Schisandracean leaf-derived triterpenoids and uses thereof

Abstract

The present application relates to schisandracean leaf-derived triterpenoids and their use. The schisandracean leaf-derived triterpenoids are extracted from the leaves of Schisandra chinensis, have excellent neuroprotective effects, and are expected to be used for the prevention and treatment of neurodegenerative diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, and more specifically, to the triterpenoid compounds of Schisandra chinensis leaves and their applications. Background Technology

[0002] Schisandra chinensis (Turcz.) Baill. is a deciduous woody vine belonging to the genus Schisandra in the family Magnoliaceae. It prefers slightly acidic humus soil and has poor drought tolerance. Young branches are reddish-brown, while older branches are grayish-brown, often wrinkled and peeling off in flakes. The entire plant is nearly hairless, with angular tips. Petioles are 1-4 cm long. Leaves are membranous, broadly elliptic, obovate, or nearly orbicular, 5-10 cm long and 3-5 cm wide. The apex is acute, the base cuneate, and the margins are dentate. Flowers are mostly unisexual and dioecious, occasionally monoecious, solitary or clustered in leaf axils. The perianth segments are pinkish-white or pink. Male flowers have pedicels 5-25 mm long with nearly obovate stamens; female flowers have pedicels 17-38 mm long with nearly ovate stamens. The aggregate berries are 1.5-8.5 cm long, red, and nearly spherical or obovate. The pulp has a slightly sour taste and a unique aroma when crushed. Mature fruits are deep red and contain 1-2 seeds. The seeds are light brown, kidney-shaped, 4-5 mm long and 2.5-3 mm wide, with a smooth seed coat and a distinct U-shaped hilum. Flowering occurs from May to July, and fruiting from July to October. As one of my country's traditional major medicinal materials, the fruit is often used medicinally. It has astringent, qi-tonifying, fluid-generating, kidney-tonifying, heart-calming, and tranquilizing effects, and can be used to treat symptoms such as lung deficiency cough, dry mouth and thirst, spontaneous sweating and night sweats, nocturnal emission, insomnia, dreaminess, forgetfulness, and fatigue.

[0003] Schisandra chinensis leaves contain lignans, flavonoids, and phenolic acids. Wu Yanan, Wen Sihua, Wu Xu, Zhao Chongbo, Liu Hang, Sun Jing, et al., compared seven components in Schisandra chinensis fruits and leaves (Northwest Pharmaceutical Journal, 2022, Vol. 37, No. 4, pp. 15-19); Jin Yinping, Qu Zhengyi, Cui Lili, Pu Xiangmin, Guo Jing, Wang Yingping, et al., determined the content and antioxidant activity of flavonoids and phenolic acids in different parts of Schisandra chinensis (Chinese Journal of Experimental Traditional Medical Formulae, 2018, Vol. 24, No. 19, pp. 79-84). However, there are few reports on the systematic isolation, identification, and application of triterpenoids contained in Schisandra chinensis leaves. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention provides schisandra leaf triterpenoid compounds of compounds 1-9 or their pharmaceutically acceptable salts, prodrugs, stereoisomers, solvates, and their use for neuroprotection.

[0005] In one aspect, the present invention provides the following Schisandra chinensis leaf triterpenoid compounds 1-9, or pharmaceutically acceptable salts, prodrugs, stereoisomers, and solvates thereof:

[0006]

[0007] In this invention, the prodrug refers to a compound that is converted into an active compound as a result of a reaction with enzymes, gastric acid, etc. in vivo. Many types of prodrugs are known, such as esters, including methyl esters and ethyl esters, and suitable prodrugs can be selected from known literature and synthesized by known methods.

[0008] In this invention, the solvate refers to a compound that forms a solid or liquid complex by coordinating with solvent molecules.

[0009] The present invention also provides a pharmaceutical composition comprising the said compound or a pharmaceutically acceptable salt, prodrug, stereoisomer, or solvate thereof.

[0010] The present invention also provides pharmaceutical compositions of the said compound or pharmaceutically acceptable salts, prodrugs, stereoisomers, or solvates thereof, or the use of said pharmaceutical compositions in the preparation of a medicament for neuroprotection.

[0011] Preferably, the drug is used to protect SH-SY5Y cells.

[0012] The present invention also provides pharmaceutical compositions of the said compound or pharmaceutically acceptable salts, prodrugs, stereoisomers, or solvates thereof, or the use of said pharmaceutical compositions in the preparation of a medicament for the prevention or treatment of neurodegenerative diseases.

[0013] Preferably, the neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS).

[0014] Preferably, the pharmaceutical composition or drug of the present invention further comprises a pharmaceutically acceptable carrier or excipient.

[0015] As pharmaceutically acceptable carriers or excipients, any carrier or excipient known to be suitable for the preparation of a particular drug or drug composition may be used. Examples include, but are not limited to, solvents, excipients, dispersants, emulsifiers, solubilizers, gelling agents, ointment bases, antioxidants, preservatives, stabilizers, carriers, fillers, binders, thickeners, complexing agents, disintegrants, buffers, penetration enhancers, polymerizers, lubricants, coating agents, propellants, tension modifiers, surfactants, colorants, flavoring agents, sweeteners, and dyes. Excipients of a type suitable for the desired formulation and route of administration are used in particular.

[0016] Furthermore, the pharmaceutically acceptable carrier or excipient may be selected from, for example, water, starch, compressible starch, dextrin, sucrose, lactose, fructose, glucose, xylitol, mannitol, microcrystalline cellulose, calcium carbonate, magnesium carbonate, calcium phosphate, calcium hydrogen phosphate, calcium sulfate, magnesium oxide, aluminum hydroxide, calcium carboxymethyl cellulose, carboxymethyl cellulose, sodium carboxymethyl starch, hydroxypropyl starch, croscarmellose sodium, croscarmellose, low-substituted hydroxypropyl methylcellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, starch Powder paste, glucose syrup, sucrose syrup, fructose syrup, sorbitol, gelatin paste, gum arabic paste, tragacanth paste, microcrystalline cellulose, methylcellulose, sodium carboxymethyl cellulose, ethylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, calcium carboxymethyl cellulose, polymethyl acrylate, alginic acid, sodium alginate, polyethylene glycol, colloidal magnesium aluminum silicate, stearic acid, calcium stearate, magnesium stearate, zinc stearate, talc, glyceryl monostearate, glyceryl palmitate, magnesium dodecyl sulfate, polyethylene glycol, sodium stearate.

[0017] The pharmaceutical composition or drug is presented in a unit dosage form, such as tablets, pills, capsules, powders, granules, suspensions, oral liquids, aerosols or liquid sprays, or drops; dosage forms for oral, parenteral, intranasal, sublingual, or rectal administration, or for inhalation or spray administration. Preferably, the pharmaceutical dosage form is an oral preparation, more preferably tablets, pills, capsules, granules, suspensions, powders, or oral liquids.

[0018] The pharmaceutical compositions or drugs of the present invention can be manufactured using methods well known in the art, such as conventional mixing, dissolving, granulation, sugar-coated pill making, grinding, emulsification, freeze-drying, etc. Pharmaceutically, the content of the active ingredient in the pharmaceutical compositions of the present invention should be in the range of 0.05% to 90% by weight, preferably 0.1% to 50% by weight, of the total composition.

