Cassane diterpenoid compound and application thereof in preparation of anti-neuroinflammation drugs

Abstract

The invention provides a cassane diterpenoid compound and an application of the cassane diterpenoid compound in preparation of an anti-neuroinflammation medicine. Six novel cassane diterpenoid compounds are separated from mysorethorn, and a preparation method of the six compounds is provided. According to the present invention, the inhibition effect of lipopolysaccharide (LPS)-induced BV-2 microglial cells on NO production is determined, and the six compounds have anti-neuroinflammatory activity, and particularly, the compounds 4 and 5 have good anti-inflammatory effect compared with other compounds, and have the potential of anti-neuroinflammatory drug preparation.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a kaxane diterpenoid compound and its application in the preparation of anti-neuroinflammatory drugs. Background Technology

[0002] *Caesalpinia*, a genus of legumes, comprises approximately one hundred species widely distributed in tropical and subtropical Asia, with seventeen species commonly found in southern and southwestern China. Some plants in this genus have been used in traditional medicine to treat bruises, dysentery, eczema, and carbuncles. Previous chemical studies of this genus have shown that it contains caesalpinia diterpenoids, flavonoids, and lignans. Caesalpinia diterpenoids are the main secondary metabolites in *Caesalpinia* plants, and their basic molecular skeleton consists of three cyclohexane rings fused with a furan ring or an α,β-butenolide ring. Some of these compounds have exhibited broad biological activities, including anti-neuroinflammatory, antibacterial, and cytotoxic effects.

[0003] *Caesalpinia macrocarpa* is mainly distributed in Guangdong, Guangxi, Yunnan, and Guizhou provinces of China. To the best of current knowledge, only one study has identified kasane diterpenoids from the seeds of *Caesalpinia macrocarpa*. These findings have sparked our interest in searching for kasane diterpenoids with novel structures from *Caesalpinia macrocarpa* seeds. Therefore, this paper reports the isolation, structural identification, and bioactivity of novel kasane diterpenoids from *Caesalpinia macrocarpa*. Summary of the Invention

[0004] In view of this, one object of the present invention is to provide a kasane diterpenoid compound comprising the structure shown in main body ①:

[0005] ①;

[0006] Compound 1 has the molecular formula C 24 H 36 O6, named 12α,14β-dimethoxy-6β-acetoxy-cass-13(15)-en-16,12-olide,

[0007] Compound 1;

[0008] Compound 2 has the molecular formula C 23 H 34 O6, named 12α-methoxy-14β-hydroxy-6β-acetoxy-cass-13(15)-en-16, 12-olide,

[0009] Compound 2;

[0010] Compound 3 has the molecular formula C 24 H 36 O5, named 12α-ethoxy-6β-acetoxy-cass-13(15)-en-16,12-olide,

[0011] Compound 3;

[0012] Compound 4 has the molecular formula C 24 H 36 O6, named 12α-ethoxy-14β-hydroxy-6β-acetoxy-cass-13(15)-en-16, 12-olide,

[0013] Compound 4;

[0014] Compound 5 has the molecular formula C 26 H 40 O6, named 12α,14β-diethoxy-6β-acetoxy-cass-13(15)-en-16,12-olide,

[0015] Compound 5;

[0016] Or for

[0017] Compound 6 has the molecular formula C 22 H 30 O6, named 14β-hydroxy6β-acetoxy-cass-11(12), 13(15)-ene-16, 12-olide,

[0018] Compound 6;

[0019] Furthermore, it includes the structure shown in main body ②:

[0020] ②;

[0021] Among them, compound 4 has the molecular formula C 24 H 36 O6, named 12α-ethoxy-14β-hydroxy-6β-acetoxy-cass-13(15)-en-16, 12-olide,

[0022] Compound 4;

[0023] Compound 5 has the molecular formula C 26 H 40 O6, named 12α,14β-diethoxy-6β-acetoxy-cass-13(15)-en-16,12-olide,

[0024] Compound 5.

[0025] A second objective of this invention is to provide a method for preparing the aforementioned compound, comprising the following steps:

[0026] 1) The seeds of *Cephalotaxus fortunei* were air-dried and ground into powder, then soaked in 95% ethanol for 72 hours. This process was repeated three times. The extracts were combined and concentrated under reduced pressure to obtain the extract.

[0027] 2) The extract was subjected to silica gel column chromatography, and eluted sequentially with petroleum ether-acetone 100:0, 7:1, 4:1, 1:1 and 95% ethanol to obtain five fractions: Fr A, Fr B, Fr C, Fr D and Fr E.

[0028] 3) Fr. C was prepared by HPLC, acetonitrile-water, 50%-100%, 0-60 min, 100%, 30 min, to separate 13 components Fr. C-1 to Fr. C-13;

[0029] 4) Fr. C-6 was purified by reversed-phase semi-preparative HPLC, COSMOSIL cholester, acetonitrile-water, 65:35, to obtain compounds 1 and 6;

[0030] ;

[0031] Compound 2 was obtained by reverse-phase semi-preparative HPLC with Fr. C-4, Phenomenex Luna C18, acetonitrile-water, 60:40;

[0032] ;

[0033] Or / and Fr. C-7 were separated and purified by reversed-phase semi-preparative HPLC, COSMOSIL cholester, acetonitrile-water, 75:25, to obtain compound 3;

[0034] ;

[0035] Compound 4 was obtained by reverse-phase semi-preparative HPLC with Fr. C-5, Phenomenex Luna C18, acetonitrile-water, 65:35;

[0036] ;

[0037] Or / and Fr. C-8 were separated and purified by reversed-phase semi-preparative HPLC, COSMOSIL NAP, acetonitrile-water, 65:35, to obtain compound 5;

[0038] .

