CN110305081B - Dihydro agarofuran type sesquiterpene and preparation and application thereof - Google Patents

Dihydro agarofuran type sesquiterpene and preparation and application thereof Download PDF

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CN110305081B
CN110305081B CN201910200913.0A CN201910200913A CN110305081B CN 110305081 B CN110305081 B CN 110305081B CN 201910200913 A CN201910200913 A CN 201910200913A CN 110305081 B CN110305081 B CN 110305081B
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宋少江
黄肖霄
卢丽威
周乐
何青军
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Shenyang Pharmaceutical University
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Abstract

The invention belongs to the technical field of medicines, relates to dihydroagarofuran sesquiterpenes and preparation and application thereof, and relates to a preparation method of dihydroagarofuran sesquiterpene compounds of dried leaves of tripterygium wilfordii of tripterygium of Celastraceae, and application of the compounds in preparation of anti-inflammatory drugs. The structure of the dihydroagarofuran type sesquiterpene compound is as follows: the compound is obtained by separating the Tripterygium wilfordii leaves through silica gel column chromatography, HP-20 macroporous resin, open ODS column chromatography, high performance liquid phase and the like, and the prepared compound has obvious anti-inflammatory activity.

Description

Dihydro agarofuran type sesquiterpene and preparation and application thereof
The technical field is as follows:
the invention belongs to the technical field of medicines, relates to dihydroagarofuran sesquiterpenes and preparation and application thereof, and relates to a preparation method of dihydroagarofuran sesquiterpene compounds of dried leaves of tripterygium wilfordii of tripterygium of Celastraceae, and application of the compounds in preparation of anti-inflammatory drugs.
Background art:
tripterygium wilfordii HooK.f., a perennial vine shrub belonging to the genus Tripterygium of the family Celastraceae, which is mainly distributed in various regions of the Yangtze river basin and in the southwest region. Has antiinflammatory, analgesic, antitumor, and immunoregulatory effects, and can be used for treating rheumatoid arthritis, skin eczema, and malignant tumor. At present, the research on chemical components and pharmacological action of tripterygium wilfordii is mainly focused on roots of tripterygium wilfordii, and tripterygium wilfordii preparations prepared by taking root extracts of tripterygium wilfordii as raw materials are widely applied to clinic and are mainly used for treating diseases such as rheumatoid arthritis, systemic lupus erythematosus and the like. But the research on the chemical components and the biological activity of the tripterygium wilfordii leaves is less.
Through research on compounds in the tripterygium wilfordii leaves, the dihydroagarofuran type sesquiterpene compound has more chiral carbon and a changeable structure, and can easily form an ester compound with various acyl groups. Different substituent groups, different sites and different configurations will likely lead to differences in pharmacological activity. The dihydroagarofuran type sesquiterpenoids related by the invention are prepared from the tripterygium wilfordii leaf extract, have good anti-inflammatory activity, and solve the problem that the existing medical application of tripterygium wilfordii leaf in treating inflammation is not found. The compounds and the activities thereof related to the present invention have not been reported in patents or literatures so far.
The invention content is as follows:
the invention provides 15 dihydro agarofuran type sesquiterpenoids and a preparation method thereof.
The invention also provides application of the dihydroagarofuran type sesquiterpene compound in preparation of anti-inflammatory drugs.
The 15 dihydroagarofuran type sesquiterpene compounds separated from the tripterygium wilfordii leaves are named as tripterfordin A-O (1-15) respectively, and the structures of the compounds are shown as follows:
Figure BDA0001995905780000011
the extraction and preparation method of the dihydroagarofuran sesquiterpenoids comprises the following steps:
(1) extracting aerial part of Tripterygium wilfordii (Tripterygium wilfordii) with flash extractor, mixing extractive solutions, and concentrating under reduced pressure to obtain total extract;
(2) suspending the total extract with water, sequentially extracting with ethyl acetate and n-butanol to obtain ethyl acetate layer and n-butanol layer respectively.
(3) Separating ethyl acetate layer sample by gel column chromatography, silica gel column chromatography, HP-20 column chromatography, ODS column chromatography and preparative HPLC to obtain 15 compounds.
Wherein the content of the first and second substances,
the extraction method in the step (1) is flash extraction, and the extraction solvent is as follows: 70% -80% of ethanol, preferably 75%, extracting for 3-5 times, wherein the weight volume ratio of the medicinal materials to the extraction solvent is 2: 5-8, preferably 2: 5.
the elution solvent of the gel column chromatography in the step (3) is a solvent prepared by mixing the following components in a volume ratio of 100: 1-1: 1, the volume ratio of the petroleum ether-EtOAc mixed system can be (100: 1, 30: 1, 15: 1 and 10: 1, 1: 1, v/v);
the elution solvent of the silica gel column chromatography in the step (3) is petroleum ether-ethyl acetate (25: 1-1: 1).
The structure of all compounds is determined by analyzing the physicochemical properties of the compounds and combining identification means such as IR, UV, OR, MS, NMR and the like. The structure of the 15 novel compounds of the invention is analyzed as follows:
compound 1 tripterester A
White powder, HR-EIS-MS gave an excimer ion peak of 655.2398[ M + Na ]]+(cal.655.2361), and determining the molecular formula C by combining carbon spectrum32H40O13The unsaturation was calculated to be 13.1H-NMR(400MHz,CDCl3) In the low field region, there is a typical set of trans-cinnamoyl signals: deltaH7.60(1H, d, J ═ 16.0), 7.48(2H, m),7.36(3H, m), 6.22(1H, d, J ═ 16.0); 5 continuous oxygen methine signals deltaH5.48(1H, d, J ═ 3.4, H-1),5.41(1H, overlapped, H-2), 5.13(1H, overlapped, H-6),5.42(1H, overlapped, H-8),5.74(1H, d, J ═ 9.6, H-9); 2 continuous oxygen methylene signal deltaH5.10,4.51(each1H, d, J ═ 13.2, H-15); in the high field region 7 methyl signals occur, 4 of which are acetoxymethyl signals, respectively deltaH1.77(OAc-2), 1.94(OAc-8), 2.09(OAc-9), 2.23 (OAc-15); the other 3 are methyl groups bonded to the ring, each being. deltaH 1.57(3H,s,H-12)，1.63(3H,s,H-13)，1.77(3H,s,H-14)。13C-NMR(100MHz,CDCl3) In low, in4 acetoxycarbonyl signals delta appear in the fieldC169.6(OAc-2), 170.1(OAc-8), 169.3(OAc-9), 170.3 (OAc-15); a typical set of trans-cinnamoyl signals δC165.0(C ═ O), 145.7(C- β), 134.1(C-4 '), 130.6 (C-1'), 129.0(C-2 ', 6'), 128.2(C-3 ', 5'), 116.9(C- α); 9 continuous oxygen carbon signals deltaC91.4(C-5), 84.6(C-11), 76.9(C-6), 75.2(C-9), 74.8(C-1), 74.1(C-8), 72.2(C-4), 67.8(C-2),60.5 (C-15); 1 methine signal deltaC53.7 (C-7); 1 quaternary carbon signal deltaC51.6 (C-10); 1 methylene signal deltaC41.1 (C-3); there are also 7 methyl signals 30.0(C-12), 26.4(C-13), 24.6(C-14), 21.5(OAc-15), 21.2(OAc-9), 21.0(OAc-8), 21.7 (OAc-2). From the above NMR data, it can be concluded that compound 1 may be a dihydroagarofuran type sesquiterpene containing 1 trans-cinnamoyl group, 4 acetoxy groups, and 2 hydroxyl groups. And performing direct hydrocarbon correlation attribution on the compound according to an HSQC spectrum, performing hydrocarbon correlation attribution in an HMBC spectrum, and determining the relative configuration of the compound according to a NOESY spectrum and a coupling constant. The absolute configuration of the compound is determined by X-single crystal diffraction, the configuration is determined to be 1R,2S,4S,5S,6R,7S,8S,9S and 10S, and the absolute configuration is searched by Scifinder scholar and is a novel compound which is not reported in the literature and is named tripterester A1The H-NMR data are shown in Table 1,13the C-NMR data are shown in Table 3, and the detailed spectrum is shown in the attached drawing.
