Terpenoid compounds in Daphne koreana and preparation method and application thereof

Abstract

The present invention belongs to the field of pharmaceutical technology, and specifically relates to extracting and separating the 9-oxa-tetracyclic [6.6.1.0 2,6 .0 8,13 ]pentadecane and tetracyclo[5.3.0.1 2,5 .2 4,11 ] two types of heteroterpene compounds of tridecane. The compounds are separated by silica gel column chromatography, ODS column chromatography and HPLC. The present invention also relates to investigating the inhibitory activity of the compounds on acetylcholinesterase. The experimental results show that compared with the positive drug donepezil, IC 50 The value was 1.85±0.03μM. Compounds 1 and 2 showed stronger inhibitory activity against acetylcholinesterase than positive drugs, IC 50 The values ​​were 0.02±0.02μM and 0.31±0.18μM respectively. The present invention enriches the medical use of Daphne koreana in fighting Alzheimer's disease. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and relates to a terpene compound with a new skeleton, a preparation method and an application thereof, and in particular to a terpene compound with a new skeleton in Daphne koreana and a preparation method thereof, and an application thereof in the preparation of an anti-Alzheimer's disease drug. Background Art

[0002] Daphne altaica Pall. is a plant of the genus Daphne in the family Thymelaeaceae, distributed in areas north of the Junggar Basin in Xinjiang. It is warm in nature and pungent in taste, and has the effects of dispelling wind and cold, removing phlegm and relieving cough, and promoting qi and relieving pain. It is often used for colds, coughs with phlegm, and stomachache caused by qi stagnation.

[0003] Alzheimer's disease (AD) is a progressive neurodegenerative disease with an insidious onset. Clinically, it is characterized by global dementia such as memory impairment, aphasia, apraxia, agnosia, visual-spatial skill impairment, executive dysfunction, and personality and behavioral changes. The cause is still unknown. According to data from the International Alzheimer's Association, one patient is born every three seconds in the world, and this number continues to rise with the aging of the population. It is estimated that by 2050, the number of Alzheimer's patients worldwide will reach 152 million. The disease seriously endangers people's health and brings a serious burden to the social economy. However, the pathogenesis of the disease is not yet fully understood, and there is no good clinical treatment drug. As one of the treatment methods, acetylcholinesterase inhibitors can improve the efficiency of cholinergic transmission by preventing the hydrolysis of released acetylcholine, and are considered to be a major therapeutic target that can change the course of AD.

[0004] The inventors conducted research on Daphne koreana and found that it contained a variety of heteroterpenoid compounds with novel structures and exhibited good anti-AD activity. Summary of the invention

[0005] The purpose of the present invention is to solve the deficiencies of the prior art and provide a terpenoid compound with a new skeleton in Daphne koreana and a preparation method thereof and an application thereof in the preparation of an anti-Alzheimer's disease drug.

[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 present invention provides two heteroterpenoid compounds with new skeletons, the structures of which are shown below:

[0008]

[0009] In a second aspect, the preparation of the heteroterpenoid compound having a novel skeleton according to the present invention comprises the following steps:

[0010] (1) extracting the dried whole plant of Daphne koreana with 80% industrial ethanol by reflux;

[0011] (2) concentrating the extract under reduced pressure to obtain an extract, and extracting the extract with n-butanol;

[0012] (3) Separating the n-butanol layer sample through a silica gel vacuum column to obtain Fr.A to Fr.F;

[0013] (4) Fr.A is separated by a reverse ODS column to obtain Fr.A1;

[0014] (5) Fr.A1 was subjected to silica gel column chromatography with a petroleum ether-ethyl acetate system = 100:1-1:1 for gradient elution, and the resulting fractions were subjected to preparative and semi-preparative HPLC with acetonitrile-water = 60:40-65:35 for gradient elution to obtain Compound 1 and Compound 2.

[0015] in,

[0016] The concentration of industrial ethanol in step (1) is 80%; the extraction times is 3 times.

[0017] The temperature of the reduced pressure concentration in step (2) is 55-65°C.

[0018] The gradient elution condition of the reduced pressure silica gel column chromatography in step (3) is dichloromethane-methanol=100:1-1:1.

[0019] The isocratic elution condition of the reversed-phase ODS column in step (4) is ethanol:water=80:20.

[0020] The gradient elution condition of the silica gel column chromatography in step (5) is petroleum ether-ethyl acetate = 100:1-1:1; the gradient elution condition of the semi-preparative HPLC is acetonitrile-water = 60:40-65:35.

