Sesquiterpenoid derivatives, their preparation methods and applications

Abstract

The sesquiterpene derivatives of the present invention, their preparation methods and applications belong to the field of pharmaceutical technology. The following compounds are obtained by solvent reflux extraction to obtain an ethanol extract, and after recovering the alcohol solvent, they are successively extracted with petroleum ether, dichloromethane, ethyl acetate and n-butanol. A variety of spectroscopic means are used to direct the separation of the active components contained in the above-mentioned extract. The petroleum ether extract rich in the target active sesquiterpene is obtained by separation methods such as silica gel column chromatography, ODS column chromatography and high performance liquid chromatography, and the structure of the sesquiterpene derivative is determined by combining various spectroscopic techniques such as nuclear magnetic resonance spectroscopy. The prepared compounds and Croton tiglium L. extract can inhibit the release of NO from LPS-induced BV-2 cells, and they have great application prospects in the preparation of anti-neuritis drugs. The compounds and extracts with neuritis activity disclosed in the present invention can be extracted from Croton tiglium L. and its congeners, or from other biological resources, or can be obtained by chemical synthesis methods.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology and relates to sesquiterpene derivatives, their preparation methods and applications. Background Art

[0002] Sesquiterpenes have small molecular weights, rich structures and remarkable activities, and are important sources for the research and development of drug candidate drugs. These components are widely present in plants, marine organisms, microorganisms and certain insects. Among the terpene compound libraries, the number and types of structural skeletons of sesquiterpene compounds are the most. These molecules have various biological activities, including anti-inflammatory, anti-tumor, anti-malaria, etc., which greatly expand the medical application fields of sesquiterpenes. Croton olivaceus Y.T.Changet P.T.Li is a plant of the genus Croton in the Euphorbiaceae family, growing in sparse forests at low altitudes and is an endemic species in Hainan Province, China. Currently, there is no literature report on the chemical constituents and pharmacological activities of Croton olivaceus. The research group of the present invention adopted an activity-guided separation strategy to search for novel sesquiterpene small molecule compounds from Croton olivaceus.

[0003] The nervous system is an important control center in the human body. When affected by external stimuli or internal factors, an inflammatory response may occur. This inflammatory state is a key mechanism in many neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), etc. Nervous system inflammation refers to the inflammatory response occurring in the central nervous system and is a common feature of various neurodegenerative diseases. Regulating inflammatory cytokines such as interleukin 1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), chemokines (CXCL2, CXCL5, CXCL1) and improving the distribution of immune cells in the nervous system and regulating the activities of immune cells can slow down or prevent the development of these neurodegenerative diseases. The sesquiterpene compounds involved in the present invention are extracted and separated from the dry branches and leaves of Croton olivaceus, and an in vitro LPS-induced BV-2 microglial cell inflammation model is established to evaluate the anti-neuritis activity of the obtained sesquiterpene compounds. There is no patent or literature report on the compounds involved in the present invention and their anti-neuritis activity tests. Summary of the Invention

[0004] The purpose of the present invention is to provide sesquiterpene derivatives, preparation methods and their applications in the preparation of anti-neuritis drugs.

[0005] The present invention adopts an in vitro LPS-induced BV-2 cell inflammation model for evaluating the anti-neuritis activities of the extracts of Croton olivaceus and the sesquiterpene compounds isolated therefrom.

[0006] The present invention provides sesquiterpene derivatives represented by the following formulas I-VI or pharmaceutically acceptable salts thereof:

[0007]

[0008] Among them, Formulas I-IV are guaiane-type sesquiterpene derivatives; V-VII are C13 nor-sesquiterpene derivatives;

[0009] In Formula I, Ring A is a cycloheptane ring, and Ring B is a cyclopentane ring;

[0010] In Formula II, Ring A is an aromatic cycloheptatrienone ring, Ring B is a cyclopentane ring, R 1 , R 2 , R 3 and R 4 are each independently -H or -OH, or R 1 and R 4 are -H or -OH, and R 2 or R 3 is =O;

[0011] In Formula III, Ring A is a cycloheptane ring, and Ring B is a cyclopentane ring;

[0012] In Formula IV, Ring A is a benzene ring, Ring B is a cyclopentane ring, R 1 , R 2 are each independently -H, -OH, -COOH or -CH(CH 3 ) 2 ;

[0013] In Formula V, Ring A is a cyclohexane ring, Ring B is a three-membered peroxide ring, and there is 1 trans double bond in the side chain;

[0014] In Formula VI, Ring A is a cyclohexane ring, and there is 1 axial chiral allene in the side chain;

[0015] In Formula VII, both Ring A and Ring B are cyclohexane rings.

