الفهرس 
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Abstract
This study focuses on the development and synthesis of novel conjugates that merge 3-cyano-2-pyridone derivatives with either chalcones (series I), pyrazoline (series II), or a combination of two pyrazoline moieties (series III) in a compact structure and aims to assess their antiproliferative effects. The thesis comprises four principal segments, which include an introduction, the objective of the research, results and discussion, and experimental sections. In addition, references and a summary are also incorporated.
1- Introduction
In the first section of the introductory chapter, a comprehensive introduction to the pyrazoline nucleus is presented, which includes its chemistry, various synthetic methods, and biological activities. The second section provides a literature review focused on the 3-cyano-2-pyridone moiety, including its synthesis and biological activities, either alone or in combination with other structures. Finally, the third section offers an overview of the biological activities of chalcones.
2- Aim of the work
In this section, the primary objectives and underlying motivations for the research are outlined. The focus is on the development of novel hybrids that combine 3-cyano-2-pyridone and pyrazoline structures, as well as the investigation of the biological activity of the synthesized compounds. Additionally, the research aims to explore the potential anticancer activity of most of the synthesized derivatives and investigate their mechanisms of action.
3- Results and discussion
The results and discussion section is subdivided into three various parts:
A-The first part
The chemistry section contains a description of the synthetic routes and methods used to prepare the intermediates and their corresponding targeted derivatives. In addition, the structural elucidation of these derivatives by various spectroscopic techniques such as 1HNMR, 13CNMR, mass, and elemental analysis has been presented.
Forty-nine novel final compounds and thirty-one intermediates were synthesized in this work.
B-The second part
Biology section which is subdivided into five different parts:
I. In vitro antiproliferative activity and structure activity relationships of title compounds
The anticancer activity of the prepared compounds in series I (8-20), series II (22-31), and series III (35-44) was evaluated as a single dose (10 µM) against four different cell lines, including Panc-1, MCF-7, HT-29, and A-549, using the MTT assay. Doxorubicin was used as a reference drug. The results showed that derivatives 8, 12, 13, 16, 18, 39, 41, 42, and 44 exhibited the highest antiproliferative, with IC50 values ranging from 0.72 µM to 1.30 µM, which was higher than the cytotoxicity of doxorubicin (IC50= 1.10 µM).
II- Cell viability assay
The MTT assay was used to test the cell survival of the synthesized compounds in series I (8-20), series II (22-31), and series III (35-44) using MCF-10A cells. The results showed that most of the compounds tested had no cytotoxic effects, with cell viability greater than 86% at a concentration of 50 µM.
III- EGFR inhibitory activity
The EGFR-TK assay was used to evaluate the inhibitory effect of compounds 8, 12, 13, 16, 18, 39, 41, 42, and 44 on EGFR. The results of this assay support the findings of the cancer-cell-based investigation, showing that all tested compounds inhibited EGFR with IC50 values ranging from 81 nM to 145 nM. Among them, hybrids 39 and 44 exhibited the most potent EGFR inhibitory effects, with IC50 values of 81 ± 05 nM and 87 ± 06 nM, respectively, which are comparable to the positive reference erlotinib (IC50 = 80 ± 05 nM).
IV- BRAF V600E inhibitory activity
An in vitro assay was conducted to assess the efficacy of newly synthesized compounds, including 8, 12, 13, 16, 18, 39, 41, 42, and 44 against BRAF V600E. The results showed that compounds 8, 12, and 13 exhibited the strongest inhibitory activity against BRAF V600E, with IC50 values ranging from 58 to 76 nM. These values were comparable to the positive reference erlotinib, which had an IC50 of 60 nM.
V- Apoptosis assay
To investigate their proapoptotic potential, the highly active compounds 39 and 44, which exhibited EGFR/BRAF V600E inhibition effects, were assessed for their ability to induce the apoptosis cascade.
Va- Activation of proteolytic caspases cascade
The findings indicated that compounds 39 and 44 resulted in a 7.2 to 8.5-fold increase in active caspase-3 levels compared to the control cells. Notably, compounds 39 and 44 exhibited the highest activity, inducing significant overexpression of caspase-3 protein levels (543.50 ± 4 and 469.50 ± 5 pg/mL, respectively) compared to staurosporine (503.00 ± 4 pg/mL).
