CN113754625A - Sesquiterpene coumarin compound and preparation method and application thereof - Google Patents

Sesquiterpene coumarin compound and preparation method and application thereof Download PDF

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CN113754625A
CN113754625A CN202010483912.4A CN202010483912A CN113754625A CN 113754625 A CN113754625 A CN 113754625A CN 202010483912 A CN202010483912 A CN 202010483912A CN 113754625 A CN113754625 A CN 113754625A
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sesquiterpene
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李宁
周地
侯悦
陈刚
郭婷婷
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Shenyang Pharmaceutical University
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Abstract

The invention belongs to the technical field of medicines, relates to a preparation method and application of a sesquiterpene coumarin compound, and particularly relates to a sesquiterpene coumarin compound structure, a preparation method and application of the sesquiterpene coumarin compound structure in the field of preparation of medicines for preventing and treating neurodegenerative diseases, wherein the compound has a structural general formula shown in the specification, wherein R is1–R7As described in the claims and specification.

Description

Sesquiterpene coumarin compound and preparation method and application thereof
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a sesquiterpene coumarin compound, and a preparation method and application thereof.
Background
Resina Ferulae is resin of Ferula Sinkiangensis K.M.Shen (Ferula sinkiangensis K.M.Shen) or Ferula fukanensis K.M.Shen (Ferula fukanensis) belonging to Ferula genus (Ferula) of Umbelliferae family. Ferula species have over 150 species in the world, mainly distributed in the southern mediterranean region of Europe and northern Africa, and also distributed in the Central Asia region of Iran, Africa, Soviet Union and Siberian regions, as well as in India, Pakistan, etc. The Chinese asafetida is mainly distributed in Xinjiang.
The resina Ferulae has effects of resolving food stagnation, eliminating abdominal mass, dispersing pathogenic accumulation, and killing parasite, and can be used for treating food stagnation, blood stasis, abdominal mass, and abdominal pain due to parasitic accumulation. Clinically, the compound containing asafetida is mainly used for treating some gastrointestinal related diseases: such as asafetida pill, for treating meat stagnation; the asafetida and galangal pills are taken for a long time to treat middle-jiao cold accumulation, regurgitation and vomiting and diet reduction; the asafetida middle energizer regulating pill is used for treating cold attacking stabbing pain, heart and abdomen fullness, stomach cold vomiting and regurgitation, and umbilical and abdomen pinching pain. Modern pharmacological research shows that the asafetida has wide pharmacological activities of resisting tumor, resisting inflammation, etc. The compound has various chemical components such as coumarins, sesquiterpenes, sulfur-containing compounds and aromatics, wherein the sesquiterpene coumarins are characteristic components of the compound.
Disclosure of Invention
The invention aims to provide a series of sesquiterpene coumarin compounds, and a preparation method and medical application thereof.
The sesquiterpene coumarin compound provided by the invention and pharmaceutically acceptable salts and isomers thereof have the following structural general formula:
Figure BDA0002518235580000011
wherein the content of the first and second substances,
R1is hydrogen, hydroxy, C1-C4 acyloxy, fragment A or fragment B;
R2is hydrogen or hydroxy;
R3is hydroxy, C1-C4 acyloxy or fragment B;
R4is hydrogen, C1-C4 alkyl or hydroxy;
R5is hydrogen, C1-C4 alkyl or hydroxy;
R6is hydrogen or hydroxy;
R7is hydrogen or hydroxy.
The invention preferably selects the sesquiterpene coumarin compound with the following structure and the pharmaceutically acceptable salt and isomer thereof,
wherein the content of the first and second substances,
R1is hydrogen, hydroxy, acetoxy, propionyloxy, fragment A or fragment B;
R2is hydrogen or hydroxy;
R3is hydroxy, acetoxy, propionyloxy or fragment B;
R4is hydrogen, methyl or hydroxy;
R5is hydrogen, methyl or hydroxy;
R6is hydrogen or hydroxy;
R7is hydrogen or hydroxy.
The invention specifically discloses the following ten specific compounds:
Figure BDA0002518235580000021
the invention also provides a preparation method of the sesquiterpene coumarin compound 1-10, which comprises the following steps:
(1) extracting resina Ferulae with solvent such as methanol, ethanol, chloroform or dichloromethane, and recovering extractive solution to obtain crude extract;
(2) separating the crude extract obtained in the step (1) by silica gel column chromatography, and performing gradient elution by using a petroleum ether-ethyl acetate mixed solvent, or a petroleum ether-acetone mixed solvent, or a dichloromethane-ethyl acetate mixed solvent, or a dichloromethane-acetone mixed solvent, or a chloroform-ethyl acetate mixed solvent, or a chloroform-acetone mixed solvent to obtain eluates with different polarities;
(3) performing ODS column chromatography on the eluate with different polarities obtained in the step (2), and performing gradient elution by using a methanol-water mixed solvent or an acetonitrile-water mixed solvent as a mobile phase;
(4) and (4) further separating the methanol-water or acetonitrile-water eluate obtained in the step (3) by HPLC, and carrying out gradient elution by using a methanol-water mixed solvent or acetonitrile-water mixed solvent as a mobile phase to obtain the sesquiterpene coumarin 1-10.
The invention provides a preparation method of 1-10 sesquiterpene coumarin compounds, wherein the extraction method in the step (1) is heating reflux extraction or heating ultrasonic extraction for 2-5 times. The volume concentration of the methanol used is 60% to 100%, preferably 80% to 90%. The volume concentration of the ethanol used is 60-100%, preferably 80-95%. The material-liquid ratio is 1: 5-1: 30g/mL, preferably 1: 10-1: 15.
The preparation method of the sesquiterpene coumarin compound 1-10 provided by the invention comprises the steps of (1) mixing the petroleum ether-ethyl acetate mixed solvent or the petroleum ether-acetone mixed solvent in the step (2) in a volume ratio of 100: 0-0: 1, preferably 100: 5-2: 1; the volume ratio of the dichloromethane-ethyl acetate mixed solvent, the dichloromethane-acetone mixed solvent, the chloroform-ethyl acetate mixed solvent or the chloroform-acetone mixed solvent is 100: 0-1: 1, preferably 100: 2-5: 1.
According to the preparation method of the sesquiterpene coumarin compound 1-10, the volume ratio of the methanol-water mixed solvent in the step (3) is 50: 50-100: 0, preferably 60: 40-90: 10; the volume ratio of the acetonitrile-water mixed solvent is 30: 70-90: 10, preferably 40: 60-80: 20.
According to the preparation method of the sesquiterpene coumarin compound 1-10, the volume ratio of the methanol-water mixed solvent in the step (4) is 60: 40-90: 10, preferably 70: 30-90: 10; the volume ratio of the acetonitrile-water mixed solvent is 50: 50-80: 20, preferably 60: 40-80: 20.
The anti-inflammatory activity test is carried out by taking BV-2 cells in vitro as a model, and the neuroinflammation inhibitory activity of the prepared sesquiterpene coumarin compounds 1-10 is evaluated. The results show that the sesquiterpene coumarins have remarkable anti-neuritis activity and can be used for developing chemopreventive agents or therapeutic drugs for neuroinflammation.
The invention provides a method for preparing and identifying 10 sesquiterpene coumarins by taking ferula asafetida as a raw material for the first time, systematically evaluates the activity of the sesquiterpene coumarins in resisting neuritis and explains the application of the sesquiterpene coumarins in developing chemoprevention and treatment medicines related to neuroinflammation.
Detailed Description
The following examples further illustrate the invention but are not intended to limit the invention thereto.
Example 1
(1) Extracting 1000g resina Ferulae with 95% ethanol under reflux for 3 times (10L), and recovering the crude extract under reduced pressure;
(2) subjecting the crude extract of 95% ethanol obtained in the step (1) to silica gel column chromatography, and performing gradient elution with petroleum ether-ethyl acetate mixed solvent 100:5, 10:1, 2:1, 0:1 to obtain eluates with different polarity parts;
(3) separating the eluate of the petroleum ether-ethyl acetate mixed solvent 100:5 in the step (2) by silica gel column chromatography, and sequentially eluting with petroleum ether-ethyl acetate mixed solvent 100:0, 100:1, 100:2, 100:3, 100:5, 100:8, 10:1, 8:1, 6:1, 4:1, 2:1 and 1: 1;
(4) the petroleum ether obtained in the step (3): subjecting the ethyl acetate 100: 5-2: 1 flow to ODS chromatography, and performing gradient elution by using a mixed solvent of methanol-water of 50:50, 60:40, 70:30, 80:20, 90: 10;
(5) separating the 80: 20-90: 10 fraction of the methanol-water obtained in the step (4) by HPLC-UV chromatography at a flow rate of 3mL/min, wherein the mobile phase is methanol: water 90:10 to give sesquiterpene coumarin 3 (t)R102min) (yield 0.0003%), sesquiterpene coumarin 4 (t)R80min) (yield 0.0031%), sesquiterpene coumarin 5 (t)R9min) (yield 0.0010%), sesquiterpene coumarin 6 (t)R24min) (yield 0.0010%) to yield sesquiterpene coumarin 7 (t)R69min) (yield 0.0016%), sesquiterpene coumarin 9 (t)R13min) (yield 0.0004%).
