Tổng hợp và đánh giá tác dụng sinh học các dẫn xuất của indirubin TT TIENG ANH

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY …………***………… NGUYEN TRONG DAN SYNTHESIS AND EVALUATE BIOLOGICAL ACTIVITIES OF INDIRUBIN DERIVATIVES Major: Chemistry of natural compounds Code: 44 01 17 SUMMARY OF CHEMICAL DOCTORAL THESIS HA NOI - 2021 This thesis was completed at Graduate University of Science and Technology - Vietnam Academy of Science and Technology Supervisors: Dr Luu Van Chinh Institute of Natural Products Chemistry – Vietnam Academy of Science and Technology Dr To Dao Cuong Phenikaa University Examiner 1: Examiner Examiner 3: The thesis defense was monitored by the Graduate University level Board of Examiners, held at: Graduate University of Science and Technology - 18 Hoang Quoc Viet - Cau Giay - Ha Noi At ……… , ….………………… 2021 The thesis is available in Vietnam National Library and Library of Graduate University of Science and Technology INTRODUCTION The urgency of the thesis Natural compounds made up from biosynthetic processes in plants and animals, microorganisms usually exhibit specific biological activities, highly selective, easily tolerated by the body and have few side effects Therefore, many of them have been widely used in practice to treat various diseases such as cancer, infections, etc., whose therapeutic effects are determined by their chemical structure The chemical structures of natural compounds are often very complex The process of obtaining these compounds using total synthesis pathway will not be economical due to many reaction steps, low yield, high environmental treatment cost, and long reaction time Thus, taking advantage of available resources from from nature to synthesize and isolate lead compounds, then convert them into derivatives with better biological activity than the initial lead compounds are attracting the attention of scientists Recently, scientists have discovered many biological activities of indirubin compounds and derivatives, a bisindole alkaloid that can be obtained in large quantities from Strobilanthes cusia, which is abundant in Vietnam or by the reaction between isatin and indoxyl acetate This bioactive compound and its derivatives exhibit many valuable biological activities such as toxicity to many cancer cell lines, antiplatelet effects, anti-inflammatory, antiviral, antioxidant, and antifungal effects, anti-psoriatic with an obvious mechanism Until now, the investigation of these activities is still ongoing However, to apply this compound in practice is still difficult due to its poor solubility in water, a number of studies have been conducted using the first direction to improve the solubility of indirubin For the second direction, several derivatives have also been synthesized but the publications are still limited Following this direction, the thesis topic refers to the synthesis of some new derivatives of indirubin, which are hybrid combinations of indirubin with heterocyclic compounds containing S, N, O; hydroxychalcone compounds and amine compounds, and then evaluated the antitumor activity in vitro, investigate their mechanism of action in order to provide chemical and biological activity data of these derivatives as the basis for further studies in the search for new drugs from plant resources available in Vietnam Based on the above arguments, we selected the topic: "Synthesis and evaluate biological activities of indirubin derivatives" The research objectives of the thesis - Synthesized at least 40 new derivatives of indirubin, derived from the starting material indirubin obtained from Strobilanthes cusia - Evaluate anticancer activities in vitro of the obtained derivatives The main research contents of the thesis Based on the above objectives, we have implemented the research content of the thesis as follows: + Synthesize new derivatives of indirubin which are hybrid combinations of indirubin with compounds 1,3,4-oxadiazole, 1,3,4thiadiazole, 6-mercaptopurine, hydroxychalcone compounds and amine compounds; + Determine the structure of the newly synthesized derivatives using modern spectroscopic methods; + Evaluate anticancer activity in vitro (colon cancer SW480, lung cancer LU-1, liver cancer HepG2, blood cancer HL-60) of newly