VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY INSTITUTE OF CHEMISTRY RESEARCH ON THE SYNTHESIS AND EVALUATION OF CYTOTOXIC ACTIVITY OF QUINAZOLINE COMPOUNDS Speciality : Organic Chemistry Code : 9.44.27.01 Students : Đinh Thuy Van DISSERTATION SUMMARY Ha Noi – 2019 The work was completed at the Vietnam academy of Science and Technology  Supervisors: Supervisor 1: Pro.Doc Nguyen Van Tuyen Supervisor 2: Doc Dang Thi Tuyet Anh Reviewer 1: Reviewer 2: Reviewer 3: The thesis will be defended before the Doctoral Dissertation Council, at the Academy of Science and Technology - Vietnam Academy of Science and Technology No 18 - Hoang Quoc Viet, Cau Giay, Hanoi At time … 2019 A INTRODUCTION The urgency, scientific and practical significance of the thesis Quinazoline is a potential class in the design of synthetic anti-cancer drugs according to the kinase enzyme inhibition mechanism [1-4] Gefitinib (Iressa), erlotinib (Tarceva), lapatinib (Tykerb) and vandetanib (Caprelsa) are typical quinazoline compounds that have been introduced into the production of cancer drugs Among them Gefitinib and Erlotinib are the first epidermal growth factor receptor(EGFR) chemotherapy drugs used to treat non-small cell lung cancer Erlotinb is a derivative of quinazoline with the trade name Tarceva, produced by Hoffmann pharmaceutical company - La Roche The drug is highly effective for the treatment of non-small cell lung cancer (NSCLC) with EGFR activating mutation This is a breakthrough method in treating NSCLC that creates an opportunity to prolong life time with higher quality of life In Vietnam, erlotinib hydrochloride Tarceva drug has not been widely used; first of all because the cost of treatment with Tarceva is very high, 2,000 USD per treatment cycle (one cycle = month), price in Vietnam market is about 42 million VND per bottle of 30 150mg tablets Therefore, the thesis "Research on synthesis and evaluation of cytotoxic activity of quinazoline compounds" is a scientifically and practically significant research direction Objectives of the dissertation Research to improve the synthesis process of erlotinib hydrochloride drugs Research on the synthesis and determination of quinazoline derivative structure Research on the synthesis and determination of the structure of hybrid compounds of quinazoline derivatives and azides via triazole bridges Research on cytotoxic activity of hybrid compounds synthesized on three human cancer cell lines including KB (carcinoma, Hep-G2 (liver cancer) and Lu (non-small cell lung cancer) New points of the dissertation a Successfully synthesized erlotinib hidrocloride according to the new improvement process b Synthesis of 23 new quinazoline derivatives in which there were 19 derivatives containing triazole rings: * derivatives of erlotinib and different azides via triazole bridges * derivatives of quinazoline-4- amine containing crown ether group in position C-6, C-7 following a completely new path These derivatives are used to hybridize with other active azides via triazole bridges by click reaction * 15 hybrid compounds of quinazoline crown ether and azides via triazole bridges c The structure of new hybrid compounds has been confirmed from the results of analysis of infrared spectral data (IR), nuclear magnetic resonance spectroscopy (1H-NMR and 13C-NMR, HMBC, HSQC) and mass spectroscopy (HRMS) d Evaluation of activity of 19 new quinazoline derivatives on three human cancer cell lines including KB (carcinoma, Hep-G2 (liver cancer) and Lu (non-small cell lung cancer) in which there are 13 substances that can cause investigated cancer cell toxicity