In vitro Anti Leukemia Cancer Activity of Some Novel Pyrazole Derivatives and Pyrazoles Containing Thiazole Moiety

The design and syntheses of several novel pyrazole derivatives (2, 5, 6 and 7) and pyrazole derivatives (3 and 4) containing thiazole moiety via by ethyl β-(p-chlorophenyl)–α-cyanoacrylate (1) and thiosemicarbazide as starting materials. Pyrazole derivatives (3 and 4) containing thiazole moiety were synthesized via cyclization of pyrazole derivative (2) with bromomethyl arylketones, to give compound 3, followed by acetylation. N(3methoxy-2-hydroxybenzal) -3(pchlorophenyl)-4cyano-5-oxopyrazol-1-thiocarboxamide (6) was synthesized via reaction of compound 2 with 3-methoxy-2hydroxybenzaldehyde. Structures of all compounds were confirmed by elemental analysis, FT-IR, 1 H-NMR, 13 C-NMR and mass spectrometry. The cytotoxic activity of all the synthetic compounds were evaluated against Leukemia HL-60 compared with Doxorubicicn. The cytotoxic activity was checked in vitro for the recently prepared compounds by using the MTT assay. Compounds 4, 6 and 9 were the most active against Leukemia HL-60. The IC50 values of them were less than 5 μM in the range of 1.35-4.78 μM. In addition, compounds 3 and 5 showed less antiproliferative activity against Leukemia HL-60 cells with IC50 values in the range 5.39-8.82 μM. Compound 6 was the most potent cytotoxic activity. The studies biological activity includes cell cycle analysis, apoptosis detection assay and Topoisomerase II inhibition activity assay explained that compound 6 is a strong Topo II inhibitor.

Further, derivatives of pyrazoles have shown significant pharmacological activities, such as antitumor [56][57][58][59] antileukemic [60][61][62] and antiproliferative [63,64] agents. In addition to their ability to apply remarkable anticancer effects throughout the inhibition of different types of enzymes which play significant roles in cell division [65][66][67]. As an expansion of our preceding work [68][69][70], this research reported the syntheses of some novel heterocyclic compounds including thiazole moiety attached to pyrazolinone ring at N-1 using ethyl β-(p-chlorophenyl) -αcyanoacrylate (1) as a key starting material. These arrangements were suggested as a trial to increase the activity against leukemia cancer cells. Hoping to find out a new guide combination structure which that would have a considerable anti-leukemia potential with small concentrations.

Chemistry
The structural diversity and biological importance of pyrazole and thiazole derivatives have made them attractive targets for synthesis. The syntheses of the new heterocyclic compounds contain thiazole and pyrazole moieties (3 and 4) are described in " Figure 1".
In the 1 H-NMR spectrum of compound 2 " Figure 3" displayed two singlet signals at δ 11.43 and 7.46 ppm assignable to the protons of OH and NH 2 groups respectively, beside two doublet signals at δ7.84 & 8.02 ppm revealed to adjacent two protons of aromatic ring. IR spectrum of compound 5 displayed the lack of the absorption bands of NH 2 , but the absorption bands observed at 3216 cm -1 attributed to NH group. Bands at 1606, 1583 were attributed to C=C. Bands at 1715, 1695 were due to C=O group. Absorption band at 1625 cm -1 was due to C=N group, and band at 2252 cm -1 was due to CN group and bands 1098, 1071cm -1 revealed the presence of C-O group. 1 H-NMR spectrum of compound 5 " Figure 4" gave cut evidence singlet signal at δ 11.73 ppm attributed to the proton of NH group and two singlet signals at δ 2.21, 2.05 ppm due to two acetyl groups. The outstanding proton signals are appearing in the predictable area of basic nucleus. In 13 C-NMR spectrum of compound 5 " Figure 5" showed signal at δ 193.55 was due to C=S group. Two signals appeared in δ 169.90 and 167.94 were assignable to C=O group, in addition to two signals showed at 23 and 22.28 ppm characteristic of two methyl groups. Also, displayed signals in the range δ 154. .83 ppm corresponding to carbons of aromatic, pyrazole and cyano groups.

