A daphnane type macrocyclic diterpenoid compound with 4,7-oxo bridge structure and a preparation method and application thereof

Abstract

The application discloses a Daphnane type macrocyclic diterpenoid compound with a 4,7-oxygen bridge structure and a preparation method and application thereof. The monomer compound is obtained by repeatedly separating and purifying an ethyl acetate part extract of Daphne giraldii by using various separation fillers and separation technologies. The new compound is subjected to structural identification by using various 1D and 2D nuclear magnetic resonance spectrometry and high resolution mass spectrometry (HR-ESI-MS) and the like, and a molecular formula and a structural formula thereof are deduced. Further in-vitro cell activity researches show that the Daphnane type macrocyclic diterpenoid compound with the 4,7-oxygen bridge structure separated from the Daphne giraldii has good inhibitory activity on melanoma cells. The application is helpful to development and utilization of natural compounds of the Daphne giraldii.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of separation and purification of natural products and application, and particularly relates to a Daphnane type macrocyclic diterpenoid compound with 4,7-oxo bridge structure and a preparation method and application thereof. BACKGROUND

[0002] Wikstroemia Chamaedaphne Meisn. of Thymelaeaceae Wikstroemia medicinal plant river shore Wikstroemia dry flower bud yellow Janhua, the original place is Shanxi, Shaanxi, Hebei, etc. Yellow Janhua was first recorded in "Tujing Bencao", included in "Shanxi Province Chinese Medicine Standards" 1987 edition and "Chinese Pharmacopoeia" 1977 edition. Yellow Janhua is also known as north Janhua, pungent, warm; small toxic. Belongs to lung meridian, kidney meridian. Used for edema, chest and abdominal water retention, phlegm accumulation, cough and fullness, difficulty in urination and defecation; external treatment of scabies, tinea, frostbite, diarrhea, water, and defecation.

[0003] In China, the research of yellow Janhua is mostly the traditional effect of anti-fertility, and the research on chemical composition is less. Recent research shows that the main chemical components of yellow Janhua are diterpenoids, flavonoids, phenolic acids, glycosides, etc. Modern pharmacological and clinical research shows that the diterpenoids in yellow Janhua have anti-fertility, anti-hepatitis B virus, anti-tumor activities, such as pimelotide A, pimelotide B and pimelotide C have strong anti-proliferative activity on human promyelocytic leukemia HL-60, human hepatoma SMMC-7721, lung cancer A549, breast cancer MCF-7 and colon cancer HT-29; Wikstroelide E has anti-hepatitis B virus activity; Wikstroemia Chamaedaphne is used for mid-term induced abortion, and has good anti-fertility effect; Janhua ester A inhibits the growth of bladder cancer and colon cancer cells by up-regulating the expression of p21. These diterpenoids of daphnane type as the effective material basis for exerting effect have attracted widespread attention of natural product chemists and pharmacologists.

[0004] Journal article (Wang, C. R., Huang, H. Z., Han, J., et al. Isolation and identification of wusho wissamia rootlet extract [J]. Chinese Pharmaceutical Journal, 1981, (06): 51-2. Research methods: Take the plant seed 14 kg after crushing, cold soak with ethanol, dilute the concentrated solution with water, extract with chloroform, and evaporate to get 725 g of crude extract. The sample was chromatographed on silica gel column, and the effective part obtained was further chromatographed on low pressure column, and silica gel G was used as filler, eluted with benzene solution containing different proportions of acetic acid ethyl ester, and 430 mg of pure product was obtained, with a yield of 0.0031%.)(Zhang, Z. Q. Diterpenoid components of wissamia rootlet and their anti-HBV activity [D]; Shanxi University, 2017; This paper adopts traditional separation method to study the chemical components of wissamia rootlet, and 18 compounds are separated, including 8 diterpenoids of daphnane type, 2 diterpenoids of tigliane type, 6 diterpenoids of truxane type and 2 flavonoid glycosides.)(Niu, Z., Chen, L. L., He, W. D., et al. Study on the chemical constituents of wissamia rootlet [J]. Chinese traditional patent medicine, 2019, 41(07): 1586-91. Methods; The 70% ethanol extract of wissamia rootlet was separated and purified by silica gel column, gel column, recrystallization and high performance preparative liquid chromatography, and the structures of the obtained compounds were identified according to the physicochemical properties and spectral data. They are piperitol (1), forsythialan A (2), eudesmin (3), sesamin (4), torilin (5), 3,4-dihydroxytoluene (6), syringaresinol (7), lariciresinol (8), pinoresinol (9), pinoresinol-4-O-β-D-glucoside (10), eriodictyol (11), eriodictyol-7,3'-dimethyl ether (12), ethyl caffeate (13), methyl caffeate (14), blumenol B (15), 5,7-dihydroxychromone (16), 5-hydroxy-7-methoxychromone (17), naringenin (18), trans-p-hydroxycinnamic acid ethyl ester (19), luteolin (20)).

