Cytotoxicity of Hydrazones of Morpholine Bearing

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Cytotoxicity of Hydrazones of Morpholine Bearing Mannich Bases Towards Huh7 and T47D Cell Lines and Their Effects on Mitochondrial Respiration Kaan Kucukoglua, Halise Inci Gula,*, Mustafa Gulb, Rengul Cetin-Atalayc, Yosra Baratlid and Bernard Genyd a

Ataturk University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Erzurum, Turkey

b

Ataturk University, Faculty of Medicine, Department of Physiology, Erzurum, Turkey

c

Cancer Systems Biology Lab. Graduate School of Informatics, METU TR-06800 Ankara, Turkey

d

Université de Strasbourg, Faculté de Médecine-EA 3072, Fédération de Médecine Translationnelle, Strasbourg, France Abstract: N,N’-bis[1-(substitutedphenyl)-3-(morpholine-4-yl)propylidene]hydrazine dihydrochloHalise I. Gul rides, N1-N11 were designed and synthesized as cytotoxic agents. These compounds were synthesized by the reaction of 2 moles of 1-(substitutedphenyl)-3-(morpholine-4-yl)-1-propanone hydrochlorides with 1 mole of hydrazine hydrate. The compounds reported here are new, except N1 and N4. The cytotoxicity of the compounds was tested against human hepatoma (Huh7) and breast cancer (T47D) cell lines. 5-Fluorouracil (5-FU) was used as a reference compound. It was found that N3, which has 4-methoxy substituent on phenyl ring, was the most cytotoxic compound towards both cell lines. Its cytotoxicity was 5.6 times higher than 5-FU. Representative compounds N2 at 144, 264 and 424 µM and N3 at 401 µM concentrations significantly inhibited mitochondrial respiration in a dose dependent manner in liver homogenates. This suggests that the inhibition of mitochondrial respiration may be one of the contributing mechanisms to the cytotoxicity of the compounds. N3 may serve as a candidate compound for further studies.

Keywords: Cytotoxic activity, Huh7, hydrazone, Mannich base, mitochondrial respiration, T47D. INTRODUCTION Breast cancer is the most common cancer in women, and it is one of the five common cancers with an estimated 1.15 million new cases worldwide in 2002 [1]. However, the treatment of breast cancer with chemotherapeutics induces significant side effects and drug resistance [2]. Hepatocellular carcinoma (HCC) is also a major health problem and causes morbidity and mortality worldwide. During the past two decades, incidence rates of HCC have nearly been tripled [3]. Some factors such as hepatitis viral infection (hepatitis B or C virus, HBV/HCV), chronic alcoholism and long-term exposure to aflatoxin B1 are wellknown risk factors for HCC [4]. Tumor resection and liver transplantation are the standard treatment procedures of HCC [5]. Chemotherapy is one of the options, however, HCC cells have high resistance to the present chemotherapeutic agents [6]. Thus, there is an urgent need to develop new compounds, which can be effective in breast cancer and hepatocellular carcinoma. Hydrazones are the condensation compounds of aldehyde or ketones with hydrazine and show several bioactivities *Address correspondence to this author at the Ataturk University Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 25240 Erzurum, Turkey; Tel: +90-442-2315219; Fax: +90-442-2315201; E-mail: [email protected] 1570-1808/16 $58.00+.00

such as antimicrobial, antimalarial, cytotoxic and anticancer activities [7]. Compounds containing a reactive hydrogen atom, formaldehyde and secondary amines react to form aminomethylated compounds, Mannich bases, in general [8]. Mannich bases are a group of compounds known with several bioactivities such as antifungal [9], anti-inflammatory [10, 11], anticonvulsant [12, 13] cytotoxic and anticancer [14-22] activities. Mannich bases may undergo deamination process to liberate α,β-unsaturated ketone moiety, which is responsible for their cytotoxic activities by alkylating cellular nucleophiles, thiols [23-27]. There are some studies trying to explain the mechanism of action of Mannich bases. These are focused on alkylation of cellular thiols [23-27], inhibition of mitochondrial respiration [19], and, inhibition of topoisomerase enzyme I [20] or II [28]. In tumour cells, the pH value was determined to change from 6.83 to 6.43 due to the accumulation of acidic metabolites [29]. Tumour cells produce large quantities of lactic acid from glucose via metabolic pathways such as glycolytic pathway [30]. Therefore, the design of pH-selective anticancer agents or acid-labile prodrugs against a wide variety of human cancers can be important. While hydrazone bond is relatively stable under neutral conditions in blood plasma (pH 7.4), it is cleavable under slightly acidic conditions (pH ca. 5) as in the interstitial space of most solid tumours and in the endosomal environment [31]. Adriamycin, which is an anthracycline antibiotic used in cancer treatment, is conju©2016 Bentham Science Publishers

