Terpenoid in tripterygium wilfordii as well as preparation, application and medicine of terpenoid

Abstract

The invention relates to a diterpenes type compound, a triterpenes type compound, a preparation method of the diterpenes type compound and the triterpenes type compound, and an application of the diterpenes type compound and the triterpenes type compound. The compounds 1 to 10 provided by the invention are novel diterpene and triterpene compounds which are obtained by extracting and separating from a vine shrub plant tripterygium wilfordii Hook.f. Of a winged euonymus tripterygium genus, and the novel diterpene and triterpene compounds are novel in structure, and the compounds 1 to 10 provided by the invention are novel diterpene and triterpene compounds which are obtained by extracting and separating from the vine shrub plant tripterygium wilfordii Hook.f. Of the winged euonymus tripterygium genus. Bioactivity experiments show that the compound 5 has significant ATP citrate lyase (ACLY) inhibitory activity, and has application potential in research and development of drugs for cancer, hyperlipidemia, atherosclerotic cardiovascular diseases and the like.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry and relates to a diterpenoid and triterpenoid compound from Tripterygium wilfordii. More specifically, it relates to the preparation and resolution methods of 10 diterpenoids and triterpenoids, and their use as ACLY-inhibiting active ingredients for the treatment of related diseases such as cancer, hyperlipidemia, and atherosclerotic cardiovascular diseases. In vitro activity studies have shown that compound 5 has significant ACLY-inhibiting activity and can serve as a lead compound for the development of new drugs for treating cancer and cardiovascular diseases. Background Technology

[0002] Tripterygium wilfordii Hook.f., a plant belonging to the genus Tripterygium in the family Celastraceae, has attracted widespread attention due to its diverse active ingredients. Extracts from this plant have been used in traditional medicine to treat various diseases, including inflammation, rheumatoid arthritis, and immune system-related disorders. Active ingredients in Tripterygium wilfordii, such as tripterine and tripterygium glycosides, have been shown to possess anti-inflammatory, immunomodulatory, and antitumor properties [1,2]. In recent years, researchers have conducted in-depth studies on the chemical composition and bioactivity of Tripterygium wilfordii, discovering its potential application value in modern drug development [3,4]. As a plant with a variety of active ingredients, it demonstrates its importance in both traditional and modern medicine and warrants further research and development.

[0003] Adenosine triphosphate-citrate lyase (ACLY) is highly expressed in the liver and adipose tissue of various tumor tissues, and can therefore be used as an intervention target to lower blood lipids and treat cardiovascular diseases. ACLY plays a key role in lipid metabolism, catalyzing the conversion of citrate and coenzyme A (CoA) into oxaloacetate and acetyl-CoA, the latter being important precursors for fatty acid, cholesterol, and protein acetylation [5]. Since many cancer cells rely on ACLY activity for proliferation, ACLY has also become a target for anticancer drugs [5,6]. In addition, ACLY inhibitors have shown promising potential in the treatment of hyperlipidemia and related diseases [7,8]. Studies have shown that ACLY inhibition can effectively reduce blood lipid levels and improve metabolic-related disease states [9,10]. Therefore, therapeutic strategies targeting ACLY may provide new ideas for the development of new drugs for cardiovascular diseases.

[0004] This invention describes a multidimensional liquid chromatography method for the separation of diterpenoids and triterpenoids from Tripterygium wilfordii. Ten terpenoid compounds, including two novel diterpenoids and eight novel triterpenoids, were isolated from the supercritical fluid extract of Tripterygium wilfordii. Pharmacological activity tests showed that compound 5 exhibited significant ACLY inhibitory activity, demonstrating important research value and application potential in drug development for cancer, hyperlipidemia, and atherosclerotic cardiovascular diseases.

[0005] References

[0006] [1]S.Cao,Q.Feng,Asian medicine:exploitation of plants,Science 335(6073)(2012)1168-1169. https: / / doi.org / 10.1126 / science.335.6073.1168-b .

[0007] [2] R.Kouda, F.Yakushiji, Recent Advances in Iridoid Chemistry: Biosynthesis and Chemical Synthesis, ChemAsian J 15(22)(2020)3771-3783. https: / / doi.org / 10.1002 / asia.202001034 .

[0008] [3]SAPirintsos,M.Bariotakis,M.Kampa,G.Sourvinos,C.Lionis,E.Castanas,The Therapeutic Potential of the Essential Oil ofThymbra capitata(L.)Cav.,Origanum dictamnus L.and Salvia fruticosa Mill.And a Case of Plant-Based Pharmaceutical Development,Front Pharmacol 11(2020)522213. https: / / doi.org / 10.3389 / fphar.2020.522213 .

[0009] [4]B.Medeiros-Neves,HFTeixeira,GLvon Poser,The genus Pterocaulon(Asteraceae)-A review on traditional medicinal uses,chemical constituents andbiological properties,Journal ofEthnopharmacology224(2018)451-464. https: / / doi.org / 10.1016 / j.jep.2018.06.012 .

[0010] [5]J.Wei,S.Leit,J.Kuai,E.Therrien,S.Rafi,HJHarwood,Jr.,B.DeLaBarre,L.Tong,An allosteric mechanism for potent inhibition of human ATP-citratelyase,Nature 568(7753)(2019)566-567. https: / / doi.org / 10.1038 / s41586-019-1094- 6 .

[0011] [6]KHGVerschueren,C.Blanchet,J.Felix,A.Dansercoer,D.De Vos,Y.Bloch,J.Van Beeumen,D.Svergun,I.Gutsche,SNSavvides,K.Verstraete,StructureofATP citrate lyase and the origin of citrate synthase in theNature cycle of Krebs 568(7753)(2019)571-575. https: / / doi.org / 10.1038 / s41586-019-1095-5 . [7]JJLiang,XFZhou,H.Long,CYLi,J.Wei,XQYu,ZYGuo,YQZhou,ZSDeng,Recent advance of ATP citrate lyase inhibitors for the treatment ofcancer and related diseases,Bioorganic Chemistry 142(2024)1. https: / / doi.org / 10.1016 / j.bioorg.2023.106933 .

[0012] [8] C. Granchi, ATP-citrate lyase(ACLY)inhibitors as therapeutic agents: a patenting perspective, Expert Opin Ther Pat 32(7)(2022)731-742. https: / / doi.org / 10.1080 / 13543776.2022.2067478 .

[0013] [9]JPSamsoondar,ACBurke,BGSutherland,DETelford,CGSawyez,JYEdwards,SLPinkosky,RSNewton,MWHuff,Prevention of Diet-InducedMetabolic Dysregulation,Inflammation,and Atherosclerosis in Ldlr(- / -)Mice byTreatment With the ATP-Citrate Lyase Inhibitor Bempedoic Acid,ArteriosclerThromb Vasc Biol 37(4)(2017)647-656. https: / / doi.org / 10.1161 / atvbaha.116.308963 .

[0014]

[10] S. Filippov, SLPinkosky, RS Newton, LDL-cholesterol reduction inpatients with hypercholesterolemia by modulation of adenosine triphosphate-citrate lyase and adenosine monophosphate-activated protein kinase, Curr OpinLipidol 25(4)(2014)309-315. https: / / doi.org / 10.1097 / mol.0000000000000091 . Summary of the Invention

[0015] This invention provides 10 terpenoid compounds, including 2 new diterpenoid compounds and 8 new triterpenoid compounds, or their crystal forms, chiral isomers, glycosides, pharmaceutically acceptable salts, solvates, prodrugs, or metabolites, with the following general structural formula (I):

[0016]

[0017] in:

[0018] In compound 1, R1-R2 are selected from α-hydrogen, R3 is selected from β-methyl, R4 is selected from hydroxyl, and R5 is selected from hydroxyl.

[0019] In compound 2, R1-R6 are independently β-hydrogen, methyl, carboxyl, β-hydrogen, α-methyl, and hydroxyl groups, respectively.

[0020] In compound 3, R1-R2 are selected from hydroxyl groups, R3 from methyl groups, R4 from β-hydrogen groups, R5 from α-methyl groups, R6 from β-methyl groups, R7 from α-methyl groups, R8 from α-methyl groups, R9 from α-hydrogen groups, and R... 10 Selected from α-methyl;

[0021] In compound 4, R1 is selected from hydroxyl, R2 from carbonyl, R3 from methyl, R4 from β-hydrogen, R5 from β-methyl, R6 from α-methyl, R7 from α-aldehyde, R8 from β-hydrogen, R9 from α-methyl, and R... 10 Selected from carboxyl groups;

[0022] In compound 5, R1 is selected from hydroxyl, R2 from hydroxyl, R3 from aldehyde, R4 from methyl, R5 from α-methyl, R6 from β-methyl, R7 from β-methyl, R8 from β-hydrogen, R9 from methyl, and R... 10 Selected from carboxyl groups;

[0023] In compound 6, R1 is selected from hydroxyl group, R2 is selected from β-methyl, R3 is selected from β-methyl, R4 is selected from α-methyl, R5 is selected from carboxyl, R6 is selected from α-methyl, and R7 is selected from β-methyl;

[0024] In compound 7, R1 is selected from hydroxyl, R2 from carbonyl, R3 from α-hydrogen, R4 from β-methyl, R5 from hydrogen, R6 from β-methyl, R7 from α-methyl, R8 from β-methyl, R9 from β-hydrogen, and R... 10 Selected from β-methyl;

[0025] In compound 8, R1 is selected from hydroxyl, R2 is selected from hydroxyl, R3 is selected from methyl, R4 is selected from methyl, R5 is selected from α-methyl, R6 is selected from β-methyl, R7 is selected from β-methyl, R8 is selected from β-hydrogen, and R9 is selected from β-methyl.