1-(Thiazolyl)-Pyrazole Derivatives (3 and 4)
The 1 H-NMR spectra of compound 3a " Figure 6" displayed a sharp singlet signal at δ 12.20 ppm due to the proton of OH group for the pyrazole ring at C-5. Protons of the aromatic and thiazole ring of compound 3a were showed at δ 7.39-8.05 ppm. 13 C-NMR spectrum of compound 3a " Figure 7" showed carbons of aromatic and thiazole ring noticed within the chemical shift regions and demonstrated integral values.  The 1 H-NMR spectra of compound 3b " Figure 8" showed a sharp singlet signal at δ 11.43 ppm characteristic to the proton of OH group for the pyrazole ring at C-5. Protons of the aromatic and thiazole ring of compound 3b were assignable at δ 7.45-8.33 ppm. The 1 H-NMR spectra of compound 3c " Figure 9" displayed a sharp singlet signal at 12.16 ppm due to the proton of OH group for the pyrazole rings at C-5. Protons of the aromatic and thiazole ring of compound 3c were due to δ 6.97-8.04 ppm. 1 H-NMR spectrum of compound 3c explained a sharp singlet signal at δ 3.79 ppm due to the protons of methoxy group (OCH 3 ). In the 1 H-NMR spectra of compounds 4a-c showed the absence of singlet signals in the region at δ 12.33-11.91 ppm represented to the protons of hydroxyl groups (OH), while the 1 H-NMR spectra of compounds 4a-c showed the new singlet signals in the region at δ 2.32-2.50 ppm assignment to acetyl groups (CH 3 CO). The 1 H-NMR spectrum of compound 4a " Figure 10" as example displayed two singlet signals at δ 8.80 and 2.42 ppm due to the protons of Hthiazole and methyl group. In addition, viewing multiplet signals in the region at δ 7.50-8.15 ppm corresponding to the protons of aromatic rings. In the 13 C-NMR spectrum of compound 4a " Figure   multiplets at around δ 7.44-8.18 ppm, singlet signal showed at δ 3.88 ppm due to -OCH 3 and signal showed at δ 11.35 indicating the presence of OH group.   On the other hand, 1 H-NMR spectrum of compound 7 " Figure 13" exhibited signal at δ 2.21ppm due to CH 3 CO group, a singlet signal showed at δ 3.88 ppm represented -OCH 3 group, in addition the aromatic multiplets in the region δ 6.84-8.07 ppm, signal showed at δ 11.73 due to OH group. 13 C-NMR spectrum of compound 7 " Figure 14" displayed signal at δ 167.94 ppm represented to C=O group, signals showed around δ 154.24-127.65 ppm corresponding to aromatic and pyrazole ring, signal resonating at δ 65.72 due to -OCH 3 and signal showed at δ 23 ppm stand for CH 3 group.

In vitro Anti-leukemia Activity
Compounds 3-9 were checked for their anti-proliferative activity against leukemia HL-60 human cancer cell line by means of MTT assay method. Doxorubicin (Dox) was employed as positive reference. The IC 50 values (µM) of the tested compounds and reference compound are mentioned in " Table 1

Analysis of Cell Cycle
Compound 6 had the highest promising cytotoxicty against Leukemia HL-60 cell line. So it was selected for extra investigation concerning its mechanism of cell proliferation inhibition mechanism. To discover the correlation between the cell cycle progression and anticancer effect, DNA flow cytometric assay by means of propidium iodode (PI) in Leukemia HL-60 cells after handling with compound 6 on its IC 50 concentration dose for 24 hrs. " Figure 16". It is observed that compound 6 increased the percentage of G2/M phase from 11.05% to 39.22% compared with the untreated control. This increase was accompanied by a decrease in the other states of the cell cycle profile in Leukemia HL-60. This information recommended that, compound 6 evidently increased the percentage of G2/M phase. This leads to cell growth arrest in leukemia HL-60 cells.

