CN106928170B - Dihydrofuranbiphenyl compound and preparation method and application thereof - Google Patents
Dihydrofuranbiphenyl compound and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a dihydrofuran biphenyl compound, a preparation method and application thereof, wherein the molecular formula of the biphenyl compound is C18H18O4The compound is named as 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxypropyl) -dihydrobenzofurane and has the following structural formula:
the preparation method takes dried branches, leaves and/or fruits of trees of Guttiferae as raw materials, and comprises the steps of extract extraction, organic solvent extraction, silica gel column chromatography and high performance liquid chromatography separation. The application is the application of biphenyl compounds in preparing anti-rotavirus medicaments. By the experiment of anti-rotavirus activity, 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxyphenyl) -dihydrobenzofran is selected as a reference to control the CC of rotavirus50And EC50Values of 164.2 and 12.9 respectivelyμmol/L, it has better anti-rotavirus activity. The compound has simple structure and good activity, can be used as a leading compound of an anti-rotavirus medicament, and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of extraction of effective components of plants, and particularly relates to a dihydrofuran biphenyl compound and a preparation method and application thereof.
Background
Guttiferae (Guttiferae)Garcinia L.) The plants are about 450 types in the whole world, produced in Asia, south Africa and west Borini, and 21 types are distributed in southern provinces such as Guangdong, Guangxi and Yunnan China. The gambogic plant is also one of main resources of natural xanthone (xanthones) components, is rich in isopentenyl substituted xanthone (xanthones), has novel and various structures and wide pharmacological activity, particularly has the most representative gambogic acid (gambogic acid) and broad-spectrum and strong anti-tumor activity, is one of the research hotspots of anti-tumor natural products in recent years, and Chinese scholars areThe injection is a new anti-tumor drug. In addition to xanthones, benzophenones, biflavonoids and biphenyls are characteristic components of plants of this family and have various biological activities. In order to more effectively utilize the gambogic plant resources in China and search for active ingredients with development prospects, systematic active ingredient research work is selected to be carried out on the gambogic plants.
Disclosure of Invention
The first purpose of the invention is to provide a biphenyl compound; the second purpose is to provide a preparation method of the biphenyl compound; the third purpose is to provide the application of the biphenyl compound in preparing anti-rotavirus medicaments.
The first purpose of the invention is realized by that the biphenyl compound is obtained by using dried branches, leaves or fruits of arbors of Guttiferae as raw materials and performing extract extraction, organic solvent extraction, silica gel column chromatography and high performance liquid chromatography separation, and the molecular formula of the compound is C18H18O4Named as 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxypropyl) -dihydrobenzofurans, has the following structural formula:
the second purpose of the invention is realized by that the preparation method of biphenyl compounds is obtained by using dried branches, leaves and/or fruits of arbors of Guttiferae as raw materials and performing extract extraction, organic solvent extraction, silica gel column chromatography and high performance liquid chromatography separation, and specifically comprises the following steps:
A. extracting the extractum: coarsely crushing branches, leaves or fruits of arbors of the family Guttiferae to 20-40 meshes, ultrasonically extracting for 40-60 min each time for 2-4 times by using an organic solvent, and mixing extracting solutions; filtering the extracting solution, concentrating the extracting solution under reduced pressure to 1/4-1/2 volume, standing, filtering out precipitates, and concentrating to obtain an extract a;
B. organic solvent extraction: adding 1-2 times of water by weight into the extract a, extracting for 3-5 times by using an organic solvent with the same volume as the water, combining organic solvent extraction phases, and concentrating under reduced pressure to obtain an extract b;
C. silica gel column chromatography: dissolving the extract b by using an organic solvent with the weight ratio of 1.5-3 times, mixing the sample by using 100-200 meshes of silica gel with the weight of 0.8-1.2 times of that of the extract, and performing silica gel column chromatography, wherein the silica gel filled in the column is 100-200 meshes, and the using amount of the silica gel is 6-8 times of that of the extract b; gradient eluting with a mixed organic solvent with a volume ratio of 1: 0-0: 1, collecting gradient eluent, concentrating, monitoring by TLC, and combining the same parts;
D. reversed-phase column chromatography: carrying out reverse phase column chromatography on eluent obtained by eluting with organic solvent in the ratio of 7:3, wherein the reverse phase column is filled with reverse phase materials of C-8, C-18, ODS or MCI; performing gradient elution by using a methanol aqueous solution with the volume content of 20-100%, collecting eluent of each part, concentrating, monitoring by TLC, and combining the same parts;
E. high performance liquid chromatography separation: separating and purifying eluent obtained by eluting with 55-80% methanol water solution by volume through high performance liquid chromatography to obtain the biphenyl compound 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxyphenyl) -dihydrobenzofuran;
F. and E, performing high performance liquid chromatography separation and purification by taking 50-70% methanol as a mobile phase, taking a Zorbax PrepHT GF reversed-phase preparation column with the flow rate of 10-14 mL/min and the flow rate of 21.2 x 250mm and the thickness of 5um as a stationary phase, taking an ultraviolet detector with the detection wavelength of 254nm, feeding 45-60 uL of sample each time, collecting chromatographic peaks for 16-32 min, and evaporating to dryness after multiple accumulation. Thus obtaining the biphenyl compound 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxyphenyl) -dihydrobenzofurans.
