Sesquiterpenes and sesquiterpenoid dimers from Daphne koreana and their preparation method and application in anti-Parkinson's disease

Abstract

The present invention belongs to the field of medical technology, and relates to five new guaiacane and carotenoid compounds extracted and separated from Daphnepenicillata Rehd., including two novel guaiacane dimers bidaphnenicillataA and bidaphnenicillataB, two new guaiacane compounds daphneaineE and daphneaineF, and a new carotenoid sesquiterpene minjiangdaphneB. The test results show that it has a protective effect on H2O2-induced oxidative damage of human SH-SY5Y nerve cells, wherein the cell survival rates after treatment with compounds 4 and 5 are increased to 72.78% and 69.43%, respectively, while the positive drug Trolox is only 60.00%.

Description

Technical field:

[0001] The invention belongs to the field of medical technology, and particularly relates to a method for preparing guaiacane compounds and carotenoid sesquiterpenes from the plant Daphne koreana and application of the compounds in anti-Parkinson's disease (PD). Background technology:

[0002] Parkinson's disease is a common neurodegenerative disease. Its main pathological change is the degeneration and death of dopaminergic neurons in the substantia nigra of the midbrain, which eventually leads to motor and cognitive dysfunction in patients, threatening human health and causing a huge economic burden. At present, the treatment research for neurodegenerative diseases mainly focuses on neuroprotection, anti-neuroinflammation, and related receptor agonists and inhibitors in the brain. The exact cause of this pathological change is still unclear. Genetic factors, environmental factors, aging, oxidative stress, etc. may all be involved in the degeneration and death of dopaminergic neurons, but a lot of evidence shows that the increase in cell apoptosis caused by oxidative stress is closely related to neuronal degeneration.

[0003] Daphne penicillata Rehd. is a plant of the genus Daphne in the family Thymelaeaceae. It is an evergreen upright shrub that is mainly distributed and grown in Sichuan, my country. A few Daphne plants have good medicinal value, mainly used to treat arthritis, rheumatic joint pain and other diseases. However, as a rare species in the genus Daphne, the chemical composition and biological activity of Daphne penicillata Rehd. have been blank at home and abroad.

[0004] The inventors conducted a study on Daphne koreana for the first time and found that it contained a variety of sesquiterpenoid compounds with novel structures and exhibited good anti-PD activity. Summary of the invention:

[0005] The purpose of the present invention is to solve the deficiencies of the prior art and provide sesquiterpenes and sesquiterpenes dimers in Daphne koreana and their preparation methods and applications in anti-Parkinson's disease.

[0006] In order to achieve the purpose of the present invention, the present invention adopts the following technical scheme:

[0007] In the first aspect, the structures of five guaiacane-type sesquiterpenes, guaiacane-type sesquiterpenes dimers, and carotene-type sesquiterpenes isolated for the first time from Daphnepenicillata Rehd. of the Thymelaeaceae family are shown in the figure:

[0008]

[0009] In a second aspect, the present invention provides a pharmaceutical composition comprising the sesquiterpene or sesquiterpene dimer derivative described in the first aspect, and a pharmaceutically acceptable carrier.

[0010] In a third aspect, the present invention provides a method for preparing the sesquiterpene or sesquiterpene dimer derivative described in the first aspect, the preparation method of the present invention comprising the following steps:

[0011] (1) The whole plant of Daphne koreana was used to prepare guaiacyl sesquiterpenes. The dried whole plant of Daphne koreana was extracted with 70% industrial ethanol for three times under reflux, each time for 2.5 hours.

[0012] (2) After the solvent is recovered, the total extract is concentrated to obtain a total extract, which is vortexed with water and extracted with ethyl acetate and n-butanol in sequence. The extract is then chromatographed on a silica gel column using a dichloromethane-methanol system as an eluent at a gradient elution ratio of 100:0-5:1. The obtained fractions are analyzed by TLC thin layer chromatography and HPLC liquid phase analysis and then combined into two fractions, FrA and FrB.

[0013] (3) Fraction A was further subjected to silica gel column chromatography using a dichloromethane-methanol system as the eluent with a gradient elution of 100:0-5:1 to obtain two fractions FrA1-FrA2.

[0014] (4) FrA2 was eluted through polyamide using a dichloromethane-methanol system as an eluent at a ratio of 100:0-5:1 to obtain two fractions, FrA2-I and FrA2-II.

[0015] (5) The FrA2-Ⅱ stream was eluted with HP-20 using a 90% ethanol-water system to remove small polar pigments, and then gradient eluted using an alcohol-water system of 20:80-90:10 using ODS column chromatography. The combined bottle was further analyzed by thin layer chromatography and HPLC to obtain 6 fractions N1-N6.

[0016] (6) The obtained fraction N2 was subjected to silica gel column chromatography with a petroleum ether-ethyl acetate system of 50:1-0:1 and gradient elution to obtain 6 fractions 2.1-2.6. The fraction 2.2 was separated by HPLC with a methanol-water system of 70:30 to obtain 2.2.1-2.2.6.

[0017] (7) Semi-preparative HPLC was used to elute 2.2.3 with an acetonitrile-water system of 53:47 to obtain compounds 1 and 2. Using the same method, N1 was separated to obtain compounds 3-5.

[0018] Preferably, the extraction is reflux extraction, and the extraction is performed 3-5 times, each time for 2-3 hours.

