Synthesis and identification biological activity of some indenoisoquinoline derivatives

MINISTRY OF EDUCATION & TRAINING VIET NAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY ------------------ NGO HANH THUONG SYNTHESIS AND IDENTIFICATION BIOLOGICAL ACTIVITY OF SOME INDENOISOQUINOLINE DERIVATIVES Major: Organic chemistry Code: 9.44.01.14 SUMMARY OF CHEMISTRY DOCTORAL THESIS Ha Noi - 2018 This thesis was completed at: Laboratory of Medicinal Chemistry - Institute of Chemistry - Vietnam Academy of Science and Technology Scientific Supervisors: Prof. Dr. Nguyen Van Tuyen 1 st Reviewer: 2 nd Reviewer: 3 rd Reviewer: The dissertation were defended at Graduate University of Science and Technology, No.18 Hoang Quoc Viet, Cau Giay District, Ha Noi City. At .. The dissertation can be found in National Library of Vietnam and the library of Graduate University of Science and Technology. 1 A. INTRODUCTION TO THESIS 1. Graduation, scientific and practical significance of thesis topic Indenoisoquinoline has some advantages over camptothecin. First, the DNA cleavage site of NSC314622 (4) is different from that of camptothecin (1), so it is possible for different cancer resistance. Secondly, the indoeritol-soluble Top1-DNA complexes of indenoisoquinoline are more stable. Third, the indenoisoquinoline is chemically stable. Fourthly, the indenoisoquinoline was less affected by the top1-resistant mutants of R364H and N722S. Two indenoisoquinolines toxic Top1: indotecan (5), indimitecan (6), were phase I clinical trials for cancer treatment and are undergoing Phase II clinical trials. Figure 1 In addition, research into hybrid compounds is gaining much interest among researchers, recent studies have shown that hybridizations can increase activity and some cases avoidance Drug resistance versus one component. Thus, the synthesis of new derivatives and cytotoxic activity assays for the detection of antitoxin-active agents of Topoisomerase 1 is of great scientific and practical significance. Therefore, we chose to study "Synthesis and identification biological activity of some indenoisoquinoline derivatives". 2. The task of the thesis 1. Synthesis of new derivatives of indenoisoquinoline is based on changes in B ring groups by modifying branching atoms of nitrogen atom and binding of triazole-containing hybrid compounds. 2. Synthesis of new derivatives of indenoisoquinoline based on the change of substituents in D ring with metoxyl groups or methylendioxy. Simultaneously change the substituents on B ring through the triazole bridge by click reaction. 3. Tested for anti-cancer activity of synthetic compounds 2 3. The contribution of the thesis 1. Successfully synthesized 41 new indenoisoquinoline compounds. 2. The estimated tumorigenic activity of 45 indenoisoquinoline compounds on two epithelial (KB) and liver (Hep-G2) cell lines, 26 of which are highly pathogenic There are 12 compounds exhibiting strong cytotoxic cytotoxic activity with IC50 <10 μM. 3. In 45 compounds, there are four indenoisoquinoline compounds with IC50 value equivalent to the Ellipticine standard: 142a, 142m, 142n, 149. 4. In 45 compounds, there are four indenoisoquinoline compounds had IC50 value higher than the Ellipticine standard: 142l, 145, 148b, 148d. 5. In 45 compounds, there are 6 compounds contained substitution of methylendioxy or metoxy on the highly active D ring: 162d, 162e, 163b, 163d, 164c, 164d. 4. The composition of the thesis The thesis has 111 pages including: Introduction: 2 pages Target: 1 page Chapter 1: Overview of 25 pages Chapter 2: Experiment 34 pages Chapter 3: Results and discussion 48 pages Conclusion: 1 page There are 126 references to the subject area of the dissertation, updated to 2017 The annex includes 40 pages of spectral types of synthetic substances 5. Research Methods Substances synthesized by modern organic synthesis methods are known, modified and adapted for specific cases. The reaction product is purified by column chromatography and recrystallized. The chemical structures of the products were confirmed using 1 H NMR, 13 C NMR, MS and IR techniques. Biological activity was investigated by Mossman's method on two cell lines: KB, Hep-G2. 