[0039] A third objective of this invention is to provide an anti-neuroinflammatory drug comprising a compound with the structure described above.

[0040] A fourth objective of this invention is to provide the use of compounds with the above-described structure in the preparation of anti-neuroinflammatory drugs.

[0041] This invention provides a kazoline diterpenoid compound and its application in the preparation of anti-neuroinflammatory drugs. Six new kazoline diterpenoid compounds were isolated from *Ligustrum lucidum*. NO is a well-known inflammatory factor in the art. The six compounds isolated in this invention can inhibit NO production induced by lipopolysaccharide (LPS) in BV-2 microglia. Compounds 4 and 5, in particular, exhibit significant anti-inflammatory activity compared to the other compounds, showing potential for the preparation of anti-neuroinflammatory drugs. Attached Figure Description

[0042] Figure 1 The structures of compounds 1-6 of this invention are shown.

[0043] Figure 2 The main structures of compounds 1-5 of this invention are shown.

[0044] Figure 3 This is the main structure of compounds 4 and 5 of the present invention. Detailed Implementation

[0045] The present invention will be described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of the present invention. The present invention is not limited to the following embodiments or examples. Any modifications and variations made without departing from the spirit of the present invention should be included within the scope of the present invention. Unless otherwise specified, the experimental materials or reagents used in the following embodiments are commercially available.

[0046] Example 1:

[0047] The seeds of *Cynanchum macrocephalum* were collected in Baise City, Guangxi Zhuang Autonomous Region, China, and stored in the Key Laboratory of Modern Chinese Medicine Preparations of the Ministry of Education, Jiangxi University of Traditional Chinese Medicine, under the number 201718.

[0048] 10 kg of seeds of *Caesalpinia macrophylla* were air-dried and ground into powder. The powder was then soaked in 25 L of 95% ethanol for 72 hours, repeated three times. The three extracts were combined and concentrated under reduced pressure to obtain 404 g of extract. The extract was subjected to silica gel column chromatography (200-300 mesh), eluted sequentially with petroleum ether-acetone (100:0, 10:1, 7:1, 4:1) and 95% ethanol, yielding five fractions: Fr. A (112 g), Fr. B (108 g), Fr. C (48 g), Fr. D (25 g), and Fr. E (112 g). Fr. C was further separated by preparative HPLC (acetonitrile-water, 50%-100%, 0-60 min, 100%, 30 min) to obtain 13 fractions: Fr. C-1 to Fr. C-13. Among them, Fr. C-6 was separated and purified by reversed-phase semi-preparative HPLC (COSMOSIL cholester, acetonitrile-water, 65:35) to obtain compound 1 (t). R =36 min, 2.0 mg) and compound 6 (t R =20 min, 1.3 mg); Fr. C-4 was separated and purified by reversed-phase semi-preparative HPLC (Phenomenex Luna C18, acetonitrile-water, 60:40) to obtain compound 2 (t R =26 min, 2.1 mg); Fr. C-7 was separated and purified by reversed-phase semi-preparative HPLC (COSMOSIL cholester, acetonitrile-water, 75:25) to obtain compound 3 (t R =18 min, 1.2 mg); Fr. C-5 was separated and purified by reversed-phase semi-preparative HPLC (Phenomenex Luna C18, acetonitrile-water, 65:35) to obtain compound 4 (t R =26 min, 2.4 mg); Fr. C-8 was prepared by reversed-phase semi-precipitation HPLC (COSMOSIL) Compound 5 (t) was obtained by separation and purification using acetonitrile-water (65:35). R =42 min, 3.7 mg).

[0049] The structures of compounds 1-6 are as follows: Figure 1 As shown, compound 1 is a white powder, and HRESIMS analysis reveals a quasi-molecular ion peak at m / z 421.2581 [M+H]. + (Calculated value is C) 24 H 37 (O6, 421.2590), which determines the molecular formula of this compound to be C. 24 H 36 O6 has an unsaturation degree of 7. 1The 1H NMR spectrum shows: five methyl groups (δ) H 0.95, 0.97, 1.07, 1.40, 2.03), two methoxy groups (δ H 3.13, 3.33), one hydroxymethyl group (δ-methyl group) H 5.55), one olefinic hydrogen (δ H 5.98). 13 C NMR combined with HSQC spectrum showed 24 carbon signals: five methyl carbons (δ) C 17.2, 19.4, 22.0, 23.5, 33.7), seven methylene carbons (δ C 19.0, 31.2, 35.6, 41.9, 43.8, 51.5, 52.3), four methylene carbons (δ C 45.1, 44.9, 54.6, 69.6), two quaternary carbons (δ C 34.0, 37.7), two oxygen-linked quaternary carbons (δ C 75.7, 108.8), one carbonyl carbon (δ C 169.2), one acetoxy carbon (δ C 170.6), two olefinic carbons (δ C 119.7, 168.6). By δ H 5.98 (H-15) and δ C The values ​​108.8 (C-12), 119.7 (C-15), 169.2 (C-16), and 168.6 (C-13) suggest that this compound contains an unsaturated butenoic acid lactone structural fragment. In the HMBC spectrum, H-15 (δ...) H 5.98) and C-12 (δ) C 108.8), C-16 (δ) C (169.2) Related to this, it was determined that the butenoic acid lactone ring is fused with C-12 and C-13. In summary, it is speculated that this compound is a Cassane diterpene with a butenoic acid lactone ring. Comparison of the NMR data of this compound with those of Neocaesalpin WF indicates that the structure of this compound is similar to that of Neocaesalpin WF, with the most significant difference being: 1 The compound showed an additional methoxy signal δ in its H NMR spectrum. H 3.13; in 13 In the CNMR spectrum, the C-14 carbon signal is generated by δ C 37.6 towards the lower field δ C 75.7 shift, and based on the molecular formula and OCH3-14 (δ) in the HMBC spectrum. H 3.13) and C-14 (δ)C Based on the above, it is speculated that there is an extra methoxy group at position C14.