The biphenyl compound is separated for the first time, is determined to be the biphenyl compound by a nuclear magnetic resonance and other spectrum technology determination method, and is characterized in that the specific structure is as follows:
the compound 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxyphenyl) -dihydrobenzofurane as an orange yellow gum; ultraviolet spectrum (solvent)The agent is methanol),λ max（logε): 570 (1.79),266 (3.69), 226 (3.82), 204 (3.96) nm; infrared spectrum (Potassium bromide tablet)V max3432, 2924,2852, 1630, 1503, 1450, 1374, 1243, 1173, 1096, 1054, 1027, 905, 855, 824,591 cm–1(ii) a HRESIMS shows the peak of the excimer of the compound of the inventionm/z298.1202 [M]+(calculated 298.1205), combined13C and1h NMR spectrum (FIG. 1 and FIG. 2, attribution of hydrogen spectrum data of carbon spectrum in FIG. 4) gives the molecular formula C18H18O4。1H NMR（C5D5N, 500 MHz) and13C NMR（C5D5n, 125 MHz) data, see fig. 4.
HRESIMS showed that its excimer peak was 298.1202 [ M ]]+(calculated 298.1205), combined13Determining molecular formula as C by CNMR spectrum18H18O4The unsaturation degree was 10. The infrared absorption spectrum is 3432cm-1Indicating the presence of hydroxyl groups at 1630, 1503 cm-1Indicating the existence of the benzene ring, and the existence of the benzene ring is also confirmed by the maximum absorption of the ultraviolet spectrum at 204, 226 and 266 nm.13C NMR and1the H NMR spectral data show the characteristic signals of the biphenyl structure.1H NMR data showed an aromatic protonδ H6.81 (1H, s), indicating that the compound is a biphenyl structure with penta-substitution. Aromatic protonδ H6.81 (1H, s) is H-5 because it is shown in HMBC as well as C-4 (C: (1H, s))δ C128.8)、C-9 (δ C118.7)、C-7 (δ C132.1)、C-6 (δ C150.0) and C-1 ″ (δ C132.5) are relevant.1H NMR data show a para-substituted benzene ring [ 2 ]δ H7.58 (2H, d, J = 8.5 Hz, H-2 ", H-6") and 7.33 (2H, d,J= 8.5 Hz, H-3′′,5′′)]an isopropenyl coumarone partial signal [ alpha ]δ H3.44 (1H, dd,J= 15.2, 9.0 Hz, H-3),3.23 (H, dd,J= 15.2, 8.6 Hz, H-3), 5.30 (1H, t,J= 8.6 Hz, H-2), 4.88 (1H, s, H-2 '), 5.22 (1H, s, H-2 '), and 1.72 (1H, s, H-2 '), (ii)3H, s, H-3′)]And also a methoxy group (δ H3.84,3H, s, 6-OMe). In HMBC, H-3 and C-4, C-9, C-8 (C-3)δ C149.1) related to H-2 and C-8 and C-9, it is known that isopropenylcoumarone is linked to C-8 and C-9. Methoxy group (A), (B), (Cδ H3.84) at the C-6 position, as can be confirmed by its association with HMBC at C-6. According to the molecular formula, two hydroxyl groups are respectively located at C-7 and C-4'. The structure of the compound is determined and named as 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxypropyl) -dihydrobenzofurans.
The third purpose of the invention is realized by the application of the biphenyl compound in preparing anti-rotavirus medicaments. By the experiment of anti-rotavirus activity, 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxyphenyl) -dihydrobenzofran is selected as a reference to control the CC of rotavirus50And EC50Values of 164.2 and 12.9 respectivelyμmol/L, it has better anti-rotavirus activity. The compound has simple structure and good activity, can be used as a leading compound of an anti-rotavirus medicament, and has good application prospect.