3 B. CONTENTS OF THE THESIS Chapter 1. OVERVIEW The overview of the thesis presents the following contents: - Synthesis methods of indenoisoquinoline - Biological activity of indenoisoquinoline compounds Chapter 2. EXPERIMENT The experiment consists of 32 pages, detailing research methods, synthetic processes, refining, physical properties of the products received such as flow point, morphology, color, yield and detailed data of spectra: IR, HRMS, 1 H-NMR, 13 C-NMR. From compound 142 we proceeded to synthesize 14 new derivatives of indenoisoquinoline 142a-n, then from compound 142a,e we synthesized 2 derivatives 143a, b From lactone 50, we synthesized 6 indenoisoquinoline-AZT derivatives and 1 hybrid contained IM5 We have synthesized 3 new indenoisoquinoline frames 153, 154, 155. From these three compounds, 15 indenoisoquinoline-triazole derivatives were synthesized. 4 Chapter 3. RESULTS AND DISCUSSION 3.1. Synthesize indenoisoquinoline with different substituents in B ring Indenoisoquinoline is known to be a class of cytotoxic agents, topoisomerase I (top 1). Compared to camptothecin, indenoisoquinoline has several advantages, such as: the cleavage site of indenoisoquinoline 4 differs from that of camptothecin (1) so that it may give a different anti- cancer spectrum, the top1-DNA cleavage complex Indenoisoquinoline is more stable, the indenoisoquinoline is chemically stable and less affected by resistance mutations Top1. These advantages have promoted the synthesis of new derivatives of indenoisoquinoline. In addition, in previous research by GS. Nguyen Van Tuyen and colleagues point out that indenoisoquinoline derivatives contain propan-2- ol branched vessels that bind to the N-atom of the B ring in combination with pyrolidinyl, piperazine and piperidine units with high cytotoxic activity for two KB and HepG2 cell lines. In addition, the 1,2,3-triazole ring is known to be part of the biological active agent. In addition, triazole- derived derivatives are easily synthesized by the click reaction. Thus, the hybridization of 1,2,3-triazole with other drugs has become one of the exciting studies for the development of new drugs. For these reasons, it has led to the hypothesis that the introduction of triazole loops into the tributary veb contains three carbon atoms on the B ring of indenoisoquinoline, in particular indenoisoquinolin-propan-2-ol, which can produce compounds have promising biological activity. Figure 3. 1 5 The reaction between the azide and the ankin was discovered a century ago by Artu Michael, then Huisgen was systematically studying this reaction for the synthesis of imiazolic compounds. Figure 3. 2 Based on the idea of the click reaction, we proceed to synthesize new indenoisoquinoline derivatives by attaching substituents containing ankin to join the click reaction with the azide. At the same time, we also conducted a total of new indenoisoquinoline derivatives through a triazole bridge with imatinib, a tyrosine kinase inhibitor used in the treatment of multiple cancer, most notably chronic myelogenous leukemia. Figure 3. 3 3.2. Synthesis results for triazol-indenoisoquinoline hybrid 142a-n Imidazoles are extremely interesting structurally heterologous heterocyclic compounds, some imidazole compounds have been used in pharmaceuticals such as 5-nitroimidazole, fungal anti-fungal muconazole, midazolam. 6 Figure 3. 