الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
 |  | from | 150 |  |  |
| | | from | 150 | | |
Abstract
The present work deals with the design and synthesis of new conjugates combining quiazolin-4-(3H)-one derivatives and chalcones (series I), or oximes as nitric oxide (NO)-releasing moiety (series II) or nicotinonitriles (series III) in one compact structure and evaluating their antiproliferative activities. The thesis is divided into four main sections, including introduction, aim of the work, results and discussion, and experimental sections. References and the summary have also been included.
This section presents the main objectives and rationale for this work. The ideas used in designing novel quinazolin-4(3H)-one hybrids, as well as the biological study of the synthesized compounds and the mechanistic study of the potential anticancer activity of most of the synthesized derivatives.
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 new final compounds and fourteen 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 compounds
The prepared compounds of series I (7-36), series II (38b,c, 39b,c) and series III (40-54) were evaluated for their anticancer activity as a single dose (10 µM) against four different cell lines, including the pancreatic cell line (Panc-1), the breast cancer cell line (MCF-7), the colon cancer cell line (HT-29), and the epithelial cancer cell line (A-549) using the -(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and doxorubicin as a control drug. The obtained results revealed that the derivatives 29, 32, 41, 42, 51 and 52 have the highest cytotoxicity (GI50= 1.16- 1.85 µM) and showed significant potency against MCF-7 breast cancer cell line (IC50 =1.00-1.60 µM) and against A-549 human lung adenocarcinoma cell line (IC50= 1.10-1.90 µM)
II- Cell viability assay
The synthesized compounds of series I (7-45), series II (38b-c, 39b-c), and series III (40-54) were cultured with MCF-10A cells and tested for cell survival by MTT assay. The compounds tested had no cytotoxic effects, and cell viability for most of the compounds tested was greater than 84% at a concentration of 50 µM.
III- EGFR inhibitory activity
Compounds 9-12, 29-32, 36, 39c, 41, 42, 51 and 52 were evaluated using the EGFR-TK assay to assess the inhibitory effect of these compounds on EGFR. The results of this assay complement the findings of the cancer-cell-based investigation. All tested compounds inhibited EGFR, with IC50 values ranging from 0.11 µM to 6.25 µM. Hybrids 29 and 51 were identified as the most potent, with EGFR inhibitory effects (IC50 = 0.11±0.2, and 0.11 ± 0.1µM, respectively) comparable to the positive control erlotinib (IC50 = 0.08 ± 0.04 µM).
IV- BRAF V600E inhibitory activity
An in vitro assay was performed to evaluate the activity of the newly synthesized compounds 9-12, 29-32, 36, 39c, 41, 42, 51 and 52 against BRAF V600E. The obtained results revealed that 3-allyl quinazolin-4-one/3-cyanopyridone derivatives 41, 42, 51 and 52 had the highest inhibitory activity against BRAFV600E (IC50 = 0.14 - 0.29 μM) comparable to the positive control erlotinib (IC50 = 0.06 μM).
V- Apoptosis assay
Most active compounds 29, 30 and 32 as EGFR inhibition effect were examined for their ability to induce the apoptosis cascade to reveal their proapoptotic potential.
Va- Activation of proteolytic caspases cascade
The results showed that tested compounds 29, 30 and 32 increased the level of active caspase-3 by 6.6-to 9.2-fold compared with the control cells. Compounds 29 and 32 were the most active ones with remarkable overexpression of caspase-3 protein level (602.4±4.50 and 519.7±3.50 pg/mL, respectively) when equated with doxorubicin (503.2 ± 4.22 pg/mL). Compared with control cells, the most active derivative 29 increased the amount of activated caspase-3 ninefold.
Vb- Apoptotic mechanism
The results showed that compound 29 increased the levels of caspase-8 and 9 by 14.4 - and 22.8-fold, respectively, while compound 32 increased the levels by 10.8- and 20.1-fold, respectively, compared with control cells, indicating activation of both the intrinsic and extrinsic pathways, although the effect on the intrinsic pathway was greater because the levels of caspase-9 were higher.
Vc- Cytochrome C assay
Cytochrome c activity of hybrids 29, 30 and 32 against the human pancreatic cancer cell line (Panc-1) resulted in an 10.8-13.5 fold increase in cytochrome c expression compared to untreated control cells.
Vd- Bax and Bcl-2 levels assay
Hybrids 29, 30 and 32 were further evaluated for their effects on Bax and Bacl-2 levels, and the results showed that compound 29 induced Bax levels (318.5 pg/mL) 38.6-fold compared with doxorubicin (276.19 pg/mL) in untreated control cells, followed by compound 32 (287.65 pg/mL and 34.8-fold change). Finally, compound 29 induced a decrease in Bcl-2 protein level (0.815 ng/mL) compared with doxorubicin (0.983 ng/mL), followed by compound 32 (0.978 ng/mL).
VI- Cell cycle analysis
Compound 29 was selected for cell cycle analysis investigation and the results showed that hybrid 29 primarily exhibited cell cycle arrest at the G0/G1 phase, G2/M phase, and preG1 apoptosis.
C-The third part
Molecular docking, the results obtained in this section elucidate the binding mode and anti-proliferative activity of the most potent hybrids 29 and 51 on the active site of the crystal structure of the EGFR tyrosine kinase domain (PDB code: 1M17) and BRAF kinase domain (PDB code: 2FB8).
4- Experimental
The experimental section displays the detailed procedures of the various experiments used, and includes three parts:
i. The first Part
Chemistry section which describes the procedures used for the synthesis of the target derivatives series I (7-36), series II (38b-c, 39b-c) and series III (40-54) . Also, it contains all the detailed analytical and spectroscopic data of the synthesized derivatives.
ii. The second part
Biology section, which outlines the procedures used to test the anticancer activity of the prepared derivatives, EGFR inhibitory assay, Caspase-3, 8 and 9 activation assay, cytochrome c assay, evaluation of Bax and Bcl-2 expressions and cell cycle analysis.
iii. The third part
Molecular docking, which describes the software and methodology used for running the molecular docking of the analyzed compounds to investigate their binding mode within EGFR-TK active site (pdb: 1M17) and BRAF kinase domain (PDB code: 2FB8).