Monocyclic sesquiterpenoids and uses thereof
1. Monocyclic sesquiterpene compounds, isomers or salts thereof:
2. the monocyclic sesquiterpene compound of claim 1, isomer or salt thereof:
3. the method for preparing the monocyclic sesquiterpene compound or the salt thereof of claim 1, wherein the method comprises the following steps:
(1) extracting herba Erigerontis with ethanol under heating and refluxing, mixing extractive solutions, concentrating to obtain extract, and extracting with dichloromethane or chloroform to obtain dichloromethane or chloroform layer and water layer extract;
(2) subjecting the water layer extract to macroporous adsorbent resin column, performing rapid gradient elution with ethanol-water system 50% -80%, and separating into three fractions A, B, C by thin layer silica gel chromatography;
(3) subjecting fraction C to rapid pressure-reducing silica gel column chromatography, performing rapid gradient elution with dichloromethane/chloroform-methanol-water system, and combining by silica gel thin layer chromatography to obtain three fractions C1、C2、C3；
(4) Fraction C1Performing polyamide column chromatography, performing gradient elution with 60% -90% ethanol-water system, and collecting three components C1-1、C1-2、C1-3。
(5) Component C obtained1-1Subjecting to gel column chromatography, isocratic eluting with 60% ethanol water to obtain three groups C1-1-1、C1-1-2、C1-1-3。
(6) Component C1-1-2Performing gradient elution with dichloromethane/chloroform-methanol system 50:1-1:1 by silica gel column chromatography, and mixing by thin layer chromatography and HPLC analysis to obtain five components C1-1-2-1-C1-1-2-5。
(7) Component C obtained1-1-2-2Performing gradient elution by 20% -90% methanol-water by ODS column chromatography to obtain six components C1-1-2-2-1-C1-1-2-2-6。
(8) Fraction C by semi-preparative HPLC1-1-2-2-5Elution was performed with a 25% acetonitrile water system to give a pair of epimers.
4. The method according to claim 3, wherein the ethanol in step (1) is 70-80% industrial ethanol, and is extracted 2-3 times; extracting with dichloromethane or chloroform at a volume ratio of 1:1 for 3-4 times.
5. The method according to claim 3, wherein the volume ratio of dichloromethane/chloroform-methanol-water in the step (3) is 9.0:1.0:0.0-1.0:1.0: 0.8.
6. The method according to claim 3, wherein the dry whole plant of Erigeron breviscapus (Erigeron brevicapus (Vant.) hand. -Mazz.) is dried whole plant of Erigeron breviscapus (Erigeron brevicapus, Compositae).
7. The method of claim 3, wherein the compounds 1a-1d and 2a-2d are obtained using a Daicel Chiralpak AD-H and a Daicel Chiralpak IC chiral chromatography column with n-hexane-isopropanol mixed solvent as the mobile phase in volume ratios of 8:1 and 5:1, respectively.
8. A pharmaceutical composition comprising the monocyclic sesquiterpene compound of claim 1 or 2 or a salt thereof and a pharmaceutically acceptable carrier or excipient.
9. Use of the monocyclic sesquiterpene compound of claim 1 or 2 or a salt thereof or the pharmaceutical composition of claim 8 for the preparation of a medicament against ADP-induced platelet aggregation.
10. Use of the monocyclic sesquiterpene compound or salt thereof of claim 1 or 2 in the preparation of an antithrombotic agent.
Background
Erigeron breviscapus, also known as Erigeron breviscapus, and Erigeron breviscapus, which are dry whole herbs of hand-Mazz, a plant of Compositae, and are mostly found in sparse forests, grassy clusters, or sunny slopes. The traditional Chinese medicine is mainly distributed in southwest areas of Yunnan, Guangxi, Sichuan, Guizhou, Tibet and the like in China, has cold property, pungent, slightly bitter and warm taste, and heart and liver meridians entering, is prepared from whole herbs, has the effects of dissipating heat, relieving exterior syndrome, relaxing meridians and treating paralysis, promoting blood circulation, removing obstruction in channels, relieving pain, dispelling wind and removing cold, and is received in the 2015 edition of Chinese pharmacopoeia. Modern pharmacological research shows that erigeron breviscapus has pharmacological effects of protecting heart and cerebral vessels, protecting nerves, resisting oxidation, resisting cancer and the like.