[0026] In compound 9, R1 is selected from hydroxyl, R2 is selected from hydroxyl, R3 is selected from methyl, R4 is selected from methyl, R5 is selected from methyl, R6 is selected from methyl, R7 is selected from β-methyl, and R8 is selected from β-hydrogen;

[0027] In compound 10, R1 is selected from hydroxyl, R2 from carbonyl, R3 from hydroxyl, R4 from methyl, R5 from methyl, R6 from methyl, R7 from methyl, R8 from β-methyl, R9 from β-hydrogen, and R... 10 Selected from β-methyl, R 11 Selected from carbonyl;

[0028] The compound is shown in formula (II):

[0029]

[0030] This invention also provides a method for preparing the above-mentioned compound (II), comprising the following steps: The roots of *Tripterygium wilfordii* are pulverized into powder with a particle size of approximately 50 mesh, extracted using supercritical carbon dioxide fluid, and dynamically extracted for 4 hours using anhydrous ethanol as an additive. The extract is then concentrated to obtain a solid sample. The obtained sample is separated and purified by five-dimensional high-performance preparative liquid chromatography, including normal-phase, reversed-phase, and supercritical fluid chromatography, to obtain compounds 1–10.

[0031] The present invention tested the ACLY inhibitory activity of the obtained 10 terpenoid compounds. The results showed that compound 5 exhibited low micromolar inhibitory activity against ACLY and could be a lead compound for the treatment of cancer and cardiovascular diseases.

[0032] The compounds described in this invention can be obtained by isolation and purification from plants; they can also be synthesized by chemical methods well known to those skilled in the art.

[0033] The compounds described in this invention can be used alone or in combination, or combined with pharmaceutically suitable carriers or excipients, and formulated into oral or non-oral dosage forms according to conventional methods.

[0034] Obviously, based on the above description of the present invention, and in accordance with common technical knowledge and conventional methods in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention.

[0035] The compounds 1-10 provided in this invention are terpenoids extracted and isolated from *Tripterygium wilfordii* Hook.f., a vine-like shrub belonging to the genus *Tripterygium* in the family Celastraceae, and all are novel compounds with novel structures. Bioactivity experiments show that the obtained triterpenoid compound 5 possesses ACLY inhibitory activity and can be used in the preparation of drugs for cancer and cardiovascular diseases.

[0036] The present invention has the following advantages: the target compound is a novel terpenoid compound with a novel structure; compound 5 has significant ACLY inhibitory activity and has broad application prospects for the development of drugs for various cancers, hyperlipidemia, and cardiovascular diseases. Attached Figure Description

[0037] Figure 1 Compound 1 1 1H NMR (hydrogen nuclear magnetic resonance) spectrum

[0038] Figure 2 Compound 1 13 C NMR (carbon nuclear magnetic resonance) spectrum

[0039] Figure 3 H,H-COSY (hydrogen-hydrogen correlation spectrum) and key HMBC (hydrogen heteronuclear multicarbon correlation spectrum) of compound 1

[0040] Figure 4 Experimental circular dichroism (ECD) and calculated ECD spectra of compound 1

[0041] Figure 5 Compound 2 1 1H NMR (hydrogen nuclear magnetic resonance) spectrum

[0042] Figure 6 Compound 2 13 C NMR (carbon nuclear magnetic resonance) spectrum

[0043] Figure 7 H,H-COSY (hydrogen-hydrogen correlation spectrum) and key HMBC (hydrogen heteronuclear multicarbon correlation spectrum) of compound 2

[0044] Figure 8 Experimental circular dichroism (ECD) and calculated ECD spectra of compound 2

[0045] Figure 9 Compound 3 1 1H NMR (hydrogen nuclear magnetic resonance) spectrum

[0046] Figure 10 Compound 3 13 C NMR (carbon nuclear magnetic resonance) spectrum

[0047] Figure 11 H,H-COSY (hydrogen-hydrogen correlation spectrum) and key HMBC (hydrogen heteronuclear multicarbon correlation spectrum) of compound 3

[0048] Figure 12 Experimental circular dichroism (ECD) and calculated ECD spectra of compound 3

[0049] Figure 13 Compound 4 1 1H NMR (hydrogen nuclear magnetic resonance) spectrum

[0050] Figure 14 Compound 4 13C NMR (carbon nuclear magnetic resonance) spectrum

[0051] Figure 15 H,H-COSY (hydrogen-hydrogen correlation spectrum) and key HMBC (hydrogen heteronuclear multicarbon correlation spectrum) of compound 4

[0052] Figure 16 Experimental circular dichroism (ECD) and calculated ECD spectra of compound 4

[0053] Figure 17 Compound 5 1 1H NMR (hydrogen nuclear magnetic resonance) spectrum

[0054] Figure 18 Compound 5 13 C NMR (carbon nuclear magnetic resonance) spectrum

[0055] Figure 19 H,H-COSY (hydrogen-hydrogen correlation spectrum) and key HMBC (hydrogen heteronuclear multicarbon correlation spectrum) of compound 5

[0056] Figure 20 Experimental circular dichroism (ECD) and calculated ECD spectra of compound 5

[0057] Figure 21 Compound 6 1 1H NMR (hydrogen nuclear magnetic resonance) spectrum

[0058] Figure 22 Compound 6 13 C NMR (carbon nuclear magnetic resonance) spectrum

[0059] Figure 23 H,H-COSY (hydrogen-hydrogen correlation spectrum) and key HMBC (hydrogen heteronuclear multicarbon correlation spectrum) of compound 6

[0060] Figure 24 Experimental circular dichroism (ECD) and calculated ECD spectra of compound 6

[0061] Figure 25 Compound 7 1 1H NMR (hydrogen nuclear magnetic resonance) spectrum

[0062] Figure 26 Compound 7 13 C NMR (carbon nuclear magnetic resonance) spectrum

[0063] Figure 27 H, H-COSY (hydrogen-hydrogen correlation spectrum) and key HMBC (hydrogen heteronuclear multicarbon correlation spectrum) of compound 7

[0064] Figure 28 Compound 8 11H NMR (hydrogen nuclear magnetic resonance) spectrum

[0065] Figure 29 Compound 8 13 C NMR (carbon nuclear magnetic resonance) spectrum

[0066] Figure 30 H, H-COSY (hydrogen-hydrogen correlation spectrum) and key HMBC (hydrogen heteronuclear multicarbon correlation spectrum) of compound 8

[0067] Figure 31 Experimental circular dichroism (ECD) and calculated ECD spectra of compound 8

[0068] Figure 32 Compound 9 1 1H NMR (hydrogen nuclear magnetic resonance) spectrum

[0069] Figure 33 Compound 9 13 C NMR (carbon nuclear magnetic resonance) spectrum

[0070] Figure 34 H,H-COSY (hydrogen-hydrogen correlation spectrum) and key HMBC (hydrogen heteronuclear multicarbon correlation spectrum) of compound 9

[0071] Figure 35 Experimental circular dichroism (ECD) and calculated ECD spectra of compound 9

[0072] Figure 36 Compound 10 1 1H NMR (hydrogen nuclear magnetic resonance) spectrum

[0073] Figure 37 Compound 10 13 C NMR (carbon nuclear magnetic resonance) spectrum

[0074] Figure 38 H, H-COSY (hydrogen-hydrogen correlation spectrum) and key HMBC (hydrogen heteronuclear multicarbon correlation spectrum) of compound 10

[0075] Figure 39 Experimental circular dichroism (ECD) and calculated ECD spectra of compound 10

[0076] Figure 40 ACLY inhibitory activity of compound 5

[0077] Figure 41 A schematic diagram of the structure of compound II in a preferred embodiment of the present invention. Detailed Implementation

[0078] The following examples are intended to illustrate the invention and not to further limit it. The invention can be implemented in any of the ways described in the invention description.

[0079] Examples of preparation of compounds of formula (II) of the present invention:

[0080] Compound preparation and structural identification:

[0081] In the following preparation examples, the liquid chromatography system included a Waters Alliance HPLC system with an e2695 separation unit and a 2998 PDA detector; data processing was performed using an Empower 3 system; a Waters AutoP automated purification system with a 2545 separation unit, a 2767 sample manager, and a 2489 dual-wavelength detector; a Changzhou Ruixi high-performance liquid chromatography system and a YMC fully automated purification system; and a Waters supercritical fluid preparative chromatograph equipped with a 2489 dual-wavelength detector. Reagents included chromatographic grade methanol and acetonitrile purchased from Thermo Fisher Scientific (Loughborough, UK); chromatographic grade formic acid purchased from Bailingwei Technology (Hebei, China); laboratory water from a Milli-Q ultrapure water purification system (Billerica, MA, USA); and preparative grade methanol, acetonitrile, and dichloromethane purchased from Shanghai Xingke High Purity Solvent Co., Ltd. (Shanghai, China). All chromatographic columns used in the experiments were from Beijing Huapu Scientific Instruments Co., Ltd.