[0019] For adult patients, the compounds of the present invention can be administered orally or non-orally in doses of 0.001–500 mg, once daily or divided into several doses. It should be noted that the dosage may be adjusted according to the type of disease, age, weight, symptoms, etc., of the patient being treated.

[0020] The present invention also provides a method for preparing compounds 1-9, wherein Schisandra chinensis leaves are extracted with a solvent to obtain a total extract of Schisandra chinensis, and then compounds 1-9 are separated from the total extract of Schisandra chinensis.

[0021] Preferably, the solvent extraction includes extraction by heating with an organic solvent.

[0022] The organic solvent is selected from at least one of methanol and ethanol, preferably 60-95% ethanol, more preferably 70-85% ethanol; it can be heated to reflux; the mass of the solvent during extraction is 5-20 times the mass of the Schisandra chinensis leaves; the number of extractions can be 1-5 times, preferably 2-4 times.

[0023] After extraction, the extracts were filtered, combined, and the solvent was removed by vacuum evaporation to obtain the total extract of Schisandra chinensis.

[0024] Preferably, the separation of compounds 1-9 from the total extract of Schisandra chinensis includes separating the total extract of Schisandra chinensis through a macroporous resin, followed by separation by silica gel column chromatography, MCI column chromatography and ODS column chromatography, gel column chromatography, and preparative HPLC to obtain compounds 1-9.

[0025] The macroporous adsorption resin separation includes loading the total extract of Schisandra chinensis onto a macroporous adsorption resin column, performing gradient elution with water-ethanol (1:0 to 5:95, v / v), and collecting the eluent fraction containing 95% ethanol.

[0026] Preferably, the macroporous adsorption resin includes HPD-100 macroporous adsorption resin, AB-8 macroporous adsorption resin, and X-5 macroporous adsorption resin.

[0027] Preferably, the water-ethanol gradient elution includes eluting sequentially with water, 15-25% ethanol, 35-45% ethanol, and 95% ethanol; more preferably, it includes eluting sequentially with water, 20% ethanol, 40% ethanol, and 95% ethanol. During elution, the volume of each eluent is 1-6 BV, which can be 1 BV, 2 BV, 3 BV, 4 BV, 5 BV, or 6 BV.

[0028] Preferably, the silica gel column separation includes loading the eluent of 95% ethanol onto a silica gel column, performing gradient elution with dichloromethane-methanol (1:0 to 0:1, v / v), identifying the eluent by thin-layer chromatography, and combining the eluents to obtain 8 fractions: Fr.A to H.

[0029] In one embodiment, Fr.C is separated into Fr.C1 to Fr.C8 by MCI column chromatography (mobile phase: methanol-water system, methanol to water volume ratio: 8:2) followed by ODS column chromatography (mobile phase: methanol-water system, methanol to water volume ratio: 3:7-9:1). Fr.C4 is further separated into Fr.C4-1 to Fr.C8 by Sephadex LH-20 gel column chromatography (mobile phase: dichloromethane-methanol system, dichloromethane to methanol volume ratio: 1:1). Fr.C4-4 is then separated into compound 2 (t) by preparative HPLC (methanol / water = 62%, 5 mL / min). R =29.3 min, 5.2 mg).

[0030] In one embodiment, Fr.D is separated into Fr.D1 to D10 by MCI column chromatography (mobile phase: methanol-water system, methanol to water volume ratio: 8:2) followed by ODS column chromatography (mobile phase: methanol-water system, methanol to water volume ratio: 3:7-9:1). Fr.D4 is separated into Fr.D4-1 to Fr.D7 by Sephadex LH-20 gel column chromatography (mobile phase: dichloromethane-methanol system, dichloromethane to methanol volume ratio: 1:1). Fr.D4-4 is separated into Fr.D4-4-1 to Fr.D4-18 by silica gel column chromatography (mobile phase: petroleum ether-acetone system, petroleum ether to acetone volume ratio: 5:1-0:1). Fr.D4-4-7 is separated into compound 1 (t) by preparative HPLC (methanol / water = 60%, 5 mL / min). R =27.2min, 2.5mg), compound 4 (t R =30.7min, 4.0mg), compound 9 (t R =34.6 min, 6.2 mg); Fr.D4-4-9 was separated by preparative HPLC (methanol / water = 60%, 5 mL / min) to obtain compound 3 (t R =26.4 min, 3.8 mg); Fr.D4-4-10 was separated by preparative HPLC (methanol / water = 60%, 5 mL / min) to obtain compound 6 (t R =27.0 min, 7.2 mg); Fr.D4-4-11 was separated by preparative HPLC (methanol / water = 58%, 5 mL / min) to obtain compound 5 (t R =28.2min, 3.2mg), compound 7 (t R =32.0 min, 8.1 mg).

[0031] Preferably, Fr.D6 was separated into Fr.D6-1 to Fr.D6-8 by Sephadex LH-20 gel column chromatography (mobile phase: dichloromethane-methanol system, dichloromethane to methanol volume ratio: 1:1), and Fr.D6-5 was separated into Fr.D6-5-1 to Fr.D6-12 by silica gel column chromatography (mobile phase: petroleum ether-acetone system, petroleum ether to acetone volume ratio: 5:1-0:1). Fr.D6-5-6 was separated into compound 8 (t) by preparative HPLC (methanol / water = 62%, 5 mL / min). R =33.5 min, 4.3 mg); Fr.D6-5-7 was separated by preparative HPLC (methanol / water = 60%, 5 mL / min) to obtain compound 9 (t). R =34.6 min, 6.2 mg).

[0032] In this invention, the percentage of ethanol refers to the volume fraction.

[0033] Beneficial effects

[0034] This invention provides a triterpenoid compound extracted from Schisandra chinensis leaves, which has excellent neuroprotective effects and is expected to be used for the prevention and treatment of neurodegenerative diseases. Attached Figure Description

[0035] Figure 1 Flowchart for isolating compounds 1-9 from Schisandra chinensis leaves;

[0036] Figure 2 HMBC of compound 1 1 H- 1 Key correlation signals in H COSY and NOESY spectra;

[0037] Figure 3 ECD spectrum of compound 1;

[0038] Figure 4 HMBC of compound 2 1 H- 1 Key correlation signals in H COSY and NOESY spectra;

[0039] Figure 5 Crystallographic structure of compound 2;

[0040] Figure 6 HMBC of compound 3 1 H- 1 Key correlation signals in H COSY and NOESY spectra;

[0041] Figure 7 Crystallographic structure of compound 3;

[0042] Figure 8 HMBC of compound 4 1 H- 1 Key correlation signals in H COSY and NOESY spectra;

[0043] Figure 9 HMBC of compound 5 1 H- 1 Key correlation signals in H COSY and NOESY spectra;

[0044] Figure 10 HMBC of compound 6 1 H- 1 Key correlation signals in H COSY and NOESY spectra;

[0045] Figure 11 Crystallographic structure of compound 6;

[0046] Figure 12 HMBC of compound 7 1 H- 1 Key correlation signals in H COSY and NOESY spectra;

[0047] Figure 13 HMBC of compound 8 1 H- 1 Key correlation signals in H COSY and NOESY spectra;

[0048] Figure 14 ECD spectrum of compound 8;

[0049] Figure 15 HMBC of compound 9 1 H- 1 Key correlation signals in H COSY and NOESY spectra;

[0050] Figure 16 ECD spectrum of compound 9. Detailed Implementation

[0051] The invention is described in more detail below to aid in understanding it.