[0005] Currently, the commonly used method in extraction and separation is to use appropriate solvents (such as ethanol, methanol, etc.) to extract wissamia rootlet and obtain the extract. Through chromatographic technology (such as column chromatography, liquid chromatography, etc.), the target compounds are separated and purified. In terms of structure identification: for the daphnane type macrocyclic diterpenoids isolated from wissamia rootlet, nuclear magnetic resonance (NMR) technology, mass spectrometry (MS) technology and other spectroscopic techniques are used to determine the molecular structure and functional groups of the compounds. Daphnane type macrocyclic diterpenoids in wissamia rootlet have diverse structures, and different compounds may have different biological activities. Therefore, the method of preparing and identifying specific target compounds may need to be adjusted according to their structural characteristics, increasing the complexity of the research.

[0006] However, there has been no report on the Daphnane-type macrocyclic diterpenoid compound with a 4,7-oxygen bridge structure and its preparation method and application. Summary of the Invention

[0007] The purpose of the present invention is to address the deficiencies in the prior art and to provide a Daphnane-type macrocyclic diterpenoid compound with a 4,7-oxygen bridge structure, a preparation method thereof, and an application thereof.

[0008] In a first aspect, the present invention provides a Daphnane-type macrocyclic diterpenoid compound having a 4,7-oxygen bridge structure or a pharmaceutically acceptable salt thereof, wherein the diterpenoid compound has a structure as shown in formula (I):

[0009]

[0010] In a second aspect, the present invention provides a method for preparing a Daphnane-type macrocyclic diterpenoid compound having a 4,7-oxygen bridge structure, comprising the following steps: extracting the flower bud powder of the yellow scutellaria baicalensis using ethanol, obtaining the extract, and then separating and purifying the extract to obtain the diterpenoid compound.

[0011] As a preferred embodiment, the preparation method comprises the following steps:

[0012] ① 10 kg of bud powder of Scutellaria baicalensis was extracted three times with 95% EtOH (50 L, each for one month) at room temperature. After removing the solvent, a crude extract (2.5 kg) was obtained. The extract was suspended in water (3 L) and extracted sequentially with petroleum ether (3 × 3 L), ethyl acetate (3 × 3 L), and n-butanol (3 × 3 L) to obtain three corresponding fractions.

[0013] ② 800 g of the ethyl acetate extract obtained in step ① was preliminarily fractionated using an XDA-7 macroporous resin column, and gradient eluted using 30%, 60%, 90%, 95%, and 100% methanol-water. Similar fractions were combined to obtain Fr.AD;

[0014] Fr.CIIc3 was purified by semi-preparative HPLC (Agilent Eclipse XDB-C18 column, 5 μM, 250 x 9.4 mm, MeOH / H2O, 7.4:2.6, 3 mL / min) to give compound 7 (3.2 mg, t R 8 min); Fr.CIIe was chromatographed on Sephadex LH-20 with CH2Cl2-MeOH (1 :1) to give Fr.CIIel-Fr.CIIe3 as three sub-fractions; Fr.CIIel was chromatographed on silica gel (CHCl3 / MeOH, 100:1 to 5:1, v / v) to give Fr.CIIel a-Fr.CIIelc; Fr.CIIelb was purified by semi-preparative HPLC (Eclipse XDB-C18 column, MeOH / H2O, 7.5:2.5, 3 mL / min) to give compound 2 (24.2 mg, t R 9.5 min); Fr.CIIe2 was chromatographed on silica gel (petroleum ether / EtOAc, 10:1 to 1 :1, v / v) to give Fr.CIIe2a-Fr.CIIe2c; Fr.CIIe2a was purified by semi-preparative HPLC (Eclipse XDB-C18 column, MeOH / H2O, 7.4:2.6, 3 mL / min) to give compound 3 (4.2 mg, t R 12 min) and compound 4 (2.1 mg, t R 13 min); Fr.CIIe2b was also purified by semi-preparative HPLC (Eclipse XDB-C18 column, MeOH / H2O, 7.9:2.1, 3 mL / min) to give compound 5 (2.6 mg, t R 11 min) and compound 9 (1.5 mg, t R 12 min); Fr.CIIe3 was chromatographed on MCI gel with MeOH / H2O (30% to 100%, v / v) to give Fr.CIIe3a and Fr.CIIe3b; Fr.CIIe3a was purified by semi-preparative HPLC (Eclipse XDB-C18 column, MeOH / H2O, 7.4:2.6, 3 mL / min) to give compound 7 (3.2 mg, tR 14 min) and compound 8 (4.3 mg, t R 15 min).

[0015] In a third aspect, the present application provides the use of the diterpenoid compound or the pharmaceutically acceptable salt thereof in the preparation of a medicament for treating melanoma.

[0016] In a fourth aspect, the present application provides an extract containing the diterpenoid compound as described above.

[0017] In a fifth aspect, the present application provides a pharmaceutical composition containing the diterpenoid compound or the pharmaceutically acceptable salt thereof as described above.

[0018] As a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0019] The pharmaceutically acceptable carrier is selected from diluents, preservatives, fillers, flow regulators, penetration enhancers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, antibacterial agents, antifungal agents, lubricants and dispersants.