[0021] The obtained compound 1-2 was subjected to systematic structural identification, and the results are as follows ( Figure 1-Figure 8 shown):

[0022] Compound 1: pale yellow solid; 91.7(c 0.1,MeOH);UV(MeOH)λ max (logε)205(2.28),239(1.97); HRESIMS gave the quasi-molecular ion peak m / z 499.2482[M+H] + (calcd for C 32 H 35 O5,499.2479), combined 1 H-NMR, 13C-NMR suggests that its molecular formula is C 32 H 34 O5, calculated unsaturation is 16.

[0023] 1 In the H NMR (600 MHz, Benzene-d6) spectrum, δ H 0.81 (3H, s, H3-31), 0.54 (3H, d, J = 6.9 Hz, H3-32) indicate the presence of two methyl hydrogen proton signals; δ H 7.85 (2H, m, H-19, 23), 7.03 (2H, o, H-20, 22), 7.10 (1H, m, H-21), 7.03 (2H, o, H-26, 30), 7.08 (2H, m, H-27, 29), 6.99 (1H, m, H-28) indicate two groups of monosubstituted benzene ring hydrogen signals; δ H 6.04 (1H, s, H-14) indicates an olefinic hydrogen proton signal.

[0024] exist 13 The C-NMR (150 MHz, Benzene-d6) spectrum gave a total of 32 carbon signals, including 2 ketone carbonyl carbon signals, 1 ester carbonyl carbon signal, 12 benzene ring carbon signals, 2 olefin carbon signals, 2 methyl carbon signals, 7 methylene carbon signals, 2 sp 3 Hybridized methine carbon signal, 4 sp 3 Hybridized quaternary carbon signal.

[0025] In the HMBC spectrum, H3-32 correlated with C-3 and C-5, H-3 correlated with C-1 and C-5, and H-2 correlated with C-4, confirming the structural fragment of ring A. H-6 correlated with C-1, C-4, and C-11, H3-31 correlated with C-1, and H-2 correlated with C-10, confirming the structure of ring B, and confirming that ring A and ring B are fused through a C1-C5 bond. H3-31 correlated with C-9 and C-11, H-9 correlated with C-1, C-7, and C-11, and H-6 correlated with C-8, confirming the structure of ring C, and confirming that ring B and ring C are fused through a C10-C11-C7 bond. H-14 correlated with C-7, C-12, and C-9, combined with δ C 162.8 (C-12) and δ C88.1 (C-7), the structural fragment of the D ring was determined, and it was confirmed that the C ring and the D ring were fused through the C7-C8 bond. In summary, the structure of the A part composed of the A, B, C, and D rings (Unit A) was determined. H-15 is related to C-9, C-14, C-7, and C-17, H-16 is related to C-8, H-19 and H-23 are related to C-21, H-19 is related to C-23, and H-19 and H-23 are related to C-17, thereby determining the structural fragment of the B part (Unit B). H-26 and H-30 are related to C-28, H-26 is related to C-30, H-24 is related to C-26 and C-30, and H-14 is related to C-24, thereby determining the structure of the C part (Unit C). In summary, the planar structure of compound 1 was determined to be a 9-oxa-tetracyclic [6.6.1.0 2,6 .0 8,13 ]Heteroterpenoid compounds with a pentadecane skeleton.

[0026] In the NOESY spectrum, H-5 correlated with H-9α, H-9β correlated with H3-31, H-9β correlated with H-14, H-14 correlated with H3-31, H-9α correlated with 1-OH, and H-15 correlated with H-6α. The relative configurations of C-1, C-5, C-7, C-8, and C-10 were determined to be 1R*, 5R*, 7S*, 8R*, and 10R*. In order to further determine the relative configuration of C-4, quantum chemical NMR calculations were performed for all possible configurations of the structure [(1R*, 4S*, 5R*, 7S*, 8R*, 10R*)-1(1-1) and (1R*, 4R*, 5R*, 7S*, 8R*, 10R*)-1(1-2)]. Various statistical analysis methods (R 2 , DP4+ probability analysis), comparing the correlation coefficient and confidence probability between the compound and the two possible configurations, it was found that the calculated results of the 1-2 configuration (1R*, 4R*, 5R*, 7S*, 8R*, 10R*) were the best. The DP4+ probability analysis of the hydrocarbon data was 100% and the calculated R 2 The highest value (R 2 =0.9966), so the relative configuration of the compound was determined to be (1R*, 4R*, 5R*, 7S*, 8R*, 10R*). By comparing the calculated ECD spectrum with the measured ECD spectrum, the absolute configuration of the compound can be determined to be 1R, 4R, 5R, 7S, 8R, 10R. According to Scifinder search, the compound is a new compound that has not been reported in the literature and is named daphnaltaicanoid A.