Apoptosis Detection Assay
According to cell death caused by compound 6, a biparamertric cytofluorimetric was analyzed using PI and Annexin-V-FITC which was in Leukemia HL-60 cells subsequent to treatment with compound 6 at its IC 50 value for 24 hrs " Figure 17". It's revealed that compound 6 increases the value of total apoptosis compared with unprocessed control group. The value of early apoptosis was increased from 1.11% to 6.74% compared with the non-treated control group. Additionally, compound 6 can increase the percentage of late apoptosis from 0.31% to 4.31% compared with untreated control. This result recommended the interest of compound 6 in the apoptosis of Leukemia HL-60 cellsinduced cell death.

Assay of Topoisomerase II
Topoisomerase II (Topo II) is an enzyme which affects on the topology of DNA and acting a vital during cell division involving DNA replication, transcription, segregation and recombination. In this study, compound 6 was evaluated for Topo II inhibition activity IC 50 value to evaluate whether the most active molecule was strong Topo II inhibitor. Five dose Topo II inhibition attempt was achieved by human DNA Topoisomerase II (Topo II) ELISA screening kit. Etoposide served as positive control in this study. As illustrated in " Figure 18", the Topo II inhibition activity assay revealed that compound 6 was efficient in inhibiting the Topo II activity with IC 50 value 56.04 nM compared with etoposide which had IC 50 value 41.11 nM. This result indicated that, compound 6 cytotoxic activity is mainly due to its potent DNA Topo II inhibition activity.

Chemistry
Melting points were measured in open capillary tubes by Electrothermal digital melting point apparatus and are uncorrected. 1 H-NMR and 13 C-NMR (400 MHz) spectra were recorded in DMSO-d6 on a Bruker 400 DRX-Avance NMR spectrometer using of TMS as internal standard and chemical shifts are expressed in δ (ppm) values. The IR spectra were recorded as KBr pellets on a Bruker (Tensor 37) FT-IR Spectrophotometer. Mass spectra were recorded on LCMS-QQQ instruments. Elemental analyses were achieved on Carlo Erba 1108 Elemental analyzer. All compounds were within ± 0.35% of the theoretical values. The reactions were monitored by thin-layer chromatography (TLC) on silica gel F254 aluminum sheets (Merck), and spots were visualized by UV lamp at 254-365 nm. Syntheses of N-(3-methoxy-2-hydroxybenzal) -3-(pchlorophenyl)-4-cyano-5-oxopyrazol-1-thiocarboxamide (6) A mixture of equimolar quantity of compound 2 (0.01mol) and 3-methoxy-2-hydroxybenzaldehyde (0.01mol) in 30mL acetic acid was heated under reflux for 2hrs. The reaction mixture was cooled and added to water and neutralized with dilute sodium bicarbonate (2%). The product formed was General Procedure for acetylation reactions A solution of compounds 2, 3, and 6 (0.01mol) in 30mL acetic anhydride was heated under reflux for 2 hrs, after that cooled & poured into ice-water. The reaction mixture was kept for 24hrs, and the producing solid was filtered, washed by water, dried and finally crystallized from ethanol to give 4, 5 and 7.