Among them there are substances exhibiting strong anti-cancer cell activity with the value of IC50 from to µM e Using protein docking simulation to predict the target activity of compounds 120d, 122a, 122b, 123c f Synthesized Compound 122a which has the strongest inhibitory activity for all three KB cell lines, Hep-G2 and Lu with IC 50 values of 0.04 µM, 0.14 µM and 1.03 µM, respectively, 100 times higher than erlotinib The 123c compound has IC50 value (1.49; 1.61; 1.81 µM) equivalent to the Ellipticine standard (IC50 is 1.95; 2.72; 1.38 µM, respectively) Structure of the dissertation The dissertation consists of 129 pages including: Introduction: pages Chapter Literature review: 31 pages Chapter Experiment: 25 pages Chapter Results and discussion: 56 pages The reference section has 122 documents on the relevant areas of the dissertation, updated to 2018 The appendix consists of 62 pages, including the spectroscopy of synthesized substances Research methodology The substances were synthesized according to known modern organic synthesis methods, improved and applied appropriately in specific cases Reaction products were cleaned by column chromatography and recrystallization The structure of the product was determined by modern spectral methods such as IR, HRMS, ESI-MS, 1H-NMR, 13 C-NMR, HMBC, HSQC, DEPT Biological activity was explored according to the method of Mossman on three cancer cell lines, KB, Hep-G2 and Lu Protein docking simulation was used to predict the target activity of synthesized compounds B CONTENTS OF THE DISSERTATION CHAPTER LITERATURE REVIEW This chapter presents the following contents: - The quinazoline synthesis methods - The erlotinib synthesis methods - Anti-cancer activity of quinazoline derivatives - Click reaction - Protein docking technique CHAPTER EXPERIMENT The experiment section consists of 25 pages, detailing the research methods, synthesis process, refining process, physical properties of received products such as melting point, shape, color, reaction performance and detailed data of IR, HRMS, 1H-NMR, 13C-NMR, HMBC, HSQC, DEPT CHAPTER 3: RESULTS AND DISCUSSION 3.1 OBJECTIVES OF THE DISSERTATION This dissertation focused on the development of an optimal procedure for erlotinib hydrochloride synthesis (diagram 3.1) to produce a synthesis process of erlotinib hydrochloride that can be applied into production in Vietnam, synthesizing new quinazoline derivatives (diagram 3.2) and hybrid compounds of quinazoline frame and triazole group (diagram 3.3) to search for new compounds with interesting biological activity Diagram 3.1: Synthesis process of erlotinib hydrochloride (93) o (a) BrCH2CH2OCH3, K2CO3, Bu4NHSO4, DMF, 110°C; (b) H2O, CH3OH, KOH, 30 C; (c) Urea, 210-220°C; (d) P2O5, xylene, Reflux; (e) HNO3, acid acetic ice, 0°C; (f) Na2S2O4, H2O, HCl; (g) DMF-DMA, acid acetic, toluen, 105°C; (h) 3-ethynylaniline, acid acetic, toluen, 60o 110 C; (i) HCl gas, CH3OH, 15-20°C Diagram 3.2: Synthesis of quinazoline derivatives containing crown ether group in position C-6, C-7 Reagents and conditions: (a) NH2OH.HCl, NaOH, MeOH, H2O, mix, 30-60 minutes, 95-98%; o (b) Ac2O, reflux, 8-12 h, 90-95%; (c) Na2S2O4, H2O, 50-65 C, 3-4 h, 80-85%; (d) 1,2dicloethan, or 1,3-dibrompropan, K2CO3, Bu4NHSO4, acetone, reflux, 10 h; (e) H2O, MeOH, o KOH, 30 C, h; (f) Urea, 150-160 °C, h; (g) P 2O5, xylene, reflux, h; (h) HNO 3, acid acetic ice, 0°C, h (i) DMF-DMA, acid acetic, toluene, reflux, 4-6 h; (k) 3-etynylaniline, acid o o acetic, toluene, 60 C-110 C, 4-6 h, 50-63% Diagram 3.3: Synthesis of hybrid compounds of quinazoline 119a-d derivatives and azides via