Compound 2 tripterine B
White powder (methanol), HR-EIS-MS gave an excimer peak of 639.2399[ M + Na ]]+(calcd for C32H40O12Na,639.2412), and determining the molecular formula as C by combining carbon spectrum32H40O12The unsaturation was calculated to be 13. IR (KBr) at 3541cm-1There was absorption, suggesting the presence of hydroxyl groups in the structure, 1748 and 1716cm-1There was an absorption, suggesting the presence of an ester group in the structure.1H-NMR(400MHz,CDCl3) In the low field region, δH7.62(1H, d, J ═ 16.2Hz),7.50(2H, overlapped),7.38(3H, overlapped),5.88(1H, d, J ═ 16.2Hz) suggested the presence of a typical set of trans cinnamoyl cues in this compoundNumber; deltaH5.48(1H, d, J ═ 3.3, H-1),5.41(1H, overlapped, H-2),5.44(1H, overlapped, H-8),5.74(1H, d, J ═ 9.6, H-9) shows the presence of four vicinal oxymethylene signals; further, δH5.08,4.57(each1H, d, J ═ 13.2, H-15) suggested that it also contained a set of signals for the ortho-coupled oxymethylene group; in the high field region 7 methyl signals occur, 4 of which are acetoxymethyl signals, respectively deltaH1.96(OAc-2),2.08(OAc-8),1.80(OAc-9),2.23 (OAc-15); another 3 methyl groups each being. deltaH 1.19(3H,s,H-12),1.51(3H,s,H-13),1.29(3H,s,H-14)。13C-NMR(100MHz,CDCl3) In the high field region, delta appearsC170.5(OAc-2),169.3(OAc-8),169.7(OAc-9),170.3(OAc-15) further confirmed 4 acetoxy groups; deltaC165.3(C ═ O),146.5(C- β),133.9(C-4 '), 131.0 (C-1'), 129.2(C-2 ', 6'), 128.4(C-3 ', 5'), 116.9(C- α) also confirmed that this compound contains a typical set of trans cinnamoyl signals; in addition to this, the present invention is,138 continuous oxygen carbon signals delta also appear in the C spectrumC90.8(C-5),83.3(C-11),75.6(C-9),75.1(C-8),75.0(C-8),69.3(C-4),68.7(C-2),61.0(C-15), wherein 90.8 and 83.3 are the sign signals of the dihydro agarofuran type sesquiterpene skeleton; 1 methine signal deltaC55.6 (C-7); 1 quaternary carbon signal deltaC47.0 (C-10); 1 methylene signal deltaC40.6 (C-3); there are also 3 methyl signals 30.2(C-12),24.9(C-14),24.5 (C-13). From the above NMR data, it can be inferred that Compound 2 is a dihydroagarofuran type sesquiterpene polyol ester containing 1 trans-cinnamoyl group, 4 acetoxy groups, and 1 hydroxyl group. And performing direct hydrocarbon correlation attribution on the compound according to an HSQC spectrum, performing hydrocarbon correlation attribution in an HMBC spectrum, and determining the relative configuration of the compound according to a NOESY spectrum and a coupling constant. The absolute configuration of the compound is determined by calculating the comparison of the ECD to the measured ECD. The actually measured spectrogram is well matched with 1R,2S,4S,5R,7S,8S,9S and 10S configurations, so that the absolute configuration of the compound is determined. The Scifinderscholar search shows that the compound is a novel compound which is not reported in the literature and is named tripterine B1The H-NMR data are shown in Table 1,13the C-NMR data are shown in Table 3, and the detailed spectrum is shown in the attached drawing.
Compound 3 tripterine C
White powder (methanol), HR-EIS-MS gave an excimer peak of 623.2463[ M + Na ]]+(calcd for C32H40O12Na,623.2463), and determining the molecular formula as C by combining carbon spectrum32H40O11The unsaturation was calculated to be 13. IR (KBr)1747cm-1There was an absorption, suggesting the presence of an ester group in the structure.1H-NMR(400MHz,CDCl3) In the low field region, δH7.61(1H, d, J ═ 16.2Hz),7.49(2H, overlapped),7.36(3H, overlapped),6.26(1H, d, J ═ 16.2Hz) suggested a typical set of trans cinnamoyl signals in this compound; deltaH5.55(1H, d, J ═ 3.4Hz, H-1),5.49(1H, dd, J ═ 6.5,3.4Hz, H-2),5.36(1H, dd, J ═ 9.6,3.4Hz, H-8),5.73(1H, overlapped, H-9) show the presence of four vicinal oxygen methine signals; further, δH5.11,4.56(each 1H, d, J ═ 13.0Hz, H-15) suggested that it also contained a set of signals for the ortho-coupled oxymethylene group; in the high field region 7 methyl signals occur, 4 of which are acetoxymethyl signals, respectively deltaH1.95(OAc-2),1.78(OAc-8),2.07(OAc-9),2.22 (OAc-15); another 3 methyl groups each being. deltaH 1.19(3H,s,H-12),1.51(3H,s,H-13),1.29(3H,d,J＝7.9Hz,H-14)。13C-NMR(100MHz,CDCl3) In (5), it was confirmed that the above-mentioned group is contained.1H NMR and13the C NMR spectrum data can infer that the compound 3 is a dihydroagarofuran type sesquiterpene containing 1 trans-cinnamoyl group and 4 acetoxy groups. The relative configuration of the compound is determined from the NOESY spectra and the coupling constants, based on the hydrocarbon-related assignments in the HMBC spectra. The absolute configuration of the compound is determined by calculating the comparison of the ECD to the measured ECD. The actual measured spectrogram is well matched with 1R,2S,4R,5S,7S,8S,9S and 10S configurations, so that the absolute configuration of the compound is determined. Finally, the structure of the compound is determined, and the Scifinder scholar searches the compound to obtain a novel compound which is not reported in the literature and is named tripterine C1The H-NMR data are shown in Table 1,13the C-NMR data are shown in Table 3, and the detailed spectrum is shown in the attached drawing.