[0082] Structural identification was performed using a Bruker AVIII-600 nuclear magnetic resonance spectrometer (Bruker, German). Compounds were dissolved in deuterated chloroform (CDCl3). Mass spectrometry (MS) analysis was conducted using an Agilent 1290 Infinity LC / 6540Q-TOF MS system, including a high-pressure binary pump, autosampler, column isotherm system, diode array detector, electrospray ionization source, and Agilent MassHunter 10.0 software. Positive ion scanning mode was used, with a scan range of m / z MS: 100-1000. Other conditions were as follows: nitrogen flow rate 8 L / min; nitrogen temperature 350 °C; nebulizer gas pressure 35 psi; capillary voltage 3.5 kV; collision voltage 75 V; collision energy 30 eV.

[0083] The preparation and compound identification steps are as follows:

[0084] (1) Extraction of medicinal materials: The dried Tripterygium wilfordii root was pulverized into 50-mesh powder (the component passing through a 50-mesh sieve), with a mass of 18.6 kg. Dynamic extraction was carried out using supercritical carbon dioxide at 50℃ and 50 MPa. Anhydrous ethanol was used as an additive, and extraction was carried out for 4 hours at a material-to-liquid ratio of 1:4 (g / ml). The extract was concentrated to obtain 322 g of solid crude extract.

[0085] (2) Dissolve the crude extract obtained in step (1) in 3.2 L of dichloromethane and perform first-dimensional separation and purification by normal phase chromatography. The stationary phase is a silica gel column (50 mm × 250 mm (inner diameter × length, the same below), 10 μm (particle size, the same below)). The mobile phase consists of (A) dichloromethane and (B) methanol, with the following gradient (volume ratio): 0.0–15.0 min: 0% B, 15.0–38.0 min: 3% B, 38.0–48.0 min: 5% B, 48.0–60.0 min: 90% B. The flow rate was 80 mL / min, and the detection wavelengths were 230 and 268 nm. Four sub-fractions, F1 to F4, were collected according to the UV absorption peaks: F1 (3.0–11.0 min), F2 (11.0–25.0 min), F3 (25.0–43.0 min), and F4 (43.0–60.0 min).

[0086] (3) The Tripterygium wilfordii fraction F3 obtained in step (2) was purified by second-dimensional separation using a C18HD (200mm×250mm, 10μm) reversed-phase column. The mobile phase was (A) acetonitrile (v / v) and (B) 0.1% formic acid-water (volume concentration, the same below). The flow rate was 1200mL / min, the detection wavelengths were 230 and 268nm, the injection volume was 700mL, and the elution gradient (volume ratio) was 0–13.0min: 40.0%B, 13.0–25.0min: 37.0%B, 25.0–35.0min: 20.0%B, and 35.0–55.0min: 5.0%B. Ten fractions were collected (F3-1 to F3-10): F3-1, 3.0–6.0 min; F3-2, 6.0–9.0 min; F3-3, 9.0–10.5 min; F3-4, 10.5–16.0 min; F3-5, 16.0–19.0 ​​min; F3-6, 19.0–21.0 min; F3-7, 21.0–28.0 min; F3-8, 28.0–34.0 min; F3-9, 34.0–40.5 min; and F3-10, 40.5–55.0 min.

[0087] (4) The fractions F3-6, F3-8, F3-9 and F3-10 obtained in step (3) were separated and purified in the third dimension using a QUIS (50mm×250mm, 10μm) reversed-phase column. The mobile phase was (A) acetonitrile (v / v) and (B) 0.1% formic acid-water. The flow rate was 80mL / min, and the detection wavelengths were 230 and 268nm. The elution gradient (volume ratio) for F3-6 was 0–20.0 min: linear gradient 65%–30%B, 20.0–25.0 min: linear gradient 30.0–10%B, and 25.0–30.0 min: 10.0%B. Five fractions were collected (F3-6-1 to F3-6-5): F3-6-1, 4.5–7.5 min; F3-6-2, 7.5–11.0 min; F3-6-3, 11.0–16.0 min; F3-6-4, 16.0–21.0 min; F3-6-5, 21.0–30.0 min; and F3-8. The elution gradient (volume ratio) was 0–50.0 min: linear gradient 70%–30% B; 50.0–52.0 min: linear gradient 30.0–10% B; and 52.0–60.0 min: 10.0% B. Six sub-fractions were collected (F3-8-1 to F3-8-6): F3-8-1, 5.0–18.0 min; F3-8-2, 18.0–28.0 min; F3-8-3, 28.0–32.0 min; F3-8-4, 32.0–35.5 min; F3-8-5, 35.5–41.0 min; F3-8-6, 41.0–50.0 min; and F3-9. The elution gradient (volume ratio) was 0–50.0 min: linear gradient 70%–40% B, 50.0–52.0 min: linear gradient 40.0–10% B, and 52.0–60.0 min: 10.0% B. Nine fractions were collected (F3-9-1 to F3-9-9): F3-9-1, 5.0–14.0 min; F3-9-2, 14.0–24.0 min; F3-9-3, 24.0–30.5 min; F3-9-4, 30.5–34.5 min; F3-9-5, 34.5–39.0 min; F3-9-6, 39.0–45.0 min; F F3-9-7, 45.0~50.5min; F3-9-8, 50.5~52.0min; F3-9-9, 52.0~60.0min; F3-10 elution gradient (volume ratio) is 0~50.0min: linear gradient 65%~40%B, 50.0~52.0min: linear gradient 40.0~10%B, 52.0~60.0min: 10.0%B.Nine fractions were collected (F3-10-1 to F3-10-8): F3-10-1, 3.5–9.0 min; F3-10-2, 9.0–14.5 min; F3-10-3, 14.5–20.0 min; F3-10-4, 20.0–24.5 min; F3-10-5, 24.5–30.0 min; F3-10-6, 30.0–40.0 min; F3-10-7, 40.0–53.0 min; and F3-10-8, 53.0–62.0 min.