triazole bridges Reagents and conditions: equiv 4-anilinoquinazoline 119a-d, 1,1 azide equiv, 12 equiv DIPEA, 0,2 equiv CuI, THF, rt, 1-2 days, 70-90% 3.2 SYNTHESIS OF ERLOTINIB HYDROCLORIDE From the synthesis methods of erlotinib hydrochloride mentioned in the reference as described in the diagrams 1.15-1.23 and the initial research results of the authors, it was found that each method has its advantages and disadvantages The two biggest difficulties of the methods are the reduction of the nitro group into the amino group and the 4-chloroquinazoline intermediate synthesis reaction In order to choose a path of synthesis of this drug in accordance with the conditions in Vietnam, we carefully studied the advantages and disadvantages of each method combined with the initial research, we chose an appropriate method to study and improve the synthesis of erlotinib hydrochloride as shown in diagram 3.1 Product 93 was structured by modern spectral methods 1H-NMR, 13 C- NMR Erlotinib hydrocloride 93 is a yellow solid with the melting point 228229oC IR 3277, 3053, 3021, 2922, 2896, 2820, 2745, 2710, 1667, 1564, 1510, 1446, 1284, 1122, 8920 cm-1 1H-NMR (DMSO-d6) 11,45 (s, 1H, NH); 8,81 (s, 1H, H-Ar); 8,30 (s, 1H, H-Ar); 7,90-7,72 (m, 2H, H-Ar); 7,537,33 (m, 3H, HAr); 4,45-4,25 (m, 4H, CH 2O); 3,79-3,70 (m, 4H, CH2O), 3,40 (s, 1H, C≡CH), 3,25 (s, 6H, OCH3) 13 C-NMR (DMSO-d6) 170,2; 159,1; 155,1; 151,2; 147,3; 142,3; 130,9; 125,8; 124,0; 122,3; 117,65; 114,2; 108,8; 100,9; 87,1; 80,6; 76,7; 73,5; 51,3 3.3 SYNTHESIS OF HIBRID COMPOUNDS OF ERLOTINB-TRIAZOLE The synthesis of hybrid structured compounds between two or more bioactive substances is also a very interesting and new issue, now attracting attention of many scientists Synthesis of a hybrid compound from two compounds with antitumor activity, especially those that act according to different mechanisms of action, may increase activity or improve the disadvantages of the original compounds On the other hand, the hybrid structured compounds when introduced into the body will be gradually hydrolyzed by the enzymes in the body to produce the original substance, thus reducing the side effects and increasing efficiency due to the long halflife In order to find and expand interesting new activities of erlotinib derivatives, we studied and synthesized the hybrid compounds of erlotinib and azides via triazole bridges with click reaction Results were new derivatives which were 105a-d HN HN N HC O O H3C O N O H3C O N N O2N N H3C O O O N N N N NO2 N 105b 1210C, 83% 105a 1240C, 75% CN CF3 HN HN H3C O O H 3C O O N N N NO2 HC N N H3 C O O N O O 105c 1020C, 86% N N N N 105d 1400C, 90% Figure 3.19: Chemical structure and some physical characteristics of compounds 105a-d The expected structure of hybrid compounds 105a-d is confirmed by their IR, MS, 1H-NMR and 13C-NMR spectral data 6,7-Bis(2-methoxyethoxy)-N-(3-(1-(3-nitro-phenyl)-1H-1,2,3-triazol-4yl)phenyl) quinazoline-4-amine 109b HN H 3C O O H3C O N N N N NO2 O N Colorless solid MP 121oC Yield 83% IR (KBr) cm-1: 2930, 1623, 1583, 1535, 1507, 1442, 1350, 1238, 1034, 928 H-NMR (DMSO-d6, 500 MHz) δ: 9.61 (1H, s, NH), 9.56 (1H, s), 8.81 (1H, t, J = Hz), 8.51-8.47 (2H, m), 8.40 (1H, s), 8.35-8.33 (1H, m), 7.95-7.92 (3H, m), 7.67 (1H, d, J = 5.5 Hz), 7.53 (1H, t, J = Hz), 7.23 (1H, s), 4.334.28 (4H, m, CH3OCH2CH2O), 3.81-3.75 (4H, m, CH3OCH2CH2O), 3.38 (3H, s, OCH3), 3.36 (3H, s, OCH3) 13C-NMR (DMSO-d6, 125 MHz) δ: 156.4, 153.6, 152.9, 148.6, 148.1, 147.7, 140.2, 137.2, 131.6, 103.2, 129.2, 125.9, 123.1, 122.4, 120.6, 120.1, 119.1, 114.6, 108.2, 103.3, 70.1, 70.0, 68.4, 68.1, 58.4, 58.3 LC-MS/MS (m/z) Calc for: C28H28N7O6: 558.2023 [M+H]+, found: 558.2061 3.4 SYNTHESIS OF HIBRID COMPOUDS OF QUINAZOLINE DERIVATIVES CONTAINING CROWN ETHER GROUP IN POSITION C-6, C-7 Studies on the relationship between structure and biological activity (SAR) of EGFR inhibitors showed that the 4-anilinoquinazoline frame is important for EGFR inhibitory activity, and substituents at the position C-6 and C-7 mainly contributing to their physical and chemical properties with good compatibility with bulky branches [15,16,104,105] With these advantages, in recent years, many 4-anilinoquinazoline derivatives have been designed and synthesized consecutively Among them, anilinoquinazoline Figure 3.26: Structure of 4-aminoquinazoline compounds containing crown ether group at position C-6, C-7 119a-d The structure of compounds 119a-d was determined simply based on spectral data analysis, including IR and 1H-NMR, HRMS 3.5 SYNTHESIS OF HIBRID COMPOUNDS OF QUINAZOLINE-TRIAZOLE The synthesis of hybrid compounds of 4-anilinoquinazoline and azides via triazole bridge, the results were a number of new hibrid compounds with components 4-anilinoquinazoline was the skeleton, triazole cycle and aryl were chained with variable substituents Most EGFR-tyrosine kinase inhibitors have the same set of 4-anilinoquinazoline, only substituents and side chains changed Therefore, the replacement of the acetylene moiety at the C3 position of the phenyl ring by a triazole nucleus could rigid the structure of the nucleus Thus hydrogen bond between triazole ring and peptide backbone of EGFR receptors could afford specifc conformations, thereby improving inhibitory activities of hybrid compounds Besides, with respect to the triazolyl substituent, we consider their influence on bioactive function including nitrophenyl and cyanotrifluoromethylphenyl Due to the 10 specifc chemical and physical properties of nitrogen and fluorine, the introduction of a NO2, CF3, and CN moieties in pharmacologically active compounds is known to convey beneficial biological effects to the resulting molecules Hence organic and medicinal chemists are increasingly interested in polyfunctional NO2–, CF3-, and CNsubstituted scaffolds In that respect, copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) 119a-d with nitrophenyl- and cyanotrifluoromethylphenylazides generating the target 4anilinoquinazoline–substituted triazole hybrid compounds 120–123a-d in 70–90% yields (diagram 3.3) The13structure of hybrid compounds 11–14 was determined by their H NMR, C NMR, and MS (ESI) spectroscopy Notably, the 1H NMR spectroscopy showed a pic singlet at 9.17–9.65 ppm corresponding to the triazolyl proton, while the 13C NMR spectroscopy showed peaks at 120–123 ppm and 147–149 ppm corresponding to characteristics CH and Cq of the triazole core unit Synthesis reaction of hybrid compounds according to diagram 3.3 The obtained results were ranges of hybrid compounds of compounds 119a-d 3.5.1 Synthesis of hybrid compounds of derivatives 119a Hybrid compounds of 120a-d are all colorless crystals MP: 186-267 oC Yield 72%-90% 120a, 212oC, 87% 120b, 242-243oC, 87% 11 120c, 267 o C 72% 120d, 186oC, 90% Figure 3.32: Chemical structure and physical characteristics of hybrid compounds 120a-d The structure of compound 120a was proved by IR, NMR spectroscopy N-(3-(1-(2-nitrophenyl)-1H-1,2,3-triazol-4-yl)phenyl)quinazolin-4-amine (120a) Colorless solid MP: 212oC Yield 87% IR (KBr) 3132, 2924, 2853, 1611, 1568, 1538, 1492, 1411, 1354, 1033, 924, 888, 773 cm -1 1H-NMR (DMSO-d6, 500 MHz) δ 9.96 (1H, s, NH), 9.19 (1H, s), 8.65 (1H, s), 8.62 (1H, d, J = Hz), 8.50 (1H, t, J = 1.5 Hz), 8.26 (1H, d, J = 7.5 Hz), 8.037.95 (3H, m), 7.88 (2H, t, J = Hz), 7.82 (1H, d, J = 7.5 Hz), 7.68-7.65 (2H, m), 7.54 (1H, t, J = Hz) 13C-NMR (DMSO-d6, 125 MHz) δ 157.9, 154.5, 149.7, 147.1, 144.1, 139.9, 134.5, 133.1, 131.3, 130.2, 129.3, 129.1, 127.8, 127.5, 126.4, 125.6, 123.0, 122.9, 122.5, 121.0, 119.3, 115.2 ESI-MS (m/z) Calc for: C22H16N7O2: 410.1287 [M+H]+; Found: 410.3196 The structure of substances 120b-d was similarly demonstrated to compound 120a by IR, NMR, and MS spectroscopy 3.5.2 Synthesis of hybrid compounds of derivative 119b 12 121a 121b 121c 121d Structure of hybrid compounds of substance 119b The structure of hybrid compound 121d was proved by IR, NMR, HSQC, HMBC, DEPT, MS spectra 5-(4-(3-([1,3]Dioxolo[4,5-g]quinazolin-8-ylamino)phenyl)-1H1,2,3-triazol-1-yl)-2-(trifluoro-methyl)benzonitrile (121d) Light yellow solid MP: 256-257oC Yield 90% IR (KBr) 3282, 3132, 2238 (CN), 1615, 1580, 1529, 1493, 1470, 1439, 1386, 1315, 1271, 1242, 1217, 1183, 1135, 1030, 911, 845, 790, 688 cm-1 1H-NMR (DMSO-d6, 500 MHz) δ 9.60 (1H, s, H-triazole), 9.53 (1H, s, NH), 8.59 (1H, s, H-19), 8.54 (1H, d, J = 8.5 Hz, H-23), 8.49 (1H, s, H-2), 8.47 (2H, m, H-11, H-22), 8.14 (1H, s, H-5), 7.93 (1H, d, J = Hz, H-15), 7.63 (1H, d, J = 7.5 Hz, H-13), 7.52 (1H, t, J = Hz, H-14), 7.19 (1H, s, H9), 6.25 (2H, s, OCH2) 13C-NMR (DMSO-d6, 125 MHz) δ 156.9 (C-4), 153.0 (C-2), 152.4 (C-6), 148.6 (C-9a), 148.0 (C-16), 147.3 (C-8), 140.3 (C13 10), 139.7 (C-18), 137.5 (C-22), 132.7 (q, J = 32.5 Hz, C-21), 129.8 (C-12), 129.2 (C-14), 123.7 (C-23), 122.2 (C-15), 120.4 (C-13), 120.2 (C-17), 118.9 (C-11), 1181 (C-19), 115.0 (C≡N), 110.2 (C-4a), 107.7 (C-20), 104.6 (C-9), 102.3 (C-7), 98.9 (C-5) HRMS calc for : C 25H15F3N7O2: 502.1161 [M+H]+; Found: 502.1233 The structure of substances 121a-c was proved similarly to 121d by IR, NMR, MS spectra 3.5.3 Synthesis of hybrid compounds of derivative 119c The synthesized results of hybrid compounds of compound 119c were hybrid compounds 122a-d The structure of compound 122a was proved by IR, NMR, HSQC, HMBC, DEPT, MS spectra N-(3-(1-(2-nitrophenyl)-1H-1,2,3-triazol-4-yl)phenyl)-7,8-dihydro dioxino [2,3-g] quinazolin-4-amine (122a) [1,4] Light yellow solid Yield: 80% MP: 195oC IR (KBr) 3134, 1603, 1568, 1531, 1505, 1415, 1348, 1289, 1220, 1066, 901 cm-1 1H-NMR (DMSO-d6, 500 MHz) δ 9.67 (1H, br.s, NH), 9.17 (1H, s), 8.49 (2H, s), 8.26 (1H, d, J = Hz), 8.14 (1H, s), 8.02-7.97 (2H, m), 7.95 (1H, d, J = Hz), 7.88 (1H, t, J = Hz), 7.63 (1H, d, J = 7.5 Hz), 7.51 (1H, t, J = Hz), 7.19 (1H, s), 4.42 (4H, d, J = 3.5 Hz, OCH2) 13C-NMR (DMSO-d6, 125 MHz) δ 156.7, 152.9, 149.2, 147.1, 145.7, 144.1, 143.7, 140.2, 134.5, 131.3, 130.1, 129.2, 129.1, 127.5, 125.6, 122.8, 122.1, 120.6, 14 118.9, 112.3, 110.0, 108.5, 64.5, 64.2 HRMS (ESI+) m/z calc for: C24H18N7O4 [M+H]+ 468.1342, Found: 468.1416 The structure of the remaining compounds is proved by IR, NMR, MS spectra 3.5.4 Synthesis of hybrid compounds of derivative 119d The structure of the compounds 123a-c was proved by IR, NMR, MS spectra N-(3-(1-(2-nitrophenyl)-1H-1,2,3-triazol-4-yl)phenyl)-8,9-dihydro7H-[1,4] dioxepino[2,3-g]quinazolin-4-amine (123a) Colorless solid MP: 278oC Yield 79% IR (KBr) 2926, 1609, 1574, 1537, 1479, 1415, 1350, 1330, 1064, 991 cm-1 1H-NMR (DMSO-d6, 500 MHz) δ 9.81 (1H, s, NH), 9.17 (1H, s), 8.58 (1H, s), 8.48 (1H, s), 8.26 (1H, dd, J = Hz, J = Hz), 8.18 (1H, d, J = Hz), 8.02-7.97 (2H, m), 7.91 (1H, d, J = Hz), 7.89 (1H, td, J = 8.5 Hz, J = Hz), 7.65 (1H, d, J = Hz), 7.51 (1H, t, J = Hz), 7.26 (1H, d, J = Hz), 4.36-4.31 (4H, m, OCH2), 2.24 (2H, t, J = 5.5 Hz, OCH2CH2CH2O) 13 