Compound 4 tripterester D
White powder, HR-EIS-MS gave an excimer peak of 617.2592[ M + H [ ]]+(cal.617.2593), and determining the molecular formula C by combining carbon spectrum32H40O12The unsaturation was calculated to be 13.1H-NMR(400MHz,CDCl3) In the low field region, there is a typical set of trans-cinnamoyl signals: deltaH7.59(1H, d, J ═ 16.0), 7.48(2H, m), 7.35(3H, m), 6.24(1H, d, J ═ 16.0); 5 continuous oxygen methine signals deltaH5.72(1H, d, J ═ 3.4, H-1),5.43(1H, m, H-2), 5.04(1H, br s, H-6), 5.39(1H, dd, J ═ 9.6,3.1, H-8),5.73(1H, d, J ═ 9.6, H-9); 2 continuous oxygen methylene signal deltaH5.09,4.51(each1H, d, J ═ 13.0, H-15); set of methylene signals deltaH2.31,1.75(each1H, m, H-3), 1 methine signal deltaH2.47(1H, overlap, H-7); in the high field region 7 methyl signals occur, 4 of which are acetoxymethyl signals, respectively deltaH2.06(OAc-2), 1.93(OAc-8), 1.75(OAc-9), 2.19 (OAc-15); the other 3 are methyl groups bonded to the ring, each being. deltaH 1.48(3H,s,H-12)，1.51(3H,s,H-13)，1.36(3H,d,J＝7.5,H-14)。13C-NMR(100MHz,CDCl3) In the middle, 4 acetoxycarbonyl signals delta appear in the low field regionC170.5(OAc-8), 170.3(OAc-15), 169.7(OAc-2), 169.7 (OAc-9); a typical set of trans-cinnamoyl signals δC165.1(C ═ O), 145.2(C- β),134.2 (C-4 '), 131.5 (C-1'), 129.0(C-2 ', 6'), 128.2(C-3 ', 5'), 117.4(C- α); 8 continuous oxygen carbon signals deltaC91.8(C-5), 82.6(C-11), 76.2(C-1), 75.6(C-9), 74.8(C-6), 74.7(C-8), 69.8(C-4), 60.6 (C-2); 2 methine signals deltaC32.9(C-4), 54.1 (C-7); 1 quaternary carbon signal deltaC50.7 (C-10); 1 methylene signal deltaC31.0 (C-3); there are also 7 methyl signals 31.0(C-12), 26.6(C-13), 21.5(OAc-15), 21.4(OAc-15), 21.0(OAc-15), 20.7(OAc-2), 18.4 (C-14). From the above NMR data, it can be concluded that compound 4 may be a dihydroagarofuran type sesquiterpene polyol ester containing 1 trans-cinnamoyl group, 4 acetoxy groups, and 1 hydroxyl group. Performing direct hydrocarbon related attribution on the compound according to HSQC spectrum, wherein HMBC spectrum isThe assignment of the relation of hydrocarbon is carried out, and the compound is further determined to contain a skeleton of dihydroagarofuran type sesquiterpene. The relative configuration of the compound was determined from the NOESY spectra and the coupling constants. The absolute configuration was determined to be (1R,2S,4R,5R,7S,8S,9S,10S) by X-ray single crystal diffraction, and in conclusion, the structure of the compound was finally determined and searched by Scifinderscholar to be a novel compound which is not reported in the literature and is named tripterester D1The H-NMR data are shown in Table 1,13the C-NMR data are shown in Table 3, and the detailed spectrum is shown in the attached drawing.
Compound 5 tripterine E
White powder, HR-ESI-MS gave an excimer peak of 655.2325[ M + Na ]]+(cal.656.2361), and determining the molecular formula C by combining carbon spectrum32H40O13The unsaturation was calculated to be 13.1H-NMR(600MHz,CDCl3) In the low field region, delta appearsH7.62(1H, d, J ═ 16.2Hz),7.50(2H, overlapped),7.38(3H, overlapped),6.26(1H, d, J ═ 16.2Hz) suggested the presence of a typical set of trans cinnamoyl signals in this compound; 6 continuous oxygen methine signals deltaH5.67(1H, d, J ═ 3.6Hz, H-1),5.39(1H, m, H-2),3.82(1H, m, H-3),5.12(1H, br s, H-6),1.49(1H, dd, J ═ 9.7Hz,3.2, H-8),5.61(1H, d, J ═ 9.7Hz, H-9); 2 continuous oxygen methylene signal deltaH4.79,4.37(each 1H, d, J ═ 13.2Hz, H-15); 2 methine signals deltaH2.37(1H, q, J ═ 7.9Hz, H-4),2.51(1H, d, J ═ 3.2Hz, H-7); in the high field region 7 methyl signals occur, 4 of which are acetoxymethyl signals, respectively deltaH2.09(OAc-2),1.98(OAc-8),1.79(OAc-9),2.20 (OAc-15); the other 3 being methyl groups bound to the quaternary carbon, each being deltaH 1.57(3H,s,H-12),1.61(3H,s,H-13),1.40(3H,d,J＝7.9Hz,H-14)。13C-NMR(125MHz,CDCl3) In the low field region, delta appearsC169.0(OAc-2),170.5(OAc-8),169.5(OAc-9),170.1(OAc-15) further confirmed the presence of 4 acetyl groups; deltaC165.3(C ═ O),146.5(C- β),133.9(C-4 '), 131.0 (C-1'), 129.2(C-2 ', 6'), 128.4(C-3 ', 5'), 116.9(C- α) also further demonstrate the presence of nicotinoyl groups; in addition, 9 continuous oxygen carbon signals delta appearC93.9(C-5) 84.9(C-11),75.5(C-6),75.1(C-9),74.3(C-8),72.9(C-1),72.9(C-3),72.7(C-2),60.3 (C-15); 2 methine signals deltaC53.1(C-7),39.5 (C-4); there are also 7 methyl signals 30.9(C-12),26.3(C-13),21.5(OAc-15),21.1(OAc-2),21.0(OAc-8),20.3(OAc-9),15.9 (C-14). From the above NMR data in combination with mass spectrum data, it can be concluded that compound 5 may be a dihydroagarofuran type sesquiterpene containing 1 trans-cinnamoyl group, 4 acetoxy groups, and 2 hydroxyl groups. And performing direct hydrocarbon correlation attribution on the compound according to an HSQC spectrum, performing hydrocarbon correlation attribution in an HMBC spectrum, and determining the relative configuration of the compound according to a NOESY spectrum and a coupling constant. The absolute configuration of the compound is determined to be (1R,2S,3R,4R,5S,6R,7S,8S,9R,10R) by calculating ECD, and in conclusion, the structure of the compound is finally determined and is a new compound which is not reported in the literature through Scfin scholar search, named tripterine E, which is1The H-NMR data are shown in Table 1,13the C-NMR data are shown in Table 3, and the detailed spectrum is shown in the attached drawing.