[0019] In addition, preferably, in the preparation method, the plant used is a whole plant collected in Wenchuan County, Aba Prefecture, Sichuan Province (N31°29'51.52'' north latitude, E103°38'14.35'' east longitude) in July 2019, and identified as Daphne penicillata Rehd. by Yunnan Plant Research Co., Ltd.

[0020] The obtained compounds were systematically identified and the results are as follows:

[0021] High-resolution mass spectrometry, one-dimensional NMR, two-dimensional NMR and computational ECD techniques were used to identify the structures of compounds 1 and 2. Bidaphnenicillata A (1): a white powdery compound, UV(MeOH)λ max (logε): 204nm (1.52), 248nm (0.87); ECD (methanol) λ max (Δε)227(+126.69),275(-141.4)nm.;The 1 H(600MHz,CDCl 3 )and 13 C NMR data (150 MHz, CDCl 3 ), HRESIMS gave a quasi-molecular ion peak of m / z 519.2719, and combined with 13 The molecular formula calculated by C NMR is C 22 H 24 O 9 Na([M+Na] + Calculate for C 30 H 40 O 6 Na,519.2723). The calculated unsaturation is 11. 1 HNMR (600MHz, CDCl 3 ) spectrum, showing four double bond proton signals δ H 4.80 (1H, brs, H-13a), 4.72 (1H, t, J = 1.62 Hz, H-13b), 4.65 (1H, t, J = 1.52 Hz, H-13′a), 4.64 (1H, brs, H-13′b); four methyl proton signals δ H 1.74(3H,t,J=1.00Hz,H 3 -12),1.67(3H,t,J=1.13Hz,H 3 -12′),1.60(3H,s,H 3 -14),1.42(3H,s,H 3 -14′).13 C NMR (150 MHz, CDCl 3 ) spectrum gives a total of 30 carbon signals. Combined with HSQC spectrum analysis, it is inferred that the compound includes two carbonyl carbon signals δ C 204.9 (C-3), 200.1 (C-3′), eight double bond carbon signals δ C 169.2 (C-5), 144.2 (C-4), 141.7 (C-1′), 141.4 (C-5′), 150.5 (C-11), 150.0 (C-11′), 109.8 (C-13), 109.5 (C-13′); Four sp3 hybridized quaternary carbon signals δ C 61.8 (C-1), 77.0 (C-10), 76.6 (C-10′), 59.1 (C-4′); four methine carbon signals δ C 54.1(C-2′),54.5(C-2),44.8(C-7′),40.3(C-7); Four methyl carbon signals δ C 20.6 (C-12′), 20.1 (C-12), 11.4 (C-14), 8.5 (C-14′). Eight methylene carbon signals δ C 63.4 (C-15′), 64.2 (C-15), 38.9 (C-6), 37.2 (C-9′), 33.9 (C-9), 32.3 (C-8′), 31.8 (C-6′), 31.1 (C-8). Combining the above NMR information and 11 unsaturated groups, it is speculated that the structure may be composed of two guaiacyl sesquiterpenes.

[0022] HMBC spectrum H 1.74(CH 3 -12) and δ C 40.3 (C-7), 109.8 (C-13) and 150.5 (C-11) are related, δ H 4.80,4.72(H 2 -13) and δ C 20.1 (C-12), 40.3 (C-7) and 150.5 (C-11) are related, indicating that C-7, C-11, C-12, CH 3 -13 forms a side chain fragment. H 1.67(CH 3 -12′) and δ C 44.8 (C-7′), 109.5 (C-13′) and 150.0 (C-11′) are related, δ H 4.64,4.65(H 2 -13′) and δC 20.6 (C-12′), 44.8 (C-7′) and 150.0 (C-11′) are related, indicating that C-7′, C-11′, C-12′, CH 3 -13' forms the presence of a side chain fragment. H 2.99(H-7) and δ C 31.1 (C-8) and 38.9 (C-6) are related, δ H 2.72,2.37(H 2 -6) and δ C 40.3 (C-7), 31.1 (C-8), 61.8 (C-1), 144.2 (C-4) and 169.2 (C-5) are related, δ H 3.39,3.30(H 2 -15) and δ C 33.9 (C-9), 61.8 (C-1) and 77.0 (C-10) are related, combined with δ H 2.99(H-7) and δ H 1.52 (H-8) and 2.37 (H-7),δ H 1.52(H-8) and δ H 2.15(H-9), exists 1 H- 1 H COSY correlation, from which the seven-membered ring fragment formed by C-1, C-5, C-6, C-7, C-8, C-9 and C-10 was determined. H 1.83(H-7′) and δ C 31.8 (C-6′), 37.2 (C-9′) and 32.3 (C-8′) are related, δ H 2.17,1.95(H 2 -6′) and δ C 44.8 (C-7′), 32.3 (C-8′), 59.1 (C-4′), 141.4 (C-4′) and 141.7 (C-5′) are related. H 2.08(H-9) and δ C 32.3 (C-8′), 61.8 (C-1′), 141.7 (C-5′) and 76.6 (C-10′), δ H 2.93(H-15′) and δ C 37.2 (C-9′) and 76.6 (C-10′) are related. A seven-membered ring fragment formed by C-1′, C-5′, C-6′, C-7′, C-8′, C-9′ and C-10′ was determined. H 1.60(CH 3-14) and δ C 144.2 (C-4), 169.2 (C-5) and 204.9 (C-3) were correlated, confirming the presence of an α,β-unsaturated ketone fragment consisting of C-3, C-4 and C-5. H 1.42(CH 3 -14′) and δ C 54.5(C-2), 59.1(C-4′), 200.1(C-3′) and 141.4(C-5′) are related, δ H 2.88(H-2) and δ C 61.8(C-1),59.1(C-4′),77.0(C-10),169.2(C-5),204.9(C-3) and 141.4(C-5′) are correlated. H 4.13(H-2′) and δ C The carbonyl groups of 54.5(C-2), 59.1(C-4′), 77.0(C-10), 76.6(C-10′), 200.1(C-3) and 141.7(C-1′) were correlated, thereby confirming the existence of two five-membered ring fragments composed of C-1, C-2, C-3, C-4, C-5 and C-1′, C-2′, C-3′, C-4′, C-5′ respectively. It was also determined that the two five-membered ring fragments were bridged together by carbon-carbon bonds formed by C-4′-C-2 and C-2′-C-1, thus confirming that the whole molecule has a sesquiterpene dimer skeleton with the five-ring characteristics of 7 / 5 / 5 / 5 / 7.