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R2

R1 O

R2

R2

R1 O CH3

R3

a

R1 O .HCl

N

R3 R4

N

R4 R4

O R4

N

R3 b

.2HCl

N

R3

N O R2

N1m-N11m

R1 N1-N11

Scheme (1). Synthesis of N,N’-Bis[1-(substituted phenyl)-3-(morpholine-4-yl)propylidene]hydrazine dihydrochlorides, N1-N11. Reagents and conditions: (a) Paraformaldehyde, Morpholine HCl, HCl (37%) and EtOH, 1-8 h, for N1m-N11m; (b) Ethanolic acetic acid (3% w/v), Hydrazine hydrate. R1 = R2 = R3 = R4 = H (N1); R1 = R2 = R4 = H, R3 = CH3 (N2); R1 = R2 = R4 = H, R3 = OCH3 (N3); R1 = R2 = R4 = H, R3 = OH (N4); R1 = R2 = R4 = H, R3 = Cl (N5); R1 = OH, R2 = R3 = R4 = H (N6); R1 = R3 = R4 = H, R2 = OCH3 (N7); R1 = R2 = R4 = H, R3 = F (N8); R1 = R2 = R4 = H, R3 = Br (N9); R1 = R3 = R4 = H, R2 = OH (N10); R1 = OCH 3, R2 = R3 = R4 = H (N11).

gated to the coreforming segments via the hydrazone linkers in case they are cleaved under acidic intracellular environments in endosomes and lysosomes (pH 5-6) in the literature [32]. In this study, first we aimed to synthesize hydrazones of mono Mannich bases as prodrugs, which can liberate Mannich bases in acidic environment of tumour cells, and secondly, we have evaluated their cytotoxicity towards human hepatoma (Huh7) and breast cancer (T47D) cell lines. We also tested some representative compounds to see their effects on mitochondrial respiration. MATERIALS AND METHODS Material and Instruments Chemicals used in the synthesis of N and Nm series compounds were purchased from J.T. Baker Chemical Company (Phillipsburg, NJ), Fluka AG (Buchs, Switzerland), Merck-Schuchardt (Hohenbrunn, Germany), Sigma-Aldrich Chemical Co. (St. Louis, MO), Acros Organics Chemical Co. (Fair Lawn, NJ) and Riedel-de Haën (Seelze, Germany). Uncorrected melting points were measured on an Electrothermal 9100 melting point apparatus (IA9100, UK). 1 H NMR (400 MHz) and 13C NMR (100 MHz) spectra of N series compounds were recorded employing a Varian 400 MHz FT spectrometer (Danbury, USA), and 1H NMR (60 MHz) spectra of Nm series compounds were recorded on a Varian EM-360. Chemical shifts were reported in (δ) ppm. TMS, were used as an internal standard. Coupling constants (J) are reported in Hertz. High resolution mass spectra were taken on a V6 Waters Micromass ZQ (Waters Corporation, USA) using TOF method. Chemistry General Procedure for the Synthesis of Precursor Mono Mannich Bases, 1-(Substituted phenyl)-3-(morpholine-4yl)-1-propanone Hydrochlorides (N1m-N11m, Scheme 1) The mixture of corresponding acetophenone (1 mol), paraformaldehyde (1.5 mol) and morpholine hydrochloride (1 mol) was dissolved in ethanol. HCl (37%) was added into the reaction mixture dropwise. Reaction content was re-