synthesized derivatives; + Study the quantitative structure-anticancer activity relationship of newly synthesized derivatives using AutoDock 4.2 software Chapter OVERVIEW The literature review is a collection of national and international studies on the following issues: 1.1 Introduction to indirubin and its isomers 1.2 Obtaining and synthesizing indirubin 1.3 Synthesis of derivatives of indirubin 1.4 Biological activity of indirubin and its derivatives 1.5 Overview of compounds 1,3,4-oxadiazole, 1,3,4-thiadiazole, 6mercaptopurine 1.6 Overview of chalcone 1.7 Introduction to molecular docking 1.8 Enzyme glycogen synthase kinase 3β (GSK-3β) Chapter MATERIALS AND METHODS This section describes in detail the origin of indirubin used for research, the reactions used in the thesis, modern techniques used to analyze the structure, chemicals, equipment, and cell lines used for research, bioactivity evaluation methods, ligand and target protein preparation, interaction simulation using AutoDock 4.2.6 software 2.1 Reseach objects The research object of the thesis is the compound indirubin obtained from Strobilanthes cusia Indirubin: Provided by Assoc Dr Nguyen Manh Cuong, Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology (purity ≥ 95%) 2.2 Research Methods The method used in the thesis consists of three main parts: Method of synthesizing indirubin derivatives using modern techniques such as NMR-1D, NMR-2D, HR-MS(ESI) to determine the structure of the obtained compounds Method to evaluate biological activity in vitro (cytotoxicity and anti-proliferative) and method to simulate interactions of compounds on GSK-3β enzyme targets using AutoDock 4.2.6 software 2.3 Chemicals, research equipment The chemicals and TLC (thin layer chromatograp used for the study were purchased from chemical companies such as SigmaAldrich, Scharlau, Prolabo The Bruker ADVANCE 500 MHz is utilized to analyze NMR spectrum; Agilent 6530 Accurate-Mass Q-TOF LC/MS is used to record HR-MS(ESI) spectra; Bruker D8 QUEST at 273 K with MoKα (λ = 0.71073 Å) with a TRIUMPH monochromator is used to record X-ray diffraction spectra; Buchi 530 is used to measure melting point 2.4 Biological activity evaluation methods The cytotoxicity assay was applied on Hep-G2 (ATCC-HB-8065), LU-1 (ACTT-HTB-57), SW480 (ATCC-CCL-228), HEK cell lines 293 (ATCC-CRL-1573) The anti-proliferative assay was applied on the HL-60 cell line (ATCC-CCL-240) 2.5 Ligands and protein preparation The two-dimensional structures of the studied compounds were built using Chemdraw19.1 software (ChemOffice 2019) and then converted to three-dimensional form using Chem3D The energy minimization was carried out using Gabedit2.5.0 The main chain of compounds were determined automatically by the AutoDockTools software (MGLTools) The 3D structure of GSK-3β protein was obtained from the protein data bank (PDB) and prepared using AutoDockTools 2.6 Docking using AutoDock4.2.6 AutoDock Tools software (MGLTools) was utilized to prepare input data for docking simulation The position and size of the grid box used for the simulation was determined to ensure that all the key residues participated in the binding interaction with the reference ligands indirubin-3′-oxime ligand, CHIR- 98014 and BIO-acetoxime are included The obtained results will be analyzed using output data including: binding free energy, the number of interaction formed within the active site of targeted protein Chapter EXPERIMENTAL The empirical section described in detail the synthesis pathway of new indirubin derivatives and biological activities assays of the new derivatives 3.1 Synthesis of hybrid combinations of indirubin with compounds 1,3,4-oxadiazole, 1,3,4-thiadiazole, 6-mercaptopurine via trimethylene linker (metabolized at the hydroxyl group of C3ʹ-oxime) The synthesis scheme: Scheme 3.1 Synthesis of hybrid combinations of indirubin with compounds 1,3,4-oxadiazole, 1,3,4-thiadiazole, 6-mercaptopurine via trimethylene linker (metabolized at the hydroxyl group of C-3ʹ-oxime) Table 3.1 Structure of thiols 41a-41i No Compound RSH No Compound 41a 41f 41b 41g 41c 41h 41d 41i RSH 3.2 Synthesis of hybrid combinations of indirubin with compounds 1,3,4-oxadiazole, 1,3,4-thiadiazole, 6-mercaptopurine via 2-ol-1,3-propylene