Erigeron breviscapus is a traditional Chinese medicine and is mainly used for treating cerebral apoplexy and sequela thereof. Since the 80 s in the 20 th century, tablets and injections have been developed for clinically treating cerebral embolism, cerebral thrombosis, paralysis caused by cerebral thrombosis, coronary heart disease, angina pectoris, acute renal failure and the like, and have very definite curative effects clinically. In the present study, scutellarin (scutellarin) and dicaffeoylquinic acid ester compounds are the main active ingredients. The diversity of the structure of the terpene natural product determines the diversity of the biological activity, and we find out a few studies from the terpene natural product to report the important role played by terpene components in cardiovascular diseases. The most effective terpenes include the potential hypertension treatment drugs citronellol, linalol, (+) -nootkatone and caryophyllene oxide, which prevent thrombosis, and triptolide, which prevents or ameliorates myocardial infarction. Some clinical trials have also provided great potential for terpenoid components such as sterol glycosides, ginsenosides and carotenoid, etc., to play in the cardiovascular medicine field. However, the research on the compounds is limited and needs to be further researched.
The invention content is as follows:
in order to overcome the defects of the prior art, the invention provides a series of monocyclic sesquiterpenoids, isomers or salts thereof, which have the following structures:
further, the present invention provides the following monocyclic sesquiterpene compound, isomer or salt thereof:
the monocyclic sesquiterpenoids, isomers or salts thereof are simultaneously separated from Erigeron breviscapus (Vant.) hand-Mazz of Erigeron of Compositae.
The invention provides a preparation method of the monocyclic sesquiterpenoids, the isomers or the salts thereof, which comprises the following steps:
(1) extracting herba Erigerontis with ethanol under heating and refluxing, mixing extractive solutions, concentrating to obtain extract, and extracting with dichloromethane or chloroform to obtain dichloromethane or chloroform layer and water layer extract;
(2) subjecting the water layer extract to macroporous adsorbent resin column, performing rapid gradient elution with ethanol-water system (50% -80%), and separating into three fractions A, B, C by thin layer silica gel chromatography;
(3) subjecting fraction C to rapid vacuum silica gel column chromatography, performing rapid gradient elution with dichloromethane/chloroform-methanol-water system (9.0:1.0:0.0-1.0:1.0:0.8), and mixing by silica gel thin layer chromatography to obtain three fractions C1、C2、C3；
(4) Fraction C1Performing polyamide column chromatography, performing gradient elution with ethanol-water system (60% -90%), and collecting three components C1-1、C1-2、C1-3。
(5) Component C obtained1-1Subjecting to gel column chromatography and isocratic elution with ethanol-water (60:40) to obtain three groups C1-1-1、C1-1-2、C1-1-3。
(6) Component C1-1-2Performing gradient elution with dichloromethane/chloroform-methanol system (50:1-1:1) by silica gel column chromatography, and combining by thin layer chromatography and HPLC analysis to obtain five components C1-1-2-1-C1-1-2-5。
(7) Component C obtained1-1-2-2Performing gradient elution with methanol-water (20% -90%) by ODS column chromatography to obtain six components C1-1-2-2-1-C1-1-2-2-6。
(8) Fraction C by semi-preparative HPLC1-1-2-2-5Elution with a 25% acetonitrile water system gave a pair of epimers 1 and 2, the following planar structures:
wherein the ethanol in the step (1) is 70-80% industrial ethanol, and is extracted for 2-3 times. Extracting with dichloromethane or chloroform at a volume ratio of 1:1 for 3-4 times.
