Summary of Chemistry doctoral thesis: Chemical research and biological activity of two species of tai chua (Garcinia cowa Roxb. Ex Choisy) and dang hoang (Garcinia hanburyi Hook. F) growing in Vietnam

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With the aim of searching for bioactive compounds from plants of the genus Garcinia in order to contribute to the scientific basis for further research in the pharmaceutical field, the thesis focuses on studying two species of the genus Garcinia is Garcinia cowa and Garcinia hanburyi.

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Nội dung Text: Summary of Chemistry doctoral thesis: Chemical research and biological activity of two species of tai chua (Garcinia cowa Roxb. Ex Choisy) and dang hoang (Garcinia hanburyi Hook. F) growing in Vietnam

MINISTRY OF EDUCATION AND VIETNAM ACADEMY TRAINING OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY …………***………… NGUYEN THI KIM AN RESEARCH ON CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF TWO SPECIES GARCINIA COWA ROXB. EX CHOISY AND GARCINIA HANBURYI HOOK. F GROWING IN VIETNAM Major: Chemistry of natural compounds Code: 9.44.01.17 SUMMARY OF CHEMICAL DOCTORAL THESIS HA NOI – 2020
This thesis was completed at Graduate University of Science and Technology - Vietnam Academy of Science and Technology. Supervisors: 1. Assoc.Prof. PhD. Ngo Dai Quang Institute of Natural Products Chemistry – Vietnam Academy of Science and Technology 2. Prof. PhD. Tran Thi Thu Thuy Institute of Natural Products Chemistry – Vietnam Academy of Science and Technology Examiner 1: Assoc.Prof. PhD. Phan Minh Giang University of Science – Vietnam National University Examiner 2: Prof. PhD. Le Mai Huong Institute of Natural Products Chemistry – Vietnam Academy of Science and Technology The thesis defense was monitored by the Graduate University level Board of Examiners, held at: Graduate University of Science and Technology - 18 Hoang Quoc Viet - Cau Giay - Ha Noi. At ……….. , ….………………….. 2020 The thesis is available in Vietnam National Library and Library of Graduate University of Science and Technology.
1 INTRODUCTION 1. The urgency of the thesis Today, natural health protection and treatment aids are increasingly popular because they are safe to use because they have fewer side effects than synthetic products. Many natural compounds have been studied and isolated, determined their chemical structures and proven to have many important biological activities. These studies not only contribute to the knowledge of compounds in nature, but also contribute to the detection of potential compounds, thereby building a conservation plan and development of suitable species. with the climate and soil of Vietnam. With a predominantly hot and humid climate, Vietnam is a suitable habitat for many valuable medicinal plants that have been used in folklore. Many species of the genus Garcinia have been used as medicinal herbs, for example, dried yellow gamboge is used to treat cancer, hemostasis, deworming, respiratory inflammation ... Many Research on the chemical composition of species of the genus Garcinia has shown that their main chemical composition is xanthone with many valuable biological activities such as cancer cell inhibitory activity, antioxidant activity, and antibacterial properties, ... In Vietnam, the two trees of the genus Garcinia are the plant of the genus Garcinia (Garcinia cowa Robx. ex Choisy) and the tree gooseberry (Garcinia hanburyi Hook. f) of the Guttiferae family grow and develop very well, distributed in many localities across the country [1]. In the world, there have been many studies on the chemical composition and biological activity of these two species, but the sour ear tree (Garcinia cowa) and the rhubarb (Garcinia hanburyi) growing in Vietnam have not been collaborated. fake any research. 2. The research objectives of the thesis With the aim of searching for bioactive compounds from plants of the genus Garcinia in order to contribute to the scientific basis for further research in the pharmaceutical field, the thesis focuses on studying two species of the genus Garcinia is Garcinia cowa and Garcinia hanburyi. Therefore, the thesis: "Chemical research and biological activity of two species of tai chua (Garcinia cowa Roxb. Ex Choisy) and dang hoang (Garcinia hanburyi Hook. F) growing in Vietnam." is implemented with the following main contents: - Isolation of compounds from Garcinia cowa latex. - Isolation of compounds from the latex and stems of Garcinia hanburyi. - Determination of the chemical structure of isolated compounds. - Investigate the kinetic and thermodynamic properties of gambogic acid as a basis for semi-synthesis of some derivatives of gambogic acid. - Investigate some biological activities of the compounds obtained. 3. The main research contents of the thesis - Isolation and structural determination of compounds from Garcinia cowa latex and resin, tree trunks Garcinia hanburyi. - Investigate some of the kinetic and thermodynamic properties of gambogic acid - Synthesize some derivatives of gambogic acid on the basis of esterification and amide reaction - Evaluation of antioxidant activity of compounds isolated by ABTS and DPPH methods. - Evaluation of enzyme α-glucosidase inhibitory activity of some substances isolated from Garcinia cowa plant