C-NMR (DMSO-d6, 125 MHz) δ 157.7, 154.3, 153.0, 147.2, 144.2, 140.1, 134.7, 131.5, 130.2, 129.4, 129.2, 127.6, 125.7, 122.9, 122.6, 121.1, 121.0, 119.4, 117.5, 70.8, 70.7, 30.9 HRMS calc for: C25H20N7O4: 482.1499 [M+H]+; Found: 482.1573 3.6 INVESTIGATION OF CYTOTOXIC ACTIVITY OF QUINAZOLINE, QUINAZOLINE-TRIAZOLE COMPOUNDS 3.6.1 Results of cytotoxic activity test The process of investigating cytotoxic activity was performed at the Applied Biochemistry Department of the Institute of Chemistry The compounds after synthesis were evaluated cytotoxic activity with human 15 cancer cell lines, including KB (carcinoma, Hep-G2 (liver cancer) and Lu (non-small cell lung cancer) We used Erlotinib, erlotinib hydrochloride (non-small cell lung cancer preparation material) and ellipticine as a positive control As shown in Table 3.5, in general, synthesized compounds showed good cytotoxic inhibitory effects in most cases and had higher inhibitory activity than erlotinib referred and erlotinib hydrochloride drugs No 10 11 12 13 14 15 16 17 18 19 20 21 22 Table 3.5: Cytotoxic activity of synthesized compounds comp IC50 IC50 IC50 R1,R2 R ound (KB), (HepG2) (Lu), s µM , µM µM 5.46 4.16 4.16 119a 120a 5.50 4.64 4.76 2-NO2 H 104.20 286.75 259.74 120b 3-NO2 5.47 8.11 9.16 120c 4-NO2 1.46 1.86 4.50 120d 3-CN-4-CF3 119b 75.60 26.17 64.88 121a 193.33 207.01 216.84 2-NO2 6.35 6.88 6.66 121b -OCH2O3-NO2 30.10 11.29 44.48 121c 4-NO2 4.59 6.10 27.46 121d 3-CN-4-CF3 119c 2.60 2.84 3.36 122a 0.04 0.14 1.03 2-NO2 3.51 0.88 5.67 122b -O(CH2)2O3-NO2 79.09 230.02 54.77 122c 4-NO2 0.27 6.09 4.44 122d 3-CN-4-CF3 119d 54.83 69.89 80.67 123a 20.44 43.35 14.75 2-NO2 -O(CH2)3O53.17 76.81 230.40 123b 3-NO2 1.49 1.61 1.81 123c 3-CN-4-CF3 Erlotinib 13.01 25.01 99.76 Erlotinib.HCl 49.62 14.17 31.15 Ellipticine 1.95 2.72 1.38 16 Substitution by different oxygen substituent heterocycles on the positions and of quinazolines skeleton affected the cytotoxic inhibition differently Compound 119a and the dioxane derivative 119c are clearly more preferred than dioxolane and dioxepine derivatives 119b, 119d The effect of these unfavorable substitutions may result from steric hindrance Moreover, compounds 119a and 119c were found to be more potent cytotoxic inhibitors against all three cancer cell lines than erlotinib and erlotinib hydrochloride with IC 50-values ranging from to µM The coupling of substituted 1,2,3-triazolyl groups with targeted dioxygenated ring fused quinazolines 119a–d at the anilino side chain was found to greatly enhance the cytotoxicity of the resulting hybrid compounds 120-123 It is important to note that these separate pharmacophores and the reference drugs display considerably less potent cytotoxic activities (IC 50values ranging from to 100 µM) as compared to the most promising conjugates 120a, c, d, 121b, d, 122a, b , d and 123c (IC50-values ranging from 0.04 µM to 25 µM) showing a reasonable activity against these cancer cell lines Preliminary investigation of the structure–activity relationships (SAR) of these synthesized hybrid compounds 120-123 revealed that the nature of the oxygen substituent heterocycles and the aryl group which connected to the triazole influenced the cytotoxicity activity remarkably For instance, the result revealed that the cytotoxic activity of dioxane substituted analogues 122a-d are more potent than those of the corresponding dioxolane, dioxepine counterparts, and the analogues without oxygen substituent heterocycles (IC50: 122a> 120a> 123a> 121a, 122b> 121b> 123b> 120b, 122d> 123c ≈ 120d> 121d) Especially, compound 122a displayed the most potent inhibitory activity 