[0023] The stereochemistry of the compound was then determined using NOESY spectroscopy. H 2.17(H-6′a) and δ H 1.42(CH 3 -14′) and 4.13(H-2′) have NOESY correlation, δ H 1.95 (H-6′b) and 1.67 (CH 3 -12′) and δ H 3.59 (H-15′) has NOE SY correlation, which confirms that C-15′ is β-oriented, and H-7′ is closely related to 14′-CH 3 is the α direction. H 2.88(H-2) and δ H 3.30(H-15),δ H 4.13(H-2′) and δ H 2.99 (H-7) has NOESY correlation, which confirms that H-2 and C-15 are β-oriented, and H-2′ and 7-CH 3The absolute configuration of the compound was confirmed by comparing the calculated ECD with the measured ECD. First, the Spartan software was used to perform conformational search in the MMFF94 molecular dynamics force field, and the conformations with Boltzmann distribution greater than 1% were selected for optimization at the B3LYP / 6-31G(d) basis set level. Then, the ECD calculation was performed at the B3LYP / 6-311++G(2d,p) basis set level, and the ECD spectrum was generated by fitting with the SpecDis software. By comparing the calculated ECD spectrum with the measured ECD spectrum, the absolute configuration of compound 1 was determined to be 1R, 2S, 7R, 10R, 2′S, 4′S, 7′S, 10′S.

[0024] A Scifinder search showed that the compound was a new compound that had not been reported in the literature, and compound 1 was the first reported sesquiterpene dimer compound with a [5.5.3.2.2.1] eicosane skeleton and a 7 / 5 / 5 / 5 / 7 pentacyclic ring system, named bidaphnenicillata A.