Drawings
FIG. 1 shows NMR carbon spectrum of 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxyphenyl) -dihydrobenzofurans (NMR)13C NMR）。
FIG. 2 shows the NMR spectrum of 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxyphenyl) -dihydrobenzofurans (1H NMR）。
FIG. 3 is a graph of the primary HMBC (→) correlation of the compound 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxypropyl) -dihydrobenzofurans.
FIG. 4 shows attribution of carbon spectrum and hydrogen spectrum data of the compound.
Figure 5 is the anti-rotavirus activity of the compounds.
Detailed Description
The invention is further described with reference to the accompanying drawings, but the invention is not limited in any way, and any alterations or modifications based on the teaching of the invention are within the scope of the invention.
The biphenyl compound is obtained by taking dried branches, leaves or fruits of arbors of Guttiferae as raw materials and performing extract extraction, organic solvent extraction, silica gel column chromatography and high performance liquid chromatography separation, and the molecular formula of the compound is C18H18O4Named as 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxypropyl) -dihydrobenzofurans, has the following structural formula:
the preparation method of the biphenyl compounds is characterized in that dried branches, leaves and/or fruits of arbors of Guttiferae are used as raw materials, and the biphenyl compounds are obtained by extractum extraction, organic solvent extraction, silica gel column chromatography and high performance liquid chromatography separation, and specifically comprise the following steps:
A. extracting the extractum: coarsely crushing branches, leaves or fruits of arbors of the family Guttiferae to 20-40 meshes, ultrasonically extracting for 30-60 min each time for 2-4 times by using an organic solvent, and mixing extracting solutions; filtering the extracting solution, concentrating the extracting solution under reduced pressure to 1/4-1/2 volume, standing, filtering out precipitates, and concentrating to obtain an extract a;
B. organic solvent extraction: adding 1-2 times of water by weight into the extract a, extracting for 3-5 times by using an organic solvent with the same volume as the water, combining organic solvent extraction phases, and concentrating under reduced pressure to obtain an extract b;
C. silica gel column chromatography: dissolving the extract b by using acetone with the weight ratio of 1.5-3 times, mixing the sample by using 80-100 meshes of silica gel with the weight ratio of 0.8-1.2 times of the extract, and performing silica gel column chromatography, wherein the silica gel filled in the column is 100-200 meshes, and the using amount of the silica gel is 6-8 times of the weight of the extract b; gradient eluting with a mixed organic solvent with a volume ratio of 1: 0-0: 1, collecting gradient eluent, concentrating, monitoring by TLC, and combining the same parts;
D. reversed-phase column chromatography: carrying out reverse phase column chromatography on eluent obtained by eluting with organic solvent in the ratio of 7:3, wherein the reverse phase column is filled with reverse phase materials of C-8, C-18, ODS or MCI; performing gradient elution by using a methanol aqueous solution with the volume content of 20-100%, collecting eluent of each part, concentrating, monitoring by TLC, and combining the same parts;
E. high performance liquid chromatography separation: separating and purifying eluent obtained by eluting with 55-80% methanol water solution by volume through high performance liquid chromatography to obtain the biphenyl compound 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxyphenyl) -dihydrobenzofuran;
F. and E, performing high performance liquid chromatography separation and purification by taking 50-70% methanol as a mobile phase, taking a reverse phase preparation column with the flow rate of 10-14 mL/min and the flow rate of 21.2X 250mm and the thickness of 5um as a stationary phase, taking an ultraviolet detector with the detection wavelength of 254nm, feeding 45-60 uL of sample each time, collecting chromatographic peaks of 16-32 min, accumulating for multiple times and then evaporating to dryness. Obtaining the biphenyl compound 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxyphenyl) -dihydrobenzofurans:
the organic solvent in the step A is 70-100% of acetone, ethanol or methanol;
the organic solvent in the step B is ethyl acetate, chloroform, diethyl ether, petroleum ether or benzene;
the mixed organic solvent in the step C is n-hexane-acetone, chloroform-methanol, petroleum ether-acetone or petroleum ether-ethyl acetate;
the volume ratio of the mixed organic solvent in the step C is 1:0, 20:1, 9:1, 8:2, 7:3, 3:2, 1:1, 1:2 and 0: 1.
The application of the biphenyl compound in preparing anti-rotavirus medicaments is provided.
The gambogic plant of the invention is not limited by regions and varieties, and can be realized.