Further, the air conditioner is provided with a fan,
chiral resolution of the pair of epimers 1 and 2 gives the compounds 1a-1d and 2a-2 d. The chiral preparation and resolution method comprises the following steps: by using Daicel Chiralpak AD-H and Daicel Chiralpak IC chiral chromatographic columns, a mobile phase is a n-hexane-isopropanol mixed solvent, the ratio is 8:1 and 5:1(v: v), the flow rate is 2.00 and 0.45mL/min respectively, and the detection wavelength of an ultraviolet detector is 210 nm.
The chiral resolution conditions described in the present invention apply to compounds 1 and 2.
The structure identification result of the compound obtained by the invention is as follows:
the planar structures and relative configurations of compounds 1, 2 were determined using high resolution mass spectrometry, one-dimensional and two-dimensional NMR techniques. And determining the absolute configurations of the optical pure compounds 1a-1d and 2a-2d after splitting by comparing the actually measured ECD with the calculated ECD spectrogram through a Mosher's method.
Compound 1, a clear oily compound, HRESI-MS gave an excimer peak M/z 293.1769[ M + Na ]]+(calcd for C15H26O4293.1723), in combination1H,13C NMR data confirmed its molecular formula to be C15H26O4The unsaturation degree is 3.1H NMR(600MHz,CDCl3) In the spectrum, the low field region is seen as deltaH6.41(1H, dd, J ═ 15.2,10.9,1.8Hz),6.12(1H, d, J ═ 10.9Hz),5.74(1H, d, J ═ 15.2Hz) are intercoupled alkene hydrogen signals, δH4.17(1H, dd, J ═ 7.8,3.5Hz),3.80(1H, t, J ═ 7.2Hz) are the two vicinal oxymethylene hydrogen signals, δH3.65(1H, dd, J ═ 11.2,3.9Hz),3.54(1H, dd, J ═ 11.2,7.8Hz) is a vicinal oxymethylene hydrogen signal. 4 methyl hydrogen signals delta can be seen in the high field regionH 1.77(3H,m),1.33(3H,s),1.22(3H,s),1.13(3H,s)And the remainder is deltaH1.90(1H, m),1.77(1H, m) and 1.83(2H, m) are aliphatic hydrogen signals.13C NMR(100MHz,CDCl3) And HSQC spectra were presumed to contain a 15 carbon signal, suggesting that compound 1 is a sesquiterpene. Including two sets of conjugated double bond signals deltaC140.0,135.9,125.7,122.3 five continuous oxygen carbon signal deltaC85.8,83.0,77.0,71.4,65.4, six sp3Hybrid carbon deltaC38.2,27.3,27.3,26.6,24.3,13.6. The compound was further resolved by HMBC spectroscopy in combination with HSQC.
Directly connected hydrocarbon signals were assigned by HSQC spectra. In HMBC spectra, H-8 (delta)H1.90) and C-6 (. delta.))C 140.0),C-10(δC85.8) there is a remote correlation; h-9 (delta)H1.83) and C-7 (. delta.)C 83.0),C-11(δC71.4) there is a long-range correlation, presumably one furan ring. H-1 (delta)H3.54) and C-2 (. delta.))C 77.0),C-3(δC135.9) there is a remote correlation; h-2 (delta)H4.17) and C-1 (. delta.))C 65.4),C-4(δC125.7) there is a remote correlation; h-4 (delta)H6.12) and C-5 (. delta.)C122.3),C-6(δC140.0) there is a remote correlation; h-6 (delta)H5.74) and C-7 (. delta.))C 83.0),C-8(δC38.2) there is a remote correlation; h-5 (delta)H6.41) and C-7 (. delta.))C83.0) there is a remote correlation. H3-12(δH1.13) and C-10 (. delta.))C 85.8),C-11(δC 71.4),C-13(δC27.3) there is a remote correlation; h3-13(δH1.22) and C-10 (. delta.))C 85.8),C-11(δC71.4),C-12(δC24.3) there is a remote correlation; h3-14(δH1.33) and C-6 (. delta.))C 140.0),C-7(δC 83.0),C-8(δC38.2) there is a remote correlation; h3-15(δH1.77) and C-2 (. delta.))C 77.0),C-3(δC 135.9),C-4(δC 125.7),C-5(δC122.3) there is a remote correlation, presumably to the attachment position of the four methyl groups. The above correlations collectively speculate the planar structure of compound 1.