4 To follow up previous studies by Professor Nguyen Van Tuyen and his colleagues, in search of new derivatives of indenoisoquinoline has exciting anti-cancer activity, we used the raw material as compound 142 (synthesized as previously published), for the synthesis of derivatives 142a-n by the Click reaction obtained product 142a-n, yield 60-80%. Compound 142a is orange crystal, melting point is 240 °C. IR (KBr) cm -1 : 3420, 3226, 2872, 1660, 1601, 1580, 1502, 1340, 1415, 1306, 1264, 1056, 971, 838, 756. 1 H-NMR (DMSO-d6, 500 MHz): 8.56 (1H, d, J = 8.5 Hz); 8.2 (1H, d, J = 8.0 Hz); 7.99 (1H, s); 7.79-7.82 (1H, m); 7.50-7.73 (2H, m); 7.41-7.44 (3H, m); 5.75 (1H, bs, OH); 5.25 (1H, bs, OH); 4.59-4,69 (3H, m); 4.55 (2H, s), 4.40-4.44 (2H, m). 13 C-NMR (DMSO-d6, 125 MHz): 190.1; 162.8; 157.4; 147.7; 137.1; 134.3; 134.0; 133.4; 131.9; 130.9; 128.0; 127.0; 124.7; 124.1; 122.8; 122.5; 122.2; 107.1; 67.0; 55.0; 53.0; 47.9. HRMS Calc. For: C22H19N4O4: 403.1401 [M+H] + ; found: 403.1402. 7 The chemical structures of indenoisoquinolines 142b-n were confirmed using 1 H NMR, 13 C NMR, MS and IR techniques. Thus, we have synthesized 14 new indenoisoquinoline derivatives with substituents in B ring via triazole bridge. These substances will continue to be tested for anti-cancer activity 3.3. Synthesized results of hybrid compounds 143a, b To extend the study, we proceeded to esterify the hydroxyl group of compound 142a, 142e by treating with acetic anhydride to obtain the corresponding esters 143a, 143b. Reactive yields were 75% and 77%. The chemical structures of the product were confirmed using 1 H NMR, 13 C NMR, MS and IR techniques. Figure 3. 5. Synthesis of ester-indenoisoquinoline Compound 143a is orange crystal, melting point is 264-265 °C. IR (KBr) cm -1 : 3056, 2924, 2853, 1735, 1663, 1502, 1425, 1375, 1223, 1032, 799, 756, 700. 1 H-NMR (DMSO-d6, 500 MHz): 8.55 (1H, d, J = 8.0 Hz); 8.24 (1H, s); 8.20 (1H, d, J = 4.0 Hz); 7.80-7.83 (1H, td, J = 8.0, 1.0 Hz); 7.51-7.56 (2H, m); 7.43-7.50 (3H, m); 5.59-5.64 (1H, m); 5.15 (2H, s); 4.92-5.00 (2H, m); 4.82-4.86 (1H, dd, J =15.0, 3.0 Hz); 4.66- 4.71 (1H, dd, J = 15.0, 4.0 Hz); 2.02 (3H, s); 1.53 (3H, s). 13 C-NMR (DMSO-d6; 125 MHz):190.0; 170.1; 169.1; 162.6; 156.4; 142.1; 136.6; 134.2; 134.0; 133.3; 131.7; 131.2; 128.0; 127.3; 126.1; 123.9; 122.6; 122.5; 122.5; 107.3; 69.1; 57.0; 49.9; 45.1; 20.5; 19.9. HRMS Calc. For: C26H23N4O6: 487.1612 [M+H] + ; found: 487.1615. The chemical structures of the product 143b were confirmed using 1 H NMR, 13 C NMR, MS and IR techniques. Thus, we have synthesized two new indenoisoquinoline derivatives with esterified hidroxy groups. These substances will continue to be tested for anti-cancer activity. 3.4. Synthesis of hybrid compounds via triazole bridges 8 As we know, 3'-Azid-3'-deoxytymidin (AZT, zidovudin) has been exhibited pronounced anticancer activity, especially in combination with other antitumor agents, for example, such as 5-fluorouracil, cisplatin, paclitaxel and triterpenoids. Thus, considering the documented anticancer activity of indenoisoquinolines, AZT and functionalized triazole, it is reasonable to suggest that the construction of triazole-indenoisoquinoline– AZT hybrids might show good cytotoxicity activities. 3.4.1. Synthesis result of compound 145 From the idea, we have synthesized the indenoisoquinoline-triazol- AZT hybrid compound from compound 144 to produce a 1,2,3-triazol- indenoisoquinolin-AZT 145 hybrid compound in good yield 75%. Figure 3. 6 Compound 145 is orange crystal, melting point is 189-190 o C. IR (KBr) cm -1 : 3402, 3149, 3072, 2951, 1714, 1689, 1656, 1606, 1546, 1500, 1429, 1259, 1093, 956, 754, 698. 1 H-NMR (DMSO-d6, 500 MHz): 11.29 (1H, s, NH); 8.59 (1H, s); 8.36 (1H, s); 8.23 (1H, d, J = 8.0 Hz); 7.90 (1H, d, J = 7.5 Hz); 7.84 (1H, t, J = 7.0 Hz); 7.75 (1H, s); 7.45- 7.56 (4H, m); 6.36 (1H, s); 5.77 (2H, s); 5.31 (1H, s, OH); 5.21 (1H, s), 4.01 (1H, s); 3.54-3.62 (4H, m); 1.77 (3H, s). 