Nghiên cứu tổng hợp các hợp chất lai của một số triterpenoid có chứa nhóm benzamide và hydroxamate tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF 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 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, MGCD0103 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 (LBH589) 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 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 The new contribution of this thesis 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 Has synthesized new amide derivatives 91, 93, 95, has not been published in any documents 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 Has confirmed the structure of new compounds from the results of spectral data analysis: IR, 1H-NMR, 13C-NMR and LC-MS/MS For the first time, assessing the cytotoxic activity of 29 new compounds on cancer cell lines in humans, cell KB (Human epidemic carcinoma) and cell Hep-G2 (Hepatocellular carcinoma), inside, there are new compounds 89c, 89e, 89f, 92a, 96b has strong cytotoxic activity with IC50 values < 10 µM The layout of the thesis The thesis has 127 pages including: Introduction pages Chapter 1: Overview, 25 pages Chapter 2: Experiment, 35 pages Chapter 3: Results and discussion, 61 pages Conclusions: 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 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 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 Chapter 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, 13CNMR, LC-MS/MS Going from derivatives of some triterpenoids, we have synthesized reaction sequences: sequence of triterpenoid hybrid compounds containing benzamide group and 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 : 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 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: 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 this absorption pattern On the 1H-NMR proton resonance spectrum Scheme 3.2: Sơ đồ tổng hợp chất 77a-e the doublet doublet resonance signal of the H-3 proton (3.19 ppm) with J = 11 and 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, 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 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 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 Scheme 3.10:Mechanism of product formation 89a Figrue 3.14: 1H-NMR spectrum of 89b compound For 89b compound on 1H-NMR spectrum in addition to the signal of lupan frame, there is also a 3H singlet signal at 3.72 ppm 22 which is typical for the -NMe group and a 3H singlet signal at 3.17 ppm belongs to the group - OMe (Figure 3.14) On the 13C-NMR spectrum in addition to the -C = O group of esters (C-28) at the signal of 173.3 ppm, there is also an additional signal at 171.1 ppm of the C = O group in the -CONMeOMe group (figure 3.15) On IR spectrum, there are two signals at 1733 and 1667 cm-1 belong to these two groups -C = O Figrue 3.15: 13C-NMR spectrum of 89b compound C-B28 #4569 RT: 11.49 AV: NL: 1.59E5 T: FTMS + p ESI SIM ms [584.5000-587.5000] 586.2869 100 95 90 85 80 75 70 Relative Abundance 65 60 55 50 45 40 35 30 25 20 15 10 586.0152 586.7262 585.5 585.6 585.7 585.8 585.9 586.0 586.1 586.2 586.3 m/z 586.4 586.5 586.6 586.7 586.8 586.9 587.0 587.1 587.2 Figrue 3.16: LC-MS/MS spectrum of 89b compound 23 Compound 89b is also proved by high resolution mass spectra, on mass spectra found fragments m / z [M + H] +: 586,2869 (Figure 3.16) in accordance with the theoretical calculated mass for CTPT C36H60NO5 is 586,2866 Comparing with spectral data in some previously published documents [62, 63], it is possible to confirm the expected structure of compound 89b as shown on the graph The structure of other compounds is similarly confirmed 3.3.2 Results of hybrid compounds of some other triterpenoids containing hydroxamate group via amide bridge The first is the synthesis of amide derivatives 91, 93, 95: Betulinic acid (2), 3 -acetoxy-21-oxolup-18-ene-28-oic acid (5), and compound 81 is reacted with 6-aminohexanoic acid with a mol ratio of 1: in DMF solvent, in the presence of BOP and DMAP catalysts within a 24 hour period, obtained amide derivatives 91, 93, 95.) Figrue 3.17: 1H-NMR spectrum of 91 compound On the 1H-NMR spectrum of these compounds, in addition to the signals of the lupane frame, there is also a 1H triplet-patterned spectral signal corresponding to the displacement of 5.67-5.80 ppm (in CDCl3 solvent), This shows that the carboxylic groups of acids 2, and 81 have been converted into amide groups (Figure 3.17) 24 Compounds 91, 93, 95 are then reacted with H2NOH.HCl or HNMeOMe.HCl with a mol ratio of 1: in DMF solvent in the presence of BOP / DMAP to obtain 92a-b, 94a-b and 96a-b hydroxamate compounds (figrue 3.11; 3.12 and 3.13) Scheme 3.11: Synthesized of 90a-b, 92a-b compounds 25 Scheme 3.12: Synthesized of 94a-b compounds Scheme 3.13: Synthesized of 96a-b compounds The structure of these compounds is also proved by modern spectroscopic methods In the 1H-NMR spectrum of compound 92a, in addition to the signals of the lupan frame, there is also a 1H singlet signal at 10.30 ppm which is a characteristic of the -NH group and a 26 1H singlet