2 - Evaluation of cytotoxic activity of substances isolated and synthesized on some cancer cell lines such as liver cancer (Hep-G2), lung (LU-1), rhabdomyosarcoma cell line (RD), colorectal (HT-29), HeLa cells (HeLa). CHAPTER 1. OVERVIEW An overview of domestic and international researches on the following issues: 1.1. General introduction to the genus Garcinia 1.1.1. Plant characteristics of the genus Garcinia 1.1.2. Uses 1.1.3. Chemical composition of genus Garcinia 1.1.4. Biological activity of substances isolated from genus Garcinia 1.1.5. Chemical research on Garcinia genus in Vietnam 1.2. Overview of the tai chua Garcinia cowa 1.2.1. Morphological and distributional characteristics 1.2.2. Chemical research and biological activities 1.3. Overview of the dang hoang Garcinia hanburyi 1.3.1. Morphological and distributional characteristics 1.3.2. Chemical research and biological activities 1.4. Overview of gambogic acid 1.4.1. Chemical structure 1.4.2. Gambogic acid inhibitory activity of cancer cells 1.4.3. Semi-synthetic and bioactive test of GA derivatives CHAPTER 2. SUBJECTS AND METHODOLOGY This section describes in detail the sample handling, extraction residue method, chromatographic procedure and compound isolation; methods for determining the chemical structure of compounds; methods of examining some kinetics and thermodynamics of gambogic acid and testing methods of biological activity. 2.1. Research subjects Garcinia cowa latex was collected in Quy Chau district - Nghe An and Phu Quoc - Kien Giang in December 2015. Template number GC2015128 is kept at Institute of Natural Products Chemistry. Garcinia hanburyi resin and stem barks were collected in Phu Quoc district - Kien Giang in December 2015. Template number GH2015129 is kept at Institute of Natural Products Chemistry. Both species were species identified by TS. Nguyen Quoc Binh - Vietnam Museum of Nature - Vietnam Academy of Science and Technology. 2.2. Research Methods 2.2.1. Methods of isolation of substances The isolation of substances from extracts of plant parts is done by different chromatographic methods such as thin layer chromatography (TLC), normal column chromatography (CC) with a stationary phase of silica gel (Merck), inversion column chromatography with stationary phase RP-18 (Merck) and molecular sieve chromatography for stationary phase is sephadex LH-20 (Merck). 2.2.2. Structure determination method The structure of isolated and semi-synthetic compounds was determined by combining physical parameters with modern spectroscopic methods. • High resolution mass spectrometry HRESIMS
3 • One-dimensional and two-dimensional nuclear magnetic resonance spectra • Melting temperature • Pole rotation angle [α]D 2.2.3. Methods of kinetic investigation of amorphous materials The most common methods for examining mirror-state kinetics of amorphous materials are differential scanning calorimetry (DSC) and broadband dielectric spectroscopy (BDS). 2.2.4. Activity evaluation methods 2.2.4.1. Assessment method of antioxidant resistance ABTS and DPPH • Methods of assessment of antioxidant activity ABTS: Antioxidant activity by ABTS of a reagent conducted according to the method of Saeed N. with a small change. • DPPH antioxidant activity assessment Antioxidant activity by DPPH was conducted according to the method of Brand Williams [219] with modified. The antioxidant capacity of ABTS and DPPH method of research samples is calculated as follows: % Free radical clearance = (OD control - OD sample) * 100 / OD control (%) In which: OD Control: Well absorbance does not contain reagents OD sample: Absorbance at the reagent well 2.2.4.2. Method of assessment of enzyme α-glucosidase inhibitory activity • Principle: Based on the p-nitrophenyl-a-D-glucopyranoside cleavage reaction under the action of a- glucosidase enzyme, the yellow p-nitrophenol product is produced: p-nitrophenyl-a-D-glucopyranoside a-D- glucose + p-nitrophenol The absorbance of the reaction mixture at 410 nm at 30 minutes after the reaction indicates the amount of p-nitrophenol product produced, which in turn reflects the enzyme a-glucosidase activity. • The test sample's ability to inhibit a- glucosidase enzyme is determined by the formula: % inhibition = [A (control) - A (test sample)] / A (control) x 100% IC50 is the reagent concentration that inhibits 50% of the activity of the enzyme a-glucosidase, calculated using Tablecurve software. 2.2.4.3. In vitro cytotoxic activity evaluation method • Methods of screening MTT activity • Active screening method of SRB
4 CHAPTER 3. EXPERIMENTAL The experimental section describes in detail the processing of extract sediment samples, isolation of clean substances from G. cowa latex and from G. hanburyi resin and stem. This section also describes the process of investigating some of the kinetic and thermodynamic properties of gambogic acid, the synthesis of derivatives of gambogic acid. Spectral data and the physical numbers for isolates and synthesizers are also presented here. 3.1. Isolation of substances from G. cowa plants G. cowa latex (3,0 kg) Đập nhỏ, sấy ở 45oC trong 3 ngày Dried G. cowa latex (2,8 kg) Ngâm MeOH (3 L x 3 times) ở nhiệt độ phòng, kết hợp siêu âm Residue (500,0 g) Ngâm DCM (3 L x 3 times)
5 DCM residue (96,7 MeOH residue g) CC-SiO2, DCM-MeOH (100:0 to 0:100, v/v) GCN1 (22,4 g) GCN2 (37,5 g) GCN3 (15,9 g) GCN3.1 GCN3.2 GC5 GC4 GC2 14,2 mg 9,8 mg 13,5 mg GCN1.3 GCN1.4 GCN1.6 GCN1.8 GC18 GC8 GC7 GC9 GC12 GC10 160 mg 230 mg 40 mg 120 mg 1,43 g 260 mg GCN2.2 GCN2.4 GCN2.6 GCN2.8 GCN2.10 GCN2.11 GC11 GC13 GC14 GC16 GC1 GC3 GC15 GC17 GC6 20 mg 45 mg 850 mg 37 mg 80 mg 8,3 mg 28 mg 12,1 mg 25,8 mg Figure 3.1. Diagram of isolation of substances from DCM extract of G. cowa latex G. cowa latex (3.0 kg) is a brown solid, which, after being purchased, is crushed into small lumps and dried in an oven at 45°C for three days to remove moisture. The result was 2.8 kg of dry plastic. Extracted dried G. cowa latex in MeOH (3 L x 3 times) at room temperature using conventional ultrasound-assisted technique for two days. Perform the extraction again 3 times, each time 3 L MeOH. The extract is filtered through a filter paper, collected and stored at low pressure solvent to obtain 500 g of total residue in the form of black brown resin. The total residue extracted with DCM solvent (500 mL x 3) at room temperature combined with ultrasound obtained 96.7 g of DCM residue and the insoluble residue was MeOH residue. 3.2. Isolation of substances from G. hanburyi plant
