition of the original control tubulin. Moreover, the mechanism of tubulin polymerization inhibition was confirmed by immunofluorescence assay, flow cytometry, and docking study.

Keywords: Anticancer, immunofluorescence, 1,3,4-oxadiazole, tubulin.

INTRODUCTION

Cancer is a major cause of death around the world. By the year 2030, the WHO estimates 12 million deaths by cancer with existing therapeutics. Despite the shortcomings of current cancer therapeutics, chemotherapy has turned out to be one of the most significant treatments in cancer management [1]. Moreover, the discovery of new anti-cancer targets in the recent years paved the way for the development of new selective agents not associated with the serious toxicities of conventional cytotoxic drugs [2]. Since tubulin is the target of the most successful currently marketed anti-cancer agents, there is a much growing interest of medicinal chemists in developing anti-tubulin agents. Alteration of microtubule dynamics results in major cellular consequences including disturbance of mitotic spindle function and consequently inhibition of the cancer cell proliferation and induction of cell apoptosis [3]. In addition, microtubule-targeting drugs disrupt cell signaling pathways associated with regulation and maintenance of endothelial cells cytoskeleton in the tumor vasculature. Combretastatin A4 (I; Chart 1), is one of the potent cytotoxic agents that strongly inhibits tubulin polymerization through binding to the colchicine-binding site of the β-tubulin subunit [4]. Literature survey revealed that combretastatin A4 (CA4) was the subject of extensive research efforts in the few past decades that resulted in the development of numerous analogues as potential anti-cancer candidates, some of which have been reviewed [5-13]. Furthermore, many reports appeared in the literature suggest cancer cell vasculature as a target for CA4 analogues [14-21].

A series of methoxy substituted 2-(3',4',5'-trimethoxybenzoyl)-benzo[b]thiophene (II) [22-24], and indole derivatives (III) [25] were found to exert significant cytotoxic effect against a panel of cancer cell lines through inhibition of tubulin polymerization by binding to the colchicine binding site resulting in cell cycle arrest in the G2-M phase (Chart 1). In addition, certain 1,3,4-oxadiazole derivatives and their Mannich bases were reported to possess anti- inflammatory [26], antitubercular [27], antifungal [28], and

*Address correspondence to this author at the Postal address: Medicinal Chemistry Department, Faculty of Pharmacy, Minia University, Minia 61519, Egypt; Tel: +201065607771; Fax: +2086-2369075; E-mails: omarsokkar@yahoo.com; omarsokkar@mu.edu.eg

anticancer activities [29-32]. Based on the above findings and as a continuation of our research interest in the synthesis of novel tubulin inhibitors [33, 34], we were interested in the synthesis and biological evaluation of novel series of Mannich bases of [1,3,4]oxadiazole-2-thione derivatives having various substituted 3- and 5-aryl rings with the objective of discovering novel and potent tubulin inhibitors. The target compounds were evaluated for in vitro cytotoxic activity against three cancer cell lines using the widely accepted MTT assay. Additionally, this novel class of compounds was mechanistically studied through cell cycle analysis and tubulin polymerization inhibition assay.

MATERIAL AND METHODS

Synthesis

Melting points were determined using Stuart electrothermal melting point apparatus and were uncorrected. IR spectra were done on Nicolet iS5 FT-IR spectrometer at Minia University. NMR spectra were obtained using a Bruker avance 300 MHz NMR spectrometer, using TMS as internal reference. Chemical shifts values are given in parts per million (ppm) relative to CDCl3 (7.29 for proton and 76.9 for carbon) or DMSO-d6 (2.50 for proton and 39.50 for carbon) and coupling constants (J) in Hertz. Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublet; m, multiplet. High resolution mass spectra (HRMS) were carried out using a JEOL JMS D-300 mass spectrometer. The progress of reactions and the purity of the prepared compounds were checked by using Merck 9385 pre-coated aluminum plate silica gel (Kieselgel 60) 5 x 20 cm TLC plates with a layer thickness of 0.2 mm. The spots were detected by UV-lamp at λ = 254 nm.

Compounds 1a-c, 2a-c and 3a-c[38], 4a [35], 4b [36], and 4c [37] were synthesized according to reported procedure.