[0050] In the HMBC spectrum, OCH3-12 (δ H 3.33) and C-12 (δ C (108.8) is related, suggesting that a methoxy group is attached at the C12 position; H-6 (δ H 5.55) and COCH3-6 (δ C Related to 170.6), it was determined that an acetoxy group exists at the C6 position; methyl H-17 (δ H 2.03) and C-13 (δ) C 168.6), C-14 (δ) C 75.7) related, methyl H-18 (δ H 0.95) and C-3 (δ) C 43.8), C-4 (δ) C 34.0), C-5 (δ) C 54.6), C-19 (δ) C 23.5) related, methyl H-19 (δ H 0.97) and C-3 (δ) C 43.8), C-4 (δ) C 34.0), C-5 (δ) C 54.6) related, methyl H-20 (δ H 1.07) and C-1 (δ) C 41.9), C-5 (δ) C 54.6), C-9 (δ) C 44.9), C-10 (δ) C 37.7) Related, in summary, the four methyl groups are located at positions 14, 4, 4, and 10, respectively. The NOESY spectrum shows: H-20 / H-8 is related, confirming that H-20 and H-8 are β configurations; H-6 / H19, H-6 / H-9, H-9 / H-5, H-17 / H-9, and H-9 / OCH3-12 are related, confirming that H-19, H-6, H-9, H-5, H-17, and OCH3-12 are α configurations. After a structural search of the SciFinder database, it was identified as a new compound and named 12α,14β-dimethoxy-6β-acetoxy-cass-13(15)-en-16, 12-olide.

[0051] Compound 2 is a white powder, and HRESIMS analysis shows a quasi-molecular ion peak at m / z 407.2429 [M+H]. + (Calculated value is C) 23H 35 (O6, 407.2434), which determines the molecular formula of this compound to be C. 23 H 34 O6 has an unsaturation degree of 7. 1 The 1H NMR spectrum shows: five methyl groups (δ) H 0.98, 0.99, 1.10, 1.35, 2.04), one methoxy group (δ H 3.20), a hydroxymethyl group (δ-methyl group) H 5.56), one olefinic hydrogen (δ H 6.06). 13 C NMR combined with HSQC spectrum showed 23 carbon signals: five methyl carbons (δ¹⁸ C⁻¹). C 17.2, 20.5, 22.0, 23.5, 33.9), six methylene carbons (δ C 19.0, 31.6, 36.9, 42.2, 43.8, 51.0), four methylene carbons (δ C 44.7, 47.0, 55.4, 68.69), two quaternary carbons (δ C 34.1, 38.1), a tertiary carbon oxide (δ) C 74.3), a quaternary carbon atom (δ) C 107.6), one carbonyl carbon (δ C 169.3), one acetoxy carbon (δ C 170.1), two olefinic carbons (δ C 115.3, 173.1). From the hydrogen signal δ H 6.06 (H-15) and carbon signal δ C The values ​​107.6 (C-12), 115.3 (C-15), 169.3 (C-16), and 173.1 (C-13) suggest that this compound contains an unsaturated n-butenoic acid lactone structural fragment. In the HMBC spectrum, H-15 (δ...) H 6.06) and C-12 (δ) C 107.6), C-13 (δ) C 173.1), C-16 (δ) C (169.3) Related to this, it was determined that the butenoic acid lactone ring is fused with C-12 and C-13. In summary, it is speculated that this compound is a Cassane diterpene with a butenoic acid lactone ring. The NMR data of this compound are similar to those of Neocaesalpin WF, with the main difference being: in 1 The HNMR spectrum of this compound showed an additional s peak and a methyl signal at δ. H 1.35 (3H, s, H-17), lacking the d-peak methyl signal δH 1.12 (3H, d, J = 7.3 Hz, H-17); in 13 The C-14 carbon signal in the C NMR spectrum is determined by δ C 37.6 (C-14) towards the lower field δ C 74.3 (C-14) shift, and based on the molecular formula and C-14 (δ) in the HMBC spectrum. C 74.3) and H15 (δ) H (6.06) Related; In summary, it is speculated that there is an extra OH at position C-14.

[0052] In the HMBC spectrum, OCH3-12 (δ H 3.20) and C-12 (δ C 107.6) is related, confirming the presence of a methoxy group at C12; H-6 (δ H 5.56) and COCH3-6 (δ C Related to 170.1), it was determined that an acetoxy group exists at the C-6 position. Methyl H-17 (δ H 1.35) and C-8 (δ) C 44.7), C-13 (δ) C 173.1), C-14 (δ) C 74.3) related, methyl H-18 (δ H 0.98) and C-3 (δ) C 43.8), C-4 (δ) C 34.1), C-5 (δ) C 55.4), C-19 (δ) C 23.5) related, methyl H-19 (δ H 0.99) and C-3 (δ) C 43.8), C-4 (δ) C 34.1), C-5 (δ) C 55.4) related, methyl H-20 (δ H 1.10) and C-1 (δ) C 42.2), C-5 (δ) C 55.4), C-9 (δ) C 47.0), C-10 (δ) C38.1) Related, in summary, the four methyl groups are located at positions 14, 4, 4, and 10, respectively. The NOESY spectrum shows: H-20 / H-8 is related, confirming that H-20 and H-8 are β configurations; H-6 / H19, H-6 / H-9, H-17 / H-9, H-9 / H-5, and H-17 / OCH3-12 are related, confirming that H-19, H-6, H-9, H-17, H-5, and OCH3-12 are α configurations. After a structural search of the SciFinder database, it was identified as a new compound and named 12α-methoxy-14β-hydroxy-6β-acetoxy-cass-13(15)-en-16, 12-olide.