6 3.2.1. Isolation of substances from stem materials The material obtained from G. hanburyi (2.5 kg) stems were cylindrical, straight or crooked segments of 10-30 cm long, 0.5-1.0 cm in diameter. The collected materials were cut into small pieces, dried for three days in an oven at a temperature of 45oC to completely remove water, and obtained 2.1 kg of dry material. Then the material is ground into powder, extracted with MeOH (3 L × 3) at room temperature using conventional ultrasound- assisted technique at 40ºC. The extract is filtered and collected and then vacuum-distilled at low pressure to obtain 325.0 g of total MeOH residue in dark brown resin. This residue is dissolved and extracted with DCM (500 mL × 3). After evaporation to remove solvents at low pressure, DCM extraction residue (71.9 g) was obtained, the remaining insoluble in DCM was MeOH residue. G. hanburyi sterm barks (2,5 kg) Cut int small pieces, dry at 45oC in 3 days Dried G. hanburyi sterm barks (2,1 kg) Extracted with MeOH (3 L x 3 times) at room temperature using conventional ultrasound-assisted technique at 40ºC Residue (325 g) Extracted with DCM (500 mL x 3 times) DCM residue (71,9 g) MeOH residue CC-SiO2, n-hexane-EtOAc (100:0 to 3:1, v/v), DCM-EtOAc (15:1 to 3:1, v/v) and DCM-MeOH (9:1 to 1:2, v/v)) GHT1 GHT2 GHT3 GHT4 GHT5 GHT6 GHT7 GHT8 3,4 g 11,9 g 7,4 g 7,5 g 9,5 g GH6 GH1 GH8 30 GH3 10 GH2 30 mg 820 mg mg mg 470 mg Figure 3.2. Diagram of isolation of substances from DCM extracts of G. hanburyi stem barks 3.1.2. Isolation of substances from resin of Garcinia hanburyi The resin is in the form of pale yellow aqueous suspension, weighing 500 g. The resin was added to a flask, acetone was added and concentrated in vacuo to remove the water from the sample, resulting in 356.0 grams of dry resin. Extracted the dry resin in MeOH (3 L x 3 times) at room temperature using conventional ultrasound- assisted technique. Extracts were filtered through a filter paper, collected and distilled the solvent at low pressure
7 to obtain 257.0 g of total residue in the form of a yellow-brown resin. The total residue extracted with DCM solvent (500 mL x 3) at room temperature combined with ultrasound obtained 89.0 g of DCM residue and the remaining insoluble in DCM was MeOH residue. Liquid G. hanburyi resin (400 g) Add acetone, concentrated in vacuo Dried G. hanburyi resin (356 g) Extracted with MeOH (3 L x 3 times) at room temperature using conventional ultrasound-assisted technique Residue (257 g) Extracted with DCM (500 mL x 3 lần) DCM residue (89,0 MeOH residue g)CC-SiO2, n-hexane-EtOAc (100:0 to 3:1, v/v), DCM-EtOAc (15:1 to 3:1, v/v) and DCM-MeOH (9:1 to 1:2, v/v)) GHN4 GHN6 GHN8 GHN10 23,4 g 29,6 g 10,8 g 15,1 g GH5 GH7 GH1 GH2 GH4 38 mg 300 mg 930 mg 270 mg 750 mg Figure 3.3. Diagram of isolation of substances from DCM extract of G. hanburyi latex 3.3. Synthesis of GA derivatives 3.3.1. Investigation of thermodynamic and kinetic properties of gambogic acid in the state of the mirror and in the state of super-cold solution Before conducting the fusion, the thermodynamic and kinetic properties of gambogic acid in the glass state and the supercooled state were investigated according to the method described in section 2.2.3 at the Institute of Physics, University of Silesia, Poland aims to evaluate the response of GA to the properties of the active ingredient that can be used as a medicine. The thermodynamic properties of gambogic acid were measured on a Mettler-Toledo differential scanning calorimeter using 1 STARe software. The instrument is equipped with a ceramic sensor with 120 thermocouples (thermocouples) and a cooling system using liquid nitrogen. The instrument is calibrated for temperature and entanpi using standard indium and zinc. Samples were examined in aluminum crucibles, 40 µL in size. All measurements were made in a temperature range of 273-373 K with a heating rate of 10 K / min. The wide-spectrum dielectric spectrum (BDS) of gambogic acid was measured on a Novo-Control GmbH Alpha high-performance frequency analyzer operating in the frequency range 10−1 to 106 Hz and within the temperature range 153-411 K The Quattro thermal controller can control the heating process with an error of less
8 than 0.1 K. The diameter of the samples is 15 mm and the distance between the glass state gambogic acid molecules is 0.1 mm. 3.3.2. Synthesis of GA derivatives The synthesis reactions of GA ester and amide derivatives are carried out according to the figure 3.4 diagram between GA and R-H agent, alcohol or amine, using DDC / DMAP catalysts to activate the acid group. The alcohols involved in the reaction include methanol and ethanol; The amines involved in the reaction include diallylamine, piperidine, morpholine, 1- (4-trifluoromethyl-phenyl) -piperazine, 1- (2,5-difluoro-benzyl) - piperazine, thiophene-2-ethylamine, furfurylamine. O O 30 29 HO 34 33 35 R 28 24 27 25 O 32 31 26 O 23 19 22 40 37 20 17 13 O O O R-H 38 O 18 16 O 14 O 36 2 12 21 DCC/DMAP 39 3 9 11 5 7 8 4 6 10 OH O OH O Figure 3.4. GA ester / amide derivative scheme 3.3.2.1. Synthesis of GA ester derivatives Mixture of GA (100 mg; 0.16 mmol), DMAP (2,925 mg; 0.024 mmol), DCC (49.5 mg; 0.24 mmol) and MeOH or EtOH (1.6 mmol) in THF (3 mL) are stirred at room temperature for 3 h. The reaction solution is poured into water (10 mL), extracted with EtOAc (3 × 10 mL). The organic phases are pooled, anhydrous and concentrated for the raw product. Purify the raw product on a silica gel column (particle size 40-63 μm, column diameter Φ 20 mm, column length L = 50 cm) using the n-hexane-EtOAc dissolution solvent system to obtain two symbol esters. are GA1 and GA2. 3.3.2.2. Synthesis of GA amide derivatives The mixture of GA (100 mg; 0.16 mmol), DMAP (2,925 mg; 0.024 mmol), DCC (49.5 mg; 0.24 mmol) and amine (0.24 mmol) in THF (3 mL) was stir at room temperature for 10-24 h (test by TLC). The reaction solution is poured into water (10 mL) and extracted with EtOAc (3 × 8 mL). The organic phases are pooled, anhydrous and concentrated, and purified on a silica gel column (particle size 40-63 μm, column diameter Φ 20 mm, column length L = 50 cm) using the solute solution n -hexane-EtOAc, the results obtained 6 amide products, denoted GA3-GA8. 3.4. Test the biological activity of substances 3.4.1. Antioxidant activity ABTS and DPPH The compounds GC7-GC16, GH1-GH8 were evaluated for their antioxidant activity ABTS and DPPH according to the method described in section 2.2.3.1, performed at the Institute of Biotechnology - Vietnam Academy of Science and Technology. Male. 3.4.2. Enzyme inhibitory activity α-glucosidase The compounds were assessed for α-glucosidase inhibitory activity according to the method described in section 2.2.3.2, performed at the Department of Applied Biochemistry - Institute of Chemistry - Vietnam Academy of Science and Technology. 3.4.3. Cytotoxic activity in vitro The compounds GC1-GC18, GH1-GH8 were assessed for cytotoxic activity on two cell lines HT-29 and HeLa according to MTT method described in section 2.2.3.3, performed at the Compounds Research Center. nature - Korea Institute of Science and Technology (KIST), Gangneung, Korea. The compounds GA1-GA9 and GA were tested for cytotoxic activity on three cancer cell lines Hep-G2, LU-1 and RD according to the SRB method