linker (metabolized at group hydroxyl of C3ʹ-oxime) The synthesis scheme: Scheme 3.2 Synthesis of hybrid combinations of indirubin with compounds 1,3,4-oxadiazole, 1,3,4-thiadiazole, 6-mercaptopurine via 2-ol1,3-propylene linker (metabolized at group hydroxyl of C-3ʹ-oxime) 3.3 Synthesis of hybrid combinations of indirubin with compounds 1,3,4-oxadiazole, 1,3,4-thiadiazole, 6-mercaptopurine via 2-ol-1,3-propylene linker (metabolized at N-1) The synthesis scheme: Scheme 3.3 Synthesis of hybrid combinations of indirubin with compounds 1,3,4-oxadiazole, 1,3,4-thiadiazole, 6-mercaptopurine via 2-ol1,3-propylene linker (metabolized at N-1) 3.4 Synthesis of hybrid combinations of indirubin with hydroxychalcone compounds via methylene 1,2,3-triazole linker (metabolized at the hydroxyl group of C-3ʹ-oxime) The synthesis scheme: Scheme 3.4 Synthesis of hybrid combinations of indirubin with hydroxychalcone compounds via methylene 1,2,3-triazole linker (metabolized at the hydroxyl group of C-3ʹ-oxime) 3.5 Synthesis of hybrid combinations of indirubin with hydroxychalcone compounds via methylene 1,2,3-triazole linker (metabolized at C-3ʹ and N-1 positions) The synthesis scheme: Scheme 3.5 Synthesis of hybrid combinations of indirubin with hydroxychalcone compounds via methylene 1,2,3-triazole linker (metabolized at C-3ʹ and N-1 positions) 3.6 Synthesis of hybrid combinations of indirubin with amine compounds via methylene 1,2,3-triazole linker (metabolized at the C-3ʹ and N-1) The synthesis scheme: Scheme 3.6 Synthesis of hybrid combinations of indirubin with amine compounds via methylene 1,2,3-triazole linker (metabolized at the C-3ʹ and N-1) 3.7 Biological activity evaluation of new indirubin derivatives 4.1.4 Synthesis of hybrid combinations of indirubin with hydroxychalcone compounds via methylene 1,2,3-triazole linker (metabolized at the hydroxyl group of C-3ʹ-oxime) Compounds 53a-53i were synthesized according to scheme 3.4, where the click reaction was used to couple 4-(2-azidoethoxy)-2hydroxychalcone 51a-51l with indirubin-3ʹ-[O-(prop-) 2-ynyl)oxime] 52 in the presence of CuI First, the 4-(2-azidoethoxy)-2-hydroxychalcone 51a-51l was synthesized from the initial compound 2,4-dihydroxyacetophenone (47) by a 3-step process with high yield as follows: Ketone 47 was Oalkylated selectively -OH group at position with 1,2-dichloroethane in DMF solvent at temperature 55oC, tác K2CO3 catalyzed for 10 hours At the end of the reaction, the crude product was purified by column chromatography/silica gel, and the solvent system eluted nhexane/acetone (3/1) to obtain the intermediate compound 4-(2chloroethoxy)-2- hydroxyacetophenone (48) with a yeild of 61%, where the -OH group at position does not participate in O-alkylation because of the formation of an intramolecular hydrogen bond between the H atom of this group and the O atom of group C=O in the 1-acetyl group Next, this alkylchloride derivative was reacted with NaN3 in DMSO solvent at 60oC in 24 hours giving the intermediate 4-(2azidoethoxy)-2-hydroxyacetophenone (49) with a yeild of 81% Finally, this ketone compound was condensed Claisen-Schmidt with aldehydes including benzaldehyde (50a), 3,4,5trimethoxybenzaldehyde (50b), 2-methoxybenzaldehyde (50c), 3methoxybenzaldehyde (50d), 4-methoxybenzaldehyde (50e), 2ethoxybenzaldehyde (50f), -ethoxybenzaldehyde (50g), 4ethoxybenzaldehyde (50h), 2-propoxybenzaldehyde (50i), 3propoxybenzaldehyde (50k) and 4-propoxybenzaldehyde (50l) in MeOH catalysed by KOH to form 4-(2-azidoethoxy)-2hydroxychalcone 51a-51l with yield of 78-87% Among them, there are new chalcones including 51a, 51f, 51g, 51h, 51i, 51k, 51l and 11 known chalcones 51b, 51c, 51d, 51f described in previous literature The obtained 4-(2-azidoethoxy)-2-hydroxychalcones are solid, yellow colored solids characteristic of chalcones The structures of intermediates 48, 49 and (2-azidoethoxy)-2-hydroxychalcone were determined by NMR and HR-MS(ESI) spectroscopy methods Next, the key intermediate indirubin-3ʹ-[O-(prop-2-ynyl)oxime] (52) was prepared from indirubin in two steps First, indirubin-3ʹoxime (25) is formed by condensation reaction of indirubin with hydrochloric hydroxylamine in pyridine solvent, refluxing for hours Next, this compound was O-alkylated with