[0052] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Where specific techniques or conditions are not specified in the embodiments, they should be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions.

[0053] Experimental materials and instruments

[0054] Experimental materials

[0055]

[0056] Experimental instruments

[0057]

[0058] Extraction and separation

[0059] 50 kg of dried Schisandra chinensis leaves were extracted three times by heating and reflux with 10 times their weight of 80% ethanol for 2 hours each time. The extracts were filtered, the filtrates were combined, and the solvent was recovered under reduced pressure to obtain 10.15 kg of total extract (yield 20.3%). The total extract of Schisandra chinensis leaves was then passed through HPD-100 macroporous adsorption resin at 1 BV·h. -1The column chromatography elution was performed at a flow rate of [flow rate missing]. The eluents were water (2 BV), 20% ethanol (2 BV), 40% ethanol (2 BV), and 95% ethanol (4 BV) in sequence. The corresponding eluents were collected and concentrated under reduced pressure to obtain 2.74 kg of water eluent, 0.91 kg of 20% ethanol eluent, 1.82 kg of 40% ethanol eluent, and 1.64 kg of 95% ethanol eluent.

[0060] The 95% ethanol eluent (0.55 kg) was subjected to silica gel column chromatography with a dichloromethane-methanol gradient elution (1:0 to 0:1, v / v). The eluent was identified by thin-layer chromatography, and eight fractions were obtained by combining the eluents: Fr. A to H. Fr. B to D were systematically separated using a combination of silica gel, MCI, ODS column chromatography, and preparative HPLC. For detailed separation procedures, please refer to [link to procedure]. Figure 1 The steps are as follows:

[0061] Fr.C was separated into Fr.C1 to Fr.C8 by MCI column chromatography (mobile phase: methanol-water system, methanol to water volume ratio: 8:2) followed by ODS column chromatography (mobile phase: methanol-water system, methanol to water volume ratio: 3:7-9:1). Fr.C4 was further separated into Fr.C4-1 to Fr.C8 by Sephadex LH-20 gel column chromatography (mobile phase: dichloromethane-methanol system, dichloromethane to methanol volume ratio: 1:1). Fr.C4-4 was separated into compound 2 (t) by preparative HPLC (methanol / water = 62%, 5 mL / min). R =29.3 min, 5.2 mg).

[0062] Fr.D was separated into Fr.D1 to D10 by MCI column chromatography (mobile phase: methanol-water system, methanol to water volume ratio: 8:2) followed by ODS column chromatography (mobile phase: methanol-water system, methanol to water volume ratio: 3:7-9:1). Fr.D4 was further separated into Fr.D4-1 to Fr.D7 by Sephadex LH-20 gel column chromatography (mobile phase: dichloromethane-methanol system, dichloromethane to methanol volume ratio: 1:1). Fr.D4-4 was separated into Fr.D4-4-1 to Fr.18 by silica gel column chromatography (mobile phase: petroleum ether-acetone system, petroleum ether to acetone volume ratio: 5:1-0:1). Fr.D4-4-7 was separated into compound 1 (t) by preparative HPLC (methanol / water = 60%, 5 mL / min). R =27.2min, 2.5mg), compound 4 (t R =30.7min, 4.0mg), compound 9 (t R =34.6 min, 6.2 mg); Fr.D4-4-9 was separated by preparative HPLC (methanol / water = 60%, 5 mL / min) to obtain compound 3 (tR =26.4 min, 3.8 mg); Fr.D4-4-10 was separated by preparative HPLC (methanol / water = 60%, 5 mL / min) to obtain compound 6 (t R =27.0 min, 7.2 mg); Fr.D4-4-11 was separated by preparative HPLC (methanol / water = 58%, 5 mL / min) to obtain compound 5 (t R =28.2min, 3.2mg), compound 7 (t R =32.0 min, 8.1 mg);

[0063] Fr.D6 was separated into Fr.D6-1 to Fr.D6-8 by Sephadex LH-20 gel column chromatography (mobile phase: dichloromethane-methanol system, dichloromethane to methanol volume ratio: 1:1). Fr.D6-5 was separated into Fr.D6-5-1 to Fr.D6-12 by silica gel column chromatography (mobile phase: petroleum ether-acetone system, petroleum ether to acetone volume ratio: 5:1-0:1). Fr.D6-5-6 was separated into compound 8 (t) by preparative HPLC (methanol / water = 62%, 5 mL / min). R =33.5 min, 4.3 mg); Fr.D6-5-7 was separated by preparative HPLC (methanol / water = 60%, 5 mL / min) to obtain compound 9 (t). R =34.6 min, 6.2 mg).

[0064] The structures of nine triterpenoid compounds were identified by physicochemical constant determination and various spectroscopic data analysis (UV, NMR, MS, etc.), as shown below:

[0065]

[0066] The 1H and 1C NMR spectra of compounds 1-9 are shown in Table 1-3:

[0067] Table 1. Compounds 1-3 1 H NMR (600MHz) and 13 C10 NMR (150MHz) data (C5D5N, δin ppm, JinHz)

[0068]

[0069]

[0070] Table 2. Compounds 4-6 1 H NMR (600MHz) and 13 C10 NMR (150MHz) data (C5D5N, δin ppm, JinHz)

[0071]

[0072]

[0073] Table 3. Compounds 7-9 1 H NMR (600MHz) and 13 C10 NMR (150MHz) data (C5D5N, δin ppm, JinHz)

[0074]

[0075]

[0076] Structural identification of compound 1

[0077] Compound 1 is a white amorphous powder, soluble in methanol. HR-ESI-MS shows [M+NH4] at m / z 562.2648 (calcd. for 562.2647). + Ion peak, combination 1 H-NMR, 13 Based on C1-NMR and DEPT spectra, its molecular formula is deduced to be C10. 29 H 36 O 10 Its degree of unsaturation is calculated to be 12.

[0078] Compound 1 in 1 In the 1H-NMR (600MHz, C5D5N) spectrum, a single olefin proton signal was clearly observed at δ 7.52 (1H, brs); oxomethyl proton signals were observed at δ 4.22 (1H, d, J = 5.4) and 5.30 (1H, brs). In the high-field region, four methyl proton signals were observed at δ 1.77 (3H, s), 1.34 (3H, d, J = 7.0), 1.68 (3H, s), and 1.15 (3H, s); typical methylene proton signals characteristic of two groups of triterpenoids were observed at δ 2.62 (1H, d, J = 18.1), 2.89 (1H, dd, J = 18.1, 5.3) and 1.92 (1H, ABd, J = 15.8), 2.33 (1H, ABd, J = 15.8).

[0079] Compound 1 in 13 The C-NMR (150 MHz, C5D5N) spectrum showed 29 carbon signals. Combined with DEPT-135 and HSQC spectra, these included one ketone carbonyl signal at δ 223.4; two ester carbonyl carbon signals at δ 175.4 and 174.3; and one double bond carbon signal at δ 149.5 and 129.8. Excluding the aforementioned sp... 2The four unsaturation levels occupied by the hybrid carbon signals indicate that compound 1 contains eight rings. Additionally, this compound also exhibits four methyl carbon signals (δ 27.8, 14.9, 10.4, 16.8), six methylene carbon signals (one oxidized), eight methine carbon signals (two oxidized), and six quaternary carbon signals (five oxidized). This information suggests that compound 1 is a highly oxidized nortriterpenoid.

