Guaiane type sesquiterpenoids in whole plant of daphne aurantii as well as preparation method and application of guaiane type sesquiterpenoids

Abstract

The invention discloses guaiane type sesquiterpenoids in a whole plant of daphne aurantiaca as well as a preparation method and application of the guaiane type sesquiterpenoids, and belongs to the technical field of medicines, 11 new guaiane type sesquiterpenoids are extracted and separated from the whole plant of daphne aurantiaca which is a daphneeacaca plant of daphneeacaca, and the compounds have good A beta aggregation inhibition activity. The preparation method disclosed by the invention is simple and easy to implement, relatively good in reproducibility and relatively high in purity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to 11 guaiacol-type sesquiterpenoid compounds prepared from daphne neroli and the application of these compounds in inhibiting Aβ aggregation activity. Background Technology

[0002] The genus *Daphne* belongs to the family Thymelaeaceae and comprises over 90 species, mainly distributed in parts of Asia, Europe, and North Africa. Recent studies have revealed that plants in this genus possess a rich variety of secondary metabolites and exhibit beneficial biological activities, including neuroprotection.

[0003] Daphne aurantiaca, a small shrub belonging to the genus Daphne in the family Thymelaeaceae, is mainly distributed in northwestern Yunnan and northwestern Sichuan in my country. Currently, there are very few reports on the characteristic chemical components of the entire Daphne aurantiaca plant. The novel compounds and activity data involved in this invention have not yet been reported in any patents or literature. Summary of the Invention

[0004] The primary objective of this invention is to provide 11 guaiacane-type sesquiterpenoid compounds isolated from the whole plant of Daphne aurantiaca, a plant belonging to the Thymelaeaceae family and the genus Daphne, and their preparation methods.

[0005] The present invention also provides the application of the sesquiterpenoid compounds in inhibiting Aβ aggregation activity.

[0006] The 11 guaiacol-type sesquiterpenoid compounds described in this invention have the following structures:

[0007]

[0008] The method for preparing guaiacane-type sesquiterpenoid compounds from the whole plant of Daphne odora of the present invention includes the following steps:

[0009] The dried whole plant of Daphne odora was extracted with ethanol, and the extracts were combined and concentrated to obtain an extract. The extract was extracted with ethyl acetate, and the obtained components were subjected to silica gel column chromatography with gradient elution using a dichloro-methanol system of 1:0-0:1. A total of 10 components Fr.A-Fr.J were collected.

[0010] Using HP20 column chromatography and ODS column chromatography, the fraction Fr.C was eluted with an ethanol-water system of 20:80-100:0 to obtain 12 fractions Fr.C1-Fr.C12.

[0011] Fr.C4 was separated by silica gel column chromatography with a petroleum ether-ethyl acetate system of 100:1-1:1 and by preparative reversed-phase high-performance liquid chromatography using an acetonitrile-water mobile phase to obtain compounds 1-11.

[0012] The above-mentioned method for preparing guaiacane-type sesquiterpenoid compounds from the whole plant of Daphne aurantiaca uses Daphne aurantiaca, a plant of the genus Daphne in the family Thymelaeaceae.

[0013] The preparation method of the guaiacane-type sesquiterpenoid compounds in the whole plant of Daphne odora is as follows: take the dried whole plant of Daphne odora and reflux extract it with 70%-90% industrial ethanol 2-3 times, each time for 2-3 hours.

[0014] The method for preparing guaiacane-type sesquiterpenoids from the whole plant of Daphne odora mentioned above involves separating Fr.C4 using an acetonitrile-water mobile phase of 80:20-20:80.

[0015] The Aβ aggregation activity of the 11 novel compounds described in this invention was investigated. Most of the compounds exhibited good inhibitory activity against Aβ aggregation. Therefore, the guaiacol-type sesquiterpenoid compounds described in this invention have the potential for further development in the prevention and treatment of neurodegenerative diseases with Aβ abnormalities.