Compound 6 tripterine F
White powder, HR-ESI-MS gave an excimer peak of 671.2310[ M + Na ]]+(cal.671.2316) and determining the molecular formula C by combining carbon spectrum32H40O14The unsaturation was calculated to be 13.1H-NMR(600MHz,CDCl3) In the low field region, delta appearsH7.62(1H, d, J ═ 16.2Hz),7.50(2H, overlapped),7.38(3H, overlapped),6.26(1H, d, J ═ 16.2Hz) suggested the presence of a typical set of trans cinnamoyl signals in this compound; 6 continuous oxygen methine signals deltaH5.53(1H, d, J ═ 3.6Hz, H-1),4.15(1H, m, H-2),3.82(1H, m, H-3),5.12(1H, br s, H-6),4.09(1H, dd, J ═ 9.7,3.2Hz, H-8),5.63(1H, d, J ═ 9.7Hz, H-9); 2 continuous oxygen methylene signal deltaH5.05,4.74(each 1H, d, J ═ 13.2Hz, H-15); 2 methine signals deltaH2.61(1H, q, J ═ 7.9Hz, H-4),2.54(1H, d, J ═ 3.2Hz, H-7); in the high field region 7 methyl signals occur, 4 of which are acetoxymethyl signals, respectively deltaH2.09(OAc-2),1.98(OAc-8),1.79(OAc-9),2.20 (OAc-15); the other 3 being methyl groups bound to the quaternary carbon, each being deltaH 1.57(3H,s,H-12),1.61(3H,s,H-13),1.40(3H,d,J＝7.9Hz, H-14). Carbon spectrum further confirms the existence of the groups, and the compound 6 can be presumed to be dihydroagarofuran type sesquiterpene containing 1 trans-cinnamoyl group, 4 acetoxyl groups and 2 hydroxyl groups by combining mass spectrum data. And performing direct hydrocarbon correlation attribution on the compound according to an HSQC spectrum, performing hydrocarbon correlation attribution in an HMBC spectrum, and determining the relative configuration of the compound according to a NOESY spectrum and a coupling constant. The absolute configuration of the compound is determined to be (1R,2S,3S,4S,5S,6R,7S,8S,9S and 10R) by calculating ECD, and in conclusion, the structure of the compound is finally determined and is a novel compound which is not reported in the literature through Scfin scholar search and is named tripterine F1The H-NMR data are shown in Table 1,13the C-NMR data are shown in Table 3, and the detailed spectrum is shown in the attached drawing.
Compound 7 tripterester G
White powder, HR-EIS-MS gave an excimer ion peak of 571.2204[ M + Na ]]+(cal.571.2155), determining molecular formula C by combining carbon spectrum28H36O11The unsaturation was calculated to be 11.1H-NMR(400MHz,CDCl3) In the low field region, there is a typical set of trans-cinnamoyl signals: deltaH7.73(1H, d, J ═ 13.0Hz),7.54(2H, m),7.41(3H, m),6.38(1H, d, J ═ 13.0 Hz); 5 continuous oxygen methine signals deltaH5.57(1H, d, J ═ 3.1Hz, H-1),5.43(1H, dd, J ═ 6.3,3.1Hz, H-2),5.00(1H, br s, H-6),5.35(1H, dd, J ═ 9.3,3.4Hz, H-8),5.69(1H, d, J ═ 9.3Hz, H-9); 2 continuous oxygen methylene signal deltaH5.08,4.63(each 1H, d, J ═ 13.2Hz, H-15); set of methylene signals deltaH2.10,2.01(each 1H, m, H-3), 1 methine signal deltaH2.43(1H, d, J ═ 3.4Hz, H-7); 4 hydroxyl signals deltaH2.07(1H, overlaid, 2-OH),3.10(1H, br s,3-OH),5.28(1H, br s,6-OH),2.30(1H, overlaid, 8-OH); in the high field region, 5 methyl signals occur, 2 of which are acetoxymethyl signals, respectively deltaH1.90(OAc-9),2.14 (OAc-15); the other 3 being methyl groups bound to the quaternary carbon, each being deltaH1.60(3H,s,H-12),1.69(3H,s,H-13),1.81(3H,s,H-14)。13C-NMR(100MHz,CDCl3) In the middle, 2 acetoxycarbonyl signals delta appear in the low field regionC 170.1(OAc-9) 171.8 (OAc-15); a typical set of trans-cinnamoyl signals δC165.3(C ═ O),146.5(C- β),133.9(C-4 '), 131.0 (C-1'), 129.2(C-2 ', 6'), 128.4(C-3 ', 5'), 116.9(C- α); 9 continuous oxygen carbon signals deltaC92.0(C-5),84.3(C-11),79.0(C-9),77.4(C-1),77.0(C-6),75.5(C-8),72.6(C-4),67.7(C-2),61.4 (C-15); 1 methine signal deltaC55.6 (C-7); 1 quaternary carbon signal deltaC51.7 (C-10); 1 methylene signal deltaC43.1 (C-3); there are also 5 methyl signals 30.3(C-12),26.6(C-13),24.6(C-14),21.7(OAc-15),21.2 (OAc-2). From the above NMR data, it can be concluded that compound 7 may be a dihydroagarofuran type sesquiterpene polyol ester containing 1 trans-cinnamoyl group, 2 acetoxy groups, and 4 hydroxyl groups. And performing direct hydrocarbon correlation attribution on the compound according to an HSQC spectrum, performing hydrocarbon correlation attribution in an HMBC spectrum, and determining the relative configuration of the compound according to a NOESY spectrum and a coupling constant. The absolute configuration of the compound is determined to be (1R,2S,3S,4S,5S,6R,7R,8S,9S and 10S) by calculating ECD, and in conclusion, the structure of the compound is finally determined and is a novel compound which is not reported in the literature through Scfin scholar search and is named tripterester G1The H-NMR data are shown in Table 1,13the C-NMR data are shown in Table 3, and the detailed spectrum is shown in the attached drawing.
Compound 8, tripterester H
White powder, HR-ESI-MS gave an excimer peak of 659.2411[ M + Na ]]+(cal.659.2468) determining molecular formula C by combining carbon spectrum35H40O11The unsaturation was calculated to be 16.1H-NMR(400MHz,CDCl3) In the low field region, there are 1 group of H signals deltaH7.94(1H, d, J ═ 7.3Hz),7.17-7.40(7H, overlaid), 6.93(1H, d, J ═ 7.3Hz), and 1 alkene hydrogen signal δH5.72(1H, d, J ═ 16.0Hz), presumably the compound may contain a benzoyl group and a trans cinnamoyl group; furthermore, there are 5 vicinal oxymethylene signals deltaH5.49(1H, d, J ═ 3.2Hz, H-1),5.40(1H, dd, J ═ 6.4,3.2Hz, H-2),6.64(1H, s, H-6),5.42(1H, dd, J ═ 5.6,4.0Hz, H-8),5.72(1H, d, J ═ 5.6Hz, H-9); 2 continuous oxygen methylene signal deltaH 5.12,4.50(each 1H,d,J＝12.7Hz,H-15); in the high field region, 5 methyl signals occur, 2 of which are acetoxymethyl signals, respectively deltaH2.06(OAc-2),2.11 (OAc-6); the other 3 being methyl groups bound to the quaternary carbon, each being deltaH 1.43(3H,s,H-12),1.53(3H,s,H-13),1.31(3H,s,H-14)。13C-NMR(100MHz,CDCl3) In the low field region, delta appearsC169.8(OAc-2),170.0(OAc-6) further confirmed the presence of 2 acetoxy groups; deltaC165.2(C ═ O),133.1(C-4 '), 129.3 (C-1'), 129.9(C-2 ', 6'), 128.5(C-3 ', 5') further illustrate that the compound contains one benzoyl group; further, δC165.8(C ═ O), 145.4(C- β),134.2(C-1 "), 130.2 (C-4"), 128.6(C-2 ", 6"), 128.1(C-3 ", 5"), 117.2(C- α), further indicating the definite presence of benzoyl and trans-cinnamoyl groups in the compound; in addition, 8 continuous oxygen carbon signals delta are containedC90.4(C-5),81.3(C-11),76.5(C-6),76.1(C-1),74.8(C-2),70.0(C-6),68.3(C-2),59.2 (C-8); 1 methylene signal deltaC31.3 (C-3); 2 methine signals deltaC33.1(C-3),56.4 (C-7); 1 quaternary carbon signal deltaC54.7 (C-10); there are also 5 methyl signals 30.8(C-12),24.6(C-13),17.6(C-14),21.5(OAc-2),21.6 (OAc-6). From the above NMR data and mass spectrum data, it can be inferred that compound 8 may be dihydroagarofuran type sesquiterpene polyol ester containing 1 benzoyl, 1 trans-cinnamoyl, 2 acetoxy and 2 hydroxyl groups. And performing direct hydrocarbon correlation attribution on the compound according to an HSQC spectrum, performing hydrocarbon correlation attribution in an HMBC spectrum, and determining the relative configuration of the compound according to a NOESY spectrum and a coupling constant. The absolute configuration of the compound is determined to be (1R,2S,4R,5S,6R,7R,8R,9S and 10R) by calculating ECD, and in conclusion, the structure of the compound is finally determined and is a novel compound which is not reported in the literature through Scfin scholar search and is named tripterester H1The H-NMR data are shown in Table 1,13the C-NMR data are shown in Table 3, and the detailed spectrum is shown in the attached drawing.