fluxed for 1-8 h. The solvent was removed in vacuo. The residue obtained was crystallized from suitable solvent, and if necessary reaction content was kept at 4°C. The crystals formed were filtered, washed and then dried. Acetophenones used in N1m-N11m and their corresponding hydrazones N1N11 can be find out from Scheme (1). The amount of suitable ketone (mmol), heating period (h), melting point (°C), crystallization solvent and yield (%) of the compounds N1mN11m (Scheme 1) were as follows: N1m (40 mmol, 8 h, 165-169°C, ethanol, 14%), N2m (30 mmol, 8 hours, 213217°C, ethanol, 46%), [33], N3m (20 mmol, 8 hours, 215216°C, ethanol, 64%), [34], N4m (20 mmol, 1 h, 217-218°C, methanol, 25%) [35], N5m (10 mmol, 3 h, 208-209°C, methanol, 43%) [36], N6m (20 mmol, 8 h, 202-205°C, methanol, 37%) [37], N7m (20 mmol, 8 h, 155-157°C, methanol, 47%) [38], N8m (10 mmol, 8 h, 215-217oC, ethanol, 68%) [33], N9m (10 mmol, 2 h, 214-217oC, ethanol, 44%) [39], N10m (20 mmol, 3.5 h, 195-198oC, ethanol, 51%) [40], N11m (10 mmol, 8 h, 135-137oC, ethanol, 64%) [41, 42]. All the synthesized precursor mono Mannich bases were already available in the literature so NMR data of them are not reported here. General Procedure for the Synthesis of Hydrazone Compounds, N,N’-bis[1-aryl-3-(morpholine-4-yl)propylidene] Hydrazine Dihydrochlorides (N1-N11, Scheme 1) A solution of hydrazine hydrate in ethanol was added to a solution of suitable compound of N1m-N11m in ethanolic acetic acid (3% w/v) in 1:2 mol ratios, respectively. The mixture was stirred at room temperature for 18-24 h. The precipitated compound was filtered, dried and then crystallized from suitable solvent to give the corresponding hydrazone of N1-N11 (Scheme 1) [43]. Experimental data of N1N11 are shown in Table 1. N,N’-Bis[3-(morpholine-4-yl)-1-(phenyl)propylidene]hydrazine Dihydrochloride (N1) 1

H NMR (CD3OD) δ [ppm]: 3.00-3.50 (m, 8H), 3.603.66 (m, 8H), 3.82-3.99 (m, 8H), 7.51-7.56 (m, 6H), 8.058.08 (m, 4H); 13C NMR (CD3OD) δ [ppm]: 23.4, 52.0, 53.7, 63.8, 127.5, 129.0, 131.1, 136.0, 163.3; HRMS (m/z):

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Table 1. Experimental data of N-series hydrazone compounds, N1-N11. Compound

Formula

MW

Time (h)

Crystallization Solvent

Yield (%)

Melting Point (oC)

N1

C26H 36Cl2N4O 2

506.22

24

Methanol

24

184-6*

N2

C28H 40Cl2N4O 2

534.25

24

Chloroform/Methanol

26

198-201

N3

C28H 40Cl2N4O 4

566.24

20

Methanol

67

214-6

N4

C26H 36Cl2N4O 4

538.21

20

Ethanol

56

218-9

N5

C26H 34Cl4N4O 2

574.14

22

Chloroform/Methanol

60

199-200

N6

C26H 36Cl2N4O 4

538.21

24

Ethanol

16

114-7

N7

C28H 40Cl2N4O 4

566.24

20

Ethanol

9

149-51

N8

C26H 34Cl2F2N4O 2

542.2

22

Ethanol

52

207-9

N9

C26H 34Br 2Cl2N4O 2

662.04

22

Chloroform/Methanol

56

209-10

N10

C26H 36Cl2N4O 4

538.21

20

Methanol

86

184-5

N11

C28H 40Cl2N4O 4

566.24

18

Methyl t-buthyl ether

74

133-4

* Reported melting point of N1 was 173-175°C [43].