[0053] Compound 3 is a white powder, and HRESIMS analysis shows a quasi-molecular ion peak at m / z 405.2630 [M+H]. + (Calculated value is C) 24 H 37 (O5, 405.2641), which determines the molecular formula of this compound to be C. 24 H 36 O5 has an unsaturation degree of 7. 1 The H NMR spectrum shows: six methyl groups (δ) H 0.97, 0.99, 1.08, 1.10, 1.17, 2.03), two hydroxymethylene groups (δ H 3.26, 3.54), one hydroxymethyl group (δ-methyl group) H 5.50), one olefinic hydrogen (δ H 5.80). 13 The C NMR spectrum combined with the HSQC spectrum showed 24 carbon signals: six methyl carbons (δ¹⁸O₂). C 15.0, 21.9, 23.5, 18.9, 12.1, 34.0), six methylene carbons (δ C 17.3, 35.6, 37.5, 42.1, 43.8, 59.2, five methylene carbons (δ C 36.5, 44.9, 55.3, 69.5, 36.4), two quaternary carbons (δ C 33.9, 37.9), one ketal carbon (δ C 108.1), one carbonyl carbon (δ C 170.3), one acetoxy carbon (δ C 107.7), two olefinic carbons (δ C 115.4, 171.7). From δ H 5.80 (H-15) and δ CThe values ​​108.1 (C-12), 115.4 (C-15), 170.3 (C-16), and 171.7 (C-13) suggest that this compound contains an unsaturated butenoic acid lactone structural fragment. In the HMBC spectrum, H-15 (δ...) H 5.80) and C-12 (δ C 108.1), C-16 (δ) C (169.5) Related to this, it was determined that the butenolate ring is fused with C-12 and C-13. In summary, it is speculated that this compound is a butenolate-lactone-type Cassane diterpene. Comparison of the NMR data of this compound with those of Neocaesalpin WF indicates that the structure of this compound is similar to that of Neocaesalpin WF, the most significant difference being that this compound has an ethoxy group at the C-12 position instead of a methoxy group.

[0054] Among them, 1 In the H NMR spectrum, δ H 3.26 (1H, dq, J = 9.1, 7.0 Hz, H-1′), δ H 3.54 (1H, dq, J = 9.1, 7.0 Hz, H-1′) belongs to the characteristic signal of the hydroxymethylene group; in 1 H- 1 In the H COSY spectrum, the hydroxymethylene δ H 3.26 (1H, dq, J = 9.1, 7.0 Hz, H-1′), δ H 3.54 (1H, dq, J = 9.1, 7.0 Hz, H-1′) and methyl δ H 1.17 (3H, t, J = 7.0 Hz, H-2′) correlation; in the HMBC spectrum, H-1′ (δ H 3.26, 3.54) and C-2 (δ C 15.0), C-12 (δ) C 108.1) related, H-2 (δ) H 1.17) and C-1 (δ C 59.2) related; in summary, it is speculated that an ethoxy group is attached at position C-12. Furthermore, in the HMBC spectrum, H-6 (δ H 5.50) and COCH3-6 (δ C The correlation (170.3) indicates that an acetoxy group is attached at the C-6 position. In the HMBC spectrum, the methyl δ... H 1.10 (3H, d, J = 7.4 Hz, H-17) and C14 (δ C36.4), C-8 (δ) C 36.5), C-13 (δ) C 171.7) related; methyl δ H 0.99 (3H, s, H19) and C-4 (δ) C 33.9), C-3 (δ) C 43.8), C-5 (δ) C 55.3) related; methyl δ H 0.97 (3H, s, H-18) and C-19 (δ C 23.5), C-4 (δ) C 33.9), C-3 (δ) C 43.8), C-5 (δ) C 55.3) related; methyl δ H 1.08 (3H, s, H-20) and C-10 (δ C 37.9), C-1 (δ) C 42.1), C-5 (δ) C 55.3), C-9 (δ) C 44.9) related; methyl δ H 1.17 (3H, t, J=7.0 Hz, H-2′′) and C-1 (δ C 59.2) Related; In summary, the five methyl groups are located at positions 14, 4, 4, 10, and 1, respectively. The NOESY spectrum shows that H-8 / H-20 is related, and H-8 and H-20 are determined to be β configurations; H-6 / H19, H-6 / H-9, H-9 / H-5, H-9 / H-17, and H-17 / H-1 are determined to be α configurations. After a structural search of the SciFinder database, it was identified as a new compound and named 12α-ethoxy-6β-acetoxy-cass-13(15)-en-16, 12-olide.

