Synthesis and Evaluation of New Pyrazoline Derivatives as Potential Anticancer Agents

New pyrazoline derivatives were synthesized and evaluated for their cytotoxic effects on AsPC-1 human pancreatic adenocarcinoma, U87 and U251 human glioblastoma cell lines. 1-[((5-(4-Methylphenyl)-1,3,4-oxadiazol-2-yl)thio)acetyl]-3-(2-thienyl)-5-(4-chlorophenyl)-2-pyrazoline (11) was found to be the most effective anticancer agent against AsPC-1 and U251 cell lines, with IC50 values of 16.8 µM and 11.9 µM, respectively. Tumor selectivity of compound 11 was clearly seen between Jurkat human leukemic T-cell line and human peripheral blood mononuclear cells (PBMC). Due to its promising anticancer activity, compound 11 was chosen for apoptosis/necrosis evaluation and DNA-cleavage analysis in U251 cells. Compound 11-treated U251 cells exhibited apoptotic phenotype at low concentration (1.5 µM). DNA-cleaving efficiency of this ligand was more significant than cisplatin and was clearly enhanced by Fe(II)-H2O2-ascorbic acid systems. This result pointed out the relationship between the DNA cleavage and the cell death.


Introduction
Cancer is not a single disease, but a large group of diseases characterized by uncontrolled, rapid, and pathological proliferation of abnormally transformed cells. Despite recent advances in cancer therapy, cancer is still the second leading cause of death after cardiovascular disorders throughout the world [1][2][3][4].
Resistance to chemotherapeutic agents remains a key challenge in the fight against cancer. Another challenge for chemotherapy is lack of selectivity. Generally anticancer drugs destroy normal cells as well as cancer cells and often cause serious adverse effects. Therefore, new antineoplastic agents are continually under development to selectively destroy tumour cells or at least limit their proliferation [1][2][3][4].
Prompted by the aforementioned findings and in the continuation of our ongoing research in the field of design, synthesis, and biological evaluation of pyrazoline derivatives [30][31][32][33][34][35], herein we described the synthesis and evaluation of a new series of heteroaryl substituted pyrazolines as potential anticancer agents against AsPC-1 human pancreatic adenocarcinoma and two glioblastoma cell lines, U87 and U251 cell lines. Furthermore, tumor selectivity test on blood cells (PBMC and Jurkat cells) and the apoptotic, necrotic, and DNA-cleavage analysis against U251 cells were carried out using the most effective compound.
In the 1 H-NMR spectra of the compounds, the CH2 protons of the pyrazoline ring resonated as a pair of doublets at δ 3.16-3.24 ppm (C4-HA), 3.89-3.94 ppm (C4-HB). The CH proton appeared as a doublet of doublets at δ 5.57-5.63 ppm (HX) due to the vicinal coupling with two magnetically non-equivalent protons of the methylene group at position four of the pyrazoline ring (JAB = 17.60-18.00 Hz, JAX = 4.00-4.80 Hz, JBX = 11.20-12.00 Hz). The CH2 protons of the acetyl group at position 1 of the pyrazoline ring were observed at 4.32-4.83 ppm as a doublet (J = 15. . This geminal coupling resulted from the steric structure of the compound. These geminal protons were observed as a doublet due to two different possible conformations since rigid protons occurred ( Figure 1). All the other aromatic and aliphatic protons were observed at expected regions. In the 13 C-NMR spectra of the compounds, the signal due to the carbonyl carbon appeared at 163.92-166.37 ppm. The 13 C-NMR chemical shift values of the carbon atoms at 43.34-43.42 ppm (C4), 60.04-60.24 ppm (C5) and 151.47-152.38 ppm (C3) corroborate 2-pyrazoline character deduced from the 1 H-NMR data. The signal due to the S-CH2 carbon was observed in the region 34.30-39.72 ppm. The other aromatic and aliphatic carbons were observed at expected regions. The mass spectral data of the synthesized compounds were found in full agreement with the proposed structures. All compounds gave satisfactory elemental analysis.
