الفهرس 
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Abstract
The present study deals with the design, synthesis and biological evaluation of new 1,5-diarypyrazole esters and carboxamides (series 5 and series 6–11, respectively), in addition to the synthesis of twenty-five intermediates. The newly synthesized compounds were assessed for different biological activities such as anticancer and anti-inflammatory activities. Additionally, molecular mechanistic studies were conducted for the most active derivatives as anticancer agents.
The thesis includes four main parts; introduction, the aim of the work, results & discussion and the experimental section in addition to the reference and abstract.
1. Introduction
This part illustrates an overview of the synthetic procedures used for the preparation of pyrazoles as well as a synopsis on their various biological activities concentrating on anti-inflammatory and anticancer activities. It clarifies various mechanisms encountered by pyrazoles as COX-2 inhibitors and protein kinase inhibitors among others.
2. Aim of the work
This part outlines the rationale for e the design of the target compounds. It also brings out the steps to be taken in the current dissertation including synthesis of new derivatives of 1,5-diarypyrazole, evaluation of their biological activities as anticancer and anti-inflammatory potential agents, in addition to molecular mechanism study of anticancer activity of some of the novel compounds.
3. Results and discussion
This section includes a detailed description of the results obtained during the various stages of the study, synthesis, structural elucidation and biological evaluation of the target compounds. This part is divided into two major sections.
A. Chemistry section
The chemistry section covers the distinct results obtained from the synthesis of the intermediate as well as the novel compounds and their structural elucidations using different spectral analysis such as IR, 1H-NMR, 13C-NMR and ESI-HRMS spectroscopy. The current research project reports the synthesis of known twenty-three intermediates, two new intermediates 4j and 4e in addition to fifty-two new final compounds 5a–j, 6a-j, 7a–e, 8a-e, 9a–d, 10a-g and 11a–k.
B. Biological evaluation
The second section illustrates various results obtained from different biological examinations done using the synthesized compounds. The biological evaluation encompasses the following:
i. Evaluation of the anticancer activity:
Anticancer activity evaluation section entailed the results obtained from experiments conducted at the Faculty of Engineering, Yamagata University, Yonezawa, Japan. The results incorporates a preliminary screening of the synthesized compounds at a concentration of 100 µM against five human cancer cell lines namely human colorectal adenocarcinoma cell line (DLD-1), human cervical cancer cell line (Hela), myelogenous leukemia cell line (K-562), pancreatic cancer cell line (SUIT-2) and human hepatocellular carcinoma cell line (HepG2) using a standard water soluble tetrazolium-8 (WST-8) assay and daunorubicin was used as the reference drug. The section goes on to list IC50 results revealed upon testing the most active compounds against the same cell lines using different concentrations. The promising anti-proliferative activity were judged based on having a superior or comparable activity to the positive control at 100 µM doses, thus compounds 5a-d, 6a, b, c, d, f-i, 7a, b, 8a, b, d, e, 9a-c, 10a, c, d, f, g, 11a and 11d-k were selected for the IC50 evaluation using WST-8 assay at different five concentrations.
This section continues to describe the anticipated anti-cancer activity of the tested compounds by demonstrating the results obtained from cell cycle analysis and measurement of apoptosis experiments. selected compounds with low IC50 values (5b, 5g, 6h and 6h) were further involved in cell cycle analysis. The results section describes their ability to increase cell population at S phase and exhibit cell cycle arrest mainly at the S phase, except compound 5g that cause accumulation of the cell in G2/M phase.
The investigation continues to support the activity via defining the compounds ability to induce apoptosis in Hela cancer cell line. The flowcytometric analysis results and microscopic examination of apoptotic effect is listed in this section revealing that the programmed cell death and cell cycle arrest are the principle mechanisms of cell death caused by the newly synthesized derivatives.
ii. Molecular mechanism study of anticancer activity of the target compounds:
This section describes and analyses data obtained from experiments carried out to fully understand the mechanism of anticancer activity that is exhibited by the new compounds, including:
a. Evaluation of EGFR inhibitory activity
Conducting of this experiment to investigates the molecular anticancer mechanism of the synthesized compounds using enzyme linked immunosorbent assay (ELISA) and the kinase inhibitor drug sorafenib as a reference drug. Compounds 5b, d, 6b, c, g, h, 9a and 11g, h, k were tested for their ability to inhibit EGFR-TK. Generally, the test compounds demonstrated a reasonable EGFR inhibitory activity and showed inhibitory activity with IC50s ranged from 1.70-1000 µM.
b. Evaluation of JNK2 inhibitory activity
The follow up of the anticancer mechanism continues to profile the selectivity of the most potent compounds toward other kinase via evaluation of compounds 5b, 5d, 6c, 6g and 6h for their ability to inhibit JNK2 enzyme. The result from this assay revealed that the vanillin derivatives 5d was discovered to be the most potent and selective JNK2 inhibitor agent, Compound 5b exhibited outstanding JNK2 inhibitory activity. Meanwhile compound 6c showed dual inhibitory activity against both JNK2 and EGFR enzymes.
c. Molecular docking studies on protein kinase enzymes
This study clarifies the possible binding mode of the newly synthesized compounds within EGFR and JNK2 active sites. Some of the most active compounds docked onto the crystal structures of EGFR and JNK2. The results of the docking study of the most active compounds 6c and 6h into EGFR revealed a good fitting of the tested compounds into the ATP binding site with the key amino acids bonded with erlotinib. The tested compounds could fit into the binding site with docking energy scores - 10.41, - 11.70 kcal/mol respectively. Also, analysis of the molecular docking into JNK2 showed that compounds 5b and 5d could fit into the binding site with docking energy scores in the range of -16.79 and - 19.46 kcal/mol, respectively.
iii. Evaluation of anti-inflammatory activity:
This experiment is performed to evaluates the anti-inflammatory activity of the novel compounds and their ability to inhibit COX-1 and COX-2 enzymes using a fluorometric COX inhibitor screening assay kits. Twenty-five compounds (5b, 6b, 7,8a-e, 9a–d, 10a-g, 11g and 11k) were selected for this assay and to study their structure activity relationship. Many of the tested compounds exhibited moderate inhibitory activity against COX-2 with IC50S values ranged from 9.00-50 µM with COX-2 selectivity indices (SI) ranged from 3.8- >16. However, for all compounds the resultant inhibitory activities were lower than that of the reference drug celecoxib (COX-2, IC50 = 0.45 µM).
4. Experimental
This section illustrates in details the different methodologies and experiments used in this thesis. This section is divided into two parts:
The first portion: Chemistry, this part covers the various procedures used for synthesis of the target compounds 5a–j, 6a-j, 7a–e, 8a-e, 9a–d, 10a-g and 11a–k. This section includes also full physical, spectroscopic and analytical data of the synthesized compounds.
The second portion was a biology section that explains how the anticancer activity of the produced compounds 5a–j, 6a–j, 7a–e, 8a–e, 9a–d, 10a–g, and 11a–k was evaluated.
This section also describes the different methods for experiments applied to investigate the molecular mechanisms of anticancer activity of the test compounds such as assay of EGFR and JNK2 inhibitory activity, cell cycle analysis, apoptosis induction by test compounds as well as molecular docking on EGFR and JNK2 enzymes. Additionally, this part illustrates protocols of anti-inflammatory screening.