[0020] According to the present application, the compound, the extract of the present application can be used alone or in the form of a pharmaceutical composition, and the administration mode can be determined according to the specific circumstances, and can be prepared into a dosage form suitable for oral, rectal administration, intramuscular injection, etc. according to the conventional method in the field of pharmacy, such as tablets, capsules, ointments, patches, injections, etc.

[0021] The dosage of the compound of the present application or the pharmaceutically acceptable salt thereof or the extract depends on the age, weight and type and severity of the disease of the subject.

[0022] The present application has the advantages of:

[0023] The present application uses various separation fillers and separation techniques to repeatedly separate and purify the ethyl acetate part extract of Daphne giraldii to obtain a monomer compound. Various 1D and 2D nuclear magnetic resonance spectra and high-resolution mass spectrometry HR-ESI-MS are used to identify the structure of the new compound, and the molecular formula and structural formula are deduced. Further in vitro cell activity studies show that the Daphnane type macrocyclic diterpenoid compound with 4,7-oxo bridge structure isolated from Daphne giraldii has good inhibitory activity on melanoma cells. The present application is helpful for the development and utilization of natural compounds of Daphne giraldii.

[0024] It should be noted that: because the drawings of the specification are presented in gray scale, Fig. 3-10 The dotted arrow is red, and the solid arrow is blue. BRIEF DESCRIPTION OF DRAWINGS

[0025] Fig. 1Extraction and isolation flow chart

[0026] Fig. 2 Structure of compound

[0027] Fig. 3 HMBC of (A) compound 2 (→) and related; (B) HMBC of compound 2 related;

[0028] Fig. 4 HMBC of (A) compound 3 (→) and related; (B) HMBC of compound 3 related;

[0029] Fig. 5 HMBC of (A) compound 4 (→) and related; (B) HMBC of compound 4 related;

[0030] Fig. 6 HMBC of (A) compound 5 (→) and related; (B) HMBC of compound 5 related;

[0031] Fig. 7 HMBC of (A) compound 6 (→) and related; (B) HMBC of compound 6 related;

[0032] Fig. 8 HMBC of (A) compound 7 (→) and related; (B) HMBC of compound 7 related;

[0033] Fig. 9 HMBC of (A) compound 8 (→) and related; (B) HMBC of compound 8 related;

[0034] Fig. 10 HMBC of (A) compound 9 (→) and related; (B) HMBC of compound 9 related. DETAILED DESCRIPTION

[0035] The separation and purification method of the present application will be further described below in conjunction with specific examples, but the scope of the present application is not limited thereto.

[0036] Example 1 Preparation of novel diterpenoid compounds of the present application

[0037] 1. Experiment

[0038] 1.1 Instruments and reagents

[0039] 1.1.1 Main instruments

[0040] High performance liquid chromatograph (Agilent Technologies 1260 series); semi-preparative high performance liquid chromatograph (Agilent Technologies 1260 series); XDA-7 macroporous resin adsorption resin (Shaanxi Leibo Biochemical Technology Co., Ltd.); ultrasonic cleaner (Shanghai Yiqing Ultrasonic Instrument Co., Ltd.); Sephadex LH-20 (GE healthcare Bio-science AB Co.); rotary evaporator (IKA Co.); GF254 thin layer chromatography silica gel (Qingdao Marine Chemical Co.); column chromatography silica gel (Yantai Jiangyou Silica Gel Development Co., Ltd.); automatic receiver (Shanghai Huxi Co.); and the like.

[0041] 1.1.2 Main reagents

[0042] Reagents: 95% ethanol for extraction was an industrial reagent; petroleum ether, ethyl acetate, dichloromethane, chloroform, methanol, acetone, and the like were all of analytical purity, and were purchased from the National Pharmaceutical Group Chemical Reagent Co., Ltd.; methanol and acetonitrile used for high performance liquid chromatography were of chromatographic purity, and were purchased from Fisher Scientific Co.; water used for high performance liquid chromatography was Wahaha purified water; TLC developer: 10% H2SO4-ethanol solution.

[0043] 1.1.3 Medicinal materials

[0044] Wikstroemia chamaedaphne Meissn. (about 10 kg) was purchased from the medicinal material market in Bozhou, Anhui Province, and was produced in Taiyuan, Shanxi Province. It was identified by Vice Chief Pharmacist Zhu Jianyong of the Seventh People's Hospital Affiliated to Shanghai University of Traditional Chinese Medicine as the flower bud of Wikstroemia (Wikstroemia) of the Thymelaeaceae (Thymelaeaceae) plant Wikstroemia chamaedaphne Meissn.

