阅读说明：本技术 黄瑞香中的异戊烯基黄酮类化合物及其用途 (Isopentene flavonoid compound in daphne giraldii nitsche and application thereof ) 是由 李玲芝 宋少江 刘莹 于 2020-04-29 设计创作，主要内容包括：本发明属于医药技术领域,涉及异戊烯基黄酮类化合物及其用途,具体涉及从瑞香科瑞香属植物黄瑞香(Daphne giraldii Nitsche.)根茎皮中提取分离的八个异戊烯基黄酮类化合物。将黄瑞香干燥根茎皮用乙醇提取,将粗提物依次经硅胶柱色谱,聚酰胺柱色谱,MCI柱色谱,ODS柱色谱,凝胶柱色谱及HPLC分离的色谱方法进行分离。通过核磁技术对化合物结构进行鉴定,利用手性HPLC对化合物4和5进行手性拆分,最终得到两对对映异构体(4a,4b和5a,5b)。本发明所述的化合物对Hep3B细胞系的具有细胞毒作用和促凋亡作用。本发明的化合物对成纤维生长因子受体1(FGFR1)具有抑制活性。<Image he="391" wi="700" file="DDA0002472491350000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention belongs to the technical field of medicines, relates to isopentenyl flavonoid compounds and application thereof, and particularly relates to eight isopentenyl flavonoid compounds extracted and separated from root bark of Daphne giraldii Nitsche (Daphne giraldii) of Daphne of Thymelaeaceae. Extracting dried cortex Daphne Giraldii Nitsche with ethanol, and sequentially subjecting the crude extract to silica gel column chromatography, polyamide column chromatography, and MCThe compound has cytotoxic effect and apoptosis promoting effect on a Hep3B cell line, and has inhibitory activity on fibroblast growth factor receptor 1(FGFR 1).)

1. Isopentenyl flavonoid compound or salt thereof:

2. the method for producing an isopentenyl flavonoid compound or a salt thereof according to claim 1, wherein the method comprises the following steps:

(1) cold soaking dried cortex daphne giraldii nitsche with ethanol, mixing extractive solutions, concentrating to obtain extract, sequentially extracting the extract with ethyl acetate and n-butanol to obtain ethyl acetate layer, n-butanol layer and water layer extract;

(2) subjecting the ethyl acetate layer extract to silica gel chromatography column with dichloromethane-methanol system (100:1-8:1) for rapid gradient elution, and separating into four fractions A, B, C, D by silica gel thin layer chromatography;

(3) subjecting fraction B to rapid polyamide column chromatography, performing rapid gradient elution with ethanol-water system (40% -100%), and mixing by silica gel thin layer chromatography to obtain five fractions B₁、B₂、B₃、B₄、B₅；

(4) Fraction B₂、B₃、B₄Subjecting to MCI column chromatography, gradient eluting with ethanol-water system (20% -80%), mixing by silica gel thin layer chromatography, and collecting five components B_(2-4)-1、B_(2-4)-2、B_(2-4)-3、B_(2-4)-4、B_(2-4)-5；

(5) Component B obtained_(2-4)-5Performing ODS column chromatography with methanol-water (20% -80%) for isocratic elution to obtain six groups B_(2-4)-5-1～B_(2-4)-5-6。

(6) The half-prepared HP L C was used to separately identify component B_(2-4)-5-1～B_(2-4)-5-6Elution with acetonitrile in water system provided compounds 1-6.

3. The method according to claim 2, wherein the ethanol in step (1) is 70-95% industrial ethanol, and the extraction is performed 2-3 times.

4. The preparation method according to claim 2, wherein the volume ratio of the ethyl acetate, the n-butanol and the extract in the step (1) is 1:1, and the extraction is performed for 3-4 times.

5. The method according to claim 2, wherein the dried bark of Daphne giraldii is a dried bark of Daphne giraldii Nitsche (Daphne giraldii Nitsche.) belonging to the genus Daphne of the family daphneceae.

6. The method of claim 2, wherein compounds 4 and 5 are subjected to chiral resolution using a Daicel Chiralpak AD-H chiral column, and the mobile phase is a mixed n-hexane-isopropanol solvent in the ratio of 3:1 and 5:1, respectively, to give compounds 4a,4b and 5a,5 b.