Example 1
Collecting dried branch, leaf and/or fruit of arbor of Guttiferae 5.7 kg, coarse pulverizing to 40 mesh, ultrasonic extracting with 70% acetone for 60min for 5 times, and mixing extractive solutions; filtering the extractive solution, and concentrating under reduced pressure to 1/4; standing, filtering out precipitates, and concentrating to obtain 368g of extract a; adding 600g of water into the extract a, extracting for 4 times by using chloroform with the same volume as the water, combining extract phases, and concentrating under reduced pressure to 247g of extract b; filling 1600g of 200-mesh silica gel into a column, adding 600g of acetone into the extract b for dissolving, then adding 250g of 100-mesh silica gel for sample mixing, and loading the mixture into the column after sample mixing; gradient eluting with chloroform-methanol mixed organic solvent at volume ratio of 1:0, 20:1, 9:1, 8:2, 7:3, 3:2, 1:1, 1:2, 0:1 respectively, collecting gradient eluate, concentrating, monitoring by TLC, mixing the same parts to obtain 9 parts, and eluting with chloroform-methanol mixed organic solvent at volume ratio of 7:3 to obtain eluate c 67 g; loading the eluate C on a reversed-phase column by using a reversed-phase material C-18, performing gradient elution by using a methanol aqueous solution with the volume content of 20-100%, collecting and concentrating the eluate of each part, monitoring by TLC, and combining the same parts; and (2) taking an eluent obtained by eluting with a methanol aqueous solution with the volume content of 50-70%, taking 60% methanol as a mobile phase, taking a Zorbax PrepHT GF reversed-phase preparation column with the flow rate of 8mL/min and the thickness of 21.2 x 250mm and the thickness of 5um as a stationary phase, taking an ultraviolet detector with the detection wavelength of 254nm, feeding 50uL of sample each time, collecting a chromatographic peak for 15min, accumulating for multiple times, and evaporating to dryness to obtain the biphenyl compound 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxypropyl) -dihydrobenzofurans.
Example 2
Collecting dried branches, leaves and/or fruits of arbor of Guttiferae 3kg, coarse pulverizing to 20 mesh, ultrasonic extracting with 100% ethanol for 3 times (60 min each time), and mixing extractive solutions; filtering the extractive solution, and concentrating under reduced pressure to 1/3; standing, filtering out precipitate, and concentrating to 189g of extract a; adding 360g of water into the extract a, extracting for 4 times by using chloroform with the same volume as the water, combining extract phases, and concentrating under reduced pressure to obtain 158g of extract b; loading 900g of 100-mesh silica gel into the column, adding 260g of acetone into the extract b for dissolving, and then adding 260g of 100-mesh silica gelgMixing the sample, and putting the sample on a column after mixing the sample; gradient eluting with n-hexane-acetone mixed organic solvent at volume ratio of 1:0, 20:1, 9:1, 8:2, 3:2, 1:1, 1:2, and 0:1, collecting gradient eluate, concentrating, monitoring by TLC, and mixing the same fractions; 76g of eluent c of the n-hexane-acetone mixed organic solvent with the volume ratio of 9: 1; loading the eluate C on a reversed-phase column by using a reversed-phase material C-8, performing gradient elution by using a methanol aqueous solution with the volume content of 20-100%, collecting and concentrating the eluate of each part, monitoring by TLC, and combining the same parts; and (3) taking an eluent obtained by eluting with 50-70% methanol aqueous solution by volume, and taking 68% methanol as a mobile phase at a flow rate of 14mL/min, 21.2 x 250mm, 5um Zorbax PrepHT GF reversed phase preparation column is stationary phase, the ultraviolet detector detects wavelength is 254nm, 50uL of each sample introduction, 21min chromatographic peak is collected, after multiple accumulation, evaporation is carried out, and the biphenyl compound 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxyphenyl) -dihydrobenzofurane is obtained.
Example 3
Pulverizing dried branch, leaf and/or fruit of arbor of Guttiferae 6kg into 30 mesh, ultrasonic extracting with 80% methanol for 30min for 4 times, and mixing extractive solutions; filtering the extractive solution, and concentrating under reduced pressure to 1/2; standing, filtering out precipitates, and concentrating to obtain 392g of extract a; adding 700g of water into the extract a, extracting for 4 times by using ether with the same volume as the water, combining extract phases, and concentrating under reduced pressure to obtain 242g of extract b; filling 1600g of 100-mesh silica gel into a column, adding 320g of acetone into the extract b for dissolving, then adding 260g of 100-mesh silica gel for sample mixing, and putting the mixture into the column after sample mixing; gradient eluting with chloroform-acetone mixed organic solvent at volume ratio of 1:0, 20:1, 9:1, 8:2, 7:3, 3:2, 1:1, 1:2, and 0:1 respectively, collecting gradient eluate, concentrating, monitoring by TLC, and mixing the same fractions; the volume ratio of chloroform-acetone mixed organic solvent eluent c is 9:1 is 45 g; loading a reversed-phase material ODS into a column, loading the eluent c into the reversed-phase column, performing gradient elution by using a methanol aqueous solution with the volume content of 20-100%, collecting and concentrating the eluent of each part, monitoring by TLC, and combining the same parts; taking an eluent obtained by eluting with a methanol aqueous solution with the volume content of 50-70%, taking 55% methanol as a mobile phase, taking a Zorbax PrepHTGF reversed-phase preparation column with the flow rate of 12mL/min and the size of 21.2 x 250mm and the size of 5um as a stationary phase, taking an ultraviolet detector with the detection wavelength of 254nm, feeding 50uL of sample each time, collecting a chromatographic peak for 19min, accumulating for multiple times, and evaporating to dryness to obtain the biphenyl compound 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxypropyl) -dihydrobenzofurane.