propargyl bromide in DMF solvent at room temperature, catalyzed with triethylamine in 48 hours At the end of the reaction, the crude product was purified by column chromatography/silica gel, the solvent system eluted nhexane/acetone (3/1) to obtain the key intermediate compound indirubin-3ʹ-[O-( prop-2-ynyl)oxime (52) with an yield of 79% The structure of compound 52 was determined by NMR and HR-MS(ESI) spectroscopy methods The final step in the synthesis of indirubin hybrids with hydroxychalcone compounds via a 1,2,3-triazole methylene bond is for each of the above 4-(2-azidoethoxy)-2-hydroxychalcone to react with the indirubin compound indirubin-3ʹ-[O-(prop-2-ynyl)oxime (52) in DMF solvent at room temperature, catalyzed with CuI for 24 hours The reaction progress was controlled by TLC, at the end of the reaction, the mixture was diluted with ethyl acetate, then washed with water, dried with anhydrous Na2SO4, the crude product was purified by column chromatography/silicagel, eluent system n-hexane/acetone (3/2) obtained eleven new derivatives of indirubin 53a-53l with yield of 54-67% The structures of the compounds were determined by modern spectroscopic methods such as: NMR -1D, 2D and HRMS(ESI) 12 4.1.5 Synthesis of hybrid combinations of indirubin with hydroxychalcone compounds via methylene 1,2,3-triazole linker (metabolized at C-3ʹ and N-1 positions) According to the scheme 3.5, the hybrid combinations 56a-56l of indirubin-3ʹ-oxime with hydroxychalcone compounds via methylene 1,2,3-triazole linkage are obtained through three reaction steps First, carried out N-alkylation between indirubin with propargyl bromide in anhydrous DMF solvent at room temperature, using K2CO3, KI, 1(butyl)triethylammonium bromide catalyst system for 48 hours At the end of the reaction, the crude product was purified by column chromatography/silica gel, elution system n-hexane/acetone (4/1) obtained compound 54 with yeild of 59% The structure of this compound was determined by NMR, HR-MS(ESI) spectroscopy Similar to the N-alkylation of indirubin with epichlorohydrin, the Npropargylation of indirubin only occurs at the N-1 position Next, the key intermediate 55 was formed with an efficiency of 79% by the condensation reaction between 54 with hydroxylamine hydrochloride in a pyridine solvent The single-crystals 55 was formed in the nhexane/acetone (2/1) solvent system and used for single-crystal X-ray diffraction spectroscopy The structure of 55 was determined by NMR, HR-MS(ESI) spectroscopy combined with single crystal X-ray diffraction spectroscopy The crystallographic data of this compound have been sent to the UK crystallography data center and given the number CCDC 1917485, confirming the structure of the synthesized compound 55 is correct and has an absolute configuration of (2ʹZindirubin, 3ʹEoxime) (Figure 4.27) From the single-crystal structure of compound 55, it is confirmed that indirubin has absolute configuration 2ʹZ and oxime 25 is formed with 3ʹE configuration Finally, a 1,2,3-triazole cyclization reaction was performed between compound 55 with with each 4-(2-azidoethoxy)-2- 13 hydroxychalcone 51a-51l in DMSO solvent at room temperature, catalyzed with CuI for 24 hours The crude product was purified by column/silica gel chromatography, the n-hexane/acetone (3/2) elution system obtained eleven compounds 56a-56l with yeild of 57-70% The structures of the compounds were determined by modern spectroscopic methods such as: NMR -1D, 2D and HR-MS(ESI) Figure 4.27 Crystal structure of compound 55 4.1.6 Synthesis of hybrid combinations of indirubin with amine compounds via methylene 1,2,3-triazole linker (metabolized at C-3ʹ and N-1 positions) Indirubin and its derivatives have very poor water solubility resulting in low bioavailability To improve the solubility of indirubin derivatives, this compound can be coupled to amines via a 1,2,3triazole methyl bond via click chemistry (Figure 3.6) The amine component was selected starting from the halogen derivatives: 2chloroethyldimethylamine, 1-(2-chloroethyl)piperidine, 1-(2chloroethyl)imidazole and N-(2-chloroethyl)morpholine These chloro amine derivatives were converted to azidos of the respective amines by NaN3/K2CO3 in in DMSO at 60°C After centrifugation to remove