[0080] Comparing compound 1 with the 1D-NMR data of lancifodilactone I, a known compound reported in literature 1, revealed that both have the same AC, G, and H rings. 1 H- 1 The relevant signals in the H COSY and HMBC spectra were verified. However, analysis of the 1D-NMR and 2D-NMR data of the two compounds revealed some significant differences in the structural data. The ketone carbonyl group at C-14 in lancifodilactone I disappeared, providing one degree of unsaturation, and the methylene group at C-12 was replaced by a methine group, suggesting that the oxidative rearrangement of the eight-membered ring was changed in compound 1. The two five-membered rings D and E in compound 1 were fused (5 / 5 [12,14]), and C-9 and C-15 formed an oxygen bridge structure. Based on DEPT-135, C-12 is a methine, and C-14 is a quaternary carbon. According to the correlation signals in the HMBC spectrum, H-8 is correlated with C-15, H-12 with C-9, C-14 with C-15, H-16 with C-15, CH3-18 with C-12, C-13 with C-16, H2-19 with C-8 and C-9, and H-22 with C-15. 1 H- 1 In the 1H COSY spectrum, the relevant signals in H2-11 / H-12 and H-16 / H-22 revealed that compound 1 possesses rare five-membered D and E rings, fused through carbon-carbon single bonds between C-12 and C-14. Furthermore, the chemical shifts of C-14 and C-15, along with the remaining degree of unsaturation, combined with the chemical formula given by high-resolution mass spectrometry, indicate that C-14 and C-15 form an epoxide structure. Combined with... 13 C-NMR and DEPT spectra revealed that C-30 is an oxidized methylene group. Based on the HMBC spectrum correlation signals, H2-30 showed correlations with C-4, C-5, and C-29, confirming that C-30 is substituted with a hydroxyl group. Thus, the planar structure of compound 1 was determined, and the eight-membered D-ring structure underwent skeletal rearrangement, forming a novel skeletal triterpenoid with two five-membered D and E rings fused together. This type of triterpenoid skeleton is the first of its kind isolated from nature.

[0081] In the NOESY spectrum of compound 1, the correlation signals of H-5 / H-8 / H2-30, CH3-21 / H-22, CH3-29 / H-1, CH3-18 / H-12 / H-16 / H-20, and H-16 / H-23 indicate that H-5, H-8, CH3-21, H-22, and H2-30 are in the α configuration; conversely, H-1, H-12, H-16, CH3-18, H-20, H-23, and CH3-29 are in the β configuration. Using quantum chemical calculations, its ECD spectrum was calculated, ultimately determining 23S.

[0082] By comprehensively analyzing the 2D-NMR spectrum of compound 1 and assigning its 1D-NMR data in detail, the structure of compound 1 was determined and named schinensilactone G.

[0083] Structural identification of compound 2

[0084] Compound 2 is a colorless crystal, soluble in methanol. HR-ESI-MS shows [M-H+H2O] at m / z 591.2092 (calcd. for 591.2083). - Ion peak, combination 1 H-NMR, 13 Based on C1-NMR and DEPT spectra, its molecular formula is deduced to be C10. 29 H 34 O 12 Its degree of unsaturation is calculated to be 13.

[0085] Compound 2 in 1 In the 1H-NMR (600MHz, C5D5N) spectrum, α-methyl proton signals were observed at δ 3.97 (1H, t-like, J = 6.7), 5.29 (1H, brs), and 5.06 (1H, brs). Five methyl proton signals were observed in the high-field region at δ 0.96 (3H, s), 1.16 (3H, d, J = 7.0), 2.15 (3H, s), 1.33 (3H, s), and 1.30 (3H, s). Typical AB-type methylene proton signals characteristic of triterpenoids were observed at δ 3.18 (H, ABd, J = 17.9), 3.22 (H, ABd, J = 17.9), 2.51 (1H, ABd, J = 16.4), and 2.69 (1H, ABd, J = 16.4).

[0086] Compound 2 in 13 The C-NMR (150 MHz, C5D5N) spectrum showed 29 carbon signals. Combined with DEPT-135 and HSQC spectra, these included two ketone carbonyl groups (δ 208.3, 220.1) and two ester carbonyl groups (δ 173.0, 177.6). Excluding the aforementioned sp...2 The four unsaturation levels occupied by the hybrid carbon signals suggest that compound 1 contains nine rings. Simultaneously, this compound also exhibits five methyl carbon signals (δ 26.5, 14.6, 17.8, 24.8, 29.3); five methylene carbon signals (δ 43.2, 27.9, 36.5, 31.9, 37.6); seven methine carbon signals (three oxidized); and eight quaternary carbon signals (seven oxidized). This information indicates that compound 2 is a highly oxidized nortriterpenoid.

[0087] Comparing the 1D-NMR data of compound 2 with that of schindilactone G reported in reference 2 revealed a significant difference in chemical shifts. The most notable difference lies in the shift of C-25 in the H ring, which shifts to a lower field at δ 77.1, suggesting hydroxyl substitution. Combined with DEPT-135, C-25 is identified as a quaternary carbon. Further analysis of the HMBC correlation spectrum revealed correlations between H-23 and C-25 and C-26, and between CH3-27 and C-25 and C-26. This indicates that C-25 in compound 2 is hydroxyl-substituted. Thus, the planar structure of compound 2 is determined.

[0088] In the NOESY spectrum of compound 2, the correlation signals of H-5 / H-7 / CH3-30, CH3-18 / H-16 / H-22, H-16 / CH3-21, and H-20 / H-23 / H-24 indicate that H-5, H-7, H-20, H-23, H-24, and CH3-30 are α-configurations; conversely, H-16, CH3-18, CH3-21, H-22, and CH3-29 are β-configurations. This structure contains multiple chiral carbons. To determine the absolute configuration, we attempted to grow single crystals, which were finally obtained in methanol solvent at 23 °C. The results were analyzed by Ga KαX- single crystal diffraction [orthorhombic, space group P41 (no. 76)]. Z=4, T=193.00K, μ(GaKα)=0.601mm -1 Dcalc = 1.412 g / cm³ 3 ,44112reflections measured(5.626°≤2θ≤121.09°),6311unique(R int =0.0505,R sigma=0.0338)which were used in all calculations. The final R1 was 0.0355 (I>2σ(I)) and wR2 was 0.0836 (all data). Flack: -0.02 (7)] determined its absolute configurations as 1R, 5S, 7S, 8S, 9S, 10R, 13S, 15S, 16S, 20S, 22S, 23R, 24S and 25R.

[0089] The 2D-NMR spectrum of compound 2 was comprehensively analyzed, and its 1D-NMR data were assigned in detail. Combined with the results of single-crystal diffraction, the structure of compound 2 was determined and named schinensilactone H.

[0090] Structural identification of compound 3

[0091] Compound 3 is a white amorphous powder, soluble in methanol. HR-ESI-MS shows [MH] at m / z 559.2192 (calcd. for 559.2185). - Ion peak, combination 1 H-NMR, 13 Based on C1-NMR and DEPT spectra, its molecular formula is deduced to be C10. 29 H 36 O 11 Its degree of unsaturation is calculated to be 12.