Compounds 1-12 were evaluated for their cytotoxic effects on these cell lines by MTT assay ( Figure 2 and Table 1), to determine their anticancer potential and selectivity. The activity of the tested compounds was influenced considerably by the nature of the aryl group. Compounds 1, 10, 11, and 12 were found to possess IC50 values lower than 500 μM against all three cell lines. Tetrazole-substituted compound 2 did not show activity, whereas triazole-substituted compound 1 was active. Generally, oxadiazole-substituted compounds 10, 11, and 12 exhibited good activity. The most effective cytotoxic agent against AsPC-1 and U251 cancer cell lines was found to be compound 11 with IC50 values of 16.8 µM, and 11.9 µM respectively, followed by compound 12 with respective IC50 values of 62.1 µM, and 70.1 µM. On the other hand, these compounds showed no significant cytotoxicity at the concentrations used for the U87 cell line.     AsPC-1, U87 and U251 cell lines ( Figure 3a). Swelling ratio curves of AsPC-1 and U251 cell lines suggested biphasic mechanism of compound 11. Moreover, the activity of compound 11 against peripheral blood mononuclear cells (PBMC) (IC50 = ~1000 μM) and Jurkat human leukemic T-cells (IC50 = 90 μM) indicated significant tumor selectivity in blood cells (more than 10 times) as shown in Figure 3b. This outcome pointed out the importance of 5-(4-methylphenyl)-1,3,4-oxadiazol-2-yl moiety for anticancer activity. According to the MTT assay results, the most active anticancer compound 11 was chosen for the evaluation of apoptosis and necrosis in U251 cells, which was carried out with annexin V/ethidium homodimer III staining method. U251 cell lines were incubated with compound 11 or cisplatin at the IC50 concentrations. U251 cells were stained and observed by fluorescence microscope (Figure 4). If the cells are colored green with annexin V, and not stained red with ethidium homodimer III, the cells are judged to be apoptotic. On the other hand, the completely opposite results indicate necrosis. The apoptotic and necrotic effects of compound 11 were compared with cisplatin. The results indicate that cisplatin has only apoptotic effects at 24 h ( Figure 4b). In contrast, U251 cell lines treated with compound 11 at 24 h showed late stage apoptotic or necrotic effect (almost all cells were colored yellow, data not shown). Therefore, the apoptotic/necrotic effects of compound 11 were tested against U251 cell line in earlier time (3h) at IC50 (11.9 µM) and low (1.   The DNA cleavage activities of compound 11 and cisplatin at the IC50 concentrations in water and Tris/boric acid buffer in the presence and absent of the iron complex, H2O2 and ascorbic acid as an activator were studied using supercoiled pUC19 DNA ( Figure 5). The reaction mixture was incubated at 37 °C for 1.5 h and then agarose gel electrophoresis was performed at 100 V for 40 min. DNA was visualized by photographing the fluorescence of intercalated ethidium bromide under a UV illuminator. Control experiments using compound 11 with and without FeSO4, H2O2, and ascorbic acid showed that the DNA cleavage efficiency was clearly enhanced in the case of iron (II) complex system ( Figure 5b). Thus, it is considered that DNA-cleavage was caused by oxygen activation. The DNA-cleaving efficiency of compound 11 was much greater than cisplatin. Compound 11 disintegrated pUC 19 DNA and these results suggest the relationship between the DNA cleavage and the cell death.

Chemistry
All reagents were purchased from commercial suppliers and were used without further purification. Melting points were determined on an Electrothermal 9100 melting point apparatus (Weiss-Gallenkamp, Loughborough, UK) and were uncorrected. IR spectra were recorded on a Shimadzu 8400 FT-IR spectrophotometer (Shimadzu, Tokyo, Japan). 1 H-NMR and 13 C-NMR spectra were recorded on a Varian Mercury-400 FT-NMR spectrometer (Agilent, Palo Alto, CA, USA). Mass spectra were recorded on an Agilent LC-MSD-Trap-SL Mass spectrometer (Agilent Technologies). Elemental analyses were performed on a Perkin Elmer EAL 240 elemental analyzer (Perkin-Elmer, Norwalk, CT, USA). Thin Layer Chromatography (TLC) was performed on TLC silica gel 60 F254 aluminium sheets (Merck, Darmstadt, Germany) using petroleum ether:ethyl acetate (3:1 v/v) as an eluent.