13 C-NMR (DMSO-d6; 125 MHz): 190.1; 163.8; 162.2; 156.2; 150.5; 141.4; 136.3; 136.2; 134.6; 133.8; 133.7; 131.8; 131.4; 128.3; 127.6; 124.8; 122.9; 122.8; 122.7; 122.4; 109.6; 107.3; 84.5; 83.6; 60.6; 59.4; 47.7; 37.1; 12.4. HRMS Calc. For: C29H25N6O6: 553.1830 [M+H] + ; found: 553.1833. Thus, we have successfully synthesized a new indenoisoquinoline- AZT-triazole. It continues to be tested for anti-cancer activity. 9 3.4.2. Synthesis result of compound 148a-e On the other hand, in order to investigate the substituted group on N- lactam side chain and containing triazole bridge, we have synthesized compounds 148a-e from compound 50, yield 75-80%. Figure 3. 7 Compound 148a is orange crystal, melting point is 155-156 o C. IR (KBr) cm -1 : 3460, 3069, 2928, 2840, 1700, 1666, 1610, 1551, 1503, 1425, 1196, 1097, 1061, 757. 1 H-NMR (DMSO-d6, 500 MHz): 11.29 (1H, s, NH); 8.59 (1H, s, J = 8.0 Hz); 8.36 (1H, s); 8.23 (1H, d, J = 8.0 Hz); 7.90 (1H, d, J = 7.5 Hz); 7.84 (1H, t, J = 7.0 Hz); 7.75 (1H, s); 7.45-7.56 (4H, m); 6.36 (1H, s); 5.77 (2H, s); 5.31 (1H, d, J = 5.5 Hz, OH); 5.21 (1H, s); 4.01 (1H, s); 3.54-3.62 (4H, m); 1.77 (3H, s). 13 C-NMR (DMSO-d6; 125 MHz): 189.9; 167.8; 163.8; 162.2; 156.2; 150.5; 141.4; 136.3; 136.2; 134.6; 133.8; 133.7; 131.8; 131.4; 128.3; 127.6; 124.8; 122.9; 122.8; 122.7; 122.4; 109.6; 107.3; 84.4; 83.9; 60.7; 59.3; 45.7; 37.1; 12.2. HRMS Calc. For: C31H27N6O8: 611.1885 [M+H] + ; found: 611.1889 The chemical structures of the product 148b-e were confirmed using 1 H NMR, 13 C NMR, MS and IR techniques. Thus, we have synthesized 5 new compounds of indenoisoquinolin- AZT and containing triazole bridge with the substituted group on N-lactam side chain. These substances will continue to be tested for anti-cancer activity. 10 3.5. Synthesis result of compound 149 With the idea, in continuation of our interest on hybridization of indenoisoquinolines via triazole linker, another bioactive coupling partner concerns imatinib and analogs, tyrosine-kinase inhibitors used in the treatment of multiple cancers, most notably chronic myelogenous leukemia, have been taken into account, we conducted synthetic hybrid compounds between 144 and 144a. Figure 3. 8 Compound 149 is orange crystal, melting point is 208-210 o C. IR (KBr) cm -1 : 3438, 3123, 3068, 2925, 2870, 1702, 1648, 1579, 1530, 1454, 1399, 1318, 1259, 1121, 876, 808, 762. 1 H-NMR (DMSO-d6, 500 MHz): 10.1 (1H, s, NH); 9.0 (1H, bs); 8.75 (1H, bs); 8.59 (2H, d, J = 8.5 Hz); 8.30 (1H, s); 8.23 (1H, d, J=7.5 Hz); 7.98-8.04 (2H, m); 7.84-7.86 (4H, s); 7.67-7.69 (1H, m); 7.65 (3H, m); 7.45-7.55 (5H, m); 7.35-7.36 (2H, m); 7.21 (1H, s); 5.78 (2H, s); 5.63 (2H, s); 2.21 (3H, s). 13 C-NMR (DMSO-d6; 125 MHz): 189.6; 164.8; 162.3; 161.5; 161.0; 159.4; 156.0; 151.3; 148.1; 141.8; 139.0; 137.7; 137.0; 136.0; 135.7; 134.8; 134.5; 134.3; 133.4; 133.2; 132.1; 131.5; 131.0; 130.0 (2xC); 128.0; 127.8 (2xC); 127.7; 127.6; 127.5 (2xC); 126.7; 125.0; 123.7; 122.6; 117.1; 116.7; 107.5; 107.3; 58.3; 38.0; 17.5. HRMS Calc. For: C43H32N9O3: 722.2623 [M+H] + ; found: 722.2623. Thus, we have successfully synthesized a new derivative of indenoisoquinolin-imatinib. This substance will continue to be tested for anti-cancer activity 3.6. Synthesis of new indenoisoquinoline derivatives which have a methylendioxy or methoxy substituent on D ring and the different substituents on B ring. Mark Cushman et al, compounds that have a methylendioxy substituent or two methoxyl groups on the A or D ring are highly active. From there, we synthesized new derivatives of indenoisoquinoline with 11 methylendioxy or methoxy groups on D ring. On the other hand, we synthesize indenoisoquinoline derivatives which have branching vessels attached to N atom that bridge triazole through the click reaction, in the hope that the synthetic derivatives are cytotoxically active on two test lines: KB and HepG2. Figure 3. 