(6) Separating and preparing the 90:10 fraction of methanol-water obtained in the step (4) by HPLC-UV chromatography at the flow rate of 3mL/min, wherein the mobile phase is methanol: water 82:18 to give sesquiterpene coumarin 2 (t)R23min) (yield 0.0002%).
(7) Separating and preparing the 90:10 fraction of methanol-water obtained in the step (4) by HPLC-UV chromatography at the flow rate of 3mL/min, wherein the mobile phase is methanol:water 79:21 to give sesquiterpene coumarin 1 (t)R44min) (yield 0.0003%), sesquiterpene coumarin 10 (t)R39min) (yield 0.0012%).
(8) And (3) separating the methanol-water fraction obtained in the step (4) with a ratio of 60: 40-70: 30 by HPLC RID-10A chromatography at a flow rate of 3mL/min, wherein the mobile phase is methanol: water 70:30 to yield sesquiterpene coumarin 8 (t)R24min) (yield 0.0005%).
The structure of the compound is identified according to the physicochemical properties and the spectrum data of the compound 1-10.
The structural identification data of sesquiterpene coumarin 1 are as follows:
yellow oil (MeOH), HRESIMS gave the excimer peak M/z 449.2307[ M + Na ]]+:(calcd.449.2304 for C26H34O5Na) and the molecular formula thereof is presumed to be C26H34O5The unsaturation degree was 10.1H NMR(600MHz,CDCl3) The characteristic hydrogen signal of 7-O-substituted coumarin parent nucleus is given by a low field region in the spectrum: 7.63(1H, d, J ═ 9.4Hz, H-4),7.37(1H, d, J ═ 8.5Hz, H-5),6.86(1H, dd, J ═ 8.5,2.2Hz, H-6),6.83(1H, d, J ═ 2.2Hz, H-8) and δH6.25(1H, d, J ═ 9.4Hz, H-3); the high field region gives 5 methyl hydrogen signals: deltaH2.04(3H, s, Me-2 "), 1.79(3H, s, Me-11'),1.68(3H, s, Me-15'),1.66(3H, s, Me-14'), and 0.97(3H, d, J ═ 6.9Hz, Me-12'). In addition, an olefinic hydrogen signal is also observed in the hydrogen spectrum: deltaH5.49(1H, t, J ═ 6.5Hz, H-2'); two sets of vicinal oxymethylene signals: deltaH4.60(2H, d, J ═ 6.5Hz, H-1') and 4.06(2H, m, H-10').13C NMR(150MHz,CDCl3) The spectrum gives a total of 26 signals, except for 9 characteristic carbon signals of 7-O-substituted coumarin parent nucleus: deltaC162.3(C-7),161.5(C-2),156.0(C-9),143.6(C-4),128.8(C-5),113.4(C-6),113.1(C-3),112.6(C-10) and 101.7 (C-8); one set of acetyl carbon signals: deltaC171.4(C-1 '), 21.2 (C-2'), there are still 15 carbon signals, which are sesquiterpene skeleton carbon signals, including two sets of alkene carbon signals: deltaC143.2(C-3'), 118.0 (C-2'), and δC134.9(C-6 '), 126.2 (C-13'); 2 vicinal oxygen carbon signals: deltaC65.6(C-1'),65.0 (C-10'). Bound hydrogenAnd (3) spectrum and carbon spectrum data, and the compound is supposed to be sesquiterpene coumarin. Since 1 coumarin unit (7 unsaturations), 1 carbonyl group and two olefin bonds in the structure of the compound constitute 10 unsaturations of the compound, the sesquiterpene moiety of the compound is presumed to be a chain structure. All hydrogen carbon data were assigned according to HSQC (tables 1 and 2).
1H-1H COSY shows that the compound has 3 spin coupling systems which are respectively H2-1′/H-2′,H2-4′/H2-5', and H3-12′/H-7′/H2-8′/H2-9′/H210' indicating the presence of three fragments C-1 '/C-2 ', C-4 '/C-5 ', and C-7' (C- ' 12 ')/C-8 '/C-9 '/C-10 '. In HMBC spectra, deltaH 4.60(H2-1') and δC118.0(C-2 '), 143.2(C-3'), deltaH5.49 (H-2') and δC143.2(C-3'), 40.2(C-4 '), 17.0(C-11 '), deltaH 2.08(Ha-5') and 2.02 (H)b-5') and δC143.2(C-3'), 134.9 (C-6'), 35.6(C-7 '), 126.2 (C-13'), deltaH 1.34(H2-8') and δC134.9 (C-6'), deltaH 1.66(H3-14') and 1.68 (H)3-15') and δC126.2(C-13 '), 134.9 (C-6'). And (3) integrating the HMBC related signals to establish a planar structure of the sesquiterpene unit. Further, δH 4.06(H2-10') and δC171.4(C-1 ') together, indicating that the acetyl group is attached at the C-10' position. DeltaH 4.60(H2-1') and δC162.3(C-7) remote correlation, suggesting that sesquiterpene fragments are linked to the C-7 position of the coumarin parent nucleus. In NOESY spectrum, H314 'is related to H-7', suggesting that Me-14 'is ipsilateral to H-7'; h3-15' and Ha,b5 'correlation, suggesting Me-15' with Ha,b-5' on the same side. The absolute configuration of compound 1 was determined by comparing the measured electron circular dichroism spectra (ECD) and calculating ECD data. The compound is a novel compound which is not reported through literature search and is named as (7R) -ferusingensine A.
The structural identification data of sesquiterpene coumarin 2 are as follows:
yellow oil (MeOH), HRESIMS gave the excimer peak M/z 407.2200[ M + Na ]]+:(calcd.407.2198for C24H32O4Na) and the molecular formula thereof is presumed to be C24H32O4. The hydrogen spectrum and carbon spectrum data are similar to those of the compound 1, except that the substituent on C-10' is different, and the combination data conjecture that-OAc of the compound 1 is possibly replaced by-OH.1H NMR(600MHz,CDCl3) In the spectrum, the characteristic proton signals of 5 7-O-substituted coumarin parent nuclei are given by a low field region: deltaH7.64(1H, d, J ═ 9.5Hz, H-4),7.37(1H, d, J ═ 8.6Hz, H-5),6.86(1H, dd, J ═ 8.6,2.4Hz, H-6),6.83(1H, d, J ═ 2.4Hz, H-8),6.25(1H, d, J ═ 9.5Hz, H-3); the high field region provides 4 methyl hydrogen signals: deltaH1.79(3H, s, Me-11'),1.68(3H, s, Me-15'),1.66(3H, s, Me-14'), and 0.97(3H, d, J ═ 6.9Hz, Me-12'). In addition, there is a double bond hydrogen signal in the hydrogen spectrum: deltaH5.49(1H, t, J ═ 6.5Hz, H-2'); two sets of vicinal oxymethylene signals: deltaH4.60(2H, d, J ═ 6.5Hz, H-1') and δH 3.61(2H,t,J＝6.6Hz,H-10')。13C NMR(150MHz,CDCl3) The spectrum has 24 signals in total, and the low field region gives 9 characteristic carbon signals of 7-O-substituted coumarin parent nucleus: deltaC162.3(C-7),161.5(C-2),156.0(C-9),143.6(C-4),128.8(C-5),113.4(C-6),113.1(C-3),112.6(C-10) and 101.7(C-8), the remaining 15 carbon signals being sesquiterpene backbone carbon signals, comprising two sets of double bond carbon signals: deltaC143.3(C-3 '), 118.0 (C-2'), and δC135.1(C-6 '), 125.9 (C-13'); 2 vicinal oxygen carbon signals: deltaC65.7(C-1'),63.5 (C-10'). All hydrogen carbon data were assigned according to HSQC (tables 1 and 2).
In HMBC spectra, deltaH 4.60(H2-1') and δC118.0(C-2 '), 143.3 (C-3'), deltaH5.49 (H-2') and δC143.3(C-3 '), 40.2(C-4 '), 17.0(C-11 '), deltaH 2.08(Ha-5') and 2.02 (H)b-5') and δC143.3(C-3 '), 135.1 (C-6'), 35.7(C-7 '), 125.9 (C-13'), deltaH 1.34(H2-8') and δC135.1 (C-6') correlation, δH 1.66(H3-14') and 1.68 (H)3-15') and δC125.9(C-13 '), 135.1 (C-6'). And (4) integrating the HMBC related signals to determine the planar structure of the sesquiterpene part. By deltaH 4.60(H2-1') and δC162.3(C-7) remote correlation, determination of sesquiterpene fragments through ether bond and coumarin nucleus C-7 phase connection. The absolute configuration of compound 2 was determined by comparing the measured ECD and calculated ECD data. The compound is a novel compound which is not reported through literature search and is named as (7R) -ferusingensine B.