9 described in section 2.2.3.3, performed at the delivery room. Experimental Study - Institute of Natural Products Chemistry - Vietnam Academy of Science and Technology. CHAPTER 4. RESULTS AND DISCUSSION This section describes how to determine the structure of isolated and synthesized compounds, the results of investigating some thermodynamic and kinetic properties of gambogic acid and the results of testing the biological activity of the fertilizers. and synthesized 4.1. Research results on the chemical composition of G. cowa plants Research results on the chemical composition of DCM extract of G. cowa latex obtained 18 substances, including 17 xanthone: cowaxanthone IK (GC1-GC3), norcowanol AB (GC4-GC5), garcinone F (GC6), fuscaxanthone A (GC7), 7-O-methylgarcinone E (GC8), cowagarcinone A (GC9), cowaxanthone (GC10), rubraxanthone (GC11), cowanin (GC12), norcowanin (GC13), cowanol (GC14), kaennacowanol A (GC15), garcinone D (GC16), fuscaxanthone I (GC17) and 01 tocotrienol compound: parvifoliol F (GC18). Of which, 06 GC1-GC6 compounds were identified as new compounds. The structure of the compounds is shown below: R6 O OH 9' 8' 8 1 R5 8a 9a R1 9 A B C 5a O 4a OH R4 5 4 10' R3 R2 R1 R2 R3 R4 R5 R6 1' O OH 1 7'-OH-Ge H H OH OCH3 H 8 1 10 MeO 11 7'-OH-Ge H H OH OH H 9 2 13 3 H 7'-OH-Ge H H OH H HO 5 O 4 O 4 4'-OH-Pr H H OH OH 7''-OH-Ge 14 7 5 3'-OH-Pr H H OH OH 7''-OH-Ge 6 3'-OH-Pr H H OH OCH3 3''-OH-Pr 8 prenyl H prenyl OH OCH3 prenyl 9 prenyl H prenyl OH OCH3 geranyl 26 5 4 geranyl H OH OCH3 H 4a 24 23 21 10 H 6 25 11 H H H OH OCH3 geranyl 12 16 20 8a O 2 22 12 prenyl H H OH OCH3 geranyl 8 9 11 15 19 prenyl H H OH OH geranyl OH 13 18 14 4'-OH-Pr H H OH OCH3 geranyl 15 4'-OH-Pr H H OH OCH3 7''-OH-Ge 16 prenyl H H OH OCH3 3''-OH-Pr 17 4'-OH-Pr H H OH OCH3 3''-OH-Ge 4' 10' 8' CH2OH 1' OH 1' 7'-OH-Ge = 9' 4'-OH-Pr = 5' 10'' 8'' 4' 1'' OH 1' OH 7''-OH-Ge = 9'' 3'-OH-Pr = 5' 10'' 4'' 8'' 1'' OH 1'' OH 3''-OH-Ge = 9'' 3''-OH-Pr = 5'' Figure 4.1. Structure of GCx compounds (x = 1-18) isolated from G. cowa plant All the obtained xanthone were substituted tetraoxygen xanthone with oxygen carrying positions C-1, C- 3, C-6 and C-7, except for GC3 compound which is potential trioxygen. On 1H NMR spectrum of isolated xanthone, characteristic signals of 1-OH phenol group associated with carbonyl group at δH 13.00-14.00 except for compounds measured in CD3OD solvent. The signal of aromatic protons in the weak field is the signal of protons belonging to the xanthone framework, in which the H-8 proton is influenced by the electron attraction effect of the conjugated carbonyl group at C-9, so it usually appears in the weaker field. with the signal of the remaining aromatic protons with a chemical shift of δH 7.45-7.53. The signal of proton H-2, H-4, H-6 usually appears at δH 6.19-6.33; meanwhile, the signal of proton H-5 usually appears in the weaker field than δH 6.68-6.86.
10 Particularly, GC3 compound with H-5 and H-6 signals appeared in very weak fields at δH 7.41 and 7.28, respectively, close to the displacement signal of H-8 at δH 7.51. On the 13C NMR spectrum of the compounds isolated, there appeared characteristic carbon signals of xanthone frame containing 1-3 prenyl or geranyl substituents. The results of synthesis of carbon signals in xanthone frame of GC1-GC17 compounds are summarized in Table 4.1 below. Table 4.1. Signals of the displacement of carbon in the xanthone frame δCaC Position C-C or C- Note C-O C-H prenyl/geranyl 1 160.0-162.7 - - GC7: δC 158.0 (C-O) 2 - 98.3-98.4 104.5-112.2 3 161.5-164.5 - - GC7: δC 159.9 (C-O) 4 - 92.2-93.9 107.8 4a 155.1-157.1 - - 5 - 100.9-103.9 111.4-113.9 GC3: δC 119.8 (C-H) 5a 152.9-157.9 - - 6 152.3-157.8 125.3 - GC3: δC 125.3 (C-H) 7 142.6-147.2 - - GC3: δC 151.4 (C-O) 8 - 105.0-109.4 129.2-139.8 8a - - 109.5-113.8 GC3: δC 121.9 (C-C) 9 179.9-183.5 - - 9a - - 101.7-103.9 a Measured in CDCl3, c125 MHz. The structure of substances was determined based on the NMR, HRESIMS spectral data combined with comparison with the published compounds in the reference. The results determined the structure of 17 xanthone, including 06 new xanthone: cowaxanthone IK (GC1-GC3), norcowanol AB (GC4-GC5), garcinone F (GC6) and 03 compounds were isolated for the first time from G. cowa plants: garcinone D (GC16), fuscaxanthone I (GC17) and parvifoliol F (GC18). The following presents the results of structural elucidation of two compounds GC1 and GC4. 4.1.1. GC1 Compound: Cowaxanthone I (New Compound) GC1 compounds are isolated in the form of yellow iridescent needles with a melting point of 204-205 oC. On the HRESIMS spectrum (Figure 4.2), the protonated molecular ion peak [M + H] + at m / z 429,1907 (theoretical calculation for C24H29O7 is 429,1908), so the CTPT of GC1 is determined as C24H28O7. Spectra 1H and 13C NMR of GC1 appeared signals suggesting GC1 has the structure of a monogeranylated xanthone. In the low field on 1H NMR spectrum there are resonance signals of three aromatic protons at δH 7.50 (1H; s; H-8); 6.80 (1H; s; H-5) and 6.32 (1H; s; H-4). There was also a signal of a methoxy group oscillating at δH 3.96 (3H; s; 7-OCH3) and a hydrated geranyl group (figure 4.3). On 13C NMR spectrum there are signals of 15 Csp2 with specific signals for xanthone frame. That is the signal of a carbonyl group at δC 181.0 (C-9) and the signal of a phenolic carbon conjugated to the carbonyl group at δC 161.1 (C-1). 13C NMR spectra also showed the signal of 6 aromatic carbon attached to oxygen at δC 161.1 (C-1); 164.1 (C-3); 157.2 (C-4a); 155.7 (C-5a); 153.9 (C-6) and 147.2 (C-7). The signal of methoxy carbon appears at δC 56,7 (7-OCH3) and the signal of a third Csp3 linked to oxygen at δC 71.5 (C-7 ') (Figure 4.4).