[0027] Compound 2: pale yellow solid; (c 0.1,MeOH); UV(MeOH)λ max (logε)233(1.71),275(1.21); HRESIMS gave the quasi-molecular ion peak m / z 517.2586[M+H] + (calcd for C 32 H 37 O6,517.2585), combined 1 H-NMR, 13 C-NMR suggests that its molecular formula is C 32 H 36 O6, calculated unsaturation is 15.

[0028] 1 In the H NMR (600 MHz, Benzene-d6) spectrum, δ H 1.22 (3H, s, 23-CH3), 0.60 (3H, d, J = 6.9 Hz, 24-CH3) indicates the presence of two methyl hydrogen signals; δ H 7.10 (2H, o, H-18, H-22), 7.10 (2H, o, H-19, H-21), 7.01 (1H, o, H-20), 8.15 (2H, m, H-3', H-7'), 7.01 (2H, o, H-4', H-6'), 7.08 (1H, m, H-5') indicate two groups of monosubstituted benzene ring hydrogen signals; δ H 9.71 (1H, s, 25-CHO) indicates an aldehyde hydrogen signal.

[0029] exist 13 The C-NMR (150 MHz, Benzene-d6) spectrum gave a total of 32 carbon signals, including one ketone carbonyl carbon signal, one aldehyde carbon signal, one ester carbonyl carbon signal, two benzene ring carbon signals, two methyl carbon signals, seven methylene carbon signals, three methine carbon signals, five sp 3 Hybridized quaternary carbon signal.

[0030] In the HMBC spectrum, H3-24 correlated with C-3 and C-5, H-3 correlated with C-1 and C-5, H-2 correlated with C-4 and C-5, and the A ring structure fragment was determined. H-4 correlated with C-6, H-2 correlated with C-10, H3-23 correlated with C-1 and C-11, H-6 correlated with C-11 and C-1, and the combination 1 H- 1The H-5 / H-6 correlation in the H COSY spectrum determined the structural fragment of the B ring and confirmed that the A and B rings were fused through the C1-C5 bond. H-9 was correlated with C-11, C-7, and C-1, H-6 was correlated with C-8, and H3-23 was correlated with C-9, which determined the structure of the C ring and confirmed that the B and C rings were fused through the C10-C11-C7 bond. H-12 was correlated with C-10, C-7, and C8, and H-13 was correlated with C-9 and C7. 1 H- 1 The H-12 / H-13 correlation in the H COSY spectrum determined the D ring structure fragment, and confirmed that the D ring and C ring were fused through the C8-C7-C11 bond. According to the HMBC correlation of H-12 and C-25, and the correlation of 25-CHO and C-11, it was determined that the aldehyde group was connected to C-11. In summary, the structure of the A part (Unit A) composed of A, B, C, and D rings was determined. H-12 was correlated with C-14, H-15 was correlated with C-17, H-16 and C-18, C-22 and C-14 were correlated, H-18 and H-22 were correlated with C-20, H-18 was correlated with C-22, and combined with 1 H- 1 The correlation of H-15 / H-16 and H-18 / H-19 / H-20 / H-21 / H-22 in the H COSY spectrum was used to determine the partial structural fragment of compound B (Unit B). The correlation of H-3' and H-7' with C-1' was combined with 1 H- 1 The H-3' / H-4' / H-5' / H-6' / H-7' correlation in the H COSY spectrum confirmed the presence of a benzoyl group. C 98.5) compared to C-7 (δ C 83.9) shifted greatly toward the downfield direction, so it was determined that the benzoyloxy group was connected to C-8, and thus the partial structural fragment of compound C (Unit C) was determined. In summary, the planar structure of compound 2 was determined to be a tetracyclic [5.3.0.1 2,5 .2 4,11 ]Tridecane skeleton heteroterpenoid compounds.

