Research on the synthesis and determination of the structure of hybrid compounds of quinazoline derivatives and azides via triazole bridges... Synthesis of 23 new quinazoline derivatives
Trang 1VIETNAM ACADEMY OF SCIENCE AND
TECHNOLOGY INSTITUTE OF CHEMISTRY
Trang 2The 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 ofScience and Technology No 18 - Hoang Quoc Viet, Cau Giay,
Trang 3A INTRODUCTION
1 The urgency, scientific and practical significance of the thesis
Quinazoline is a potential class in the design of synthetic anti-cancerdrugs according to the kinase enzyme inhibition mechanism [1-4] Gefitinib(Iressa), erlotinib (Tarceva), lapatinib (Tykerb) and vandetanib (Caprelsa) aretypical quinazoline compounds that have been introduced into the production ofcancer drugs Among them Gefitinib and Erlotinib are the first epidermal growthfactor receptor(EGFR) chemotherapy drugs used to treat non-small cell lungcancer Erlotinb is a derivative of quinazoline with the trade name Tarceva,produced by Hoffmann pharmaceutical company - La Roche The drug is highlyeffective for the treatment of non-small cell lung cancer (NSCLC) with EGFRactivating mutation This is a breakthrough method in treating NSCLC thatcreates an opportunity to prolong life time with higher quality of life InVietnam, erlotinib hydrochloride Tarceva drug has not been widely used; first ofall because the cost of treatment with Tarceva is very high, 2,000 USD pertreatment 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 cytotoxicactivity of quinazoline compounds" is a scientifically and practicallysignificant 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
Trang 44 Research on cytotoxic activity of hybrid compounds synthesized on threehuman 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 positionC-6, C-7 following a completely new path These derivatives are used tohybridize with other active azides via triazole bridges by click reaction
* 15 hybrid compounds of quinazoline crown ether and azides via triazolebridges c The structure of new hybrid compounds has been confirmed from theresults of analysis of infrared spectral data (IR), nuclear magnetic resonancespectroscopy (1H-NMR and 13C-NMR, HMBC, HSQC) and mass
spectroscopy (HRMS)
d Evaluation of activity of 19 new quinazoline derivatives on three humancancer cell lines including KB (carcinoma, Hep-G2 (liver cancer) and Lu(non-small cell lung cancer) in which there are 13 substances that can causeinvestigated 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 Ellipticinestandard (IC50 is 1.95; 2.72; 1.38 µM, respectively)
Trang 54 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 thedissertation, updated to 2018
The appendix consists of 62 pages, including the spectroscopy ofsynthesized substances
5 Research methodology
The substances were synthesized according to known modern organicsynthesis methods, improved and applied appropriately in specific cases.Reaction products were cleaned by column chromatography andrecrystallization The structure of the product was determined by modernspectral methods such as IR, HRMS, ESI-MS, 1H-NMR, 13C-NMR,HMBC, HSQC, DEPT Biological activity was explored according to themethod of Mossman on three cancer cell lines, KB, Hep-G2 and Lu.Protein docking simulation was used to predict the target activity ofsynthesized 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
Trang 6CHAPTER 2 EXPERIMENT
The experiment section consists of 25 pages, detailing the research methods,synthesis process, refining process, physical properties of received productssuch 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) BrCH 2 CH 2 OCH 3 , K 2 CO 3 , Bu 4 NHSO 4 , DMF, 110°C; (b) H 2 O, CH 3 OH, KOH, 30 o C; (c) Urea, 210-220°C; (d) P 2 O 5 , xylene, Reflux; (e) HNO 3 , acid acetic ice, 0°C; (f) Na 2 S 2 O 4 , H 2 O,
HCl; (g) DMF-DMA, acid acetic, toluen, 105°C; (h) 3-ethynylaniline, acid acetic, toluen,
60-110 o C; (i) HCl gas, CH 3 OH, 15-20°C.
