الفهرس 
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Abstract
The current study is concerned with the design, synthesis and biological evaluation of new pyranoquinolinone heteroannulated to triazolopyrimidine derivatives, new 2’-aminospiro[pyrano[3,2-c]quinoline]-3’-carbonitrile derivatives, new quinoline-2-one thiosemicarbazides, new 4-thiazolidinoquinoline-2-ones and new quinolone-triazole hybrids. The synthesized compounds were tested for various biological activities such as, anticancer, anti-bacterial and urease inhibitory activity. In addition, molecular mechanistic studies for some selecte compounds were performe for selected compounds were performed as urease inhibitory agents and as ATP non-competitive Src kinases inhibitors.
The current study is divided into five sections: introduction, aim of the work, results and discussion, experimental and references.
1- Introduction
The first section provides an overview about past and current synthetic methods for the preparation of heteroannulated quinolone architcture. In addition, it includes literature reports about the biological activities of such scaffold, such as anti-cancer, anti-bacterial, anti-inflammatory, anti-diabetes, alzheimer’s disease, CNS, antioxidant and diuretic activities.
2- Aim of the work
The target of the work involves the design and synthesis of new quinoline-2-one derivatives as well as the assessment of various biological activities of these new derivatives including anti-cancer, anti-bacterial and urease inhibitory activity.
3- Results and discussion
This part includes detailed discussion of the results obtained from various stages of synthesis, structural elucidation and biological evaluation of the target compounds.
This part is divided into two main subsections:
The first: Chemistry, which includes an overview of the various methods used in the synthesis of intermediate and final compounds as well as structural confirmation. Various spectral analysis techniques were implemented, such as IR, 1H NMR, 13C NMR, 2D NMR as well as MS spectroscopy and elemental analyses, beside X-ray structure analyses which are used to verify the structure of some compounds. In this study, 61 final target compounds were synthesized (Schemes II, V, VI, VII, VIII).
The second: Biology, which includes various methods and results obtained from various biological evaluations of the synthesized compounds. It is subdivided into two parts:
1- Evaluation of anticancer activity
a- Anticancer screening
The anticancer activity of the synthesized compounds were evaluated against a panel of 60 cancer cell lines derived from different tumors by NCI. Screening results showed that the series 5a-e, 8a-e and 9a-e have a weak to moderate activities on most cell lines especially, leukemia and breast cancer cell lines. Moreover, the results revealed that, the open chain formimidic acid ethyl ester 5a-e exhibited variable degrees of growth inhibitory activity towards a majority of the tested cancer cell lines than the cyclized triazolo-pyrimidine derivatives 8a-e and 9a-e. In addition, 5a with N-methyl quinoline showed more antiproliferative activity on a majority tested leukemia and breast Cancer cell lines.
Interestingly, the second series 11a-f, 13a,c,f,g and 15a,b,e,f,g revealed that, 11d induced a broad tumor cell death for most cancer cell lines. Compound 11d overall potency was considerably higher than all the other tested compounds. The distinguishable inhibitory activity caused by 11d encouraged the NCI to select it for advanced five dose testing. Compound 11d showed anti-cancer activity against all the tested tumor sub-panels with GI50 values mostly between 1-3 µM except three cell lines that exhibited some resistance, including the Leukemia (HL-60), Melanoma (SK-MEL-5) and Ovarian Cancer (NCI/ADR-RES) showed GI50 of 19, 10 and 7 µM respectively. TGI values were less than 10 µM except for the same resistant cell lines. The estimated LC50 values of 11d against the tested cell lines are considerably higher if compared to the corresponding GI50 values, suggesting lower lethal potency of 11d if compared to its ability to cause growth arrest.
b- The new compounds inhibited Src kinases activity
The newly synthesized pyrano[3,2-c]quinoline derivatives were evaluated as Src kinase inhibitors in an in vitro kinase assay. Compound 11d showed the most potent inhibition with IC50s of 0.9 µM.
c- Inhibition of Src by 11d is non-competitive with ATP.
Compound 11d did not impact the affinity of Src towards MgATP, i.e., MgATP (Km) remains unchanged at different doses of 11d, suggesting that 11d does not compete with MgATP for the Src active site.
d- Cellular studies in breast cancer cell lines
1- Compound 11d impairs cell migration of the breast cancer cell line MDA-MB-231.
MDA-MB-231 cells treated with most of the tested compounds, especially 11d, showed a reduced capacity to migrate into the wounded area compared with DMSO-treated cells.