7. A pharmaceutical composition comprising the prenyl flavonoid or salt thereof of claim 1 and a pharmaceutically acceptable carrier or excipient.

8. Use of the prenyl flavonoid or the salt thereof according to claim 1 or the pharmaceutical composition according to claim 7 in the preparation of an antitumor drug.

9. The use of claim 8, wherein the tumor is liver cancer.

10. Use of the prenylflavonoid compound or the salt thereof according to claim 1 for the preparation of a fibroblast growth factor receptor 1 inhibitor.

The technical field is as follows:

the invention belongs to the technical field of medicines, relates to isopentenyl flavonoid compounds and application thereof, and particularly relates to 8 isopentenyl flavonoid compounds (comprising two pairs of enantiomers) separated from medicinal plant daphne giraldii root bark and application thereof in preparing antitumor drugs.

Background

Daphne giraldii Nitsche, also called flos buddlejae and new flos buddlejae, which are Daphne plants in Daphne of daphneceae, are shrubs with upright fallen leaves, mainly distributed in provinces such as shanxi, gansu, sichuan, Qinghai and Ningxia, and also distributed in Henan, Jiangxi and Shanxi, and mostly grow in forests in mountainous regions with an elevation of about 1600 m and in the negative wetlands under broadleaf trees. The root bark and stem bark of the medicine can be used as a girald daphne herb medicine, has warm property, pungent and bitter taste and small toxicity, has the effects of dispelling wind and eliminating dampness, relieving pain and dissipating blood stasis, is mainly used for treating diseases such as rheumatic arthralgia, traumatic injury, arthritis and the like, is widely used in northwest China and folks, and has good effects of treating rheumatoid arthritis and lumbocrural pain.

The daphne giraldii nitsche tablet, the daphne giraldii nitsche joint pain relieving ointment, the daphne giraldii nitsche injection and the like in national important protection varieties issued by the ministry of health of China all use daphne giraldii nitsche as a main raw material. Modern pharmacological research shows that the daphne giraldii nitsche has the effects of resisting inflammation, easing pain, resisting tumor, resisting malaria and the like. In recent years, a plurality of active ingredients such as coumarins, flavonoids, diterpenes, lignans and the like are separated from daphne giraldii, the coumarins are characteristic ingredients and effective ingredients of daphne giraldii and generally exist in daphne plants, wherein the content of daphne giraldii (daphne methyl element) and daphne glycoside are the highest, daphne is a protein kinase inhibitor, and the daphne is capable of inhibiting the growth of liver cancer cells SMMC-7721, sarcoma cells S180 and kidney cancer cells RCC to different degrees in an effective concentration range and obviously enhancing the cellular immunity, nonspecific immunity and erythrocyte immunity functions of S180 tumor-bearing mice. The flavonoid compound is another main chemical component in daphne giraldii, is mostly from the root bark of plants, and the isopentene flavonoid component has good selective cytotoxicity on human liver cancer cell lines HepG2 and Hep3B and has activity on a fibroblast growth factor FGFR 1.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a series of isopentene group flavonoid compounds or salts thereof, which have the following structures:

the isopentenyl flavonoid compound is obtained by simultaneously separating Daphne giraldii nitsche (Daphne giraldii nitsche.) of Daphne of Thymelaeaceae.

The invention provides a preparation method of an isopentene flavonoid compound, which comprises the following steps:

(1) cold soaking dried cortex daphne giraldii nitsche with ethanol, mixing extractive solutions, concentrating to obtain extract, sequentially extracting the extract with ethyl acetate and n-butanol to obtain ethyl acetate layer, n-butanol layer and water layer extract;

(2) subjecting the ethyl acetate layer extract to silica gel chromatography column with dichloromethane-methanol system (100:1-8:1) for rapid gradient elution, and separating into four fractions A, B, C, D by silica gel thin layer chromatography;

(3) subjecting fraction B to rapid polyamide column chromatography, performing rapid gradient elution with ethanol-water system (40% -100%), and mixing by silica gel thin layer chromatography to obtain five fractions B₁、B₂、B₃、B₄、B₅；