[0016] Furthermore, in Formula I, Ring A is a seven-membered cycloheptane ring, and Ring A has an internal peroxide bridge, Ring B is a cyclopentane ring, and C8 and C-9 form a conjugated double bond and form a conjugated system with the carbonyl group at the C-7 position.

[0017] Furthermore, in Formula II, Ring A is an aromatic seven-membered cycloheptatrienone ring, Ring B is a cyclopentane ring, R 1 is a hydroxyl group, R 2 is a carbonyl group, R 3 , R 4 are both hydrogen at the same time; or R 2 is a hydroxyl group, R 1 , R 3 , R 4 are all hydrogen at the same time; or R3 is a hydroxyl group, R 1 , R 2 , R 4 are simultaneously hydrogen; or R 2 is a carbonyl group, R 1 , R 3 , R 4 are simultaneously hydrogen; or R 2 is a carbonyl group, R 4 is a hydroxyl group, R 1 , R 3 are simultaneously hydrogen; or R 1 , R 2 , R 3 , R 4 are simultaneously hydrogen.

[0018] Furthermore, in the formula III, the A ring is a seven-membered cycloheptane ring, the B ring is a cyclopentane ring, and C4 and C-10 form a double bond and form a conjugated system with the carbonyl group at the C-1 position.

[0019] Furthermore, in the formula IV, the A ring is an aromatic benzene ring, the B ring is a cyclopentane ring, there is a carbonyl substitution at the C1 position, R 1 is an isopropyl group, R 2 is a carboxyl group; or R 1 is a carboxyl group, R 2 is hydrogen; or R 1 is a hydroxyl group, R 2 is hydrogen.

[0020] Furthermore, in the formula V, the A ring is a cyclohexane ring, the B ring is a peroxide spiro ring, and C7 and C8 form a double bond and form a conjugated system with the carbonyl group at the C9 position.

[0021] Furthermore, in the formula VI, the A ring is a cyclohexane ring, there is a hydroxyl substitution at the C3 position, there is a methoxy substitution at the C5 position, and C6, C7, and C8 form an allene and form a conjugated system with the carbonyl group at the C9 position.

[0022] Furthermore, in the formula VII, both the A and B rings are cyclohexane rings, there is a hydroxyl substitution at the C9 position, and C4 and C5 form a double bond and form a conjugated system with the carbonyl group at the C3 position.

[0023] The present invention finally preferably provides 13 sesquiterpene derivatives, and their structures and names are as follows, where those marked with "*" are 9 new structures:

[0024] Compounds 1-13 are croton-guaiane A (1*), croton-guaiane B (2*), croton-guaiane C (3*), croton-guaiane D (4*), pernambucone (5), ganyearmcaoone A (6), orobanone (7), pancherione (8), croton-guaiane E (9*), croton-guaiane F (10*), croton-megastigmene A (11*), croton-megastigmene B (12*), croton-megastigmene C (13*), respectively, and all of them are isolated from Croton virgatus Baill. for the first time.

[0025]

[0026] The spectroscopic data of Compounds 1-13 are as follows:

[0027] Compound 1 croton-guaiane A

[0028] Light brown oil (methanol), 1 H-NMR (600 MHz, CDCl 3 ) δ H : 5.84 (1H, d, J = 1.5 Hz, H-8), 5.70 (1H, d, J = 2.6 Hz, H-5), 2.62 (1H, m, H-3), 2.53 (1H, hept, J = 6.8 Hz, H-13), 2.25 (1H, m, H-1a), 2.03 (1H, dd, J = 7.3, 2.2 Hz, H-1b), 2.01 (3H, s, H 3 -11), 1.95 (1H, dtd, J = 12.1, 6.9, 2.2 Hz, H-2a), 1.33 (1H, m, H-2b), 1.19 (3H, d, J = 6.4 Hz, H 3 -12), 1.06 (3H, d, J = 6.8 Hz, H 3 -15), 0.96 (3H, d, J = 6.8 Hz, H 3 -14). 13 C-NMR (150 MHz, CDCl 3 ) δ C: 198.3 (C-7), 161.6 (C-4), 152.4 (C-9), 129.2 (C-8), 114.4 (C-5), 90.7 (C-6), 89.4 (C-10), 39.3 (C-3), 31.9 (C-1), 31.7 (C-13), 31.4 (C-2), 22.4 (C-11), 16.7 (C-14), 16.3 (C-15), 16.3 (C-12).

