Summary of Chemisttry doctoral thesis: Research on the synthesis of hybrid compounds of some triterpenoids containing benzamide and hydroxamate groups

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Study on synthesis of hybrid compounds of some triterpenoids containing benzamide and hydroxamate groups in order to search for hybrid compounds with high biological activity, as a scientific basis for further research to create anti-cancer drugs, contribute to health care for the community.

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Nội dung Text: Summary of Chemisttry doctoral thesis: Research on the synthesis of hybrid compounds of some triterpenoids containing benzamide and hydroxamate groups

MINISTRY OF EDUCATION VIETNAM ACADEMY OF AND TRAINING SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY ------------------ DINH THI CUC SUMMARY OF CHEMISTTRY DOCTORAL THESIS STUDY ON SYNTHESIS OF HYBRID COMPOUNDS OF SOME TRITERPENOIDS CONTAINING BENZAMIDE AND HYDROXAMATE GROUPS Major : Organic chemistry Code: 9.44.01.14 Hanoi-2020
The thesis was completed in Graduate University Science and Technology, Vietnam Academy of Science and Technology.  Supervisor: Prof.Dr. Nguyen Van Tuyen Dr. Le Nhat Thuy Giang Reviewer 1: Reviewer 2: Reviewer 3: The thesis will be presented before the Council for Evaluation of Ph.D. thesis at the Academy, meeting at Graduate University Science and Technology, Vietnam Academy of Science at …. am, ….March, , 2020.
A. INTRODUCTION OF THE THESIS 1. The urgency, scientific and practical significance of the thesis topic. Cancer is one of the most threatening diseases in the world. Globally an estimated 8.2 million people died from cancer in 2012, and lung cancer (bronchial and tracheal cancer) increased significantly has become the fifth leading cause of death in 2012, killing 1.1 million men and 0.5 million women. Currently, hybrid structured drugs are increasingly being studied and synthesized by scientists to create hybrid compounds with high biological activity that are superior to the original ones. Combining biologically active components to create new structures with exciting activities is currently a research direction that attracts the attention of many scientists. Triterpenoids are a group of natural compounds or their derivatives that have received significant research interest from scientists in recent years. Many tritecpenoid compounds such as betulin, betulinic acid, ursolic acid, oleanolic acid have been declared to have anti-HIV, antibacterial, antifungal, anti-inflammatory and anti-cancer activity. Besides, benzamide compounds such as MS-275, MGCD- 0103 are interesting bioactive compounds, has been approved by the US Food and Drug Administration (US-FDA) for the treatment of solid organs, leukemia, and metastatic melanoma of late stage [7-11]. Hydroxamic compounds such as vorinostat, also known as zolinza (SAHA) were also approved by the FDA in 2006 for the treatment of T-cell skin lymphoma, trichosatin A (TSA) and belinostat (PXD-101) for therapeutic use. donation and blood cancer, panobinostat (LBH- 589) in the treatment of solid organs, AML, ALL, MDS [12-16]. However, hybrid compounds of triterpenoid containing benzamide and hydroxamate groups have not been studied much. The research on the synthesis of triterpenoid hybrid compounds 1
containing benzamide group, hydroxamate group and testing of cytotoxic activity to search for compounds with anticancer activity is a new research direction. Based on such ideas, we chose the topic: "Research on the synthesis of hybrid compounds of some triterpenoids containing benzamide and hydroxamate groups" is a new, interesting and highly practical scientific matter. 2.Thesis objectives Study on synthesis of hybrid compounds of some triterpenoids containing benzamide and hydroxamate groups in order to search for hybrid compounds with high biological activity, as a scientific basis for further research to create anti-cancer drugs, contribute to health care for the community. 