The structural identification data of sesquiterpene coumarin 3 are as follows:
yellow oil (MeOH), HRESIMS gave the excimer peak M/z 573.3553[ M + Na ]]+:(calcd.573.3556for C35H50O5Na) and the molecular formula thereof is presumed to be C35H50O5. The hydrogen spectrum and carbon spectrum data are similar to those of the compound 1, and the difference is that the substituent on C-10' is different.1H NMR(600MHz,CDCl3) In the spectrum, the low field region indicates 5 characteristic proton signals of the 7-O-substituted coumarin parent nucleus: deltaH7.64(1H, d, J ═ 9.4Hz, H-4),7.36(1H, d, J ═ 8.5Hz, H-5),6.86(1H, dd, J ═ 8.5,2.2Hz, H-6),6.83(1H, d, J ═ 2.2Hz, H-8) and 6.25(1H, d, J ═ 9.4Hz, H-3); the high field region provides 5 methyl hydrogen signals deltaH1.79(3H, s, Me-11'),1.68(3H, s, Me-15'),1.66(3H, s, Me-14'),0.97(3H, d, J ═ 6.8Hz, Me-12'), and 0.87(3H, t, J ═ 6.8Hz, Me-10 "). In addition, the hydrogen spectrum also has a double bond hydrogen signal deltaH5.49(1H, t, J ═ 6.5Hz, H-2'), two sets of coherent oxymethylene signals δH4.60(2H, d, J ═ 6.5Hz, H-1') and 4.05(2H, t, J ═ 6.7Hz, H-10').13C NMR(150MHz,CDCl3) The spectrum gives 35 signals in total, and the low field region gives 9 characteristic carbon signals of 7-O-substituted coumarin parent nucleus: deltaC162.3(C-7),161.4(C-2),156.1(C-9),143.6(C-4),128.8(C-5),113.4(C-6),113.2(C-3),112.6(C-10) and 101.7 (C-8). The remaining 26 carbon signals, compared with the compound 1 carbon spectrum data, are presumed to be sesquiterpene unit carbon signals, including two groups of double-bond carbon signals: deltaC143.2(C-3'),118.1(C-2'), and δC 135.0(C-6′),126.1(C-13′)；2Single vicinal oxygen carbon signal: deltaC65.7(C-1'),64.8 (C-10'). The remaining 11 carbon signals are those of the C-10' substituent. All hydrogen carbon data were assigned according to HSQC (tables 1 and 2).
1H-1In the H COSY spectrum, a spin coupling system of a C-10' position substituent is given: h-2 '/H-3' (H-11 ')/H-4'/H-5 '/H-6'/H-7 '/H-8'/H-9 '/H-10'. In HMBC spectra, deltaH 0.70(Ha-11 ") and-0.15 (H)b-11 ") and δCThe signals 34.2(C-2 '), 11.7 (C-3'), 15.7(C-4 '), 29.0 (C-5'), correlate, suggesting the presence of a cyclopropane fragment. In addition, delta is made ofH0.87 (H-10') and deltaC22.8(C-9 '), 32.0 (C-8'), deltaH1.30 (H-7') and deltaC32.0 (C-8'), deltaH 1.37(Ha-6 ") and 1.26 (H)b-6 ") and δC29.4 (C-7'), deltaH 1.36(Ha-5 ") and 1.16 (H)b-5 ") and δC11.7(C-3 '), 15.7(C-4 '), 30.0(C-6 '), and deltaH 2.35(Ha-2 ") and 2.24 (H)b-2 ") and δC174.0(C-1 '), 11.7 (C-3'), 15.7(C-4 '), define the planar configuration of the substituent at the C-10' position. According to1High field region signal delta in H NMR spectrumH 0.79(1H,m,H-4”),0.70(1H,dt,J＝8.4,4.7Hz,Ha-11”),-0.15(1H,q,J＝4.7Hz,Hb11 ") the relative configuration of the cyclopropane; the absolute configuration of compound 3 was determined by comparing the measured ECD and calculated ECD data. The compound is a novel compound which is not reported through literature search and is named as (7' S, 3' R,4' R) -ferusingensine C.
The structural identification data of sesquiterpene coumarin 4 are as follows:
yellow oil (MeOH), HRESIMS gave the excimer peak M/z 559.3396[ M + Na ]]+:(calcd.559.3399for C34H48O5Na) and the molecular formula thereof is presumed to be C34H48O5The hydrogen and carbon spectra data are similar to compound 1, except that the substituent at the C-10' position is different.1H NMR(600MHz,CDCl3) In the spectrum, the low field region gives 7-O-substituted coumarin parent nucleus5 characteristic proton signals: deltaH7.63(1H, d, J ═ 9.5Hz, H-4),7.36(1H, d, J ═ 8.6Hz, H-5),6.86(1H, dd, J ═ 8.6,2.2Hz, H-6),6.82(1H, d, J ═ 2.2Hz, H-8) and 6.24(1H, d, J ═ 9.5Hz, H-3); the high field region provides 5 methyl proton signals: deltaH1.79(3H, s, Me-11'),1.68(3H, s, Me-15'),1.65(3H, s, Me-14'),0.96(3H, d, J ═ 6.8Hz, Me-12'), and 0.87(3H, t, J ═ 6.6Hz, Me-10 '). In addition, there are 3 double bond hydrogen signals in the hydrogen spectrum: deltaH5.49(1H, t, J ═ 6.5Hz, H-2'),5.40(1H, m, H-5 "), and 5.31(1H, m, H-4"); two groups of vicinal oxymethylene hydrogen signals: deltaH4.59(2H, d, J ═ 6.5Hz, H-1') and 4.02(2H, t, J ═ 6.7Hz, H-10').13C NMR(150MHz,CDCl3) The spectrum gives a total of 34 signals, the low field region gives 9 characteristic carbon signals of 7-O-substituted coumarin parent nucleus: deltaC162.3(C-7),161.4(C-2),156.0(C-9),143.6(C-4),128.8(C-5),113.3(C-6),113.1(C-3),112.6(C-10),101.7 (C-8). By comparison with compound 1 carbon spectrum data, the 15 carbon signal is presumed to be a sesquiterpene unit carbon signal, which includes two groups of double bond carbon signals: deltaC143.2(C-3'),118.1(C-2'), and δC135.0(C-6'),126.1 (C-13'); 2 vicinal oxygen carbon signals: deltaC65.6(C-1'),64.8 (C-10'). The remaining 10 carbon signals are those of the C-10' substituent and comprise a set of double bond carbon signals: deltaC131.7(C-5'),127.5 (C-4'). All hydrogen carbon data were assigned according to HSQC (tables 1 and 2).
1H-1In the H COSY spectra, 1 spin-coupled system H-3 '/H-4 '/H-5 '/H-6 '/H-7 '/H-8 '/H-9 '/H-10 ' giving a substituent at the C-10' position, indicates the presence of the enoate fragment. Combining signals in HMBC spectra, deltaH 2.35(Ha-2 ") and 2.27 (H)b-2 ") and δC173.5(C-1 '), 22.7 (C-3'), deltaH2.33 (H-3') and deltaC127.5(C-4'), 131.7(C-5'), deltaH2.04 (H-6') and deltaC127.5(C-4'), 131.7(C-5'), 29.4(C-7 '), 31.6 (C-8'), and deltaH0.87 (H-10') and deltaC23.0(C-9 '), 31.6(C-8 '), the planar structure of the 4-decenoate substituent at the C-10' position was determined. By comparing measured ECD and calculated ECD data, determiningThe absolute configuration of compound 4 is shown. The compound is a novel compound which is not reported through literature search and is named as (7R) -ferusingensine D.
The structural identification data of sesquiterpene coumarin 5 are as follows:
yellow oil (MeOH), HRESIMS gave the excimer peak M/z 405.2045[ M + Na ]]+:(calcd.405.2042 for C24H30O4Na) and the molecular formula thereof is presumed to be C24H30O4Contains 10 unsaturations.1H NMR(600MHz,CDCl3) In the spectrum, the low field region shows 5 proton signals characteristic of the 7-O-substituted coumarin parent nucleus: deltaH7.63(1H, d, J ═ 9.5Hz, H-4),7.36(1H, d, J ═ 8.6Hz, H-5),6.85(1H, dd, J ═ 8.6,2.3Hz, H-6),6.82(1H, d, J ═ 2.3Hz, H-8) and 6.24(1H, d, J ═ 9.5Hz, H-3); the high field region gives 4 sets of methyl proton signals: deltaH1.76(3H, s, Me-13'),1.60(3H, s, Me-14'),1.08(3H, d, J ═ 6.9Hz, Me-15'), and 1.08(3H, d, J ═ 6.9Hz, Me-12'). In addition, there are 2 double bond proton signals in the hydrogen spectrum: deltaH5.46(1H, t, J ═ 6.5Hz, H-2') and 5.09(1H, t, J ═ 6.9Hz, H-6'); and group 1 oxymethylene proton signals: deltaH 4.60(2H,d,J＝6.5Hz,H-1′)。13C NMR(150MHz,CDCl3) The spectrum shows 24 carbon signals, the low field region gives 9 7-O-substituted coumarin parent carbon signals: deltaC162.3(C-7),161.5(C-2),156.0(C-9),143.6(C-4),128.8(C-5),113.4(C-6),113.1(C-3),112.6(C-10),101.7 (C-8). The remaining 15 carbon signals are those of sesquiterpene units, comprising 2 sets of double bond carbon signals: deltaC142.3(C-3'),118.7(C-2'), and δC134.6(C-7'),124.1 (C-6'); 1 carbonyl carbon signal: deltaC214.6 (C-10'); 1 oxygen carbon signal: deltaC65.6 (C-1'). All hydrogen carbon data were assigned according to HSQC (tables 1 and 2).