[0088] (5) The fractions F3-6-2, F3-6-3, F3-8-3, F3-9-4, F3-10-5, F3-10-6 and F3-10-7 obtained in step (4) are separated and purified in the fourth dimension. The mobile phase is (A) acetonitrile and (B) 0.1% formic acid-water (v / v). For F3-6-2, separation was performed using a PHN (30 mm × 250 mm, 10 μm) reversed-phase column. The elution gradient (volume ratio) was 0.0–30.0 min, 46% A; 30.0–38.0 min, linear gradient 46%–90% A; 38.0–45.0 min, 90% A. The flow rate was 30 mL / min. Eleven fractions were collected (F3-6-2-1 to F3-6-2-11): F3-6-2-1, 3.0–9.5 min; F3-6-2-2, 9.5–12.7 min; F3… -6-2-3,12.7~15.5min; F3-6-2-4,15.5~18.6min; F3-6-2-5,18.6~21.2min; F3-6-2-6,21.2~27.0min; F3-6-2-7,27.0 ~29.2min; F3-6-2-8, 29.2~31.9min; F3-6-2-9, 31.9~36.0min; F3-6-2-10, 36.0~38.6min; F3-6-2-11, 38.6~45.0min;. For F3-6-3, separation was performed using a C18ME (50 mm × 250 mm, 10 μm) reversed-phase column. The elution gradient (volume ratio) was 0.0–30.0 min, 55% A; 30.0–38.0 min, linear gradient 55%–90% A; 38.0–45.0 min, 90% A. The flow rate was 80 mL / min. A total of 13 fractions were collected (F3-6-3-1 to F3-6-3-13): F3-6-3-1, 3.0–6.2 min; F3-6-3-2, 6.2–8.5 min; F3-6-3-3, 8.5–12.4 min; F3 -6-3-4,12.4~15.1min; F3-6-3-5,15.1~18.7min; F3-6-3-6,18.7~21.2min; F3-6-3-7,21.2~25.5min; F3-6-3-8,25.5~28.9min; F3- 6-3-9, 28.9~33.2min; F3-6-3-10, 33.2~35.7min; F3-6-3-11, 35.7~39.5min; F3-6-3-12, 39.5~42.8min; F3-6-3-13, 42.8~50.0min.For F3-8-3, separation was performed using a PHN (50 mm × 250 mm, 10 μm) reversed-phase column. The elution gradient (volume ratio) was 0.0–35.0 min, 47% A; 35.0–45.0 min, linear gradient 47%–90% A; 45.0–60.0 min, 90% A. The flow rate was 80 mL / min. A total of 13 fractions were collected (F3-8-3-1 to F3-8-3-13): F3-8-3-1, 5.3–9.0 min; F3-8-3-2, 9.0–14.3 min; F3-8-3-3, 14.3–18.7 min; F3 -8-3-4,18.7~22.4min; F3-8-3-5,22.4~30.4min; F3-8-3-6,30.4~33.8min; F3-8-3-7,33.8~36.3min; F3-8-3-8,36.3~41.4min; F3- 8-3-9, 41.4~42.8min; F3-8-3-10, 42.8~44.4min; F3-8-3-11, 44.4~45.9min; F3-8-3-12, 45.9~47.9min; F3-8-3-13, 47.9~55.0min. For F3-9-4, separation was performed using a C18ME (50 mm × 250 mm, 10 μm) reversed-phase column. The elution gradient (volume ratio) was 0.0–30.0 min, 70% A; 30.0–35.0 min, linear gradient 70%–90% A; 35.0–50.0 min, 90% A. The flow rate was 80 mL / min. A total of 12 fractions were collected (F3-9-4-1 to F3-9-4-12): F3-9-4-1, 4.0–8.7 min; F3-9-4-2, 8.7–11.0 min; F3-9-4-3, 11.0 min. 0~13.6min; F3-9-4-4, 13.6~16.2min; F3-9-4-5, 16.2~19.9min; F3-9-4-6, 19.9~25.8min; F3-9-4-7, 25.8~29.8min; F3-9-4-8 , 29.8~35.9min; F3-9-4-9, 35.9~38.4min; F3-9-4-10, 38.4~40.4min; F3-9-4-11, 44.4~43.6min; F3-9-4-12, 43.6~50.0min;.For F3-10-5, separation was performed using a C18ME (50 mm × 250 mm, 10 μm) reversed-phase column. The elution gradient (volume ratio) was 0.0–30.0 min, 70% A; 30.0–35.0 min, linear gradient 70%–90% A; 35.0–50.0 min, 90% A. The flow rate was 80 mL / min. Eleven fractions were collected (F3-10-5-1 to F3-10-5-11): F3-10-5-1, 3.4–4.9 min; F3-10-5-2, 4.9–7.7 min; F… 3-10-5-3,7.7~9.6min; F3-10-5-4,9.6~12.8min; F3-10-5-5,12.8~18.0min; F3-10-5-6,18.0~21.4min; F3-10-5-7,21.4 ~26.1min; F3-10-5-8, 26.1~28.9min; F3-10-5-9, 28.9~36.3min; F3-10-5-10, 36.3~38.0min; F3-10-5-11, 38.0~45.0min.For F3-10-6, separation was performed using a C18ME (50 mm × 250 mm, 10 μm) reversed-phase column. The elution gradient (volume ratio) was 0.0–30.0 min, with a linear gradient of 76%–90% A; 30.0–55.0 min, 90% A. The flow rate was 80 mL / min. A total of 22 fractions were collected (F3-10-6-1 to F3-10-6-22), with F3-10-6-1 collected from 3.0 to 7.0 min. F3-10-6-2,7.0~9.7min; F3-10-6-3,9.7~11.4min; F3-10-6-4,11.4~13.3min; F3-10-6-5,13.3~1 5.5min; F3-10-6-6, 15.5~16.8min; F3-10-6-7, 16.8~18.2min; F3-10-6-8, 18.2~21.0min; F3-10-6 -9,21.0~23.6min; F3-10-6-10,23.6~25.1min; F3-10-6-11,25.1~28.2min; F3-10-6-12,28.2~29 .7min; F3-10-6-13, 29.7~32.0min; F3-10-6-14, 32.0~34.2min; F3-10-6-15, 34.2~35.8min; F3-1 0-6-16, 35.8~38.0min; F3-10-6-17, 38.0~40.5min; F3-10-6-18, 40.5~43.5min; F3-10-6-19,43. 5~46.1min; F3-10-6-20, 46.1~48.2min; F3-10-6-21, 48.2~50.5min; F3-10-6-22, 50.5~55.0min;.For F3-10-7, separation was performed using a C18ME (50 mm × 250 mm, 10 μm) reversed-phase column. The elution gradient (volume ratio) was 0.0–30.0 min, with a linear gradient of 76%–90% A; 30.0–40.0 min, 90% A. The flow rate was 80 mL / min. A total of 22 fractions were collected (F3-10-7-1 to F3-10-7-22). For F3-10-7-1, the elution gradient was 3.0–4.5 min. ;F3-10-7-2,4.5~7.0min; F3-10-7-3,7.0~9.0min; F3-10-7-4,9.0~11.2min; F3-10-7-5,11.2~13 .0min; F3-10-7-6, 13.0~14.4min; F3-10-7-7, 14.4~16.5min; F3-10-7-8, 16.5~18.0min; F3-10-7 -9,18.0~22.0min; F3-10-7-10,22.0~24.0min; F3-10-7-11,24.0~25.0min; F3-10-7-12,25.0~28 .2min; F3-10-7-13, 28.2~29.8min; F3-10-7-14, 29.8~30.4min; F3-10-7-15, 30.4~32.6min; F3-1 0-7-16, 32.6~34.4min; F3-10-7-17, 34.4~36.2min; F3-10-7-18, 36.2~38.4min; F3-10-7-19,38. 4~40.0min; F3-10-7-20, 40.0~42.4min; F3-10-7-21, 42.4~49.6min; F3-10-7-22, 49.6~55.0min.

[0089] (6) The fractions F3-6-2-6, F3-6-2-11, F3-6-3-3, F3-8-3-8, F3-9-4-9, F3-10-5-9, F3-10-6-10, F3-10-6-20, F3-10-7-10, and F3-10-7-11 obtained in step (5) are separated and purified in the fifth dimension. For the reversed-phase mode, the mobile phase is (A) methanol and (B) 0.1% formic acid-water (v / v), and the flow rate is 3.3 mL / min. For the SFC mode, the mobile phase is (A) CO2 and (B) 0.1% formic acid-methanol (v / v), and the flow rate is 10.0 mL / min. For F3-6-2-6, separation was performed using a BH (10 mm × 250 mm, 10 μm) (SFC column) with an elution gradient (volume ratio) of 0–12.0 min, linear gradient 8–30% B; 12.0–16.0 min, linear gradient to 55% B; and 16.0–20.0 min, linear gradient to 10% B, yielding compound 1 (8.5–9.5 min). For F3-6-2-11, separation was performed using a C18ME (10 mm × 250 mm, 10 μm) (reversed-phase column) with an elution gradient (volume ratio) of 8–30% B; 12.0–16.0 min, linear gradient to 55% B; and 16.0–20.0 min, linear gradient to 10% B, yielding compound 1 (8.5–9.5 min). Compound 7 was obtained (27.0–28.5 min) with an elution gradient (volume ratio) of 0–60 min and 80% A. For F3-6-3-3, separation was performed using a C18ME (10 mm × 250 mm, 10 μm) reversed-phase column with an elution gradient (volume ratio) of 0–60 min and 45% A, yielding compound 2 (15.5–16.8 min). For F3-8-3-8, separation was performed using a C3HC (10 mm × 250 mm, 10 μm) reversed-phase column with an elution gradient (volume ratio) of 0–60 min and 65% A. %A, yielding compound 4 (7.2–7.6 min); for F3-9-4-9, separation was performed using an X5H (10 mm × 250 mm, 10 μm) (SFC column) with an elution gradient (volume ratio) of 0–12.0 min, linear gradient 5–30%B; 12.0–16.0 min, linear gradient to 55%B; 16.0–20.0 min, linear gradient to 5%B, yielding compound 3 (7.5–8.5 min); for F3-10-5-9, separation was performed using a C18HD (10 mm × 250 mm, 10 μm) column. For F3-10-6-10, separation was performed using a BH (10 mm × 250 mm, 10 μm, SFC column) with an elution gradient (volume ratio) of 0–60 min, 81% A, yielding compound 9 (31.5–34.0 min).For F3-10-6-20, separation was performed using an X5H (10 mm × 250 mm, 10 μm) (SFC column) with an elution gradient (volume ratio) of 0–12.0 min, linear gradient 5–25% B; 12.0–16.0 min, linear gradient to 55% B; and 16.0–20.0 min, linear gradient to 5% B, yielding compound 6 (4.8–5.5 min). For F3-10-7-10, separation was performed using a C18 column. Separation was performed using an HD (10 mm × 250 mm, 10 μm) reversed-phase column with an elution gradient (v / v) of 0–60 min and 82% A, yielding compound 10 (34.5–35.5 min). For F3-10-7-11, separation was performed using a PHN (10 mm × 250 mm, 10 μm) reversed-phase column with an elution gradient (v / v) of 0–60 min and 70% A, yielding compound 5 (51.5–55.5 min).