The relative configuration of compound 1 was determined by Noesy spectra and coupling constants. In NOESY spectra，H-10(δH3.80) with H3-14(δH1.33) No NOESY correlation, indicating H-10 and H3-14 is not coplanar, on the opposite side of the furan ring; h3-15(δH1.77) and H-1 (. delta.))H 3.54),H-5(δH6.41) had a correlation, presumably Δ3,4The configuration of the double bond is E configuration. In addition, J of H-5 and H-65,6Delta is proved by 15.2Hz5,6The configuration of the double bond is E configuration.
Compound 2: clear oil, HRESI-MS gave the excimer peak M/z 293.1724[ M + Na ]]+(calcd for C15H26O4293.1723), in combination1H,13C NMR data confirmed its molecular formula to be C15H26O4The unsaturation degree is 3.1H NMR(600MHz,CDCl3) In the spectrum, the low field region is seen as deltaH6.43(1H, dd, J ═ 15.4,10.9Hz),6.12(1H, d, J ═ 10.9Hz),5.83(1H, d, J ═ 15.4Hz) are the intercoupled olefinic hydrogen signals. Further, δH4.17(1H, dd, J ═ 7.7,3.5Hz),3.65(1H, dd, J ═ 11.2,3.6Hz),3.54(1H, dd, J ═ 11.2,7.6Hz),3.86(1H, t, J ═ 7.2Hz) are 3 vicinal oxygen hydrocarbon signals. High field region visible deltaH1.76(3H, s),1.35(3H, s),1.23(3H, s),1.13(3H, s) are the four methyl hydrogen signals, the remainder being δH1.92(1H, m),1.82(1H, m) and 1.87(2H, m) are aliphatic hydrogen signals.13C NMR(100MHz,CDCl3) And HSQC spectra were presumed to contain a 15 carbon signal, suggesting that compound 2 is a sesquiterpene. Including two sets of conjugated double bond signals deltaC140.2,136.4,125.7,122.7 five continuous oxygen carbon signal deltaC85.7,82.8,77.1,71.5,65.4, six sp3Hybrid carbon signal deltaCSp for 38.6,27.5,26.8,26.5,24.5,13.63Hybrid carbon. The compound was further resolved by combining HSQC and HMBC spectra.
Directly connected hydrocarbon signals were assigned by HSQC spectra. In HMBC spectra, H-8 (delta)H1.92) and C-6 (. delta.))C 140.2),C-10(δC85.7) there is a remote correlation; h-9 (delta)H1.87) and C-7 (. delta.))C 65.4),C-11(δC125.7) there is a long-range correlation, presumably one furan ring. H-1 (delta)H3.54) and C-2 (. delta.))C 77.1),C-3(δC136.4) there is a remote correlation; h-2 (delta)H4.17) and C-1 (. delta.))C 65.4),C-4(δC125.7) there is a remote correlation; h-4 (delta)H6.12) and C-5 (. delta.)C 122.7),C-6(δC140.2) there is a remote correlation; h-5 (delta)H6.43) and C-7 (. delta.))C82.8) there is a remote correlation; h-6 (delta)H5.83) and C-7 (. delta.)C 82.8),C-8(δC38.6) there is a remote correlation. H3-12(δH1.13) and C-10 (. delta.))C85.7),C-11(δC 71.5),C-13(δC27.5) there is a remote correlation; h3-13(δH1.23) and C-10 (. delta.))C 85.7),C-11(δC 71.5),C-12(δC24.5) there is a remote correlation; h3-14(δH1.35) and C-6 (. delta.))C 140.2),C-7(δC82.8),C-8(δC38.6) there is a remote correlation; h3-15(δH1.76) and C-2 (. delta.))C 77.1),C-3(δC 136.4),C-4(δC 125.7),C-5(δC122.7) there is a remote correlation, presumably to the attachment position of the four methyl groups. The above correlations collectively speculate the planar structure of compound 2.