[0025] Bidaphnenicillata B(2): White powdery compound, UV(MeOH)λ max (logε):204nm(1.52),248nm(0.87); ECD(MeOH)λ max (Δε)227(+126.69),275(-141.4)nm.The 1 H(600MHz,CDCl 3 )and 13 C NMR data (150 MHz, CDCl 3 ).HRESIMS(m / z):519.2719, combined with 13 The molecular formula calculated by C NMR is C 30 H 40 O 6 ([M+Na] + Calculate for C 30 H 40 O 6 Na, 519.2723), the degree of unsaturation is 11. 1 HNMR (600 MHz, DMSO-d 6 ) spectrum, showing five double bond proton signals δ H6.38 (1H, s, H-2), 4.72 (1H, brs, H-13a), 4.67 (1H, t, J = 1.76 Hz, H-13b), 4.62 (1H, t, J = 1.82 Hz, H-13′a), 4.64 (1H, brs, H-13′b); four methyl proton signals δ H 1.69(3H,s,H 3 -12),1.66(3H,s,H 3 -12′),0.82(3H,s,H 3 -14),1.00(3H,s,H 3 -14′). 13 C NMR (150 MHz, DMSO-d 6 ) spectrum gives a total of 30 carbon signals. Combined with HSQC spectrum analysis, it is inferred that the compound includes two carbonyl carbon signals δ C 207.7 (C-3), 200.1 (C-3′), eight double bond carbon signals δ C 184.7 (C-1), 134.8 (C-2), 142.5 (C-1′), 139.4 (C-5′), 150.4 (C-11), 150.8 (C-11′), 109.1 (C-13), 108.7 (C-13′); three methine carbon signals δ C 56.0(C-2′),46.2(C-7′),42.9(C-7); five quaternary carbon signals δ C 54.9 (C-4), 54.1 (C-5), 75.6 (C-10), 58.8 (C-4′), 76.5 (C-10′); four methyl carbon signals δ C 20.4 (C-12′), 20.8 (C-12), 13.8 (C-14), 7.9 (C-14′); eight methylene carbon signals δ C 64.9 (C-15′), 67.7 (C-15), 35.2 (C-6), 36.4 (C-9′), 32.9 (C-9), 29.7 (C-8′), 32.5 (C-6′), 28.6 (C-8). The comparative analysis of the NMR data of compounds 1 and 2 showed that they had similar guaiacyl-type sesquiterpene dimer skeletons. The main difference was that compound 2 had an additional double bond proton signal, but lacked the methyl signal at the H-2 position in 1. H 1.69(CH 3 -12) and δ C 42.9 (C-7), 109.1 (C-13) and 150.4 (C-11) are related, δ H 4.67,4.72(H2 -13) and δ C 20.8 (C-12), 42.9 (C-7) and 150.4 (C-11) are related, indicating that C-7, C-11, C-12, 13-CH 3 The presence of side chain fragments. H 1.66(12′-CH 3 ) and δ C 46.2 (C-7′), 108.7 (C-13′) and 150.8 (C-11′) are related, δ H 4.64,4.62(H 2 -13′) and δ C 20.4 (C-12′), 46.2 (C-7′) and 150.8 (C-11′) are related, indicating that C-7′, C-11′, C-12′, CH 3 -13' forms the presence of a side chain fragment. H 1.57,1.37(H 2 -6) and δ C 42.9 (C-7), 28.6 (C-8), 54.1 (C-5) and 184.7 (C-1) are correlated, δ H 3.52,3.45(H 2 -15) and δ C 32.9 (C-9), 184.7 (C-1) and 75.6 (C-10) are related, combined with δ H 2.13(H-9) and δ H 1.50(H-8),δ H 2.55(H-7) and δ H 1.50 (H-9) and 1.37 (H-6), exist 1 H- 1 H COSY correlation, the seven-membered ring fragment formed by C-1, C-5, C-6, C-7, C-8, C-9 and C-10 was determined. H 2.08,1.99(H 2 -6′) and δ C 46.2 (C-7′), 29.7 (C-8′), 139.4 (C-5′) and 142.5 (C-1′) are related, δ H 3.28,3.34(H 2 -15′) and δ C 36.4 (C-9′), 139.4 (C-5′) and 76.5 (C-10′) are related, δ H 1.28(H 2 -9′) and δC There is a correlation between 46.2 (C-7′), 29.7 (C-8′), 64.9 (C-15′) and 76.5 (C-10′), from which the seven-membered ring fragment formed by C-1′, C-5′, C-6′, C-7′, C-8′, C-9′ and C-10′ was determined. H 0.82(CH 3 -14) and δ C 207.7 (C-3), 54.9 (C-4), 54.1 (C-5) and 58.8 (C-4′) are related, δ H 1.0(CH 3 -14′) and δ C 54.9 (C-4), 58.8 (C-4′), 200.1 (C-3′) and 139.4 (C-5′) are related, δ H 6.38(H-2) and δ C 184.7 (C-1), 75.6 (C-10), 54.1 (C-5), 207.7 (C-3) and 54.9 (C-4) are correlated. H 3.82(H-2′) and δ C 54.1(C-5),35.2(C-6),58.8(C-4′),76.5(C-10′),142.5(C-5′),200.1(C-3′) and 139.4(C-1′) were correlated, which confirmed the existence of two five-membered ring fragments composed of C-1,C-2,C-3,C-4,C-5 and C-1′,C-2′,C-3′,C-4′,C-5′, respectively. At the same time, it was also determined that the two five-membered ring fragments were bridged by C-4′-C-4 and C-2′-C-5 to form a carbon-carbon bond, thus confirming that the whole molecule has a sesquiterpene dimer skeleton with 7 / 5 / 5 / 5 / 7 five-ring characteristics. The relative configuration of compound 2 was determined based on the NOESY spectrum data, 14′-CH 3 and 12′-CH 3 There is NOESY correlation between H-6′, H-15′, H-6′ and H-7 and H-2′, and H-2′, 14′-CH 3 The orientation of H-6 and 14-CH 3 , 13-CH 3 There is NOESY correlation with H-15, which confirms that 14-CH 3 , C-15 and 12-CH 3The absolute configuration of compound 2 was determined by comparing the calculated ECD spectrum with the measured ECD spectrum, and its absolute configuration was determined to be 4S, 5R, 7S, 10S, 2′R, 4′R, 7′R, 10′R, and named bidaphnenicillata B. The NMR data of compounds 1-2 are shown in Table 1.

[0026] Table 1 1-2(a: CDCl 3 and b: DMSO) 1 H(600MHz) and 13 C (150 MHz) NMR data

[0027]

[0028] Daphneaine E(3): Pale yellow oily compound, UV(MeOH)λ max (logε):239nm(3.86); ECD(MeOH)λ max (Δε)230(+28.3),304(-34.1)nm.The 1 H(600MHz,CDCl 3 )and 13 C NMR data (150 MHz, CDCl 3 ).HRESIMS(m / z):275.1621, combined with 13 The molecular formula calculated by C NMR is C 15 H 24 O 3 ([M+Na] + Calculate for C 15 H 24 O 3 Na, 275.1623), the degree of unsaturation is 4. 1 H NMR (600 MHz, CDCl 3 ) spectrum shows a proton signal on the olefin carbon δ H 6.14 (1H, ddq, J = 4.81, 2.73, 1.29 Hz), 1 proton signal on the oxymethylene carbon δ H 3.58 (1H, d, J = 10.63 Hz), 3.58 (1H, d, J = 10.63 Hz), and 3 methyl hydrogen signals δ H 1.85 (3H, m), 1.30 (3H, s), 0.95 (3H, s), where δ H 1.85 of the methyl groups are attached to the double bond carbon. 13 CNMR (150MHz, CDCl3 ) spectrum showed 15 carbon signals in total. Combined with HSQC spectrum analysis, it was inferred that the compound contained 1 carbonyl carbon signal (δ C 205.3), 2 olefin carbon signals (δ C 138.1, 135.8), two sp3 hybridized quaternary carbon signals (δ C 74.6,43.5), 2 methine carbon signals (δ C 49.3,42.5), 5 methylene carbon signals (δ C 69.3, 46.9, 43.9, 42.4, 26.8) and three methyl carbon signals (δ C 25.2,22.2,20.3).