[0045] 1.1.4 Extraction and separation

[0046] Flower bud powder of W. chamaedaphne (10 kg) was extracted with 95% EtOH (50 L, one month each) at room temperature. The crude extract (2.5 kg) was obtained after removal of the solvent. The extract was suspended in water (3 L) and successively extracted with petroleum ether (3 x 3 L), ethyl acetate (3 x 3 L) and n-butanol (3 x 3 L) to give three corresponding fractions. The ethyl acetate fraction of W. chamaedaphne (800 g) was applied to a XDA-7 macroporous resin column for preliminary fractionation, and eluted with 30%, 60%, 90%, 95% and 100% methanol-water, respectively. Similar fractions were combined to give Fr. A-D. Fr. C was further fractionated on a MCI column and eluted with 50%-100% gradient methanol-water to give two fractions, Fr. CI and CII. Fr. CII (87 g) was subjected to silica gel (CHCl3 / MeOH, 1 :0 to 0:1, v / v) to give Fr. CIIa-Fr. CIIg. Fr. CIIc was chromatographed on Sephadex LH-20 with CH2Cl2-MeOH (1 :1) to give three sub-fractions, Fr. CIIc1-Fr. CIIc3. Fr. CIIc3 was purified by semi-preparative HPLC (Agilent Eclipse XDB-C18 column, 5 μM, 250 x 9.4 mm, MeOH / H2O, 9:1, 3 mL / min) to give 6 (3.2 mg, t R 8 min). Fr. CIIe was chromatographed on Sephadex LH-20 with CH2Cl2-MeOH (1 :1) to give three sub-fractions, Fr. CIIe1-Fr. CIIe3. Fr. CIIe1 was subjected to silica gel (CHCl3 / MeOH, 100:1 to 5:1, v / v) to give Fr. CIIe1a-Fr. CIIe1c. Fr. CIIe1b was purified by semi-preparative HPLC (Eclipse XDB-C18 column, MeOH / H2O, 7.5:2.5, 3 mL / min) to give 2 (24.2 mg, t R 9.5 min). Fr. CIIe2 was subjected to silica gel (petroleum ether / EtOAc, 10:1 to 1 :1, v / v) to give Fr. CIIe2a-Fr. CIIe2c. Fr. CIIe2a was purified by semi-preparative HPLC (Eclipse XDB-C18 column, MeOH / H2O, 7.4:2.6, 3 mL / min) to give 3 (4.2 mg, t R 12 min) and 4 (2.1 mg, t R13 min). Fr. CIIe2b was also purified by semi-preparative HPLC (Eclipse XDB-C18 column, MeOH / H2O, 7.9:2.1, 3 mL / min) to give 5 (2.6 mg, t R 11 min) and 9 (1.5 mg, t R 12 min). Fr. CIIe3 was chromatographed on MCI gel with MeOH / H2O (30% to 100%, v / v) to give Fr. CIIe3a and Fr. CIIe3b. Fr. CIIe3a was purified by semi-preparative HPLC (Eclipse XDB-C18 column, MeOH / H2O, 7.4:2.6, 3 mL / min) to give 7 (3.2 mg, t R 14 min) and 8 (4.3 mg, t R 15 min). See Fig. 1 .

[0047] 2. Structure identification of compounds

[0048] Wikstdaphnein B-I (2-9) with 4,7-oxo bridge structure, see Fig. 2 .

[0049] 2.1 Structure identification of new compounds

[0050] 2.1.1 Structure identification of Wikstdaphnein B (2)

[0051]

[0052] Compound 2 was white amorphous powder, its structure was identified by HR-ESI-MS m / z 634.2778 [M+Na] + (calcd for C 36 H 42 O 10 Na, 657.2671) and 13 C NMR deduced structure was C 36 H 42 O 10 , with unsaturation Ω=16. Compared with Wikstdaphnein A, there was one more C2H2, and one more unsaturation, which was considered to be one more double bond. According to HMBC, H-3 was correlated with the carbonyl carbon (δ C 167.4) of this ester, and H-2' was correlated with the carbon (δ C129.8) has relevant, preliminary inference of the benzoyl and double bond connection position. The structure is verified by 1D NMR, HMBC, HMQC. In the NOESY spectrum, according to the NOE correlation of H-2 / H-3, H-2 / H-18, it shows that H-2, H-3 and CH3-18 are in the a configuration, and according to the NOE correlation of H-8 / H-16, H-14 / H-8 and H-8 / H-1, it shows that CH-16, H-14, H-8 and H-1 are in the β configuration. The absolute configuration is determined to be 2S, 3S, 4R, 5R, 6S, 7R, 8R, 9R, 10S, 11R, 13R, 14R (ECD), and named Wikstdaphnein B. Fig. 3

[0053] 2.1.2 Wikstdaphnein C (3) structure identification

[0054]

[0055] Compound 3 is a white amorphous powder, and the structure is verified by HR-ESI-MS m / z 634.2778 [M+Na] + (calcd for C 36 H 42 O 10 Na, 657.2671) and 13 C NMR structure formula is C 36 H 42 O 10 , which is completely consistent with compound 2. According to the HMBC, H-3 has relevant with the ester carbonyl carbon (δ C 167.4), H-2' has relevant with the quaternary carbon (δ C 134.3), and H-5' / H-9' has relevant with the tertiary carbon (δ C 146.2), which indicates that the trans-cinnamoyl is connected with O at 3 position. In the NOESY spectrum, according to the NOE correlation of H-2 / H-3, H-2 / H-18, it shows that H-2, H-3 and CH3-18 are in the a configuration, and according to the NOE correlation of H-8 / H-16, H-14 / H-8 and H-8 / H-1, it shows that CH-16, H-14, H-8 and H-1 are in the β configuration. The absolute configuration is determined to be 2S, 3S, 4S, 5R, 6R, 7R, 8R, 9R, 10S, 11R, 13R, 14R (ECD), and named Wikstdaphnein C. Fig. 4