[0029] Compound 2 croton-guaiane B

[0030] Pale yellow oil (methanol), 1 H-NMR (600 MHz, CD 3 OD) δ H : 7.44 (1H, s, H-7), 7.23 (1H, s, H-4), 4.93 (1H, d, 1.6, H-9a), 4.90 (1H, d, 1.4, H-9b), 3.42 (1H, m, H-11), 3.38 (1H, m, H-3), 2.87 (1H, dd, 19.0, 2.3, H-2a), 2.23 (1H, m, H-2b), 1.37 (3H, d, J = 7.1 Hz, H 3 -10), 1.27 (3H, d, J = 6.8 Hz, H 3 -13), 1.24 (3H, d, J = 6.8 Hz, H 3 -12). 13 C-NMR (150 MHz, CDCl 3 ) δ C : 208.7 (C-1), 189.1 (C-6), 172.6 (C-3a), 164.8 (C-5), 148.3 (C-8a), 136.6 (C-8), 134.2 (C-4), 131.3 (C-7), 63.4 (C-9), 44.6 (C-2), 38.3 (C-3), 32.4 (C-11), 22.7 (C-12), 22.6 (C-13), 21.7 (C-10).

[0031] Compound 3 croton-guaiane C

[0032] Pale yellow oil (methanol), 1 H-NMR (600 MHz, CD 3 OD) δ H : δ H7.32(1H, s, H-4), 6.98(1H, s, H-7), 5.16(1H, dd, J = 7.3, 2.4Hz, H-1), 3.39(1H, m, H-11), 3.13(1H, pd, J = 7.3, 3.0Hz, H-3), 2.54(1H, ddd, 13.9, 8.5, 7.3Hz, H-2a), 2.47(3H, s, H 3 -9), 1.62(1H, dt, J = 13.9, 2.8Hz, H-2b), 1.38(3H, d, J = 7.2Hz, H 3 -10), 1.21(3H, d, J = 6.9Hz, H 3 -12), 1.17(3H, d, J = 6.9Hz, H 3 -13). 13 C-NMR(150MHz, CDCl 3 ) δ C : 187.5(C-6), 160.6(C-5), 153.7(C-3a), 148.8(C-8), 147.0(C-8a), 139.8(C-7), 132.9(C-4), 77.9(C-1), 44.6(C-3), 41.2(C-2), 31.4(C-11), 22.7(C-13), 22.7(C-12), 24.1(C-9), 23.1(C-10).

[0033] Compound 4 croton-guaiane D

[0034] Pale yellow oil (methanol), 1 H-NMR(600MHz, CD 3 OD) δ H : 7.35(1H, s, H-4), 7.00(1H, s, H-7), 5.18(1H, d, J = 6.8Hz, H-2), 3.41(1H, m, H-11), 3.50(1H, pd, J = 7.3, 3.0Hz, H-3), 2.20(1H, ddd, 13.9, 8.5, 7.3Hz, H-1a), 2.47(3H, s, H 3 -9), 1.85(1H, dt, J = 13.9, 2.8Hz, H-1b), 1.30(3H, d, J = 7.2Hz, H 3 -10), 1.20(3H, d, J = 6.9Hz, H 3 -13), 1.20(3H, d, J = 6.9Hz, H 3 -12). 13 C-NMR(150MHz, CDCl3 ) δ C : 187.4 (C-6), 160.8 (C-5), 153.5 (C-3a), 148.6 (C-8), 147.6 (C-8a), 139.9 (C-7), 132.2 (C-4), 77.0 (C-2), 44.6 (C-3), 42.9 (C-1), 31.4 (C-11), 22.7 (C-13), 22.7 (C-12), 24.1 (C-9), 23.1 (C-10).

[0035] Compound 5 pernambucone

[0036] Brown oil (methanol), 1 H-NMR (600 MHz, CDCl 3 ) δ H : 7.16 (1H, s, H-4), 6.81 (1H, s, H-7), 3.46 (1H, m, H-11), 3.22 (1H, pd, J = 7.1, 2.1 Hz, H-3), 2.83 (1H, dd, J = 18.9, 7.4 Hz, H-2a), 2.54 (3H, s, H 3 -9), 2.23 (1H, dd, J = 18.9, 2.4 Hz, H-2b), 1.36 (3H, d, J = 7.1 Hz, H 3 -10), 1.23 (3H, d, J = 6.8 Hz, H 3 -12), 1.20 (3H, d, J = 6.9 Hz, H 3 -13). 13 C-NMR (150 MHz, CDCl 3 ) δ: 206.5 (C-1), 186.4 (C-6), 169.6 (C-3a), 164.4 (C-5), 143.4 (C-8), 139.1 (C-7), 136.6 (C-8a), 129.0 (C-4), 44.1 (C-2), 36.5 (C-3), 30.9 (C-11), 23.7 (C-9), 22.6 (C-13), 22.5 (C-12), 21.8 (C-10).