3. The new contribution of this thesis 1. For the first time successfully designed and synthesized 13 new compounds of some triterpenoids containing benzamide group and 16 new compounds of some triterpenoids containing hydroxamate group by ester and amide bridge. 2. Has synthesized 3 new amide derivatives 91, 93, 95, has not been published in any documents. 3. A method of improving the synthesis of benzamide and hydroxamate derivatives by BOP activating agent instead of using the old activating agent is DCC or CDI, thereby opening a new direction to effectively synthesize these compounds. 4. Has confirmed the structure of new compounds from the results of spectral data analysis: IR, 1H-NMR, 13C-NMR and LC-MS/MS. 5. For the first time, assessing the cytotoxic activity of 29 new compounds on 2 cancer cell lines in humans, cell KB (Human epidemic carcinoma) and cell Hep-G2 (Hepatocellular carcinoma), inside, there are 5 new compounds 89c, 89e, 89f, 92a, 96b has strong cytotoxic activity with IC50 values < 10 µM. 2
4. The layout of the thesis The thesis has 127 pages including: Introduction 2 pages Chapter 1: Overview, 25 pages Chapter 2: Experiment, 35 pages Chapter 3: Results and discussion, 61 pages Conclusions: 1 page. References: include 100 references relating to close area research of thesis, updated until 2018. Appendix: 51 pages, including all the spectra of the synthesized compounds. 5. Research methods Substances synthesized by known modern organic synthesis methods, are modified and applied appropriately in specific cases. The reaction product is cleaned by column chromatography. The structure of the product is determined by modern spectroscopic methods such as: IR, 1H-NMR, 13C-NMR, LC-MS/MS. Biological activity was assessed by Mossman's method on two cancer cell lines in humans, KB and Hep-G2. B- THE THESIS CONTENT Chapter 1. OVERVIEW The overview of the thesis consists of 25 pages with the following main contents: - Triterpenoid derivatives and their biological activity. - Synthesis and biological activity of benzamide class - Synthesis and biological activity of the hydroxamate class 3
Chapter 2. EXPERIMENT The experiment consists of 35 pages, detailing the research methods, synthesis and refining processes, the physical properties of the products received are: melting point, morphology, color, reaction performance and detailed data of spectra IR, HRMS, 1H-NMR, 13C- NMR, LC-MS/MS. Going from derivatives of some triterpenoids, we have synthesized 2 reaction sequences: 1 sequence of triterpenoid hybrid compounds containing benzamide group and 1 sequence of triterpenoid hybrid compounds containing hydroxamate group. The optimal method of using these compounds is to use a carboxylic group activator, BOP and a catalyst, DMAP, in a weak base medium, Et3N, and a reactive agent are amines in DMF solvent. We evaluated the cytotoxic activity of synthetic compounds on two human cancer cell lines, KB and Hep-G2. Chapter 3 : RESULTS AND DISCUSSION 3.1. The goal of the subject First perform the –OH group transformations at C-28 of some triterpenoids to form ester and amide derivatives, then react with different amines to form new compounds containing the group. benzamide and hydroxamate. Some triterpenoid compounds are directly reacted at C-28 with different amines as shown in figure 3.1. . 4
Scheme 3.1: The goal of the thesis 3.2. Synthesized results of hybrid compounds of some triterpenoids containing benzamide group 3.2.1.Synthesized results of betulin-containing hybrid compounds containing benzamide group via ester bridge To synthesize benzamide derivatives via ester bridges, the thesis first synthesized ester derivatives of betulin. Betulin (1) is reacted with carboxylic acid anhydride with a molar ratio of 1: 4 in anhydrous CH2Cl2 solvent with a alkaline catalyst of triethyl amine, in a reaction time of 24 hours. The 76a-f acid derivatives are white crystals, with a synergistic efficiency of 60% to 79%. The infrared (IR) spectrum of 76a compounds showed a absorption pattern at 1732 and 1642 cm-1, which was characteristic of the -C = O group in the ester and acid functional groups while the infrared spectrum of betulin did not appear 5