[0092] Compound 3 in 1 In the 1H-NMR (600MHz, C5D5N) spectrum, α-methyl proton signals were observed at δ 4.79 (1H, brs), 4.59 (1H, brs), and 4.64 (1H, brs). Five methyl proton signals were observed in the high-field region at δ 0.91 (3H, s), 1.21 (3H, d, J = 6.9), 1.35 (3H, d, J = 7.2), 1.44 (3H, s), and 1.33 (3H, s). Typical AB-type methylene proton signals characteristic of triterpenoids were observed at δ 2.93 (1H, ABd, J = 16.3) and 3.71 (1H, ABd, J = 16.3).

[0093] Compound 3 in 13 The C-NMR (150 MHz, C5D5N) spectrum showed 29 carbon signals. Combined with DEPT-135 and HSQC spectra, these included two ketone carbonyl groups (δ 209.2, 222.1) and two ester carbonyl groups (δ 172.7, 178.0). Excluding the aforementioned sp... 2The four unsaturation levels occupied by the hybrid carbon signals suggest that compound 3 contains eight rings. Simultaneously, this compound also exhibits five methyl carbon signals (δ 26.8, 14.4, 8.1, 25.7, 30.9); four methylene carbon signals (δ 31.4, 37.5, 31.8, 48.6); ten methine carbon signals (three oxidized); and six quaternary carbon signals (five oxidized). This information indicates that compound 3 is a highly oxidized schisanartane-type nortriterpenoid.

[0094] In the HMBC correlation spectrum of compound 3, H-2 correlates with C-1 and C-10, H-5 with C-4, H2-6 with C-10, and CH3-29 with C-4 and C-5, while also exhibiting correlation signals. 1 H- 1 In the H COSY spectrum, the correlation signals in H-2 / H-7 / H2-6 / H-5 revealed that compound 3 possesses a rare six-membered A ring. Simultaneously, correlation signals were observed in the HMBC correlation spectra of H-16 with C-13 and C-15, CH3-18 with C-12, C-13, C-16 and C-17, CH3-21 with C-17, H-22 with C-24, H-24 with C-23 and C-26, and CH3-27 with C-26. 1 H- 1 In the HCOSY spectrum, the correlation signals of H-11 / H-12, H-16 / H-22, H-21 / H-20 / H-22 / H-23, and H-24 / H-25 / H-27 confirm the presence of the DH ring. Furthermore, the HMBC correlation signals of H-8 with C-1, C-11, and C-14, and H2-19 with C-1, C-9, and C-10 confirm the presence of a five-membered C ring in the compound. The HMBC correlation signal of H-2 / C-3, combined with the chemical shifts and remaining unsaturation of C-1 (δ90.6), C-2 (δ51.7), C-3 (δ174.7), and C-10 (δ100.2), indicates that the B ring is a five-membered lactone ring. Furthermore, the HMBC correlation signals between H-8 and C-2, and H2-19 and C-1 and C-10, suggest that rings B and C are fused through carbon-carbon single bonds between C-1 and C-10. This information indicates that compound 3 has the same planar structure as the known compound chinorlactone A reported in reference 3.

[0095] Compound 3 differs from chinorlactone A in that the chemical shifts at the C-2 and C-7 positions are significantly different, leading us to speculate that it is a diastereomer of chinorlactone A. Combined with NOESY spectra, the correlation signals of CH3-18 / H-16 / H-22, H-22 / CH3-21, H-23H-20 / H-23 / H-24, and H-23 / H-25 indicate that H-20, H-23, H-24, and H-25 are α-configurations; conversely, H-16, CH3-21, H-22, and CH3-27 are β-configurations. This structure contains multiple chiral carbons. To determine the absolute configuration, we attempted to grow single crystals, which were finally obtained at 23°C in methanol solvent. Cu KαX- single-crystal diffraction [tetragonal, space group P43212(no.96)] was used to determine the configuration. Z=8, T=170.00K, μ(GuKα)=0.763mm -1 Dcalc = 1.189 g / cm³ 3 ,30752reflections measured(8.118°≤2θ≤149.174°),6258unique(R int =0.0742,R sigma =0.0476)which were used in all calculations. The final R1 was 0.0429 (I>2σ(I)) and wR2 was 0.1196 (all data). Flack: 0.13 (8)] determined its absolute configurations as 1R, 2S, 5S, 7R, 8R, 9S, 10R, 13S, 15S, 16S, 20S, 22S, 23R, 24R and 25S.

[0096] By comprehensively analyzing the 2D-NMR spectrum of compound 3 and assigning its 1D-NMR data in detail, and combining the results of single-crystal diffraction, the structure of compound 3 was determined and named schinensilactone I.

[0097] Structural identification of compound 4

[0098] Compound 4 is a white amorphous powder, soluble in methanol. HR-ESI-MS shows [M+FA-H] at m / z 587.2142 (calcd. for 587.2134). - Ion peak, combination 1 H-NMR, 13 Based on C1-NMR and DEPT spectra, its molecular formula is deduced to be C10. 29 H34 O 10 Its degree of unsaturation is calculated to be 13.

[0099] Compound 4 in 1 In the 1H-NMR (600MHz, C5D5N) spectrum, a single olefin proton signal was clearly observed at δ 6.19 (1H, s); oxomethyl proton signals were observed at δ 4.76 (1H, brs), 4.72 (1H, brs), and 4.81 (1H, brs). In the high-field region, five methyl proton signals were observed at δ 1.20 (3H, s), 1.26 (3H, d, J = 7.0), 1.37 (3H, d, J = 7.1), 1.58 (3H, s), and 1.66 (3H, s); typical AB-type methylene proton signals characteristic of triterpenoids were observed at δ 1.58 (1H, ABd, J = 13.8) and 2.53 (1H, ABd, J = 13.8).

[0100] Compound 4 in 13 The C-NMR (150 MHz, C5D5N) spectrum showed 29 carbon signals. Combined with DEPT-135 and HSQC spectra, these included one ketone carbonyl group (δ 221.0); three ester carbonyl groups (δ 172.7, 173.6, 177.8); and a set of double bond carbon signals (δ 178.4, 117.6). Excluding the aforementioned sp... 2 The five unsaturation levels occupied by the hybrid carbon signals indicate that compound 1 contains eight rings. Simultaneously, this compound also exhibits five methyl carbon signals (δ 27.2, 14.3, 8.1, 25.0, 31.1); four methylene carbon signals (δ 30.3, 35.9, 31.7, 42.4); nine methine carbon signals (three oxidized); and five quaternary carbon signals (four oxidized). This information suggests that compound 5 is a highly oxidized nortriterpenoid.

[0101] Comparison of the NMR spectrum data of compound 4 with that of schinensilactone A, a known compound reported in reference 4, revealed that they share the same parent nucleus structure. Further comparison of their NMR data showed a difference in the C-20-22 chemical shift, suggesting a possible configurational change. In the NOESY spectrum, the correlation signals of H-5 / H-14 / CH3-30, CH3-30 / H-24, H-20 / H-23, H-23 / H-25, CH3-18 / H-16 / H-22, and H-22 / CH3-21 indicate that H-14, H-20, H-23, H-24, H-25, and CH3-30 are α-configurations; conversely, H-16, CH3-21, H-22, and CH3-27 are β-configurations.

[0102] By comprehensively analyzing the 2D-NMR spectrum of compound 4 and assigning its 1D-NMR data in detail, the structure of compound 4 was determined and named schinensilactone J.