[0016] A pharmaceutical composition comprising the above-described guaiacol-type sesquiterpenoid compound prepared from the whole plant of Daphne odora or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

[0017] The present invention also provides the use of the guaiac sesquiterpene compounds prepared from the whole plant of Daphne odora or pharmaceutical compositions containing the above compounds in the preparation of drugs with Aβ aggregation inhibitory activity.

[0018] The advantages of this invention are that the compounds provided by this invention are all new compounds with novel structures, and all are optically pure compounds with defined stereoconfigurations. At the same time, most of the compounds have good activity in inhibiting Aβ aggregation and have value for further development. Attached Figure Description

[0019] Figure 1 HRESIMS of compound 1 1 H, 13 C-NMR and HMBC spectra;

[0020] Figure 2 HRESIMS of compound 2 1 H, 13 C-NMR and HMBC spectra;

[0021] Figure 3HRESIMS of compound 3 1 H, 13 C-NMR and HMBC spectra;

[0022] Figure 4 HRESIMS of compound 4 1 H, 13 C-NMR and HMBC spectra;

[0023] Figure 5 HRESIMS of compound 5 1 H, 13 C-NMR and HMBC spectra;

[0024] Figure 6 HRESIMS for compound 6 1 H, 13 C-NMR and HMBC spectra;

[0025] Figure 7 HRESIMS for compound 7 1 H, 13 C-NMR and HMBC spectra;

[0026] Figure 8 HRESIMS for compound 8 1 H, 13 C-NMR and HMBC spectra;

[0027] Figure 9 HRESIMS for compound 9 1 H, 13 C-NMR and HMBC spectra;

[0028] Figure 10 HRESIMS of compound 10 1 H, 13 C-NMR and HMBC spectra;

[0029] Figure 11 HRESIMS of compound 11 1 H, 13 C-NMR and HMBC spectra;

[0030] Figure 12 This indicates the Aβ aggregation inhibition activity of compounds 1-11. Detailed Implementation

[0031] The embodiments listed below are intended to help those skilled in the art better understand the present invention, but do not limit the invention in any way.

[0032] Example 1

[0033] Preparation of compounds 1-11:

[0034] The dried whole plant of Daphne odora was extracted three times by reflux with 80% ethanol (v / v) for 2-3 hours each time. The extracts were combined and concentrated to obtain an extract. The extract was extracted with ethyl acetate and the obtained components were subjected to silica gel column chromatography with gradient elution using a dichloro-methanol system of 1:0-0:1 (v / v). A total of 10 components Fr.A-Fr.J were collected.

[0035] The pigments of the component Fr.C were removed by HP20 column chromatography using an ethanol-water system of 20:80-100:0.

[0036] The fraction Fr.C was eluted by gradient elution with an ethanol-water system of 20:80-100:0 using ODS column chromatography to obtain 12 fractions Fr.C1-Fr.C12.

[0037] Fr.C4 was separated by silica gel column chromatography with a petroleum ether-ethyl acetate system of 100:1 to 1:1 and by preparative reversed-phase high performance liquid chromatography using an acetonitrile-water mobile phase of 80:20 to 20:80 to obtain compounds 1-11.

[0038] The results of systematic structural identification of the obtained compounds are as follows:

[0039] The structures of compounds 1-11 were identified using high-resolution mass spectrometry, one-dimensional NMR, two-dimensional NMR, computer-aided structure analysis, rhodium salt-induced CD analysis, quantum chemical calculations, and biomimetic synthesis. The corresponding spectra are shown below. Figure 1-11 As shown.

[0040] Dapurant A(1): Yellow oily substance; (c 0.11,MeOH); HRESIMS: m / z 503.2765[M+Na] + (calcd for C 30 H 40 The molecular formula of O5Na, 503.2768) is determined to be C. 30 H 40 O5; By analyzing the nuclear magnetic resonance spectral data of dapurant A, the structure of dapurant A was determined using computer-aided structural analysis, quantum chemical calculations, and biomimetic synthesis methods, identifying it as a new compound.