[0055] Compound 4 is a white powder, and HRESIMS analysis shows a quasi-molecular ion peak at m / z 421.2581 [M+H]. + (Calculated value is C) 24 H 37 (O6, 421.2590), suggesting the molecular formula of this compound is C. 24 H 36 O6 has an unsaturation degree of 7. 1 The H NMR spectrum shows: six methyl groups (δ) H 0.98, 0.99, 1.10, 1.17, 1.34, 2.03), two hydroxymethylene groups (δH 3.27, 3.56), one hydroxymethyl group (δ-methyl group) H 5.56), one olefinic hydrogen (δ H 6.03). 13 The C NMR spectrum combined with the HSQC spectrum showed 24 carbon signals: six methyl carbons (δ¹⁸O₂). C 15.0, 19.0, 20.6, 21.9, 23.8, 33.8), six methylene carbons (δ C 17.2, 31.6, 37.1, 42.2, 43.8, 59.8, four methylene carbons (δ C 44.7, 47.4, 55.4, 68.9), two quaternary carbons (δ C 34.0, 38.1), a tertiary carbon oxide (δ C 74.1), one ketal carbon (δ C 107.6), one carbonyl carbon (δ C 169.5), one acetoxy carbon (δ C 107.7), two olefinic carbons (δ C 115.5, 173.7). From the hydrogen signal δ H 6.03 (H-15) and carbon signal δ C The values ​​107.6 (C-12), 115.5 (C-15), 169.5 (C-16), and 173.7 (C-13) suggest that this compound contains an unsaturated butenoic acid lactone structural fragment. In the HMBC spectrum, H-15 (δ...) H 6.03) and C-12 (δ) C 107.6), C-13 (δ) C 173.7), C-16 (δ) C (169.5) Related to this, it was determined that the butenoic acid lactone ring is fused with C-12 and C-13. In summary, it is speculated that this compound is a Cassane diterpene with a butenoic acid lactone ring. The NMR data of this compound are similar to those of compound 4, with the main difference being: in 1 The compound showed an additional s peak and a methyl signal in its δ H NMR spectrum. H 1.34 (3H, s, H-17), lacking the d-peak methyl signal δ H 1.10 (3H, d, J = 7.4 Hz, H-17); in 13 The C-14 carbon signal in the C NMR spectrum is determined by δ C 36.4 (C-14) towards the lower field δ C 74.1 (C-14) shift, and based on the molecular formula and C-14 (δ) in the HMBC spectrum.C 74.1) and H-7 (δ) H (1.71-1.80) Related; In summary, it is speculated that there is an extra OH at position C-14.

[0056] Among them, 1 In the H NMR spectrum, δ H 3.27 (1H, dq, J = 9.1, 7.0 Hz, H-1), δ H 3.56 (1H, dq, J = 9.1, 7.0 Hz, H⁻¹) is a characteristic signal of the hydroxymethylene group; in 1 H 1 In the H COSY spectrum, the hydroxymethylene δ H 3.27 (1H, dq, J = 9.1, 7.0 Hz, H-1′), δ H 3.56 (1H, dq, J = 9.1, 7.0 Hz, H-1′) and methyl δ H 1.17 (3H, t, J = 7.0 Hz, H-2′) correlation; in the HMBC spectrum, H-1 (δ H 3.27, 3.56) and C-2 (δ C 15.0), C-12 (δ) C 107.6) related, H-2 (δ) H 1.17) and C-1 (δ C 59.8) is related; in summary, it is speculated that an ethoxy group is attached at position C-12. Furthermore, in the HMBC spectrum, H-6 (δ) H 5.56) and COCH3-6 (δ C The correlation (170.7) indicates that an acetoxy group is attached at the C-6 position. In the HMBC spectrum, the methyl δ... H 1.34 (3H, s, H-17) and C-8 (δ C 44.7), C-14 (δ) C 74.1), C-13 (δ) C 173.7) related; methyl δ H 0.99 (3H, s, H-19) and C-18 (δ C 33.8), C-4 (δ) C 34.0), C-3 (δ) C 43.8), C-5 (δ) C 55.4) related; methyl δ H 0.98 (3H, s, H-18) and C-19 (δ C23.5), C-4 (δ) C 34.0), C-3 (δ) C 43.8), C-5 (δ) C 55.4) related; methyl δ H 1.10 (3H, s, H-20) and C-10 (δ C 38.1), C-1 (δ) C 42.2), C9 (δ) C 47.4), C-5 (δ) C 55.4) related; methyl δ H 1.17 (3H, t, J=7.0 Hz, H-2′′) and C-1 (δ C 59.8) Related; In summary, the five methyl groups are located at positions 14, 4, 4, 10, and 1, respectively. The NOESY spectrum shows that H-8 / H-1 and H-20 / H-1 ​​are related, and H-8 and H-20 are identified as β configurations; H-6 / H-19, H-6 / H-9, H-5 / H-9, H-9 / H-17, and H-17 / H-1 are related, and H-19, H-6, H9, H-5, H-17, and H-1 are identified as α configurations. Through a structural search of the SciFinder database, no literature reports were found, and it was identified as a new compound, named 12α-ethoxy-14β-hydroxy-6β-acetoxy-cass-13(15)-en-16, 12-olide.