(4) Fraction B₂、B₃、B₄Subjecting to MCI column chromatography, gradient eluting with ethanol-water system (20% -80%), mixing by silica gel thin layer chromatography, and collecting five components B_(2-4)-1、B_(2-4)-2、B_(2-4)-3、B_(2-4)-4、B_(2-4)-5；

(5) Component B obtained_(2-4)-5Performing ODS column chromatography with methanol-water (20% -80%) for isocratic elution to obtain six groups B_(2-4)-5-1～B_(2-4)-5-6。

(6) The half-prepared HP L C was used to separately identify component B_(2-4)-5-1～B_(2-4)-5-6Elution with acetonitrile in water system provided compounds 1-6.

Wherein the ethanol in the step (1) is 70-95% industrial ethanol, and is extracted for 2-3 times. Extracting for 3-4 times with ethyl acetate, n-butanol and extract at volume ratio of 1: 1;

the acetonitrile water system in the step (6) is 50-70%.

Further, chiral resolution is carried out on the compounds 4 and 5 to obtain the compounds 4a,4b and 5a,5b, and the chiral preparation resolution method comprises the steps of utilizing a Daicel Chiralpak AD-H chiral chromatographic column, wherein a mobile phase is a n-hexane-isopropanol mixed solvent, the ratio is 3:1 and 5:1, the flow rate is 1.5m L/min, and the detection wavelength of an ultraviolet detector is 210 nm.

The chiral resolution conditions described in the present invention apply to compounds 4 and 5.

The structure identification result of the compound obtained by the invention is as follows:

the planar structure and relative configuration of the compound are determined by high resolution mass spectrometry and one-dimensional and two-dimensional NMR techniques. The absolute configuration of resolved optically pure compounds 4a-4b and 5a-5b was determined by comparing measured CD with literature experience.

Compound 1: yellow solid (methanol). HRESI-MS gave an excimer peak 543.2266[ M + Na ]]⁺(calcdfor 543.2353). Bonding of¹H,¹³C-NMR data confirm that the molecular formula is C₃₁H₃₆O₇The unsaturation degree was 14.¹H-NMR(400MHz CD₃OD) shows that: 6.72(1H, s),6.25(1H, s) are two aromatic ring proton signals, and the hydrogen spectrum also gives proton signals for the tri-isoamylene substituent group 5.10(1H, overlaid), 5.07(1H, overlaid), 4.93(1H, m),3.44(2H, d, J ═ 6.34Hz),3.33(2H, d, J ═ 4.76Hz),3.26(2H, d, J ═ 5.78Hz),1.74(3H, s),1.69(3H, s),1.58(3H, s),1.47(3H, s),1.45(3H, s),1.30(3H, s), and furthermore, 3.57(3H, s) is a set of methoxy proton signals.¹³C-NMR(100MHz CD₃OD) spectrum gives a 31 carbon signal, with 180.4,163.5,162.1,160.7,156.3,146.9,143.7,140.4,132.8,129.0,122.5,115.3,108.0,106.2,99.4 suggesting a 15 carbon signal for the flavone mother nucleus, 132.3,131.9,131.8,125.1,124.6,123.429.4,26.3,26.0 × 2,25.7,22.4,18.1,17.8 × 2 suggest a triosopentyl carbon signal and 61.0 a methoxy carbon signal.

The direct connected hydrogen-carbon signals are subjected to attribution through HSQC spectrum related signals. In the HMBC spectrum, there was observed a long-range correlation between 6.25 and 163.5(C-7),160.7(C-5),108.0(C-8),106.2(C-10), 3.33 and 163.5(C-7),156.3(C-9),108.0(C-8), indicating that 6.25 is the proton signal at position 6 of the flavone A ring and that position 8 is substituted with isopentenyl. 6.72 is remotely related to 162.1(C-2),146.9(C-4 '), 143.7(C-3 '), indicating that 6.72 is the proton signal at the 2 ' position of the B ring of flavone. The presence of remote associations of 3.44 with 146.9(C-4 '), 129.0 (C-5'), 3.26 with 129.0(C-5 '), 122.5 (C-1'), indicates that the 5', 6' position of the B ring is substituted by isopentenyl. The presence of 3.57 in remote relation to 140.4(C-3) indicates that the 3-position of the C ring is substituted by methoxy. And C-5,7,3 ', 4' is judged to be substituted by-OH by combining HMBC related signals and mass spectrum information. Thus defining the planar structure of the compound.

