الفهرس 
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Abstract
aim of the work” outlines the main goal and rationale of this work, including biological activities of some diarylpyrazoles, NO-donors, amino acids derivatives, thiazole derivatives, benzothiazole derivatives, benzamide and hydroxamate derivatives and the idea of these combining 1,5-diarylpyrazoles derivatives by evaluation of anticancer and anti-inflammatory activities for the purpose of synergistic effect, in addition to mechanistic study of anticancer activity of target compounds.
The third part “results and discussion” discuses and describe the data obtained from various steps of synthesis of derivatives of 1,5-diarylpyrazole amino acids, oxime, thiazole, benzothiazole, benzamide and hydroxamate derivatives. This part is subdivided into three categories:
A) The process employed in the synthesis of 1,5-diarylpyrazole-amino acids derivatives (7a-j) and (8a-j), 1,5-diarylpyrazole-oxime derivatives (10a-j) and (12a-c), 1,5-diarylpyrazole benzamide derivatives (13a-e), 1,5-diarylpyrazole thiazole derivatives (14a-e), 1,5-diarylpyrazole benzothiazole-derivatives (15a-d) and 1,5-diarylpyrazole N,O dimethylhydroxamte derivatives (16a-d) . Structural confirming of all newly synthesized compounds is based on different spectral methods including IR, 1H-NMR, 13C-NMR and HRM spectroscopy.
B) Nitric oxide measurements of 1,5-diarylpyrazole-oxime hybrids 10a, 10b, 10d-i and 12a-c using Griess colorimetric method to investigate their anticancer activity.
C) The evaluation of the biological activities of the synthesized compounds which is included four sections:
1- Evaluation of anticancer activity.
All synthesized compounds 7a-j, 8a-j, 9a-j, 10a-j, 11a-c, 12a-c, 13a-e, 14a-e, 15a-f and 16a-f were evaluated for in vitro anticancer activities against five cancer cell lines namely, human colorectal adenocarcinoma cell lines DLD-1, human cervical cancer cell line Hela, human pancreatic cancer cell line SUIT-2, human myelogenous leukemia cell line K562 and human hepatocellular carcinoma cell line HepG2 using WST-8 assay at concentration 100 µM to investigate the growth inhibition percent (GI%) of each compound using daunorubicin as reference drug and for further investigation, compounds with reasonable antiproliferative activity against five cancer cell lines (DLD-1, Hela, K562, SUIT-2 and HepG2 ) at 100 µM were selected to measure growth inhibition percentage using WST-8 assay at different six concentration for majority of compounds and eight concentrations for highly cytotoxic compounds for calculating their IC50 using daunorubicin as reference.
2-Different mechanistic study of anticancer activity of the target compounds:
Different experiments have been performed to investigate the mechanism of anticancer activity that was exerted by the new target compounds including:
A- Evaluation of EGFR inhibitory activity
Human EGFR-TK Elisa kit assay was performed to evaluate the in vitro EGFR-inhibitory potency of the compounds 7i, 10b, 10d, 10g, 10i, 12c, 13b, 13d, 15f and 16a using the multi-target kinase inhibitor drug sorafenib as a reference using quantitative sandwich enzyme immunoassay technology. Compound 16a showed potent EGFR inhibitory activity with IC50 of 4.00 µM as well as, compound 10d exhibited excellent EGFR inhibitory activity with IC50 of 8.00µM, while compounds 10g, 10i and 12c exhibited good EGFR inhibitory activity with IC50 of 18, 21 and 12 µM respectively, in comparison to sorafenib of IC50 3.5 µM.
B- In silico molecular docking on EGFR
Compounds 10d, 10g, 12c, 13b, 13d and 16a, were investigated for their in silico simulation studies targeting EGFR kinase domain (PDB ID: 4ZAU) using sorafenib, a multitarget kinase inhibitor drug as reference. All docking results were matched with EGFR-TK Elisa kit assay.
C- Evaluation of JNK-2 inhibitory activity
JNK-2 inhibitory activity of the selected compounds7i, 10d, 10g, 10i and 15f was performed in vitro using simple step ELISA kit for quantitative measurement of JNK-2 (pT138/Y185) protein in human cells using sorafenib as reference drug. Compound 10i showed a potent inhibition of JNK-2 with an IC50 of 1 μM as the activity of sorafenib, while compound 10d showed good JNK-2 inhibitory activity with IC50 of 29μM.
D- In silico molecular docking on JNK-2
Compounds 7i, 10d and 10i were investigated for their in silico simulation studies targeting JNK-2 binding pocket (PDB ID: 3E7O) using sorafenib as reference drug. Like sorafenib, compound 10i bound the active site by the same mode forming hydrogen bonds, while compounds 7i and 10d showed weaker affinity to JNK-2 binding pocket than sorafenib.
E- Cell cycle analysis
Selected compounds were investigated on Hela cancer cell cycle distribution and cell-death associated DNA fragmentation. compounds 7i and 10g showed G2/M arrest, while compounds 10i and 15f showed a combined S phase and G2 phase arrest.
F- Apoptosis as a second mechanism of cell death
Compounds 7i, 10g, 10i and 15f were selected for studying of apoptotic changes after treatment of Hela cell using fluorescent microscope and flowcytometry and their results observed indicated that apoptotic cells were increased after treatment of these compounds as result of antiproliferative activity not due to cytotoxicity.
G- Evaluation of cytotoxicity towards normal cell line PC12
To investigate the selectivity of the target compounds towards cancer cells, the cytotoxicity of compounds 7i, 10g, 10i, and 15f was evaluated against the normal cell line PC12 using a WST-8 in comparison to daunorubicin as reference drug. compounds 10i observed no cytotoxicity against PC12 cell line with CC50 >100 μM. Also, compounds 7i and 15f showed low cytotoxicity towards PC12 cell line with CC50 of 57.9 and 35.5 μM respectively, as well as compound 10g showed a moderate cytotoxicity with CC50 of 16.1 μM in comparison to daunorubicin.
3-Evaluation of anti-inflammatory activity
i) In vitro cyclooxygenase inhibition assay
Target compounds 7b, 7c, 7g, 7h, 8a-j, 14a-e and 16a-f were evaluated in vitro for COX-2 inhibitory activity as well as COX-1 inhibitory activity to determine their COX-2 selectivity index using SC560 as reference COX-1 inhibitor and celecoxib as reference COX-2 inhibitor drug using fluorometric inhibitory screening kits (Biovision, Switzerland) according to the manufacturer’s instructions. Compounds 7b and 7h showed significant COX-2 inhibitory activity with IC50 of 9 and 8.3 µM and S.I. > 22.9 and >24.09 respectively. Also, compounds 8a, 8f, 8i and 8j demonstrated high selectivity and inhibitory activity against COX-2 enzyme with IC50 of 9.5, 9, 9 and 11 µM respectively, and S. I. ranging from > 18.18 to > 22.2. Moreover, compounds 8g, 14a, 14c, 16b and 16e showed good COX-2 inhibitory activity with IC50 of 13, 13, 12, 18 and 12.5 µM respectively.
ii) In silico molecular docking on COX-1 and COX-2 enzymes
Compounds 7b, 7c, 7g, 7h, 8a-j, 14a-e and 16a-f were investigated for their in silico simulation studies targeting Superimposed molecular structures of COX-1 (PDB ID: 3KK6) and COX-2 (PDB ID: 3LN1). All docking results were compatible with COX-1 and COX-2 assay kits results.