[0103] Structural identification of compound 5

[0104] Compound 5 is a white amorphous powder, soluble in methanol. HR-ESI-MS shows [M-H+H2O] at m / z 577.2300 (calcd. for 577.2291). - Ion peak, combination 1 H-NMR, 13 Based on C1-NMR and DEPT spectra, its molecular formula is deduced to be C10. 29 H 36 O 11 Its degree of unsaturation is calculated to be 12.

[0105] Compound 5 in 1 In the 1H-NMR (600MHz, C5D5N) spectrum, α-methyl proton signals were observed at δ 5.39 (1H, brs), 4.71 (1H, brs), and 4.82 (1H, brs). Five methyl proton signals were observed in the high-field region at δ 1.21 (3H, s), 1.29 (3H, d, J = 6.8), 1.35 (3H, d, J = 7.0), 1.51 (3H, s), and 1.69 (3H, s). Typical AB-type methylene proton signals characteristic of triterpenoids were observed at δ 3.13 (1H, ABd, J = 18.1) and 3.30 (1H, ABd, J = 18.1).

[0106] Compound 5 in 13 The C-NMR (150 MHz, C5D5N) spectrum showed 29 carbon signals. Combined with DEPT-135 and HSQC spectra, this included one ketone carbonyl group at δ 222.6; three ester carbonyl carbon signals at δ 174.7, 175.5, and 178.2; excluding the aforementioned sp... 2 The four unsaturation levels occupied by the hybrid carbon signals indicate that compound 1 contains eight rings. Simultaneously, this compound also exhibits five methyl carbon signals (δ 27.3, 14.4, 8.0, 24.9, 31.1); five methylene carbon signals (δ 47.0, 31.6, 36.9, 31.9, 44.5); nine methine carbon signals (three oxidized); and six quaternary carbon signals (four oxidized). This information suggests that compound 4 is a highly oxidized nortriterpenoid.

[0107] Comparison of the NMR spectrum data of compound 5 with that of schinensilactone A reported in reference 4 revealed that they share the same parent nucleus structure. The main difference lies in the significant changes in the C-1 and C-2 chemical shifts in schinensilactone A. Combined with DEPT-135, it can be inferred that it is composed of a double sp bond. 2 Hybridization resulted in a single oxidized quaternary carbon (C-1) and a methylene group (C-2). Further analysis of the HMBC correlation spectrum revealed correlation signals between H2-2 and C-1, C-3, and C-10, and between H-8 and C-1. This indicates that C-1 of compound 4 is substituted with a hydroxyl group and C-2 is a methylene group. Further comparison of its NMR data revealed differences in the C-20-22 chemical shifts, suggesting a possible configurational change. In the NOESY spectrum, the correlation signals of H-5 / H-14 / CH3-30, CH3-30 / H-24, H-20 / H-23, H-23 / H-25, CH3-18 / H-16 / H-22, and H-22 / CH3-21 indicate that H-14, H-20, H-23, H-24, H-25, and CH3-30 are in the α configuration; conversely, H-16, CH3-21, H-22, and CH3-27 are in the β configuration.

[0108] By comprehensively analyzing the 2D-NMR spectrum of compound 5 and assigning its 1D-NMR data in detail, the structure of compound 5 was determined and named schinensilactone K.

[0109] Structural identification of compound 6

[0110] Compound 6 is a colorless crystal, soluble in methanol. HR-ESI-MS shows [MH] at m / z 559.2195 (calcd. for 559.2185). - Ion peak, combination 1 H-NMR, 13 Based on C1-NMR and DEPT spectra, its molecular formula is deduced to be C10. 29 H 36 O 11 Its degree of unsaturation is calculated to be 12.

[0111] Compound 6 in 1In the 1H-NMR (600MHz, C5D5N) spectrum, α-methyl proton signals are observed at δ 4.39 (1H, s) and 4.73 (1H, s). Five methyl proton signals are visible in the high-field region at δ 0.92 (3H, s), 1.15 (3H, d, J = 7.2), 1.54 (3H, d, J = 7.5), 1.08 (3H, s), and 1.21 (3H, s). Typical AB-type methylene proton signals characteristic of triterpenoids are observed at δ 2.61 (1H, ABd, J = 16.0) and 2.73 (1H, ABd, J = 16.0).

[0112] Compound 6 in 13 The C-NMR (150 MHz, C5D5N) spectrum showed 29 carbon signals. Combined with DEPT-135 and HSQC spectra, these included two ketone carbonyl groups (δ 214.8, 219.4) and two ester carbonyl groups (δ 176.8, 178.5). Excluding the aforementioned sp... 2 The four unsaturation levels occupied by the hybrid carbon signals suggest that compound 6 contains eight rings. Simultaneously, this compound also exhibits five methyl carbon signals (δ 26.1, 18.2, 17.2, 21.0, 27.8); six methylene carbon signals (δ 19.2, 32.2, 35.8, 31.6, 43.4, 37.9); seven methine carbon signals (two oxidized); and seven quaternary carbon signals (six oxidized). This information indicates that compound 6 is a highly oxidized nortriterpenoid.

[0113] Comparing the 1D-NMR data of compound 6 with that of schigrandilactone A reported in reference 5 revealed chemical shifts. The main difference lies in schigrandilactone A, which lacks one oxidized methylene and one methyl group, and has one oxidized methine and one methyl group. Combined with DEPT-135, C-2 is identified as methine and C-30 as methyl. Further analysis of the HMBC correlation spectrum showed correlation signals between H-2 and C-1, C-10, and between CH3-30 and C-4, C-5, and C-29. This indicates that C-2 in compound 6 is substituted with a hydroxyl group and C-30 is a methyl group. Thus, the planar structure of compound 6 was determined.

[0114] In the NOESY spectrum of compound 6, the correlation signals of H-5 / CH3-30, CH3-29 / H-1, CH3-18 / H-16 / H-22, H-22 / CH3-21, and H-20 / CH3-27 indicate that H-5, H-20, CH3-27, and CH3-30 are α-configurations; conversely, H-16, CH3-18, CH3-21, H-22, H-25, and CH3-29 are β-configurations. This structure contains multiple chiral carbons. To determine the absolute configuration, we attempted to grow single crystals, which were finally obtained in methanol solvent at 23 °C. The results were obtained by Cu KαX- single crystal diffraction [orthorhombic, space group P21(no.4)]. Z=4, T=193.00K, μ(CuKα)=0.917mm -1 Dcalc = 1.364 g / cm³ 3 ,45999reflections measured(7.892°≤2θ≤159.188°),12158unique(R int =0.0642,R sigma =0.0519)which were used in all calculations. The final R1 was 0.1127 (I>2σ(I)) and wR2 was 0.3095 (all data). Flack: 0.11(5)] determined its absolute configurations as 1R, 2R, 5S, 8S, 9S, 10R, 13S, 15R, 16S, 20S, 22S, 23R and 25S.

[0115] By comprehensively analyzing the 2D-NMR spectrum of compound 6 and assigning its 1D-NMR data in detail, and combining the results of single-crystal diffraction, the structure of compound 6 was determined and named schinensilactone L.

[0116] Structural identification of compound 7

[0117] Compound 7 is a white amorphous powder, soluble in methanol. HR-ESI-MS shows [MH] at m / z 559.2192 (calcd. for 559.2185). - Ion peak, combination 1 H-NMR, 13 Based on C1-NMR and DEPT spectra, its molecular formula is deduced to be C10. 29 H 36 O 11 Its degree of unsaturation is calculated to be 12.