1H-1H COSY spectrum, giving 4 coupling systems for sesquiterpene fragments: h-1 '/H-2', H-4 '/H-5'/H-6 ', H-8'/H-9 ', and H-13'/H-11 '/H-14'. In HMBC spectra, deltaH 2.09(H2-4') and δC118.7(C-2'), 142.3(C-3'), 26.3(C-5 '),124.1(C-6'), deltaH 2.22(H2-8') and δC124.1(C-6'), 134.6(C-7'), 39.2(C-9 '), 214.6(C-10'), deltaH 1.08(H3-12') and 1.08 (H)3-15') and δC41.0(C-11 '), 214.6(C-10'), and the planar structure of sesquiterpene fragments was determined. DeltaH 4.60(H2-1') and δC162.3(C-7) correlation, suggesting that the sesquiterpene unit is linked to the C-7 position of the coumarin parent nucleus through an ether bond. The natural product is found by literature search and is named as ferusingensine E.
The structural identification data of sesquiterpene coumarin 6 are as follows:
white needle crystals (MeOH), HRESIMS gave the excimer peak M/z 463.2459[ M + Na ]]+:(calcd.463.2460for C27H36O5Na) and the molecular formula thereof is presumed to be C27H36O5This suggests that there are 10 unsaturations in the structure.1H NMR(600MHz,CDCl3) 5 proton signals in the spectrum that characterize the 7-O-substituted coumarin nucleus: deltaH7.63(1H, d, J ═ 9.4Hz, H-4),7.34(1H, d, J ═ 8.6Hz, H-5),6.81(1H, dd, J ═ 8.6,2.3Hz, H-6),6.75(1H, d, J ═ 2.3Hz, H-8) and 6.23(1H, d, J ═ 9.4Hz, H-3); group 2 continuous oxygen methylene proton signal: deltaH 4.06(2H,m,H-3′),3.88(1H,d,J＝8.3Hz,Ha-11') and 3.69(1H, d, J ═ 8.3Hz, H)b-11'). The high field region gives 5 methyl proton signals: deltaH1.62(3H, s, Me-14'), 1.45(3H, d, J ═ 1.7Hz, Me-13'), 1.16(1H, t, J ═ 7.6Hz, Me-3 "), 1.12(3H, s, Me-15') and 0.91(3H, d, J ═ 7.0Hz, Me-12').13C NMR(150MHz,CDCl3) The spectrum shows 27 carbon signals, 9 are characteristic carbon signals delta of 7-O-substituted coumarin parent nucleusC163.1(C-7),161.5(C-2),156.1(C-9),143.7(C-4),128.7(C-5),113.3(C-6),112.9(C-3),112.4(C-10),101.3 (C-8). 15 are carbon signals of sesquiterpene units, comprising a 1-group double-bond carbon signal: deltaC130.3(C-5'),125.4 (C-4'); 2 vicinal oxygen carbon signals: deltaC71.8(C-11'),65.0 (C-3'). 3 are propionyl signals: deltaC174.8(C-1 "), 27.8 (C-2") and 9.4(C-3 "). Since the coumarin nucleus, 1 carbonyl group and 1 double bond together provide 9 unsaturations, the sesquiterpene moiety of the compound is presumed to provideIs a single ring structure. All hydrogen-carbon data were then assigned according to HSQC (tables 1 and 3).
In HMBC spectra, deltaH 1.45(H3-13') and 1.62 (H)3-14') and δC125.4(C-4'), 130.3(C-5'), related to δC65.0(C-3') is irrelevant, suggesting the A-ring cleavage of sesquiterpene units. DeltaH 0.91(H3-12') and δC32.2(C-7 '), 35.0(C-8 '), 40.9(C-9 '), relative, suggesting that Me-12' is attached at the C-8' position; deltaH 1.12(H3-15') and δC35.0(C-8 '), 40.9 (C-9'), 43.1(C-10 '), 71.8(C-11'), suggesting that Me-15 'is attached at the C-9' position; deltaH 1.16(H3-3 ") and δC27.8(C-2 '), 174.8 (C-1'), deltaH4.06(H-3') and δC174.8(C-1 ') indicate that propionyl is attached at the C-3' position. Further, δH 3.88(Ha-11') and 3.69 (H)b11') and deltaCRemote correlation at 163.1(C-7) suggested that the sesquiterpene fragment was linked to the C-7 position of the coumarin nucleus via an ether linkage.
In NOESY spectrum, H3-12' and Hb11 'related, H-10' with Ha11' correlation, H3-15' and H2-1 'correlation, suggesting Me-12' and Ha,bThe-11 'is in the β -orientation, the H-1' and Me-15 'are in the α -orientation, and the relative configuration of the chiral centers is presumed to be 8' S,9'S,10' S. By comparing the measured ECD and calculated ECD data, the absolute configuration of compound 6 was determined to be 8' S,9' S,10' S. The compound is a new compound which is not reported in the literature through searching and is named as (8' S,9' S,10' S) -propionyl fekrynol.
The structural identification data of sesquiterpene coumarin 7 are as follows:
yellow oil (MeOH), HRESIMS gave the excimer peak M/z 559.3387[ M + Na ]]+:(calcd.559.3399 for C34H48O5Na) and the molecular formula thereof is presumed to be C34H48O5The hydrogen and carbon spectra data are similar to compound 6, except that the substituent at the C-3' position is different. By comparing 1D NMR spectrum data of Compound 7 with that of Compound 4 (see tables 1 to 3), it was found that the substituent at the C-3' position of Compound 7 was present together with CompoundThe hydrogen spectrum and carbon spectrum data of the substituent at the C-10' position of 4 are the same.1H NMR(600MHz,CDCl3) In the spectrum, the low field region shows 5 proton signals characteristic of the 7-O-substituted coumarin parent nucleus: deltaH7.62(1H, d, J ═ 9.5Hz, H-4),7.34(1H, d, J ═ 8.6Hz, H-5),6.81(1H, dd, J ═ 8.6,2.2Hz, H-6),6.75(1H, d, J ═ 2.2Hz, H-8) and 6.22(1H, d, J ═ 9.5Hz, H-3); group 1 alkene hydrogen signals: deltaH5.42(1H, m, H-5') and deltaH5.34(1H, m, H-4 "); group 2 continuous oxygen methylene proton signal: deltaH 4.06(2H,m,H-3′),3.88(1H,d,J＝8.3Hz,Ha-11') and 3.69(1H, d, J ═ 8.3Hz, H)b-11'). The high field region gives 5 methyl proton signals: deltaH1.62(3H, s, Me-14'), 1.45(3H, d, J ═ 1.5Hz, Me-13'), 1.11(3H, s, Me-15'), 0.90(3H, d, J ═ 7.0Hz, Me-12') and 0.88(1H, t, J ═ 7.1Hz, Me-10 ").13C NMR(150MHz,CDCl3) The spectrum shows 34 carbon signals, 9 are characteristic carbon signals delta of 7-O-substituted coumarin parent nucleusC163.1(C-7),161.4(C-2),156.1(C-9),143.6(C-4),128.7(C-5),113.3(C-6),112.9(C-3),112.4(C-10),101.3 (C-8). 15 are carbon signals of sesquiterpene units, comprising a 1-group double-bond carbon signal: deltaC130.3(C-5'),125.3 (C-4'); 2 vicinal oxygen carbon signals: deltaC71.9(C-11'),65.1 (C-3'). 10 are decenoate fragments: contains 1 set of double bond carbon signals: deltaC131.7(C-5 "), 127.5 (C-4"). All hydrogen carbon data were assigned according to HSQC (tables 1 and 3).
In HMBC spectra, deltaH4.06(H-3') and δC173.5(C-1 ') indicating that the decenoate fragment is attached at the C-3' position of the sesquiterpene unit. DeltaH 3.88(Ha-11') and 3.69 (H)b11') and deltaCRemote association of 163.1(C-7) suggested that the sesquiterpene fragment was linked to the C-7 position of the coumarin nucleus via an ether linkage. In NOESY spectrum, H3-12' and Hb11 'related, H-8' with H3-15' correlation, H3-15' and Ha,b-1 'correlation, suggesting Me-12' and Ha,b11' is in the beta-orientation, Ha,b-1' and Me-15' are in α -orientation, presumably with relative configuration of chiral centers at 8' S,9' S,10' S. By comparing the measured ECD and calculated ECD data, the identity of Compound 7 was determinedThe absolute configuration is 8' S,9' S,10' S. The compound is a new compound which is not reported in the literature through searching and is named as (8' S,9' S,10' S) -ferusingensine F.