Compound 2: yellow solid (methanol).¹H-NMR (400MHz DMSO) showed: 6.63(1H, s),6.29(1H, s) are two aromatic ring proton signals, and the hydrogen spectrum also gives proton signals for the three isopentene substituents of 5.28(1H, t, J ═ 7.35Hz),5.02(1H, t, J ═ 7.11Hz),4.96(1H, t, J ═ 6.88Hz),3.26(4H, overlaid), 3.21(2H, d, J ═ 7.01Hz),1.67(3H, s),1.65(3H, s),1.54(3H, s),1.44(3H, s),1.41(3H, s),1.25(3H, s), furthermore, 3.56(3H, s) is a set of methoxy proton signals, and 12.60(1H, brs) is a 5-position hydroxyl signal.¹³The C-NMR (150MHz DMSO) spectrum gave 31 carbon signals, of which 178.2,161.8,159.5,158.9,154.2,145.3,143.2,138.5,126.1,125.9,121.4,121.1,105.8,104.4,98.3, indicated 15 carbon signals for the flavone mother nucleus, 131.7,130.8,130.0,123.0,122.4,122.0,27.9,25.6,25.5,25.4,25.2,21.0,17.6,17.4,17.3 indicated three groups of isopentenyl carbon signals, and 59.8 indicated one group of methoxy carbon signals. The structure of the compound is further determined by combining two-dimensional spectrum HSQC and HMBC data.

The direct connected hydrogen-carbon signals are subjected to attribution through HSQC spectrum related signals. In HMBC spectra, a long-range correlation between 6.29 and 158.9(C-5),105.8(C-8),104.4(C-10), 3.21 and 161.8(C-7),154.2(C-9) was observed, indicating that 6.29 is the proton signal at position 6 of the flavone A ring and that position 8 is substituted with isopentenyl. The presence of 6.63 and 159.5(C-2),145.3(C-4 '), 126.1(C-5 ') correlated remotely, indicating that 6.72 is the proton signal at the 6' position of the flavone B ring. The long-range associations of 3.27 with 143.2(C-3 '), 3.26 with 145.3 (C-4'), 6.63 with 25.6(C-1 "") exist, indicating that the 2 ',5' position of the B ring is substituted with isopentenyl. The presence of 3.56 in remote relation to 138.5(C-3) indicates that the 3-position of the C ring is substituted by methoxy. And C-5,7,3 ', 4' is judged to be substituted by-OH by combining HMBC related signals and mass spectrum information. Thus defining the planar structure of the compound.

Compound 3: yellow solid (methanol). HRESI-MS gave an excimer peak 541.2175[ M + Na ]]⁺(calcdfor 541.2197). Bonding of¹H,¹³C-NMR data confirm that the molecular formula is C₃₁H₃₄O₇The unsaturation degree was 15.¹H-NMR (600MHz DMSO) showed: the hydrogen spectrum also gives the proton signals for the isopentene substituent group of two aromatic rings 5.08(1H, t, J ═ 7.25Hz),5.00(1H, t, J ═ 6.97Hz),3.39(2H, d, J ═ 6.85Hz),3.32(2H, overlaid), 1.57(3H, s),1.46(3H, s),1.42(3H, s),1.30(3H, s), the proton signals for the substituent group of 2 "", 2 "" -dimethylpyran 6.34(1H, d, J ═ 9.81Hz),5.08(1H, d, J ═ 9.78Hz),1.49(6H, s), and furthermore, 3.59(3H, s) is the proton signal for the proton substituent group of one methoxy group.¹³C-NMR (150MHz DMSO) spectrum gives 31 carbon signals, wherein 180.4,164.0,161.2,160.8,156.5,144.7,143.0,140.5,129.9,124.3,120.6,119.7,108.1,106.2,99.6 indicates 15 carbon signals of a flavone mother nucleus, 132.3 × 2,124.0,123.5,26.8,26.1,25.8,22.5 and 17.8 × 2 indicates a isopentenyl carbon signal, 132.1,122.8,78.6 and 28.2 × 2 indicate proton signals of a group of 2 ', 2' -dimethyl pyran substituents, and 61.1 indicates a group of methoxy carbon signals.