11 Figure 4.3. 1H NMR spectra of GC1 compound Figure 4.4. 13C NMR spectra of GC1 compound The aromatic proton signal at low field δH 7.50 is attributable to H-8 due to the electron attraction effect of the conjugated carbonyl group at C-9. HMBC spectrum also shows interaction of H-8 with C-9, C-8a (δC 113,6) and C-7. The methoxy group is attributed to the C-7 position by the HMBC interaction of the methoxy group proton and H-8 with C-7. The remaining two aromatic protons are attributed to H-5 (δH 6.80) and H-4 (δH 6,32) due to HMBC interaction of H-5 proton with C-9, C-7, C- 6 and the proton H-4 with C-9, C-2 (δC 111,8), C-3. The presence of the hydrated geranyl group was determined by signals on the spectrum 1H, 13C NMR, HSQC and HMBC. On the HMBC spectrum appears interactions of proton H-1 '(δH 3.33) with phenolic carbon C-1 and with C-2, C-3; interaction of proton alken at δH 5.27 (1H; t; 6.0; H-2 ') with two methylene carbon C-1' (δC 22,1), C-4 '(δC 41 , 3) and a C-10 'methyl carbon (δC 16,1); interaction of the H-4 'methylene proton (δH 1.98) with a quaternary alken carbon C-3' (δC 135.6) and two carbon methylene C-5 '(δC 23,7), C-6 '(δC 44,3). The position of the hydroxy group on the geranyl group was determined at C-7 'due to the HMBC interaction of the two methylene proton groups H-5', -6 '(δH 1.47 and 1.40, respectively) and the proton of the two. methyl group H-8 ', -9' (δH 1.15) with C-7 '. The spectral data of GC1 are given in Table 4.2, the molecular structure and the main interactions on the HMBC spectrum of GC1 compounds are shown in Figure 4.5. 10' 9' H O OH O OH OH 8 1 1' 3' 5' 7' OH H3CO H3CO 7 9a 8a9 2 2' 4' 6' 8' 5a 3 HO 6 O 4a OH HO O OH 5 4 H H Figure 4.5. Chemical structure and main HMBC interaction of GC1 compound Table 4.2. NMR spectral data of GC1 and GC2 compounds Position GC1 GC2 H (mult; J) ab C C a HMBC (HC) H (mult; J) ab CaC HMBC (HC) 1 161,1 161,1 2 111,8 111,6 3 164,1 164,0 4 6,32 (s) 94,0 2, 3, 4a, 9a, 9 6,33 (s) 93,9 2, 3, 4a, 9a, 9 4a 157,2 157,2 5 6,80 (s) 103,7 5a, 8a, 7, 6, 9 6,79 (s) 103,4 5a, 8a, 7, 6, 9 5a 155,7 155,3 6 153,9 153,2 7 147,2 144,7 8 7,50 (s) 105,7 5, 5a, 6, 7, 8a, 9 7,45 (s) 109,1 5, 5a, 6, 7, 9 8a 113,6 113,8 9 181,0 181,1 9a 103,2 103,2 1’ 3,33 (m) 22,1 3, 1, 3’, 2’, 2 3,33 (m) 22,1 3, 1, 3’, 2’, 2
12 2’ 5,27 (t; 6,0) 123,8 1’, 4’, 10’ 5,27 (t; 7,0) 123,8 1’, 4’, 10’ 3’ 135,6 135,6 4’ 1,98 (m) 41,3 2’, 3’, 5’, 6’, 10’ 1,98 (t; 7,0) 41,3 2’, 3’, 5’, 6’, 10’ 5’ 1,47 (m) 23,7 3’, 7’, 6’, 4’ 1,47 (m) 23,6 3’, 7’, 6’, 4’ 6’ 1,40 (m) 44,3 5’, 8’, 9’, 4’, 7’ 1,39 (m) 44,2 5’, 8’, 9’, 4’, 7’ 7’ 71,5 71,5 8’ 1,15 (s) 29,2 6’, 7’, 9’ 1,15 (s) 29,1 6’, 7’, 9’ 9’ 1,15 (s) 29,2 6’, 7’, 8’ 1,15 (s) 29,1 6’, 7’, 8’ 10’ 1,80 s) 16,1 2’, 3’, 4’ 1,79 (s) 16,1 2’, 3’, 4’ OCH3 3,96 (s) 56,7 7 - - - a Measured in CD3OD, b 500 MHz, C 125 MHz Based on the analysis of HRESIMS spectrum and 1D, 2D NMR spectra of GC1 compound, we determined GC1 to be 1,3,6-trihydroxy-7-methoxy-2- (7-hydroxy-3,7-dimethyloct- 2-enyl) xanthone. This is a new compound, isolated from nature for the first time and named cowaxanthone I. 4.1.2. GC4 Compound: Norcowanol A (New Compound) GC4 compounds isolated as pale yellow powder. On the HRESIMS spectrum (Figure 4.14) appears protonated molecular ion peak [M + H] + at m/z 499,2324 (theoretical calculation for CTPT C28H35O8 is 499,2326), so the CTPT of GC4 is determined. is C28H34O8. Figure 4.15. 1H NMR spectra of GC4 compound Figure 4.16. 13C NMR spectra of GC4 compound 1 13 GC4's H and C NMR spectra showed signals that suggest the GC4 has the structure of a xanthone containing a hydrated geranyl group and a hydrated prenyl group. In the low field on the spectrum 1H and 13C NMR there are resonance signals of two aromatic CH groups at δH 6.27 (1H; s; H-4) / δC 93.1 and 6.69 (1H; s; H- 5) / δC 101.0. The signals of two doublet methylene groups appear at δH 3.41 (2H; d; 7.5; H-1 ') / δC 21.8 and 4.15 (2H; d; 6.5; H -1”) / δC 26.5 and singlet signal of 4 methyl groups suggest the existence of two substituent prenyl or geranyl groups on xanthone frame. The geranyl group was identified as 7-hydroxy-3,7-dimethyloct-2-enyl group based on NMR spectrum and HC interactions on HSQC and HMBC spectrum, in particular the signals of two methylene groups have the same displacement. chemically metabolized at δH 1.12 (3H; s; H-8 ", -9") / δC 29.1 and interacted on the HMBC spectrum of these two methyl groups with a tertiary Csp3 binding to oxygen at δC 71.5 (C-7”). There is also an HC interaction on the HMBC spectrum of the H-1 proton H-1” with the two C-2” carbon alkanes (δC 125,0), C-3” (δC 135,5) and the interaction of the alpha-alpha proton. -2” with 2 carbon methylene C-1”, C-4” (δC 41,3) and 1 methyl carbon C-10” (δC 16,5). The low field shift signal of the CH2-1” methylene group suggests that the geranyl group binds to C-8. On the HMBC spectrum also appears the interaction of H-1” with the carbon of the xanthone frame, namely C-7 (δC 142,5), C-8 (δC 129,2) and C-8a (δC 112, first). The prenyl group was defined as a 4-hydroxy-3-methylbut-2-enyl group based on the HMBC interaction of the proton H-1 'with C-2' carbon (δC 126.8) and C-3 '(δ C 135,1) and interaction of the singlet methylene proton binding to oxygen at δH 4.33 (3H; s; H-4 ') / δC 61.8 with C-2', C-3 carbon 'and C-5' (δC 21,7). The position of the prenyl group was determined at C-2 due to the HMBC interaction of H-1 'with C-1 (δC 161,5), C-2 (δC 110,3) and C-3 (δC 163.2). HMBC interactions of proton H-4 with C-2, C-3, C-4a (δC 156,4), C-9a (δC 103,9), C-9 (δC 183,