[0031] In the NOESY spectrum, 25-CHO and H3-23 have NOESY correlation, H3-23 and H-12β have NOESY correlation, H-12β and 25-CHO have correlation, H-12α and H-13 have correlation, H-15 and H3-23 have NOESY correlation, and 25-CHO and H-2α have NOESY correlation. Therefore, the relative configurations of C-1, C-7, C-8, C-10, C-11, and C-13 of the compound were determined to be 1S*, 7S*, 8R*, 10R*, 11R*, and 13S*. In order to further determine the relative configurations of C-4 and C-5, quantum chemical NMR calculations were performed on all possible configurations of the structure [(1S*,4S*,5R*,7S*,8R*,10R*,11R*,13S*)-2(2-1),(1S*,4R*,5R*,7S*,8R*,10R*,11R*,13S*)-2(2-2),(1S*,4S*,5S*,7S*,8R*,10R*,11R*,13S*)-2(2-3)and(1S*,4R*,5S*,7S*,8R*,10R*,11R*,13S*)-2(2-4)]. Various statistical analysis methods (R 2 , DP4+ probability analysis), comparing the correlation coefficient and confidence probability between the compound and the four possible configurations, it was found that the calculated results of the 2-3 configuration (1S*, 4S*, 5S*, 7S*, 8R*, 10R*, 11R*, 13S*) were the best. The DP4+ probability analysis of the hydrocarbon data was 100% and the calculated R 2 The highest value (R 2 =0.9978), so the relative configuration of the compound was determined to be 1S*, 4S*, 5S*, 7S*, 8R*, 10R*, 11R*, 13S*. By comparing the calculated ECD spectrum with the measured ECD spectrum, the absolute configuration of the compound can be determined to be 1S, 4S, 5S, 7S, 8R, 10R, 11R, 13S. According to Scifinder search, the compound is a new compound not reported in the literature, and it is named daphnaltaicanoid B.

[0032] The NMR data of compound 1-2 are shown in Table 1.

[0033] Table 1 Compound 1-2 (Benzene-d6) 1 H (600 MHz) NMR and 13 C (150 MHz) NMR data

[0034]

[0035]

[0036] The inhibitory activity of the heteroterpenoid compounds on acetylcholinesterase was investigated. The experimental results showed that compared with the positive drug donepezil (IC 50 The value was 1.85±0.03μM), and compound 1 and compound 2 showed stronger inhibitory activity against acetylcholinesterase than positive drugs, IC 50 The values ​​were 0.02±0.02 μM and 0.31±0.18 μM, respectively.

[0037] like Fig. 9 and Fig.10 As shown, the molecular docking experiment results show that the 12-CO group of compound 1 interacts with the amino acid residue PHE288 through hydrogen bonds, and the two benzene ring fragments of compound 1 can interact with the amino acid residues TRP 279 and TYR 334 through π-π bonds, respectively. The 1-OH and 7-OH of compound 2 can interact with the amino acid residues TRP 279 and ASP 285 through hydrogen bonds, respectively, and one benzene ring fragment of compound 2 interacts with the residue TRP 279 through π-π bonds.

[0038] In the third aspect, the present invention also provides a pharmaceutical composition comprising the heteroterpenoid compound 1-2 having a new skeleton or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

[0039] In a fourth aspect, the present invention provides a use of an extract of Daphne koreana L. in the preparation of an anti-Alzheimer's disease drug, wherein the extract comprises the terpene compound having a novel skeleton.

[0040] In addition, the present invention provides the use of heteroterpenoid compounds having a new skeleton or pharmaceutically acceptable salts thereof in the preparation of anti-Alzheimer's disease drugs.

[0041] In addition, the present invention also provides the use of the above-mentioned pharmaceutical composition in the preparation of anti-Alzheimer's disease drugs.

[0042] Compared with the prior art, the present invention has the following beneficial effects: The advantage of the present invention is that the heteroterpenoid compounds 1-2 of the present invention are all optically pure compounds with a novel skeleton with a definite stereochemical configuration, and compounds 1-2 have strong acetylcholinesterase inhibitory activity and are worthy of further development. BRIEF 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 Key HMBC correlation and COSY correlation of compounds 1-2;

[0048] Figure 6 Key NOESY correlation of compounds 1-2;

[0049] Figure 7 Linear correlation diagram of measured and calculated carbon chemical shifts of compound 1-2;

[0050] Figure 8 Comparison of measured and calculated ECD of compound 1-2;

[0051] Fig. 9 Molecular docking diagram of compound 1 and acetylcholinesterase;

[0052] Fig.10 Molecular docking diagram of compound 2 and acetylcholinesterase. DETAILED DESCRIPTION

[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 1-2

[0055] The preparation method of compound 1-2 specifically comprises the following steps:

[0056] 50 kg of the whole plant of Daphne altaica Pall. was selected and extracted three times with 80% industrial ethanol by heating and refluxing. The obtained ethanol extracts were concentrated under reduced pressure and combined to obtain 3.6 kg of total extract. It was extracted with n-butanol solvent to obtain 2.8 kg of n-butanol layer. The n-butanol layer sample was subjected to silica gel vacuum column chromatography with dichloromethane: methanol gradient elution (100:1-1:1) (v / v) to obtain 6 components (Fr.A~Fr.F). Fr.A was eluted isocratically with 80:20 ethanol water through a reverse ODS column to obtain Fr.A1. Fr.A1 was subjected to silica gel column chromatography with a petroleum ether-ethyl acetate system 100:1-1:1 for gradient elution, and the obtained components were subjected to preparative and semi-preparative HPLC with acetonitrile-water (60:40-65:35) to obtain compounds 1 (2.2 mg) and 2 (1.6 mg).

[0057] Example 2: Study on the inhibitory activity of compound 1-2 on acetylcholinesterase

[0058] The acetylcholinesterase inhibitory activity of compound 1-2 was investigated by adding 50 μL of PBS (0.1 M, pH 7.4), 12.5 μL of acetylcholinesterase (0.2 U / mL), 125 μL of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) (0.1 U / mL) and 25 μL of compound (100 μM) to a 96-well plate and letting stand overnight. Then 50 μL of iodinated thioacetylcholine (ATCI) was added and detected at a wavelength of 412 nm using an ELISA reader.

[0059] The results are shown in Table 2. Compared with the positive drug donepezil (IC 50 The value was 1.85±0.03μM), and compounds 1 and 2 showed stronger inhibitory activity against acetylcholinesterase than positive drugs, IC 50 The values ​​were 0.02±0.02 μM and 0.31±0.18 μM, respectively.

[0060] Table 2 Acetylcholinesterase inhibitory activity of compound 1-2

[0061]

[0062] In addition, if Fig. 9 and Fig.10 As shown, the molecular docking experiment results show that the 12-CO group of compound 1 interacts with the amino acid residue PHE 288 through hydrogen bonds, and the two benzene ring fragments of compound 1 can interact with the amino acid residues TRP 279 and TYR 334 through π-π bonds, respectively. The 1-OH and 7-OH of compound 2 can interact with the amino acid residues TRP 279 and ASP285 through hydrogen bonds, respectively, and a benzene ring fragment of compound 2 interacts with the residue TRP 279 through π-π bonds.

[0063] 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 terpene compound, characterized in that: The compound has the structure shown below: 。 2. The method for preparing the heteroterpenoid compound according to claim 1, characterized in that: The following steps are involved: (1) extracting the dried whole plant of Daphne koreana with 80% industrial ethanol by reflux; (2) concentrating the extract under reduced pressure to obtain an extract, and extracting the extract with n-butanol; (3) Separate the n-butanol layer sample through a silica gel vacuum column to obtain Fr. A to Fr. F; (4) Fr. A is separated by a reverse ODS column to obtain Fr. A1; (5) Fr. A1 was subjected to silica gel column chromatography with a petroleum ether-ethyl acetate system = 100:1-1:1 for gradient elution, and the resulting fractions were subjected to preparative and semi-preparative HPLC with a gradient elution of acetonitrile-water = 60:40-65:35 to obtain compound 1 and compound 2.

3. The preparation method according to claim 2, characterized in that: The ethanol concentration in step (1) is 80%; the number of extractions is 3 times; and the temperature of reduced pressure concentration in step (2) is 55-65°C.

4. The preparation method according to claim 2, characterized in that: The gradient elution condition of the reduced pressure silica gel column chromatography in step (3) is dichloromethane-methanol = 100:1-1:

1.

5. The preparation method according to claim 2, characterized in that: The isocratic elution condition of the reverse ODS column in step (4) is ethanol:water = 80:

20.

6. The preparation method according to claim 2, characterized in that: In step (5), the gradient elution condition of silica gel column chromatography is petroleum ether-ethyl acetate = 100:1-1:1; the gradient elution condition of semi-preparative HPLC is acetonitrile-water = 60:40-65:

35.

7. A pharmaceutical composition, characterized in that: The composition comprises the heteroterpenoid compound according to claim 1 and a pharmaceutically acceptable carrier or excipient.

8. Use of the heteroterpenoid compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an anti-Alzheimer's disease drug.

9. Use of the pharmaceutical composition according to claim 7 in the preparation of anti-Alzheimer's disease drugs.

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