calculated for C26H34N4O2: 435.2760 (M+1)+, Found: 435.2763. N,N’-Bis[1-(4-methylphenyl)-3-(morpholine-4-yl)propylidene]hydrazine Dihydrochloride (N2) 1

H NMR (CD3OD) δ [ppm]: 2.42 (s, 6H), 3.00-3.50 (m, 8H), 3.50-3.75 (m, 8H), 3.75-4.02 (m, 8H), 7.35 (d, 4H, J = 7.6 Hz), 7.95 (d, 4H, J = 8.0 Hz); 13C NMR (CD3OD) δ [ppm]: 20.4, 32.3, 43.5, 52.5, 63.8, 127.4, 128.2, 129.4, 133.6, 145.2; HRMS (m/z): calculated for C28H38N4O2 : 463.3073 (M+1)+, Found: 463.3063. N,N’-Bis[1-(4-methoxyphenyl)-3-(morpholine-4-yl)propylidene]hydrazine Dihydrochloride (N3) 1

H NMR (DMSO-d6) δ [ppm]: 3.00-3.70 (m, 16H), 3.743.97 (m, 8H), 3.84 (s, 6H), 7.07 (d, 4H, J = 8.8 Hz), 7.97 (d, 4H, J = 8.8 Hz), 11.27 (bs, 2H); 13C NMR (DMSO-d6) δ [ppm]: 32.8, 51.8, 51.9, 56.3, 63.9, 114.7, 129.6, 131.1, 164.2, 195.6; HRMS (m/z): calculated for C28H38N4O4: 495.2971 (M+1)+, Found: 495.2952. N,N’-Bis[1-(4-hydroxyphenyl)-3-(morpholine-4-yl)propylidene]hydrazine Dihydrochloride (N4) 1

H NMR (CD3OD) δ [ppm]: 3.10-3.61 (m, 4H), 3.743.88 (m, 12H), 4.01-4.12 (m, 8H), 6.87 (d, 4H, J = 8.8 Hz), 7.95 (d, 4H, J = 8.8 Hz); 13C NMR (CD3OD) δ [ppm]: 31.9, 52.4, 52.9, 63.8, 115.3, 127.9, 130.8, 163.3, 195.0; HRMS (m/z): calculated for C26H34N4O4: 465.2326 (M+1)+, Found: 465.2322. N,N’-Bis[1-(4-chlorophenyl)-3-(morpholine-4-yl)propylidene]hydrazine Dihydrochloride (N5) 1

H NMR (CD3OD) δ [ppm]: 3.10-3.45 (m, 4H), 3.503.61 (m, 12H), 3.61-4.06 (m, 8H), 7.56 (d, 4H, J = 8.8 Hz),

8.04 (d, 4H, J = 8.8 Hz); 13C NMR (CD3OD) δ [ppm]: 32.6, 52.5, 52.5, 63.8, 129.0, 129.8, 134.6, 140.1, 195.4; HRMS (m/z): calculated for C26H32Cl2N4O2: 503.1981 (M+1)+, Found: 503.2002. N,N’-Bis[1-(2-hydroxyphenyl)-3-(morpholine-4-yl)propylidene]hydrazine Dihydrochloride (N6) 1

H NMR (CD3OD) δ [ppm]: 3.13 (t, 4H, J = 8.6 Hz), 3.26 (t, 4H, J = 9.2 Hz), 3.28-3.34 (m, 8H), 3.46-3.49 (m, 8H), 6.80-7.00 (td, 2H, J = 8.6 and 1.5 Hz), 7.28 (dd, 2H, J = 8.1 and 1.5 Hz), 7.20-7.50 (td, 2H, J = 8.6 and 1.5 Hz), 7.96 (dd, 2H, J = 8.1 and 1.5 Hz); 13C NMR (CD3OD) δ [ppm]: 32.9, 36.5, 51.3, 52.8, 115.9, 117.9, 119.1, 127.7, 130.2, 130.4, 136.3; HRMS (m/z): calculated for C26H34N4O4: 467.18 88 (M+1)+, Found: 467.1880 N,N’-Bis[1-(3-methoxyphenyl)-3-(morpholine-4-yl)propylidene]hydrazine Dihydrochloride (N7) 1