Compound 9 tripterester I
White powder, HR-EIS-MS gave an excimer ion peak of 655.2370[ M + Na ]]+(cal.655.2361), and determining the molecular formula C by combining carbon spectrum32H40O13The unsaturation was calculated to be 13.1H-NMR(400MHz,CDCl3) In the low field, there is a typical set of cis-cinnamoyl signals: deltaH7.62(2H, d, J ═ 7.0), 7.35(3H, m), 6.97(1H, d, J ═ 12.6), 5.65(1H, d, J ═ 12.6); 5 continuous oxygen methine signals deltaH5.42(1H, overlapped, H-1), 5.38(1H, overlapped, H-2), 5.13(1H, d, J ═ 9.7, H-6), 5.41(1H, overlapped, H-8),5.74(1H, d, J ═ 9.7, H-9); 2 continuous oxygen methylene signal deltaH4.95,4.51(each1H, d, J ═ 13.2, H-15); in the high field region 7 methyl signals occur, 4 of which are acetoxymethyl signals, respectively deltaH1.92(OAc-2), 1.97(OAc-8), 2.11(OAc-9), 2.23 (OAc-15); the other 3 are methyl groups bonded to the ring, each being. deltaH 1.57(3H,s,H-12)，1.62(3H,s,H-13)，1.77(3H,s,H-14)。13C-NMR(100MHz,CDCl3) The above groups were further confirmed. In addition, there are 9 continuous oxygen carbon signals deltaC91.4(C-5), 84.6(C-11), 77.0(C-6), 75.0(C-9), 74.8(C-1), 74.1(C-8), 72.2(C-4), 67.8(C-2),60.8 (C-15); 1 methine signal deltaC53.7 (C-7); 1 quaternary carbon signal deltaC51.4 (C-10); 1 methylene signal deltaC41.2 (C-3); there are also 7 methyl signals 30.1(C-12), 26.5(C-13), 21.5(OAc-15), 21.3(OAc-15), 21.1(OAc-8), 21.0(OAc-6), 20.9 (C-2). From the above NMR data, it was concluded that the compound was a dihydroagarofuran type sesquiterpene polyol ester containing 1 cis cinnamoyl group, 4 acetoxy groups, and 2 hydroxyl groups. And performing direct hydrocarbon correlation attribution on the compound according to an HSQC spectrum, performing hydrocarbon correlation attribution in an HMBC spectrum, and determining the relative configuration of the compound according to a NOESY spectrum and a coupling constant. The absolute configuration of the compound is determined to be (1R,2S,4S,5S,6R,7S,8S,9S,10S) through X-single crystal diffraction, and in conclusion, the structure of the compound is finally determined and is a novel compound which is not reported in the literature through Scfin scholar search and is named tripterester I1The H-NMR data are shown in Table 2,13the C-NMR data are shown in Table 3, and the detailed spectrum is shown in the attached drawing.
Compound 10 tripterester J
White colourPowder, HR-EIS-MS gave an excimer peak of 617.2611[ M + H ]]+(cal.617.2593), and determining the molecular formula C by combining carbon spectrum32H40O12The unsaturation was calculated to be 13.1H-NMR(400MHz,CDCl3) In the low field, there is a typical set of cis-cinnamoyl signals: deltaH7.63(2H, d, J ═ 7.0), 7.33(3H, m), 6.94(1H, d, J ═ 12.6), 5.67(1H, d, J ═ 12.6); 5 continuous oxygen methine signals deltaH5.52(1H, d, J ═ 3.2, H-1), 5.42(1H, dd, J ═ 5.6,3.2, H-2), 5.05(1H, br s, H-6), 5.34(1H, dd, J ═ 9.7,3.1, H-8), 5.71(1H, d, J ═ 9.7, H-9); 2 continuous oxygen methylene signal deltaH4.92,4.64(each1H, d, J ═ 13.0, H-15); set of methylene signals deltaH2.28,1.76(each1H, m, H-3), 1 methine signal deltaH2.44(1H, overlap, H-7); in the high field region 7 methyl signals occur, 4 of which are acetoxymethyl signals, respectively deltaH2.08(OAc-2), 1.98(OAc-8), 1.91(OAc-9), 2.20 (OAc-15); the other 3 are methyl groups bonded to the ring, each being. deltaH 1.48(3H,s,H-12)，1.53(3H,s,H-13)，1.35(3H,d,J＝7.5,H-14)。13C-NMR(100MHz,CDCl3) In the middle, 4 acetoxycarbonyl signals delta appear in the low field regionC169.9(OAc-8), 170.6(OAc-15), 169.9(OAc-2), 170.2 (OAc-9); a typical set of cis cinnamoyl signals δC164.1(C ═ O), 145.5(C- β), 134.5(C-4 '), 129.3 (C-1'), 130.1(C-2 ', 6'), 128.1(C-3 ', 5'), 118.2(C- α); 8 continuous oxygen carbon signals deltaC91.8(C-5), 82.6(C-11), 76.2(C-1), 75.3(C-9), 75.0(C-6), 74.8(C-8), 69.7(C-4), 61.0 (C-2); 2 methine signals deltaC32.9(C-4), 54.1 (C-7); 1 quaternary carbon signal deltaC50.5 (C-10); 1 methylene signal deltaC31.0 (C-3); there are also 7 methyl signals 31.1(C-12), 26.4(C-13), 21.6(OAc-15), 21.5(OAc-15),21.1 (OAc-15), 20.9(OAc-2), 18.4 (C-14). From the above NMR data, it can be concluded that compound 10 may be a dihydroagarofuran type sesquiterpene polyol ester containing 1 cis-cinnamoyl group, 4 acetoxy groups, and 1 hydroxyl group. Performing direct hydrocarbon correlation attribution on the compound according to HSQC spectrum, performing attribution on hydrocarbon correlation in HMBC spectrum, and performing attribution on the compoundThe relative configuration was determined from the NOESY spectra and the coupling constants. The absolute configuration of the compound is determined to be (1R,2S,4R,5R,7S,8S,9S,10S) through X-single crystal diffraction, and in conclusion, the structure of the compound is finally determined and is a novel compound which is not reported in the literature through Scfin scholar search and is named tripteresterJ1The H-NMR data are shown in Table 2,13the C-NMR data are shown in Table 3, and the detailed spectrum is shown in the attached drawing. Compound 11 tripterester K
White powder, HR-EIS-MS gave an excimer ion peak of 697.2369[ M + Na ]]+(cal.697.2359) and determining molecular formula C by combining carbon spectrum34H42O14The unsaturation was calculated to be 14.1H-NMR(400MHz,CDCl3) In the low field, there is a typical set of cis-cinnamoyl signals: deltaH7.62(2H, m),7.36(3H, m),6.98(1H, d, J ═ 12.6 Hz); 5 continuous oxygen methine signals deltaH5.43(1H, d, J ═ 3.2Hz, H-1),5.36(1H, dd, J ═ 6.3,3.2Hz, H-2),6.47(1H, br s, H-6),5.53(1H, dd, J ═ 9.6,3.5Hz, H-8),5.73(1H, d, J ═ 9.6Hz, H-9); 2 continuous oxygen methylene signal deltaH4.81,4.69(each1H, d, J ═ 13.2Hz, H-15); in the high field region, 8 methyl signals appear, of which 5 are acetoxymethyl signals, respectively deltaH1.96(OAc-2),1.98(OAc-6),2.10(OAc-8),2.10(OAc-9),2.30 (OAc-15); the other 3 being methyl groups bound to the quaternary carbon, each being deltaH 1.53(3H,s,H-12),1.64(3H,s,H-13),1.47(3H,s,H-14)。