[0057] Compound 5 is a white powder, and HRESIMS analysis shows a quasi-molecular ion peak at m / z 449.2893 [M+H]. + (Calculated value is 449.2903), suggesting the molecular formula of this compound is C. 26 H 40 O6 has an unsaturation degree of 7. 1 The 1H NMR spectrum shows: seven methyl groups (δ) H 0.98, 0.98, 1.09, 1.10, 1.21, 1.39, 2.03), four hydroxymethylene groups (δ H 3.18, 3.34, 3.58, 3.65), one hydroxymethyl group (δ-hydroxymethyl group) H 5.55), one olefinic hydrogen (δ H 5.89). 13 The combined 1C NMR spectrum and HSQC spectrum showed 26 carbon signals, including seven methyl carbons (δ¹⁸O). C 15.5, 15.5, 17.2, 22.0, 20.2, 23.6, 33.9), seven methylene carbons (δ C19.0, 31.3, 35.0, 41.9, 43.8, 59.5, 59.6), five methylene carbons (δ C 44.9, 45.1, 54.6, 69.8, 75.1), two quaternary carbons (δ C 34.0, 37.7), one ketal carbon (δ C 108.8), one carbonyl carbon (δ C 169.8), one acetoxy carbon (δ C 170.7), two olefinic carbons (δ C 118.7, 170.3). From δ H 5.89 (H-15) and δ C The values ​​108.8 (C-12), 118.7 (C-15), 169.8 (C-16), and 170.3 (C-13) suggest that this compound contains an unsaturated butenoic acid lactone structural fragment. In the HMBC spectrum, H-15 (δ...) H 5.89) and C-12 (δ C 108.8), C-13 (δ) C 170.3), C-16 (δ) C (169.8) Related to this, it was determined that the butenoic acid lactone ring is fused with C-12 and C-13. In summary, it is speculated that this compound is a Cassane diterpene with a butenoic acid lactone ring. The NMR data of this compound are similar to those of compound 4, with the main difference being: in 1 The 1H NMR spectrum showed two additional dq signal peaks, indicating the presence of four dq peak signals: δ H 3.18 (1H, dq, J= 8.5, 7.0Hz, H-3), δ H 3.34 (1H, dq, J = 8.5, 7.0 Hz, H-3′′) and δ H 3.58 (1H, dq, J= 8.7, 7.0Hz, H-1), δ H 3.64 (1H, dq, J = 8.7, 7.0 Hz, H⁻¹); An additional t-peak methyl signal was observed, resulting in two t-peak methyl signals: δ H 1.10 (3H, t, J= 6.9 Hz, H-4), δ H 1.21 (3H, t, J = 7.0 Hz, H⁻²). In 1 H 1 In the H COSY spectrum, the hydroxymethylene δ H 3.58 (1H, dq, J= 8.7, 7.0 Hz, H-1), δH 3.64 (1H, dq, J = 8.7, 7.0 Hz, H⁻¹) and methyl δ H 1.21 (3H, t, J = 7.0 Hz, H-2') correlation; in the HMBC spectrum, H-2 (δ H 1.21) and C-1 (δ C 59.5), C-12 (δ) C (108.8) Related; In summary, it is speculated that an ethoxy group is attached to the C-12 position.

[0058] exist 1 H- 1 In the H COSY spectrum, the hydroxymethylene δ H 3.18 (1H, dq, J= 8.5, 7.0 Hz, H-3), δ H 3.34 (1H, dq, J = 8.5, 7.0 Hz, H-3) and methyl δ H 1.10 (3H, t, J = 6.9 Hz, H-4) correlation; in the HMBC spectrum, H-3 (δ H 3.34, 3.18) and C-4 (δ C 15.5), C-14 (δ) C 75.1) Related, H-4 (δ) H 1.21) and C-3 (δ) C 59.5) related; in 13 In the C NMR spectrum, the C-14 carbon signal is represented by δ C 36.4 (C-14) towards the lower field δ C 75.1 (C-14) shift, and based on the molecular formula and C-14 (δ) in the HMBC spectrum. C 75.1) and H-15 (δ) H (5.89) is related, suggesting an extra ethoxy group at C-14. Furthermore, in the HMBC spectrum, H-6 (δ) H 5.55) and COCH3-6 (δ C The correlation (170.7) indicates that an acetoxy group is attached at the C-6 position. In the HMBC spectrum, the methyl δ... H 0.98 (3H, s, H-17) and C-8 (δ C 44.9), C-13 (δ) C 170.3), C-14 (δ) C 75.1) Related; Methyl δ H 1.07 (3H, s, H-19) and C-3 (δ C43.8), C-4 (δ) C 34.0), C-5 (δ) C 54.6), methyl δ H 0.98 (3H, s, H-18) and C-3 (δ C 43.8), C-4 (δ) C 34.0), C-5 (δ) C 54.6), C-19 (δ) C 20.2), methyl δ H 1.39 (3H, s, H-20) and C-1 (δ C 41.9), C-5 (δ) C 54.6), C-9 (δ) C 45.1), C-10 (δ) C 37.7), methyl δ H 1.21 (3H, t, J = 7.0 Hz, H-2′) and C-1 (δ C 59.5), methyl δ H 1.10 (3H,t, J = 6.9 Hz, H-4′′) and C-3 (δ C 59.5) Correlation, the six methyl groups were determined to be located at positions 14, 4, 4, 10, 1, and 3, respectively. In the NOESY spectrum, H-8 / H-20 was correlated, and H-20 and H-8 were determined to be β configurations; H-6 / H-19, H-6 / H-9, H-9 / H-5, H-9 / H-17, and H-17 / H-1 were determined to be α configurations. A search of the SciFinder database revealed no literature reports, thus identifying it as a new compound, named 12α,14β-diethoxy-6β-acetoxy-cass-13(15)-en-16, 12-olide.