H NMR (CDCl3) δ [ppm]: 3.10-3.81 (m, 16H), 3.85 (s, 6H), 3.91-4.40 (m, 8H), 7.15 (d, 2H, J = 8.2 Hz), 7.39 (t, 2H, J = 7.8 Hz), 7.48 (s, 2H), 7.59 (d, 2H, J = 7.8 Hz), 10.19 (bs, 2H); 13C NMR (CDCl3) δ [ppm]: 33.1, 43.3, 52.6, 55.7, 63.9, 112.5, 120.9, 121.2, 130.1, 136.9, 160.2, 195.9; HRMS (m/z): calculated for C28H38N4O4: 495.2971 (M+1)+, Found: 495.2947. N,N’-Bis[1-(4-fluorophenyl)-3-(morpholine-4-yl)propylidene]hydrazine Dihydrochloride (N8) 1

H NMR (CD3OD) δ [ppm]: 3.10-3.40 (m, 4H), 3.453.75 (m, 12H), 3.75-4.05 (m, 8H), 7.26 (t, 4H, J = 8.8 Hz), 8.11-8.15 (m, 4H); 13C NMR (CD3OD) δ [ppm]: 32.5, 52.4, 52.5, 63.8, 131.0, 131.1, 132.7, 167.7, 195.0; HRMS (m/z): calculated for C26H32F2N4O2: 470.2502 (M+1)+ Found: 470.2501.

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N,N’-Bis[1-(4-bromophenyl)-3-(morpholine-4-yl)propylidene]hydrazine Dihydrochloride (N9) 1

H NMR (D2O) δ [ppm]: 3.10-3.40 (m, 4H), 3.40-3.70 (m, 12H), 3.70-4.20 (m, 8H), 7.65 (d, 4H, J = 8.4 Hz), 7.80 (d, 4H, J = 8.1 Hz); 13C NMR (D2O) δ [ppm]: 32.7, 52.3, 52.4, 64.0, 129.3, 130.0, 132.4, 134.5, 198.8; HRMS (m/z): calculated for C26H32Br2N4O2: 591.0970 (M+1)+, Found: 591.0977. N,N’-Bis[1-(3-hydroxyphenyl)-3-(morpholine-4-yl)propylidene]hydrazine Dihydrochloride (N10) 1

H NMR (CD3OD) δ [ppm]: 3.10-3.40 (s, 4H), 3.40-3.75 (m, 12H), 3.75-4.2 (m, 8H), 7.07 (dd, 2H, J = 8.1 and 1.9 Hz), 7.35 (t, 2H, J = 7.8 Hz), 7.42 (t, 2H, J = 1.9 Hz), 7.52 (dt, 2H, J = 7.8 and 1.6 Hz); 13C NMR (CD3OD) δ [ppm]: 32.6, 52.5, 52.7, 63.8, 114.3, 119.4, 120.9, 129.8, 137.4, 158.0, 196.5; HRMS (m/z): calculated for C26H34N4O4: 467.2677 (M+1)+, Found: 467.2657. N,N’-Bis[1-(2-methoxyphenyl)-3-(morpholine-4-yl)propylidene]hydrazine Dihydrochloride (N11) 1