[0029] HMBC spectrum H 1.85(H 3 -11) and δ C 205.3 (C-7), 135.8 (C-8) and 138.1 (C-9) are related, δ H 6.14(H-9) and δ C 205.3 (C-7), 135.8 (C-8) and 22.2 (C-11) are related, indicating the presence of an α,β-unsaturated ketone fragment composed of C-7, C-8 and C-9, and the methyl CH 3 -11 is attached to C-8. H 6.14(H-9) and δ C There is a correlation between 46.9 (C-10) and 43.5 (C-1), δ H 2.45(H-10α) and δ C 138.1(C-9),135.8(C-8),43.5(C-1),42.5(C-5),42.4(C-2) and 20.3(C-12) are related. H 0.95(H 3 -12) is related to 46.9 (C-10) and 43.5 (C-1), indicating that C-10 is connected to C-9 and C-1, and C-1 is connected to C-2, C-5, C-10 and 12-CH 3 Connected. H 2.51(H-5) and δ C 49.3 (C-4), 46.9 (C-10), 43.9 (C-6), 43.5 (C-1) and 20.3 (C-12) are related. H 3.06(H-6α) and δ C205.3 (C-7), 135.8 (C-8), 49.3 (C-4), 46.9 (C-10), 43.5 (C-1), 42.5 (C-5) and 20.3 (C-12) were correlated, which confirmed the existence of a seven-membered ring fragment composed of C-1, C-5, C-6, C-7, C-8, C-9, and C-10, and methyl groups were attached to C-1 and C-8 respectively. 1 H- 1 H COSY spectrum analysis revealed that δ H 3.06 (H-6α) and δ H 2.96(H-6β) and δ H 2.51(H-5),δ H 2.51(H-5) and δ H 2.37(H-4),δ H 2.37(H-4) and δ H 1.69(H-3),δ H 1.69(H-3) and δ H 1.61 (H-2α) and δ H 1.38 (H-2β) is related, which confirms the existence of a five-membered ring composed of C-1, C-2, C-3, C-4, and C-5, and is connected to the seven-membered ring above through C-1 and C-5. H 2.37(H-4) and δ C 74.6 (C-13), 69.3 (C-14) and 25.2 (C-15) and δ H 3.58 (H-14α) and δ C 74.6 (C-13), 49.3 (C-4) and 25.2 (C-15) have HMBC correlation, which confirms that C-13 is related to C-4, C-14 and CH 3 -15 connected, combined with C-13 (δ C 74.6) and C-14 (δ C 69.3), it was inferred that C-13 and C-14 were connected to hydroxyl groups, and the planar structure of the compound was determined.

[0030] The NOESY spectrum of compound 3 was analyzed, 12-CH 3 There is NOE correlation with H-14, indicating that 12-CH 3 All of them are β-configuration, and H-4 is α-configuration. 3 -12 and H-6β(δ H 2.96), H-5 and H-6α(δ H 3.06), indicating that H-5 is also in α configuration.3 , the relative configurations of H-4 and H-5 were determined to be R*, S*, S*. The relative configuration of the side chain C-13 was determined by density functional theory (DFT) at the B3LYP / 6-311++G (2d, p) level by calculating its chemical shift, and the relative configuration of the entire molecule was finally determined to be 1R*, 4S*, 5S*, 13R*. The absolute configuration and retrograde determination of the compound were further determined by calculating ECD, and the absolute configuration of compound 3 was determined to be 1S, 4R, 5R, 13S.

[0031] Daphneaine F(4): Pale yellow oily compound, UV(MeOH)λ max (logε):239nm(3.86); ECD(MeOH)λ max (Δε)230(+28.3),304(-34.1)nm.The 1 H(600MHz,CDCl 3 )and 13 C NMR data (150 MHz, CDCl 3 ).HRESIMS(m / z):275.1619, combined with 13 The molecular formula calculated by C NMR is C 15 H 24 O 3 ([M+Na] + Calculate for C 15 H 24 O 3 Na, 275.1623), the degree of unsaturation is 4. 1 H NMR (600 MHz, DMSO-d 6 ) spectrum showed a double bond proton signal of 6.89 (1H, dt, J = 8.3, 2.7 Hz) and an aldehyde hydrogen signal δ H 9.28 (1H, s), and 2 methyl carbon signals δ H 1.19 (3H, s) and 0.84 (3H, s). 13 CNMR (150MHz, DMSO-d 6 ) spectrum shows 15 carbon signals, including 3 sp 2 Hybridized carbon signal (δ C 157.2,143.4,196.0), 2 methyl carbon signals (δ C 26.2,17.0), 4 methine carbon signals (δ C 48.6,48.4), 6 methylene carbon signals (δ C68.4, 41.7, 28.9, 25.4, 20.1, 39.6) and two quaternary carbon signals (δ C 73.9,44.0).

