الفهرس 
Summary
ChemistryOnly 14 pages are availabe for public view
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Abstract
Novel quinazoline derivatives were designed, synthesized, characterized by using several spectroscopic techniques, and evaluated as potential multi-target anticancer agents through dual inhibition of EGFR/HDAC and EGFR/VEGFR-2 enzymes. The synthesized compounds were categorized according to the rationale design and based on the general chemical structure of each series to: (a) compounds 5a-e and 8a-j were designed as quinazoline/chalcone hybrids as EGFR/HDAC dual inhibitors; (b) compounds 13a-e, 16a-c, 19a-c, 22a-c, 25a-c, 29a-c, and 33a-c were designed as quinazoline hybrids as EGFR/VEGFR-2 dual inhibitors. All the synthesized compounds were screened against a panel of 60 cancer cell lines at NCI, USA to determine their antiproliferative potenial.
Among the first category as EGFR/HDAC inhibitors, compound 5e bearing a 3,4,5-trimethoxyphenyl chalcone moiety, showed the most effective growth inhibition value against the panel of NCI 60 human cancer cell lines. Thus, it was selected for further investigation for NCI five doses assay. Interestingly, the trimethoxy substituted analogue 5e inhibited the proliferation of RPMI-8226 cell line by 96 %, at 10 µM with an IC50 = 9.09±0.34 µM and SI = 7.19 against normal blood cells. In order to confirm the selectivity of compound 5e, it was evaluated against a panel of enzymes. Mechanistically, 5e successfully and selectively inhibited EGFR, HDAC-6, and HDAC-8 enzymes with IC50 = 90± 4 nM, 410± 15 nM, and 610± 27 nM, respectively, if compared to the reference drugs with IC50 = 70±3 nM (Laptinib), 1000±18 nM (Entinostat), and 1000±31 nM (Entinostat), respectively. Furthermore, the selected derivative 5e induced apoptosis via the mitochondrial apoptotic pathway by raising the Bax/Bcl-2 ratio and activating caspases 3, 7, and 9.
While the second series of the designed compounds as EGFR/VEGFR-2 dual inhibitors, five compounds showed excellent growth inhibition values against the tested panel of NCI 60 human cancer cell lines, namely the flouro substituted amide derivative 13b, hydrazone derivatives 19b (methoxy substituted), 19c (chloro substituted), and amide chalcone derivatives 22a (unsubstituted), 22b (methoxy substituted) with GI% mean of 57%, 78%, 73%, >100%, and 100%, respectively. The most active compounds were subjected to antiproliferative MTT assay against liver cancer HepG-2, colon cancer HCT-116, and breast cancer MCF-7 cell lines as well as the normal fibroblasts cell line WI-38 to estimate their cytotoxic activity in IC50 and selectivity properties in selectivity index (SI). The five compounds showed promising IC50 values against the tested cell lines, especially compound 13b that revealed inhibition against breast cancer cell lines with IC50 of 6.32 ±0.4 µM and SI = 7 and compound 22a that revealed cytotoxic activity against colon cancer HCT-116 cell lines with IC50 of 2.37 ±0.1 µM and SI = 13. Also the most active compounds 13b, 19b, 19c, 22a, and 22b successfully inhibited both EGFR and VEGFR-2 at nano molar range of 271± 10 nM, 630± 26 nM, 317± 13 nM, 64± 3 nM, and 119± 5 nM, respectively for EGFR inhibition assay and 193± 8 nM, 483± 19 nM, 397± 20 nM, 74± 3 nM, and 362± 14 nM, respectively for VEGFR-2 inhibition assay. Furthermore, compound 13b induced a MCF-7 cancer cell cycle arrest pattern in the G1 and S phases. Also, 22a induced a HCT-116 cancer cell cycle arrest pattern in the G1 and S phases. Both compounds exhibited significant efficacy in induction of apoptosis in MCF-7 breast cancer cells and HCT-116 colon cancer cells.