[0059] Compound 6 is a white powder, and HRESIMS analysis shows a quasi-molecular ion peak at m / z 375.2168 [M+H]. + (Calculated value is C) 22 H 31 (O6, 375.2171), which determines the molecular formula of this compound to be C. 22 H 30 O6 has an unsaturation degree of 8. 1 The 1H NMR spectrum shows: five methyl groups (δ) H 1.00, 1.00, 1.17, 1.30, 2.05), one hydroxymethyl group (δ H 5.61), one olefinic hydrogen (δ H6.06). 13 C NMR combined with HSQC spectrum showed 22 carbon signals: five methyl carbons (δ¹⁸ C⁻¹). C 17.8, 22.0, 22.7, 23.1, 33.2), four methylene carbons (δ C 18.8, 30.7, 40.8, 43.9), four methylene carbons (δ C 41.7, 52.3, 55.4, 68.7), two quaternary carbons (δ C 34.2, 38.8), a tertiary carbon oxide (δ) C 71.7), one carbonyl carbon (δ C 169.7), one acetoxy carbon (δ C 170.6), four olefinic carbons (δ C 110.9, 117.9, 149.9, 169.1). By δ H 6.06 (H-15) and δ C The values ​​149.9 (C-12), 110.0 (C-15), 169.7 (C-16), and 163.4 (C-13) suggest that this compound contains an unsaturated butenoic acid lactone structural fragment. In the HMBC spectrum, H-11 (δ...) H 5.86) and C-12 (δ C 149.9) and C-13 (δ C 163.4) and H-15 (δ H 6.06) and C-12 (δ C 149.9), C-13 (δ) C 163.4) and C-16 (δ C The cross-signal peak at 169.7°C confirms the presence of the unsaturated γ-lactone ring conjugated double bond, establishing the fusion of the butenoic acid lactone ring with C-12 and C-13. In summary, this compound is presumed to be a butenoic acid lactone ring-type Cassane diterpene. Comparison of the NMR data of this compound with those of Neocaesalpin WB indicates that the structure of this compound is similar to that of Neocaesalpin WB, with the most significant difference being the structure of this compound... 13 In the C NMR spectrum, the C-14 carbon signal is represented by δ C 34.7 Towards lower field δ C 71.7 shift, based on the molecular formula and H-17 (δ) in the HMBC spectrum. H 1.30) and C-14 (δ) C Related to 71.7), it is speculated that a hydroxyl group exists at the C-14 position. H-6 (δ H5.61) and COCH3-6 (δ C (170.4) Related to this, it was determined that there is an acetoxy group at the C-6 position.

[0060] In the HMBC spectrum, methyl H-17 (δ) H 1.30) and C-8 (δ) C 41.7), C-13 (δ) C 163.4), C-14 (δ) C 71.7) related, methyl H-18 (δ H 1.00) and C-3 (δ) C 43.9), C-4 (δ) C 34.2), C-5 (δ) C 55.4), C-19 (δ) C 23.1) related, methyl H-19 (δ H 1.00) and C-3 (δ) C 43.9), C-4 (δ) C 34.2), C-5 (δ) C 55.4) related, methyl H-20 (δ H 1.17) and C-1 (δ) C 40.8), C-5 (δ) C 55.4), C-9 (δ) C 52.3), C-10 (δ) C 38.8) Correlation, determining that the four methyl groups are located at positions 14, 4, 4, and 10, respectively. The NOESY spectrum shows: H-8 / H-20 correlation, determining that H-8 and H-20 are β configurations; H-6 / H19, H-6 / H-9, H-9 / H-5, H-17 / H-9 correlation, determining that H-19, H-6, H-9, H-5, and H-17 are α configurations. Through a structural search of the SciFinder database, it was identified as a new compound and named 14β-hydroxy6β-acetoxy-cass-11(12), 13(15)-ene-16, 12-olide.

[0061] Compounds 1-3 1 H and 13 The C10 NMR spectral data of compounds 4-6 are shown in Table 1. 1 H and 13 The C NMR spectral data are shown in Table 2.

[0062] Table 1. Compounds 1-3 1 H and 13 C10 NMR spectral data (chemical shift δ in ppm, coupling constant J in Hz).a

[0063]

[0064] a The NMR data of compounds 1-3 were determined in CDCl3. 1 The H NMR measurement frequency was 600 MHz. 13 The C NMR measurement frequency was 150 MHz.

[0065] Table 2. Compounds 4-6 1 H and 13 C10 NMR spectral data (chemical shift δ in ppm, coupling constant J in Hz). a

[0066]

[0067] a The NMR data of compounds 4-6 were determined in CDCl3. 1 The H NMR measurement frequency was 600 MHz. 13 The C NMR measurement frequency was 150 MHz.

[0068] Example 2

[0069] Appropriate amounts of the isolated monomeric compounds 1-6 and the positive control drug quercetin were weighed and dissolved in DMSO to prepare 100 mM stock solutions. These solutions were then filtered through a 0.22 μm filter membrane for sterilization and use. A NO assay kit (Shanghai Beyotime Biotechnology Co., Ltd.) was used to determine the inhibitory effect of the compounds on NO production induced by lipopolysaccharide (LPS) in BV-2 microglia (Suzhou Haixing Biotechnology Co., Ltd.).