[0032] HMBC spectrum H 9.28(H-11) and δ C 20.1(C-9),143.4(C-8) and 157.2(C-7) are related, δ H 6.89(H-7) and δ C The presence of correlations at 20.1 (C-9), 28.9 (C-6), 48.6 (C-5), 143.4 (C-7) and 196.0 (C-11) indicates the presence of α,β-unsaturated aldehyde fragments formed by C-7, C-8 and C-11. H 1.81(H-5) and δ C 17.0(CH 3 -12), 28.9 (C-6), 44.0 (C-1), 43.4 (C-5), 48.4 (C-4), 73.9 (C-13) and 157.2 (C-7) are related. H 0.84(CH 3 -15) and δ C 41.7 (C-1), 44.0 (C-1), 40.1 (C-10) and 48.6 (C-5) are related, combined with δ H 6.89(H-7) and δ H 3.12, 2.54 (H-6), 1.74, 1.12 (H-10) and 2.58, 1.92 (H-9) exist 1 H- 1 H COSY correlation indicates the existence of a seven-membered ring fragment consisting of C-1, C-5, C-6, C-7, C-8, C-9, and C-10, and 12-CH 3 Connected to C-1. H 2.33(H-4) and δ C 48.6 (C-5), 25.4 (C-3), 68.4 (C-14) and 73.9 (C-13) are related, δ H 1.19(CH 3 -15) and δ C 48.6 (C-5), 68.4 (C-14) and 73.9 (C-13) are related, δ H 1.24,1.37(H-2) and δ C1.63, 1.57 (H-3), 1.63, 1.57 (H-3) and 2.33 (H-4) are related to the group of five-membered ring fragments composed of C-1, C-2, C-3, C-4, C-5, which are connected to the above seven-membered ring fragments through C-1 and C-5, and C-13, C-14 and 15-CH 3 The side chain of the component is connected to C-4. Combining the chemical shifts of C-13 and C-14, it is inferred that there are hydroxyl groups on C-13 and C-14.

[0033] The NOESY spectrum of compound 4 was analyzed and it was found that 12-CH 3 There are NOESY related signals with H-14, H-5 and H-4, confirming that 12-CH 3 The orientation of H-4 and H-5 of compound 4 was determined to be α by NOESY. 3 The relative configuration of the side chain C-13 was determined by density functional theory (DFT) at the B3LYP / 6-311++G (2d, p) level by calculating the chemical shift, and the relative configuration of the entire molecule was finally determined to be 1R*, 4S*, 5S*, 13R*.

[0034] minjiangdaphne B(5): a colorless oily compound, UV(MeOH)λ max (logε):204nm(1.54)268nm(0.04); ECD(MeOH)λ max (Δε)210(-7.14)nm. 1 H(600MHz,CDCl 3 )and 13 C NMR data (150 MHz, CDCl 3 ).HRESIMS(m / z):275.1620, combined with 13 The molecular formula calculated by C NMR is C 15 H 24 O 3 ([M+Na] + Calculate for C 15 H 24 O 3 Na, 275.1623), the degree of unsaturation is 4. 1 H NMR (600 MHz, DMSO-d 6 ) The spectrum shows 2 sp 2Hybrid proton signal 4.75 (1H, dt, J = 2.8, 1.4 Hz), 4.70 (1H, d, J = 2.5 Hz), 2 methyl carbon signals δ H 1.68 (3H, s) and 0.90 (3H, s) and two hydroxyl proton signals δ H 4.87 (1H, t, J = 5.8 Hz) and 3.94 (1H, dd, J = 3.1, 1.0 Hz). 13 C NMR (150 MHz, DMSO-d 6 ) spectrum shows 15 carbon signals, including 2 sp 2 Hybridized carbon signal (δ C 147.6,113.1), 2 methyl carbon signals (δ C 22.8,18.1), 4 methine carbon signals (δ C 48.9, 45.3, 63.9, 56.7), 5 methylene carbon signals (δ C 63.5, 40.7, 29.2, 27.6, 40.1) and two quaternary carbon signals (δ C 63.3,43.4).

[0035] HMBC spectrum H 1.68(CH 3 -14) and δ C 48.9 (C-2), 113.1 (C-13) and 147.6 (C-12) are related, δ H 4.75,4.70(H-13) and δ C 48.9(C-2),22.8(CH 3 -14) and 147.6 (C-12) are related to each other, which indicates that C-2, C-13, C-12, CH 3 -14 forms a terminal double bond fragment, where CH 3 -14 is linked to C-12. H 2.26(H-1) and δ C 18.1(CH 3 -15), 29.2 (C-10), 40.1 (C-6), 43.4 (C-5), 48.9 (C-2), 63.9 (C-9) and 147.6 (C-12) are related. H 0.90(CH 3 -15) and δ C 45.3 (C-1), 40.7 (C-4), 40.1 (C-6) and 43.4 (C-5) are related and δ H 1.41,1.30(H-4) and δC 27.6 (C-3), 48.9 (C-2), 43.4 (C-5), 45.3 (C-1) and 18.1 (CH 3 -15) indicates the existence of a five-membered ring fragment consisting of C-1, C-2, C-3, C-4, and C-5, and CH 3 -15 is attached to C-5. H 2.93(H-7) and δ C 40.1(C-6),63.3(C-8),63.9(C-9) and 63.5(C-11) are related, δ H 3.95(HO-9) and δ C 29.2 (C-10), 63.3 (C-8) and 63.9 (C-9) are related, δ H 3.43,3.37(H-11) and δ C The presence of 56.7 (C-7), 63.3 (C-8) and 63.9 (C-9) indicates that a seven-membered ring fragment composed of C-1, C-5, C-6, C-7, C-8, C-9 and C-10 is present, and is linked to the five-membered ring fragment above through C-1 and C-5. Combining the chemical shifts of H-8 and H-9, it is inferred that a three-membered oxygen ring is formed between C-8 and C-9. At the same time, combined with the mass spectrometry data, further judgment was made, thus determining the planar structure of compound 5.