Example 4
Pulverizing dried branch, leaf and/or fruit of arbor of Guttiferae 5.3kg into 40 mesh coarse powder, ultrasonic extracting with 90% ethanol for 45min for 3 times, and mixing extractive solutions; filtering the extractive solution, and concentrating under reduced pressure to 1/4; standing, filtering out precipitate, concentrating to 321g extract a(ii) a Adding 680g of water into the extract a, extracting for 4 times by using petroleum ether with the same volume as the water, combining extract phases, and concentrating under reduced pressure to obtain 235g of extract b; packing 1450g of 100-mesh silica gel into a column, adding 290g of acetone into the extract b for dissolving, and then adding 265 g of 100-mesh silica gelgMixing the sample, and putting the sample on a column after mixing the sample; gradient eluting with petroleum ether-acetone mixed organic solvent at volume ratio of 1:0, 20:1, 9:1, 8:2, 7:3, 3:2, 1:1, 1:2, and 0:1, collecting gradient eluate, concentrating, monitoring by TLC, and mixing the same fractions; 52g of eluent c of the petroleum ether-acetone mixed organic solvent with the volume ratio of 9: 1; loading the eluate c on a reversed-phase column by using a reversed-phase material MCI (methanol to acetic acid) column, performing gradient elution by using a methanol aqueous solution with the volume content of 20-100%, collecting and concentrating the eluate of each part, monitoring by TLC (thin layer chromatography), and combining the same parts; taking an eluent obtained by eluting with 50-70% methanol aqueous solution by volume content, taking 70% methanol as a mobile phase, taking a Zorbax PrepHT GF reversed-phase preparation column with the flow rate of 10mL/min and the size of 21.2 x 250mm and the size of 5um as a stationary phase, taking an ultraviolet detector to detect the wavelength of 254nm, collecting a chromatographic peak for 14min, and evaporating to dryness after multiple accumulation to obtain the biphenyl compound 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxyphenyl) -dihydrobenzofurans.
Example 5
Pulverizing dried branches, leaves and/or fruits of arbor of Guttiferae 10 kg into 20 mesh coarse powder, ultrasonic extracting with 70% methanol for 35 min for 4 times, and mixing extractive solutions; filtering the extractive solution, and concentrating under reduced pressure to 1/2; standing, filtering out precipitate, and concentrating to obtain 879g of extract a; adding 1700g of water into the extract a, extracting for 5 times by using benzene with the same volume as the water, combining extract phases, and concentrating under reduced pressure to 445g of extract b; 3330 g of 200-mesh silica gel is filled into a column, 900g of acetone is added into the extract b for dissolution, 580g of 100-mesh silica gel is added for sample mixing, and the mixture is loaded on the column after the sample mixing; gradient eluting with petroleum ether-ethyl acetate mixed organic solvent at volume ratio of 1:0, 20:1, 9:1, 8:2, 3:2, 1:1, 1:2, and 0:1, collecting gradient eluate, concentrating, monitoring by TLC, and mixing the same fractions; 105 g of eluent c of petroleum ether-ethyl acetate mixed organic solvent with the volume ratio of 9: 1; loading the eluate C on a reversed-phase column by using a reversed-phase material C-18, performing gradient elution by using a methanol aqueous solution with the volume content of 20-100%, collecting and concentrating the eluate of each part, monitoring by TLC, and combining the same parts; taking an eluent obtained by eluting with 50-70% methanol aqueous solution by volume content, taking 50% methanol as a mobile phase, taking a Zorbax PrepHT GF reversed-phase preparation column with the flow rate of 12mL/min and the size of 21.2 x 250mm and the size of 5um as a stationary phase, taking an ultraviolet detector to detect the wavelength of 254nm, collecting a chromatographic peak for 31 min, and evaporating to dryness after multiple accumulation to obtain the biphenyl compound 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxyphenyl) -dihydrobenzofurans.