H NMR (CDCl3) δ [ppm]: 2.95-3.05 (m, 4H,), 3.25-3.75 (m, 12H), 3.77-4.20 (m, 8H), 3.97 (s, 6H), 6.95 (d, 2H, J = 7.4 Hz), 7.00 (t, 2H, J = 7.4 Hz), 7.51 (t, 2H, J = 7.8 Hz), 7.74 (d, 2H, J = 7.4 Hz), 10.06 (bs, 2H); 13C NMR (CDCl3) δ [ppm]: 38.1, 43.4, 52.7, 56.1, 64.0, 112.1, 121.0, 126.4, 130.8, 135.1, 159.5, 197.6; HRMS (m/z): calculated for C28H38N4O4 : 495.2971(M+1)+, Found: 495.2950. Biological Activity Cytotoxic Activity Assay Against Human Hepatoma and Breast Cancer Cells Cytotoxic activity tests of the drug candidates were carried out in KANILTEK screening lab in Bilkent University, Ankara, Turkey. N Series hydrazone compounds, N1-N11, were tested with (NCI) anticancer drug screening method and identifying N1-N11 with growth-inhibitory activity was aimed against human hepatoma cells (Huh7) and breast cancer cells (T47D). 5-Fluorouracil (5-FU) was used as the reference compound [44]. T47D and Huh7 cells (5000 or 10000) were inoculated to treatment with inhibitors into 96 well microtiter plates in 100 µl of standard medium 24 hours prior. N-Series hydrazone compounds, N1-N11, with various concentrations (2.5-40 µM) were applied in additional 100 µl of cell culture medium. After the cells were treated with inhibitors for 72 hours, they were fixed by gentle addition of 50 µl of cold 50% (w/v) trichloroacetic acid (TCA) for 60 minutes at 4°C. Next, cell culture medium was discarded and distilled water was used as washing material and dried in air. Then, 100 µl of 0.4% Sulforhodamine B (SRB) solution was applied to each well and allowed to incubate for 10 minutes at room temperature. Extra unbound dye was washed five times with 200 µl of 1% acetic acid and air-dried. SRB dye, which was bound to cellular proteins, was then solubilized by adding 200 µl of 10 mM Tris-Base solution and the absorbances were acquired at 515 nm. Finally, IC50 values were calculated [21, 44].

Kucukoglu et al.

Inhibition of Mitochondrial Respiration Assay Animals Experiments were performed on adult male Wistar rats (6-11) aged 8 weeks which were housed in a thermo-neutral environment (22 ± 2ºC), on a 12:12 h photoperiod. They were provided food and water ad libitum. This investigation was performed in accordance with the Guide for the Care and Use of Laboratory which was published by the US National Institute of Health and approved by the institutional animal care committee (NIH publication No. 85-23, revised 1996). Rats were submitted to general anesthesia with 3% isoflurane and oxygen (1 L/min) in an induction chamber (Minerve, Esternay, France). The liver was excised, cleaned and then immediately used for mitochondrial respiratory parameters analysis [45]. Study of Mitochondrial Respiration Isolation of Liver Mitochondria The liver was maintained on ice finely minced in ice-cold isolation buffer (50 mM tris, 1 mM EGTA, 70 mM Sucrose, 210 mM Mannitol, pH 7.4 at 4°C), and homogenized with a Potter-Elvehjem device. The obtained homogenate was centrifuged at 1300 g for 3 min at 4°C. Then the supernatant was ultracentrifuged at 10000 g for 10 min at 4°C to sediment mitochondria. Finally, the mitochondrial pellet was washed twice. Then it was suspended in 50 mM Tris, 70 mM Sucrose, 210 mM Mannitol, pH 7.4 at 4°C [46, 47]. Protein content was routinely assayed with a Bradford assay and bovine serum albumin (BSA) was used as a standard until used mitochondria were kept on ice. Measurement of the Activities of the Mitochondrial Respiratory Chain Complexes A Clark type electrode (Strathkelvin Instruments, Glasgow, UK) was used to measure mitochondrial respiration as mentioned before [48]. Before oxygraph measurement, 3 ml of solution M (100 mM KCl, 50 mM Mops, 1 mM EGTA, 5 mM Kpi, 1 mg/ml BSA) was added to the oxygraph chambers for 10 min then, 0.15 mg of isolated liver mitochondria were introduced with 10 mM glutamate and 2.5 mM malate. The temperature was kept at 25°C. After that, succinate (25 mM) and adenosine diphosphate (2mM) were added. Mitochondrial respiration in these conditions allowed to determine the activities of the complexes I, II, III, IV, and V (Fig. 1). After that, the representative compounds N2 (a 4methyl derivative) and N3 (a 4-methoxy derivative) were added at different concentrations. Results are expressed as mean ± SEM. Statistical analyses were performed using one-way ANOVA followed by a Newman-Keuls post test (GraphPad Prism 5, Graph Pad Software, Inc., San Diego, CA, USA). Statistical significance required a p N5 (chlorine substituted, 48.93 µM) > N8 (fluorine substituted, 67.05 µM). When the cytotoxicity of the compounds N2, which had methyl substituent, and N3, which had methoxy substituent, were compared, the cytotoxicities of the compounds N2 and N3 were similar suggesting other factors different than hydrogen bonding may play a role in showing cytotoxicity. When cytotoxicities of the compounds towards T47D cells were compared, the compounds N1, which had non substituted phenyl ring, and N2, which had methyl substituent on phenyl ring at position 4, had similar and lowest cytotoxicities among all compounds. Except hydrogen and methyl substitutions at position 4, other substituents on phenyl ring at positions 2, 3 or 4 were useful in increasing cytotoxicity. Although the compounds synthesized had less cytotoxicity than 5-FU, the most cytotoxic compound towards T47D cell was N3, which had methoxy substituent on phenyl ring as it was also the case with Huh7 cells. When hydrogen acceptor methoxy substituent bearing compounds were compared in terms of cytotoxicity towards T47D and the position of substituent, the order of cytotoxic potency was N3 (which had a methoxy at position 4 of phenyl ring, 9.69 µM) > N11, (which had a methoxy group