Anti-tumor Activity Against Leukemia Cancer Cell
Line (Leukemia HL-60) The cytotoxic activity was checked in vitro for the recently prepared compounds by using the MTT assay. Cells were plated in 96-multiwall plate (10 5 cells/well) for 24 hrs before handling with the compounds. Examined compounds were dissolved in dimethyl sulfoxide. Several concentrations of the compound under assessment (10, 25, 50, and 100 µM) were added to the cell's monolayer. Triplicate wells were arranged for each individual concentration. Monolayer cells were incubated with the compound(s) for 48 hrs at 37°C and in atmosphere of 5% CO 2 . After 48 hrs, cells were fixed, washed and stained with 40µL of MTT solution (5 mg/mL of MTT in 0.9% NaCl) and was added in each well then incubated for extra 4 hrs. MTT crystals were dissolved in 180 µL of acidified isopropanol/well. The plate was shaking at room temperature, and then determining the absorbance at 570 nm by ELISA reader. The molar concentration requisite to inhibit 50% of cell capability (IC 50 ) was calculated, and compared with the reference drug doxorubicin. The surviving fractions were represented as means ± S.E.M.

Analysis of Cell Cycle for Compound 6
Leukemia HL-60 cells, (3.0x10 5 cells/well) was incubated at 37°C for 12 hrs. Compound 6 was added to target cells at its IC 50 concentration and left for 24 hrs. After treatment, cells were collected and fixed with 75% ethanol at 20°C overnight. The cells were washed with PBS followed by centrifugation and then incubated with (10 mg/mL) Rnase (Sigma, USA) and (5 mg/mL) propidium iodide (PI, Sigma) before flow cytometry analysis (FACSCalibur cytometer using Cellquest software, BD Bioscience, USA).

Annexin V-FITC/PI and Detection of Apoptosis of Compound 6
Leukemia HL-60 cells (1.5x10 5 cells/well), incubated for 12 hrs. Cells were treated with compound 6 at its IC 50 value for 24 hrs. Apoptosis was determined by staining cells with FITC-conjugated Annexin-V and Propidium Iodide, and then analyzed by a FACSCalibur cytometer by using Cellquest software (BD Bioscience).

In vitro Topoisomerase II Enzyme Inhibition Assay
Compound 6 was selected to be examined against topo II using human DNA Topoisomerase II (Topo II) ELISA screening kit according to manufacturer's instructions. 100 mL of standard and sample was added per well and incubate for 2 hrs at 37°C. Take out the liquid of each well. Add 120 mL of biotin-antibody to each well then incubate for 1 hr at 37°C. Aspirate each well and wash three times. Add 100 mL of horseradish Peroxidase (HRP-avidin) to each well and then incubate for 1 h at 37°C. Repeat the aspiration/wash process for five times. Add 90 mL of 3,3 ʹ ,5,5 ʹ -Tetramethylbenzidine (TMB) substrate to each well and incubate for 15-30 min at 37°C, save from light. Add 50 mL of stop solution to each well and find out the optical density of each well within 5 min, using a ROBONEK P2000 ELISA reader to 450 nm. The values of % activity versus a series of compound log concentrations (-2nM, -1nM, 0nM, 1nM, 2nM) were then plotted using non-linear regression analysis of sigmoidal doseresponse curve. The IC 50 values for compounds 6 against topo II was estimated by the concentration causing a half-maximal percent activity and the records were compared with etoposide as standard. All experiments were made in triplicates.

Conclusion
The purpose of the current work was to syntheses and study In vitro antileukemia cancer activity of some novel pyrazole derivatives (2 and 5) and pyrazoles carrying the biologically active thiazole moieties at 1-position. All the synthesized compounds were established by using IR, MS, 1 H, 13 C NMR and elemental analyses. All of the synthesized compounds were monitored for their cytotoxic activity against Leukemia HL-60 cell line by using Doxorubicin as positive control. The results demonstrated that compound 6 was the most potent cytotoxic activity with IC 50 value 1.35 µM. Exhaustive mechanism studies for cell cycle analysis of compound 6 were exposed cell cycle detain at G2/M phase and pre-G1 apoptosis. Topoisomerase II inhibition activity assay confirmed that compound 6 is a potent DNA Topoisomerase II inhibitor.

Declaration of Interest Section
Author has no declarations of interest to report This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.