Example 6
Collecting dried branch, leaf and/or fruit of arbor of Guttiferae 8.1 kg, coarse pulverizing to 20 mesh, ultrasonic extracting with 100% acetone for 30min for 4 times, and mixing extractive solutions; filtering the extractive solution, and concentrating under reduced pressure to 1/2; standing, filtering out precipitate, and concentrating to 638g of extract a; adding 1200g of water into the extract a, extracting for 5 times by using benzene with the same volume as the water, combining extraction phases, and concentrating under reduced pressure to obtain 362g of extract b; filling 2400 g of 200-mesh silica gel into a column, adding 500 g of acetone into the extract b for dissolving, then adding 400g of 100-mesh silica gel for sample mixing, and loading the mixture into the column after sample mixing; gradient eluting with petroleum ether-acetone mixed organic solvent at volume ratio of 1:0, 20:1, 9:1, 8:2, 3:2, 1:1, 1:2, and 0:1, collecting gradient eluate, concentrating, monitoring by TLC, and mixing the same fractions; the eluent c of the petroleum ether-ethyl acetate mixed organic solvent with the volume ratio of 9:1 is 87 g; loading a reversed-phase material ODS into a column, loading the eluent c into the reversed-phase column, performing gradient elution by using a methanol aqueous solution with the volume content of 20-100%, collecting and concentrating the eluent of each part, monitoring by TLC, and combining the same parts; and (2) taking an eluent obtained by eluting with 50-70% methanol aqueous solution by volume content, taking 62% methanol as a mobile phase, taking a Zorbax PrepHT GF reversed-phase preparation column with the flow rate of 12mL/min and the size of 21.2 x 250mm and the size of 5um as a stationary phase, taking an ultraviolet detector to detect the wavelength of 254nm, collecting a chromatographic peak for 22 min, and evaporating to dryness after multiple accumulation to obtain the biphenyl compound 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxyphenyl) -dihydrobenzofurans.
Example 7
Taking the compound 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxypropyl) -dihydrobenzofurane prepared in example 1 as orange yellow gum; the determination method comprises the following steps: nuclear magnetic resonance, combined with other spectroscopic techniques, was used to identify structures:
(1) ultraviolet spectrum (the solvent is methanol),λ max（logε）：570 (1.79), 266 (3.69), 226(3.82), 204 (3.96) nm；
(2) infrared spectrum (Potassium bromide tablet) V max3 432, 2 924, 2 852, 1 630, 1 503, 1 450,1 374, 1 243, 1 173, 1 096, 1 054, 1 027, 905, 855, 824, 591 cm–1；
(3) HRESIMS shows the peak of the excimer of the compound of the inventionm/z298.1202 [M]+(calculated 298.1205), combined13C and1h NMR spectrum (FIG. 1 and FIG. 2, attribution of hydrogen spectrum data of carbon spectrum in FIG. 4) gives the molecular formula C18H18O4。1H NMR（C5D5N, 500 MHz) and13C NMR（C5D5n, 125 MHz) data, see fig. 4;
HRESIMS showed that its excimer peak was 298.1202 [ M ]]+(calculated 298.1205), combined13Determining molecular formula as C by CNMR spectrum18H18O4The unsaturation degree was 10. The infrared absorption spectrum is 3432cm-1Indicating the presence of hydroxyl groups at 1630, 1503 cm-1Indicating the existence of the benzene ring, and the existence of the benzene ring is also confirmed by the maximum absorption of the ultraviolet spectrum at 204, 226 and 266 nm.13C NMR and1the H NMR spectral data show the characteristic signals of the biphenyl structure.1H NMR data showed an aromatic protonδ H6.81 (1H, s), indicating that the compound is a biphenyl structure with penta-substitution. Aromatic protonδ H6.81 (1H, s) is H-5 because it is shown in HMBC as well as C-4 (C: (1H, s))δ C128.8)、C-9 (δ C118.7)、C-7 (δ C132.1)、C-6 (δ C150.0) and C-1 ″ (δ C132.5) are relevant.1H NMR data show a para-substituted benzene ring [ 2 ]δ H7.58 (2H, d, J = 8.5 Hz, H-2′′, H-6′′) And 7.33 (2H, d,J= 8.5 Hz, H-3′′,5′′)]an isopropenyl coumarone partial signal [ alpha ]δ H3.44 (1H, dd,J= 15.2, 9.0 Hz, H-3),3.23 (H, dd,J= 15.2, 8.6 Hz, H-3), 5.30 (1H, t,J= 8.6 Hz, H-2), 4.88 (1H, s, H-2 '), 5.22 (1H, s, H-2 ') and 1.72 (3H, s, H-3 ')]And also a methoxy group (δ H3.84,3H, s, 6-OMe). In HMBC, H-3 and C-4, C-9, C-8 (C-3)δ C149.1) related to H-2 and C-8 and C-9, it is known that isopropenylcoumarone is linked to C-8 and C-9. Methoxy group (A), (B), (Cδ H3.84) at the C-6 position, as can be confirmed by its association with HMBC at C-6. According to the molecular formula, two hydroxyl groups are respectively located at C-7 and C-4'. The structure of the compound is determined and named as 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxypropyl) -dihydrobenzofurans.