General Procedure for the Preparation of 5-(substituted phenyl)-3-[(substitutedphenylamino)methyl]-3H-[1,3,4]oxadiazole-2-thione 5a-l

To a stirred solution of 5-(substituted phenyl)-3H-[1,3,4] oxadiazole-2-thione 4a-c (0.02 mol) in ethanol (40 mL), formalin

O.M. Aly

270 Anti-Cancer Agents in Medicinal Chemistry, 2016, Vol. 16, No. 2 Abdel-Aziz et al.

40% (1.5 mL, 0.02 mol) was added. A solution of the substituted aniline (0.02 mol) in 10.0 mL ethanol was added in a dropwise manner and the reaction mixture was allowed to stir at room temperature for 12 h. The formed precipitate was filtered, washed with cold ethanol, dried and recrystallized from ethanol.

5-(3,4,5-Trimethoxyphenyl)-3-[(3,4,5-trimethoxyphenylamino) methyl]-3H-[1,3,4]oxadiazole-2-thione 5a

Yield 77%, mp 138 °C; FT-IR (cm-1): 3392 (NH), 1577 (C=C), 1525 (C=N), 1414 (C=S), 1268 (C-O); 1H-NMR (300 MHz, CDCl3) δ (ppm): 3.78 (s, 3H, OCH3), 3.82 (s, 6H, 2OCH3), 3.88 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 4.91 (s, 1H, NH), 5.56 (s, 2H, CH2), 6.31 (s, 2H, ArH), 7.09 (s, 2H, ArH); 13C-NMR (75 MHz, CDCl3) δ (ppm): 56.12, 56.25, 60.57, 60.83, 60.88, 60.93, 65.84, 95.74, 95.86, 103.60, 103.67, 107.86, 116.89, 135.65, 138.42, 140.33, 145.34, 150.30, 153.64, 159.35, 176.54. RMS (LC-MS/MS) Calcd. for C21H25N3O7S [M-H]+ 462.13404, Found: 462.13461.

3-[(3,4-Dimethoxyphenylamino)methyl]-5-(3,4,5-trimethoxyphenyl)-3H-[1,3,4]oxadiazole-2-thione 5b

Yield 77%, mp 111-113 °C; FT-IR (cm-1): 3415 (NH), 1576 (C=C), 1523 (C=N), 1414 (C=S), 1267 (C-O);1H-NMR (300 MHz, CDCl3) δ (ppm): 1H-NMR (300 MHz, CDCl3) δ (ppm): 3.76 (s, 3H, OCH3), 3.83 (s, 6H, 2OCH3), 3.86 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 5.49 (s, 1H, NH), 6.01 (s, 2H, CH2), 6.80-6.84 (m, 1H, ArH), 6.84-6.97 (m, 1H, Ar-H), 7.04 (s, 2H, ArH), 7.06 (s, 1H, ArH); 13C-NMR (75 MHz, CDCl3) δ (ppm):55.97, 56.13, 56.20, 56.34, 60.79, 65.84, 103.52, 103.78, 109.09, 112.29, 114.03, 116.69, 116.86, 137.71, 141.68, 143.61, 149.69, 153.48, 159.36, 176.18. RMS (LC-MS/MS) Calcd. for C20H23N3O6S [M-H]+ 432.12348, Found: 432.12350.

3-[(4-Methoxyphenylamino)methyl]-5-(3,4,5-trimethoxyphenyl)-3H-[1,3,4]oxadiazole-2-thione 5c

Yield 76%, mp 117-118 °C; FT-IR (cm-1): 3381 (NH), 1576 (C=C), 1515 (C=N), 1416 (C=S), 1267 (C-O); 1H-NMR (300 MHz, CDCl3) δ (ppm): 1H-NMR (300 MHz, CDCl3) δ (ppm): 1H-NMR (300 MHz, CDCl3) δ (ppm): 3.71 (s, 3H, OCH3), 3.86 (s, 6H, 2OCH3), 3.88 (s, 3H, OCH3), 4.97 (t, 1H, J = 7.80 Hz, NH), 5.47 (d, 2H, J = 6.60 Hz, CH2), 6.78 (d, 2H, J = 9.00 Hz, ArH), 6.86 (d, 2H, J = 9.00 Hz, ArH), 7.08 (s, 2H, ArH); 13C-NMR (75 MHz, CDCl3) δ (ppm): 55.54, 56.31, 59.72, 60.94, 66.22, 103.65, 103.75, 114.83, 115.71, 117.15, 117.38, 137.49, 141.60, 153.59, 153.65, 159.25, 176.25. RMS (LC-MS/MS) Calcd. for C19H21N3O5S [M-H]+402.11291, Found: 402.11331.