13C-NMR(100MHz,CDCl3) In the middle, 2 acetoxycarbonyl signals delta appear in the low field regionC169.6(OAc-2),169.8(OAc-6),170.0(OAc-8),170.1(OAc-9),171.3 (OAc-15); a typical set of cis cinnamoyl signals δC163.9(C ═ O),146.2(C- β),134.4(C-4 '), 129.5 (C-1'), 130.1(C-2 ', 6'), 128.1(C-3 ', 5'), 117.7(C- α); 9 continuous oxygen carbon signals deltaC92.2(C-5),84.4(C-11),75.6(C-6),75.1(C-9),75.0(C-8),74.0(C-1),69.7(C-4),68.1(C-2),61.0 (C-15); 1 methine signal deltaC52.4 (C-7); 1 quaternary carbon signal deltaC52.0 (C-10); 1 methylene signal deltaC42.1 (C-3); there are also 5 methyl signals 29.8(C-12),26.0(C-13),24.9(C-14),21.0(OAc-2),21.0(OAc-6),21.3(OAc-8),21.4(OAc-9),21.6(OAc-15). From the above NMR data, it can be concluded that compound 11 may be a dihydroagarofuran type sesquiterpene containing 1 cis-cinnamoyl group, 5 acetoxy groups, and 1 hydroxyl group. And performing direct hydrocarbon correlation attribution on the compound according to an HSQC spectrum, performing hydrocarbon correlation attribution in an HMBC spectrum, and determining the relative configuration of the compound according to a NOESY spectrum and a coupling constant. The absolute configuration of the compound is determined to be (1R,2S,4S,5S,6R,7R,8S,9S,10S) by calculating ECD, and in conclusion, the structure of the compound is finally determined and is a novel compound which is not reported in the literature through Scifinderscholarer search and is named tripterester K1The H-NMR data are shown in Table 2,13the C-NMR data are shown in Table 3, and the detailed spectrum is shown in the attached drawing.
Compound 12 tripterine L
White powder (methanol), HR-EIS-MS gave an excimer peak of 623.2470[ M + Na ]]+(calcd for C32H40O12Na,623.2463), and determining the molecular formula as C by combining carbon spectrum32H40O11The unsaturation was calculated to be 13. IR (KBr) at 1747cm-1There was an absorption, suggesting the presence of an ester group in the structure.1H-NMR(400MHz,CDCl3) In the low field region, δH7.58(2H, d, J ═ 7.2Hz),7.30(3H, overlaid), 6.89(1H, d, J ═ 12.6Hz),5.64(1H, overlaid) suggested the presence of a typical set of cis cinnamoyl signals in this compound; deltaH5.42(1H, d, J ═ 3.3Hz, H-1),5.34(1H, overlapped, H-2),5.37(1H, dd, overlapped, H-8),5.64(1H, d, J ═ 9.6Hz, H-9) shows the presence of four vicinal oxymethylene signals; further, δH4.85,4.54(each 1H, d, J ═ 12.9Hz, H-15) suggested that it also contained a set of signals for the ortho-coupled oxymethylene group; in the high field region 7 methyl signals occur, 4 of which are acetoxymethyl signals, respectively deltaH1.96(OAc-2),1.86(OAc-8),2.02(OAc-9),2.16 (OAc-15); another 3 methyl groups each being. deltaH1.13(3H,s,H-12),1.46(3H,s,H-13),1.22(3H,d,J＝7.9Hz,H-14)。13C-NMR(100MHz,CDCl3) In (5), it was confirmed that the above-mentioned group is contained.1H NMR and13the C NMR spectrum data can infer that the compound 12 is a compound containing 1 cis-cinnamonAcyl, 4 acetoxy dihydroagarofuran type sesquiterpenes, compound 3, showed H-1 and cis-cinnamoyl delta in HMBC spectrumC164.0 correlation, judging that cinnamoyl is linked to position 1; h-2, H-8, H-9, H-15 and acetyl deltaC170.5,169.9,169.7,170.2 related to the confirmation of the attachment of the acetyl group to the 2,8,9,15 position respectively. The relative configuration of the compound was determined from the NOESY spectra and the coupling constants. The absolute configuration of the compound is determined by calculating the comparison of the ECD to the measured ECD. The actual measured spectrogram is well matched with 1R,2S,4R,5S,7S,8S,9S and 10S configurations, so that the absolute configuration of the compound is determined. In conclusion, the structure of the compound is finally determined, and the Scfiner scholar search shows that the compound is a novel compound which is not reported in the literature and is named tripterine L1The H-NMR data are shown in Table 2,13the C-NMR data are shown in Table 3, and the detailed spectrum is shown in the attached drawing.
Compound 13 tripterine M
White powder (methanol), HR-EIS-MS gave an excimer peak of 639.2447[ M + Na ]]+(calcd for C32H40O12Na,639.2412), and determining the molecular formula as C by combining carbon spectrum32H40O12The unsaturation was calculated to be 13. IR (KBr) at 3439cm-1There is an absorption indicating that there is a hydroxyl group in the structure, 1747cm-1There was an absorption, suggesting the presence of an ester group in the structure.1H-NMR(400MHz,CDCl3) In the low field region, δH7.61(2H, d, J ═ 7.2Hz),7.38(3H, overlaid), 6.95(1H, d, J ═ 12.6Hz),5.66(1H, d, J ═ 12.6Hz) suggested the presence of a typical set of cis cinnamoyl signals in this compound; deltaH5.41(1H, d, J ═ 3.2, H-1),5.37(1H, dd, J ═ 6.6,3.2, H-2),5.43(1H, dd, J ═ 9.6,3.2, H-8),5.75(1H, d, J ═ 9.6, H-9) show the presence of four vicinal oxymethylene signals; further, δH4.92,4.53(each 1H, d, J ═ 13.1, H-15) suggest that there is also a set of signals for the ortho-coupled oxymethylene group; in the high field region 7 methyl signals occur, 4 of which are acetoxymethyl signals, respectively deltaH2.07(OAc-2),1.97(OAc-8),1.92(OAc-9),2.20 (OAc-15); another 3 methyl groups each being. deltaH 1.28(3H,s,H-12),1.57(3H,s,H-13),1.43(3H,s,H-14)。13C-NMR(100MHz,CDCl3) In (5), it was confirmed that the above-mentioned group is contained.1H NMR and13the C NMR spectrum data can infer that the compound 13 is a dihydroagarofuran type sesquiterpene containing 1 cis-cinnamoyl group, 4 acetoxyl groups and 1 hydroxyl group, the HMBC spectrum is assigned according to the relation of hydrocarbon, and the relative configuration of the compound is determined according to the NOESY spectrum and the coupling constant. The absolute configuration of the compound is determined by calculating the comparison of the ECD to the measured ECD. The actually measured spectrogram is well matched with 1R,2S,4S,5R,7S,8S,9S and 10S configurations, so that the absolute configuration of the compound is determined. In conclusion, the structure of the compound is finally determined, and the Scfiner scholar search shows that the compound is a novel compound which is not reported in the literature and is named tripterine M1The H-NMR data are shown in Table 2,13the C-NMR data are shown in Table 3, and the detailed spectrum is shown in the attached drawing.