The relative configuration of Compound 2 was determined by Noesy spectroscopy as shown (FIG.2-4), H-10 (. delta.) (H3.86) and H3-14(δH1.35) have NOESY correlation, indicating that H-10 and H3-14 are coplanar, on the same side of the furan ring. Delta was additionally determined from NOEST spectra and coupling constants, respectively3,4And Δ5,6Relative configuration of the double bonds, H3-15(δH1.76) and H-1 (. delta.))H 3.54)，H-5(δH6.43) had a correlation, presumably Δ3,4The configuration of the double bond is E configuration; j of H-5 and H-65,6Delta is proved by 15.4Hz5,6The configuration of the double bond is E configuration.
The specific optical rotation of compounds 1 and 2 was close to zero and there was no significant CD absorption, presumably a racemic mixture. Further separation of compounds 1 and 2 was performed using Daicel Chiralpak AD-H and Daicel Chiralpak IC chiral columns, resulting in four pairs of enantiomers 1a-1d and 2a-2 d. Their absolute configuration is determined by comparing calculated and measured ECDs.
TABLE 11-2 in CDCl3In1H (600MHz) and13c (100MHz) NMR data
Description of the drawings:
HRESIMS spectra of compound 1 of figure 1;
FIG.2 HMBC spectra (600MHz, CDCl) of Compound 13)；
FIG. 3 HSQC spectra (600MHz, CDCl) of Compound 13)；
FIG. 4 NOESY spectrum (600MHz, CDCl) of Compound 13)；
FIG. 5 HRESIMS spectrum of Compound 2;
FIG. 6 HMBC spectra (600MHz, CDCl) of Compound 23)；
FIG. 7 HSQC spectra (600MHz, CDCl) of Compound 23)；
FIG. 8 NOESY spectrum (600MHz, CDCl) of Compound 23)；
FIG. 9 chiral resolution chromatograms of Compounds 1 and 2;
ECD was measured for compounds 1a-1d and 2a-2d of FIG. 10.
The specific implementation mode is as follows:
the examples set out below are intended to assist the person skilled in the art in a better understanding of the invention, but do not limit it in any way.
Example 1: preparation of Compounds 1-2.
20kg of dried whole plant of Erigeron breviscapus (Vant.) hand-Mazz of Compositae is selected as raw material, and is heated and refluxed with 80% industrial ethanol for 3 times, each time for 2 hours. Concentrating under reduced pressure to obtain 4kg of ethanol crude extract, extracting the extract with dichloromethane at a volume ratio of 1:1 for 3 times to obtain 600g of dichloromethane layer and 3.2kg of water layer extract. Subjecting the water layer extract to macroporous adsorbent resin column 5Performing rapid gradient elution with 0-80% ethanol-water system, and detecting with silica gel thin layer to obtain three fractions A (1.1kg), B (1.1kg) and C (0.9 kg). Subjecting fraction C to rapid vacuum silica gel column chromatography, performing rapid gradient elution with dichloromethane-methanol-water system (9.0:1.0:0.0-1.0:1.0:0.8), and mixing by silica gel thin layer chromatography to obtain three fractions C1(210g)、C2(243g)、C3(186g) In that respect Fraction C1Performing polyamide column chromatography, performing gradient elution with 60% -90% ethanol-water system, and collecting three components C1-1、C1-2、C1-3. Component C obtained1-1Subjecting to gel column chromatography with 60% methanol-water isocratic elution to obtain three groups C1-1-1、C1-1-2、C1-1-3. Component C1-1-2Performing gradient elution with dichloromethane-methanol system (50:1-1:1) by silica gel column chromatography, and combining by thin layer chromatography and HPLC analysis to obtain five components C1-1-2-1-C1-1-2-5(ii) a Component C obtained1-1-2-2Performing gradient elution by using 20-90% methanol-water through ODS column chromatography to obtain six components C1-1-2-2-1-C1-1-2-2-6. Component C by semi-preparative reverse phase HPLC1-1-2-2-5Elution with acetonitrile-water system (25: 75) gave a pair of epimers 1(12.3g) and 2(10.2 g). By using Daicel ChiralpakAD-H and Daicel Chiralpak IC chiral chromatographic columns, a mobile phase is a n-hexane-isopropanol mixed solvent, the ratio is 8:1 and 5:1(v: v), the flow rate is 2.00 and 0.45mL/min respectively, and the detection wavelength of an ultraviolet detector is 210 nm. Chiral resolution of the pair of epimers 1 and 2 gives the compounds 1a-1d and 2a-2 d.