Cell Culture and Drug Treatment
U251 and U87 human glioblastoma cells were incubated in Dulbecco's-modified Eagle's medium (DMEM) (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) (Equitech-Bio, Kerrville, TX, USA). AsPC-1 human pancreas adenocarcinoma and Jurkat human leukemic T-cells were incubated RPMI 1640 (Wako Pure Chemical Industries), supplemented with 10% FBS. Peripheral blood mononuclear cells (PBMC) (Precision Bioservices, Frederick, MD, USA) were incubated RPMI 1640 and supplemented with 10% human serum AB (HS) (Gemini, Woodland, CA, USA). All media were supplemented with 89 µg/mL streptomycin (Meiji Seika Pharma, Tokyo, Japan) and cells were incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Growing cells were plated at 1 × 10 5 cells/mL into 24-well microtiter tissue culture plates (Iwaki brand Asahi Glass Co., Chiba, Japan) and incubated for 24 h before the addition of the drugs (the optimal cell number for cytotoxicity assays was determined in preliminary experiments). Stock solutions (1 mM, 5 mM, 10 mM, 20 mM, and 50 mM) of compounds and cisplatin (Sigma-Aldrich, St. Louis, MO, USA) were prepared in dimethyl sulfoxide (DMSO; Wako Pure Chemical Industries) and in dimethylformamide (DMF; Wako Pure Chemical Industries) respectively, then were added to fresh culture medium. The concentration of DMSO and DMF in the final culture medium was 1%.

MTT Assay for Cytotoxicity of Compounds
The level of cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Dojindo Molecular Technologies, Kumamoto, Japan) was quantified as previously described in the literature with small modifications [38]. The tested compounds were incubated with cells to give a final concentration in the range 50-500 µM for 24 h. At the end of this period, MTT was added to cells in culture to give a final concentration of 0.275 mg/mL and incubated further for 4 h at 37 °C. The medium was removed and the formazan crystals were solubilized by addition of 100 µL DMSO to each well. After the solubilized crystals were diluted (1:10) with DMSO, 100 µL was transferred to wells of 96-well microtiter plates (Iwaki brand Asahi Glass Co.) and the absorbance at 550 nm was measured using a microplate spectrophotometer Infinitive M1000 (Tecan, Groding, Austria). Every concentration was repeated in three wells and IC50 values were defined as the drug concentrations which reduced absorbance to 50 % of control values.

Detection of Apoptotic and Necrotic Cells
U251 cells were incubated with compound 11 at IC50 concentration for 3 h and 24 h. Then, apoptotic/necrotic/healthy cells detection kit protocol was applied according to the manufacturer's instruction manual (PromoKine, Heidelberg, Germany) [39,40]. After the cells were washed twice with 1 × binding buffer, a staining solution containing 50 µL of 1 × binding buffer, 2 µL of FTIC-Annexin V solution, 2 µL of ethidium homodimer III solution and 2 µL of Hoechst 33342 solution was added and the cells were incubated for 15 min at RT, protected from light. Cells were washed with 1 × binding buffer and analyzed by all-in-one fluorescence microscope Biorevo Fluorescence BZ-9000 (Keyence, Osaka, Japan).

Conclusions
In the present paper, new pyrazoline derivatives were synthesized and investigated for their antiproliferative effects on AsPC-1 human pancreatic adenocarcinoma, U87, and U251 human glioblastoma cell lines. Detailed investigation of compound 11 against AsPC-1, U87, U251 cell lines and tumor selectivity of this compound on blood cells (PBMC and Jurkat cells) were also carried out. Among these compounds, compound 11 was the most effective anticancer agent against AsPC-1 and U251 cancer cell lines and exhibited significant tumor selectivity. Therefore, compound 11 was chosen for apoptosis/necrosis evaluation and DNA-cleavage analysis in U251 cells. Compound 11-treated U251 cells showed apoptotic activity at low concentration (1.5 µM). Interestingly, DNA-cleaving efficiency of this ligand was more significant than cisplatin. This outcome pointed out the relationship between the DNA cleavage and the cell death.
In the view of this study, further research can be carried out on the development of new effective anticancer agents by the modification of compound 11.