solids, each azido solution of the amines was obtained for click chemistry with (2ʹZ, 3ʹE)-1-(prop-2-ynyl)indirubin-3ʹ-oxime (55) in the presence of CuI catalyst at room temperature to get click products 56m-56p The crude product was purified by column chromatography/silica gel eluted with the n-hexane/acetone system 14 (1/1) yielding the target products 56m-56p with yields ranging from 56-68% The structures of the compounds were determined by modern spectroscopic methods such as: NMR -1D, 2D and HR-MS(ESI) 4.2 Evaluation of the anticancer activity in vitro of new indirubin derivatives 4.2.1 Evaluation of the antitumor activity of new indirubin derivatives 42a-42i Table 4.13 In vitro cytotoxic and antiproliferative activities of new indirubin 42a-42i derivatives and positive controls IC50 (μM) No Compound 10 11 42a 42b 42c 42d 42e 42f 42g 42h 42i 6-mercaptopurin Indirubin Indirubin-3ʹoxime Ellipticine 12 13 SW480 LU-1 HepG2 HL-60 >20 >20 >20 >20 2,56 >20 >20 >20 4,82 19,10 >20 >20 >20 >20 >20 2,92 >20 >20 >20 5,19 18,75 >20 >20 >20 >20 >20 3,69 >20 >20 >20 4,70 19,72 >20 >20 >20 >20 >20 2,54 >20 >20 >20 5,94 16,04 >20 14,26 13,11 15,87 14,90 2,19 1,91 1,79 2,40 The results of cytotoxicity and antiproliferative assays on cancer cells showed that two derivatives 42e and 42i exhibit cytotoxic activities on all four tested cell lines, IC50 values ranged from 2,545,94 µM The remaining seven derivatives did not show activity at the studied concentrations 4.2.2 Evaluation of the antitumor activity of new indirubin derivatives 43a-43i 15 Table 4.14 In vitro cytotoxic and antiproliferative activities of new indirubin 43a-43i derivatives and positive controls IC50 (μM) No 10 11 12 13 Compound 43a 43b 43c 43d 43e 43f 43g 43h 43i 6-mercaptopurin Indirubin Indirubin-3ʹoxime Ellipticine SW480 LU-1 HepG2 HL-60 19,24 >20 >20 >20 >20 16,38 15,96 >20 >20 19,10 >20 19,00 >20 >20 >20 >20 16,90 19,11 19,20 >20 18,75 >20 >20 >20 >20 >20 >20 17,67 15,34 >20 >20 19,72 >20 >20 >20 >20 >20 >20 9,52 12,03 16,40 >20 16,04 >20 14,26 13,11 15,87 14,90 2,19 1,91 1,79 2,40 The results of cytotoxicity and antiproliferative assays on cancer cells showed that two derivatives 43f and 43g exhibit cytotoxic activities on all four tested cell lines, IC50 values ranged from 9,5219,11 µM Compounds 43a and 43h exhibited weak cytotoxic activity against two cancer cell lines The remaining five derivatives did not show activity at the studied concentrations 4.2.3 Evaluation of the antitumor activity of new indirubin derivatives 45a-45h Table 4.15 In vitro cytotoxic and antiproliferative activities of new indirubin 45a-45h derivatives and positive controls IC50 (μM) No Compound 45a 45b SW480 LU-1 HepG2 HL-60 >20 >20 >20 >20 >20 >20 >20 >20 16 10 11 12 45c 45d 45e 45f 45g 45h 6-mercaptopurin Indirubin Indirubin-3ʹoxime Ellipticine >20 >20 >20 1,65 >20 4,19 19,10 >20 >20 >20 >20 2,21 >20 4,53 18,75 >20 >20 >20 >20 1,90 >20 4,47 19,72 >20 >20 >20 >20 1,35 >20 4,28 16,04 >20 14,26 13,11 15,87 14,90 2,19 1,91 1,79 2,40 The results of cytotoxicity and antiproliferative assays on cancer cells showed that two derivatives 45f and 45h exhibit cytotoxic activities on all four tested cell lines, IC50 values ranged from 1,354,53 µM The remaining six derivatives did not show activity at the studied concentrations 4.2.4 Evaluation of the antitumor activity of new indirubin derivatives 53a-53l Table 4.16 In vitro cytotoxic and antiproliferative activities of new indirubin 53a-53l derivatives and positive controls IC50 (μM) No Compound 10 11 53a 53b 53c 53d 53e 53f 53g 53h 53i 53k 53l SW480 LU-1 HepG2 HL-60 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 >20 17 12 13 14 Indirubin Indirubin-3ʹoxime Ellipticine >20 14,26 >20 13,11 >20 15,87 2,19 1,91 1,79 >20 14,90 2,40 Obtained results showed that all derivatives did not exhibit cytotoxic and anti-proliferative activities on all four cancer cell lines at the studied concentrations 4.2.5 Evaluation of the antitumor activity of new indirubin derivatives 56a-56p Table 4.17 In vitro cytotoxic and antiproliferative activities of new indirubin 54, 55, 56a-56p derivatives and positive controls IC50 (μM) No Compound 10 11 12 13 14 15 16 17 18 54 55 56a 56b 56c 56d 56e 56f 56g 56h 56i 56k 56l 56m 56n 56o 56p