Example 2: the inhibition of platelet aggregation induced by ADP in vitro was examined by compounds 1a-1d and 2a-2 d.
(1) Platelet aggregation assay procedure:
1. preparation of 3.8% sodium citrate (now available)
Taking 0.19g sodium citrate, adding distilled water to constant volume to 5ml, dissolving, filtering, bottling, and sterilizing at 121 deg.C under high pressure for 15 min.
2. Preparing the medicine:
(1) preparing the medicine: weighing a proper amount of sample, and preparing the sample with physiological saline to have the concentration of 0.25mg/ml and the DMSO content of 5%.
(2) Negative control: DMSO was measured in an amount of 20. mu.l, and the DMSO solution was prepared at a concentration of 5% with physiological saline.
(3) Positive control: 1.0mg of aspirin and 0.25mg/ml of physiological saline are weighed to prepare a solution, and the DMSO content is 5%.
3. Preparation of ADP
Weighing ADP 5.551mg, diluting with normal saline to a constant volume of 5ml, mixing, subpackaging, storing at-20 deg.C for each 1ml of 5 EP of 1.5ml, melting at present, diluting by 10 times when using, storing at-20 deg.C after use, and mixing when dissolving. Injecting the blood into a plastic tube capable of centrifuging, and adding an anticoagulant to make the blood: the anticoagulant is added at a ratio of 9:1, and the mixture is shaken up gently and is not shaken vigorously.
4. Platelet Rich Plasma (PRP) preparation: centrifuging the anticoagulated blood sample at 200 Xg (1700 rpm) for 8 minutes, taking out the sample after the centrifuge is naturally stopped, and carefully sucking the upper layer of platelet-rich plasma. (the PRP after centrifugation should not contain red blood cells, otherwise it should be re-centrifuged, and the appropriate rotation speed and centrifugation time (preferably the amount of PRP in the blood sample 1/3) should be adjusted according to the centrifuge used.
Platelet Poor Plasma (PPP) preparation: the PRP-aspirated blood was centrifuged again. 1500 Xg (3900 rpm), centrifuging for 10 minutes, taking out the specimen after the centrifuge is naturally stopped, carefully sucking the plasma of the upper layer of the platelet poor for standby, and cleaning and transparentizing the plasma.
The platelet count of platelet-rich plasma was adjusted to 4X 105And/mm 3 or so.
5. Accurately measuring PPP 400 mu L, adding into a test cup, and zeroing the instrument; precisely adding PRP 125 mu L into another test cup, then precisely adding the test sample solution 100 mu L, placing the test cup into a test hole after the test is finished, incubating for 1min at 37 ℃, adding magnetic beads, stirring uniformly, then adding 25 mu L ADP, quickly pressing a start button, recording a platelet aggregation curve, and investigating the biological activity of the drug for inhibiting platelet aggregation.
Inhibition rate (maximum aggregation of blank plasma-maximum aggregation rate of test sample)/maximum aggregation rate of blank plasma
(2) Platelet aggregation assay results:
the experimental results show that the compounds 1a,1c,2b and 2d all show significant platelet aggregation inhibition effect at the concentration of 0.25mg/mL, which is equivalent to 83.67 +/-3.25% of the positive drug, and have certain activity difference of stereoselectivity among enantiomers.
TABLE 2 inhibition of platelet aggregation [ ((S))n＝3)0.25mg/mL]