[0041] Dapurant B(2): Yellow oily; (c 0.11,MeOH); HRESIMS: m / z 503.2766[M+Na] + (calcd for C 30 H 40The molecular formula of O5Na, 503.2768) is determined to be C. 30 H 40 O5; By analyzing the nuclear magnetic resonance spectral data of dapurant B, the structure of dapurant B was determined using computer-aided structural analysis, quantum chemical calculations, and biomimetic synthesis methods, identifying it as a new compound.

[0042] Dapurant C(3): Yellow oily; (c 0.10,MeOH); HRESIMS:m / z 271.1459,273.1433[M+H] + (calcd for C 15 H 24 ClO2 (271.1459, 273.1434) has been determined to have the molecular formula C. 15 H 23 ClO2; By analyzing the nuclear magnetic resonance spectral data of dapurant C, and using rhodium salt-induced CD analysis and quantum chemical calculations, the structure of dapurant C was determined, identifying it as a new compound.

[0043] Dapurant D(4): Yellow oily; (c 0.10,MeOH); HRESIMS:m / z 271.1458,273.1433[M+H] + (calcd for C 15 H 24 ClO2 (271.1459, 273.1434) has been determined to have the molecular formula C. 15 H 23 ClO2; By analyzing the nuclear magnetic resonance spectral data of dapurant D, the structure of dapurant D was determined using rhodium salt-induced CD analysis and quantum chemical calculations, identifying it as a new compound.

[0044] Dapurant E(5): Yellow oily substance; (c 0.13,MeOH); HRESIMS: m / z 271.12982[M+Na] + (calcd for C 15 H 20 The molecular formula of O3Na, 271.13O46) is determined to be C. 15 H 20 O3; By analyzing the nuclear magnetic resonance spectral data of dapurant E and using quantum chemical calculations, the structure of dapurant E was determined, identifying it as a new compound.

[0045] Dapurant F(6): Yellow oily; (c 0.14,MeOH); HRESIMS:m / z 203.14288[M+H] + (calcd for C 14 H 19 The molecular formula (O, 203.14304) is determined to be C. 14 H 18 O; By analyzing the nuclear magnetic resonance spectral data of dapurant F and using quantum chemical calculations, the structure of dapurant F was determined, identifying it as a new compound.

[0046] Dapurant G(7): Yellow oily substance; (c 0.19,MeOH); HRESIMS:m / z 251.16351[M+H] + (calcd for C 15 H 23 The molecular formula of O3,251.16417 is determined to be C. 15 H 22 O3; By analyzing the nuclear magnetic resonance spectral data of dapurant G and using quantum chemical calculations, the structure of dapurant G was determined, identifying it as a new compound.

[0047] Dapurant H(8): Yellow oily substance; (c 0.16,MeOH); HRESIMS: m / z 303.1573[M+Na] + (calcd for C 16 H 24 The molecular formula of O4Na, 303.1572) is determined to be C. 16 H 24 O4; By analyzing the nuclear magnetic resonance spectral data of dapurant H and using quantum chemical calculations, the structure of dapurant H was determined, identifying it as a new compound.

[0048] Dapurant I(9): White amorphous powder; (c 0.10,MeOH); HRESIMS:m / z265.17923[M+H] + (calcd for C 16 H 25 The molecular formula of O3,265.17982 is determined to be C. 16 H 24O3; By analyzing the nuclear magnetic resonance spectroscopy data of dapurant I and using quantum chemical calculations, the structure of dapurant I was determined, identifying it as a new compound.

[0049] Dapurant J(10): Yellow oily; (c 0.29,MeOH); HRESIMS: m / z 277.14304[M+H] + (calcd for C 16 H 21 The molecular formula of O4,277.14344 is determined to be C. 16 H 20 O4; By analyzing the nuclear magnetic resonance spectral data of dapurant J and using quantum chemical calculations, the structure of dapurant J was determined, identifying it as a new compound.

[0050] Dapurant K(11): Yellow oily substance; (c 0.12,MeOH); HRESIMS:m / z 299.1260[M+Na] + (calcd for C 16 H 20 The molecular formula of O4Na,299.1259) is determined to be C. 16 H 20 O4; By analyzing the nuclear magnetic resonance spectral data of dapurant K and using quantum chemical calculations, the structure of dapurant K was determined, identifying it as a new compound.