[0056] 2.1.3 Wikstdaphnein D (4) structure identification

[0057] ​​

[0058] Compound 4 was a white amorphous powder, and its HR-ESI-MS at m / z 634.2778 [M+Na] + (calcd for C 36 H 42 O 10 Na,657.2671) and 13 C NMR suggested structure was C 36 H 42 O 10 , which was exactly the same as compound 3. According to HMBC, H-5 was correlated with this ester carbonyl carbon (δ C 168.6) and H-20 was correlated with quaternary carbon (δ C 166.3), which suggested that trans-cinnamoyl was connected with O at 5 position. In NOESY spectrum, according to H-2 / H-3, H-2 / H-18 NOE correlations indicated that H-2, H-3 and CH3-18 were in α configuration, and according to H-8 / H-16, H-14 / H-8 and H-8 / H-1 NOE correlations indicated that CH-16, H-14, H-8 and H-1 were in β configuration. Through 1D NMR, HMBC, HMQC verification, and ECD determination, the absolute configuration was 2S, 3R, 4R, 5R, 6S, 7R, 8R, 9R, 10S, 11R, 13R, 14R( Fig. 5 ), and named as Wikstdaphnein D.

[0059] 2.1.4 Wikstdaphnein E (5) structure identification

[0060]

[0061] Compound 5 was a white amorphous powder, and its HR-ESI-MS m / z 631.2491 [M+Na] + (calcd for C 34 H 40 O 10 Na,631.2514) with unsaturation Ω = 15, which was also the same as compound 1. First, we considered whether it was different in the position of substituents. According to HMBC, H-5 was correlated with this ester carbonyl carbon (δ C 167.8) and H-20 was correlated with this ester carbonyl carbon (δ C166.8) have NOE correlations between H-1 / H-10, H-5 / H-12, H-12 / H-8, H-1 / H-8 and H-16 / H-12, which indicate that H-1, H-5, H-8, H-12 and H-16 are in the α configuration, and H-20 / H-5", H-3" / 7" / H-5" NOE correlations indicate that H-3" / 7", H-5" and H-20 are in the β configuration. ECD calculation determines the absolute configuration as 2S, 3S, 4R, 5R, 6S, 7R, 8R, 9R, 10S, 11R, 13R, 14R Fig. 6 ), therefore, compound 5 is named Wikstdaphnein E.

[0062] 2.1.5 Wikstdaphnein F (6) structure identification

[0063]

[0064] Compound 6 is a white amorphous powder, HR-ESI-MS m / z 608.2621 [M+Na] + (calcd for C 34 H 40 O 10 Na, 608.2621) to determine the molecular formula as C 34 H 40 O 10 , with unsaturation Ω = 15, which is exactly the same as compound 1. Comparing the 1D NMR of the two compounds, it is possible that it is also because of the different positions of the substituents. According to HMBC, HMQC, it is found that the carbon signals of 14 and 20 positions of compound 2 are slightly different compared with compound 1, through 1D NMR, HMBC, HMQC, it is known that the C-14 and C-20 two positions of compound 2 shift to high field [δ C 73.5 (C-14), and 68.7 (C-20) in 1; δ C 72.4 (C-14), and 67.5 (C-20) in 2], which preliminarily indicates that the structure may be the exchange of substituents at 14 and 20 positions. According to HMBC, H-14 has a correlation with the ester carbonyl carbon (δ C 166.7), and H-3 has a correlation with the ester carbonyl carbon (δ C167.5). Based on the inference from compound 1, NOE correlations between H-1 / H-8, H-14 / H-17, H-14 / H-8, H-14 / H2-20, and H-3 / H-8 indicated that H-1, H-3, H-8, H-14, H-16, H-17, and H2-20 were α-configured. NOE correlations between H-19 / H-3' / 7' and H-3' / 7' / H-12 indicated that H-3' / 7, H-12, and H-19 were β-configured. ECD calculations determined the absolute configurations to be 2S, 3S, 4S, 5R, 6R, 7R, 8R, 9R, 10S, 11R, 13R, and 14R. Therefore, compound 6 was named Wikstdaphnein F.

[0065] 2.1.6 Structure identification of Wikstdaphnein G(7)

[0066]

[0067] Compound 7 was a white amorphous powder, and HR-ESI-MS m / z 530.2516[M+Na] + (calcd forC 29 H 38 O9Na,553.2389) and 13 C NMR suggests the structural formula is C 29 H 38 O9, the degree of unsaturation is 11. Compared with compound 5, it lacks C7H5O. 13 C NMR suggests that the benzoyl group may be missing. According to HMBC, H-3 and the carbonyl carbon of the ester (δ C The results were published online in Nature Communications. The results were 166.7 (correlation with the 3-position of the trans-cinnamoyl group), suggesting that the trans-cinnamoyl group is attached to the O at the 3-position. This was confirmed by 1D NMR, HMBC, and HMQC. In the NOESY spectrum, NOE correlations at H-2 / H-3 and H-2 / H-18 indicated that H-2, H-3, and CH3-18 were in α-configuration. Similarly, NOE correlations at H-8 / H-16, H-14 / H-8, and H-8 / H-1 indicated that CH-16, H-14, H-8, and H-1 were in β-configuration. ECD confirmed the absolute configuration to be 2S, 3S, 4S, 5R, 6R, 7R, 8R, 9R, 10S, 11R, 13R, and 14R, and the compound was named Wikstdaphnein G.