signal at 8.61 ppm belongs to the group - OH in hydroxamic group -CONHOH, also appears the 1H triplet signal at 7.37 ppm which is characteristic of -NH group in amide group at ankyl bridge (due to interaction with protons of -CH2 group at position '(the ankyl bridge part), so the spectrum is triplet-shaped and has a stronger field resonance) (Figure 3.18) Figrue 3.18: 1H-NMR spectrum of 92a compound Scheme 3.19: 13 C-NMR spectrum of 92a compound 27 On the 13C-NMR spectrum of compound 92a in addition to the signal of 206.9 ppm of the ketone group and 170.1 of CH3CO-, there was also an additional signal of carbonyl group of amide at C28 at 173.1 ppm and carbonyl group in the hydroxamic group at 172.8 ppm (Figure 3.19) On the high resolution mass spectra, we found the m / z piece [M + H] +: 641,4489 (Figure 3.20) in accordance with the theoretical calculation volume for CTPT C38H61N2O6 is 641.4429 Thus, the expected structure of 92a compound is suitable for the spectrogram The structure of other compounds is similarly proven C-44 #1146 RT: 3.90 AV: NL: 6.78E8 T: FTMS + p ESI SIM ms [639.5000-642.5000] 641.4489 100 95 90 85 80 75 70 Relative Abundance 65 60 55 50 45 40 642.4512 35 30 25 20 15 10 640.4673 639.6 639.8 640.0 640.2 640.4 640.6 640.8 641.0 m/z 641.2 641.4 641.6 641.8 642.0 642.2 642.4 Figrue 3.20: LC-MS/MS spectrum of 92a compound Experimental study of two protons in the hydroxamic functional group -CONHOH author Rachel Cold [100] showed that in a non-proton solvent such as DMSO, the H of N-H acts as a ptoton acid rather than N-OH Because of the more flexibility, -NH resonates at the weaker field (δ = 10,30-12,37 ppm) Moreover, these protons are very flexible and easily exchanged or mutually exchanged, so some of the signal -NH, -OH signals are very weak like in the 89g compound, the two resonant signals of each proton NH and -OH is separated into two doublet signals with the intensity 28 of 0.5H or in some cases does not give signals on the spectrum as 89e In addition to the above groups, another group of protons in the structure of the sequences is branched protons Most substances have enough branched protons with the chemical shift of the -CH2 groups in the range of 1.25-3.94 ppm The substances are measured in DMSO solvent, so due to the effect of proton of methyl group in incompletely deuterized DMSO solvent ( = 2.50 ppm), some protons in protons of group -CH2 cannot be observed signals at position of about 2.50 ppm Thus, we have successfully synthesized 16 hybrid compounds of some triterpenoids containing hydroxamate group via ester bridge and amide bridge The newly synthesized compounds have been proven by modern spectroscopy such as IR infrared spectroscopy, 1H-NMR and 13C-NMR nuclear magnetic resonance spectra, mass spectrometry LC-MS / MS 3.4 Anticancer activity of hybrid compounds With the desire to synthesize biologically active hybrids in search of new compounds with anticancer activity, hybrid compounds of some triterpenoids containing benzamide and hydroxamate groups after being synthesized have been developed to test in vitro cytotoxic activity against two human cancer cell lines, KB (epithelial cancer) and Hep-G2 (liver cancer), along with the activity test of Ellipticine standard The process of examining cytotoxic activity was conducted at the Department of Applied Biochemistry of Institute of Chemistry Results of activity testing of compounds are presented in Table 3.1 and Table 3.2 29 Table 3.1: Results of activity testing of hybrid compounds containing benzamide STT Compound IC50 (µM) KB IC50 (µM) Hep-G2 77a 202,2 202,2 77b 197,9 166,0 77c 193,7 115,6 77d 193,7 128,9 77e 186,9 108,0 80 214,0 234,9 83a 222,7 168,5 83b 240,2 176,8 84 15,4 12,1 10 85 234,9 234,9 11 87 216,6 158,7 12 88a 68,2 137,1 13 88b 152,5 209,0 14 Ellipticine 1,3 1,5 30 Table 3.2: Results of activity testing of hybrid compounds containing hydroxamate group STT Compound IC50 (µM) KB IC50 (µM) Hep-G2 89a 29,76 23,39 89b 55,71 93,25 89c 8,5 67,11 89d 87,71 65,77 89g 52,25 71,75 89h 58,14 113,70 89e 7,60 97,0 89f 63,84 9,13 90a 23,15 24,72 10 90b 50,09 141,03 11 92a 3,06 4,22 12 92b 68,2 137,1 13 94a 43,71 64,42 14 94b 103,76 106,66 15 96a 35,08 45,07 16 96b 5,18 6,21 17 Ellipticine 1.3 1,5 31 The activity test results of hybrid products are detailed in Table 3.1 and Table 3.2 Results showed that most hybrid compounds exhibited toxic activity for these two cell lines at different concentrations For a hybrid of some triterpenoids containing benzamide, compound 84 is the best biologically active compound on both KB and Hep-G2 cell lines with IC50 values at 12.1µM and 15, µM On the other hand, comparing the cytotoxicity of 3-acetyl compounds with 3-hydroxy compounds, it is easy to see that compounds with 3acetyl groups (83a and 88a) have stronger activity than 3-hydroxy compounds (83b and 88b) (table 3.1) For the hybrid of a number of triterpenoids containing hydroxamate group, it can be seen that most of the compounds are bioactive, including compounds 89c, 89e, 92a, 96b showing toxic activity Strong cells on KB cancer cell line and compounds 89f, 92a, 96b showed strong cytotoxic activity on Hep-G2 cancer cell line with IC50 value