الفهرس 
SummaryOnly 14 pages are availabe for public view
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Abstract
Aim of the work
This second part outlined ideas utilized in designing and synthesis of new and potent antiproliferative multiple kinase inhibitors using 2-oxoindoles as core scaffolds. The aim of this research work was achieved via the synthesis of series of new PDGFRα/ß and VEGFR-2 inhibitor hybrids (series I &II) and new EGFR/VEGFR-2 and Tubulin Polymerization inhibitor hybrids (series III) with the objective of discovering their dual action. Also, testing all synthetized target compounds’ antiproliferative activity upon NCI one dose screening and promising ones via NCI five dose testing. Moreover, detecting the effect of the most potent hybrid 13f from series III on normal cells (HSF) and this most potent hybrid 13f undergoes cell cycle analysis. Additionally, studing the mechanisms that stand behind the antiproliferative activities of compounds 6a-o and 9a-p from series I&II and the most potent hybrid 13f from series III. Also, different substitutions on both aromatic sides of synthetized hybrids were used to explore their effect on the antiproliferative activity.
Results and discussion
A) Chemistry section, which includes the description of the several methods used for synthesis of the intermediates and their corresponding, targeted hybrids. Also, it displayed structural elucidations of these newly synthesized scaffolds by various spectroscopic techniques including 1H NMR, 13C NMR spectroscopy, LC-MS/ MS, GC/MS, as well as elemental microanalysis. In this work, the following compounds were synthesized:
Forty-five reported intermediates: (2a-h) and (4a-h) in synthesis of series I, (8a-g) in synthesis of series II, (11a-c and 12a-c) in synthesis of series III, (15a-e, 16a-e, and 17a-e) in synthesis of series IV, and (20) in synthesis of series V.
Five reported final compounds (6a, 6b, 6h, 18a, and 18e).
New sixty-four final compounds (6c-6g, 6i-6o, 9a-p, 13a-j, 18b-d, 18f-w, and 21a-e) as follow:
Thirty-one PDGFRα/ß and VEGFR-2 inhibitor hybrids (series I&II) (6a-o and 9a-p).
Ten EGFR/VEGFR-2 and Tubulin Polymerization inhibitor hybrids (series III) (13a-j).
B) Biology section, this section is subdivided into ten parts:
a) Anticancer screening for all the final compounds (6a-o, 9a-p, 13a-j, 18a-w, and 21a-e) by NCI
Sixty-nine compounds were screened for their anticancer activity via NCI one dose testing. Fortunately, eleven compounds (6f, 9f, 18d, 18k, 18n, 18p, 18q, 18r, 21a, 21b, and 21c) showed remarkable anticancer activity with complete cell death in different cell lines tested., so they are selected for five dose testing protocol.
b) Antiproliferative activity of compounds 6a-o and 9a-p on Pancreatic Ductal Adenocarcinoma cells (PDAC)
To elucidate the inhibitory activity of our target compounds 6a-o and 9a-p compounds versus PDGFRα, PDGFRβ, and VEGFR-2 kinases affecting the signaling cascades in PDAC, the growth inhibitory activities of the synthesized compounds 6a-o and 9a-p were evaluated on the pancreatic ductal adenocarcinoma cell lines (MDA-PATC53 and PL45) using MTT assay.
c) Kinase profiling of the target compounds 6a-o and 9a-p
The percentage of inhibition of the target compounds 6a-o and 9a-p was tested versus a panel of eleven kinases representing various signaling cascades including ABL1, BTK, SRC, FRK, JAK2, EGFR, FGFR, IGF1R, PDGFRα, PDGFRβ, and VEGFR-2 kinases in the presence of 10 µM of tested derivatives 6a-o and 9a-p.
d) PDGFRα/β and VEGFR-2 kinase inhibitory activity of the target compounds 6a-o and 9a-p
Because of the highly promising and specific inhibitory activities of most of the synthesized compounds 6a-o and 9a-p against PDGFRα, PDGFRβ, and VEGFR-2, the IC50 values of these compounds have to be evaluated versus these three mentioned kinases. The synthetized 2-oxoindole scaffolds 6a-o and 9a-p were examined for their potential IC50 values against PDGFRα, PDGFRβ, and VEGFR-2. Sunitinib was used as a reference for PDGFRα/β and VEGFR-2 kinases.
e) PDGFRα, PDGFRβ, and VEGFR-2 mRNA expressions’ detection of the target compounds 6a-o and 9a-p in MDA-PATC53 cells using RT-PCR technique
The PDGFRα, PDGFRβ, and VEGFR-2 mRNA expressions in MDA-PATC53 cells were collected after the incubation of these kinases with 10 µM of each compound (6a-o and 9a-p) for 6 h compared to DMSO as a control to ensure the efficacy of these multiple kinase agents in PDAC therapy.
f) Study the effect of the 6a-o and 9a-p hybrids cell cycle arrest via evaluation of the most potent hybrid 6f and 9f from series I&II.
g) Cytotoxicity of compounds 13f and 13g versus MCF-7 Cell Line from series III.
Compounds 13f and 13g from series III showed the uppermost observed antiproliferative activity versus the human breast cancer MCF-7 cell lines, so they were selected to detect the concentration required to hamper 50% of cellular growth (IC50) versus the MCF-7 cell lines compared to 5-fluorouracil (5FU) using the MTT assay.
h) Study the effect of the 13a-j hybrids on normal cells (HSF), and cell cycle arrest via evaluation of the most potent hybrid 13f from series III.
i) RTK Kinases’ Circumvention of compound 13f from series III
Due to the fundamental role of RTKs in cancer pathogenesis, the inhibitory IC50 values of compound 13f on three types of RTKs (EGFR, VEGFR-2 (KDR), and PDGFR_ẞ receptors were investigated using the kinase kit assay protocol.
j) Tubulin Polymerization Inhibition assays of compound 13f from series III
The tubulin polymerization hampering activity for the most potent compound 13f from series III, was examined to investigate the circumvention of the tubulin polymerization process using combretastatin A-4 (CA-4) as a positive control.
C) Molecular modelling and docking study, this section is subdivided into five parts:
i) Molecular modeling studies were carried out for all synthesized compounds (6a-o and 9a-p) from series I&II to explain the bindings modes inside the active binding sites of human VEGFR-2, PDGFRα, and PDGFRβ enzymes (PDB IDs: 4ASD, 6JOK, and 3MJG, respectively).
ii) Molecular modeling studies for the target hybrids 13a-j from series III was carried out to explain the bindings modes of all synthesized compounds (13a-j) inside the binding sites of human VEGFR-2, EGFR, and tubulin (PDB IDs: 1YWN, 4HJO, and 5LYJ, respectively).
iii) Molecular dynamics (MD) simulations are utilized to forecast the movements of the protein-ligand complex within the atomic level. In relation, the stability, and movements of VEGFR-2, PDGFRα, and PDGFRβ docked protein ligand complexes of the most active compounds 6f and 9f from series I&II were analyzed for 100 ns.
iv) In silico drug likeness and ADMET studies for the most potent hybrids 6f and 9f from series I& II.
v) In silico drug likeness and ADMET studies for the most potent hybrids 13f and 13g from series III.
In conclusion, these newly dual PDGFRα/β and VEGFR-2 (specifically; compounds 6f and 9f) and EGFR/VEGFR-2 and Tubulin Polymerization (specifically; compound 13f) hybrid inhibitors could be utilized as lead candidates that requires further developments and optimization.