[0068] 2.1.7 Structure identification of Wikstdaphnein H(8)

[0069]

[0070] Compound 8 was a white amorphous powder, and its structure was confirmed by HR-ESI-MS m / z 530.2516 [M+Na] + (calcd for C 29 H 38 O9Na,553.2386) and 13 C NMR suggested structure was C 29 H 38 O9, with 11 degrees of unsaturation. The same molecular formula as compound 7, the first consideration is the different position of substituents. According to the HMBC, H-20 was correlated with the carbonyl carbon (δ C 167.9), indicating that the trans-cinnamoyl group was connected to O at position 20. Verified by 1D NMR, HMBC, HMQC. In the NOESY spectrum, according to the NOE correlations of H-2 / H-3, H-12 / H-3, it showed that H-2, H-3 and CH-12 were in α configuration, and according to the NOE correlations of H-8 / H-14, H-17 / H-14, H-17 / H-18, H-17 / H-5, H-17 / H-16 and H-8 / H-1, it showed that CH-17, H-14, H-8, H-18, H-5, H-16 and H-1 were in β configuration. The absolute configuration was determined to be 2S, 3S, 4R, 5R, 6R, 7R, 8R, 9R, 10S, 11R, 13R, 14R by ECD, and named as Wikstdaphnein H.

[0071] 2.1.8 Wikstdaphnein I (9) structure identification

[0072]

[0073] Compound 9 was a white amorphous powder, and its structure was confirmed by HR-ESI-MS m / z 634.2778 [M+Na] + (calcd for C 36 H 43 O 10 Na,635.2841) and 13 C NMR suggested structure was C 36 H 42 O 10 , which was exactly the same as compound 4. According to the HMBC, H-14 was correlated with the carbonyl carbon (δ C 167.6) of this ester, and H-20 was correlated with the quaternary carbon (δ C169.0) has a correlation, suggesting that trans-cinnamoyl is connected with O at position 20. In the NOESY spectrum, according to the NOE correlations of H-2 / H-3, H-2 / H-18, it indicates that H-2, H-3 and CH3-18 are in α configuration, and according to the NOE correlations of H-8 / H-16, H-14 / H-8 and H-8 / H-1, it indicates that CH-16, H-14, H-8 and H-1 are in β configuration. The absolute configuration is determined to be 2S, 3S, 4R, 5R, 6R, 7R, 8S, 9R, 10S, 11R, 13R, 14R by 1D NMR, HMBC, HMQC verification, and ECD, and named as Wikstdaphnein I.

[0074] 2.2 Spectroscopic and physicochemical data of new compounds

[0075] Wikstdaphnein B (2): white, amorphous powder; [α] 25 D -20 (c 0.1, MeOH); UV (MeOH) λ max (log ε) 220 (3.78), 275 (3.24) nm; ECD (c 1.2 x 10 -4 M, MeOH) λ max (Δε) 199 (-17.56), 219 (+0.40) 239 (-3.60) nm nm; IR (KBr) ν max 3419, 2927, 1710, 1638, 1451, 1276, 1177, 1027, 711 cm -1 ; 1 H and 13 C NMR (CDC13) data, see Tables 2 and 3; HRESIMS m / z 657.2724 [M + Na] + (calcd for C 34 H 40 O 10 Na, 657.2670).

[0076] Wikstdaphnein C (3): white, amorphous powder; [α] 25 D -20 (c 0.1, MeOH); UV (MeOH) λ max (log ε) 222 (3.78), 275 (3.71) nm; ECD (c 1.2 x 10 -4 M, MeOH) λ max(Δε) 205 (-4.04), 218 (+5.18), 235 (-5.38) nm; IR (KBr) v max 3417, 2925, 1720, 1698, 1450, 1277, 1261, 1097, 1027, 803, 711 cm -1 ; 1 H and 13 C NMR (CDC13) data, Tables 2 and 3; HRESIMS m / z 657.2690 [M + Na] + (calcd for C 34 H 40 O 10 Na, 657.2670).

[0077] Wikstdaphnein D (4): white, amorphous powder; [a] 25 D -27 (c 0.1, MeOH); UV (MeOH) λ max (log e) 222 (3.96) 275 (3.56) nm; ECD (c 0.8 x 10 -4 M, MeOH) λ max (Δε) 202 (+2.32), 226 (-4.46), 277 (+4.07) nm; IR (KBr) v max 3417, 2927, 1716, 1637, 1451, 1273, 1117, 1025, 711 cm -1 ; 1 H and 13 C NMR (CDC13) data, Tables 2 and 3; HRESIMS m / z 657.2690 [M + Na] + (calcd for C 34 H 40 O 10 Na, 657.2670).