The structural identification data of sesquiterpene coumarin 8 are as follows:
yellow oil (MeOH), HRESIMS gave the excimer peak M/z 419.1832[ M + Na ]]+:(calcd.419.1834 for C24H28O5Na) and the molecular formula thereof is presumed to be C24H28O5Indicating that there are 11 unsaturations in the structure.1H NMR(600MHz,CDCl3) In the spectrum, the low field region shows 5 proton signals characteristic of the 7-O-substituted coumarin parent nucleus: deltaH7.64(1H, d, J ═ 9.5Hz, H-4),7.35(1H, d, J ═ 8.6Hz, H-5),6.84(1H, dd, J ═ 8.6,2.3Hz, H-6),6.81(1H, d, J ═ 2.3Hz, H-8) and 6.25(1H, d, J ═ 9.5Hz, H-3); 3 double bond hydrogen signals: deltaH6.64(1H, d, J ═ 10.2Hz, H-1'), 5.82(1H, d, J ═ 10.2Hz, H-2') and 5.51(1H, t, J ═ 6.6Hz, H-9 '); group 1 continuous oxygen methylene proton signal: deltaH4.60(2H, m, H-11'). The high field region gives 4 methyl proton signals: deltaH1.80(3H, s, Me-12'), 1.37(3H, s, Me-15'), 1.18(3H, s, Me-13') and 1.04(3H, s, Me-14').13C NMR(150MHz,CDCl3) The spectrum shows 24 carbon signals, 9 are characteristic carbon signals delta of 7-O-substituted coumarin parent nucleusC162.1(C-7),161.4(C-2),156.0(C-9),143.6(C-4),128.9(C-5),113.4(C-6),113.2(C-3),112.7(C-10),101.7 (C-8); the remaining 15 are carbon signals of sesquiterpene units, containing 2 sets of double bond carbon signals: deltaC154.8(C-1'),125.1(C-2'), and δC142.9(C-8'),119.3 (C-9'); 1 carbonyl carbon signal: deltaC204.2 (C-3'); 2 vicinal oxygen carbon signals: deltaC72.1(C-10'),65.4 (C-11'). Since 10 unsaturations are provided by the coumarin parent nucleus (7 unsaturations), 1 carbonyl group and 2 double bonds, the compound sesquiterpene moiety is presumed to be a monocyclic structure. All hydrogen-carbon data were then assigned according to HSQC (tables 1 and 3).
In HMBC spectra, deltaH6.64(H-1') and δC204.2(C-3') correlation, δH5.82(H-2') and δC46.2 (C-4') correlation, suggesting the presence of alpha, beta-unsaturated ketone fragments；δH 1.18(H3-13') and 1.04 (H)3-14') and δC204.2(C-3'), 46.2 (C-4'), 53.2(C-5 '), relative, suggesting that Me-13', Me-14 'is attached at the C-4' position; deltaH 1.37(H3-15') and δC154.8(C-1'), 72.1(C-10'), 53.2(C-5 '), related to δC65.4(C-11') is irrelevant, suggesting that Me-15' is connected at C-10' position and B ring of sesquiterpene unit is cleaved; deltaH 1.80(H3-12') and δC40.5(C-7 '), 142.9(C-8'),119.3(C-9'), indicating that Me-12' is attached at the C-8' position; deltaH 4.60(H211') and deltaC142.9(C-8'),119.3(C-9') are related, suggesting that another double bond is located at C-8 '/C-9'. Further, δH 4.60(H211') and deltaC162.1(C-7) remote correlation suggests that the sesquiterpene fragment is linked to the C-7 position of the coumarin nucleus via an ether linkage.
In NOESY spectrum, H3-14' and H3-15', H-5' related, H26 'and H-1', H3-13' correlation, suggesting that Me-14', Me-15' and H-5 ' are in α -orientation, Me-13' and-OH are in β -orientation, assuming relative configuration of chiral centers 5' S,10' R. By comparing the observed and calculated ECD data, the absolute configuration of compound 8 was determined to be 5'S,10' R. The compound is a new compound which is not reported in the literature through searching and is named as (5'S,10' R) -ferusingensine G.
The structural identification data of sesquiterpene coumarin 9 are as follows:
yellow oil (MeOH), HRESIMS gave the excimer peak M/z 383.2216[ M + H ]]+:(calcd.383.2222for C24H31O4) The molecular formula is presumed to be C24H30O4This suggests that there are 10 unsaturations in the structure.1H NMR(600MHz,CDCl3) In the spectrum, the low field region shows 5 proton signals characteristic of the 7-O-substituted coumarin parent nucleus: deltaH7.61(1H, d, J ═ 9.4Hz, H-4),7.32(1H, d, J ═ 8.6Hz, H-5),6.83(1H, dd, J ═ 8.6,2.3Hz, H-6),6.75(1H, d, J ═ 2.3Hz, H-8) and 6.22(1H, d, J ═ 9.4Hz, H-3); 1 double bond hydrogen signal: deltaH5.43(1H, t, J ═ 3.8Hz, H-6'); group 2 continuous oxygen methylene hydrogen signal: deltaH 3.66(1H,dt,J＝12.5,3.2Hz,Ha-3′),3.32(1H,m,Hb-3′),3.81(1H,d,J＝8.3Hz,Ha-11') and 3.76(1H, d, J ═ 8.3Hz, H)b-11'); the high field region gives 4 methyl proton signals: deltaH1.31(3H, s, Me-14'), 1.19(3H, s, Me-15'), 0.98(3H, J ═ 7.9Hz, Me-12'), and 0.97(3H, s, Me-13').13C NMR(150MHz,CDCl3) The spectrum shows 24 carbon signals, 9 are characteristic carbon signals delta of 7-O-substituted coumarin parent nucleusC163.0(C-7),161.5(C-2),156.1(C-9),143.6(C-4),128.7(C-5),113.5(C-6),112.9(C-3),112.4(C-10),100.9 (C-8); 15 are carbon signals of sesquiterpene units, comprising a 1-group double-bond carbon signal: deltaC148.7(C-5'),120.3 (C-6'); 3 continuous oxygen carbon signal deltaC78.0(C-4'),71.6(C-11'),64.3(C-3'), combined with the hydrogen profile suggested that C-4' is 1 vicinal quaternary carbon signal in sesquiterpene fragments, except C-3 'and C-11'. Since the coumarin parent nucleus and 1 double bond together provide 8 unsaturations, the structure has the remaining 2 unsaturations, and the sesquiterpene part of the compound is supposed to be a bicyclic structure. All hydrogen carbon data were assigned according to HSQC (tables 1 and 3).
In HMBC spectra, deltaH 1.31(H3-14') and 0.97 (H)3-13') and δC78.0(C-4'), related to δC64.3(C-3') is irrelevant, δH 3.66(Hα-3') and δH 3.32(Hβ-3') and δC78.0(C-4') is relevant, indicating that C-3'/C-4 'are linked by an ether linkage, while Me-13', Me-14 'are linked at the C-4' position; deltaH 1.19(H3-15') and δC30.8(C-8 '), 38.7 (C-9'), 41.2(C-10 '),71.6(C-11'), suggesting that Me-15 'is attached at the C-9' position; deltaH 0.98(H3-12') and δC33.0(C-7 '), 30.8(C-8 '), and 48.7(C-9 '), indicating that Me-12' is attached at the C-8' position; deltaH 3.81(Ha-11') and 3.76 (H)b11') and deltaC163.0(C-7) suggests that the sesquiterpene fragment is linked to the C-7 position of the coumarin nucleus via an ether linkage.
In NOESY spectrum, H3-12' and Hb11 'related, H-10' with Ha-11′,H3-12' correlation with H3-15′In relation to H-8 ', it is suggested that Me-12', H211', H-10' is in the beta-orientation, H-8 ', Me-15' is in the alpha-orientation; h-10' and H3-13 'correlation, suggesting that Me-13' is β -oriented and Me-14 'is α -oriented, assuming relative configuration of chiral centers at 8' S,9'S,10' R. By comparing the measured ECD and calculated ECD data, the absolute configuration of compound 9 was determined to be 8' S,9' S,10' R. The compound is a new compound which is not reported in the literature through searching and is named as (8' S,9' S,10' R) -ferusingensine H.