[0090] (7) The structure identification of the compound and related information are as follows:

[0091] Compound 1, based on its HR-ESIMS (m / z) 371.1852 [M+H-H2O], has the following molecular formula: C 20 H 28 O2 has an unsaturation degree of 7. According to... 1 H NMR data indicate the presence of an isopropyl group [δ] H 1.23 (3H,d,J=6.7Hz), 1.25 (3H,d,J=6.7Hz), 3.14 (1H,m)], 2 olefin protons δ H 7.00 (3H,d,J=8.2Hz), δ H 6.88 (3H,d,J=8.2Hz), 4 aliphatic methylene groups, 1 hydroxymethylene group, and 3 aliphatic methine groups. 13 The C NMR spectrum shows one aromatic quaternary carbon and four aromatic quaternary carbons, all of which are carbon signals from the same benzene ring.

[0092] Based on the HMBC spectral correlation signal H-15(δ) H 3.14) / C-16(δ C 22.9), C-17(δ) C 22.5), C-13(δ) C 130.4), C-12(δ C 117.8), C-14(δ) C 150.5); H-12 (δ) H 7.00) / C-14(δ C 150.5), C-9(δ) C145.3) indicates the structural unit of the isopropyl-substituted benzene ring. (Combined) 1 H- 1 The H-COSY spectrum showed three spin coupling systems, H-1 / H-2 / H-3, H-5 / H-6, and H-11 / H-12, confirming that compound 1 has a rosinane diterpenoid skeleton. Based on H2-19 (δ... H 3.53) / C-3(δ C 17.5), C-4(δ) C 17.9), C-18(δ) C The correlation signal in the HMBC spectrum of 23.8) confirmed that C-4, C-5, and C-18 form a three-membered ring structure, and that C-18 is connected to the oxygen-bound methylene group. The H-3 / H-4 correlation signal in the NOESY spectrum indicated that the two proton hydrogens are located on the same side of the planar structure. Combining the experimental CD and calculated ECD curves, the absolute configuration of compound 1 was determined to be 3S, 4S, 10S. Figure 3 As shown. The new compound was named triptomethanol.

[0093] Compound 2, according to HR-ESIMS (m / z) 361.1654 [M+H] + The molecular formula of compound 2 is presumed to be C. 20 H 24 O6 shows 9 degrees of unsaturation. 1 H NMR data also indicate the presence of one isopropyl substituent [δ] H 1.03 (3H,d,J=6.8Hz), 1.08 (3H,d,J=6.8Hz), 2.94 (1H,m)], 1 aromatic proton [δ H [6.25(1H,s)], 3 aliphatic methylene groups, 1 aliphatic methine group [δ] H 2.15 (1H,m)] and 1 methyl signal [δ H 1.31(3H,s)]. 13 The C NMR spectrum also showed the presence of one carbonyl carbon, one carboxyl carbon, six unsaturated quaternary carbons, one oxygen-bound quaternary carbon, and one oxygen-bound tertiary carbon signal.

[0094] According to HMBC signal H-16(δ) H 1.03) / C-13(δ C 147.2), C-15(δ) C 26.6); H-17 (δ) H 1.08) / C-13(δ C 147.2), C-15(δ) C 26.6) Determine that the isopropyl group is attached at the C-13 position, H-12 (δH 6.25) / C-13(δ C 147.2), C-14(δ) C 185.1); H2-8(δ) H 2.27) / C-14(δ C 185.1),C-1(δ C 46.2), C-10(δ) C The relevant signal (148.4) indicates that the carbon-carbon double bonds in the C ring are located at C-9 / C-10 and C-12 / C-13 positions, respectively, and the ketone carbonyl group is located at C-14. According to H-19 (δ... H 2.12) / C-2(δ C 148.9), C-3(δ) C 122.3), C-18(δ) C 171.9); H-5 (δ) H 4.29) / C-6(δ C 35.9), C-1(δ C 46.2), C-20(δ) C 14.2) HMBC related signals and 1 H- 1 The H-4 / H-5 correlation signal in the HCl COSY spectrum determined that the carboxyl group was attached at the C-3 position, the double bond was located between C-2 and C-3, and the two methyl groups were located at the C-2 and C-6 positions, respectively. Figure 3-34 As shown, the relative configuration of compound 2 was confirmed based on the correlation signals of H-1 / H-7 in the NOESY spectrum. Figure 8 The results showed that the experimentally measured CD curve matched the calculated ECD curve, confirming the absolute configuration of compound 2 as 1S,5R,6S,11S, and naming the compound triptotinA-1.

[0095] Compound 3, according to HR-ESIMS (m / z) 425.3051 [M+H] + It is inferred that its molecular formula is C 28 H 40 O3 contains 9 degrees of unsaturation. 1 The H NMR spectrum shows the presence of one aromatic proton signal [δ]. H [6.67(1H,s)], 6 methyl groups, 8 aliphatic methylene groups, and 3 aliphatic methine signals. In 13 The C NMR spectrum shows the presence of one carbonyl δ group. C 215.0 (C-21), 6 aromatic carbons are assigned to carbon atoms on the benzene ring, and 6 methyl carbons.

[0096] According to H-28(δ) H0.99) / C-19(δ C 32.0), C-20(δ) C 42.5), C-21(δ C 215.0); H2-19(δ) H 2.22,1.68) / C-21(δ C 215.0); H2-22(δ) H 2.95, 1.84) / C-21(δ C The correlation signal from the HMBC spectrum at 215.0 indicates the presence of a carbonyl group at the C-21 position. 1 H- 1 The H COSY spectrum showed correlation signals for H-6 / H-7, H-12 / H-13, H-15 / H-16, and H-18 / H-19 / H-20, suggesting the presence of connecting segments for C-6 / C-7, C-12 / C-13, C-15 / C-16, and C-18 / C-19 / C-20. Further analysis based on H-24 (δ...) H 1.22) / C-10(δ C 143.9), C-8(δ) C 43.6), C-12(δ C 34.1); H-26 (δ) H 1.26) / C-13(δ C 30.4), C-14(δ) C 40.2), C-18(δ) C 44.1) Correlation signals from the HMBC spectrum determined the planar configuration of compound 3. Correlation signals from H-24 / H-26 / H-27 / H-28 in the NOESY spectrum indicated that these protons were located on the same side of the plane, thus determining their relative configuration. Comparison of experimental CD and calculated ECD curves yielded the following results: Figure 12 As shown, the absolute configuration of compound 3 was determined to be 8S,9S,13R,14R,17R,18S,20S, and the new compound was named regeol D.

[0097] Compound 4, according to HR-ESIMS (m / z) 483.3129 [M+H] + It is inferred that its molecular formula is C 30 H 42 O5 contains 10 degrees of unsaturation. From 1 Two olefin proton signals [δ] can be detected in the H NMR spectrum. H 6.32(1H,s),δ H [5.45(1H,s)], 6 methyl groups, 8 aliphatic methylene groups, and 3 aliphatic methine signals. In 13The C10 NMR spectrum showed three carbonyl signals, four olefin carbon signals, six methyl carbon signals, and six aliphatic quaternary carbon signals.

[0098] Based on the correlation signal H-1(δ) of the HMBC spectrum H 6.32) / C-2(δ C 143.9), C-3(δ C 201.2); H-16 (δ) H 2.01) / C-28(δ C 206.6); H2-21(δ) H 1.86, 1.59) / C-29(δ C 183.3), C-19(δ) C 39.2); H-30(δ) H 1.28) / C-20(δ C 42.1), C-29(δ) C 183.3) demonstrates that the three carbonyl groups are located at C-3, C-28, and C-29, respectively. 1 H- 1 The presence of H-11 / H-12 / H-13, H-15 / H-16, H-18 / H-19, and H-21 / H-22 spin-coupled systems in the H COSY spectrum proves the existence of C-11 / C-12 / C-13, C-15 / C-16, C-18 / C-19, and C-21 / C-22 connection segments in the structure. Combined with the H-23 (δ) in the HMBC spectrum... H 1.21) / C-3(δ C 201.2), C-5(δ C 53.9); H-26 (δ) H 0.81) / C-8(δ C 40.4), C-7(δ) C 32.6); H-27 (δ) H 1.14) / C-8(δ C 40.4), C-14(δ) C 42.0), C-15(δ) C The correlation signals in 6.7) confirmed the planar structure of compound 4, and the correlation signals of H-25 / H-26 / H-18 in the NOESY spectrum indicated that these protons have the same spatial orientation. Combining the experimental CD curves and calculated ECD curves, as shown... Figure 16As shown, its absolute configuration was determined to be 5R,8R,10R,14S,18S,20S, and the compound was named 2-hydroxy-3-oxo-18β-oleana-1,12-dien-28-al-30-oic acid.

[0099] Compound 5, according to HR-ESIMS (m / z) 465.2650 [M+H] + It is inferred that its molecular formula is C 29 H 36 O5, with an unsaturation degree of 12. From 1 Two aromatic protons [δ] can be detected in the H NMR spectrum. H 7.74 (1H, s), δ H 7.27 (1H, s)], 1 aldehyde hydrogen signal [δ H [10.69(1H,s)], 5 methyl groups, 7 aliphatic methylene groups, 1 aliphatic methine signal. In 13 The C10 NMR spectrum shows two carbonyl signals, ten aromatic carbon signals (attributed to two fused benzene ring carbon signals), five methyl carbon signals, and four aliphatic quaternary carbon signals.