Compound 14 tripterinoid N
White columnar crystals (MeOH). HR-ESI-MS shows that the peak of the quasi-molecular ion is 595.2157[ M + Na ]]+(cal.595.2150) and determining the molecular formula C by combining with carbon spectrum30H36O11。1H-NMR(600MHz,CDCl3) In the low field region, delta appearsH7.69(1H, d, J ═ 16.0), 7.53(2H, m), 7.39(3H, m), 6.34(1H, d, J ═ 16.0), suggesting that the compound contains a trans cinnamoyl group; having 3 continuous oxygen methine signals deltaH5.69(1H, d, J ═ 3.2, H-1),5.39(1H, d, J ═ 6.4,3.2, H-2),5.66(1H, s, H-9); 2 methylene signals connected to oxygen deltaH5.23,4.35(each1H, d, J ═ 12.8, H-15); 6 methyl signals appear in the high field region, 3 of which are acetoxymethyl signals, respectively deltaH1.90(OAc-2),2.12(OAc-9),1.96 (OAc-15); and additionally methyl radicals bound to the quaternary carbon, each being deltaH 1.41(1H,s,H-12),1.51(1H,s,H-13),1.52(1H,s,H-14)。13C-NMR(125MHz,CDCl3) The above groups are further confirmed; in addition, 7 continuous oxygen carbon signals delta appearC91.2(C-5),83.6(C-11),79.7(C-9),73.9(C-1),69.6(C-2),69.6(C-2),60.8 (C-15); 2 methylene signals deltaC40.7(C-3),31.9 (C-6); 1 is provided withMethine signal 59.2 (C-7); 2 quaternary carbon signal δC69.6(C-4),55.3 (C-10); there are also 6 methyl signals 28.8(C-12),23.4(C-13),25.4(C-14),20.3(OAc-1),21.4(OAc-2),20.7 (OAc-15). From the above NMR data, it was concluded that compound 14 may be a dihydroagarofuran type sesquiterpene containing 1 trans-cinnamoyl group, 3 acetoxy groups. And performing direct hydrocarbon correlation attribution on the compound according to an HSQC spectrum, performing hydrocarbon correlation attribution in an HMBC spectrum, and determining the relative configuration of the compound according to a NOESY spectrum and a coupling constant. The absolute configuration of the compound is determined to be (1R,2S,4S,5R,7R,9S,10S) through calculating ECD, the structure of the compound is finally determined, and the compound is a novel compound which is not reported in the literature through Scifinder scholar search and is named as tripterinoid N1The H-NMR data are shown in Table 2,13the C-NMR data are shown in Table 3, and the detailed spectrum is shown in the attached drawing.
Compound 15 tripterinoid O
White columnar crystals (MeOH). HR-ESI-MS shows that the peak of the quasi-molecular ion is 611.2046[ M + Na ]]+(cal.611.2207), and determining the molecular formula C by combining with carbon spectrum30H36O12。1H-NMR(600MHz,CDCl3) In the low field region, delta appearsH7.69(1H, d, J ═ 16.0), 7.53(2H, m), 7.39(3H, m), 6.33(1H, d, J ═ 16.0), suggesting that the compound contains a trans cinnamoyl group; having 4 continuous oxygen methine signals deltaH5.69(1H, d, J ═ 3.2, H-1),5.39(1H, d, J ═ 6.4,3.2, H-2),5.28(1H, m, H-6),5.66(1H, s, H-9); 2 methylene signals connected to oxygen deltaH5.20,4.38(each1H, d, J ═ 12.8, H-15); 6 methyl signals appear in the high field region, 3 of which are acetoxymethyl signals, respectively deltaH1.90(OAc-2),2.15(OAc-9),1.97 (OAc-15); and additionally methyl radicals bound to the quaternary carbon, each being deltaH 1.55(1H,s,H-12),1.67(1H,s,H-13),1.84(1H,s,H-14)。13C-NMR(125MHz,CDCl3) The above groups are further confirmed; in addition, 7 continuous oxygen carbon signals delta appearC92.8(C-5),85.3(C-11),79.5(C-9),76.5(C-6),74.0(C-1),72.4(C-4),68.5 (C-2); 1 methylene signal deltaC41.2 (C-3); 1 methine signal 66.6 (C-7); 1 quaternary carbon signal deltaC55.3 (C-10); there were also 6 methyl signals 30.0(C-12),24.8(C-13),25.3(C-14),20.3(OAc-2),21.3(OAc-9),20.7 (OAc-15). From the above NMR data, it was concluded that compound 15 may be a dihydroagarofuran type sesquiterpene containing 1 trans-cinnamoyl group and 3 acetoxy groups. And performing direct hydrocarbon correlation attribution on the compound according to an HSQC spectrum, performing hydrocarbon correlation attribution in an HMBC spectrum, and determining the relative configuration of the compound according to a NOESY spectrum and a coupling constant. The absolute configuration of the compound is determined to be (1R,2S,4S,5S,6R,7R,9S,10S) by calculating ECD, and in conclusion, the structure of the compound is finally determined and is a novel compound which is not reported in the literature through Scfin scholar search and is named tripterinoid O1The H-NMR data are shown in Table 2,13the C-NMR data are shown in Table 3, and the detailed spectrum is shown in the attached drawing.
Figure BDA0001995905780000111
Figure BDA0001995905780000121
Figure BDA0001995905780000131
The applicant verified by anti-inflammatory experiments that compounds 1,4,9,10 and 13 significantly inhibited Nitric Oxide (NO) production in Lipopolysaccharide (LPS) -induced RAW 264.7 macrophages with IC50 values ranging from 11.93 to 30.97 μ M.
Drawings
FIG. 1: x-single crystal diffractogram of Compound 1
FIG. 2: ECD spectrum of Compound 2
FIG. 3: ECD spectrum of Compound 3
FIG. 4: x-single crystal diffractogram of Compound 4
FIG. 5: ECD spectrum of Compound 5
FIG. 6: ECD spectrum of Compound 6
FIG. 7: ECD spectrum of Compound 7
FIG. 8: ECD spectrum of Compound 8
FIG. 9: x-single crystal diffractogram of Compound 9
FIG. 10: x-single crystal diffractogram of Compound 10
FIG. 11: ECD spectrum of Compound 11
FIG. 12: ECD spectrum of Compound 12
FIG. 13: ECD spectrum of Compound 13
FIG. 14: ECD spectrum of Compound 14
FIG. 15: ECD profile of compound 15.