The direct connected hydrogen-carbon signals are subjected to attribution through HSQC spectrum related signals. In HMBC spectra, the presence of long range correlations between 6.26 and 160.8(C-5),108.1(C-8), 3.32 and 164.0(C-7),156.5(C-9),108.1(C-8) was observed, indicating that 6.26 is the proton signal at position 6 of the flavone A ring and that position 8 is substituted with isopentenyl. The existence of remote correlation between 6.63 and 161.2(C-2) indicates that 6.63 is the proton signal at the 6' position of the B ring of flavone. 3.39 is remotely related to 144.7(C-3 '), 129.9(C-2 '), indicating that the 2 ' position of the B ring is substituted with isopentenyl. The proton signal 6.34 at the 4 "" position in the 2 "" dimethylpyran substituent is remotely related to 126.6(C-5'),119.7(C-6'), indicating that the 2 "" dimethylpyran substituent is attached at the 4',5' position of the B ring. The presence of 3.59 in remote relation to 140.5(C-3) indicates that the 3-position of the C ring is substituted by methoxy. And C-5,7,3 ', 4' is judged to be substituted by-OH by combining HMBC related signals and mass spectrum information. Thus defining the planar structure of the compound.

Tables 11 to 3¹H and¹³c NMR data

Compound 4: yellow solid (methanol). HRESI-MS gave an excimer peak 515.2339[ M + Na ]]⁺(calcd.515.2404). Bonding of¹H,¹³C-NMR data confirm that the molecular formula is C₃₀H₃₆O₆The unsaturation degree was 13.¹H-NMR(600MHz CD₃OD) shows that: 4.88(1H, d, J ═ 11.27Hz),4.44(1H, d, J ═ 11.19Hz) are characteristic proton signals at positions 2 and 3 of flavanonols, 5.94(1H, s) are proton signals at position 6 of flavanonol a ring, 7.07(2H, s) are proton signals at positions 2 ', 6' of B ring, and furthermore, proton signals for three groups of isopentene substituents are given in the hydrogen spectrum 5.34(2H, m),5.12(1H, m),3.35(4H, d, J ═ 7.27Hz),3.12(2H, d, J ═ 7.27Hz),1.74(6H, s),1.72(6H, s),1.59(3H, s),1.49(3H, s).¹³C-NMR(150MHz CD₃OD) spectrum, wherein 197.9,169.0,163.2,161.2,153.9,130.1 × 2,129.8,127.8 × 2,109.8,101.4,97.4,85.2,73.8 suggests a 15 carbon signal for the mother core of flavanonol, 133.6 × 2,131.5,123.8 × 2,124.1,29.8 × 2,26.1 × 3,22.5,18.0 × 3 suggests three different groupsPentenyl carbon signal. The structure of the compound is further determined by combining two-dimensional spectrum HSQC and HMBC data.

The direct connected hydrogen-carbon signals are subjected to attribution through HSQC spectrum related signals. In the HMBC spectrum, the long-range correlation between 4.88 and 161.2(C-10), between 3.12 and 169.0(C-7), between 161.2(C-9) and 109.8(C-8) is observed, indicating that the 8-position of the A ring is substituted by isopentenyl, and the long-range correlation between 5.94 and 169.0(C-7), between 163.2(C-5), between 109.8(C-8) and between 101.4(C-10) is observed, indicating that the 5.94 is the 6-position proton signal of the A ring. There is a long-range correlation between 7.07(2H, s) and 153.9(C-5 '), 130.1(C-3 ', 4 '), 85.2(C-2),29.8(C-1 ', 1 '), indicating that 7.07 is a proton signal at the 2 ', 6' position of the B ring, and that the 3 ', 4' positions are substituted with isopentenyl groups, respectively. And C-5,7, 4' is judged to be substituted by-OH by combining HMBC related signals and mass spectrum information. Thus defining the planar structure of the compound.