[0100] Based on the correlation signal H-1(δ) of the HMBC spectrum H 7.74) / C-3(δ C 155.2), C-5(δ C 125.5), C-9(δ) C 128.2); H-23 (δ) H 10.69) / C-3(δ C 155.2), C-5(δ C 125.5); H-7 (δ) H 7.27) / C-5(δ C 125.5), C-9(δ) C 128.2), C-24(δ) C 27.1), C-14(δ) C 41.7) The A and B ring structures of the compound were determined, and the aldehyde group was located at the C-4 position. 1 H- 1 The presence of H-11 / H-12, H-15 / H-16, H-18 / H-19, and H-21 / H-22 correlation signals in the H COSY spectrum indicates the presence of C-11 / C-12, C-15 / C-16, C-18 / C-19, and C-21 / C-22 connection segments within the structure. Based on H-26(δ... H 1.15) / C-13(δ C 37.3), C-5(δ)C 27.4); H-30 (δ) H 1.24) / C-19(δ C 30.9), C-21(δ) C 29.5), C-29(δ) C The correlation signal (184.0) confirmed the planar structure of compound 5. The presence of cross signals (H-26 / H-28 / H-18 / H-30) in the NOESY spectrum indicates that these proton hydrogen signals have the same spatial orientation. For example... Figure 20 By comparing the experimental CD curve and the calculated ECD curve, the absolute configuration of the new compound was determined to be 13S,14S,17S,18R,20R, and it was named wilforic acid G.

[0101] Compound 6, according to HR-ESIMS (m / z) 439.3568 [M+H-COOH] + It is inferred that its molecular formula is C 31 H 48 O4 has an unsaturation degree of 8. 1 The H NMR spectrum shows the presence of one olefin proton [δ]. H [5.28 (1H,t,J=3.1)], 10 aliphatic methylene groups, 3 aliphatic methine groups, 1 hydroxymethine group, and 7 methyl signals. In 13 The C NMR spectrum shows that in addition to the carbon signals corresponding to the proton spectrum, there are two carbonyl signals and six quaternary carbon signals.

[0102] exist 1 H- 1 The H COSY spectrum contains self-selected coupling systems H-1 / H-2 / H-3, H-5 / H-6 / H-7, H-9 / H-11 / H-12, H-15 / H-16, H-18 / H-19, and H-21 / H-22. According to H-31(δ... H 8.11) / C-31(δ C 161.3), C-3(δ C 81.2); H2-16(δ) H 1.98, 1.61) / C-28(δ C 183.3); H2-22(δ) H 1.77,1.57) / C-28(δ C 183.3), C-17(δ) C The relevant signal from 46.7) determined that the aldehyde group was located at the C-3 position, which is bonded to oxygen, and the carboxyl group was bonded at the C-17 position. Further analysis based on H-23 (δ...) H 0.89) / C-4(δ C37.8), C-3(δ) C 81.2); H-27 (δ) H 1.13) / C-8(δ C 39.4), C-14(δ) C 41.8), C-15(δ) C 27.8); H-29 (δ) H 0.93) / C-20(δ C 30.8), C-21(δ) C The correlation signal (33.9) confirmed the planar structure of compound 6. The cross signals of H-24 / H-25 / H-26 / H-30 in the NOESY spectrum indicated that these protons were located on the same side of the molecular plane, thus confirming the relative configuration of the compound. (As shown...) Figure 24 As shown, by comparing the experimental CD curve and the calculated ECD curve, its absolute configuration was confirmed to be 3S,8R,10R,14S,17S, and it was named 3-O-al-oleanolic acid.

[0103] Compound 7, according to HR-ESIMS (m / z) 467.3160 ​​[M+H] + It is inferred that its molecular formula is C 30 H 42 O4 has an unsaturation degree of 10. 1 Two olefin proton signals were present in the H NMR spectrum [δ] H 6.34 (1H, s), δ H [5.36(1H,t,J=3.4)], 7 methyl groups, 7 aliphatic methylene groups, 3 aliphatic methine groups, and one aliphatic oxymethine signal. In 13 The C10 NMR spectrum showed two carbonyl signals, four olefin carbon signals, six methyl carbon signals, and six aliphatic quaternary carbon signals.

[0104] Based on the correlation signal H-1(δ) of the HMBC spectrum H 6.34) / C-2(δ C 144.0), C-3(δ C 201.2); H-23(δ) H 1.23) / C-3(δ C 201.2); H-24 (δ) H 1.13) / C-3(δ C 201.2) confirmed that the carbonyl group is located at the C-3 position. Based on H-30 (δ... H 1.22) / C-19(δ C 39.8), C-21(δ) C 34.0), C-29(δ)C 182.5); H-22 (δ) H 4.15) / C-18(δ C 43.7), C-29(δ) C 182.5); H-28 (δ) H 0.88) / C-22(δ C The relevant signal from 83.1) proves that the carboxyl group is located at C-29 and forms a lactone structure through esterification with the C-22 position. 1 H- 1 The presence of H-5 / H-6 / H-7, H-9 / H-11, H-15 / H-16, H-18 / H-19, and H-21 / H-22 correlation signals in the H COSY spectrum proves that the structure contains connection segments of C-5 / C-6 / C-7, C-9 / C-11, C-15 / C-16, C-18 / C-19, and C-21 / C-22. Based on H-25 (δ) in the HMBC spectrum... H 1.25) / C-5(δ C 54.0), C-9(δ) C 43.1); H-26 (δ) H 1.01) / C-8(δ C 40.1), C-7(δ) C The relevant signals from 33.1) confirmed the planar structure of compound 7. The cross signals of H-24 / H-25 / H-26 / H-28H in the NOESY spectrum indicated that these protons were located on the same side of the molecular plane, thus confirming the relative configuration of the compound, which was named 2-hydroxy-olean-12-ene-22,29-lactone.

[0105] Compound 8, according to its HR-ESIMS (m / z) 451.2842 [M+H] + Analysis shows that the molecular formula is C 29 H 38 O4 has an unsaturation degree of 11. 1 The H NMR spectrum contained three aromatic proton signals [δ] H 7.13(1H,s),δ H 7.63 (1H, d, J = 8.4 Hz), δ H 7.13(1H,d,J=8.4Hz)], 6 methyl groups, 7 aliphatic methylene groups. 13 The C NMR spectrum provided signals for 7 aromatic quaternary carbons, 3 aliphatic quaternary carbons, 1 oxygen-linked quaternary carbon, and 1 ester carbonyl carbon.

[0106] There is a correlation signal H-30 (δ) in the HMBC spectrum. H1.10) / C-29(δ C 183.4), C-21(δ C 39.1); H2-19(δ) H 1.34) / C-30(δ C 27.6), C-13(δ) C 52.0) and 1 H- 1 The H COSY spectrum shows spin-coupled fragments at H-11 / H-12, H-15 / H-16, H-18 / H-19, and H-21 / H-22, and a methyl group at C-14 is linked to a carboxyl group at C-30 via esterification to form a lactone. The cross signals at H-26 / H-28 / H-18 / H-30 in the NOESY spectrum indicate that these groups are located on the same side of the molecular plane. Comparison of experimental CD and calculated ECD curves, as shown... Figure 31 As shown, the absolute configuration of compound 8 was determined to be 13S,14S,17R,18R,20R, and the compound was named isotripterinⅢ.

[0107] Compound 9, according to its HR-ESIMS (m / z) 451.2842 [M+H] + Analysis shows that the molecular formula is C 29 H 38 O4 has an unsaturation degree of 11. 1 The H NMR spectrum shows three aromatic proton signals [δ] H 7.26 (1H, s), δ H 7.62 (1H,d,J=8.6Hz),δ H 7.18 (1H, d, J = 8.6 Hz)], 6 methyl groups, 7 aliphatic methylene groups, and 1 aliphatic methine group. In 13 The C NMR spectrum also includes signals from 7 aromatic quaternary carbons, 3 aliphatic quaternary carbons, 1 oxygen-bound quaternary carbon, and 1 ester carbonyl carbon.

[0108] Based on the correlation signal H-28(δ) in the HMBC spectrum H 1.10) / C-16(δ C 34.0), C-17(δ) C 35.8), C-18(δ) C 90.3), C-22(δ) C 35.8); H-30 (δ) H 1.10) / C-21(δ C 31.2), C-20(δ) C 43.9), C-19(δ) C 41.7), C-29(δ)C 181.4) This study demonstrated that esterification occurred at positions C-18 and C-29 in the molecular structure of compound 30, thus determining its planar structure. The relative configuration of compound 9 was determined by the correlation signals of H-30 / H2-19 and H-28 / H2-19 in the NOESY spectrum. The experimental CD curves and the ECD-calculated curves showed good agreement, as shown in the results. Figure 35 As shown, the absolute configuration of compound 9 is 13S,17R,20S. Compound 9 is named isotripterin IV.