13 5) and the interaction of proton H-5 with carbon C-8a, C-7, C-6 and C-9 allows to locate the aromatic protons in the xanthone framework. The chemical structure and the main HMBC interactions of GC4 are presented below, the spectral data of GC4 compounds are presented in Table 4.4. 9'' OH 8'' OH 7'' 6'' 5'' 10'' 4'' 3'' OH OH 2'' 1'' 4' O OH O OH 8 1 1' 3' HO HO 7 9 9a 8a 2 2' 5' 5a 3 HO 6 O 4a OH HO O OH 5 4 H H Figure 4.17. Chemical structure and main HMBC interaction of GC4 compound The results of spectroscopic analysis of GC4 compounds showed that the structure of the compound almost coincided with the kaennacowanol A compound isolated from G. cowa [141], except for the signal dilatexpearance of the methoxy group. On the basis of analysis of HRESIMS spectrum and 1D, 2D NMR spectra of GC4 compound, GC4 compound was identified as 1,3,6,7-tetrahydroxy-2- (4-hydroxy-3-methylbut-2-enyl ) - 8- (7-hydroxy-3,7-dimethyloct-2-enyl) xanthone. This is a new compound, isolated from nature for the first time and named norcowanol A. 4.2. Research results on the chemical composition of G. hanburyi plants Research results on the chemical composition of DCM extract of G. hanburyi stem and resin obtained 8 caged xanthones GH1-GH8. The structures of the compounds are shown below. On the NMR spectrum of the compounds GH1- GH8 appeared specific signals for potential polyprenyl xanthone compounds with 4-oxotricyclo cage [4.3.1.03,7] dec-8-en-2-one - a type xanthone frames are common in G. hanburyi trees. There are also signals characteristic of a pyrano ring formed by the reaction of the –OH group and the geranyl group. R1 R2 CHO 24 25 O 23 O 19 22 20 13 O 18 1716 O 14 O O O O R3 2 A 21 D C B 12 3 9 11 5 7 8 4 6 10 OH O OH O O R1 R2 R3 R 7 R = CH3 1 COOH CH3 isoprenyl 8 R = n-butyl 2 CH3 COOH isoprenyl 3 COOH CH3 H 4 CH3 COOH H isoprenyl = 5 CH3 CHO H 6 CH3 CH3 H Figure 4.61. Chemical structure of the compounds GHx (x = 1-8) isolated from G. hanburyi resin and sterm barks 1 H NMR spectra of the caged xanthone frame of the compounds GH1-GH8 showed that the equivalent proton signals had similar shifts. It is the singlet signal of the phenolic hydroxy proton conjugate with the carbonyl group at δH 12.70-13.00 (6-OH). The weak field proton doublet signal represents the signal of proton olefin conjugate with carbonyl group at δH 7,55-7,57 (d; 6,5-7,0; H-10). Proton signaling group characteristic for cage structure includes the signal of a methylene group appearing at δH 2.31 (1H; dd; 13,0; 5.0; H-21); 1,34-1,36 (1H; overlap; H- 21); one proton methine at δH 2.51 (1H; d; 9.5; H-22) and one proton methine at δH 3.47 (1H; m; H-11) for xanthone carrying a 4-oxotricyclo cage [4.3.1.03,7] dec-8-en-2-one or at δH 2.81-2.89 for xanthone with 4-oxotricyclo cage frame [4.3.1.03,7] decan-2-one . The signal of a pair of proton doublet has separation constant J = 10.0 at δH 6.61- 6.66 (H-4) and 5.38-5.54 (H-3) characterizing the double bond of pyrano ring (ring D) (table 4.17). Table 4.17. Signals of displacement of protons and carbon in the cage xanthone
14 Position Xanthone with caged frame 4- Xanthone with caged frame 4- oxotricyclo[4.3.1.03,7]dec-8-en-2-one oxotricyclo[4.3.1.03,7]decan-2-one Hab Cac Hab Cac 2 78,4-81,3 78,4-81,3 3 5,38-5,54 (d; 10,0) 124,5-126,5 5,38-5,54 (d; 10,0) 124,5-126,5 4 6,61-6,66 (d; 10,0) 115,3-115,9 6,61-6,66 (d; 10,0) 115,3-115,9 5 102,8-103,3 102,8-103,3 6 - 157,3 -157,8 - 157,3 -157,8 7 100,4-100,6 100,4-100,6 8 178,8-179,0 178,8-179,0 9 133,2-133,8 3,07-3,18 (m) 48,5-48,6 10 7,55-7,57 (d; 6,0-6,5) 134,9-135,6 4,37-4,42 (dd; 4,5; 72,3-74,1 1,5) 11 3,47 46,8-47,0 2,81-2,89 43,7-44,2 12 202,4-203,5 208,1-208,4 13 83,7-84,7 86,0-86,4 14 90,5-90,9 88,4-88,4 16 157,3-157,7 155,5-155,7 17 107,6-108,3 107,6-108,3 18 160,9-161,5 160,9-161,5 21 2,31 (dd; 13,0; 5,0); 1,34- 25,2-25,5 2,31 (dd; 13,0; 5,0); 20,0 1,36 (overlap) 1,34-1,36 (overlap) 22 2,51 (d; 9,5) 49,0-49,2 2,51 (d; 9,5) 43,6 23 83,2-84,1 82,1-82,4 24 1,69 (s) 29,7-30,1 1,69 (s) 29,7-30,1 25 1,29 (s) 27,2-29,1 1,29 (s) 27,2-29,1 a Measured in CDCl3, b 500 MHz, c 125 MHz. Signals of carbon displacement at the same position in cage-bearing xanthone with similar structure (6 xanthone with 4-oxotricyclo cage frame [4.3.1.03,7] dec-8-en-2-one GH1- GH6 and two xanthone carrying 4- oxotricyclo cage frame [4.3.1.03,7] decan-2-one GH7-GH8) almost coincide. In addition, it can be observed that when the cage xanthone frame is oxidized to a 4-oxotricyclo [4.3.1.03,7] decan-2-one frame, the whole signal of carbon in the cage structure is shifted. The structure of substances was determined based on the associated NMR spectroscopy compared with the compounds published in the references. The results have isolated and determined structure of 08 caged xanthone, including gambogic acid (GH1), isogambogic acid (GH2), morellic acid (GH3), isomorellic acid (GH4), isomorellin (GH5), desoxymorellin (GH6), isomoreollin B (GH7) and 10α- butoxygambogic acid (GH8). The analysis results of spectral data of gambogic acid are presented below: 4.2.1. GH1 compound: Gambogic acid The compound GH1 was isolated from the resin extract and the stem of G. hanburyi tree branches in the form of an orange amorphous powder, polar angle of rotation [α] = -578o (c 0.201; CHCl3). Spectra 1H, 13C NMR and HSQC of GH1 allowed to determine the signals of 44 protons and 38 carbons, including 1 -OH group at δH 12,77; 8 methyl groups; 5 methylene groups; 6 groups -CH sp2; 2 groups of methine; 3 carbonyl carbon; 10 Csp2 does not contain C-H bonds, of which 3 carbons is bound to oxygen; 3 Csp3 of grade 4 associated with oxygen.