[0078] Wikstdaphnein E (5): amorphous solid; [a] 25 D -22 (c 0.2, MeOH); UV (MeOH) λ max (log e) 193 (4.02), 228 (3.66) nm; ECD (c 1.0 x 10 -4 M, MeOH) λ max(Δε) 192 (+3.28), 224 (-0.85), 238 (+2.45) nm; IR (KBr) v max 3425, 2924, 1717, 1643, 1451, 1267, 1118, 976, 710 cm -1 ; 1 H and 13 C NMR (CDC13) data, see Table 1; HRESIMS m / z 631.2491 [M + Na] + (calcd for C 34 H 40 O 10 Na, 631.2514).

[0079] Wikstdaphnein F (6): white, amorphous powder; [a] 25 D -24 (c 0.1, MeOH); UV (MeOH) λ max (log e) 193 (3.63) 229 (3.13) nm; ECD (c 1.2 x 10 -4 M, MeOH) λ max (Δε) 198 (+2.33), 221 (-0.55), 235 (+1.69) nm; IR (KBr) v max 3405, 2924, 1692, 1456, 1021, 801 cm -1 ; 1 H and 13 CNMR (CDC13) data, see Table 1; HRESIMS m / z 631.2485 [M + Na] + (calcd for C 34 H 40 O 10 Na, 631.2514).

[0080] Wikstdaphnein G (7): amorphous solid; [a] 25 D -16 (c 0.1, MeOH); UV (MeOH) λ max (log e) 221 (3.82) nm 275 (3.58); ECD (c 1.2 x 10 -4 M, MeOH) λ max (Δε) 213 (+1.79), 282 (0.88) nm; IR (KBr) vmax 3396, 2927, 1705, 1636, 1450, 1283, 1176, 1030, 768 cm -1 1 H and 13 C NMR (CDC13) data, see Tables 2 and 3; HRESIMS m / z 553.2389 [M + Na] + (calcd for C 34 H 40 O 10 Na, 553.2414).

[0081] Wikstdaphnein H (8): amorphous solid; [a] 25 D -19 (c 0.1, MeOH); UV (MeOH) λ max (log e) 221 (3.27) 275 (3.35) nm; ECD (c 1.2 x 10 -4 M, MeOH) λ max (De) 218 (+1.29), 281 (0.70) nm; IR (KBr) v max 3380, 2924, 1713, 1456, 1377, 1260, 1095, 1025, 801 cm -1 1 H and 13 C NMR (CDC13) data, see Tables 2 and 3; HRESIMS m / z 553.2386 [M + Na] + (calcd for C 34 H 40 O 10 Na, 553.2414)

[0082] Wikstdaphnein I (9): amorphous solid; [a] 25 D -32 (c 0.1, MeOH); UV (MeOH) λ max (log e) 221 (3.82) nm 274 (3.77); ECD (c 1.2 x 10 -4 M, MeOH) λ max (De) 228 (-5.50), 269 (+7.66) nm; IR (KBr) v max ​​3408, 2926, 1713, 1638, 1451, 1281, 1178, 1020, 801, 769, 711 cm -1 ; 1 Hand 13 C NMR (CD3OD) data, see Tables 2 and 3; HRESIMS m / z 635.2841 [M+H] + (calcd for C 36 H 43 O 10 , 635.2851).

[0083] Table 1 1 H and 13 C NMR Data for Wikstdaphneins E and F (5 and 6)

[0084]

[0085]

[0086] Table 2 1 H NMR (600 MHz, δ in ppm, J in Hz) Data for Wikstdaphneins B-D (2-4) and G-I (7-9)

[0087]

[0088]

[0089] Table 3 13 C NMR (150 MHz, δ in ppm) Data for Wikstdaphneins B-D (2-4) and G-I (7-9)

[0090]

[0091]

[0092] Example 2 In vitro Anti-melanoma Activity of Chemical Constituents of Wikstroemia indica

[0093] 1. Effects of compounds 2-9 on melanoma cells

[0094] The nine compounds isolated from the ethyl acetate extract of Wikstroemia indica were screened for cell activity by MTT method, and the IC 50 values of the compounds with better activity were calculated.

[0095] 2. Experimental materials

[0096] Test substances: compounds 2-9. Cell strains: A375 (human melanoma cells), accession number: A375-CSP-533; B16 (mouse melanoma cells), accession number: B16-SCSP-5096; HFF-1 (human fibroblast cells), accession number: HFF-1-SCSP-656, all purchased from the China Academy of Sciences Culture Collection Cell Bank.