[0109] Compound 10, according to its HR-ESIMS (m / z) 487.2816 [M+H] + Analysis shows that the molecular formula is C 29 H 38 O5 has an unsaturation degree of 11. 1 The H NMR spectrum includes two aromatic proton signals [δ] H 7.42 (1H, d, J = 7.5Hz), δ H 7.14, (1H, d, J = 7.5 Hz)], 2 olefin proton signals [δ H 7.07 (1H, s), δ H 5.15 (1H,dd,J=6.4,4.9Hz)], 6 methyl groups, 6 aliphatic methylene groups, 1 aliphatic methine group. In 13 The C NMR spectrum also includes signals from 4 aromatic quaternary carbons, 2 olefin quaternary carbons, 3 aliphatic quaternary carbons, 1 oxygen-linked quaternary carbon, and 2 carbonyl carbons.

[0110] Based on the HMBC spectral correlation signal H-1(δ) H 7.07) / C-3(δ C 200.4), C-5(δ) C 139.8), C-9(δ) C 138.3); H-7 (δ) H 7.14) / C-5(δ C 139.8); and H-25 (δ) H 2.30) / C-7(δ C 130.3), C-9(δ) C 138.3) confirmed the structural fragments of rings A and B, proving that the carbonyl group is located at C-3, a hydroxyl group is attached to each of the C-2 and C-5 positions, and the two methyl groups are located at C-4 and C-7 positions, respectively. Based on H-12 (δ... H 5.15) / C-9(δ C 138.3), C-14(δ) C 35.0), C-18(δ)C The HMBC signal in 2.71) indicates that the double bond is located between C-12 and C-13. 1 H- 1 The H COSY spectrum shows H-6 / H-7, H-11 / H-12, H-15 / H-16, H-18 / H-19, and H-21 / H-22 related signals, indicating the presence of structural fragments of C-6 / C-7, C-11 / C-12, C-15 / C-16, C-18 / C-19, and C-21 / C-22, combined with H-26 (δ H 1.13) / C-15(δ C 36.4) and H-30 (δ) H 1.36) / C-19(δ C 35.9), C-21(δ) C 29.1), C-29(δ) C The HMBC correlation signal (182.8) led to the derivation of the planar structure of compound 10. Correlation signals of H-18 / H-28 / H-30 were observed in the NOESY spectrum, indicating that these proton hydrogens are located on the same side of the plane. Comparison of experimental CD curves and calculated ECD curves, such as... Figure 29 The absolute configuration of compound 10 was confirmed to be 17S,18S,20R, and it was named isopristimerin V-1.

[0111] Activity test examples:

[0112] Experimental methods:

[0113] Activity screening experiments were performed using ADP-Glo ​​reagent (Promega Corporation, Madison, USA, catalog number: V9102), and ACLY activity was determined by the amount of ADP generated during the reaction. The enzyme reaction buffer was optimized to contain 40 mM tris (pH 8.0), 4 mM dithiothreitol (DTT), 10 mM MgCl2, 1% dimethyl sulfoxide (DMSO, v / v), 0.01% Brij35 (v / v), and 0.001% bovine serum albumin (BSA, v / v). ACLY protein and the original compound solution (i.e., the dilution buffer for compound 5) were diluted in this reaction buffer. In a 384-well plate, 5 μL of 30 nMACLY (ATP citrate lyase) solution was mixed with 5 μL of the compound solution and incubated at room temperature for 15 min. Then, 5 μL of a substrate mixture of 600 μM citrate, 300 μM ATP (adenine triphosphate), and 300 μM CoA (coenzyme A) was added. The final concentration of ACLY monomer in the reaction was 10 nM (the final concentrations of compound 5 were 100 μM, 25 μM, 6.25 μM, 1.563 μM, 0.391 μM, 0.098 μM, 0.024 μM, and 0.006 μM, respectively). The reaction was incubated at 37 °C for 55 min, then transferred to room temperature for 5 min. 10 μL of LADPGlo was added. TM The reagent was incubated at room temperature for 40 minutes to terminate the reaction and consume excess ATP. Finally, 20 μL of kinase assay reagent (Promega, ADPGlo) was added. TM (Included in the kit), incubate at room temperature for 40 min to convert ADP to ATP. Detect the luminescence signal at 562 nm using a photometer plate reader (Ensight, PerkinElmer).

[0114] Information on the ACLY inhibitory activity of compound 5 is as follows: Figure 40 As shown, the test results indicate that compound 5 has significant inhibitory activity against ACLY and can be developed as a lead compound for the treatment of cancer and cardiovascular diseases.

[0115] The triterpenoid compounds 1-10 provided in this invention are all novel terpenoid compounds extracted and isolated from *Tripterygium wilfordii* Hook.f., a vine-like shrub belonging to the genus *Tripterygium* in the family Celastraceae. Bioactivity experiments show that compound 5 possesses ATP citrate lyase (ACLY) inhibitory activity, and has broad application prospects in the development of drugs for various cancers, hyperlipidemia, and atherosclerotic cardiovascular diseases.

Claims

1. A tripterygium diterpenoid and triterpenoid compound, characterized in that: The compound is one or more of the following: a compound having the structure shown in general formula (I), a compound of general formula (II) with different crystal forms thereof, a chiral isomer thereof, a glycoside thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, a prodrug thereof, or a metabolite thereof. In compound 1, R1-R2 are selected from α-hydrogen, R3 is selected from β-methyl, R4 is selected from hydroxyl, and R5 is selected from hydroxyl. In compound 2, R1-R6 are each independently one or more of the following groups: β-hydrogen, methyl, carboxyl, β-hydrogen, α-methyl, and hydroxyl. In compound 3, R1-R2 are selected from hydroxyl groups, R3 from methyl groups, R4 from β-hydrogen groups, R5 from α-methyl groups, R6 from β-methyl groups, R7 from α-methyl groups, R8 from α-methyl groups, R9 from α-hydrogen groups, and R... 10 Selected from α-methyl; In compound 4, R1 is selected from hydroxyl, R2 from carbonyl, R3 from methyl, R4 from β-hydrogen, R5 from β-methyl, R6 from α-methyl, R7 from α-aldehyde, R8 from β-hydrogen, R9 from α-methyl, and R... 10 Selected from carboxyl groups; In compound 5, R1 is selected from hydroxyl, R2 from hydroxyl, R3 from aldehyde, R4 from methyl, R5 from α-methyl, R6 from β-methyl, R7 from β-methyl, R8 from β-hydrogen, R9 from methyl, and R... 10 Selected from carboxyl groups; In compound 6, R1 is selected from hydroxyl group, R2 is selected from β-methyl, R3 is selected from β-methyl, R4 is selected from α-methyl, R5 is selected from carboxyl, R6 is selected from α-methyl, and R7 is selected from β-methyl; In compound 7, R1 is selected from hydroxyl, R2 from carbonyl, R3 from α-hydrogen, R4 from β-methyl, R5 from hydrogen, R6 from β-methyl, R7 from α-methyl, R8 from β-methyl, R9 from β-hydrogen, and R... 10 Selected from β-methyl; In compound 8, R1 is selected from hydroxyl, R2 is selected from hydroxyl, R3 is selected from methyl, R4 is selected from methyl, R5 is selected from α-methyl, R6 is selected from β-methyl, R7 is selected from β-methyl, R8 is selected from β-hydrogen, and R9 is selected from β-methyl. In compound 9, R1 is selected from hydroxyl, R2 is selected from hydroxyl, R3 is selected from methyl, R4 is selected from methyl, R5 is selected from methyl, R6 is selected from methyl, R7 is selected from β-methyl, and R8 is selected from β-hydrogen; In compound 10, R1 is selected from hydroxyl, R2 from carbonyl, R3 from hydroxyl, R4 from methyl, R5 from methyl, R6 from methyl, R7 from methyl, R8 from β-methyl, R9 from β-hydrogen, and R... 10 Selected from β-methyl, R 11 Selected from carbonyl groups.

2. The compound according to claim 1, characterized in that: The compound is one or more of the following 10 structural compounds, or compounds of general formula (II) with different crystal forms thereof, or chiral isomers thereof, or glycosides thereof, or pharmaceutically acceptable salts thereof, or solvates thereof, or prodrugs thereof, or metabolites thereof.