[0036] The NOESY spectrum of compound 5 was analyzed and it was found that 15-CH 3 with H-7, H-1 with 9-HO, H-9 with H 2 -11 has NOESY related signals, 14-CH 3 With 15-CH 3 The presence of NOESY related signals confirmed the presence of 15-CH 3 ,H-7 and H 2 -11 is β configuration, H-1, HO-9 and H-2 are α configuration, and the relative configuration of compound 5 is determined to be 1R*, 2R*, 5S*, 7R*, 8S*, 9S*. The absolute configuration of the compound is determined by comparing the calculated ECD with the measured ECD, and the absolute configuration of the molecule is finally determined to be 1R, 2R, 5S, 7R, 8S, 9S. The NMR data of compound 3-5 are shown in Table 2.

[0037] Table 2 3-5(a: CDCl 3 and b: DMSO) 1 H(600MHz) and 13 C (150 MHz) NMR data

[0038]

[0039]

[0040] In a fourth aspect, the present invention provides use of the sesquiterpene or sesquiterpene dimer derivative described in the first aspect or the pharmaceutical composition described in the second aspect in the preparation of an anti-Parkinson's disease drug.

[0041] The neuroprotective activity of the five new compounds isolated from Daphne koreana described in the invention on SH-SY5Y neuronal cell damage induced by hydrogen peroxide was studied. The in vitro test results showed that compounds 4 and 5 showed relatively better anti-hydrogen peroxide-induced human SH-SY5Y neuronal cell damage activity at 50 μM. Their survival rates were 72.78% and 69.43% (positive drug Trolox 60.00%), respectively. Therefore, the new guaiacyl sesquiterpenes described in the present invention have the prospect of further development of drugs for the prevention and treatment of nerve damage.

[0042] Compared with the prior art, the present invention has the following beneficial effects: The advantage of the present invention is that the sesquiterpene or sesquiterpene dimer compounds described in the present invention are optically pure compounds with a definite stereoconfiguration, and at the same time they have good neuroprotective activity and are worthy of further development. Description of the drawings:

[0043] Figure 1 UV spectrum of compound 1;

[0044] Figure 2 HRESIMS spectrum of compound 1;

[0045] Figure 3 UV spectrum of compound 2;

[0046] Figure 4 HRESIMS spectrum of compound 2;

[0047] Figure 5 UV spectrum of compound 3;

[0048] Figure 6 HRESIMS spectrum of compound 3;

[0049] Figure 7 UV spectrum of compound 4;

[0050] Figure 8 HRESIMS spectrum of compound 4;

[0051] Fig. 9 UV spectrum of compound 5;

[0052] Fig.10 HRESIMS spectrum of compound 5. Specific implementation method:

[0053] The following examples are intended to help those skilled in the art to better understand the present invention, but are not intended to limit the present invention in any way.

[0054] Example 1: Preparation of Compound 1-5

[0055] Take 50 kg of dried whole plant of Daphne penicillata Rehd., extract it with 70% industrial ethanol under heating and reflux for 3 times, each time for 2.5 hours, and recover the solvent to obtain about 3.5 kg of total extract. After the total extract is suspended in water, it is extracted with ethyl acetate and n-butanol in turn, and finally about 500 g of ethyl acetate layer extract and about 1900 g of n-butanol layer extract are obtained. The ethyl acetate layer extract and the n-butanol layer extract are respectively subjected to rapid gradient elution by reduced pressure silica gel column chromatography with a dichloromethane-methanol system as the eluent at a ratio of 100:0-5:1. The obtained fractions are analyzed by TLC thin layer chromatography and HPLC liquid phase, and then combined into two fractions of FrA-B. Fraction A is further subjected to silica gel column chromatography with a dichloromethane-methanol system as the eluent at a gradient elution of 100:0-5:1 to obtain two fractions of about 50 g FrA1 and about 200 g FrA2. FrA2 was eluted with dichloromethane-methanol system as polyamide at a ratio of 100:0-5:1 to obtain two fractions, FrA2-Ⅰ about 60g and FrA2-Ⅱ about 120g. FrA2-Ⅱ fraction was eluted with HP-20 using 90% ethanol-water system to remove small polar pigments, and ODS column chromatography was used for gradient elution with alcohol-water system 20:80-90:10, and thin layer chromatography and HPLC analysis were performed to further obtain 6 fractions N1 about 20g, N2 about 20g, N3 about 13g, N4 about 10g, N5 about 14g, N6 about 15g. The obtained fraction N2 was subjected to silica gel column chromatography with petroleum ether-ethyl acetate system 50:1-0:1 and gradient elution to obtain 6 fractions 2.1-2.6. HPLC was performed using a C18 preparative column; fraction 2.2 was separated by reverse phase separation using a methanol-water system of 70:30 to obtain 2.2.1-2.2.6, and further HPLC was performed using a C18 semi-preparative column and fraction 2.2.3 was eluted using an acetonitrile-water system of 53:47 to obtain compounds 1 (5 mg) and 2 (1 mg). The N1 fraction was separated in a similar manner to obtain five fractions 1.1-1.5. Fraction 1.4 was subjected to reverse phase separation using a C18 preparative column with a methanol-water system of 75:25 by HPLC to obtain fractions 1.4.1-1.4.6. Fraction 1.4.2 was further eluted using an acetonitrile-water system of 62:38 by HPLC using a C18 semi-preparative column to obtain compound 3 (3 mg). Fraction 1.4.3 was eluted with an acetonitrile-water system of 60:40 to obtain compound 4 (2 mg); and fraction 1.4.4 was eluted with an acetonitrile-water system of 59:41 to obtain compound 5 (5 mg).