[0097] 3. Experimental methods

[0098] 3.1 Cell viability detection

[0099] MTT method was used to detect cell viability. A375 cells, B16 cells, and HFF-1 cells were inoculated in 96-well plates at a density of 5×10 3 cells / well for 24 hours. The culture solution was removed, and the experimental group was added with the prepared culture solution containing compound 2-9 at a concentration of 30 μM, the blank control group was added with fresh culture solution, and the negative control group was added with 150 μL of DMSO solution. Three replicate wells were set for each group. 20 μL of MTT (5 mg / mL, dissolved in PBS) solution was added to each well, and the incubator was incubated in the dark for 4 hours. 150 μL of DMSO was added to each well, and the absorbance OD value was measured at a wavelength of 560 nm by a microplate reader. The inhibition rate was calculated.

[0100] 4. Experimental results

[0101] 4.1 Screening results of compounds 2-9

[0102] When the concentration of the compound was 30 μM, the inhibition rate of compounds 2, 4, and 6 on B16 cells was greater than 50%, the inhibition rate of compounds 2, 4, and 6 on A375 cells was greater than 50%, and the inhibition rate of compounds 2 and 4 on HFF-1 cells was less than 30%, as shown in Table 4.

[0103] Table 4: In vitro cell activity experiment results of 8 compounds

[0104]

[0105] The above only describes the preferred embodiments of the present application. It should be noted that, for those skilled in the art, without departing from the method of the present application, several improvements and supplements can also be made, which should be considered as the protection scope of the present application.

Claims

1. A method for preparing a Daphnane-type macrocyclic diterpenoid compound having a 4,7-oxygen bridge structure, characterized in that: The structure of the Daphnane-type macrocyclic diterpenoid compound with a 4,7-oxygen bridge structure is as shown in formula (I): As shown: The preparation method of a Daphnane-type macrocyclic diterpenoid compound having a 4,7-oxygen bridge structure comprises the following steps: ① Take 10 kg of W. chamaedaphne flower bud powder and soak it in 50 liters of 95% ethanol at room temperature for three extractions. Combine the ethanol extracts and concentrate to recover the ethanol to obtain 2.5 kg of crude extract. Suspend the crude extract in 3 liters of water and extract it three times with 3 liters of petroleum ether, three times with 3 liters of ethyl acetate, and three times with 3 liters of n-butanol. Combine the extracts obtained with the same solvents and concentrate to recover the three fractions extracted with the corresponding solvents. ② 800 g of the ethyl acetate extract obtained in step ① was preliminarily fractionated using an XDA-7 macroporous resin column, and gradient eluted with 30%, 60%, 90%, 95%, and 100% methanol-water. Similar fractions were combined to obtain Fr.AD; ③ Fr.C obtained in step ② was further fractionated using an MCI column and eluted with a 50%-100% gradient of methanol-water to obtain two components, Fr.CI-CⅡ; 87 g of Fr.CII was treated with silica gel and the eluent was CHCl3 / MeOH, 1:0 to 0:1, v / v, to obtain Fr.CIIa-Fr.CIIg; Fr.CIIc was chromatographed on Sephadex LH-20 with 1:1 CH2Cl2-MeOH to obtain three subcomponents, Fr.CIIc1-Fr.CIIc3; Fr.CIIc3 was purified by semi-preparative HPLC on an Agilent Eclipse XDB-C18 column, 5 μM, 250×9.4 mm, MeOH / H2O, 9:1, 3 mL / min, to obtain compound 6, 3.2 mg, t R 8min; Fr.CIIe was chromatographed on Sephadex LH-20 with 1:1 CH2Cl2-MeOH to obtain three subfractions Fr.CIIe1-Fr.CIIe3; Fr.CIIe1 was treated with silica gel with CHCl3 / MeOH, 100:1 to 5:1, v / v as eluent to obtain Fr.CIIe1a-Fr.CIIe1c; CIIe1b was purified by semi-preparative HPLC on an Eclipse XDB-C18 column with MeOH / H2O, 7.5:2.5, 3 mL / min to obtain compound 2, 24.2 mg, t R 9.5min; Fr.CIIe2 was treated with silica gel, petroleum ether / EtOAc, 10:1 to 1:1, v / v to obtain Fr.CIIe2a-Fr.CIIe2c; Fr.CIIe2a was purified by semi-preparative HPLC, Eclipse XDB-C18 column, MeOH / H2O, 7.4:2.6, 3mL / min to obtain compound 3, 4.2mg, t R 12min and compound 4, 2.1mg, t R 13min; Fr.CIIe2b was also purified by semi-preparative HPLC, Eclipse XDB-C18 column, MeOH / H2O, 7.9:2.1, 3mL / min to give compound 5, 2.6mg, t R 11min and compound 9, 1.5mg, t R 12min; Fr.CIIe3 was chromatographed on MCI gel with 30% to 100%, v / v MeOH / H2O to give Fr.CIIe3a and Fr.CIIe3b; Fr.CIIe3a was purified by semi-preparative HPLC on an Eclipse XDB-C18 column with MeOH / H2O, 7.4:2.6, 3 mL / min to give compound 7, 3.2 mg, t R 14min and compound 8, 4.3mg, t R 15 minutes, The compound 2 is WCL-2; the compound 3 is WCL-3; the compound 4 is WCL-4; the compound 5 is WCL-5; the compound 6 is WCL-6; the compound 7 is WCL-7; the compound 8 is WCL-8; and the compound 9 is WCL-9.

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