3. A method for preparing the compound according to claim 2, characterized in that: Includes the following steps: (1) Extraction of medicinal materials: The dried roots of Tripterygium wilfordii were crushed into 30-50 mesh powder, with a mass of 15-20 kg; dynamic extraction was carried out using supercritical carbon dioxide at 40-50℃ and 40-50 MPa; anhydrous ethanol was used as an additive, and the material was extracted for 4-6 hours at a material-to-liquid ratio of 1:4-1:5 (g / ml); the extract was concentrated to obtain a solid crude extract. (2) The crude extract obtained in step (1) was dissolved in dichloromethane and purified by first-dimensional separation using normal phase chromatography. The stationary phase was a silica gel column (50 mm × 250 mm, 10 μm), and the mobile phase consisted of (A) dichloromethane and (B) methanol. The gradient was as follows: 0.0–15.0 min: 0% B, 15.0–38.0 min: linear gradient to 3% B, 38.0–48.0 min: linear gradient to 5% B, 48.0–60.0 min: linear gradient to 90% B. The flow rate was 65–80 mL / min, and the detection wavelengths were 230 nm and 268 nm. Four sub-fractions, F1–F4, were collected according to the UV absorption peaks. (3) The Tripterygium wilfordii fraction F3 (25.0–42.0 min) obtained in step (2) was purified by second-dimensional separation using a C18HD (200 mm × 250 mm, 10 μm) reversed-phase column. The mobile phase was (A) acetonitrile (v / v) and (B) 0.1%–0.5% formic acid-water (volume concentration, the same below); the flow rate was 800–1200 mL / min, the detection wavelengths were 230 and 268 nm, the injection volume was 500–700 mL, and the elution gradient was 0–13.0 min: 40.0% B, 13.0–25.0 min: linear gradient to 37.0% B, 25.0–35.0 min: linear gradient to 20.0% B, 35.0–55.0 min: linear gradient to 5.0% B; a total of 10 sub-fractions (F3-1–F3-10) were collected. (4) The fractions F3-6 (19.0-22.0 min), F3-8 (28.0-33.0 min), F3-9 (33.0-40.5 min) and F3-10 (40.5-55.0 min) obtained in step (3) were separated and purified in the third dimension using a QUIS (50 mm × 250 mm, 10 μm) (reversed phase column). The mobile phase was (A) acetonitrile (v / v) and (B) 0.1%-0.5% formic acid-water. The flow rate was 60–80 mL / min, the detection wavelengths were 230 and 268 nm, and the elution gradient for F3-6 was 0–20.0 min: linear gradient 65%–30% B, 20.0–25.0 min: linear gradient 30.0–10% B, and 25.0–30.0 min: 10.0% B; a total of 5 sub-fractions (F3-6-1 to F3-6-5) were collected. The elution gradient for F3-8 was 0–50.0 min: linear gradient 70%–30% B, 50.0–52.0 min: linear gradient 30.0–10% B, and 52.0–60.0 min: 10.0% B; a total of 6 sub-fractions (F3-8-1 to F3-8-6) were collected. The elution gradient for F3-9 was 0–50.0 min: linear gradient 70%–40% B, 50.0–52.0 min: linear gradient 40.0–10% B, and 52.0–60.0 min: 10.0% B; a total of 6 sub-fractions (F3-9-1 to F3-9-8) were collected. The elution gradient for F3-10 was 0–50.0 min: linear gradient 65%–40% B, 50.0–52.0 min: linear gradient 40.0–10% B, and 52.0–60.0 min: 10.0% B; a total of 9 sub-fractions (F3-10-1 to F3-10-9) were collected. (5) The fractions F3-6-2, F3-6-3, F3-8-3, F3-9-4, F3-10-5, F3-10-6 and F3-10-7 obtained in step (4) are separated and purified in the fourth dimension, with the mobile phase being (A) acetonitrile (v / v) and (B) 0.1% to 0.5% formic acid-water. For F3-6-2, separation was performed using a PHN (30 mm × 250 mm, 10 μm) reversed-phase column with elution gradients of 0.0–30.0 min, 46% A; 30.0–38.0 min, linear gradient 46%–90% A; 38.0–45.0 min, 90% A, at a flow rate of 20–30 mL / min. A total of 11 fractions (F3-6-2-1 to F3-6-2-11) were collected. For F3-6-3, separation was performed using a C18ME (50 mm × 250 mm, 10 μm) reversed-phase column with elution gradients of 0.0–30.0 min. The linear gradient was 55% A for 30.0–38.0 min, followed by 90% A for 38.0–45.0 min, at a flow rate of 60–80 mL / min, collecting a total of 13 fractions (F3-6-3-1 to F3-6-3-13). For F3-8-3, separation was performed using a PHN (50 mm × 250 mm, 10 μm) reversed-phase column with an elution gradient of 0.0–35.0 min, 47% A for 35.0–45.0 min, followed by 90% A for 45.0–60.0 min, at a flow rate of 60–80 mL / min. Thirteen sub-fractions (F3-8-3-1 to F3-8-3-13) were collected. For F3-9-4, separation was performed using a C18ME (50 mm × 250 mm, 10 μm) reversed-phase column with an elution gradient of 0.0–30.0 min, 70% A; 30.0–35.0 min, linear gradient of 70%–90% A; and 35.0–50.0 min, 90% A. The flow rate was 60–80 mL / min, and twelve sub-fractions (F3-9-4-1 to F3-9-4-12) were collected. For F3-10-5, separation was performed using a C18ME (50 mm × 250 mm, 10 μm) column. Separation was performed using a C18ME (50 mm × 250 mm, 10 μm) reversed-phase column with elution gradients of 0.0–30.0 min, 70% A; 30.0–35.0 min, linear gradient 70%–90% A; and 35.0–50.0 min, 90% A, at a flow rate of 60–80 mL / min. Eleven fractions (F3-10-5-1 to F3-10-5-11) were collected. For F3-10-6, separation was performed using a C18ME (50 mm × 250 mm, 10 μm) reversed-phase column with elution gradients of 0.0–30.0 min, linear gradient 76%–90% A; and 30.0–55.0 min, linear gradient 70%–90% A.For fraction F3-10-6-1 to F3-10-6-22, separation was performed using a C18ME (50 mm × 250 mm, 10 μm) reversed-phase column at a flow rate of 60–80 mL / min. The elution gradient was 0.0–30.0 min with a linear gradient of 76%–90% A. For fraction F3-10-7, separation was performed at 30.0–40.0 min with a flow rate of 60–80 mL / min at 90% A, again collecting 22 fractions (F3-10-7-1 to F3-10-7-22). (6) The fractions F3-6-2-6, F3-6-2-11, F3-6-3-3, F3-8-3-8, F3-9-4-9, F3-10-5-9, F3-10-6-10, F3-10-6-20, F3-10-7-10, and F3-10-7-11 obtained in step (5) are subjected to fifth-dimensional separation and purification. For reversed-phase mode, the mobile phase is (A) methanol (v / v) and (B) 0.1% formic acid-water, with a flow rate of 3-4 mL / min; for SFC mode, the mobile phase is (A) CO2 (v / v) and (B) 0.1% formic acid-methanol, with a flow rate of 8-10.0 mL / min; for F3-6-2-6, BH (10 mm × For compound 1, separation was performed using a C18ME (10mm × 250mm, 10μm) reversed-phase column with an elution gradient of 0–12.0 min, linear gradient to 8–30% B; 12.0–16.0 min, linear gradient to 55% B; and 16.0–20.0 min, linear gradient to 10% B. For compound 7, separation was performed using a C18ME (10mm × 250mm, 10μm) reversed-phase column with an elution gradient of 0–60 min, 80% A. For compound 8, separation was performed using a C18ME (10mm × 250mm, 10μm) reversed-phase column with an elution gradient of 0–60 min, 45% A. Compound 2 was obtained; for F3-8-3-8, separation was performed using a C3HC (10 mm × 250 mm, 10 μm) reversed-phase column with an elution gradient of 0–60 min, 65% A, to obtain compound 4; for F3-9-4-9, separation was performed using an X5H (10 mm × 250 mm, 10 μm) SFC column with an elution gradient of 0–12.0 min, 5–30% B; 12.0–16.0 min, linear gradient to 55% B; 16.0–20.0 min, linear gradient to 5% B, to obtain compound 3; for F3-10-5-9, separation was performed using a C18HD (10 mm × 250 mm, 10 μm) reversed-phase column, elution... The elution gradient was 0-60 min, 81% B, to obtain compound 9; for F3-10-6-10, separation was performed using a BH (10 mm × 250 mm, 10 μm) (SFC column) with an elution gradient of 0-12.0 min, 10-30% B; 12.0-13.0 min, linear gradient 30-55% B; 13.0-17.0 min, 55% B, to obtain compound 8; for F3-10-6-20, separation was performed using an X5H (10 mm × 250 mm, 10 μm) (SFC column) with an elution gradient of 0-12.0 min, 5-25% B; 12.0-16.0 min, linear gradient to 55% B; 16.0-20.Compound 6 was obtained by elution with a linear gradient of 0-60 min and 5% B. For F3-10-7-10, separation was performed using a C18HD (10 mm × 250 mm, 10 μm) reversed-phase column with an elution gradient of 0-60 min and 82% A. Compound 10 was obtained. For F3-10-7-11, separation was performed using a PHN (10 mm × 250 mm, 10 μm) reversed-phase column with an elution gradient of 0-60 min and 70% A. Compound 5 was obtained.

4. The use of one or more of the following compounds, crystal forms, isomers, glycosides, pharmaceutically acceptable salts, solvates, prodrugs, or metabolites as claimed in any one of claims 1-2, in the preparation of a medicament for the prevention and / or treatment of one or more of cancer, hyperlipidemia, atherosclerotic cardiovascular diseases, and central nervous system-related diseases.

5. A pharmaceutical composition comprising one or more of the compound of any one of claims 1 to 2, or its crystal form, or its isomer, or its glycoside, or its pharmaceutically acceptable salt, or its solvate, or its prodrug, or its metabolite, and any other pharmaceutically acceptable excipient, carrier, diluent, or other active ingredient.

Images