this absorption pattern. On the 1H-NMR proton resonance spectrum Scheme 3.2: Sơ đồ tổng hợp các chất 77a-e the doublet doublet resonance signal of the H-3 proton (3.19 ppm) with J = 11 and 5 Hz, the signals at Ha-28 and Hb-28 appear at 4.31 and 3.90 ppm respectively; 1H singlet signals of Ha-29 and Hb-29 appear at 4.68 and 4.58 ppm, 6 methyl groups appear fully with singlet signals at 0.75 - 1.68 ppm, signals this does not change much from the standard spectrum of betulin. In addition, on the proton spectrum of compound 76a, there are also full branched protons (2.71-2.64 ppm, 2H-2 'and 2H-3'). Particularly for 76e, the reaction agent is cis-1,2,3,6-tetrahydro phtalic anhydride when reacting with betulin to form 76e ester derivative, showing that two resonant signals of each proton Ha-28 and Hb -28 was split into two doublet signals with the intensity of 0.5H, the interaction constant J is 11.0 Hz, which confirms the cis configuration in the double connection of acid anhydride cis-1,2,3,6-tetrahydro phtalic has been converted into a trans configuration in compound 76e by reacting with this anhydride acid to betulin. Other compounds have been shown similarly. Comparison of these spectral analysis results with reference [66] can confirm that the structure of 76a-f ester derivatives is consistent with chromatography on spectroscopy. 6
Figure 3.1: Chemical structure and some physical characteristics of 76a-f compounds From the ester derivatives of acid 76a-e, continue to be reacted with 1,2-diaminobenzene (molar ratio of 1: 1.5) in DMF solvent in the presence of BOP / DMAP / Et3N received 77a-e products. The -COOH acid group is converted to the amide group, this reaction occurs quickly and has high efficiency, the product of a very selective reaction. The structure of 77a-e products is confirmed by spectral data. On the IR spectrum of compound 77c, the absorption peak at 3373 cm-1 is typical of the -NH group and there is the strong absorption peak of the -C = O group on the amide group at 1655 cm-1. On the 1H-NMR spectrum of compound 77c, in addition to the signals of the lupan frame, there are additional signals of the 7
benzamide group as in the singlet 1H signal (7.55 ppm) of the -NH group. The signal in the range of 7.18 - 6.76 ppm is of the aromatic ring, specifically the doublet doublet signal at 7.18 ppm (1H), the constant J = 1.5 Hz is of the H-6 proton; 7.06 ppm (1H, td, J = 7.5; 1.5 Hz, H-4 ”); 6.78 (1H, dd, J = 7.5; 2.0 Hz, H-3 ”) and 6.76 (1H, td, J = 7.5; 1.5 Hz, H-5”) ( figure 3.2) Figure 3.2: 1H-NMR relaxation spectrum of 77c compound On the 13C-NMR spectrum of 77c compound appears to push enough signals of the carbon atoms present in the molecule. In addition to the signals of the lupane frame, there are also signals of the carbonyl group of esters and amides and aromatic rings, specifically, as the signal 175,6 ppm is of the carbonyl group of esters (C-1 ’); at signal 172.7 ppm is of the carbonyl group of amide (C-4 ’); signals of carbon atoms in the aromatic ring are as follows: at signal 142.0 ppm, it is of C-2 ”; 127.5 is C-1 ”; 123,5 is C-6 ”; at signal 118.9 is C-5 ”; 117.2 is C-3 ”(figure 3.3). On the high resolution mass spectra of compound 77c found the m / z fragment [M + H] + is 661,4883 (figure 3.4) in accordance with the theoretical 8
calculated mass for C42H65N2O4 molecular formula of the compound 77c is 661.4866. Comparing these spectral analysis results with previously published references [38, 41, 42, 88-90] can confirm that the structure of 77c compound is consistent with the spectral data. The diagram and structure of 77a-b and 77d-e compounds were similarly confirmed. Figure 3.3: 13C-NMR relaxation spectrum of 77c compound C_92 #1377 RT: 4.68 AV: 1 NL: 3.64E7 T: FTMS + p ESI Full ms [50.0000-750.0000] 661.48834 100 90 80 70 Relative Abundance 60 50 664.49823 40 30 20 10 683.46967 699.44305 556.83197 578.81519 600.79724 622.78101 644.75861 706.54669 0 560 580 600 620 640 660 680 700 m/z Figure 3.4: Mass spectrometry LC-MS/MS of 77c compound 9