The structural identification data of sesquiterpene coumarin 10 are as follows:
white amorphous powder (MeOH), HRESIMS gave the excimer ion peak M/z 449.2301[ M + Na [ ]]+:(calcd.449.2226 for C26H34O5Na) and the molecular formula thereof is presumed to be C26H34O5This suggests that there are 10 unsaturations in the structure.1H NMR(600MHz,CDCl3) In the spectrum, the low field region shows 5 proton signals characteristic of the 7-O-substituted coumarin parent nucleus: deltaH7.62(1H, d, J ═ 9.4Hz, H-4),7.35(1H, d, J ═ 8.6Hz, H-5),6.83(1H, dd, J ═ 8.6,2.3Hz, H-6),6.79(1H, d, J ═ 2.3Hz, H-8) and 6.23(1H, d, J ═ 9.4Hz, H-3); group 1 continuous oxygen methylene proton signal: 3.72(1H, d, J ═ 8.7Hz, H)a-11') and 3.65(1H, d, J ═ 8.7Hz, H)b-11'); 1 continuous oxygen methine hydrogen signal: deltaH4.67(1H, td, J ═ 11.1,5.1Hz, H-3'); the high field region gives 5 methyl proton signals: deltaH2.04(3H, s, Me-2 ") 1.06(3H, s, Me-15'), 0.94(3H, d, J ═ 7.1Hz, Me-12'), 0.91(3H, s, Me-14') and 0.80(3H, d, J ═ 6.6Hz, Me-13').13C NMR(150MHz,CDCl3) The spectrum shows 26 carbon signals, 9 are characteristic carbon signals delta of 7-O-substituted coumarin parent nucleusC162.6(C-7),161.4(C-2),156.0(C-9),143.5(C-4),128.8(C-5),113.1(C-6),113.1(C-3),112.6(C-10),101.6 (C-8). 15 carbon signals for sesquiterpene units, including 2 vicinal carbon signals: deltaC76.1(C-11'),74.7 (C-3'). The 2 carbon signal is an acetyl signal: deltaC171.1(C-1 "), 21.5 (C-2"). Since the coumarin parent nucleus and 1 carbonyl group provide 8 unsaturations, the sesquiterpene moiety of the compound is suggested to be a bicyclic structure. Sorting all hydrogen and carbon data according to HSQCGenus (tables 1 and 3).
In HMBC spectra, deltaH 0.80(H3-13') and δC74.7(C-3'), 49.9(C-4 '), 38.3(C-5 '), indicating that Me-13' is attached at the C-4' position; deltaH 0.91(H3-14') and δC49.9(C-4 '), 38.3 (C-5'), 32.5(C-6 '), 44.5 (C-10'), indicating that Me-14 'is attached at the C-5' position; deltaC 0.94(H3-12') and δC25.3(C-7 '), 35.5(C-8 '), 39.2(C-9 '), indicating that Me-12' is attached at the C-8' position; deltaH 1.12(H3-15') and δC35.5(C-8 '), 39.2 (C-9'), 44.5(C-10 '), 76.1(C-11'), suggesting that Me-15 'is attached at the C-9' position; deltaH 2.04(H3-2 ") and 4.67(H-3') and deltaC171.1(C-1 ') indicates that the acetyl group is attached at the C-3' position. Further, δH 3.72(Ha-11') and 3.65 (H)b11') and deltaC162.6(C-7) remote correlation suggests that the sesquiterpene fragment is linked to the C-7 position of the coumarin nucleus via an ether linkage.
In NOESY spectrum, H3-14 'and H-3', Hβ-7′,H315' related, H-10' with H-4 ', Ha,b-11′,H3-12' correlation, suggesting that Me-13', Me-14', Me-15' and H-3' are in the β -orientation, H-10' and Me-12' are in the α -orientation, and the relative configuration of the chiral centers is presumed to be 3' R,4' R,5' S,8' R,9' R,10' R. By comparing the measured ECD and the calculated ECD data, the absolute configuration of compound 10 was determined to be 3'R,4' R,5'S,8' R,9'R,10' R. The compound is a new compound which is not reported in the literature through searching and is named as (3'R,4' R,5'S,8' R,9'R,10' R) -kamolol acetate.
The NMR data of the sesquiterpene coumarins 1-10 are shown in tables 1-3.
TABLE 1 carbon Spectroscopy data (150MHz, CDCl) for Compounds 1-103)
Figure BDA0002518235580000131
TABLE 2 Hydrogen spectra data (600MHz, CDCl) for compounds 1-53)
Figure BDA0002518235580000141
a Overlapped resonances.
TABLE 3 Hydrogen spectra data (600MHz, CDCl) for compounds 6-103)
Figure BDA0002518235580000151
a Overlapped resonances.
Example 2
(1) Extracting 1500g of resina Ferulae with 95% ethanol under reflux for 3 times (30L), and recovering the crude extract under reduced pressure;
(2) subjecting the 95% ethanol crude extract obtained in the step (1) to silica gel column chromatography, and performing gradient elution with chloroform-acetone mixed solvent 100:0, 100:5, 10:1, 5:1 to obtain eluates with different polarity parts;
(3) separating the eluate of the chloroform-acetone mixed solvent 100: 0-100: 5 in the step (2) by silica gel column chromatography, and eluting with petroleum ether-ethyl acetate mixed solvent 100:4, 100:6, 100:8, 10:1, 7:1, 5:1, 2:1 and 1:1 in sequence;
(4) subjecting the petroleum ether-ethyl acetate 100: 6-2: 1 flow obtained in the step (3) to ODS chromatography, and performing gradient elution by using a mixed solvent of methanol-water of 50:50, 65:35, 80:20 and 90: 10;
(5) and (3) separating the 80: 20-90: 10 fraction of the methanol-water obtained in the step (4) by HPLC RID-10A chromatography at a flow rate of 3.5mL/min, wherein the mobile phase is methanol: water 90:10 to give sesquiterpene coumarin 3 (t)R90min) (yield 0.0003%), sesquiterpene coumarin 4 (t)R68min) (yield 0.0033%), sesquiterpene coumarin 5 (t)R6min) (yield 0.0009%), sesquiterpene coumarin 6 (t)R18min) (yield 0.0010%), sesquiterpene coumarin 7 (t)R53min) (yield 0.0015%), sesquiterpene coumarin 9 (t)R15min) (yield 0.0004%).
(6) 90:10 stream of methanol-water obtained in step (4) aboveSeparating by HPLC RID-10A chromatography at flow rate of 3mL/min, and mobile phase of methanol: water 80:20 to give sesquiterpene coumarin 2 (t)R28min) (yield 0.0002%).
(7) Separating the 80:20 fractions of methanol-water obtained in the step (4) by HPLC RID-10A chromatography at a flow rate of 3.5mL/min, wherein the mobile phase is methanol: water 80:20 to give sesquiterpene coumarin 1 (t)R34min) (yield 0.0003%), sesquiterpene coumarin 10 (t)R30min) (yield 0.0013%).
(8) And (3) separating the fraction 65: 35-80: 20 of the methanol-water obtained in the step (4) by HPLC-UV chromatography at a flow rate of 3mL/min, wherein the mobile phase is methanol: water 70:30 to yield sesquiterpene coumarin 8 (t)R26min) (yield 0.0005%).
The structural identification of sesquiterpene coumarins 1-10 is shown in example 1.
Example 3
(1) Heating and ultrasonically extracting 2000g of resina Ferulae with dichloromethane for 4 times (the dosage is 30L), and recovering the crude extract of the extractive solution under reduced pressure;
(2) performing silica gel column chromatography on the dichloromethane crude extract obtained in the step (1), and performing gradient elution by using a petroleum ether-acetone mixed solvent of 100:5, 10:1, 5:1 and 2:1 to obtain eluates of different polarity parts;
(3) separating the eluate of the petroleum ether-acetone mixed solvent 100:5 in the step (2) by silica gel column chromatography, and sequentially eluting with petroleum ether-ethyl acetate mixed solvent 100:3, 100:5, 100:8, 10:1, 6:1, 2:1, 0: 1;
(4) subjecting the petroleum ether-ethyl acetate 100: 5-2: 1 flow obtained in the step (3) to ODS chromatography, and performing gradient elution by using acetonitrile-water mixed solvent of 40:60, 60:40, 70:30, 80:20 and 90: 10;
(5) and (3) separating the acetonitrile-water fraction of 80: 20-90: 10 obtained in the step (4) by HPLC-UV chromatography at a flow rate of 4mL/min, wherein a mobile phase is methanol: water 90:10 to give sesquiterpene coumarin 3 (t)R91min) (yield 0.0003%), sesquiterpene coumarin 4 (t)R67min) (yield 0.0031%) sesquiterpene coumarin 5 (t)R= 5min) (yield 0.0008%), sesquiterpene coumarin 6 (t)R15min) (yield 0.0008%), sesquiterpene coumarin 7 (t)R49min) (yield 0.0013%), sesquiterpene coumarin 9 (t)R8min) (yield 0.0003%).
(6) And (3) separating the acetonitrile-water fraction of 70: 30-80: 20 obtained in the step (4) by HPLC RID-10A chromatography at a flow rate of 3.5mL/min, wherein the mobile phase is methanol: water 80:20 to give sesquiterpene coumarin 1 (t)R36min) (yield 0.0002%), sesquiterpene coumarin 2 (t)R23min) (yield 0.0002%), sesquiterpene coumarin 10 (t)R30min) (yield 0.0011%).
(7) And (3) separating the acetonitrile-water fraction of 40: 60-60: 40 obtained in the step (4) by HPLC RID-10A chromatography at a flow rate of 3.5mL/min, wherein the mobile phase is methanol: water 70:30 to yield sesquiterpene coumarin 8 (t)R20min) (yield 0.0004%).