[0037] Compound 6 ganyearmcaoone A

[0038] Brown oil (methanol), 1 H-NMR (600 MHz, CD 3 OD) δ H: 7.67 (1H, s, H-4), 6.87 (1H, s, H-7), 3.43 (1H, m, H-11), 2.78 (2H, s, H-2), 2.54 (3H, s, H 3 -9), 1.56 (3H, d, J = 7.1 Hz, H 3 -10), 1.27 (3H, d, J = 6.9 Hz, H 3 -12), 1.24 (3H, d, J = 6.9 Hz, H 3 -13). 13 C-NMR (150 MHz, CDCl 3 ) δ: 204.6 (C-1), 188.2 (C-6), 168.6 (C-3a), 164.9 (C-5), 145.2 (C-8), 140.5 (C-7), 136.7 (C-8a), 128.6 (C-4), 52.8 (C-2), 75.3 (C-3), 32.4 (C-11), 28.9 (C-10), 23.9 (C-9), 22.7 (C-13), 22.6 (C-12).

[0039] Compound 7 orobanone

[0040] Brown oil (methanol), 1 H-NMR (600 MHz, CDCl 3 ) δ H : 7.15 (1H, s, H-4), 7.09 (1H, s, H-7), 3.49 (1H, hept, J = 6.4 Hz, H-11), 3.21 (3H, h, J = 6.6 Hz, H-11), 2.91 (3H, dt, J = 15.4, 7.3 Hz, H-1a), 2.79 (3H, dt, J = 15.4, 7.3 Hz, H-1b), 2.25 (3H, s, H 3 -9), 2.21 (3H, ddd, J = 12.9, 7.2, 4.0 Hz, H-1a), 1.60 (3H, m, H-1b), 1.25 (3H, d, J = 7.0 Hz, H 3 -10), 1.18 (3H, d, J = 6.9 Hz, H 3 -12), 1.17 (3H, d, J = 6.9 Hz, H 3 -13). 13 C-NMR (150 MHz, CDCl 3)δ: 185.4 (C-6), 150.6 (C-3a), 158.5 (C-5), 145.6 (C-8), 138.9 (C-7), 146.4 (C-8a), 130.6 (C-4), 30.9 (C-2), 45.1 (C-3), 34.7 (C-1), 34.7 (C-11), 20.6 (C-10), 25.3 (C-9), 22.7 (C-13), 22.7 (C-12).

[0041] Compound 8 pancherione

[0042] Colorless oil (methanol), 1 H-NMR (600 MHz, CDCl 3 ) δ H : 2.88 (1H, dq, J = 7.6, 6.9, 3.3 Hz, H-8), 2.80 (1H, t, J = 5.9 Hz, H-3), 2.77 (1H, m, H-4a), 2.60 (1H, dd, J = 18.8, 6.4 Hz, H-2a), 2.35 (1H, dd, J = 15.7, 11.9 Hz, H-4b), 1.99 (1H, m, H-6a), 1.94 (1H, m, H-2b), 1.84 (1H, m, H-7a), 1.64 (1H, m, H-6b), 1.50 (1H, m, H-7b), 1.37 (1H, ddt, J = 12.4, 10.6, 2.0 Hz, H-5), 1.23 (3H, s, H 3 -12), 1.19 (3H, s, H 3 -13), 1.16 (3H, d, J = 7.1 Hz, H 3 -10), 1.01 (3H, d, J = 7.2 Hz, H 3 -9). 13 C-NMR (150 MHz, CDCl 3 ) δ: 211.0 (C-1), 181.6 (C-3a), 146.0 (C-8a), 73.7 (C-11), 50.9 (C-5), 44.0 (C-2), 39.2 (C-3), 34.0 (C-7), 33.2 (C-4), 28.6 (C-8), 27.7 (C-6), 27.6 (C-12), 25.6 (C-13), 19.4 (C-10), 18.1 (C-9).