3-[(4-Chlorophenylamino)methyl]-5-(3,4,5-trimethoxyphenyl)-3H-[1,3,4]oxadiazole-2-thione 5d

Yield 79%, mp 145-146 °C; FT-IR (cm-1): 3392 (NH), 1576 (C=C), 1515 (C=N), 1416 (C=S), 1269 (C-O); 1H-NMR (300 MHz, CDCl3) δ (ppm): 1H-NMR (300 MHz, CDCl3) δ (ppm): 3.85 (s, 6H, 2OCH3), 3.86 (s, 3H, OCH3), 5.21 (t, 1H, J = 7.80 Hz, NH), 5.45 (d, 2H, J = 8.10 Hz, CH2), 6.81 (d, 2H, J = 8.80 Hz, ArH), 7.04 (s,

2H, ArH), 7.11 (d, 2H, J = 8.80 Hz, ArH); 13C-NMR (75 MHz, CDCl3) δ (ppm): 56.20, 58.41, 60.83, 103.55, 115.22, 116.89, 124.47, 129.12, 141.56, 142.47, 153.48, 159.22, 176.15.RMS (LC-MS/MS) Calcd. for C18H18ClN3O4S [M-H]+406.06338, Found: 406.06345.

5-(3,4-Dimethoxyphenyl)-3-[(3,4,5-trimethoxyphenylamino) methyl]-3H-[1,3,4]oxadiazole-2-thione 5e

Yield 75%, mp 173-175 °C; FT-IR (cm-1): 3344 (NH), 1614 (C=N), 1597 (C=C), 1423 (C=S), 1269 (C-O); 1H-NMR (300 MHz, CDCl3) δ (ppm): 3.60 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.86 (s, 6H, OCH3), 5.53 (t, 1H, J = 7.2 Hz, NH), 5.99 (s, 2H, CH2), 6.75-6.94 (m, 3H, ArH), 7.21 (s, 1H, ArH), 7.26 (s, 1H, ArH), 7.43 (d, 1H, J = 8.10 Hz, Ar-H); 13C-NMR (75 MHz, CDCl3) δ (ppm): 55.77, 55.82, 56.46, 60.62, 65.28, 92.11, 108.52, 111.07, 114,15, 119.97, 120.37, 140.08, 149.16, 152.49, 153.66, 159.65, 176.08. RMS (LC-MS/MS) Calcd. for C20H23N3O6S [M-H]+432.12348, Found: 432.12349.

5-(3,4-Dimethoxyphenyl)-3-[(3,4-dimethoxyphenylamino)methyl]-3H-[1,3,4]oxadiazole-2-thione 5f

Yield 78%, mp 129-130 °C; FT-IR (cm-1): 2930 (NH), 1615 (C=N),1597 (C=C), 1424 (C=S), 1260 (C-O); 1H-NMR (300 MHz, CDCl3) δ (ppm): 1H-NMR (300 MHz, CDCl3) δ (ppm): 3.74 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.87 (s, 6H, 2OCH3), 5.53 (t, 1H, J = 7.2 Hz, NH), 5.99 (s, 2H, CH2), 6.75-6.94 (m, 3H, ArH), 7.21 (s, 1H, ArH), 7.26 (s, 1H, ArH), 7.43 (d, 1H, J = 8.10 Hz, Ar-H); 13C-NMR (75 MHz, CDCl3) δ (ppm): 56.00, 56.20, 56.53, 56.72, 65.76, 100.34, 106.22, 108.66, 111.09, 112.42, 114.40, 117.25, 120.43, 137.91, 143.72, 149.31, 152.62, 159.66, 176.23. RMS (LC-MS/MS) Calcd. for C19H21N3O5S [M-H]+402.11291, Found: 402.11298.