[0056] Example 2: Compounds 1-5 in vitro for H 2 O 2 Activity study of protective effect of induced human SH-SY5Y nerve cells injury

[0057] The MTT assay was used to investigate the effects of the compounds on H 2 O 2 The blank group was not added with H 2 O 2 and compounds, but inoculated with human SH-SY5Y neural cells (ATCC, Rockville, MD, USA); the negative control group was added with H 2 O 2 The drug-treated groups were pretreated with different concentrations of compounds 1-5 (12.5, 25, 50 μM) in the blank culture medium for 1 h, and then 200 μM H 2 O 2 At 5% CO 2 , and continue to culture in a 37°C incubator for 4 hours to establish an oxidative damage model. 20 μL MTT (5 mg / mL) was added to each well for 4 hours, and the formed crystals were dissolved in 150 μL DMSO. The absorbance value at a wavelength of 492 nm was measured using an enzyme marker, and the survival rate was calculated according to the following formula:

[0058] Cell survival rate (%) = [A 490(sample) –A 490(control) ] / [A 490(control) –A 490(blank) ]×100%.

[0059] The neuroprotective activity of compounds 1 to 5 was evaluated. After treatment with compounds 1 to 5, the cell survival rates were 54.95%, 44.32%, 53.68%, 72.78%, and 69.43%, respectively (positive drug Trolox 60.00%), among which compounds 4 and 5 showed relatively better neuroprotective activity and had the potential to be further developed into anti-PD drugs.

[0060] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A guaiacyl sesquiterpene or sesquiterpene dimer derivative from Daphne koreana, characterized in that: The derivative has the structure shown below: 。 2. The sesquiterpene or sesquiterpene dimer derivative according to claim 1, characterized in that: The Minjiang Daphne is a plant of the genus Daphne of the family Thymelaeaceae. Daphne penicillata Rehd.].

3. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the sesquiterpene or sesquiterpene dimer derivative according to claim 1 or claim 2 and a pharmaceutically acceptable carrier.

4. The method for preparing the guaiacane sesquiterpene or sesquiterpene dimer derivative from Daphne koreana of claim 1, characterized in that: The preparation method comprises the following steps: The whole plant of Daphne koreana was dried, and extracted with 70% industrial ethanol under heating and reflux for 3 times. The total extract was concentrated after the solvent was recovered. The total extract was vortexed with water and extracted with ethyl acetate and n-butanol successively. The obtained extract was then chromatographed on a silica gel column with a dichloromethane-methanol system as an eluent at a gradient elution ratio of 100:0-5:

1. The obtained fractions were analyzed by TLC thin-layer chromatography and HPLC liquid phase analysis and then combined into two fractions of Fr A and Fr B. Fraction A was further subjected to silica gel column chromatography using a dichloromethane-methanol system as the eluent with a gradient elution of 100:0-5:1 to obtain two fractions Fr A1- Fr A2; Fr A2 was eluted through polyamide using a dichloromethane-methanol system as an eluent at a ratio of 100:0-5:1 to obtain two fractions, Fr A2-Ⅰ and Fr A2-Ⅱ; The Fr A2-Ⅱ fraction was eluted with HP-20 using a 90% ethanol-water system to remove small polar pigments, and then eluted with an ODS column chromatography using an alcohol-water system of 20:80-90:10 for gradient elution to obtain 6 subfractions N1-N6; The obtained fraction N2 was subjected to silica gel column chromatography with a petroleum ether-ethyl acetate system of 50:1-0:1 for gradient elution to obtain 6 fractions 2.1-2.

6. Fraction 2.2 was separated by HPLC with a methanol-water system of 70:30 to obtain 2.2.1-2.2.6, and fraction 2.2.3 was separated to obtain compounds 1 and 2. Similarly, N1 was separated by silica gel column chromatography to obtain 5 fractions 1.1-1.

5. Fraction 1.4 was separated by HPLC with a methanol-water system of 75:25 to obtain 1.4.1-1.4.

6. 1.4.2, 1.4.3, and 1.4.4 were separated by C18 column acetonitrile-water 59:41-62:38 to obtain compounds 3-5.

5. The method for preparing a sesquiterpene or sesquiterpene dimer derivative according to claim 4, characterized in that: The Daphne koreana used is Daphne koreana of the genus Daphne in the family Thymelaeaceae. Daphne penicillata Rehd.].

6. The method for preparing a sesquiterpene or sesquiterpene dimer derivative according to claim 4, characterized in that: Take the dried whole plant of Daphne koreana and extract it with 70% industrial ethanol for 3 times, each time for 2-3 hours.

7. The method for preparing a sesquiterpene or sesquiterpene dimer derivative according to claim 4, characterized in that: 1.4.2, 1.4.3, 1.4.4, and 2.2.3 were separated using 75:25-50:50 acetonitrile-water mobile phase.

8. Use of the guaiacane sesquiterpene and sesquiterpene dimer derivatives from Daphne koreana as claimed in claim 1 or claim 2 or their pharmaceutically acceptable salts, or the pharmaceutical composition as claimed in claim 3, in the preparation of a medicament for preventing or treating Parkinson's disease.

9. The use according to claim 8, characterized in that: The derivative or pharmaceutical composition has neuroprotective activity against H2O2-induced oxidative damage to human SH-SY5Y cells.

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