Detailed Description
Example 1: preparation of Compounds 1-15 of formula
Extracting 30kg of dried Tripterygium wilfordii leaf with 75% ethanol by volume concentration for 3 times, and performing heating reflux extraction or ultrasonic extraction. And combining all the extracting solutions, carrying out reduced pressure concentration, recovering the solvent, and drying after the reduced pressure concentration is finished to obtain the extract. Sequentially extracting with ethyl acetate and n-butanol, eluting ethyl acetate layer with gel column chromatography and petroleum ether-EtOAc mixed system (100: 1, 30: 1, 15: 1 and 10: 1, 1: 1, v/v) to obtain fraction A-D, eluting C fraction with cross-linked dextran LH-20 column and ODS gel column to obtain 5 main fractions C1-C5, separating C2(9.5g) with silica gel column chromatography, and eluting with petroleum ether-ethyl acetate gradient (25: 1 to 1: 1) to obtain 8 sub-fractions (Fr.C2-1 to Fr.C 2-8). Semi-preparative RP-HPLC (65: 35, CH) was used3CN/H2O) subfraction C2-2 was further isolated to give 9(7.6 mg). By semi-preparative RP-HPLC (65: 35, CH)3CN/H2O) purification of subfraction C2-3 to give 2(3.1mg), 4(2.9mg), 5(4.2mg), 6(1.5mg) and 13(3.1 mg). Fraction C3 was chromatographed on silica gel CC (petroleum ether/ethyl acetate, 30: 1 → 1: 1, v/v) to give 6 subfractions (fr.c 3-1-fr.c 3-6). Subfraction C3-1 was separated by semi-preparative HPLC using YMC Pack ODS-A column to give 1(4mg) and 12(4.2 mg). Preparative RP-HPLC was used (subfraction C3-3 was further purified to give 10(6.2mg), 11(4.2mg), 14(3.1mg) and 15(5.0mg), and A semi-preparative HPLC column and YMC Pack ODS-A column (75:25，CH3CN/H2o) subfraction C3-4 was purified repeatedly to give 3(3.2mg), 7(3.2mg) and 8(4.0 mg).
Example 2: anti-inflammatory Activity of Compounds on lipopolysaccharide
1. Experimental Material
1.1 Experimental reagents: compounds 1-15 mouse mononuclear macrophages RAW 264.7(ATCCTIB-71) were purchased from Chinese academy of sciences RPMI 1640 medium, penicillin, streptomycin and fetal bovine serum were purchased from Invitrogen, USA. Lipopolysaccharide (LPS), dimethyl sulfoxide (DMSO) and hydrocortisone were purchased from Sigma.
2 test procedure
2.1 cell culture and treatment
RAW 264.7 cells were cultured in RPMI 1640 medium (5% CO2 and 95% air) containing penicillin (100U/ml), streptomycin (100g/ml) and 10% heat-inactivated fetal bovine serum, with medium changes occurring on average every two days. 264.7 cells were trypsinized to confluence and examined during exponential growth. Compounds 1-15 were dissolved in cell culture levels of dimethyl sulfoxide (DMSO) and added to the culture media to give a concentration of 0.2% (v/v). DMSO solution had no significant effect on the growth of the cell lines tested. Controls were treated with equal amounts of DMSO.
2.2 nitric oxide analysis
Cell concentration was adjusted to 106Perml, 96-well plates were inoculated with 200. mu.L per well. After 1h of cell incubation, cells were treated with 1. mu.g/mL LPS and different concentrations of test substance were added for 24 h. Determination of NO in 264.7 cell culture supernatants by Griess reaction2 -And (4) concentration. NO2 -The concentration was measured using a standard curve of 0, 1, 2, 5, 10, 20, 50, 100. mu.M sodium nitrite. By NO2-Levels LPS-induced inhibition of NO production was calculated as follows:
Figure BDA0001995905780000152
3 data analysis
Each set of experiments was repeated 3 times and the data represent the mean (± s.d) of three independent experiments.
4 results of the experiment
The results of the experiments are shown in the table, and compared with the positive control group, the compounds 1,4,9,10 and 13 significantly inhibited the production of NO induced by LPS. IC (integrated circuit)50The value was 11.93-30.97. mu.M.
Figure BDA0001995905780000151
a none of the other 9 compounds were active at a concentration of 100. mu.M.
b HSS, Positive control
Anti-inflammatory experiments showed that compounds 1,4,9,10 and 13 significantly inhibited Nitric Oxide (NO) production in Lipopolysaccharide (LPS) -induced RAW 264.7 macrophages with IC50 values ranging from 11.93 to 30.97 μ M. The other 9 compounds were inactive at a concentration of 100. mu.M.

Claims (9)

1. Dihydro agarofuran type sesquiterpene compounds and pharmaceutically acceptable salts thereof, which are shown in the following structures:
Figure DEST_PATH_IMAGE002
。
2. the compound of claim 1, wherein: the compound is extracted and separated from dried leaves of Tripterygium wilfordii hook F of Tripterygium in Celastraceae.
3. The method for preparing dihydroagarofuran type sesquiterpene compound according to claim 1, which comprises the following steps:
(1) extracting aerial part of Tripterygium wilfordii (Tripterygium wilfordii) with flash extractor, mixing extractive solutions, and concentrating under reduced pressure to obtain total extract;
(2) suspending the total extract with water, sequentially extracting with ethyl acetate and n-butanol to obtain ethyl acetate layer and n-butanol layer respectively;
(3) separating ethyl acetate layer sample by gel column chromatography, silica gel column chromatography, HP-20 column chromatography, ODS column chromatography and preparative HPLC.
4. The method according to claim 3, wherein the extraction method in step (1) is flash extraction, and the extraction solvent is: extracting for 3-5 times by using 70-80% ethanol, wherein the weight volume ratio of the medicinal materials to the extraction solvent is 2: 5-8.
5. The method according to claim 3, wherein the elution solvent for the gel column chromatography in the step (3) is a solvent having a volume ratio of 100: 1-1: 1 petroleum ether-EtOAc mixed system.
6. The process according to claim 3, wherein the eluting solvent in the silica gel column chromatography in the step (3) is a solvent having a volume ratio of 25: 1-1: 1 petroleum ether-ethyl acetate.
7. A pharmaceutical composition comprising the dihydroagarofuran type sesquiterpene compound of claim 1 and pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier or excipient.
8. Use of the dihydroagarofuran type sesquiterpene compound of claim 1 or the pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 7 in the preparation of NO inhibitors.
9. Use of the dihydroagarofuran type sesquiterpene compound of claim 1 or the pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 7 in the preparation of anti-inflammatory drugs.
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JP2005320307A (en) * 2004-05-11 2005-11-17 Nitto Denko Corp New sesquiterpene compound and method for producing the same and composition
CN103073529A (en) * 2013-01-03 2013-05-01 西北农林科技大学 2-furoacridone-beta-dihydroagarofuran sesquiterpene compound in leafy parnassia, and preparation method and application thereof

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Protective effects of β-dihydroagarofuran-type sesquiterpene against Aβ25-35-induced neuronal apoptosis and oxidative damage;Shasha Ji et al.;《Journal of Chinese Pharmaceutical Sciences》;20161231;第25卷(第8期);第582-589页 *
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