In that¹In the H-NMR spectrum, the relative configuration of the C-2, C-3 positions, i.e. H-2, -3 is in trans-di-orthosteric bond, can be determined from 4.88(1H, d, J ═ 11.27Hz),4.44(1H, d, J ═ 11.19Hz), however, since this compound lacks the optical activity of Circular Dichroism (CD), this indicates its racemic nature, then, separation of the two enantiomers 4a and 4b is caused by chiral HP L C, by the CD spectrum of 4a, a negative Cotten effect is shown at 291nm, indicating that C-2 is assigned to S configuration, by the trans-double bond between H-2 and H-3, the configuration of C-3(S) of 4a is further determined, whereas 4b is opposite to the CD curve of 4a, so the configurations of 4b are assigned to C-2(R) and C-3 (R).

Compound 5: brown solid (methanol). HRESI-MS gave an excimer peak 401.2092[ M + Na ]]⁺(calcd.401.2087). Bonding of¹H,¹³C-NMR data confirm that the molecular formula is C₂₅H₃₀O₃The unsaturation degree was 11.¹H-NMR(600MHz CD₃OD) shows that: 4.80(1H, dd, J ═ 9.69,2.32Hz),2.74(1H, m),2.56(1H, m),2.00(1H, m),1.90(1H, m) are characteristic proton signals at 2,3,4 positions of flavanoids, 6.80(1H, d, J ═ 8.24Hz),6.29(1H, dd, J ═ 8.22,2.46Hz),6.23(1H, d, J ═ 2.44Hz) are proton signals at 2 ', 6' positions of B ring, 6.91(2H, s) are proton signals at 2 ', 6' positions of B ring, and furthermore, proton signals at 5.28(2H, m),3.29 (c), which give two groups of isopentene substituents in the hydrogen spectrum4H,overlapped),1.70(6H,s),1.66(6H,s)。¹³C-NMR(150MHz CD₃OD) spectrum, wherein 157.6,157.2,153.0,134.6,131.0,129.8 × 2,126.0 × 2,114.4,109.1,104.1,79.2,31.2,25.3 suggests a 15 carbon signal for the flavan parent nucleus, 133.5 × 2,123.9 × 2,29.8 × 2,26.0 × 2,18.0 × 2 suggests two sets of isopentenyl carbon signals.

The direct connected hydrogen-carbon signals are subjected to attribution through HSQC spectrum related signals. In the HMBC spectrum, there is a long-range correlation between 6.80 and 25.5, and between 6.29 and 157.6,131.0,104.1, and the combination of the a-ring proton coupling constants indicates that 6.80,6.29 and 6.23 are proton signals at positions 5,6 and 8 of the a-ring, respectively. The presence of remote associations of 6.91(2H, s) with 153.0(C-5 '), 79.2(C-2),3.29(4H, overlapped) and 153.0,129.8,126.1 indicates that 6.90 is the proton signal at the 2 ', 6' position of the B ring and that the 3 ', 4' positions are respectively substituted with isopentenyl groups. And C-7, 4' is judged to be substituted by-OH by combining HMBC related signals and mass spectrum information. Thus defining the planar structure of the compound.

The compound has an asymmetric carbon center at C-2, since there are two equal peaks in chiral HP L C, 5a and 5b are considered to be equal amounts of the racemate, and were successfully obtained in chiral HP L C.the CD spectrum of 5a shows a positive Cotten effect at 290nm, so the absolute configuration at C-2 is designated R. while 5b shows an opposite CD curve compared to 5a, and the absolute configuration of 5b is designated C-2 (S).