Indirubin Indirubin-3ʹoxime Ellipticine 19 20 SW480 LU-1 HepG2 >20 18,58 3,36 >20 3,80 4,46 4,66 2,54 4,10 14,13 3,43 2,91 3,03 14,36 12,54 11,16 11,71 >20 15,65 >20 19,55 2,26 >20 2,99 2,85 3,28 1,30 3,96 8,74 2,07 2,50 2,07 8,74 10,08 8,80 8,61 >20 16,36 >20 >20 3,56 >20 3,38 3,76 4,62 2,01 5,68 14,23 2,28 2,95 3,28 15,31 11,70 11,68 10,61 >20 16,00 >20 15,27 2,39 >20 1,43 3,40 3,27 0,98 4,82 6,50 1,28 1,29 1,28 15,96 10,44 9,39 11,25 >20 1,76 2,50 1,93 2,19 18 HL-60 HEK-293 16,50 >20 16,22 3,21 >20 1,19 2,01 6,98 1,03 1,80 9,74 0,85 1,92 7,12 11,93 9,52 5,89 7,72 >20 14,16 0,32 Based on the results in Table 4.17, it was found that intermediate compound 55 showed weak activity against SW-480, LU-1 and HL60 lines The hybrids of indirubin with hydroxychalcone via the methylene 1,2,3-triazole 56a-56l linkage exhibited significantly stronger activity than the precursors, with the exception of compound 56b with the simultaneous presence of methoxy groups (CH3O-) at positions R2, R3, R4 did not show activity on all cancer cell lines at the studied concentrations Compound 56f exhibited the strongest cytotoxic and anti-proliferative activities on cancer cell lines SW480, LU-1, HepG2 and HL-60 with IC50 value of 2,54; 1,30; 2,01 and 0,98 μM, respectively, significantly higher than indirubin-3'-oxime Correlation analysis between the structure of compound 55, indirubin3'-oxime, the 56a-56p derivatives combined with the experimental data suggest that the presence of an oxime group (=N-OH) in the structure might play an important role in the cytotoxic and antiproliferative activities of cancer cells This hypothesis is also consistent with the results of the 53a-53l series (Table 4.16), these indirubin derivatives lack the oxime group (with the same hydoxychalcone composition) and did not show any activity It is noteworthy that three hybrid combinations 56e, 56h and 56l of indirubin-3'-oxime with hydroxychalcone via methylene 1,2,3triazole linkage possess substituents methoxy (CH3O-), ethoxy (C2H5O-), propoxy ( n-C3H7O-) at the R3 position (the substituent at the para position in the aldehyde component of hydroxychalcone) was observed to influence the selective cytotoxicity between cancer and normal cells The evidence is that the IC50 values of compounds 56e, 56h and 56l on normal renal cell line HEK-293 are 6,98; 9,74; 7,12 μM, higher than that of cancer cell lines which mean they have exhibit lower toxicity These results can be used for further studies on anticancer potential 19 The 56m-56p derivatives both exhibited stronger cytotoxic and anti-proliferative activities on cancer cell lines than indirubin-3'oxime, but in general, they exhibit weaker activities than the 56a-56l derivatives However, these compounds can form soluble salts with acids and increase their bioavailability 4.3 Study the quantitative structure–anticancer activity relationship of studied compounds To explain why amongst the above synthetic derivatives, there are substances that not exhibit anti-cancer activity at the concentrations studied meanwhile the others do, we carried out the quantitative structure–anticancer activity relationship studies Simulation of interactions of compounds on GSK-3β enzyme targets using AutoDock4.2.6 software will be used to contribute to the interpretation of the above experimental results The simulation results in Table 4.18 and Table 4.19 show that there is a good correlation between the binding free energies (dock energy or affinity) of the compounds with the GSK-3β enzyme as well as the number of hydrogen bonds formed between compounds with key residues in the active site of the enzyme (amino acids in bold) and experimental antitumor activity Table 4.18 Dock score and predicted inhibition constant values of studied compounds Compound E dock Ki, pred Compound E dock Ki, pred (Kcal/mol (M) (Kcal/mol) (M) ) 54 -8,49 600,31.10-9 -12,73 467,7.10-12 56k 55 56a 56b 56c 56d 56e -9,30 -13,64 -9,34 -13,17 -11,82 -13,18 151,8.10-9 99,73.10-12 1,39.10-6 222,3.10-12 2,18.10-9 219,2.10-12 56l 56m 56n 56o 56p 20 -13,78 -9,88 -11,55 -11,19 -11,25 -9,08 79,11.10-12 57,51.10-9 3,43.10-9 6,27.10-9 5,71.10-9 222,71.10-9 56f 56g 56h 56i -14,09 -13,72 -11,53 46,6.10-12 87,29.10-12 3,53.10-9 -12,85 380,4.10-12 25 CHIR-98014 