[0051] Table 1. Compounds 1-2 1 H NMR and 13 C NMR data (600MHz, in CDCl3, δin ppm)

[0052]

[0053]

[0054] Compounds 3-5 in Table 2 1 H NMR and 13 C NMR data (600MHz, in DMSO-d6, δin ppm)

[0055]

[0056]

[0057] Compounds 6-8 in Table 3 1 H NMR and13 C NMR data (600MHz, a. in CDCl3, b. in DMSO-d6, δ in ppm)

[0058]

[0059] Table 4. Compounds 9-11 1 H NMR and 13 C NMR data (600MHz, a. in CDCl3, b. in DMSO-d6, δ in ppm)

[0060]

[0061]

[0062] Example 2

[0063] Biomimetic synthesis of compounds 1-2:

[0064] Take 6 mg (0.022 mmol) of daphnauranol D (Ren,Q.,Zhao,WY,Shi,SC,Han,FY,Zhang,YY,Liu,QB,Yao,GD,Lin,B.,Huang,XX,Song,SJ,2019.Guaiane-type sesquiterpenoidsfrom the roots of Daphne genkwa and evaluation of their neuroprotective effects.J.Nat.Prod.82,1510-1517.https: / / doi.org / 10.1021 / acs.jnatprod.8b01049) in a 10 mL round-bottom flask, add 1 mL of anhydrous methanol to dissolve it, and then add 0.2 mL of 30% sodium hydroxide methanol solution dropwise. Stir the reaction at 55 °C for 12 h. The reaction was confirmed to be complete by HPLC. The pH of the reaction solution was adjusted to neutral. The mixture was extracted with ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The crude product was purified by preparative high-performance liquid chromatography to obtain an intermediate (5.0 mg, 30:70:0.1 acetonitrile-water-formic acid).

[0065] The intermediate obtained above was dissolved in dry dichloromethane, and the reaction was stirred in an ice-water bath. Under nitrogen protection, a triphosgene-dichloromethane solution (2.0 mg, 0.007 mmol) and triethylamine (3 μL, 0.019 mmol) were added dropwise, and the reaction was continued at room temperature until complete. The reaction was terminated by adding water, and the mixture was extracted with ethyl acetate. The organic layers were combined, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain the corresponding crude product, which was then used for the next reaction without purification.

[0066] Take wikstronone C (Liu,Z.,Dong,M.,Chang,H.,Han,N.,Yin,J.,2020.Guaianetype of sesquiterpene with NO inhibitory activity from the root of Wikstroemia indica.Bioorganic Chemistry99,103785.https: / / doi.org / 10.1016 / j.bioorg.2020.103785) (4.5 mg, 0.019 mmol) and DMAP (0.5 mg, 0.004 mmol) in a 10 mL round-bottom flask, and add 0.5 mL of dry dichloromethane under nitrogen protection. Subsequently, the crude product obtained above and triethylamine (3 μL, 0.019 mmol) were added under ice-water bath conditions, and the reaction was stirred at room temperature for 2 h. The reaction was terminated by adding 4 mL of saturated sodium citrate solution. The mixture was extracted with ethyl acetate, the organic layers were combined, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain the corresponding crude product. The final product (0.6 mg, yield 6.12%, 60:40 acetonitrile-water) was obtained by preparative high-performance liquid chromatography. Comparison showed that the product was similar to compound 1. 1 H NMR and 13 The C NMR data were completely consistent, indicating that compound 1 was successfully synthesized via the above route. The synthesis method of compound 2 was the same as that of compound 1, except that wikstronone A was used instead of wikstronone C. The final reaction yielded 1.0 mg of product (yield 5.26%).

[0067] Example 3

[0068] Investigation of Aβ aggregation inhibition activity of compounds 1-11:

[0069] Curcumin was used as a positive control. The concentration of the test compound was prepared to 20 μmol / L using PBS (pH = 7.4). The entire reaction system contained 10.0 μL of the test compound, 10.0 μL of Aβ, and 60 μL of PBS. The mixture was incubated at 37 °C for 24 h, and finally 80 μL of thioflavone T (Th-T) was added. After 5 min of reaction, the absorbance was measured at a wavelength of 485 nm.