[0043] Compound 9 croton-guaiane E

[0044] White amorphous powder (methanol), 1 H-NMR (600 MHz, CDCl3 ) δ H : 7.30 (1H, s, H-4), 3.36 (1H, td, J = 7.1, 3.7 Hz, H-3), 3.10 (1H, m, J = 6.8 Hz, H-11), 2.92 (1H, dd, J = 18.7, 7.7 Hz, H-2a), 2.67 (3H, s, H 3 -9), 2.28 (1H, dd, J = 18.7, 3.7 Hz, H-2b), 1.39 (1H, d, J = 7.1 Hz, H 3 -10), 1.31 (3H, d, J = 7.1 Hz, H 3 -12), 1.31 (3H, d, J = 7.1 Hz, H 3 -13). 13 C-NMR (150 MHz, CDCl 3 ) δ: 206.5 (C-1), 170.9 (C-6), 162.2 (C-3a), 152.6 (C-5), 135.5 (C-8), 132.9 (C-8a), 131.9 (C-7), 119.8 (C-4), 46.3 (C-2), 32.1 (C-3), 29.9 (C-11), 24.1 (C-13), 24.0 (C-12), 21.6 (C-10), 15.2 (C-9).

[0045] Compound 10 croton-guaiane F

[0046] White amorphous powder (methanol), 1 H-NMR (600 MHz, CD 3 OD) δ H : 8.00 (1H, s, H-5), 7.78 (1H, s, H-7), 3.44 (1H, m, H-3), 2.96 (1H, dd, J = 18.9, 7.6 Hz, H-2a), 2.63 (3H, s, H 3 -10), 2.29 (1H, dd, J = 18.9, 3.7 Hz, H-2b), 1.41 (1H, d, J = 7.1 Hz, H 3 -11). 13 C-NMR (150 MHz, CDCl 3 ) δ: 209.1 (C-1), 162.5 (C-4), 162.5 (C-6), 139.4 (C-8), 137.5 (C-9), 131.4 (C-7), 125.1 (C-5), 47.1 (C-2), 33.5 (C-3), 21.7 (C-11), 18.4 (C-10).

[0047] Compound 11 croton - megastigmene A

[0048] Pale yellow oil (methanol), 1 ¹H - NMR (600 MHz, CD 3 OD) δ H : 6.72 (1H, dd, J = 15.9, 10.2 Hz, H - 7), 6.21 (1H, d, J = 15.9 Hz, H - 8), δ H 2.30 (3H, s, H 3 - 10), δ H 2.06 (1H, m, H - 4a), 1.90 (1H, dd, J = 14.1, 2.7, H - 2a), 1.63 (1H, t, J = 10.2, H - 6), 1.33 (1H, m, H - 5), 1.25 (1H, m, H - 2b), 1.07 (1H, m, H - 4b), 0.95 (3H, d, J = 6.4 Hz, H 3 - 13), 0.90 (3H, s, H 3 - 11), 0.90 (3H, s, H 3 - 12). 13 ¹³C - NMR (150 MHz, CDCl 3 ) δ: 200.8 (C - 9), 151.8 (C - 7), 134.6 (C - 8), 59.6 (C - 6), 46.4 (C - 2), 42.4 (C - 4), 36.1 (C - 1), 101.1 (C - 3), 32.4 (C - 5), 30.1 (C - 12), 26.9 (C - 10), 22.2 (C - 13), 21.3 (C - 11).

[0049] Compound 12 croton - megastigmene B

[0050] Colorless oil (methanol), 1 ¹H - NMR (600 MHz, CD 3 OD) δ H : 5.95 (1H, s, H - 8), 4.00 (1H, m, H - 3), 3.28 (3H, s, 5 - OCH 3 ), 2.30 (3H, s, H 3 - 10), 2.02 (1H, dd, J = 13.9, 5.9 Hz, H - 4a), 1.95 (1H, m, H - 4b), 1.81 (1H, dd, J = 13.4, 7.4, H - 2a), 1.71 (1H, dt, J = 13.4, 4.2 Hz, H - 2b), 1.37 (3H, s, H 3-13), 1.36 (3H, s, H 3 -12), 1.17 (3H, s, H 3 -11). 13 C-NMR (150 MHz, CDCl 3 ) δ: 212.0 (C-9), 201.1 (C-7), 118.3 (C-6), 101.4 (C-8), 78.4 (C-5), 66.8 (C-3), 51.0 (5-OCH 3 ), 46.9 (C-2), 45.6 (C-4), 35.5 (C-1), 31.8 (C-11), 31.6 (C-12), 27.6 (C-10), 25.6 (C-13).