2- The pyrano[3,2-c]quinolone derivative 11d suppress activation and downstream effects of the Src-Fak Complex
The effect of the newly synthesized compounds was tested on Src and Fak phosphorylation at Y416 and Y925, respectively, in the MCF7 breast cancer cell line. Compound 11d was the most potent inhibitor, it inhibited the phosphorylation of both sites at 1.6 µM.
3- Effect of the inhibitors on breast cancer cell colony formation
Interestingly, 3.7 µM of 11d exhibited a solid tendency to inhibit MDA-MB-231 colony-forming ability. While 11 µM of 11a, 11b, 11c and 11e was needed to inhibit MDA-MB-231 colony-forming ability.
4- The effect on serum mediated activation of ERK1/2 and Akt in MCF7 breast cancer cell line
Stimulation of ERK and Akt phosphorylation by serum was entirely diminished by 1.6 µM of 11d. While, 25 µM was needed from 11a, 11b, and 11c to show enough ERK/Akt suppression.
e- Activation of Caspases
Compounds 5a,b,c have been tested for caspase 3 activation and compared with doxorubicin. The results revealed that compounds 5a-c showed an improvement in caspase 3 levels by 4.7-5.9 fold compared with untreated control cells, and that 5a was the most active compound with a remarkable over expression of caspase-3 level (388.58 ± 4.20 pg/mL) compared to doxorubicin (503.2 ± 4.22 pg/mL).
f- Bax and Bcl-2 levels assay
The results show that compounds 5a and 5b increased the Bax level by 29.21 and 20.84-fold greater than the control, compared to doxorubicin 33.43-fold. In addition, compounds 5a and 5d caused a down-regulation of Bcl-2 protein level up to 4.68 and 2.61-fold less than the control compared to doxorubicin 5.17-fold.
g- Cell cycle analysis and apoptosis
Cell cycle analysis and apoptosis assay were carried out for the most active compound 5a that showed pre G1 apoptosis and cell cycle arrest at G2/M phase (supported by Caspases-3,8, BAX and Bcl-2 studies).
h- Effect of compounds 5a and 5b on p53 transcription
The results showed an increase of 12-19 in p53 relative to the test cells and that p53 protein levels of 5a and 5b.
2- Evaluation of antimicrobial urease inhibitory activity:
A new two series of quinolone derivatives containing thiosemicarbazone and thiazole functionalities 19a-g and 21a-l, respectively were screened for their in vitro urease inhibition properties. The results showed that most of the tested compounds exhibited moderate to good inhibitory effect when compared to clinically used enzyme inhibitors, thiourea and acetohydroxamic acid. It is worth mentioning that quinolone-thiosemicarbazone hybrids 19a-g are effective molecules for urease inhibition ranking (IC50= 1.83-11.21 µM). Among 19a-g series, compounds 19a-c bearing methyl group at position-1 on the quinolone skeleton are the most active for urease inhibition ranking (IC50= 1.83-2.48 μM). Morever, 19c bearing methyl group at position-1 and thiosemicarbazide phenyl terminal was proved to be the most potent inhibitor in the present series achieving (IC50= 1.83±0.79 uM).
Moreover, the docking results exhibited the designed compounds can interact with the active site of the enzyme through multiple hydrogen bonds. Moreover, the methylation of the quinolinyl nitrogen was very important because it hindered HB formation between the quinolonyl part and the amino acid clefts. Compound 19c (N-phenylthiourea derivative) was the only compound whose thiourea functionality forms two HBs with the receptor in a chelative fashion.
4- Experimental
This section defines in details the various methods and experiments used in this study. This section is divided into two parts:
The first part: Chemistry, that illustrates the various procedures used in the synthesis of the target compounds 5a-e, 8a-e, 9a-e, 11a-f, 13a,c,f,g, 15a,b,e,f,g, 19a-g, 21a-l and 22a-e. In this section also includes all of the physical, spectroscopic and analytical data for the synthesized compounds were also described.
The second part: Biology, this part describes the method used by the National Cancer Institute to evaluate the anticancer activity of the synthesized compounds. Also, this section illustrates the methodology for various experiments used for molecular mechanism studies of anticancer activity of the tested compounds, it includes the inhibitory assay of Src kinase, cell cycle apoptosis and apoptotic detection.
Furthermore, this section demonstrates antimicrobial screening, urease inhibitory activity and molecular docking on various used protiens.
5- References
This section contains cited references on the chemical synthesis and biological methods of the current work.