الفهرس 
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Abstract
The present study deals with the design, synthesis and biological evaluation of new pyrazoloquinolin-2-ones, arylidenes of quinolin-2-one, quinoline-2-one thiosemicarbazides and 4-thiazolidinoquinoline-2-ones. The synthesized compounds were evaluated for different biological activities including anticancer, antimicrobial and urease inhibitory activity. Additionally, molecular mechanistic studies were applied for some of the active compounds as anticancer and antimicrobial urease inhibitory agents.
1- Introduction
The first part presents an overview about old and modern chemical methods of quinolones synthesis. Also, it includes literature reports about the biological activities of such nucleus including anticancer, anti-bacterial, antifungal, antiviral, antioxidant and anticonvulsant activities.
2- Aim of the work
This section includes the goal of this study, including design and synthesis of some new derivatives of quinoline-2-one and evaluation of different biological activities of these derivatives such as anticancer, antimicrobial and urease inhibitory activity,
3- Results and discussion
This section includes detailed explanation of the various results obtained from different stages of synthesis, structural elucidation and biological evaluation of the target compounds 6a-g, 7a-r, 8a-o, 9a-j, 10a-i and 11a-c.
This section is subdivided into two main subsections:
The first one: Chemistry, which includes a description for the different methods, used for synthesis of intermediate as well as final compounds and structural elucidations of these derivatives by different spectral analysis including IR, 1H NMR, 13C NMR, 2D NMR and MS spectroscopy in addition to elemental analyses, beside X-ray structure analyses which used to prove the structure of some compounds. In the present research study we reported the synthesis of 62 final target compounds.
The second subsection is: Biology, that illustrates different methods and results obtained from various biological evaluations of the synthesized compounds. It is subdivided into parts:
A- Evaluation of anticancer activity
a- Anticancer screening
Anticancer activity was evaluated at the National Cancer Institute, Bethesda, MD, USA. Among the synthesized derivatives, compounds 6a-g, 7a-r and 8a-o were selected according to the protocol of drug evaluation branch of the National Cancer Institute. The selected compounds were screened against sixty different cancer cell lines (NCI-60 cell line panel) including leukaemia, melanoma, lung, colon, CNS, ovarian, renal, prostate and breast cancers. Screening results revealed that the majority of the selected compounds possess moderate to high activity against most of cell lines tested. Pyrazoloquinoline-2-one derivatives 7a-f were more potent than the open chain analogues 6a-g. Also, 8l showed outstanding inhibition on all leukemia cell lines tested with mean GI50 of 14.94 according to NCI protocol.
Compounds 7e and 7f and 8l were selected for five doses testing against the full panel of 60 human tumor cell lines. Compound 7e showed moderate selectivity against ovarian, small cell lung, leukemia and cancer cell lines. Testing of the most active compounds 7a-f showed that 6-halogen substituted derivatives 7e and 7f are the most active against leukemia MOLT-4 and CCRF-CEM cell lines with IC50=4.34 ± 0.14 μM and 5.07 ± 0.10 μM (7e); 1.35 ± 0.08 μM and 2.42 ± 0.04 μM (7f) compared to erlotinib with IC50=7.38 ± 0.56 μM and 4.92 ± 0.24 μM, respectively.
Moreover, among its series, 8l was the most active one; with IC50 = 15.72±1.21 and 46.05±2.36 M against RPMI-8226 and normal cell lines, respectively
b- Molecular mechanistic study
Different experiments have been performed in order to adequately investigate the mechanism of antitumor activity that was exerted by the new target compounds.
Compounds 7e and 7f experienced an outstanding inhibitory activity comparable to erlotinib on the EGFR TK with IC50=247.14 nM and 208.42 nM, respectively. Moreover, 8l showed a remarkable inhibitory activity compared to gefitinib on the EGFR TK mutant, wild and on H-RAS in addition to STAT-3 with IC50 = 695.49±21.8, 263.15±15.13, 10.61±0.27 and 1.753±0.81 nM, respectively
c- Cell cycle analysis and apoptosis
Cell cycle analysis and apoptosis assay were carried out for the most active compounds 7e, 7f and 8l and tested compounds showed pre G1 apoptosis and cell cycle arrest at G2/M phase (supported by Caspases-3,8, BAX and Bcl-2 studies).
d- Molecular docking on EGFR TK enzyme
In this experiment, the binding mode of compounds 7e, 7f and 8l was investigated on the catalytic domain of EGFR (PDB code: 1M17) using Open Eye® software compounds revealed a spontaneous and good fitting with the enzyme binding sites. Also 8l was more investigated using different docking studies (modeling, ROCS and Tanimoto scores) with good fitting with the urease binding sites
B- Evaluation of antimicrobial urease inhibitory activity:
Compounds 9a-j, 10a-i and 11a-c were evaluated for urease inhibitory activity and/or its antimicrobial inhibition.
Results revealed that most of the tested compounds showed moderate to good activity against R. mucilaginosa, with 12-14 mm zone of inhibition compared to clotrimazole (25 mm). Compound 9h experienced the highest anti- P. mirabilis activity achieving MIC= 399.3 µM which represents about 300 times less activity than the reference ciprofloxacin.
Urease inhibitory activity was done using Weatherburn indophenol method, taking thiourea as a standard urease inhibitor. Most compounds showed urease inhibitory activity better than their reference thiourea.
Compounds 10e and 9b were the most active with (IC50 = 0.46±0.04 and 0.43 ± 0.09 μM). Also, 10b, 10d and 9a showed outstanding urease inhibitory potential with IC50 values of 0.92±0.17, 0.74±0.14 and 0.58 ± 0.15μM, respectively.
Moreover, selected compounds were docked into urease proteins (ID: 3LA4 and ID: 4UBP) using Open Eye® software to understand correctly about ligand–receptor interactions. The docking results revealed that the designed compounds can interact with the active site of the enzyme through multiple strong hydrogen bonds. Moreover, rapid overlay of chemical structures’ analysis was described to understand the 3D QSAR of synthesized compounds as urease inhibitors. The results emphasize the importance of polar thiosemicarbazide directly linked to 6-substituted quinolone moieties as promising antimicrobial urease inhibitors.
4- Experimental
This section illustrates in details the different methods and experiments used in this research. This section includes two main parts:
Firstly: Chemistry, this part describes the different procedures used for synthesis of the target compounds 6a-g, 7a-r, 8a-o, 9a-j, 10a-i and 11a-c. This part also comprises all detailed physical, spectroscopic and analytical data of the synthesized compounds.
The second part: Biology section that outlined the method applied by the National Cancer Institute for evaluation of the anticancer activity of the selected compounds. This part described also the methodology for different experiments applied for molecular mechanism study of anticancer activity of the test compounds including inhibitory assay of EGFR TK, H-RAS and STAT, cell cycle apoptosis and apoptotic detection.
Additionally, this part illustrated the protocols of antimicrobial screening, urease inhibitory assay and molecular docking on different used enzymes.