[0070] The Aβ aggregation inhibitory activity of compounds 1-11 is as follows: Figure 12 As shown, compounds 1-11 exhibit varying inhibitory activities. Compounds 3 and 4 show relatively high inhibition rates, significantly exceeding those of the positive control curcumin, while compound 6 shows a relatively low inhibition rate. This indicates that compounds with different structures have significantly different inhibitory effects on Aβ aggregation, suggesting a correlation between structure and activity. From the overall activity distribution, most compounds demonstrate some Aβ aggregation inhibitory activity, and further in-depth research is needed on structural optimization and mechanisms of action.

Claims

1. A guaiacane-type sesquiterpene compound from the whole plant of *Daphne odora*, characterized in that, It can be any of the following structures:

2. The guaiacol-type sesquiterpenoid compounds in the whole plant of *Daphne odora* according to claim 1, characterized in that, The orange-flowered daphne is a plant of the genus Daphne in the family Thymelaeaceae [Daphne aurantiaca].

3. A method for preparing guaiacane-type sesquiterpenoid compounds from the whole plant of *Daphne odora* as described in claim 1 or 2, characterized in that, Includes the following steps: The dried whole plant of Daphne odora was extracted with ethanol, and the extracts were combined and concentrated to obtain an extract. The extract was extracted with ethyl acetate, and the obtained components were subjected to silica gel column chromatography with gradient elution using a dichloro-methanol system of 1:0-0:1 to obtain 10 components Fr.A-Fr.J. Using HP20 column chromatography and ODS column chromatography, the fraction Fr.C was eluted with an ethanol-water system of 20:80-100:0 to obtain 12 fractions Fr.C1-Fr.C12. Fr.C4 was separated by silica gel column chromatography with a petroleum ether-ethyl acetate system of 100:1-1:1 and by preparative reversed-phase high-performance liquid chromatography using an acetonitrile-water mobile phase to obtain compounds 1-11.

4. The method for preparing guaiacane-type sesquiterpenoids from the whole plant of *Daphne odora* according to claim 3, characterized in that, The Daphne aurantiaca used is a plant of the genus Daphne in the family Thymelaeaceae.

5. The method for preparing guaiacane-type sesquiterpenoids from the whole plant of *Daphne odora* according to claim 3, characterized in that, The dried whole plant of Daphne odora and orange blossoms was extracted by reflux with 70%-90% industrial ethanol 2-3 times, each time for 2-3 hours.

6. The method for preparing guaiacane-type sesquiterpenoids from the whole plant of *Daphne odora* according to claim 3, characterized in that, Fr.C4 was separated using an acetonitrile-water mobile phase of 80:20-20:

80.

7. A method for preparing compounds 1 and 2 according to claim 1, characterized in that, Includes the following steps: Daphnauranol D was dissolved in anhydrous methanol, and sodium hydroxide or potassium hydroxide was used as a catalyst. After the reaction was complete, the mixture was purified by HPLC. The obtained intermediate was dissolved in dichloromethane, and triphosgene dichloromethane solution and triethylamine were added. The reaction was carried out at room temperature to obtain the crude product. The crude product was added to a dichloromethane solution of Wikstronone C, and DMAP was added. After the reaction was complete, saturated sodium citrate solution was added to terminate the reaction. After purification, compound 1 was obtained. Using the same method, but replacing daphnauranol D with daphnauranol A, compound 2 was obtained after the reaction.

8. A pharmaceutical composition comprising a guaiacane-type sesquiterpene compound or a pharmaceutically acceptable salt thereof from the whole plant of Daphne odora as described in claim 1 or 2, and a pharmaceutically acceptable carrier or excipient.

9. The use of guaiacane-type sesquiterpenoid compounds from the whole plant of Daphne odora according to claim 1 or 2 in the preparation of drugs with Aβ aggregation inhibitory activity.

10. Use of the pharmaceutical composition of claim 8 in the preparation of a drug with Aβ aggregation inhibitory activity.

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