[0118] Compound 7 in 1 In the 1H-NMR (600MHz, C5D5N) spectrum, α-methyl proton signals are observed at δ 4.42 (1H, s) and 4.76 (1H, s). Five methyl proton signals are observed in the high-field region at δ 0.95 (3H, s), 1.19 (3H, d, J = 6.9), 1.26 (3H, d, J = 7.1), 1.10 (3H, s), and 1.23 (3H, s). Typical AB-type methylene proton signals characteristic of triterpenoids are observed at δ 2.65 (1H, ABd, J = 16.0) and 2.77 (1H, ABd, J = 16.0).

[0119] Compound 7 in 13 The C-NMR (150 MHz, C5D5N) spectrum showed 29 carbon signals. Combined with DEPT-135 and HSQC spectra, these included two ketone carbonyl groups (δ 214.7, 219.5) and two ester carbonyl groups (δ 176.9, 178.3). Excluding the aforementioned sp... 2 The four unsaturation levels occupied by the hybrid carbon signals suggest that compound 6 contains eight rings. Simultaneously, this compound also exhibits five methyl carbon signals (δ 26.0, 18.3, 14.5, 21.0, 27.9); six methylene carbon signals (δ 19.3, 32.2, 35.7, 31.6, 43.4, 39.6); seven methine carbon signals (two oxidized); and seven quaternary carbon signals (six oxidized). This information indicates that compound 7 is a highly oxidized nortriterpenoid.

[0120] Comparison of the 1D-NMR spectra of compound 7 with those of compound 6 revealed striking similarities. Further NMR analysis revealed subtle differences in the chemical shifts of C-24, C-25, and C-27, suggesting a possible change in the C-25 configuration. Correlation signals from H-5 / CH3-30, CH3-29 / H-1, CH3-18 / H-16 / H-22, H-22 / CH3-2, CH3-21 / H-24β, and H-24β / CH3-27 indicate that H-5, H-20, H-25, and CH3-30 are α-configurations; conversely, H-16, CH3-18, CH3-21, H-22, CH3-75, and CH3-29 are β-configurations.

[0121] By comprehensively analyzing the 2D-NMR spectrum of compound 7 and assigning its 1D-NMR data in detail, the structure of compound 7 was determined and named schinensilactone M.

[0122] Structural identification of compound 8

[0123] Compound 8 is a white amorphous powder, soluble in methanol. HR-ESI-MS shows [M+FA-H] at m / z 591.2456 (calcd. for 591.2447). - Ion peak, combination 1 H-NMR, 13 Based on C1-NMR and DEPT spectra, its molecular formula is deduced to be C10. 29 H 38 O 10 Its unsaturation degree is calculated to be 11.

[0124] Compound 8 in 1 In the 1H-NMR (600MHz, C5D5N) spectrum, a single olefin proton signal was clearly observed at δ 7.15 (1H, s); oxymethyl proton signals were observed at δ 5.12 (1H, dd, J = 5.4, 1.4), 4.51 (1H, d, J = 7.5), and 5.16 (1H, m). Five methyl proton signals were observed in the high-field region at δ 1.17 (3H, s), 1.81 (3H, brs), 1.80 (3H, brs), 1.30 (3H, s), and 1.23 (3H, s). 13 In the C-NMR (150 MHz, C5D5N) spectrum, one ketone carbonyl group (δ 215.8) was clearly observed; two ester carbonyl carbons (δ 175.1, 174.8) were observed; and one set of double bond carbons (δ 148.8, 130.1) were observed. Excluding the above-mentioned sp... 2 The four unsaturation levels occupied by the hybrid carbon signals suggest that compound 8 contains seven rings. Simultaneously, this compound also exhibits five methyl carbon signals (δ 28.4, 17.6, 10.6, 21.3, 27.7); five methylene carbon signals (δ 35.8, 21.9, 21.9, 29.8, 26.2); nine methine carbon signals (four oxidized); and five quaternary carbon signals (four oxidized). This information indicates that compound 8 is a highly oxidized nortriterpenoid.

[0125] Comparison of the NMR spectrum data of compound 8 with that of preschisanartanin X, a known compound reported in reference 6, revealed that they share the same parent nucleus structure. Further comparison of their NMR data showed that compound 8 lacked one set of acetoxy carbon signals compared to preschisanartanin X, and the C-22 chemical shift showed only a minor change. Combined with DEPT-135, C-22 is identified as a methine. Based on HMBC correlation signals, CH3-21 and C-22 show a correlation signal. Furthermore, combining… 1 H- 1The correlation signals of H-17 / H-20 / H-22 / H-23 in the 1H COSY spectrum further confirm that C-22 is substituted with a hydroxyl group. Thus, the planar structure of compound 8 is determined.

[0126] In the NOESY spectrum of compound 8, the correlation signals of H-5 / H-8 / CH3-30, CH3-29 / H-1, and CH3-18 / H-16 indicate that H-5, H-8, and CH3-30 are in the α configuration; conversely, H-1, H-16, CH3-18, and CH3-29 are in the β configuration. 3 J H-16 / H-17 (9.2 Hz) suggests that they are in the same plane, and the absolute configuration of C-17 is R (see reference 7). Meanwhile, compound 8 was calculated using quantum chemical calculations to determine its ECD spectrum, which ultimately identified 20R, 22R, and 23S.

[0127] By comprehensively analyzing the 2D-NMR spectrum of compound 8 and assigning its 1D-NMR data in detail, the structure of compound 8 was determined and named schinensilactone N.

[0128] Structural identification of compound 9

[0129] Compound 9 is a white amorphous powder, soluble in methanol. HR-ESI-MS shows [M+FA-H] at m / z 573.2350 (calcd. for 573.2341). - Ion peak, combination 1 H-NMR, 13 Based on C1-NMR and DEPT spectra, its molecular formula is deduced to be C10. 29 H 36 O9 has an unsaturation degree of 12.

[0130] Compound 9 in 1 In the 1H-NMR (600MHz, C5D5N) spectrum, two olefin proton signals were clearly observed at δ 5.78 (1H, d, J = 10.4) and 7.76 (1H, brs); and δ 5.10 (1H, d, J = 5.3) and 3.87 (1H, d, J = 6.2), representing oxomethyl proton signals. In the high-field region, five methyl proton signals were observed at δ 1.00 (3H, s), 1.59 (3H, d, J = 6.7), 1.96 (3H, brs), 1.31 (3H, s), and 1.21 (3H, s). A group of methylene proton signals typical of triterpenoids were observed at δ 2.92 (1H, d, J = 18.2) and 3.03 (1H, dd, J = 18.2, 5.3).

[0131] exist 13The C-NMR (150 MHz, C5D5N) spectrum showed 29 carbon signals. Combined with DEPT-135 and HSQC spectra, one ketone carbonyl group (δ 215.6) was clearly observed; two ester carbonyl groups (δ 176.3, 171.1); and two sets of double bond carbon signals (δ 119.8, 148.1, 134.8, 130.7). Excluding the aforementioned sp... 2 The five unsaturation levels occupied by the hybrid carbon signals suggest that compound 9 contains seven rings. Simultaneously, this compound also exhibits five methyl carbon signals (δ 28.1, 24.1, 10.6, 22.0, 28.5); five methylene carbon signals (δ 35.4, 23.6, 27.6, 23.9, 25.7); seven methine carbon signals (two oxidized); and five quaternary carbon signals (four oxidized). This information indicates that compound 9 is a highly oxidized nortriterpenoid.