Bioacetoxime -9,51 -11,82 -10,69 107,82.10-9 2,18.10-9 14,06.10-9 Table 4.19 Hydrogen bond formed between studied compounds with key residues of GSK-3β enzyme Compound No of H-bond Amino acid Compound No of H-bond 54 Val135 56i 55 Val135 56n 56a Ile62, Val135, Gln185 56b Ile62 25 56c Ile62, Val135, Asn186, Cys199 56f Val135, Thr138 CHIR98014 Bioacetoxime Amino acid Asn64, Tyr134, Gln185 Ile62, Val135, Cys199 Asp133, Val135 Ile62, Asp133, Val135 Ile62, Thr138, Val135, Gln185 Asp133, Val135 CONCLUSIONS In this thesis, we have synthesized 52 new compounds which are derivatives of indirubin including: - new derivatives 42a-42i are the combination of indirubin-3'oxime with compounds 1,3,4-oxadiazole, 1,3,4-thiadiazole, 6mercaptopurine via trimethylene linker at position 3'- oxime; - new derivatives 43a-43i are the combination of indirubin-3'oxime with compounds 1,3,4-oxadiazole, 1,3,4-thiadiazole, 6mercaptopurine via 2-ol-1,3 -propylene linker at the 3'-oxime position; 21 - new derivatives 45a-45i are the combination of indirubin with compounds 1,3,4-oxadiazole, 1,3,4-thiadiazole, 6-mercaptopurine via 2-ol-1,3-propylene linker at position N-1; - 11 new derivatives 53a-53l are the combination of indirubin-3'oxime with hydroxychalcone compounds via methylene 1,2,3-triazole linker at the 3'-oxime position; - 11 new derivatives 56a-56l are the combination of indirubin-3ʹoxime with hydroxychalcone compounds via methylene 1,2,3-triazole linker at position N-1; - new derivatives 56m-56p are the combination of indirubin-3ʹoxime with amine compounds via methylene 1,2,3-triazole linker at position N-1 The target compounds and intermediate compounds in the synthesis process are determined by the modern spectroscopic methods such as 1D, 2D-NMR, HR-MS(ESI) In addition, the key intermediate compound 55 was subjected to single-crystal X-ray diffraction spectroscopy to determine the absolute configuration Evaluation of in vitro anticancer activity of new indirubin derivatives: - Evaluation of the cytotoxic and anti-proliferative assays of 52 new derivatives on four cancer cell lines SW480, LU-1, Hep-G2, HL60 resulted in: 22 new derivatives including 42e, 42i, 43a, 43f, 43g, 43h, 45f, 45h, 56a and 56c-56p exhibited cytotoxic and antiproliferative activities against cancer cell lines with IC50 values ranging from 1,3-19,24 µM Of which, 20 derivatives including 42e, 42i, 43f, 43g, 45f, 45h, 56a and 56c-56p showed cytotoxic and antiproliferative activities on all four tested cell lines In particular, there are 18 dervatives including 42e, 42i, 45f, 45h, 56a and 56c-56p exhibit cytotoxic and anti-proliferative activities on all four cancer cell lines with IC50 values ranging from 1,3-15,96 µM, this activity is stronger than that of indirubin-3ʹ-oxime, a compound that is being studied extensively for cancer studies - Evaluation of cytotoxic activity of 15 compounds 56a-56p on normal renal cell line HEK-293 resulted in: Three compounds 56e, 22 56h, 56l exhibit cytotoxic activity on normal renal cell line with the corresponding IC50 value of 6,98; 9,74; 7,12 μM, which is higher than the IC50 value in cancer cell lines Therefore, it is suggested that these three compounds are potential for further studies to find new drugs to treat cancer Quantitative structure–anticancer activity relationship of studied compounds: - Compare the experimental results to evaluate the in vitro anticancer activity of indirubin derivatives at 3'-oxime and N-1 received, especially the results of the experiments of series 53a-53l and 56a -56l, we hypotheze that the 3'-oxime group (=N-OH) in the structure of indirubin derivatives can play an important role to induce antitumor activity on SW480, LU- 1, Hep-G2, HL-60 of compounds The appearance of hydroxychalcone components at the new N-1 position increases the antitumor activity of the derivatives This result is also consistent with the results of investigation of interactions of derivatives with GSK-3β enzyme - Docking simulation of studied compounds on GSK-3β enzyme targets using AutoDock4.2.6 has contributed to elucidating anticancer activity on cell lines SW480, LU-1, Hep-G2, HL- 60 of the derivatives PROPOSALS Further expanding the trial of cardiovascular activities, especially the antiplatelet