9 12 3.6.1. The result was synthesized indenoisoquinoline derivatives with methylendioxy groups In order to receive compounds containing methylendioxy substituents in ring D, we have synthesized compound 153 from glycine and benzo[d][1,3]dioxole-5-carbaldehyde, 60% yield. Figure 3. 10. Synthesized indenoisoquinoline containing methylendioxy group Compound 153 is a reddish violet crystal, melting point 207-208 o C. IR (KBr) cm -1 : 2913, 1743, 1667, 1610, 1580, 1549, 1497, 1477, 1431, 1375, 1304, 1271, 1229, 1072, 1033, 997, 979, 929, 830, 787, 760, 725, 615, 573. 1 H-NMR (500 MHz, CDCl3) δH ppm: 8.59 (1H, d, J = 8.0 Hz, H-1); 8.29 (1H, dd, J = 1.0; 8.0 Hz, H-4); 7.70 (1H, m, H-2); 7.42 (1H, m, H-3); 7.08 (1H, s, H-7); 6.77 (1H, s, H-10); 6.07 (2H, s, H-18); 5.26 (2H, s, H-1’); 3.83 (3H, s, H-3’). 13 C-NMR (125 MHz, CDCl3) δc ppm: 189.16 (C-11); 167.99 (C-2’); 163.01 (C-5); 154.74 ; 151.48 ; 149.25 ; 134.17; 132.46 ; 132.17; 130.20 ; 128.66 ; 126.84; 123.17 ; 122.55 ; 108.15 ; 105.68 ; 103.96 ; 102.71 (C-18); 53.14 (C-3’); 45.48 (C-1’). Thus, we have successfully synthesized a new framework of indennoisoquinolin with the methylendioxi group on the D ring and the ester group attached to the N atom on the B ring. These substances will continue to be tested for anti-cancer activity and will be used as a raw material for subsequent synthesis. 3.6.2. The result was synthesized indenoisoquinoline derivatives with methoxy groups Synthesis of indenoisoquinoline derivatives containing methoxy groups the same to the synthesis of 153 compound but the starting material is the aldehyde used in this reaction is 3-methoxybenzaldehyde. But unexpectedly, the result of the reaction was a mixture of two isomers, 13 compound 154 and compound 155 (molar ratio 1: 1), the total reaction yield was 60%. Figure 3. 11. Synthesis indenoisoquinoline derivatives with methoxy groups Compounds 154, 155 are orange crystals, melting point are 207, 208 o C. In the ring D, there is a methoxy group that has the effect of pushing electrons, so that the second group of substituents enters the ring at the ortho position and the para position. If the substituent enters the para position, it receives the product 154, while the ortho position will receive the product 155. For compound 154, protons H-7 and protons H-9 separated by a methoxy group (in the meta position) have an interaction constant J = 1-3 Hz, so the H-7 signal is in the form doublet. For compound 155, the H-7, H-8 and H-9 protons are next to each other so there is no doublet signal with the interaction constant J = 1-3 Hz as compound 154 so the resonant signal of The proton will be doublet- doublet or doublet with a large interactive constant J = 6-10 Hz. When comparing the 1 H-NMR spectrum of the two compounds 154, 155 we see the difference as follows: On the 1 H-NMR spectrum of compound 154 there is a doublet resonance signal at 6.83 ppm with an interaction constant J = 1.5 Hz of the proton H-7, but on the spectrum of compound 155 there is no signal. Thus, compound 154 is the product of a closed loop reaction at the para position, while compound 155 is the product of the ortho-closed loop, this confirms that the reaction forms a mixture of the two closed products at ortho and para. Compound 154 has the spectrum: IR (KBr) cm -1 : 2922, 2851, 1740, 1696, 1658, 1611, 1576, 1550, 1500, 1467, 1419, 1368, 1322, 1274, 1221, 1082, 1036, 999, 979, 862, 784, 14 759, 725, 691, 647, 660, 647, 612, 570, 515. 1 H-NMR (500 MHz, CDCl3) δH ppm: 8.69 (1H, d, J = 8.0 Hz, H-1); 8.32 (1H, m, H-4); 7.73 (1H, m, H- 2); 7.54 (1H, d, J=8.0 Hz, H-10); 7.48 (1H, m, H-3); 6.83 (1H, d, J=1.5 Hz, H-7); 6.71 (1H, dd, J=1.5 Hz, 8.0 Hz, H-9); 5.30 (2H, s, H-1’); 3.85 (3H, s, H-18); 3.82 (3H