17 against KB, HepG2, and Lu with IC 50-values of 0.04 µM, 0.14 µM, and 1.03 µM, respectively, which was up to 100 fold higher than those of erlotinib The introduction of an electron-withdrawing groups such as NO 2, CF3, and CN of the aryl which connected to the triazole can remarkable improve the cytotixic activities Substitution by a trifluoromethyl group of the aryl seemed to be better than a nitro (IC 50: 120d> 120a> 120c> 120d, 121d> 121b> 121c> 121b, 123c> 123a> 123b) In addition, the effects of the substituent position seemed to be also dependent on the substituent nature In fact, with a nitro substituent, substitution at ortoposition was better than at meta- or para-positions in most cases 3.6.2 Docking method on synthesized compounds on EGFR receptors New compounds were designed and synthesized based on the structural framework of typical quinazoline compounds, especially erlotinib Among those compounds there were compounds 120d, 122a, 122b, 123c with very good cytotoxic activity, especially compound 122a showed the strongest inhibitory activity for all three KB cell lines, Hep-G2 and Lu with IC50 values are 0.04 µM, 0.14 µM and 1.03 µM respectively, 100 times higher than erlotinib Protein docking simulation method was used Protein docking is a modeling technique that predicts a favorable position and structure that the molecule can bind to its target targets, usually protein molecules Due to erlotinib's ability to inhibit EGFR non-activation and EGFR-TKD mutations in non-small cell lung cancer cells (non-small-cell lung cancer, NSCLC) we aimed at studying interaction between 120d, 122a, 122b, 123c with different phenotypes of EGFR To prepare protein models, we collected and processed X-ray crystalline structures of EGFR from the PDB Protein Bank (https://www.rcsb.org/): 1) 18 EGFR activated without mutation under code 1M17 and 2ITX, 2) EGFR activated with mutation L834R under code 2ITV, and 3) EGFR inactive under code 4HJO and 1XKK Compounds 120d, 122a, 122b, 123c were built using 3D structure LigPrep-Schrodinger software was used (https://www.schrodinger.com/ligprep) Docking simulation was done on ICM-pro version 3.8 (www.molsoft.com/icmpro) The docking results showed that all compounds 120d, 122a, 122b, 123c had good interaction with EGFR both in the active (mutation or not mutation) and inactive form Figure 3.47 Due to the oil body characteristics of the center activity of all three targets, the aminoquinazoline framework created a dense network of van der Waals interactions with important amino acids such as Leu844, Val726, Ala743, Lys745 of the L858R mutation In addition, the triazole group also strongly interacted with oil body amino acids at the top of the bag like Lys745 In general, the first compounds showed the ability to inhibit EGFR both in active and inactive forms However, unlike erlotinib, all four compounds exhibited weaker activity on activated EGFR targets (with or without mutations), while activity on inactive EGFR targets was stronger The binding energy calculated for Erlotinib on all three systems ranged from -7.1 to 9.9 kcal / mol Meanwhile, compounds 120d, 122a, 122b, 123c showed higher levels of interaction energy with active EGFR than erlotinib (from -10.0 to -10.6 kcal / mol) The energy calculated for inactive EGFR system is higher (from -10.6 to -12.3 kcal / mol) In summary, the molecular docking simulation results helped predict the target activity of compounds 120d, 122a, 122b, 123c, contributing to explaining the cytotoxic mechanism of these compounds Combining theoretical and experimental research on compound 122a can be