15 29 30 HOOC HOOC 34 35 28 24 33 27 25 O O COSY 32 31 26 23 19 H 13 22 40 37 20 17 O O O 38 O 18 16 O 14 O 36 2 D C B A 12 21 HMBC 39 3 9 11 H 5 7 8 H 4 6 10 H O O H OH O H Figure 4.62. Chemical structure and COSY interaction, HMBC of compound GH1 Figure 4.63. 1H NMR spectra of the compound GH1 Figure 4.64. 13C NMR spectra of the compound GH1 Interactions on COSY, HSQC and HMBC spectrum showed that GH1 has structural fragments including: three prenyl groups, one of which contains a COOH group; a double coupling CH = CH; a spin system CHsp2- CHsp3-CH2-CHsp3 (Figure 4.62). These data suggest that GH1 has the structure of a substitute polyprenyl xanthone. On the HMBC spectrum, the interaction of the double-bonded proton appears at δH 5.38 (d; 10.0; H-3) and 6.60 (d; 10.0; H-4) with the quaternary sp3 carbon at δ C 81.3 (C-2). HMBC interactions between methylene protons at δH 1,76 (1H; overlap; H-20); 1.59 (1H; m; H-20) and 2.01 (2H; m; H-36) with C-2 (δC 81,3) help confirm the structural part related to D-ring of xanthone frame cage. Interactions between olefin singlet proton at low field δH 7.55 (1H; d; 7.0; H-10) with carbon include: 2 carbonyl carbon at δC 178.9 (C-8) and 203, 3 (C-12); carbon sp3 at δC 46.8 (C-11) and the HMBC interaction between protons at δH 2.51 (1H; d; 9.0; H-22) with (C-14) and (C-23) help make Unravel the structure related to the A ring in the cage xanthone frame. HMBC interaction between methylene protons of 3-carboxylbut-2-enyl groups at δH 2.95 (2H; d; 7.0; H-26) with carbon C-12, C-13, C-14 shows position of this group on the cage xanthone frame (figure 4.70). This suggests that the chemical structure of GH1 could be acid (Z) -4 - ((2R, 11S, 13R, 14S, 23S) -6-hydroxy-2,23,23-trimethyl-17- (3- methylbut-2-en-1- yl) -2- (4-methylpent-3-en-1-yl) -8,12-dioxo-2,8,11,12,13,23-hexahydro-7H, 4H -11,22-methanofuro [3,2-g] pyrano [3,2-b] xanthen-3a-yl) -2-methylbut-2-enoic (gambogic acid) conforms to molecular formula C38H44O8. Combining analysis of COSY, HSQC and HMBC spectra, we assigned the remaining carbon and proton signals. Results of NMR spectroscopy analysis and comparison with spectral data of GH1 with gambogic acid in the reference [36] are summarized in Table 4.18, showing that the spectral data completely coincides. Therefore, we conclude that the compound GH1 is gambogic acid. 4.3. Results of GA derivative synthesis 4.3.1. Results of molecular recovery in the glass state and supercooler state of amorphous gambogic acid Among the morphological forms of the active substance, the amorphous form is of more interest than the crystalline form because of its better solubility in water and higher biological activity. The amorphous form of a material is created by rapidly cooling the drug to avoid crystallization after melting it at its melting point. The molecular movement in amorphous materials is characterized by the time for α structure recovery in the supercooler and glass states. These materials have an out-of-order and temperature dependent structure, and at high temperature amorphous materials have liquid-like properties but at low temperature molecular recovery takes place slowly. these materials are much more like solids. The investigation of molecular recovery in the glass state
16 and supercooling state of GA is to evaluate the potential of GA medicinal use. The thermal properties of GA have been investigated on the basis of differential scanning calorimetry (DSC) in the temperature range from 273-373 K with the temperature rise rate of 10 K / min. The results have determined that the glass transition temperature of GA is Tg = 338K (Figure 4.87). Figure 4.87. DSC spectrum of a) GA increment; b) GA after being heated at 373 K for 3 minutes To investigate the molecular dynamics of the amorphous GA, wide-field dielectric spectroscopy (BDS) was measured in a wide frequency range from 10-1 to 106. During the measurement, the temperature increased from 153 to 333 K. with heating rates of 10 K / min and from 333 to 411 K with heating rates of 2 K / min. The GA BDS broadband dielectric spectrum from the supercooler and the glass form is shown in Figure 4.88 below. Figure 4.88. GA's broadband dielectric spectrum is at a) higher than the mirror transfer temperature and b) lower than the mirror transfer temperature. On the broadband dielectric spectrum of GA at a temperature lower than the glass phase transition temperature, two secondary molecular recovery processes β and can be observed in conjunction with the intermolecular motion of GA. Meanwhile, on the BDS spectrum at a temperature higher than the glass transfer temperature, a peak appears corresponding to the recovery of α structure and dc conductivity. GA's molecular recovery processes shift towards higher frequencies with increasing temperature, showing an increase in the degree of molecular motion with increasing temperature. By combining the experimental data measured in the BDS spectrum, combined with the Vogel – Fulcher – Tammann equation (VFT), the glass transition temperature Tg = 333 K (determined at the temperature at which the recovery time is is equal to 100 s). This result is deviated from the DSC method, but this error is normal and acceptable. Theoretical calculation results on the basis of the dependence of α structural recovery time on τα temperature (T = 300 K) also show that GA can exist in a dynamic stable state in about 2, 31,109 days. This proves that GA is quite durable and can be stored at room temperature. In addition, based on the VFT equation, the material brittleness of GA is mp = 103 (common substances mp = 16-200 [199]). When the brittleness is between 16 and 30, eg glass (SiO2) the material is considered very hard. With a material brittleness greater than 100, the material is considered very brittle. Between 30 and 100, the brittleness is medium. Hence, the GA in the supercooled state could be classified as a brittle material. Calculation results on the ECNLE software obtained the glass transfer temperature Tg = 338 K with the heating rate of 10 K / min completely consistent with the experiment. In addition, the calculation results also show that the process is closely related to molecular recovery and the kinetic formation of individual molecules, this process is also known as Johari – Goldstein recovery. . Thus, the large kinetic stability time and relative material