The structural identification of sesquiterpene coumarins 1-10 is shown in example 1.
Example 4
(1) Extracting 500g resina Ferulae with chloroform under reflux for 3 times (10L), and recovering the crude extract under reduced pressure;
(2) performing silica gel column chromatography on the chloroform crude extract obtained in the step (1), and performing gradient elution by using a dichloromethane-ethyl acetate mixed solvent of 100:0, 100:5, 10:1, 5:1 and 2:1 to obtain eluates of different polarity parts;
(3) separating the eluate of the dichloromethane-ethyl acetate mixed solvent 100: 0-100: 5 in the step (2) by silica gel column chromatography, and eluting with petroleum ether-ethyl acetate mixed solvent 100:3, 100:5, 100:8, 10:1, 8:1, 6:1, 4:1, 2:1, 0:1 in sequence;
(4) subjecting the petroleum ether-ethyl acetate 100: 5-2: 1 flow obtained in the step (3) to ODS chromatography, and performing gradient elution by using a mixed solvent of methanol-water of 50:50, 60:40, 80:20 and 90: 10;
(5) and (3) separating the 80: 20-90: 10 fraction of the methanol-water obtained in the step (4) by HPLC RID-10A chromatography at a flow rate of 3.5mL/min, wherein the mobile phase is methanol: water 90:10 to give sesquiterpene coumarin 3 (t)R97min) (yield 0.0003%), sesquiterpene coumarin 4 (t)R74min) (yield 0.0027%), sesquiterpene coumarin 5 (t)R4min) (yield 0.0009%), sesquiterpene coumarin 6 (t)R19min) (yield 0.0009%), sesquiterpene coumarin 7 (t)R53min) (yield 0.0015%), sesquiterpene coumarin 9 (t)R9min) (yield 0.0004%).
(6) Separating the 90:10 fraction of methanol-water obtained in the step (4) by HPLC RID-10A chromatography at a flow rate of 4mL/min, wherein the mobile phase is methanol: water 80:20 to give sesquiterpene coumarin 1 (t)R34min) (yield 0.0003%), sesquiterpene coumarin 2 (t)R18min) (yield 0.0003%), sesquiterpene coumarin 10 (t)R27min (yield 0.0012%).
(7) Separating the 80:20 fractions of methanol-water obtained in the step (4) by HPLC-UV chromatography at a flow rate of 3.5mL/min, wherein the mobile phase is methanol: water 70:30 to yield sesquiterpene coumarin 8 (t)R19min) (yield 0.0005%).
The structural identification of sesquiterpene coumarins 1-10 is shown in example 1.
Example 5
(1) Heating and reflux-extracting 2500g resina Ferulae with 90% methanol for 4 times (25L), and recovering the crude extract under reduced pressure;
(2) subjecting the 90% methanol crude extract obtained in the step (1) to silica gel column chromatography, and performing gradient elution with chloroform-ethyl acetate mixed solvent 100:0, 100:5, 10:1, 5:1, 2:1 to obtain eluates with different polarity parts;
(3) separating the eluate of the chloroform-ethyl acetate mixed solvent 100: 0-100: 5 in the step (2) by silica gel column chromatography, and eluting with petroleum ether-acetone mixed solvent 100:2, 100:4, 100:6, 100:8, 10:1, 8:1, 5:1, 2:1 and 0:1 in sequence;
(4) subjecting the petroleum ether-acetone 100: 6-2: 1 flow obtained in the step (3) to ODS chromatography, and performing gradient elution by using a mixed solvent of methanol and water, wherein the mixed solvent is 50:50, 60:40, 70:30, 80:20 and 90: 10;
(5) and (3) separating the 80: 20-90: 10 fraction of the methanol-water obtained in the step (4) by HPLC RID-10A chromatography at a flow rate of 4mL/min, wherein the mobile phase is acetonitrile: water 80:20 to obtain sesquiterpeneCoumarin 3 (t)R93min) (yield 0.0003%), sesquiterpene coumarin 4 (t)R70min) (yield 0.0030%), sesquiterpene coumarin 5 (t)R4min) (yield 0.0009%), sesquiterpene coumarin 6 (t)R16min) (yield 0.0008%), sesquiterpene coumarin 7 (t)R57min) (yield 0.0016%), sesquiterpene coumarin 9 (t)R8min) (yield 0.0005%).
(6) Separating the 90:10 fractions of methanol-water obtained in the step (4) by HPLC RID-10A chromatography at a flow rate of 3mL/min, wherein the mobile phase is acetonitrile: water 70:30 to yield sesquiterpene coumarin 2 (t)R20min) (yield 0.0002%).
(7) Separating the 80: 20-90: 10 fraction of the methanol-water obtained in the step (4) by HPLC RID-10A chromatography at a flow rate of 3.5mL/min, wherein the mobile phase is acetonitrile: water 65:35 to give sesquiterpene coumarin 1 (t)R38min) (yield 0.0003%), sesquiterpene coumarin 10 (t)R30min) (yield 0.0011%).
(8) Separating the methanol-water fraction obtained in the step (4) with a ratio of 70: 30-80: 20 by HPLC RID-10A chromatography at a flow rate of 4mL/min, wherein the mobile phase is acetonitrile: water 55:45 to give sesquiterpene coumarin 8 (t)R16min) (yield 0.0005%).
The structural identification of sesquiterpene coumarins 1-10 is shown in example 1.
Example 6
(1) Extracting 1000g resina Ferulae with 90% ethanol under reflux for 4 times (15L), and recovering the crude extract under reduced pressure;
(2) subjecting the 90% ethanol crude extract obtained in the step (1) to silica gel column chromatography, and performing gradient elution with dichloromethane-acetone mixed solvent 100:0, 100:5, 10:1, 5:1, 2:1 to obtain eluates with different polarity parts;
(3) separating the eluate of the dichloromethane-acetone mixed solvent 100: 0-100: 5 in the step (2) by silica gel column chromatography, and eluting with petroleum ether-ethyl acetate mixed solvent 100:0, 100:3, 100:5, 100:8, 10:1, 8:1, 5:1, 2:1, 1:1 in sequence;
(4) subjecting the petroleum ether-ethyl acetate 100: 5-1: 1 flow obtained in the step (3) to ODS chromatography, and performing gradient elution by using a mixed solvent of methanol and water, wherein the mixed solvent is 50:50, 60:40, 70:30, 80:20, and 90: 10;
(5) separating the 80: 20-90: 10 fraction of the methanol-water obtained in the step (4) by HPLC-UV chromatography at a flow rate of 3mL/min, wherein the mobile phase is methanol: water 90:10 to give sesquiterpene coumarin 3 (t)R105min) (yield 0.0002%), sesquiterpene coumarin 4 (t)R83min) (yield 0.0031%), sesquiterpene coumarin 5 (t)R= 11min (yield 0.0009%), sesquiterpene coumarin 6 (t)R27min) (yield 0.0008%), sesquiterpene coumarin 7 (t)R71min) (yield 0.0013%), sesquiterpene coumarin 9 (t)R15min) (yield 0.0003%).
(6) Separating the 80: 20-90: 10 fraction of the methanol-water obtained in the step (4) by HPLC-UV chromatography at a flow rate of 3mL/min, wherein the mobile phase is methanol: water 80:20 to give sesquiterpene coumarin 1 (t)R43min) (yield 0.0003%), sesquiterpene coumarin 2 (t)R25min) (yield 0.0002%), sesquiterpene coumarin 10 (t)R37min) (yield 0.0011%).
(7) Separating and preparing the 70:30 fractions of the methanol-water obtained in the step (4) by HPLC-UV chromatography, wherein the flow rate is 3mL/min, and the mobile phase is methanol: water 70:30 to yield sesquiterpene coumarin 8 (t)R25min) (yield 0.0004%).
The structural identification of sesquiterpene coumarins 1-10 is shown in example 1.
EXAMPLE 7 anti-neuritic Activity test of the New sesquiterpene coumarins 1-10 prepared in examples 1-6
(1) The experimental principle is as follows:
chronic neuroinflammation mediated by excessive microglial activation plays a key role in the development and progression of neurodegenerative diseases. Under resting state, microglia can eliminate metabolic products in brain and maintain brain tissue homeostasis. When the central nervous system is damaged (e.g., inflammation), microglia are activated and over-activated, producing large amounts of inflammatory factors that damage neurons. Meanwhile, the inflammatory factors further activate microglia, so that a malignant cycle is formed in the brain, and finally, the neurodegenerative disease is generated and further deepened. Therefore, inhibition of activation of neuroinflammation mediated by activation of microglial activity is an effective strategy for prevention and treatment of neurodegenerative diseases. According to the invention, the anti-inflammatory activity of the new sesquiterpene coumarins 1-10 is evaluated by constructing a screening model of abnormal activation of BV2 microglia activated by in vitro LPS and taking NO released by activated microglia as an index.