Cơ chế hình thành hợp chất 77c đầu tiên là quá trình thế nguyên tử hydro của hợp chất 76c trong môi trường bazơ yếu là triethyl amine bằng nhóm (NMe2)3P- trong tác nhân hoạt hóa BOP để tạo thành hợp chất trung gian 76c1, tiếp theo dưới xúc tác DMAP hợp chất 76c1 được chuyển thành hợp chất trung gian 76c2 và sau đó là phản ứng thế bằng tác nhân thế ái nhân là 1,2-diaminobenzene để hình thành sản phẩm 77c (sơ đồ 3.3). Scheme 3.3: Mechanism of compound formation 77c 3.2.2. Synthesized results of hybrid compounds of pentacyclic triterpenoid diacid containing benzamide group By the same methods, the thesis synthesizes hybrid compounds of pentacyclic triterpenoid diacid containing benzamide group with the desire to search for new hybrid compounds with interesting biological activity. Diacid pentacyclic triterpenoid derivative 78a-b isolated from Cheffleraoctophylla (Ivy tree) [91] was reacted with Jone oxidant (Cr3O / H2SO4) in acetone solvent 10
which received oxidized products 79a-b [30, 31] (scheme 3.5). Compound 79b is then reacted directly with 1,2-diaminobenzen with a molar ratio of 1: 1,5 in DMF solvent in the presence of BOP / DMAP / Et3N to obtain compound 80 (scheme 3.4 ). Scheme 3.4: Synthesized of 80 compound On the 1H-NMR spectrum of compound 80, in addition to the signals of the lupan frame, there was also the signal of the -NH group Figure 3.5: 1H-NMR spectrum of 80 compound 11
at 7.47 ppm; signal of 4 aromatic ring protons at 7.08-6.78 ppm (Figure 3.5). On the 13C-NMR spectrum also appear full signal of lupan frame and aromatic ring. The two ketone groups of C-3 and C- 11 appear in the weak fields 213.2 and 210.8 ppm, the carbonyl C-28 group at 174.6 ppm, the aromatic carbon atoms appear in the region 118 , 5 - 140.9 ppm (figure 3.6). 13 Figrue 3.6: C-NMR spectrum of 80 compound C_82 #880 RT: 2.99 AV: 1 NL: 1.98E8 T: FTMS + p ESI SIM ms [542.5000-545.5000] 545.3702 100 95 90 85 80 75 70 65 60 Relative Abundance 55 50 45 40 35 30 25 20 15 10 5 542.8172 543.3550 544.8902 0 542.6 542.8 543.0 543.2 543.4 543.6 543.8 544.0 544.2 544.4 544.6 544.8 545.0 545.2 545.4 m/z Figrue 3.7: LC-MS/MS spectrum of 80 compound 12
The structure of compound 80 is also proved by mass spectra, on mass spectra of compound 80 found piece m / z [M + H] + is 545,3702 (Figure 3.7) in accordance with the calculated mass According to the theory for the molecular formula C35H49N2O3 of compound 80 is 545,3737. Compound 79a is deoxidized by reducing agent NaBH4 with molar ratio of 1: 4 in MeOH solvent at room temperature, C = O group at C-3 position is reduced to -OH group with 3β –hydroxy configuration. (compound 81) according to figrue 3.5. Figrue 3.5: Synthesized of 83a-b compounds The 1H-NMR nucleus resonance spectrum of 81 appears the signal of a proton doublet at δH 3.71 ppm (dd, J = 2.5 Hz, H-3β) typical for 3β-OH group in C-3 position. To protect this 3β-OH group, before reacting with 1,2-diaminobenzene, compound 81 was acetylated with acetic anhydride agent with mol ratio of 1: 1.5 in 13
DCM solvent, received product 3-acetyl products (82). Compound 82 was then reacted with 1,2-diaminobenzene with a mol ratio of 1: 1,5 in DMF solvent in the presence of BOP / DMAP / Et 3N and obtained benzamide 83a (figrue 3.5). To obtain a new benzamide product, compound 83a continued to be hydrolyzed by LiOH agent in MeOH solvent to obtain compound 83b (figrue 3.5). The structure of 83a-b compounds was similarly confirmed by 1H-NMR and 13C-NMR spectrum. 3.2.3. Snthesized results of hybrid compounds of betulinic acid containing benzamide group Betulinic acid (2) is also a derivative of triterpenoid with many biological activities, so the thesis continues to explore the direction Figrue 3.6: Synthesized of 84 and 85 compounds direction study on synthesis of benzamide compounds from betulinic acid. Betulinic acid was reacted with 1,2-diaminobenzene in DMF 14