[0070] BV-2 mouse microglia were cultured in DMEM medium containing 10% fetal bovine serum (FBS), penicillin (100 U / mL), and streptomycin (100 μg / mL). Logarithmic growth phase BV-2 microglia were cultured at a rate of 1 × 10⁻⁶ cells / mL. 4After incubation at 37 °C and 5% CO2 for 24 h, LPS (final concentration 1 μg / mL) and different concentrations of monomer compound test sample solutions (final concentrations of 150, 100, 50, 25, and 12.5 μM) were added to each well in the experimental group; the same amount of LPS and the same concentration of DMSO as the experimental group were added to each well in the model group; and the same amount of LPS and quercetin at the same concentration gradient as the experimental group were added to each well in the positive control group. Six replicates were set up for each concentration, and the culture was continued for another 24 h. After 24 h, the NO content in the supernatant of each group was indirectly determined using a NO assay kit based on the Griess reaction. NO is converted to NO2 in aqueous solution. - It reacts with the colorimetric reagent to form a pale red azo compound, the concentration of which is related to NO2. - The content of [a substance] exhibits a linear relationship within a certain range, and the absorbance at 540 nm (A) is determined by colorimetry. 540 This can indirectly reflect the NO content, and the inhibition rate of the drug against NO can be calculated based on the NO concentration obtained from each group. Then, the half-maximal inhibitory concentration (IC50) is obtained through nonlinear regression fitting. 50 IC 50 The value is defined as the concentration of the compound that reduces NO production by 50% compared to the model group (Table 3).

[0071] Table 3. Anti-neuritis activity of 6 compounds

[0072] Compound <![CDATA[IC 50 (μM) a ]]> 1 23.88±0.18 2 24.22±0.23 3 20.98±0.51 4 12.39±0.95 5 11.74±0.33 6 30.59±0.72

[0073] In summary, this invention has extracted six new kazoline diterpenoids from *Ligustrum lucidum*, among which compounds 1-5 possess the following properties: Figure 2 The main structure is shown, and all six compounds exhibit anti-neuroinflammatory effects; in particular, compounds 4 and 5 show significantly higher effects than compounds with other structures, possessing properties such as... Figure 3 The main structure shown indicates that the anti-inflammatory activity of the compound with this structure is approximately 2-3 times that of the other four compounds.

[0074] The conventional techniques and solutions not described in detail in the above embodiments are all well known in the art, and therefore will not be elaborated upon here. The above embodiments and / or experimental examples describe the preferred embodiments of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A cardanolipid compound, characterized by, comprising the structure shown in body ①: ①； wherein the compound 1 has a molecular formula of C 24 H 36 O6, named 12α,14β-dimethoxy-6β-acetoxy-cass-13(15)-en-16, 12-olide, Compound 1; The molecular formula of compound 2 is C 23 H 34 O6, named 12α-methoxy-14β-hydroxy-6β-acetoxy-cass-13(15)-en-16, 12-olide, Compound 2; The molecular formula of compound 3 is C 24 H 36 O5, named 12α-ethoxy-6β-acetoxy-cass-13(15)-en-16, 12-olide, Compound 3; The molecular formula of compound 4 is C 24 H 36 O6, named 12α-ethoxy-14β-hydroxy-6β-acetoxy-cass-13(15)-en-16, 12-olide, Compound 4; The molecular formula of compound 5 is C 26 H 40 O6, named 12α,14β-diethoxy-6β-acetoxy-cass-13(15)-en-16, 12-olide, Compound 5; or The molecular formula of compound 6 is C 22 H 30 O6, named 14β-hydroxy 6β-acetoxy-cass-11(12), 13(15)-ene-16, 12-olide, Compound 6.

2. The compound of claim 1, wherein comprising the structure shown in body ②: ②； wherein the compound 4 has a molecular formula of C 24 H 36 O6, named 12a-ethoxy-14β-hydroxy-6β-acetoxy-cass-13(15)-en-16, 12-olide, Compound 4; The molecular formula of compound 5 is C 26 H 40 O6, named 12α,14β-diethoxy-6β-acetoxy-cass-13(15)-en-16, 12-olide, Compound 5.

3. A process for the preparation of a compound according to claim 1, characterized in that, comprising the following steps: 1) Dry and grind the seeds of Sterculia macrophylla, soak in 95% ethanol for 72 hours, repeat three times, combine the extract and concentrate under reduced pressure to obtain the extract; 2) Silica gel column chromatography is performed on the extract, and petroleum ether-acetone 100:0, 7:1, 4:1, 1:1 and 95% ethanol are used for elution in sequence to obtain five components Fr A, Fr B, Fr C, Fr D and Fr E; 3) Fr. C is separated by preparative HPLC with acetonitrile-water, 50%-100%, 0-60 min, 100%, 30 min to obtain 13 components Fr. C-1 to Fr. C-13; 4) Fr. C-6 is separated and purified by reverse-phase semi-preparative HPLC with COSMOSIL cholester, acetonitrile-water, 65:35 to obtain compounds 1 and 6; ； or / and Fr. C-4 is separated and purified by reverse-phase semi-preparative HPLC with Phenomenex Luna C18, acetonitrile-water, 60:40 to obtain compound 2; ； or / and Fr. C-7 is separated and purified by reverse-phase semi-preparative HPLC with COSMOSIL cholester, acetonitrile-water, 75:25 to obtain compound 3; ； or / and Fr. C-5 is separated and purified by reverse-phase semi-preparative HPLC with Phenomenex Luna C18, acetonitrile-water, 65:35 to obtain compound 4; ； or / and Fr. C-8 is separated and purified by reverse-phase semi-preparative HPLC with COSMOSIL NAP, acetonitrile-water, 65:35 to obtain compound 5; 。 4. An anti-neuroinflammatory drug, characterized in that, The compound comprising the structure of claim 1 or 2.

5. Use of the compound of the structure of claim 1 or 2 in the preparation of an anti-neuroinflammatory drug.

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