5-(3,4-Dimethoxyphenyl)-3-[(4-methoxyphenylamino)methyl]-3H-[1,3,4]oxadiazole-2-thione 5g

Yield 78%, mp 149-150 °C; FT-IR (cm-1): 3329 (NH), 1619 (C=N),1572 (C=C), 1424 (C=S); 1H-NMR (300 MHz, CDCl3) δ (ppm):1H-NMR (300 MHz, CDCl3) δ (ppm): 3.67 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 4.97 (s, 1H, NH), 5.42 (s, 2H, CH2), 6.74 (d, 2H, J = 9.00 Hz, ArH), 6.82-6.87 (m, 3H, ArH), 7.14 (s, 1H, ArH), 7.43 (d, 1H, J = 8.70 Hz, Ar-H); 13C-NMR (75 MHz, CDCl3) δ (ppm): 55.44, 55.92, 59.48, 65.86, 108.55, 114,37, 114.73, 115.42, 115.55, 120.22, 137.54, 149.16, 152.36, 153.47, 159.32, 176.01. RMS (LC-MS/MS) Calcd. for C18H19N3O4S [M-H]+372.10235, Found: 372.10233.

3-[(4-Chlorophenylamino)methyl]-5-(3,4-dimethoxyphenyl)-3H-[1,3,4]oxadiazole-2-thione 5h

Yield 82%, mp 183-184 °C; FT-IR (cm-1): 3328 (NH), 1618 (C=N), 1599 (C=C), 1423 (C=S), 1261 (C-O); 1H-NMR (300 MHz, CDCl3) δ (ppm):3.84 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 5.39 (s, 2H, CH2), 6.77 (d, 2H, J = 9.00 Hz, ArH), 6.84 (d, 1H, J = 9.00 Hz, ArH), 7.05 (d, 2H, J = 8.70 Hz, ArH), 7.23-7.26 (m, 1H, ArH), 7.40 (d, 1H, J = 8.40 Hz, ArH); 13C-NMR (75 MHz, CDCl3) δ (ppm):

Chart 1. Structure of combretastatin-A4 and analogues.

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55.84, 55.88, 58.26, 108.48, 111.05, 114.40, 115.09, 120.04, 120.30, 124.16, 128.81, 129.02, 142.69, 149.15, 152.44, 159.47, 176.08.RMS (LC-MS/MS) Calcd. for C18H18ClN3O4S [M-H]+376.06737, Found: 376.06739.

5-(4-Methoxyphenyl)-3-[(3,4,5-trimethoxyphenylamino)methyl]-3H-[1,3,4]oxadiazole-2-thione 5i

Yield 76%, mp 189-190 °C; FT-IR (cm-1): 3328 (NH), 1612 (C=N),1599 (C=C), 1419 (C=S), 1257 (C-O); 1H-NMR (300 MHz, CDCl3) δ (ppm): 1H-NMR (300 MHz, CDCl3) δ (ppm): 3.80 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 5.55 (s, 1H, NH), 6.12 (s, 2H, CH2), 6.66 (s, 2H, ArH), 7.00 (d, 2H, J = 8.70 Hz, ArH), 7.86 (d, 2H, J = 8.70 Hz, ArH; 13C-NMR (75 MHz, CDCl3) δ (ppm): 55.47, 56.15, 56.76, 60.86, 65.35, 91.55, 92.08, 114.32, 114,61, 128.21, 128.31, 132.10, 140.11, 153.96, 159.75, 162.98, 176.20. RMS (LC-MS/MS) Calcd. for C19H21N3O5S [M-H]+402.11291, Found: 402.11282.

3-[(3,4-Dimethoxyphenylamino)methyl]-5-(4-methoxyphenyl)-3H-[1,3,4]oxadiazole-2-thione 5j

Yield 77%, mp 160-161 °C; FT-IR (cm-1): 3328 (NH), 1617 (C=N),1599 (C=C), 1416 (C=S), 1255 (C-O); 1H-NMR (300 MHz, CDCl3) δ (ppm):1H-NMR (300 MHz, CDCl3) δ (ppm): 3.82 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 5.50 (s, 2H, CH2), 6.58 (s, 1H, ArH), 6.72-6.77 (m, 2H, ArH), 6.95 (d, 2H, J = 9.00 Hz, ArH), 7.80 (d, 2H, J = 9.00 Hz, ArH); 13C-NMR (75 MHz, CDCl3) δ (ppm): 55.42, 55.70, 56.59, 66.10, 101.49, 103.87, 109.11, 110.03, 111.78, 114.04, 114.52, 128.44, 136.22, 143.18, 148.62, 149.53, 159.47, 162.83, 176.34.RMS (LC-MS/MS) Calcd. for C18H19N3O4S [M-H]+372.10235, Found: 372.10238.