solvent in the presence of BOP / DMAP / Et3N received product 84 (figrue 3.6). Next, betulinic acid (2) is oxidized by the agent Jone (Cr3O / H2SO4) in acetone solvent to obtain compound 69 (figrue 3.6). The - OH group in carbon position 3 in the oxidized molecule, this is confirmed on the proton spectrum when the characteristic signal of H-3 protons does not appear on the spectrum of compound 69. In addition, on the IR spectrum of compound 69, the characteristic absorption signal of the cyclic ketone group appears at a wavelength of 1701 cm-1. Such data allow us to confirm the structure of compound 69 [30,31]. Compound 69 was then reacted with 1,2- diaminobenzene in DMF solvent in the presence of BOP / DMP / Et3N and received benzamide 85 (figrue 3.6). The structure of compounds 84 and 85 are similarly demonstrated. 3.2.4. Synthesized results of other triterpenoid hybrids containing benzamide Ursolic acid (3) and 3 --acetoxy-21-oxolup-18-ene-28-oic acid (5) are also well-studied triterpenoid derivatives. Therefore, the thesis continues to study synthesis of benzamide compounds from these acids. The acetylated ursolic acid is similar to compound 81 to obtain compound 86. Then the compound 86 reacts with 1,2- diaminobenzene in DMF solvent in the presence of BOP / DMAP / Et3N received product 87 benzamide (figrue 3.7). The final compound, triterpenoid (5), was also reacted with 1,2-diaminobenzene with a mol ratio of 1: 1.5 in DMF solvent in the presence of BOP / DMAP / Et3N received 88a compound. Like compound 83a, compound 88a is also hydrolyzed by LiOH with a mol ratio of 1: 5 in MeOH solvent, receiving compound 88b (figrue 3.8), on the proton spectrum of compound 88b no longer see any signal 3H singlet effect at 2.05 ppm, this proves that the 3β-acetoxy group of compound 88a has been converted into 3β -hydroxy group in compound 88b. On the 1H-NMR spectrum of compound 87, in 15
addition to the signals of the ursan frame, there is a singlet 1H signal at 7.53 ppm of -NH group, 4 protons of the aromatic ring appear in the region from 7.13 to 6, 77 ppm (Figure 3.8). Scheme 3.7: Synthesized of 87 compound Scheme 3.8: Synthesized of 88a-b compounds On the 13C-NMR spectrum also appear full signal of carbon atoms, the carbonyl group at C-28 appears at 176.6 ppm, the carbonyl group (CH3C = O) appears at the signal of 171.0 ppm, 6 carbon atoms of the aromatic ring appears in the stronger field region, at the signal of 140.6 ppm is of C-2 '; at 126.6 ppm the signal is C-1 ’; at 16
signal 126.1 ppm is of C-4 ’; at 124.7 ppm the signal is C-6 ’; at signal 119.4 ppm is C-5 'and at signal 118.2 ppm is C-3' (Figure 3.9). Figrue 3.8: 1H-NMR spectrum 87 compound 13 Figrue 3.9: C-NMR spectrum 87 compound 17
The structure of compound 87 is also demonstrated by high resolution mass spectra. On the high resolution mass spectra of compound 87 found the m / z fragment [M + H] + is 589,4329 (Figure 3.10) in accordance with the theoretical calculated mass for the molecular formula C38H57N2O3 of the compound 87 is 589,4363. Thus, based on the above data, it can be confirmed that the structure of compound 87 is consistent with the data on the graph. The structure of 88a-b compounds has been similarly proven by modern spectroscopic methods. C-94 #1690 RT: 5.74 AV: 1 NL: 1.65E8 T: FTMS + p ESI SIM ms [587.5000-590.5000] 589.4329 100 95 90 85 80 75 70 65 60 Relative Abundance 55 50 45 40 35 590.4356 30 25 20 15 10 5 587.5426 588.4661 0 587.6 587.8 588.0 588.2 588.4 588.6 588.8 589.0 589.2 589.4 589.6 589.8 590.0 590.2 590.4 m/z Figrue 3.10: LC-MS/MS spectrum of 87 compound Thus, the thesis has successfully synthesized 13 hybrid compounds of some triterpenoids containing benzamide group and these are new and obtained compounds with high performance. The structure of the products has been demonstrated by modern spectroscopic methods such as IR, 1H-NMR, 13C-NMR and LC-MS / MS. 3.3. Synthesized results of hybridization compound of some triterpenoids containing hydroxamate group Although many derivatives of triterpenoid acid have been prepared and screened for their cytotoxic activity [92-99], the hybrid compounds of triterpenoid containing hydroxamate group are very 18