Moreover, the most active compound 5e as EGFR/HDAC dual inhibitor, was subjected to docking studies to predict its binding modes at the active domains of EGFR, HDAC-6, and HDAC-8 enzymes and interpret its enzyme inhibition activity. Also, 13b, 19b, 19c, 22a, and 22b as EGFR/VEGFR-2 dual inhibitors were ran into docking studies to predicted their binding poses at the active sites of EGFR and VEGFR-2 enzymes and explain their enzyme inhibition assay results.
Finally, the most active compounds of the study were subjected to physicochemical properties investigation to estimate their ability for being orally active molecules.
This thesis includes four main parts: introduction, rational & design, results & discussion, and the experimental sections in addition to abstract, conclusion and references sections.
1. Introduction:
This section presents a literature review of cancer, types of cancer treatment, role of quinazoline derivatives against biological cancer targets (EGFR and its consecutive mutations), benefits of multi-target agents, and role of quinazoline derivatives as multi-targeted agents in cancer treatment.
2. Rationale and Design:
It included the research objectives and the major aims that directed the theoretical and practical work for the designing of new quinazoline based hybrids and various rationales employed through this research work. The ideas utilized in designing and synthesis of new and potent antiproliferative multi-target antiproliferative agents using quianzoline as core scaffold. The aim of this research work was achieved via the synthesis of series of new EGFR/HDAC dual inhibitors as well as new EGFR/VEGFR-2 dual inhibitor hybrids with the objective of discovering their dual action, testing the antiproliferative activity of all synthetized target compounds upon NCI one dose screening and promising ones via NCI five dose testing.
3. Results and Discussion:
This part is subdivided into three main sections:
A) Chemistry:
In this part, various experimental methods and conditions of reactions adopted for the preparation of the designed compounds were discussed. In addition, it dealt with the structure elucidation of the newly synthesized compounds by different methods, including mass, elemental analyses, IR, 1H NMR, and 13C NMR spectral data. In this work, the following compounds were synthesized:
• Forty four reported intermediates: (1), (2), and (4a-e) in Scheme 1, (6a, 6b) and (7a, 7b) in Scheme 2, (9), (10), and (12a-e) in Scheme 3, (15b, 15c) in Scheme 4, (17) and (18) in Scheme 5, (20a-c) and (21a-c) in Scheme 6, (23a-c) and (24a-c) in Scheme 7, (27a-c) and (28a-c) in Scheme 8, (30) and (31a-c) in Scheme 9.
• Three new intermediates: (32a-c) in Scheme 9.
• Thirty eight final compounds as follows:
Fifteen final compounds (5a-e) and (8a-j) as EGFR/HDAC dual inhibitors.
Twenty three final compounds (13a-e), (16a-c), (19a-c), (22a-c), (25a-c), (29a-c), and (33a-c) as EGFR/VEGFR-2 dual inhibitors.
B) Biological evaluation:
This part discussed the results obtained from the study of synthesized compounds as multi-targeted anticancer agents, as illustrated below:
a) In vitro antiproliferative screening against panel of NCI 60 cancer cell lines:
All the synthesized compounds 5a-e, 8a-j, 13a-e, 16a-c, 19a-c, 22a-c, 25a-c, 29a-c, and 33a-c were screened against 60 panel of cancer cell lines at NCI, USA to determine their antiproliferative activities.
b) In vitro antiproliferative study using MTT assay against multiple myeloma RPMI-8226 cell lines and human normal blood cell PC5-800-011:
The IC50 of compound 5e was determined to assess its cytotoxic activity against multiple myeloma cell lines RPMI-8226 versus its corresponding human normal blood cell PC5-800-011 to evaluate its selectivity index (SI).
c) In vitro antiproliferative study using MTT assay against liver cancer HepG-2, colon cancer HCT-116, and breast cancer MCF-7 cell lines as well as the normal fibroblasts cell line WI-38:
The IC50 of compounds 13b, 19b, 19c, 22a, and 22b was determined to assess its cytotoxic activity against HepG-2, HCT-116, and MCF-7 cancer cell lines versus WI-38 normal cell lines to evaluate their selectivity index (SI).