[0051] Compound 13 croton-megastigmene C

[0052] Colorless oil (methanol), 1 H-NMR (600 MHz, CD 3 OD) δ H : 5.80 (1H, s, H-4), 2.43 (1H, dd, J = 13.9, 2.6 Hz, H-13a), 2.40 (1H, m, H-13b), 2.26 (1H, d, J = 15.5 Hz, H-2a), 2.17 (1H, d, J = 15.5 Hz, H-2b), 2.15 (1H, m, H-6), 1.84 (1H, m, H-8a), 1.81 (1H, m, H-7a), 1.75 (1H, m, H-8b), 1.66 (1H, m, H-7b), 1.29 (3H, s, H 3 -10), 1.1.07 (3H, s, H 3 -12), 0.98 (3H, s, H 3 -11). 13 C-NMR (150 MHz, CDCl 3 ) δ: 202.6 (C-3), 167.4 (C-5), 125.2 (C-4), 72.7 (C-9), 51.2 (C-2), 49.7 (C-6), 49.7 (C-13), 39.0 (C-8), 35.6 (C-1), 31.0 (C-10), 28.8 (C-12), 24.7 (C-11), 24.4 (C-7).

[0053] The present invention also provides a method for preparing the sesquiterpene derivative, comprising the following steps:

[0054] (1) The dry branches and leaves of Croton lachnocarpus Benth. are crushed and subjected to heating under reflux extraction with 3 - 15 times the amount of 30% - 100% ethanol or methanol. After reflux extraction, the alcohol solvent is recovered under reduced pressure and suspended in water.

[0055] (2) The suspension is successively extracted with petroleum ether, dichloromethane, ethyl acetate, and n - butanol for 1 - 5 times. The volume ratio of the extraction solvent to the suspension is 1:1 - 2:1. After recovering the solvent, petroleum ether layer extract, dichloromethane layer extract, ethyl acetate layer extract, n - butanol layer extract, and water layer extract are obtained.

[0056] (3) Spectroscopic methods are used to separate the active ingredients contained in the extracts obtained in step (2). By examining the inhibitory effect of the extracts on the production of nitric oxide (NO) in LPS - induced BV - 2 microglial cells, it can be seen that the active components are mainly concentrated in the petroleum ether layer extract. The mass spectrometry results show that the molecular ion peaks of the petroleum ether extraction layer extract are mostly concentrated in the range of 200 - 300. Combining the fact that it shows a light yellow color and has an aromatic odor with 10% sulfuric acid ethanol, it is speculated that the active substance is a sesquiterpene derivative. According to the changes of chromophores and auxochromes, its ultraviolet full - wavelength scanning shows certain regularity and characteristics. Through the above analysis, the directional separation of active sesquiterpene components is achieved.

[0057] Most of the sesquiterpene skeletons isolated from the branches and leaves of Croton lachnocarpus Benth. have conjugated alkenes or conjugated enones, as Figure 1 shown. When there is an intracyclic enone conjugation, the main characteristic of the ultraviolet absorption is the presence of an absorption band around 240 - 260 nm; when the A - ring is an aromatic cycloheptatrienone seven - membered ring, there are mainly two red - shifted absorption bands in the ultraviolet absorption: the first peak: around 240 nm, the second peak: around 330 nm; when the A - ring is a benzene ring, there is an absorption band around 260 nm. Based on this, the sesquiterpenes contained therein can be directionally separated.

[0058] (4) It is preliminarily determined from step (3) above that the active sesquiterpenes are mainly enriched in the petroleum ether layer extract. The petroleum ether layer extract is separated by silica gel column chromatography and eluted with different solvent systems composed of petroleum ether, ethyl acetate, dichloromethane, methanol, etc.

[0059] (5) The fractions containing sesquiterpene components in step (4) above are separated again by open - type silica gel or ODS column chromatography and then separated by preparative or semi - preparative HPLC chromatography, eluted with methanol - water or acetonitrile - water as the mobile phase to obtain sesquiterpene compounds.

[0060] The present invention provides a method for separating the extract of petroleum ether extract by silica gel column chromatography. The organic solvents used are one or two mixed solvents of petroleum ether, ethyl acetate, dichloromethane, and methanol, including the petroleum ether-ethyl acetate (100:1-1:1) system and the dichloromethane-methanol system (100:0-15:1). When using a GF254 silica gel plate to explore the open silica gel column elution conditions for the petroleum ether-ethyl acetate (100:1-1:1) system, the ratios include: petroleum ether-ethyl acetate (100:1), petroleum ether-ethyl acetate (20:1), petroleum ether-ethyl acetate (4:1), petroleum ether-ethyl acetate (3:1), petroleum ether-ethyl acetate (2:1), petroleum ether-ethyl acetate (1:1).

