Synthesis of anticholinesterase flavonoid derivatives with computer aided drug discovery procedures and investigation of their intraoperative cancer imaging properties

In our era people are facing with aging and dementia due to being under different stress factors. There are approximately 20 millions people are Alzheimer's Disease. Alzheimer's disease which is a neurodegenerative disease is related with anti-inflammatory, antioxidant and antiradical mechanisms. In this thesis we searched cholinesterase enzyme's inhibition ability of 3-hydroxyflavones (3HF) from flavonoid's family and designed more potential 3HFs derivatives with computer aided drug discovery procedures. We prepared a small library of synthetically accessible 3-hydroxyflavone derivatives and using acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzyme's crystal structures from protein data bank (www.pdb.org) with virtual screening procedures. Firstly, compounds were subject to docking studies using the GOLD software package to identify compounds with high docking scores. Subsequently, the high-scoring compounds were visually inspected for enzyme-inhibitor binding interactions. Compounds predicted to be inactive were screened and eliminated before synthesis, so that waste of time and unnecessary chemical consumption were prevented. After this procedures selected compounds were synthesized, spectral analysis of each other were done and cholinesterase enzymes' inhibition capacity of synthesized compounds tested with using the Ellman method. We observed that computer-aided ligand discovery results and in vitro cholinesterase inhibition test results were well in allignment with each other. As mentioned in results section, compounds with high docking scores are also showed good IC50 values in vitro tests. Synthesized compounds showed high inhibition potential to BChE enzyme more than AChE and have also low IC50 values. To these results we discovered our ligands are selectively inhibited BChE to AChE. FN34 showed best inhibiton to BChE enzyme with IC50=19.34 µM even better than standard compound galantamin and second high inhibition to AChE with IC50 =78.47 µM value. In addition FN28, FN11, FN23, FN24, FN35 and FN 38 showed high inhibition to BChE enzymes to compete with standard compound galantamine. When the results are evaluated in terms of the structure activity relationship (SAR), FN15, FN28 and FN34 which have hydroxy group at B ring, forming H-bonding with amino acids by means of these groups. These bonds increase the interactions with BChE enzyme and give high docking scores. As a matter of fact that when the results of in vitro test assays' are examined. FN34 gave best result with IC50=19.36 µM and FN38 gave second good IC50=33.30 µM value to BChE enzyme as expected. FN23 and FN38 which have dimethylamine fragments at 4' position at B ring, gave good IC50 values (IC50= 39.95 µM and IC50=38.44 µM respectively) due to hydrogen bonds with amino acids by nitrogen atom of dimethylamine. Alzheimer's disease is a disease based on oxidative stress and inflammation so that we also searched the capacity of antioxidant potential of compounds with several assays such as Beta carotene lipid peroxidation inhibitory activity, DPPH free radical-scavenging assay, ABTS cation radical decolorization assay and Cupric Reducing Antioxidant Capacity (CUPRAC). In this way our study have gained a scientific character with combination of anticholinesterase and antioxidant assays. ABTS cation radical decolorization assay gave best IC50 results. The IC50 values of FN38, FN21, FN41, FN23, FN15, FN27, FN28 and FN5 are even better than standard compounds BHT ve α-Toc's IC50 values. Quercetin is also a 3HF showed best results in standard compounds. When DPPH free radical-scavenging assay results are wieved FN23 have best IC50 =58.60 µM value even between standard compounds except quercetin. In lipid peroxidation inhibitory activity results FN5 showed best inhibition with 93.59 µM at 200 µM and gave best IC50=58.60 µM value. We thought that two methoxy groups in structure gave lipophilic character to FN 5. When the results of CUPRAC assay evaluated FN7 showed best absorbance value with 2.67 at 100 µM. It was better result than standard compounds BHA, BHT and α-Toc. Quercetin also showed best result again with 3.16 µM. As a matter of fact that literature data showed the flavonoids are the most strong antioxidant structures in the nature. 3-Hydroxyflavones have gained more antioxidant character due to having double bond between C2-C3 and OH group at C3. Conjugation gives them the best antioxidant nucleus character between flavonoids. Our new synthesized 3HF derivatives results also reaffirmed this claim one more time. FN5, FN7 and FN23 which showed best results nearly in all antioxidant tests, have methoxy groups in their structures. In addition, general observation of us the more OH groups the better antioxidant activity. Hydroxy groups at C-4' position increase both the capacity of cholinesterase enzyme's inhibiton and antioxidant activity. In the world millions of people are caught with cancer every year and this ilness resulting with dead mostly. Cancer treatement is started with removel of tumor tissue and continues with chemotherapy generally and other methods. In this thesis, we aimed to remove only timor tissue instead of all cancer organ with using colouring targeted agents. During surgery (intraoperative), to wiev the tumor tissue with photodynamically, we synthesized a 3HF compound FN15 as a starting material. We made an amphiphilic material with adding two polymeric chains to the starting material with polymerization and click reactions. We obtained a micelle from amphiphilic compound in order to be able to target only cancer tissue on the basis of "enhanced permeability and retention effect" (EPR effect) principles. Ehrlich tumor model was created in 2 mice before application. Because of the Ehrlich tumor is a very aggressive short-term tumor that grew up quickly, we checked them after one week and saw it is ready to operation. Micelles were injected to mices subcutaneously (abdominal cavity). one day before from surgery due to preventing the sudden death of mices. After a day the surgery was done carefully by general surgeon. A UV lamp (366 nm) was used during the operation and it was seen that the micelles were targeted and accumulated to the only tumor tissue. Thus, removal of only tumour tissue was achieved instead of all cancerous area. Since there is no toxic effect of flavonoids which are natural compounds, it will able to be applied on not only animals but also humans with advanced clinical studies in the future. Thus, without raising the costs of surgical intervention it will be a new epoch in the field of surgery. This study was supported by the Istanbul Technical University, Scientific Research Projects Unit (Project No: BAP-40259).