Research on the synthesis and evaluation of cytotoxic activity of quinazoline compounds

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 = 1 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.

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1 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 2 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 1 A. INTRODUCTION 1. 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 = 1 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. 2. Objectives of the dissertation 1. Research to improve the synthesis process of erlotinib hydrochloride drugs. 2. Research on the synthesis and determination of quinazoline derivative structure. 3. Research on the synthesis and determination of the structure of hybrid compounds of quinazoline derivatives and azides via triazole bridges. 2 4. 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). 3. 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: * 4 derivatives of erlotinib and different azides via triazole bridges * 4 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 ( 1 H-NMR and 13 C-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 8 substances exhibiting strong anti-cancer cell activity with the value of IC50 from 2 to 6 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 IC50 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). 3 4. Structure of the dissertation The dissertation consists of 129 pages including: Introduction: 2 pages. Chapter 1. Literature review: 31 pages Chapter 2. Experiment: 25 pages. Chapter 3. 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. 5. 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 1. 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 4 CHAPTER 2. 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) (a) BrCH2CH2OCH3, K2CO3, Bu4NHSO4, DMF, 110°C; (b) H2O, CH3OH, KOH, 30 oC; (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, 60- 110oC; (i) HCl gas, CH3OH, 15-20°C. 5 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%; (b) Ac2O, reflux, 8-12 h, 90-95%; (c) Na2S2O4, H2O, 50-65 oC, 3-4 h, 80-85%; (d) 1,2- dicloethan, or 1,3-dibrompropan, K2CO3, Bu4NHSO4, acetone, reflux, 10 h; (e) H2O, MeOH, KOH, 30oC, 4 h; (f) Urea, 150-160 °C, 5 h; (g) P2O5, xylene, reflux, 5 h; (h) HNO3, acid acetic ice, 0°C, 2 h (i) DMF-DMA, acid acetic, toluene, reflux, 4-6 h; (k) 3-etynylaniline, acid acetic, toluene, 60oC-110oC, 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: 1 equiv 4-anilinoquinazoline 119a-d, 1,1 azide equiv, 12 equiv DIPEA, 0,2 equiv CuI, THF, rt, 1-2 days, 70-90%. 6 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 1 H-NMR, 13 C- NMR. Erlotinib hydrocloride 93 is a yellow solid with the melting point 228- 229 o C. IR 3277, 3053, 3021, 2922, 2896, 2820, 2745, 2710, 1667, 1564, 1510, 1446, 1284, 1122, 8920 cm -1 . 1 H-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); 7,53- 7,33 (m, 3H, HAr); 4,45-4,25 (m, 4H, CH2O); 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 7 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 half- life . 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 4 new derivatives which were 105a-d. O O CH3 O O CH3 N N NH N N N CF 3 CN O O CH3 O O CH3 N N NH N N N O2N O O CH3 O O CH3 N N NH N N N NO 2 O O CH3 O O CH3 N N NH N N N NO 2 1240C, 75% 1210C, 83% 1020C, 86% 140 0C, 90% 105a 105b 105d105c 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, 1 H-NMR and 13 C-NMR spectral data. 8 6,7-Bis(2-methoxyethoxy)-N-(3-(1-(3-nitro-phenyl)-1H-1,2,3-triazol-4- yl)phenyl) quinazoline-4-amine 109b O O CH3 O O CH3 N N NH N N N NO 2 Colorless solid. MP 121 o C. Yield 83%. IR (KBr) cm -1 : 2930, 1623, 1583, 1535, 1507, 1442, 1350, 1238, 1034, 928. 1 H-NMR (DMSO-d6, 500 MHz) δ: 9.61 (1H, s, NH), 9.56 (1H, s), 8.81 (1H, t, J = 2 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 = 8 Hz), 7.23 (1H, s), 4.33- 4.28 (4H, m, CH3OCH2CH2O), 3.81-3.75 (4H, m, CH3OCH2CH2O), 3.38 (3H, s, OCH3), 3.36 (3H, s, OCH3). 