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Letters in Drug Design & Discovery, 2016, Vol. 13, No. 9

at position 2, 14.54 µM) > N7, which had a methoxy at position 3, 23.96 µM. When the hydroxy groups were considered in terms of cytotoxicity and substituent position, the cytotoxic potency order was N4 (which had hydroxy at 4, 10.57 µM) > N10, which had hydroxy at 3, 31.14 µM) > N6 (which had hydroxy at 2, no inhibition). It seemed that position 4 was the most suitable position showing the highest cytotoxicity towards T47D cells among the compounds having hydrogen donor hydroxy or hydrogen acceptor methoxy substituent on phenyl ring.

[4]

When the halogen bearing compounds N5, N8, and N9 were compared with each other, fluorine bearing compound N8 with 11.08 µM IC50 value was the most potent compound towards T47D cell line.

[9]

The differences in cytotoxicities with different compounds against different cell lines may result from the chemical structure of compound, the nature of cell line used, and different mechanism of action of the compound in different cell lines. There was no correlation between cytotoxicity of the compounds towards cell lines used and log P value of the compounds, which represents solubility of the compounds in both lipid and water (data not shown). The effects of the compounds N3, which had methoxy substituent and the highest cytotoxic potential against both cell lines, and N2, which was chosen randomly to test mitochondrial respiration were investigated. The compounds N2 (at 144, 264 424 µM) and N3 (at 401 µM) inhibited mitochondrial respiration significantly. This may suggest that the inhibition of mitochondrial respiration may be one of the mechanisms contributing to the cytotoxicities of the compounds. In conclusion, N3, which had methoxy group on aryl ring at position 4, had the highest cytotoxicity among the others towards breast (T47D) and human hepatoma (Huh7) cell lines and inhibited the mitochondrial respiration dose dependently. Thus, this compound (N3) can be considered for further investigation and analogue development. CONFLICT OF INTEREST

[5]

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The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS This study was supported by the Research Foundation of Ataturk University (Project Number: 2007/58) Erzurum (Turkey) and cytotoxic experiments were performed at the KANILTEK anticancer biomolecule screening facility, Bilkent University. REFERENCES [1] [2] [3]

Parkin, D. M.; Bray, F.; Ferlay, J.; Pisani, P. Global cancer statistics, 2002. CA Cancer J Clin., 2005, 55 (2), 74-108. Bange, J.; Zwick, E.; Ullrich, A. Molecular targets for breast cancer therapy and prevention. Nat Med., 2001, 7 (5), 548-551. El-Serag, H. B. Hepatocellular carcinoma. N Eng J Med., 2011, 365 (12), 1118-1127.

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Revised: September 22, 2015

Accepted: October 29, 2015