Trang 7Diagram 3.2: Synthesis of quinazoline derivatives containing crown ether group in position C-6, C-7
Reagents and conditions: (a) NH 2 OH.HCl, NaOH, MeOH, H 2 O, mix, 30-60 minutes, 95-98%; (b) Ac 2 O, reflux, 8-12 h, 90-95%; (c) Na 2 S 2 O 4 , H 2 O, 50-65 o C, 3-4 h, 80-85%; (d) 1,2- dicloethan, or 1,3-dibrompropan, K 2 CO 3 , Bu 4 NHSO 4 , acetone, reflux, 10 h; (e) H 2 O, MeOH, KOH, 30 o C, 4 h; (f) Urea, 150-160 °C, 5 h; (g) P 2 O 5 , xylene, reflux, 5 h; (h) HNO 3 , acid acetic ice, 0°C, 2 h (i) DMF-DMA, acid acetic, toluene, reflux, 4-6 h; (k) 3-etynylaniline, acid acetic, toluene, 60 o C-110 o 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: 1 equiv 4-anilinoquinazoline 119a-d, 1,1 azide equiv, 12 equiv
DIPEA, 0,2 equiv CuI, THF, rt, 1-2 days, 70-90%.
Trang 83.2 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 anddisadvantages The two biggest difficulties of the methods are the reduction
of the nitro group into the amino group and the 4-chloroquinazolineintermediate synthesis reaction In order to choose a path of synthesis of thisdrug in accordance with the conditions in Vietnam, we carefully studied theadvantages and disadvantages of each method combined with the initialresearch, 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
228-229oC 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); 7,33 (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) 13C-NMR (DMSO-d6) 170,2;
7,53-159,1; 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 morebioactive substances is also a very interesting and new issue, now attractingattention of many scientists Synthesis of a hybrid compound from twocompounds with antitumor activity, especially those that act according to
Trang 9different mechanisms of action, may increase activity or improve thedisadvantages of the original compounds On the other hand, the hybridstructured compounds when introduced into the body will be graduallyhydrolyzed 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 erlotinibderivatives, we studied and synthesized the hybrid compounds of erlotiniband azides via triazole bridges with click reaction Results were 4 new
derivatives which were 105a-d.
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
Trang 104.28 (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 isimportant for EGFR inhibitory activity, and substituents at the position C-6 andC-7 mainly contributing to their physical and chemical properties with goodcompatibility with bulky branches [15,16,104,105] With these advantages, inrecent years, many 4-anilinoquinazoline derivatives have been designed andsynthesized consecutively Among them, anilinoquinazoline
Trang 11analogues are combined with new tyfin EGFR inhibitors [17,106,107] TheSAR shows that oxygen-containing heterocyclic rings with 12 membershigher ring size are fused anilinoquinazoline, and the preferred substituents
on 4-anilino is a halogen such as chlorine, bromine, or phenyl group in themeta position [17,107] Some of them have been proved to be active inEGFR-mediated phosphorylated assays in human tumor cells A431 [17]while another showed strong activity against dissociation by inhibition ofboth tyrosine kinase receptors including EGFR, VEGFR, PDGFR, andnonreceptor TKs includes C-Src and Abl kinase with higher inhibitoryactivity against EGFR [107] According to the results mentioned above, andfrom erlotinib, we devised and synthesized the series of quinazolineincorporating dioxygenated rings containing the ethynyl group in the metaposition of aniline without rings, for the purpose of collecting the agentshowing 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 atposition C-6, C-7
Trang 12Figure 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 onspectral data analysis, including IR and 1H-NMR, HRMS
3.5 SYNTHESIS OF HIBRID COMPOUNDS OF
10
Trang 13specifc chemical and physical properties of nitrogen and fluorine, theintroduction of a NO2, CF3, and CN moieties in pharmacologically activecompounds is known to convey beneficial biological effects to the resultingmolecules 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 1H NMR, 13C NMR, and MS (ESI) spectroscopy.Notably, the 1H NMR spectroscopy showed a pic singlet at 9.17–9.65 ppmcorresponding to the triazolyl proton, while the 13C NMR spectroscopyshowed peaks at 120–123 ppm and 147–149 ppm corresponding tocharacteristics 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: 186-267oC.Yield 72%-90%
120a, 212oC, 87% 120b, 242-243oC, 87%
11
Trang 14149.7, 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
Trang 15121a 121b
Structure of hybrid compounds of substance 119b
The structure of hybrid compound 121d was proved by IR, NMR, HSQC,
9), 6.25 (2H, s, OCH2) 13C-NMR (DMSO-d6, 125 MHz) δ 156.9 (C-4),
Trang 1610), 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: 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 = 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) 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,