Example 8
The compound prepared in example 2 was taken as an orange-yellow gum; the structure determination was carried out as in example 6, with the results: the structure is the same as example 6, the molecular formula is C18H18O4. The compound prepared in example 2 was confirmed to be the biphenyl compound 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxyphenyl) -dihydrobenzofurans.
Example 9
The compound prepared in example 3 was taken as an orange-yellow gum; the structure determination was carried out as in example 6, with the results: the structure is the same as example 6, the molecular formula is C18H18O4. Confirming that the compound prepared in example 3 is the biphenyl compound 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxyphenyl) -dihydrobenzofurans;
process for preparation of the compound of FIG. 41H and13c NMR data (solvent C)5D5N）(125 and 500 MHz)。
Example 10
The compound prepared in example 4 was taken as an orange-yellow gum; the structure determination was carried out as in example 6, with the results: the structure is the same as example 6, the molecular formula is C18H18O4. The compound prepared in example 4 was confirmed to be the biphenyl compound 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxyphenyl) -dihydrobenzofurans.
Example 11
The compound prepared in example 5 was taken as an orange-yellow gum; the structure determination was carried out as in example 6, with the results: the structure is the same as example 6, the molecular formula is C18H18O4. It was confirmed that the compound produced in example 5 was 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxyphenyl) -dihydrobenzofurans, which is a biphenyl compound.
Example 12
Any one of the biphenyl compounds prepared in examples 1-5 is used for a cytotoxic activity detection test, and the test conditions are as follows:
cell line, the Rohou kidney cell line (MA-104) was provided by Shanghai Micromond Biotech Ltd.
The experimental design is that MA-104 cells and compounds with different concentrations are incubated for 72 hours, the experiment of each cell is repeated once, the results of the two experiments are used for data processing, the inhibition degree of the compounds on cell proliferation is evaluated by adopting an improved MTT method, the inhibition rate is calculated, and IC is calculated by adopting a Logit method according to the inhibition rate50In vitro antitumor activity of the compounds was compared.
EC50That is, the half effective concentration is the concentration at which 50% of the test animals are caused to produce a certain reaction or the reaction index is half inhibited.
CC50I.e., to half the cytotoxic concentration, to the concentration required to produce a toxic effect on half the cells. In this experiment, the concentration of drug required to cause 50% cell death is referred to.
Compound cytotoxicity assays
The compound is dissolved in dimethyl sulfoxide (DMSO), sterilized by microwave for 10 min, prepared into mother liquor of l mg/mL with MEM, and diluted to the required concentration. 96-well cell culture plate, add l × 105MA-104 cell suspension at concentration/mL, 100ul/well, 37 ℃ and 5% CO2Incubating for 24h, and adding l mg/mL, 0.2 mg/mL and 40 mg/mL onto the well-grown monolayer cells respectivelyug/mL、8ug/mL、1.25uCompound of g/mL 100ul/well, 3 duplicate wells per concentration, and a normal cell control. Standing at 37 deg.C for 5% CO2After the incubator is continuously cultured for 24h, the cell survival rate is detected by the MTT method.
Preventive effect of Compound on viral infection
At a concentration of 104L00 per well/mLul inoculating cells in 96-well plate, culturing for 24 hr, and making the cells grow into monolayer and grow well at 100 concentrationug/mL、75ug/mL、50ug/mL、25ug/mL、lug/mL of the compound was preincubated at 37 ℃ for 1.5 h, washed with PBS and 100 TCID 50/mL of rotavirus per welluAdsorbing for 1 hr, discarding, adding MEM medium 100uMaintaining the mixture at 37 deg.C and 5% CO2And (5) incubating, and observing the cytopathic condition every day. After 48h, the virus inhibition rate is detected by an MTT method.