effect of indirubin derivatives NEW CONTRIBUTIONS OF THE THESIS A total of 52 new indirubin derivatives have been synthesized, including: - new derivatives 42a-42i are the combination of indirubin-3'oxime with compounds 1,3,4-oxadiazole, 1,3,4-thiadiazole, 6mercaptopurine via trimethylene bond at position 3'-oxime; - new derivatives 43a-43i are the combination of indirubin-3'oxime with compounds 1,3,4-oxadiazole, 1,3,4-thiadiazole, 623 mercaptopurin via 2-ol-1,3-propylen bond at position 3'-oxime; - new derivatives 45a-45i are the combination of indirubin with compounds 1,3,4-oxadiazole, 1,3,4-thiadiazole, 6-mercaptopurin via 2-ol-1,3-propylen bond at position N-1; - 11 new derivatives 53a-53l are the combination of indirubin-3'oxime with hydroxychalcone compounds via methylen 1,2,3-triazole bond at position 3'-oxime; - 11 new derivatives 56a-56l are the combination of indirubin-3ʹoxime with hydroxychalcone compounds via methylen 1,2,3-triazole bond at position N-1; - new derivatives 56m-56p are the combination of indirubinʹ -oxime with amine compounds via methylen 1,2,3-triazole bonds at position N-1 The target products and intermediate products during the synthesis process are determined by the modern spectroscopic methods such as 1D, 2D-NMR, HR-MS(ESI) In addition, the key intermediate (2ʹZ, 3ʹE)-1-(prop-2-ynyl)indirubin-3ʹ-oxime was subjected to single-crystal X-ray diffraction spectroscopy to determine the absolute configuration Evaluated cytotoxicity and antiprofilerative activities of 52 new indirubin derivatives on four cancer cell lines SW480, LU-1, Hep-G2, HL-60 In addition, evaluated cytotoxicity of 15 compounds 56a-56p on the normal renal cell line HEK-293 The results indicated that derivatives 56e, 56h, 56l exhibit less cytotoxic activities on the normal renal cell line with the IC50 value higher than that of cancer cell lines These results suggest these compounds potential for further studies to develop new drugs to treat cancer Studied the quantitative structure–activity relationship of synthesized derivatives Simulated the interaction between compounds on GSK-3β enzyme using AutoDock 4.2.6 software Obtained results contributed important data to elucidating mechanism of action on the anti-cancer activity on SW480, LU-1, Hep-G2, HL60 cell lines of studied compounds 24 PUBLISHED SCIENTIFIC WORKS Nguyen Trong Dan, Hoang Duc Quang, Vuong Van Truong, Do Huu Nghi, Nguyen Manh Cuong, To Dao Cuong, Tran Quoc Toan, Long Giang Bach, Nguyen Huu Thuan Anh, Nguyen Thi Mai, Ngo Thi Lan, Luu Van Chinh, Pham Minh Quan, Design, synthesis, structure, in vitro cytotoxic activity evaluation and docking studies on target enzyme GSK-3β of new indirubin-3ʹ-oxime derivatives, Scientific Report, 2020, 10:11429 ( ISI uy tín, Q1) Dan Trong Nguyen, Giang Nguyen Truong, Truong Van Vuong, Tai Nguyen Van, Cuong Nguyen Manh, Cuong To Dao, Thuy Dinh Thi Thuy, Chinh Luu Van, Vu Tran Khac, Synthesis of new indirubin derivatives and their in vitro anticancer activity, Chemical Papers, 2019, 73, 1083-1092 (Quốc tế uy tín, Q2) Nguyễn Trọng Dân, Nguyễn Trường Giang, Phạm Minh Quân, Tô Đạo Cường, Nguyễn Mạnh Cường, Lưu Văn Chính, Tổng hợp đánh giá hoạt tính chống ung thư in vitro dẫn xuất indirubin, Tạp chí Hóa học, 2019, 57(4e3,4), 50-54 (Quốc gia uy tín) Nguyễn Trọng Dân, Nguyễn Trường Giang, Nguyễn Thị Kim Thoa, Tô Đạo Cường, Nguyễn Mạnh Cường, Lưu Văn Chính, Tổng hợp dẫn xuất indirubin với chalcone, Tạp chí Hóa học, 2019, 57(4e3,4), 55-60 (Quốc gia uy tín) Nguyễn Trọng Dân, Nguyễn Trường Giang, Lưu Văn Chính, Nguyễn Mạnh Cường, Tổng hợp số chalcone có chứa nhóm azide, Tạp chí Khoa học Công nghệ Nhiệt đới, 2018, 16, 95-101 (Quốc gia) ... Quân, Tô Đạo Cường, Nguyễn Mạnh Cường, Lưu Văn Chính, Tổng hợp đánh giá hoạt tính chống ung thư in vitro dẫn xuất indirubin, Tạp chí Hóa học, 2019, 57(4e3,4), 50-54 (Quốc gia uy tín) Nguyễn Trọng... Chính, Tổng hợp dẫn xuất indirubin với chalcone, Tạp chí Hóa học, 2019, 57(4e3,4), 55-60 (Quốc gia uy tín) Nguyễn Trọng Dân, Nguyễn Trường Giang, Lưu Văn Chính, Nguyễn Mạnh Cường, Tổng hợp số... the key intermediate indirubin- 3ʹ-[O-(prop-2-ynyl)oxime] (52) was prepared from indirubin in two steps First, indirubin- 3ʹoxime (25) is formed by condensation reaction of indirubin with hydrochloric