[0132] Comparison of the NMR spectrum data of compound 9 with that of pre-schisanartanin, a known compound reported in reference 7, revealed that they share the same parent nucleus structure. Further comparison of their NMR data showed that pre-schisanartanin lacked one set of acetoxy carbon signals and had one set of double bond carbon signals. Combined with DEPT-135, C-22 was identified as a methine carbon and C-23 as a quaternary carbon. Based on HMBC correlation signals, CH3-21 correlated with C-22, and H-22 correlated with C-23. Furthermore, combined with… 1 H- 1 The H-20 / H-22 correlation signal in the HCOSY spectrum further confirms that C-22 and C-23 are double sp bonds. 2 Hybridization. Thus, the planar structure of compound 9 was determined.

[0133] In the NOESY spectrum of compound 9, the correlation signals of H-5 / H-8 / CH3-30, CH3-29 / H-1, H-1 / H-19, and CH3-18 / H-16 indicate that H-5, H-8, and CH3-30 are in the α configuration; conversely, H-1, H-16, CH3-18, and CH3-29 are in the β configuration. No correlation signal was found for H-22 / H-24 in the NOESY spectrum, but a correlation signal for H-20 / H-24 was found, confirming that the double bond configuration of C-22 / C-23 is E. 3 J H-16 / H-17 The 8.9 Hz frequency indicates that they are in the same plane, and the absolute configuration of C-17 is R (see reference 7). Meanwhile, compound 9 was calculated using quantum chemical calculations to determine its ECD spectrum, which ultimately identified 20R.

[0134] By comprehensively analyzing the 2D-NMR spectrum of compound 9 and assigning its 1D-NMR data in detail, the structure of compound 9 was determined and named schinensilactone O.

[0135] Document 1: Xiao WL, Huang SX, Zhang L, et al. Nortriterpenoids from Schisandralancifolia[J]. Journal of natural products, 2006, 69(4): 650-653.

[0136] Document 2: Huang SX, Han QB, Lei C, et al. Isolation and characterization of miscellaneous terpenoids of Schisandra chinensis[J]. Tetrahedron, 2008, 64(19): 4260-4267.

[0137] Document 3: Yang YC, Zhu SY, Liu LQ, et al.Chinorlactone A:aschinortriterpenoid with a6 / 5 / 8 / 5-fused carbocyclic core from the stems andleaves ofSchisandra chinensis[J].Organic Chemistry Frontiers, 2022,9(7):1917-1923.

[0138] Literature 4: Liu Y, Liu GZ, Li XM, et al. Anti-proliferative Properties of Schinensilactone A, ASchinortriterpenoid with 7,8-Seco-1,8-cyclo Scaffoldagainst Caco-2 by Inducing Cell Apoptosis from the Leaves of Schisandrachinensis[J]. Chinese Journal of Chemistry,2022,40(11):1331-1336.

[0139] Document 5: Xiao WL, Gong YQ, Wang RR, et al. Bioactive nortriterpenoids from Schisandra grandiflora[J]. Journal of natural products, 2009, 72(9): 1678-1681.

[0140] Document 9: Kun HU, Xing-Ren LI, Tang JW, et al. Structural determination ofeleven new preschisanartane-type schinortriterpenoids from two Schisandraspecies and structural revision of preschisanartanin J using NMR computationmethod[J]. Chinese journal of natural medicines, 2019, 17(12):970-981.

[0141] Document 7: Huang SX, Li RT, Liu JP, et al. Isolation and characterization of biogenetically related highly oxygenated nortriterpenoids from Schisandrachinensis[J]. Organic Letters, 2007, 9(11):2079-2082.

[0142] Example 2: Screening for in vitro neuroprotective activity of triterpenoid compounds from Schisandra chinensis leaves

[0143] Experimental materials and instruments

[0144] instrument

[0145]

[0146] Material

[0147]

[0148]

[0149] Preparation of test solution

[0150] Test compounds: Weigh an appropriate amount (0.5 mg) of each triterpenoid compound obtained from the above-isolated Schisandra chinensis leaves, dissolve it in a small amount of DMSO to prepare a stock solution, filter it through a 0.22 μm microporous membrane for sterilization, and store it at 4℃ for later use. Dilute it to the required concentration with DMEM medium before use.

[0151] Experimental methods

[0152] 1. Cell Culture

[0153] After revival using standard methods, SH-SY5Y cells were cultured in DMEM medium containing 10% fetal bovine serum and 100 U / mL penicillin / streptomycin / amphotericidal B (37℃, 5% CO2), and passaged once every 2-3 days by repeated pipetting with the medium. All cells used in the experiment were in the logarithmic growth phase.

[0154] 2. Cell protection experiment

[0155] (1) SH-SY5Y cells were seeded in 96-well plates (1×10⁻⁶ cells per well). 4 Cells / well), cultured overnight.

[0156] (2) The cells were divided into normal control group, H2O2 770 μmol / L (model group), H2O2 770 μmol / L + N-acetylcysteine ​​(NAC) 0.5 mmol / L (positive control group), H2O2 770 μmol / L + various concentrations of compounds and blank control group.

[0157] After inoculation overnight, each drug group was diluted with complete culture medium (2-fold serial dilution), and a solvent control (DMSO) was prepared simultaneously. The original culture medium in the well plate was discarded, and the drug to be tested was added at 100 μL per well. After incubation for 22 h, the original culture medium was discarded, and 10 μL of CCK-8 solution was added to each well. The plate was incubated at 37°C with 5% CO2 for 2 h. The OD value of the samples was then measured using a microplate reader at a wavelength of 450 nm.

[0158] Cell viability % = (Experimental group - Blank group)OD / (Control group - Blank group)OD × 100%

[0159] Statistical analysis

[0160] Experimental data The results indicate that GraphPad Prism Version 8.0 (La Jolla, CA, USA) software was used, and the One-way analysis of variance (ANOVA) method was employed for intergroup comparisons.

[0161] Experimental results

[0162] Table 4. Neuroprotective effects of compounds 1-9 on H2O2-induced SH-SY5Y cells at 50 μmol / L.

[0163]

[0164] ** P<0.01, * P<0.05vs.Model(55.24±3.36%)

[0165] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, said compound being selected from compounds 1-9 listed below: 。 2. A pharmaceutical composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof.

3. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 2 in the preparation of a medicament for the purpose of protecting the nervous system.

4. The use according to claim 3, characterized in that, The drug is used to protect SH-SY5Y cells.

5. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 2 in the preparation of a medicament for the prevention or treatment of neurodegenerative diseases.

6. The use according to claim 5, characterized in that, The neurodegenerative diseases mentioned are selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS).

7. A method for preparing the compound of claim 1, characterized in that, Schisandra chinensis leaves were extracted with solvent to obtain total extract of Schisandra chinensis, and then compounds 1-9 were isolated from the total extract of Schisandra chinensis. The solvent extraction includes heating extraction with an organic solvent; the organic solvent is selected from at least one of methanol and ethanol; after extraction, the extract is filtered, combined, and the solvent is removed by vacuum evaporation to obtain the total extract of Schisandra chinensis. The separation of compounds 1-9 from the total extract of Schisandra chinensis includes separating the total extract of Schisandra chinensis through macroporous resin, followed by separation by silica gel column chromatography, MCI column chromatography and ODS column chromatography, gel column chromatography, and preparative HPLC to obtain compounds 1-9.

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