(2) The experimental method comprises the following steps:
firstly, culturing mouse microglia line BV-2
All glassware and metal instruments (culture bottles, pipettes, solution bottles, etc.) used in cell culture and model building were autoclaved at 121 ℃ for 30min to completely remove the contaminated LPS. A cell culture medium containing 10% fetal bovine serum was prepared on the basis of DMEM medium. The ratio of microglia is about 2.0X 105cells/mL at 5% CO2And subculturing in a culture bottle at 37 ℃, wherein the adherent cells account for about 70-80% of the bottom area of the culture bottle by the third day, digesting the adherent cells by pancreatin, and subculturing to another culture bottle. BV2 thawed in a refrigerator at the ultralow temperature of-80 ℃ is taken as the first generation, and BV2 cells of 3 th to 8 th generations are selected for experiments.
② process for preparing medicine
Test compounds were all solid and dissolved in DMSO. The stock solution was prepared at a concentration of 100mM and stored at-20 ℃. It was diluted with DMDM medium at the time of use to 100. mu.M, 30. mu.M, 10. mu.M and 1. mu.M in this order. The final concentration of DMSO is less than 1 ‰.
③ Griess method for detecting inhibition of compound to LPS activated microglia
Taking BV2 microglia in logarithmic growth phase, adjusting the cell density to 2.0 × 10 by using fresh DMDM culture medium containing 10% fetal calf serum5cells/mL, seeded in 96-well plates at 100. mu.L/well at 37 ℃ in 5% CO2Culturing in the incubator. And replacing the cells with serum-free fresh culture solution after 24 hours of adherent culture, and simultaneously adding drugs. Each compound was administered at a dose of 100. mu.M, 30. mu.M, 10. mu.M, 1. mu.M in combination with LPS. Blank control was also set. Final concentration of LPS in each administration groupIs 100 ng/mL. Continuously culturing for 24h after adding medicine into cells, collecting supernatant, and detecting NO in the supernatant by Griess colorimetric method2 -And (4) content.
MTT method for detecting influence of compound on survival rate of microglia cell
Taking BV2 microglia cultured in logarithmic growth phase, adjusting cell density to 2.0 × 10 by using fresh DMDM culture medium containing 10% fetal calf serum5cells/mL, seeded in 96-well plates at 100. mu.L/well at 37 ℃ in 5% CO2Culturing in the incubator. After the cells are cultured for 24 hours adherent, the cells are changed into fresh culture solution, and meanwhile, the cells are treated by adding medicine. Each compound was administered at a dose of 100. mu.M, 30. mu.M, 10. mu.M, 1. mu.M in combination with LPS. Blank control was also set. The final concentration of LPS in each administration group was 100 ng/mL. After adding the drug, the cells were cultured for 24h, MTT solution, 10. mu.L/well, was added to the cell fluid, the cells were incubated with 0.25mg/mL MTT at 37 ℃ for 3h, the culture fluid was aspirated, and 150. mu.L DMSO solution was added to determine the OD value of the optical density. And (3) processing data, namely processing the data by using corresponding software of a microplate reader, calculating an average value of OD values of 3 holes of each sample, and calculating the Cell viability (CV%) by using the average value according to the following formula.
Percent cell survival%
Fifthly, statistical method
All data were examined and analyzed using the SPSS statistical software package. Results are expressed as mean ± standard error, and the global differences were evaluated, and the means between groups was analyzed by One-Way ANOVA analysis for homogeneity of variance and by Dunnett's test analysis for comparison between groups. The multiple sample homogeneity of variance test was conducted using a Leven test, where the variances were uniform when p >0.05, the differences in mean among the groups were tested using Dunnett's two-sided T, and the differences in mean among the groups were tested using Dunnett T3 when p <0.05 and the variances were not uniform.
⑥IC50Is calculated by
Calculating IC by nonlinear regression fitting of parameters such as each dosage and inhibition rate50。
(3) The experimental results are as follows: see Table 4
TABLE 4 sesquiterpene coumarins 1-10 Experimental results for inhibiting microglial cell activation
Figure BDA0002518235580000211
Significance:*P<0.05,**P<0.01,***P<0.001 compared to LPS-induced group;###P<0.001 compared to the control group.
As a result, the new sesquiterpene coumarin compounds 1 (30. mu.M, 100. mu.M), 3 (100. mu.M), 4 (100. mu.M), 5 (100. mu.M), 6 (30. mu.M, 100. mu.M), 7 (10. mu.M, 30. mu.M, 100. mu.M), 8 (1. mu.M, 10. mu.M, 30. mu.M, 100. mu.M), 9 (30. mu.M, 100. mu.M) and 10 (100. mu.M) prepared in examples 1 to 6 were able to significantly inhibit the release of LPS-induced over-activated BV2 microglia NO.

Claims (10)

1. The sesquiterpene coumarin compound and pharmaceutically acceptable salts and isomers thereof have the following structural general formula:
Figure FDA0002518235570000011
R1is hydrogen, hydroxy, C1-C4 acyloxy, fragment A or fragment B;
R2is hydrogen or hydroxy;
R3is hydroxy, C1-C4 acyloxy or fragment B;
R4is hydrogen, C1-C4 alkyl or hydroxy;
R5is hydrogen, C1-C4 alkyl or hydroxy;
R6is hydrogen or hydroxy;
R7is hydrogen or hydroxy.
2. The sesquiterpene coumarins of claim 1, and pharmaceutically acceptable salts, isomers thereof, wherein,
R1is hydrogen, hydroxy, acetoxy, propionyloxy, fragment A or fragment B;
R2is hydrogen or hydroxy;
R3is hydroxy, acetoxy, propionyloxy or fragment B;
R4is hydrogen, methyl or hydroxy;
R5is hydrogen, methyl or hydroxy;
R6is hydrogen or hydroxy;
R7is hydrogen or hydroxy.
3. The sesquiterpene coumarin compound and pharmaceutically acceptable salts and isomers thereof are as follows:
Figure FDA0002518235570000021
4. the process for the preparation of sesquiterpene coumarins according to claim 3, and pharmaceutically acceptable salts and isomers thereof, comprising the steps of:
(1) extracting resina Ferulae with solvent such as methanol, ethanol, chloroform or dichloromethane, and recovering extractive solution to obtain crude extract;
(2) separating the crude extract obtained in the step (1) by silica gel column chromatography, and performing gradient elution by using a petroleum ether-ethyl acetate mixed solvent, or a petroleum ether-acetone mixed solvent, or a dichloromethane-ethyl acetate mixed solvent, or a dichloromethane-acetone mixed solvent, or a chloroform-ethyl acetate mixed solvent, or a chloroform-acetone mixed solvent to obtain eluates with different polarities;
(3) performing ODS column chromatography on the eluate with different polarities obtained in the step (2), and performing gradient elution by using a methanol-water mixed solvent or an acetonitrile-water mixed solvent as a mobile phase;
(4) and (4) further separating the methanol-water or acetonitrile-water eluate obtained in the step (3) by HPLC, and carrying out gradient elution by using a methanol-water mixed solvent or acetonitrile-water mixed solvent as a mobile phase to obtain the sesquiterpene coumarin 1-10.
5. The process for the preparation of sesquiterpene coumarins and pharmaceutically acceptable salts and isomers thereof according to claim 4, wherein: the extraction method in the step (1) is heating reflux extraction or heating ultrasonic extraction for 2-5 times, the volume concentration of methanol and ethanol is 60-100%, and the material-liquid ratio is 1: 5-1: 30 g/mL.
6. The process for the preparation of sesquiterpene coumarins and pharmaceutically acceptable salts and isomers thereof according to claim 4, wherein: the volume ratio of the petroleum ether-ethyl acetate mixed solvent or the petroleum ether-acetone mixed solvent in the step (2) is 100: 0-0: 1; the volume ratio of the dichloromethane-ethyl acetate mixed solvent, or the dichloromethane-acetone mixed solvent, or the chloroform-ethyl acetate mixed solvent, or the chloroform-acetone mixed solvent is 100: 0-1: 1.
7. The preparation method of the sesquiterpene coumarin compound and the pharmaceutically acceptable salts and isomers thereof according to claim 4, wherein the volume ratio of the methanol-water mixed solvent in the step (3) is 50: 50-100: 0, and the volume ratio of the acetonitrile-water mixed solvent is 30: 70-90: 10; .
8. The preparation method of the sesquiterpene coumarin compound and the pharmaceutically acceptable salts and isomers thereof according to claim 4, wherein the volume ratio of the methanol-water mixed solvent in the step (4) is 60: 40-90: 10, and the volume ratio of the acetonitrile-water mixed solvent is 50: 50-80: 20.
9. A pharmaceutical composition comprising the sesquiterpene coumarins of any one of claims 1 to 3, pharmaceutically acceptable salts, isomers thereof and a pharmaceutically acceptable adjuvant, diluent or carrier.
10. Use of the sesquiterpene coumarins of any one of claims 1 to 3 and pharmaceutically acceptable salts, isomers or pharmaceutical compositions of claim 9 for the preparation of a medicament for the prevention or treatment of neurodegenerative disorders.
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