13 C-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 9 analogues are combined with new tyfin EGFR inhibitors [17,106,107]. The SAR shows that oxygen-containing heterocyclic rings with 12 members higher ring size are fused anilinoquinazoline, and the preferred substituents on 4-anilino is a halogen such as chlorine, bromine, or phenyl group in the meta position [17,107]. Some of them have been proved to be active in EGFR-mediated phosphorylated assays in human tumor cells A431 [17] while another showed strong activity against dissociation by inhibition of both tyrosine kinase receptors including EGFR, VEGFR, PDGFR, and nonreceptor TKs includes C-Src and Abl kinase with higher inhibitory activity against EGFR [107]. According to the results mentioned above, and from erlotinib, we devised and synthesized the series of quinazoline incorporating dioxygenated rings containing the ethynyl group in the meta position of aniline without rings, for the purpose of collecting the agent showing stronger anti-cancer activities. In this study, the synthesis of quinazoline 119a-d crown ether through 5-6 steps with two different paths. One is from different benzaldehydes, two is from acid 106 3.4- dihidroxy benzoic as described in diagram 3.14. The fusion of quinazoline derivatives is showed in diagram 3.2 The results were 4 quinazoline derivatives containing crown ether group at position C-6, C-7 10 Figure 3.26: Structure of 4 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 1 H-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 3 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 11 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 4- anilinoquinazoline–substituted triazole hybrid compounds 120–123a-d in 70–90% yields (diagram 3.3). The structure of hybrid compounds 11–14 was determined by their 1 H NMR, 13 C NMR, and MS (ESI) spectroscopy. Notably, the 1 H NMR spectroscopy showed a pic singlet at 9.17–9.65 ppm corresponding to the triazolyl proton, while the 13 C 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 4 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 o C. Yield 72%-90%. 120a, 212 o C, 87% 120b, 242-243 o C, 87%. 12 120c, 267 o C. 72% 120d, 186 o C, 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: 212 o C. Yield 87%. IR (KBr) 3132, 2924, 2853, 1611, 1568, 1538, 1492, 1411, 1354, 1033, 924, 888, 773 cm -1 . 1 H-NMR (DMSO-d6, 500 MHz) δ 9.96 (1H, s, NH), 9.19 (1H, s), 8.65 (1H, s), 8.62 (1H, d, J = 8 Hz), 8.50 (1H, t, J = 1.5 Hz), 8.26 (1H, d, J = 7.5 Hz), 8.03- 7.95 (3H, m), 7.88 (2H, t, J = 7 Hz), 7.82 (1H, d, J = 7.5 Hz), 7.68-7.65 (2H, m), 7.54 (1H, t, J = 8 Hz). 13 C-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 13 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)-1H-1,2,3- triazol-1-yl)-2-(trifluoro-methyl)benzonitrile (121d) Light yellow solid. MP: 256-257 o C. 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 . 1 H-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 = 8 Hz, H-15), 7.63 (1H, d, J = 7.5 Hz, H-13), 7.52 (1H, t, J = 8 Hz, H-14), 7.19 (1H, s, H- 9), 6.25 (2H, s, OCH2). 13 C-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 (C- 14 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 : C25H15F3N7O2: 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 4 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 [1,4] dioxino [2,3-g] quinazolin-4-amine (122a) Light yellow solid. Yield: 80%. MP: 195 o C. IR (KBr) 3134, 1603, 1568, 1531, 1505, 1415, 1348, 1289, 1220, 1066, 901 cm -1 . 1 H-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 = 8 Hz), 8.14 (1H, s), 8.02-7.97 (2H, m), 7.95 (1H, d, J = 8 Hz), 7.88 (1H, t, J = 8 Hz), 7.63 (1H, d, J = 7.5 Hz), 7.51 (1H, t, J = 8 Hz), 7.19 (1H, s), 4.42 (4H, d, J = 3.5 Hz, OCH2). 13 C-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, 15 118.9, 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 hybri