[0061] The method for preparing sesquiterpene derivatives by preparative or semi-preparative HPLC provided by the present invention has a mobile phase of methanol-water (1:4-9:1) or acetonitrile-water (1:5-4:1), and preferably the methanol-water (1:4-9:1) system.

[0062] The present invention provides a pharmaceutical composition comprising one or several of the above-mentioned sesquiterpene derivatives or their pharmaceutically acceptable salts and a pharmaceutically acceptable carrier or excipient.

[0063] The petroleum ether layer extract of Croton lachnocarpus in the present invention has an anti-neuritis effect and can be used to prepare a drug for the treatment of neurodegenerative diseases related to neuroinflammation.

[0064] The sesquiterpene derivatives or their pharmaceutically acceptable salts or their pharmaceutical compositions in the present invention have an anti-neuritis effect and can be used to prepare drugs for neurodegenerative diseases related to neuroinflammation.

[0065] The beneficial effects of the present invention:

[0066] The present invention first extracts the above-mentioned sesquiterpene derivatives from the dried branches and leaves of Croton lachnocarpus, and determines the structure of the sesquiterpene derivatives by using separation methods such as silica gel column chromatography, ODS column chromatography, and high performance liquid chromatography (HPLC), combined with various spectroscopic techniques such as nuclear magnetic resonance spectroscopy. The sesquiterpene derivatives prepared by the method of the present invention can inhibit the release of NO from LPS-induced BV-2 cells, and have great application prospects in the preparation of anti-neuritis drugs. Description of the Drawings

[0067] Figure 1 Ultraviolet full wavelength scanning diagram of sesquiterpenes with different structural types;

[0068] Figure 2 Effect of sesquiterpene derivatives (50 μM) on the viability of BV-2 cells (Gal: galantamine);

[0069] Figure 3 Effect of sesquiterpene derivatives on NO release in LPS-induced BV-2 cells (#: IC 50 > 50 μM; Gal: galantamine) Detailed implementation mode

[0070] The following examples will further illustrate the present invention in detail and are not used to limit the present invention.

[0071] Example 1

[0072] 20.0 kg of dry branches and leaves of Croton tiglium were pulverized, and refluxed with 95% ethanol with a material-liquid ratio of 1:8 three times for 2 h each time. The solvent was recovered under reduced pressure, and the extract was dispersed with distilled water and extracted three times with equal volumes of petroleum ether, dichloromethane, ethyl acetate, and n-butanol to obtain 300.8 g of petroleum ether layer extract. Open silica gel column chromatography was used for separation, and the mobile phase was selected as petroleum ether-ethyl acetate (100:1 - 1:1, v / v) gradient elution. The obtained fractions were analyzed by silica gel thin layer chromatography, and six eluates were obtained after combining the same fractions. The fractions of petroleum ether-ethyl acetate (4:1)-(2:1) were collected, totaling 36.6 g, and further separated. Using ODS column chromatography, the fraction of petroleum ether-ethyl acetate 4:1 was eluted with methanol-water (1:4 - 9:1, v / v) gradient. At methanol-water 2:1, the yellow-brown viscous oil was separated by preparative HPLC with methanol-water (9:11, v / v, 3 mL / min) to obtain compounds 1, 5 - 10; the fraction of petroleum ether-ethyl acetate 3:1 was eluted with methanol-water (1:5 - 8:1, v / v) gradient. At methanol-water 1:1, the yellow-brown viscous oil was separated by preparative HPLC with methanol-water (1:1, v / v, 3 mL / min) to obtain compounds 11 - 12; the fraction of petroleum ether-ethyl acetate 2:1 was eluted with methanol-water (1:5 - 9:1, v / v) gradient. At methanol-water 1:4, the yellow-brown viscous oil was separated by preparative HPLC with methanol-water (1:5, v / v, 3 mL / min) to obtain compounds 2 - 4, 13.

[0073] Example 2

[0074] In vitro anti-neuritis activity screening of extract and sesquiterpene derivatives

[0075] (1) Cell culture

[0076] BV-2 cells were cultured in DMEM medium with 10% FBS at 37 °C and 5% CO 2 condition.