considered 19 a new hit, guiding the synthesis of new navigation compounds that inhibit EGFR, the future treatment of lung cancer Compound 120d Compound 122a Compound 122b Compound 123c Figure 3.47: 2D diagram showing docking interaction of compounds 120d, 122a, 122b, 123c with active sites of target EGFR proteins (In which atoms were classified by color, the number in the cells is the binding energy (kcal / mol) of the compounds) 120d 122a IC50 (µM): 1.46 (KB); 1.86 IC50 (µM): 0.04 (KB); 0.14 (HepG2); (HepG2); 1.03 Lu 20 119c 122b IC50 (µM): 2.6 (KB); 2.84 (HepG2); IC50 (µM): 0.88 (HepG2); 122d 123c IC50 (µM): 1.49 (KB); 1.61 (HepG2); 1.81( Lu) IC50 (µM): 0.27 (KB); Figure 3.48: Structure of some hybrid compounds with good activity 21 CONCLUSION Successfully synthesized erlotinib hydrochloride according to the improved process Synthesized 39 substances including 23 quinazoline derivatives; 19 new hybrid compounds not yet seen in previous publications including: * hybrid compounds of erlotinib –triazole * hybrid compounds of N- (3-ethynylphenyl) quinazoline-4-amine (119a) and azides via triazole bridges * hybrid compounds of N- (3-ethynylphenyl) - [1,3] dioxolo [4,5-g] quinazoline-8-amine (119b) and azides via triazole bridges * hybrid compounds of N- (3-ethynylphenyl) -7,8-dihydro- [1,4] dioxino [2,3-g] quinazoline-4-amine (119c) and azides via triazole bridges * hybrid compounds of N- (3-ethynylphenyl) -8,9-dihydro-7H- [1,4] dioxepino [2,3-g] quinazoline-4-amine (119d) and azides via triazole bridges Demonstrated the structure of 19 new hybrid compounds with modern spectral methods such as IR, 1H-NMR, 13C-NMR, DEPT, HSQC, HMBC, high-resolution mass spectra (HRMS) Tested cytotoxic activity on 19 synthesized compounds on three cancer cell lines in humans KB, Hep-G2 and Lu The results showed that many hybrid compounds have very good activity, much better than erlotinib such as compounds 120a, 120d; 121b, 121d; 122a, 122b, 122d and 123c In particular, compound 122a showed the strongest inhibitory activity for all three cell lines KB, Hep-G2 and Lu with an IC50 value of 0.04 µM, 0.14 µM and 1.03 µM, respectively, 100 times higher than erlotinib 22 Used protein docking simulation to predict the target activity of compounds 120d, 122a, 122b, 123c, contributing to explaining the cytotoxic mechanism of these compounds 23 RELATED PUBLICATIONS Research on the procedure of improving and synthesizing erlotinib Journal of Chemistry No 6e2, Volume 54, page (2016) Research on the synthesis of erlotinib derivatives N-(3-ethynylphenyl)7,8-dihydro-[1,4]dioxino[2,3-g]quinazolin-4-amin Journal of Chemistry, No 5E34, Volume 55, page 13-16 (2017) Synthesis and evaluation of cytotoxicity of novel substituted triazole– erlotinib hybrid compounds Vietnam Journnal of chemistry, volume 56, Number 4e1, (2018) Design, synthesis and evaluation of novel hybrids between 4anilinoquinazolines and substituted triazoles as potent cytotoxic agents Bioorganic & Meedicinal Chemistry Letters 28, 3741-3747.(2018) 24 ... fusion of quinazoline derivatives is showed in diagram 3.2 The results were quinazoline derivatives containing crown ether group at position C-6, C-7 Figure 3.26: Structure of 4-aminoquinazoline... OF QUINAZOLINE-TRIAZOLE The synthesis of hybrid compounds of 4-anilinoquinazoline and azides via triazole bridge, the results were a number of new hibrid compounds with components 4-anilinoquinazoline... of 23 new quinazoline derivatives in which there were 19 derivatives containing triazole rings: * derivatives of erlotinib and different azides via triazole bridges * derivatives of quinazoline-4-