17 brittleness properties of GA show that GA can meet the physical requirements of an active substance with medicinal potential. This is an important basis to conduct GA derivative fusion reactions to obtain highly active derivatives with potential for practical application. 4.3.2. Research orientation HOOC O O O O OH O Figure 4.89. Chemical and crystalline structure of GA The crystal structure of GA shows that the xanthone ring structure is on one plane and has two different upper and lower sides. The two prenyl groups and the polycyclic ring are located above, forming the hydrophobic face, while the carboxylic acid group and the carbonyl group of the lower polycyclic ring form the hydrophilic face (figure 4.89). The results of the carboxylic group transformation suggest that the hydrophilic plane does not play an important role in binding to its biological target. The carboxyl -COOH group can be converted to other functional groups such as ester, amide or other base group without much influence on apoptosis activity. The structural-activity (SAR) studies of GA have shown the importance of coupling on the D ring (conjugation with C = O group of ring C) for activity. The derivatives generated from 6-OH group (B-ring) metabolism such as methylation or acylation have similar activity as the primary agent. Therefore, this 6-OH group does not play a decisive role in activity. From the above results of SAR analysis of GA, we selected to synthesize some GA derivatives by converting carboxylic acid group to ester and amide form with the aim of preserving active structural parts of GA. The metabolic reactions use the DCC / DMAP catalyst to activate the acid group. 4.3.3. Results synthesized derivatives The transformation of the COOH group of GA is done according to the diagram in Figure 3.4. The structure of the products and their reaction efficiency are presented in Table 4.26. Table 4.26. The structure of products and the yield of the reactions Symbol R Yield (%) Physical state Weight (mg) Note GA1 -OCH 3 91 Yellow oil 220 GA2 -OC2H5 75 Yellow oil 175 GA3 70 Yellow oil 250 New compound N GA4 Yellow oil N 84 233 O GA5 Yellow oil New compound N 79 189 GA6 N N CF3 51 Yellow oil 140 New compound F GA7 Yellow oil New compound N N 68 126 F GA8 N 15 Yellow oil 52 New compound S H Gambogic acid metabolism results obtained 08 derivatives, of which 02 ester derivatives are methyl gambogate (GA1), ethyl gambogate (GA2) and 06 amide derivatives are N, N-diallylgambogamide (GA3), N- piperidinylgambogamide. (GA4), N-morpholinegambogamide (GA5), 1 (4-trifluoromethylbenzene-piperazinyl) gambogamide (GA6), 1- (2,5-difluorobenzyl) piperazinylgambogamide (GA7) and N- (2-thiophen-2-yl)
18 ethylgambogamide (GA8). In which, 05 derivatives N, N-diallylgambogamide (GA3), N- morpholinegambogamide (GA5), 1 (4-trifluoromethylbenzene-piperazinyl) gambogamide (GA6), 1- (2,5- difluorobenzyl) piperazinylgambogamide (GA7) and N- (2-thiophen-2-yl) ethylgambogamide (GA8) are new compounds. The structure of the synthetic products is determined by one-dimensional and two-dimensional NMR spectra. The clean compounds GA1-GA5 have been subjected to high resolution spectroscopy. The analytical results of spectral data of compounds GA1 and GA4 are presented below: 4.3.3.1. GA3 compounds: N, N-diallyl gambogamide On the HRESIMS spectrum of compound GA3 appeared protonated molecular peak [M + H] + at m / z 708,3883 (calculated for CTPT C44H54NO7 is 708,3900). Therefore, the CTPT of the GA3 compound is C44H53NO7. Spectrum 1H, 13C NMR and HSQC of GA3 showed proton and carbon signals corresponding to allyl group at δH 5,61 (2H; m) / δC 133,6; 132.8 (2CH = allyl); δH 5.09-5.02 (4H; m) / δC 117.6 (2CH2 = allyl); δH 3.88 (2H; m) / δC 45.5 (CH2 allyl); δH 3.71; 3.61 (2H; dd; 16.0; 5.5) / δC 49.5 (CH2 allyl). The analysis results on the COSY spectrum did not show any interaction of allyl protons with protons of GA. 30 28 34 35 24 O 33 27 29 25 O 31 N 19 32 23 26 40 37 20 17 13 38 O 18 16 O 14 O 2 22 12 36 21 39 3 9 11 5 7 8 4 6 10 OH O Figure 4.96. Chemical structure of GA3 compound The chemical shift of protons and carbon of other positions in the GA frame is almost unchanged. However, there are some changes related to proton and carbon at positions 26, 27, 28. Specifically: proton signal H-26 is split into two peaks at δH 2,22 (1H; dd; 15,0; 6.0); 2.42 (1H; dd; 15.0; 7.0); proton signals H-26, -27 were translated to higher field than GA (GH1: δH 2.95 (H-26), δH 6.09 (H-27); GA3: δH 2.42; 2, 22 (H-26), δH 5,43 (H- 27)); the C-27 carbon signal (δC 122.3) is shifted towards the higher field, while the C-28 (δC 133.9) is shifted to the lower field (GH1: δC 137.8 (C-27)) , δC 127.8 (C-28); GA3: δC 122.3 (C-27), δC 133.8 (C-28). Figure 4.97. 1H NMR spectra of GA3 compound Figure 4.98. 13C NMR spectra of the compound GA3 The results of the separation of the H-26 signal and the displacement of the H-26 and H-27 proton signals towards the higher field can be explained by the remote shielding of the N atom having a large electron density when the two conjugated double bonds C = C and C = O exist in the S-trans structure and due to the bulky structure of diallyl amine. This is probably because the C27 = C28 double-bond has the cis configuration as documented. Then the two protons H-26 could be located differently in space compared to the N atom, so they are no longer equivalent protons, resulting in two signals on the 1H NMR spectrum. Signal cleavage also occurred for the ethyl gambogate ester but not for methyl gambogate. The results of the displacement of C-27 and C-28 can be explained because the N atom has a large electron density, which can cause the conjugation effect with C = O bonds, causing the union of two double bonds. C27=C28 and C=O decrease. The variation of the signals indicated above can be considered as characteristic signals that GA has been esterified or amideized.