5-(4-Methoxyphenyl)-3-[(4-methoxyphenylamino)methyl]-3H-[1,3,4]oxadiazole-2-thione 5k

Yield 77%,mp 147-149 °C; FT-IR (cm-1): 3432 (NH), 1619 (C=N),1570 (C=C), 1413 (C=S); 1H-NMR (300 MHz, CDCl3) δ (ppm): RMS (LC-MS/MS) Calcd. for C17H17N3O3S [M-H]+342. 09179, Found: 342.09189.

3-[(4-Chlorophenylamino)methyl]-5-(4-methoxyphenyl)-3H-[1,3,4]oxadiazole-2-thione 5l

Yield 82%, mp 179-180 °C; FT-IR (cm-1): 3314 (NH), 1612 (C=N),1570 (C=C), 1405 (C=S), 1267 (C-O); 1H-NMR (300 MHz, CDCl3) δ (ppm): 1H-NMR (300 MHz, CDCl3) δ (ppm): 3.87 (s, 3H, OCH3), 4.54 (s, 1H, NH), 5.46 (s, 2H, CH2), 6.70 (d, 1H, J = 8.70 Hz, ArH), 6.91 (d, 2H, J = 8.70 Hz, ArH), 7.01-7.05 (m, 2H, ArH), 7.11 (d, 2H, J = 9.00 Hz, ArH), 7.81 (d, 2H, J = 8.70 Hz, Ar-H);

13C-NMR (75 MHz, CDCl3) δ (ppm): 54.14, 56.76, 113.15, 113.35, 113.41, 113.49, 121.36, 126.65, 126.86, 127.34, 127.54, 142.70, 157.98, 161.21, 161.51, 174.56. RMS (LC-MS/MS) Calcd. for C16H14ClN3O2S [M-H]+ 346.04225, Found: 346.04215.

BIOLOGY

Cell Culture

Human hepatocarcinoma (HepG2), breast adenocarcinoma (MCF-7), and leukemia (HL-60) cells, purchased from ATCC, USA, were used in the cytotoxicity evaluation of the test compounds. Cells were routinely cultured in DMEM (Dulbecco’s Modified Eagle’s Medium). Media were supplemented with 100 units/mL streptomycin sulfate, 100 units/mL penicillin G sodium, 2 mM L-glutamine, 250 ng/mL amphotericin B and 10% fetal bovine serum (FBS). Cells were maintained at 37 ºC in humidified air containing 5% CO2 and harvested by trypsinization. Cells were used when confluence had reached 75%. Samples were dissolved in dimethylsulfoxide (DMSO), and then diluted for the assays. All cell

culture material was purchased from Gibco®/Invitrogen, USA. All chemicals were from Sigma/Aldrich, USA, except mentioned. All experiments were repeated four times.

Cytotoxic Activity

MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) cytotoxicity assay of tested samples was measured against each cell line. The assay is based on the active mitochondrial dehydrogenase enzyme in the vital cells that reduces tetrazolium rings in MTT to form blue formazan crystals. The number of vital cells is directly proportional to the intensity of formazan blue color absorbance at 570 nm [39]. The procedure was done as reported [34] by using paclitaxel as a reference standard.

Cell Lysate Preparation

The total protein content of the lysates of treated and untreated cells was prepared and measured as reported [34,40].

Tubulin Polymerization Inhibition

Tubulin level was determined in cell lysates by ELISA as reported [34].

Immunofluorohistochemical Localization of Tubulin

According to the method originally developed by Kawahira et al. [41] and Noel et al. [42] with some modifications as reported by us [34], immunohistochemical detection of tubulin in fixed cells was done. Images were visualized using a Apotome fluorescence microscope (Axiostar plus, Zeiss, Goettingen, Germany) equipped with image analyzer and digital camera (PowerShot A20, canon, USA).