Vb- Bax and Bcl-2 levels assay
The effects of hybrids 39 and 44 on Bax and Bcl-2 levels were assessed, revealing that compound 39 led to a higher induction of Bax levels (296 Pg/mL) in untreated control cells compared to staurosporine (280 Pg/mL), followed by compound 44 (285 Pg/mL). Additionally, compound 39 caused a decrease in Bcl-2 protein levels (1.05 ng/mL) compared to staurosporine (1.10 ng/mL), followed by compound 44 (1.15 ng/mL).
Vc- Cell cycle analysis
For the cell cycle analysis investigation, compound 13 was selected, and the results indicated that this hybrid primarily caused cell cycle arrest at the G0/G1 phase.
C-The third part
In silico studies can be divided into three different components:
I-The first component of in silico studies is molecular docking, which involves the use of computational algorithms to predict the binding mode and potential anti-proliferative activity of molecules on a specific protein target. In this case, the most potent hybrids 8, 12, 13, 39 and 42 were docked onto the active site of the crystal structure of the EGFR tyrosine kinase domain (PDB code: 1M17) and the BRAF kinase domain (PDB code: 2FB8). The results obtained from this analysis provided insight into how these molecules interact with their protein targets and their potential effectiveness as anti-proliferative agents.
II-The second component of in silico studies is DFT (density functional theory) analysis, which involves the use of computational methods to calculate the electronic properties and reactivity of molecules. In this case, DFT studies were performed on the most active compounds, 39 and 42, to calculate their HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) energies, as well as their softness and hardness. These calculations provide insight into the electronic structure and reactivity of the synthesized hybrids and facilitate the understanding of their potential pharmacological activities.
III-The third component of in silico studies is ADMET (absorption, distribution, metabolism, excretion, and toxicity) prediction, which involves the use of computational models to evaluate the potential bioavailability, toxicity, and adverse effects of compounds. In this case, ADMET analysis was performed on the synthesized 35-44.
4- Experimental
The experimental section presents a comprehensive account of the methodology employed in conducting the experiments. This section is typically divided into three parts:
The first Part
In this chemistry section, we will outline the methods used to synthesize three series of target derivatives, namely series I (8-20), series II (22-31), and series III (35-44). We will also provide a comprehensive overview of the analytical and spectroscopic data obtained for the synthesized derivatives.
The second part
In the biology section, we will detail the methods used to evaluate the anticancer activity of the prepared derivatives. Specifically, we used the following assays: EGFR inhibitory assay, Caspase-3, 8, and 9 activation assay, cytochrome c assay, evaluation of Bax and Bcl-2 expressions, and cell cycle analysis.
The third part
For molecular docking, we used software such as BIOVIA I Discovery Studio 2016 to predict the binding affinity of the synthesized derivatives to target proteins. For DFT calculations, we used software such as the Gaussian 09 program package to investigate the electronic structure, molecular properties, and reactivity of the synthesized derivatives. For ADMET analysis, we used software such as BIOVIA I Discovery Studio 2016 to predict the pharmacokinetic and toxicological properties of the synthesized derivatives.
Two articles were published from this thesis:
1- Hesham A. Abou-Zied; Eman A Beshr; Gomaa, Hesham AM; Mostafa, Yaser A; Youssif, Bahaa GM; Alaa M. Hayallah; Mohamed Abdelaziz. Archiv der Pharmazie
(Impact Factor: 4.61). “ Discovery of new cyanopyridine/chalcone hybrids as dual inhibitors of EGFR/BRAFV600E with promising antiproliferative properties”, https://doi.org/10.1002/ardp.202200464
2- Hesham A. Abou-Zied, Eman A Beshr, Alaa M. Hayallah, Mohamed Abdelaziz.
Journal of advanced Biomedical and Pharmaceutical Sciences. (ID: JABPS-2212-1177 (R2)“ Recent mechanistic biological insights in chalcone scaffold : A masterpiece for medicinal chemistry” , 2023, accepted.