[0077] (2) Activity test

[0078] MTT cytotoxicity assay: Pretreat BV-2 cells with the test extract at a concentration of 0.781 - 500 μg / mL for 4 h, then add LPS and incubate for 24 h; Pretreat BV-2 cells with the test monomer compound at a final concentration of 50 μM for 4 h, then add LPS and incubate for 24 h; Finally, determine the cell viability according to the MTT reagent instruction manual.

[0079] NO inhibition activity screening: Pretreat BV-2 cells with the extract at gradient concentrations of 0 - 200 μg / mL for 4 h, then add LPS and incubate for 24 h; Pretreat BV-2 cells with the test monomer at gradient concentrations of 0 - 50 μM for 4 h, then add LPS and incubate for 24 h; Finally, determine the concentration of NO in the cell culture supernatant according to the Griess kit instruction.

[0080] The experimental results of the extract are shown in Table 1.

[0081] The experimental results of the compounds are as Figure 2 、 Figure 3 。

[0082] Table 1 Evaluation of the inhibitory activities of the extracts from different extraction layers of Croton lachnocarpus on the viability of BV-2 cells and NO release

[0083]

[0084] Note: "-" indicates that the dichloromethane layer extract has high toxicity and is not suitable for NO inhibition activity determination.

[0085] The results showed that compounds 2 - 3, 5 - 13 could significantly inhibit the release of NO from LPS-induced BV-2 cells, and compounds 3, 5 - 9, 11 had strong inhibitory activities, with IC 50 all less than 2 μM.

Claims

1. A sesquiterpene derivative or a pharmaceutically acceptable salt thereof, characterized in that: The sesquiterpene derivative is any one of the following compounds:

2. The method for preparing the sesquiterpene derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The following steps are involved: (1) The dried branches and leaves of Croton oleifera are crushed, and then refluxed and extracted with 3 to 15 times the amount of 30% to 100% ethanol or methanol, and the alcohol solvent is recovered under reduced pressure, and then suspended in water; (2) extracting the suspension with petroleum ether, dichloromethane, ethyl acetate, and n-butanol in sequence, the extraction times being times, the volume ratio of the extraction solvent to the suspension being 1:1-2:1, recovering the solvent, and obtaining a petroleum ether layer extract, a dichloromethane layer extract, an ethyl acetate layer extract, an n-butanol layer extract, and an aqueous layer extract; (3) separating the active ingredients contained in the petroleum ether layer extract of step (2), and separating the petroleum ether layer extract by open silica gel column chromatography, using a petroleum ether-ethyl acetate 100:1-1:1 system or a dichloromethane-methanol system 100:0-15:1 system as the solvent; analyzing the obtained fractions by silica gel thin layer chromatography, combining the same fractions to obtain 6 eluates, and collecting the petroleum ether-ethyl acetate 4:1-2:1 fraction; (4) The fraction containing sesquiterpenoid components in step (3) is separated again by open silica gel or ODS column chromatography, and then separated by preparative or semi-preparative HPLC chromatography, with methanol-water 1:4-9:1 or acetonitrile-water 1:5-4:1 as the mobile phase for elution to obtain sesquiterpene derivatives.

3. The method for preparing the sesquiterpene derivative or a pharmaceutically acceptable salt thereof according to claim 2, characterized in that: In the step (4), ODS column chromatography was used to elute the petroleum ether-ethyl acetate 4:1 fraction with a gradient of methanol-water 1:4-9:1, and at the methanol-water 2:1, the yellow-brown viscous oil was separated by preparative HPLC with methanol-water 9:11, 3 mL / min to obtain compounds 9-10; petroleum ether-ethyl acetate 3:1 fraction was eluted with a gradient of methanol-water 1:5-8:1, and at the methanol-water 1:1, the yellow-brown viscous oil was separated by preparative HPLC with methanol-water 1:1, 3 mL / min to obtain compounds 11-12; petroleum ether-ethyl acetate 2:1 fraction was eluted with a gradient of methanol-water 1:5-9:1, and at the methanol-water 1:4, the yellow-brown viscous oil was separated by preparative HPLC with methanol-water 1:5, 3 mL / min to obtain compounds 2-3, 13.

4. A pharmaceutical composition comprising one or more of the sesquiterpene derivatives or pharmaceutically acceptable salts thereof according to claim 1 and a pharmaceutically acceptable carrier or excipient.

5. Use of the sesquiterpene derivatives according to claim 1 or the sesquiterpene derivatives shown in the following structure in the preparation of drugs for neurodegenerative diseases related to neuroinflammation

Images