Cell viability assay
Using the MTT method, 5 mg/mL Methylthiazolyltetrazole (MTT) 20 was added to the cells cultured for 48hul, continuing to culture for 3-4 h, discarding the supernatant, adding DMSO into each well for 100 hoursul, shaking to dissolve the crystals completely in the well immediately at 490nmMeasurement of absorbance A value at wavelength:
cell viability = mean a value of drug group/a value of cell control group x 100%
Viral inhibition = [ mean a value of experimental group-mean a value of viral control group ]/[ mean a value of cell control group-mean a value of viral control group ] × 100%
Therapeutic Index (TI) = half toxic concentration (CC)50) Half maximal Inhibitory Concentration (IC)50)。
Results of the experiment
The experimental results show that: by the experiment of anti-rotavirus activity, 2-isopropenyl-6-methoxy-7-hydroxy- (4-hydroxyphenyl) -dihydrobenzofran is selected as a reference to control the CC of rotavirus50And EC50Values of 164.2 and 12.9 respectivelyμmol/L, it has better anti-rotavirus activity (as shown in figure 5).
Claims (3)
1. A dihydrofuran biphenyl compound is characterized in that: the biphenyl compound is prepared from dried branches, leaves or fruits of arbors of Guttiferae by extracting with extract, extracting with organic solvent, performing silica gel column chromatography, reverse phase column chromatography, and separating with high performance liquid chromatography, and has molecular formula of C18H18O4Having the following structural formula:
2. a preparation method of biphenyl compounds according to claim 1, wherein the biphenyl compounds are obtained by using dried branches, leaves or fruits of arbors belonging to Guttiferae as raw materials, and performing extract extraction, organic solvent extraction, silica gel column chromatography, reversed phase column chromatography and high performance liquid chromatography separation, and specifically comprise:
A. extracting the extractum: coarsely crushing branches, leaves or fruits of arbors of the family Guttiferae to 20-40 meshes, ultrasonically extracting for 2-4 times with 80-100% of acetone, ethanol or methanol for 30-60 min each time, and mixing the extracting solutions; filtering the extracting solution, concentrating the extracting solution under reduced pressure to 1/4-1/2 volume, standing, filtering out precipitates, and concentrating to obtain an extract a;
B. organic solvent extraction: adding 1-2 times of water by weight into the extract a, extracting for 3-5 times by using an organic solvent with the same volume as the water, combining organic solvent extraction phases, and concentrating under reduced pressure to obtain an extract b, wherein the organic solvent is ethyl acetate, chloroform, diethyl ether, petroleum ether or benzene;
C. silica gel column chromatography: dissolving the extract b by using an organic solvent with the weight ratio of 1.5-3 times, mixing the sample by using 100-200 meshes of silica gel with the weight of 0.8-1.2 times of that of the extract, and performing silica gel column chromatography, wherein the silica gel filled in the column is 100-200 meshes, and the using amount of the silica gel is 6-8 times of that of the extract b; gradient elution is carried out by using a mixed organic solvent with the volume ratio of 1: 0-0: 1, gradient eluent is collected, concentration is carried out, the same parts are combined by monitoring through TLC, the mixed organic solvent is n-hexane-acetone, chloroform-methanol, petroleum ether-acetone or petroleum ether-ethyl acetate, and the volume ratio of the mixed organic solvent is 1:0, 20:1, 9:1, 8:2, 7:3, 3:2, 1:1, 1:2 and 0: 1;
D. reversed-phase column chromatography: subjecting the eluate obtained by eluting with organic solvent at ratio of 7:3 or 9:1 to reverse phase column chromatography, wherein the reverse phase column is prepared by loading reverse phase material C-8, C-18, ODS or MCI into column; performing gradient elution by using 40-100% methanol aqueous solution, collecting eluates of each part, concentrating, monitoring by TLC, and combining the same parts;
E. high performance liquid chromatography separation: separating and purifying eluent obtained by eluting with 55-80% methanol water solution by volume by using high performance liquid chromatography to obtain the biphenyl compound;
F. e, performing high performance liquid chromatography separation and purification by taking 50-70% methanol as a mobile phase, taking a reverse phase preparation column with the flow rate of 10-14 mL/min and the flow rate of 21.2 x 250mm and the thickness of 5um as a stationary phase, taking an ultraviolet detector with the detection wavelength of 254nm, feeding 45-60 uL of sample each time, collecting chromatographic peaks for 16-34 min, and evaporating to dryness after multiple accumulation; thus obtaining the biphenyl compound.
3. An application of biphenyl compound of claim 1 in preparing anti-rotavirus drugs.
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