Compound 6: brown solid (methanol). HRESI-MS gave an excimer peak 417.1980[ M + Na ]]⁺(calcd.417.2036). Bonding of¹H,¹³C-NMR data confirm that the molecular formula is C₂₅H₃₀O₄The unsaturation degree was 11.¹H-NMR(600MHz CD₃OD) shows that: 4.70(1H, d, J ═ 6.59Hz),3.99(1H, dd, J ═ 11.73,7.07,2.16Hz),2.79(1H, dd, J ═ 15.81,4.83Hz),2.64(1H, dd, J ═ 15.68,7.43Hz) are characteristic proton signals at 2,3,4 positions of flavan-3-ols, 6.83(1H, d, J ═ 8.26Hz),6.33(1H, dd, J ═ 8.22,2.45Hz),6.27(1H, d, J ═ 2.41Hz) are proton signals at 2, 6.90(2H, s) at 2 ', 6' positions of the B ring, and two groups of hydrogen spectra are givenProton signals for the isoamylene substituent 5.28(2H, m),3.28(4H, overlaid), 1.70(6H, s),1.65(6H, s).¹³C-NMR(150MHz CD₃OD) spectrum, wherein 158.1,156.3,153.2,132.1,131.4,129.8 × 2,126.6 × 2,112.6,109.5,103.7,83.0,68.9,32.6 suggests a 15 carbon signal for the flavan-3-ol parent nucleus, 133.7 × 2,123.7 × 2,29.7 × 2,26.0 × 2,17.9 × 2 suggests two sets of isopentenyl carbon signals.

The direct connected hydrogen-carbon signals are subjected to attribution through HSQC spectrum related signals. In the HMBC spectrum, there is a long-range correlation between 6.83,6.27,4.70 and 156.3, 6.33 and 158.1,112.6,103.7, and the combination of the a ring proton coupling constants indicates that the 6.83,6.33 and 6.27 are proton signals at positions 5,6 and 8 of the a ring, respectively. The long-range correlation between 6.90(2H, s) and 153.2(C-5 '), 129.8(C-3 ', 4 '), 83.0(C-2),29.8(C-1 ', 1 ') indicates that 6.90 is the proton signal at the 2 ', 6' position of the B ring, and that the 3 ', 4' positions are respectively substituted by isopentenyl groups. And C-7, 4' is judged to be substituted by-OH by combining HMBC related signals and mass spectrum information. Thus defining the planar structure of the compound.

By aligning it¹Examination of the H NMR data determined the relative configuration of 6. The coupling constant for H-2 (J ═ 6.59Hz) indicates that H-2, H-3 have the same orientation. In a CD spectrum, the Cotten effect is a negative minimum value at 280-290 nm, which indicates that 6 is a single-configuration chiral compound and the absolute stereochemistry of C-2 is R. On this basis, the absolute configuration of C-3 is designated R because H-2, H-3 are located on the same side of the C-ring. Thus, the structure of compound 6 was determined.

Tables 24 to 5¹H and¹³c NMR data

Drawings

HRESIMS spectra of compound 1 of figure 1;

FIG. 2 HMBC spectra (600MHz, CD) of Compound 1₃OD)；

FIG. 3 HSQC spectra (600MHz, CD) of Compound 1₃OD)；

FIG. 4 HMBC spectra (600MHz, DMSO) of Compound 2;

FIG. 5 HSQC spectra (600MHz, DMSO) of Compound 2;

FIG. 6 HRESIMS spectrum of Compound 3;

FIG. 7 HMBC spectra (600MHz, CD) of Compound 3₃OD)；

FIG. 8 HSQC spectra (600MHz, CD) of Compound 3₃OD)；

FIG. 9 HRESIMS spectrum of Compound 4;

FIG. 10 HMBC spectra (600MHz, CD) of Compound 4₃OD)；

FIG. 11 HSQC spectra (600MHz, CD) of Compound 4₃OD)；

FIG. 12 chiral resolution chromatograms of compounds 4a and 4 b;

FIG. 13 Compounds 4a and 4b measured CD;

FIG. 14 HRESIMS spectrum of Compound 5;

FIG. 15 HMBC spectra (600MHz, CD) of Compound 4₃OD)；

FIG. 16 HSQC spectra (600MHz, CD) of Compound 5₃OD)；

Figure 17 chiral resolution chromatograms of compounds 5a and 5 b;

FIG. 18 Compounds 5a and 5b measured CD;

FIG. 19 HRESIMS spectrum of Compound 6;

FIG. 20 HMBC spectra (600MHz, CD) of Compound 6₃OD)；

FIG. 21 HSQC spectra (600MHz, CD) of Compound 6₃OD)；

Figure 22 compound 6 measured CD.

Detailed Description

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.