d) In vitro enzyme inhibition assays:
The most active compound of scaffold A, compound 5e was subjected to enzyme inhibition panel of 10 enzymes namely, HDAC1, 2, 3, 8 and 6, EGFR, CDK-2, VEGFR-2, tubulin, and topoisomerase-2 enzymes to estimate its efficacy and selectivity as EGFR/HDAC dual inhibitor. While compounds 13b, 19b, 19c, 22a, and 22b of scaffold B, were tested against EGFR and VEGFR-2 enzyme inhibition assay to estimate their ability to inhibit both enzymes. Furthermore, compound 22a was subjected to enzyme inhibition assay of mutant EGFR C797S enzyme.
e) Measuring the expression of apoptotic and anti-apoptotic markers:
The effect of compound 5e, the most active compound of scaffold A, on Bax, Bcl-2, caspase-3, caspase-7, and caspase-9 were measured as apoptotic and anti-apoptotic markers in MM RPMI-8226 cell lines.
f) Cell cycle analysis:
Flow cytometry explored the mechanistic action for compounds with the highest antiproliferative activity; 5e against MM RPMI-8226 cell lines, 13b against MCF-7 cell lines, and 22a against HCT-116 cell lines. Accordingly, the tested cell lines were treated with IC50 of these compounds for 24 h.
g) Apoptosis analysis:
Apoptosis, or programmed cell death, plays an integral part in eliminating neoplastic cells and is thus regarded as a preventive mechanism against cancer growth. After post-treatment with IC50 of compounds 5e, 13b & 22a and analyzed by flow cytometry.
C) Molecular docking:
This section is subdivided into three parts:
a) In silico molecular docking simulations results for compound 5e of scaffold A against EGFR, HDAC-6 and HDAC-8 enzymes.
b) In silico molecular docking simulations results for compounds 13b, 19b, 19c, 22a, and 22b against EGFR and VEGFR-2 enzymes.
c) Physicochemical properties prediction for compounds 5e, 13b, 19b, 19c, 22a, and 22b.
4. Experimental:
A) Chemistry:
This part presented the practical procedures for synthesizing the reported and new intermediates and final compounds 5a-e, 8a-j, 13a-e, 16a-c, 19a-c, 22a-c, 25a-c, 29a-c, and 33a-c. Also, it summarized their spectral and elemental data.
B) Biological evaluation:
This section deals with the detailed laboratory materials and procedures used in the evaluation of the biological activities of synthetic compounds.
C) Molecular docking:
This section illustrates the software and protocols used in molecular docking and preparing 2D and 3D captions of docking study. The Molecular Operating Environment MOE® software version 2022.02 was used for molecular docking study. For generation of 2D caption MOE® (v2022.02) and BIOVIA Discovery Studio Visualizer® (v2021) while the 3D captions were generated by PyMOL® (v0.99). Moreover, the method used in calculated the predicted values of the physicochemical properties of the tested compounds. Online pkCSM (https://biosig.lab.uq.edu.au/pkcsm/) pharmacokinetics prediction properties were used to calculate the pharmacokinetic properties of the investigated compounds.
Three articles were published from this thesis:
1. Mostafa A. Mansour, Asmaa M. AboulMagd, Samar H. Abbas, Hamdy M. Abdel-Rahman and Mohamed Abdel-Aziz, Insights into fourth generation selective inhibitors of (C797S) EGFR mutation combating non-small cell lung cancer resistance: a critical review, RSC advances, (2023), 13(27), 18825-18853. https://doi.org/10.1039/D3RA02347H
2. Mostafa A. Mansour, Samar H. Abbas, Asmaa M. AboulMagd, Hamdy M. Abdel-Rahman and Mohamed Abdel-Aziz, The significance of quinazoline derivatives as potential multi-target anti-cancer agents: review article, JABPS, (2023), 7(1), 1-17, doi: 10.21608/jabps.2023.234736.1203.
3- Mostafa A. Mansour, Asmaa M. AboulMagd, Samar H. Abbas, Mohamed Abdel-Aziz and Hamdy M. Abdel-Rahman, Quinazoline-chalcone hybrids as HDAC/EGFR dual inhibitors: Design, synthesis, mechanistic, and in-silico studies of potential anticancer activity against multiple myeloma, Arch. Pharm. 2024, e2300626. https://doi.org/10.1002/ardp.202300626