Cell Cycle Analysis

HepG2 cells (5×105) after collection were being treated with the test compound 5a, mixed with 4 mL of ice-cold 70% ethanol after washing twice with PBS and re-suspension in 250 µL of PBS. The cells were centrifuged and the pellets were re-suspended in 1 mL of propidium iodide (PI)/Triton X-100 staining solution (0.1 % Triton X-100 in PBS, 0.2 mg/mL RNase A and 10 µg/mL PI) and incubated for 30 min at room temperature. The stained cells were analyzed by flow cytometry.

Docking Study

Discovery Studio 2.5 software (Accelrys Inc., San Diego, CA, USA) was used for docking analysis. Fully automated docking tool using “Dockligands (CDOCKER)” protocol running on Intel(R) core(TM) i32370 CPU @ 2.4 GHz 2.4 GHz, RAM Memory 2 GB under the Windows 7.0 system. The receptor protein is prepared. The force field applied is CharmM to the receptor and the hydrogens are minimized. By selecting only the ligand part and clicking on “Define sphere from selection” so that the crystal ligand is used to define the binding site of 9 Angstroms on the receptor molecule. Now the above prepared receptor is given as input for "input receptor molecule‟ parameter in the CDOCKER protocol parameter explorer. Force fields are applied on compound 5a to get lowest energy structure. The CDOCKER energy (-(protein-ligand interaction energies) of best poses docked into the receptor.

RESULTS

Chemistry

5-(Substituted phenyl)-3H-[1,3,4]oxadiazole-2-thione derivatives 4a-c were prepared according to a previously reported method [35-37] by reaction of substituted benzoic acid hydrazide 3a-c [38] with CS2 in KOH. Reaction of compounds 4a-c with formaldehyde and

272 Anti-Cancer Agents in Medicinal Chemistry, 2016, Vol. 16, No. 2 Abdel-Aziz et al.

different substituted anilines afforded the target compounds 5a–k in good yield (Scheme 1). The chemical structure of the prepared compounds was elucidated on the basis of their IR, 1H NMR, 13C NMR and high resolution mass spectra.

Biology

The target compounds were evaluated for in vitro cytotoxic activity against a panel of three cancer cell lines namely, hepatocellular carcinoma HepG2, Leukemia HL-60, and breast cancer MCF-7 cells using the widely accepted MTT assay. Treatment of HepG2 cells with different concentrations of the tested compounds revealed that compounds 5a, 5b, 5e, 5h and 5k exhibited the highest cytotoxic effect, while the other tested compounds showed weak cytotoxic effect on HepG2 (Table 1). Compound 5a was the most cytotoxic compound against HepG2

cells. Treatment of MCF-7 cells with tested compounds indicated that compounds 5a, 5b, 5c, 5e,5f, 5h and 5k possessed the highest cytotoxic effect. Similarly, treatment of HL-60 cells with the tested compounds demonstrated that compounds 5a, 5b, 5e, 5h and 5k are the most cytotoxic among the compounds tested.

Tubulin polymerization inhibitory activity of all the target compounds was evaluated in HepG2 cancer cells after treating with 30% of the IC50 of each compound for 48 h using direct ELISA. Treatment of HepG2 cells revealed that compounds 5a, 5b, 5h and 5k showed a promising inhibitory effect of cellular tubulin, as concluded from their percentage of inhibition (around 50%), as shown in Fig. 1, while the other tested compounds showed a variable but lower inhibitory effect. Compounds 5a and 5b were the most potent tubulin polymerization inhibitors in HepG2 cells in agreement with their relatively higher cytotoxic activity.

Scheme 1. Reagents and conditions: a) Methanol, H2SO4, reflux. b) Hydrazine hydrate hydrate, ethanol, reflux. c) 1. CS2, KOH, reflux, 2. HCl. d) Formalin 40%, substituted aniline, ethanol, room temperature, 12 h.

Table 1. The effect of compounds 5a-5l on the growth of hepatocarcinoma HepG2, breast adenocarcinoma MCF-7, and leukemia HL-60 calculated as IC50 (µM) from linear equation of dose response curve for against.

Cell Line Cpd R1 R2 R3 R4 R5 R6

HepG2 MCF-7 HL-60

5a OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 12.01±0.21 7.52±0.18 9.7±0.211

5b OCH3 OCH3 OCH3 OCH3 OCH3 H 16.65±0.31 11.48±0.12 13.98±.31

5c OCH3 OCH3 OCH3 H OCH3 H 37.82±1.11 24.27±1.21 33.08±1.21

5d OCH3 OCH3 OCH3 H Cl H 78.54±2.21 63.33±2.43 70.70±2.21

5e OCH3 OCH3 H OCH3 OCH3 OCH3 22.14±1.14 17.55±1.45 19.78±1.34

5f OCH3 OCH3 H OCH3 OCH3 H 29.20±1.13 24.75±0.97 26.88±1.01

5g OCH3 OCH3 H H OCH3 H 107.67±3.12 87.15±1.96 97.09±2.17

5h OCH3 OCH3 H H Cl H 25.26±1.25 17.48±1.14 21.25±0.91

5i H OCH3 H OCH3 OCH3 OCH3 40.99±1.33 22.17±1.04 36.5±1.16

5j H OCH3 H OCH3 OCH3 H 102.60±2.71 55.41±1.43 82.98±2.45

5k H OCH3 H H OCH3 H 25.68±1.78 26.06±1.99 25.57±1.11

5l H OCH3 H H Cl H 98.90±2.11 23.28±1.16 90.21±2.49

PTX - - - - - - 0.73± 0.41 0.81±0.53 0.56±0.17 aIC50 = Concentration required to inhibit tumor cell proliferation by 50% and represented as the mean ± SE from the dose-response curves of at least three experiments.

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The localization of tubulin was explored in Hep-G2 cell line after treatment with selected promising compounds that revealed an inhibitory effect in tubulin as indicated by ELISA experiment. Cells were treated with 30% of the IC50 of each compound for 48 h, and then submitted to immunofluorescence labeling and analysis under Apotome fluorescence microscope. Treatment of HepG2 cells with compounds 5a, 5b, 5h and 5k showed a variable promising inhibitory effect of cellular localization of tubulin, as concluded from the inhibition of the fluorescence intensities (Fig. 2), while the other tested compounds showed a variable but lower inhibitory effect. Compounds, 5h, 5k>5b>5a were the most inhibitory compounds in HepG2 cells, where 5h resulted in 81.1 % inhibition of the original control tubulin. The Control untreated HepG2 cells showed a well distributed tubulin in a well distributed microtubules mesh and an intact cell-cell microtubule connection, as shown in Fig. 2. The cells treated with 5a showed impaired microtubules with impaired distribution of tubulin, which was absent in some peripheral zones and accumulated and localized in other side with a remarkable loss in the cell-cell microtubule connection. On the other hand, the cells treated with 5b showed a shrinked cellular content and spread microtubules mesh with a non homogenous distribution of tubulin with a lack in the cell-cell microtubule connection. The cells treated with 5h exhibited a reduced microtubules mesh area in relation to the total cellular content with impaired distribution and low concentration of tubulin, with noticeable absence the cell-cell microtubule connection. The cells treated with 5K revealed severely impaired microtubules with non homogenous localization of tubulin, which lacks in some cellular parts and in contrary was highly accumulated in peripheral zones in a dipolar pattern and with a remarkable absence in the cell-cell microtubule connection (Fig. 2).

Results of Cell Cycle Analysis

To explore the alteration in the cell cycle phases after the treatment with the active cytotoxic compound 5a. The cells submitted to flow cytometry analysis indicated that in HepG2 cells treated with 5a a predominated growth arrest at the S- and G2/M-phases (P<0.01) as compared with control cells, where the S-phase progression of HepG2 cells was considerably delayed (Fig. 3).

Docking of the Target Molecules to Tubulin Protein in Colchicine Binding Site

Docking of the conformation database of the target compound was done using Discovery Studio software program. The X-ray crystallographic structure of tubulin complexed with DAMA-colchicine was obtained from the Protein Data Bank through the Internet (http://www. rcsb.org/, PDB code 1SA0) [43]. Compounds 5a was docked into the empty colchicines binding site of tubulin. Compound 5a revealed CDOCKER interaction energy of -56.5548. It is obvious from Fig. 4, that compound 5a showed high interaction in the colchicine binding site located between α-tubulin specially the amino acids residues: GLN11, ASN101, PRO173, SER178, THR179, GLU183 and TYR224 and β-tubulin specially the amino acid residues: GLN247, LEU248, ASN249, ALN250, LYS254, LEU255, ASN258, MET259, ALA316, ALA317, VAL318, LYS352, THR353 and ALA 354.

DISCUSSION AND CONCLUSIONS

Chemistry

A series of novel 5-(substituted phenyl)-3-[(substituted phenylamino)methyl]-3H-[1,3,4]oxadiazole-2-thione derivatives were synthesized and characterized by different spectroscopic techniques. The IR spectra of compounds 5a-k generally showed the characteristic bands corresponding to the thione moiety at a range of 1405-1424 cm-1 in addition to the NH moiety at a range of 3328-3415 cm-1. A characteristic feature of the 1H-NMR spectra for compounds 5a-k is the appearance a singlet signal corresponding to CH2 protons at δ 5.39-6.12 ppm. For compound 5c and 5d the CH2 protons appeared as a doublet at 5.45-5.47 ppm. All aromatic and aliphatic protons were observed at expected regions. Furthermore, the 13C NMR and high resolution mass spectral data are in accordance with the expected structures of the prepared compounds.

Biology

The effect of the tested compounds on the viability of different human cancer cell lines including hepatocellular carcinoma HepG2, Leukemia HL-60, and breast cancer MCF-7 cells were evaluated

Fig. (1). Effect of compounds 5a-5l on the level of tubulin in HepG2 cells as measured by ELISA. * The significant difference from control P<0.05.

274 Anti-Cancer Agents in Medicinal Chemistry, 2016, Vol. 16, No. 2 Abdel-Aziz et al.

using MTT assay. Treatment of HepG2 cells with different concentrations of the tested compounds revealed that compound 5a was the most cytotoxic compound against HepG2 cells. The treatment of MCF-7 cells with tested compounds indicated that compounds 5a, 5b, 5c, 5e,5f, 5h and 5k possessed the highest cytotoxic effect. Similarly, the treatment of HL-60 cells with the tested compounds demonstrated that compounds 5a, 5b, 5e, 5h and 5k are the most potent cytotoxic effect. The obtained results indicated that compounds with the higher number of methoxy substituents have greater potential to exert cytotoxic activity as evidenced by the relatively higher cytotoxicity of compounds 5a, 5b, 5e, and 5h against all the tested cell lines. Additionally, compound 5a, with the highest number of methoxy groups displayed the most potent cytotoxic activity against all cell lines tested with IC50 values of 12.01, 7.52 and 9.7 µM against HepG2, MCF-7 and HL-60 cells, respectively.

Treatment of HepG2 cells revealed that compounds 5a, 5b, 5h and 5k showed a promising inhibitory effect of cellular tubulin, while the other tested compounds showed a variable but lower inhibitory effect. Compounds 5a and 5b were the most potent tubulin polymerization inhibitors in HepG2 cells in agreement with their relatively higher cytotoxic activity. Treatment of HepG2 cells with compounds 5a, 5b, 5h and 5k showed a variable promising inhibitory effect of cellular localization of tubulin. Compound 5h exhibited a reduced microtubules mesh area in relation to the total cellular content with impaired distribution and low concentration of tubulin, with noticeable absence the cell-cell microtubule connection. The compound 5a target S-phase at the G2/M phase and mitotic arrest. The mechanism of action for 5a as tubulin inhibitor involves the perturbation of microtubule dynamics during the G2/M phase of cell division, accumulation at S phase and subsequent entry into apoptosis (Fig. 3). In conclusion, most of

Fig. (2). Fluorescence intensity (IFU) of tubulin localization in HepG-2 cells after the treatment with test compounds, using Apotome fluorescence microscope (x400). The cells were stained with tubulin antibodies and further conjugated with FITC-labeled IgG antibodies.

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the prepared 1,3,4-oxadiazole-2-thione derivatives of remarkable anticancer activity showed tubulin polymerization inhibitory activity which may be the expected mechanism of action of these compounds.

CONFLICT OF INTEREST

The author(s) confirm that this article content has no conflict of interest.

ACKNOWLEDGEMENTS

The authors acknowledge the science and technology development fund (STDF) Egypt (project No. 2943 Basic and Applied Research) for funding this work.

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Received: September 27, 2014 Revised: May 19, 2015 Accepted: September 03, 2015