CN109053447B

CN109053447B - Isopentyl substituted biphenyl compound and preparation method and application thereof

Info

Publication number: CN109053447B
Application number: CN201810885754.8A
Authority: CN
Inventors: 高雪梅; 李干鹏; 蒋孟圆; 江志勇; 王闪闪; 朱鸿
Original assignee: Yunnan Minzu University
Current assignee: Yunnan Minzu University
Priority date: 2018-08-06
Filing date: 2018-08-06
Publication date: 2021-04-06
Anticipated expiration: 2038-08-06
Also published as: CN109053447A

Abstract

The invention discloses an isopentyl substituted biphenyl compound, a preparation method and application thereof, wherein the molecular formula of the isopentyl substituted biphenyl compound is C₂₀H₂₄O₆Having the following structural formula:

the preparation method takes dried branches, leaves or fruits of arbors of Guttiferae as raw materials, and comprises the steps of extract extraction, organic solvent extraction, silica gel column chromatography and high-pressure liquid chromatography separation. The application is the application of isopentyl substituted biphenyl compounds in preparing anti-rotavirus medicaments. By an anti-rotavirus activity experiment, ribavirin is selected as a control, and the compound has a TI value of more than 10 to rotavirus and 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

Isopentyl substituted biphenyl compound and preparation method and application thereof

Technical Field

The invention belongs to the technical field of extraction of effective components of plants, and particularly relates to an isopentyl substituted 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. Garcinia plant is also one of main resources of natural xanthone (xanthone) components, and is rich in isoamyleneThe xanthone (xanthones) is novel and diverse in component structure and has wide pharmacological activity, particularly gambogic acid (gambogic acid) is most representative, has broad-spectrum and strong anti-tumor activity, is one of the research hotspots of anti-tumor natural products in recent years, and Chinese scholars are developing injection thereof, and are novel anti-tumor medicines. In addition to xanthones, compounds such as biphenyls, benzophenones, biflavonoids and depsiphenones are characteristic components of plants in 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 invention provides an isopentane-substituted biphenyl compound; the second purpose is to provide a preparation method of the isopentyl substituted biphenyl compound; the third purpose is to provide the application of the isopentyl substituted biphenyl compound.

The first purpose of the invention is realized by that the isopentane group substituted biphenyl compound is obtained by using dry 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 C₂₀H₂₄O₆Having the following structural formula:

the second purpose of the invention is realized by that the preparation method of the isopentane group substituted biphenyl compound is obtained by using dry branches, leaves or fruits of arbors of Guttiferae as raw materials and performing extract extraction, organic solvent extraction, silica gel column chromatography and high-pressure 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 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 an organic solvent with the weight ratio of 1.5-3 times, then mixing the sample by using 200-300 meshes of silica gel with the weight of 0.8-1.2 times of the extract, and then performing silica gel column chromatography, wherein the silica gel filled in the column is 200-300 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: subjecting the eluate obtained by eluting with organic solvent at ratio of 4:1 to reverse phase column chromatography, wherein the reverse phase column is filled with reverse phase material C-18, C-8 or ODS; performing gradient elution by using a methanol aqueous solution with the volume content of 50-100%, collecting eluent of each part, concentrating, monitoring by TLC, and combining the same parts;

E. high performance liquid chromatography separation: and (3) separating and purifying an eluent obtained by eluting with 50-70% methanol aqueous solution by volume by using high performance liquid chromatography to obtain the isopentane group substituted biphenyl compound.

The isopentyl substituted biphenyl compound is separated for the first time, is determined to be the isopentyl substituted biphenyl compound by a nuclear magnetic resonance and other spectrum technology measuring method, and is characterized in that the specific structure is as follows:

the compound is a light yellow gum; ultraviolet spectrum (the solvent is methanol),λ _max(log ε): 570 (1.71), 270 (3.43), 208 (3.89) nm; infrared spectrum (Potassium bromide tablet) v_max 3425, 2934, 2958, 1711, 1610, 1501, 1462, 1453, 1367, 1254, 1201, 1124, 1092, 1036, 986 cm^-1(ii) a HRESIMS shows the peak of the excimer of the compound of the inventionm/z 360.1565 [M]⁺(calculated 360.1573), combined¹³C and¹the H NMR spectrum (FIGS. 1 and 2, attribution of hydrogen spectrum data in carbon spectrum in Table 1) gave a molecular formula C₂₀H₂₄O₆。¹H NMR (CD₃OD, 400 MHz) and¹³C NMR（CD₃OD, 100 MHz) data, see table 1.

HRESIMS shows that the peak of the excimer ion is the peak of the excimer ionm/z 360.1565 [M]⁺(calculated 360.1573), combined¹³C NMR spectrum to confirm the molecular formula as C₂₀H₂₄O₆The unsaturation degree was 9. The infrared spectrum showed hydroxyl groups (3425 cm)^-1) Carbonyl group (1711 cm)^-1) The absorption peak of (1). In that¹³In C-NMR, 4' -OAc: (δ _C173.6,21.0) also confirmed the presence of carbonyl groups. Of the compound¹H-and¹³C-NMR data is classified and shows that the carbon number of the carbon is 20, wherein 3 methyl groups, 3 methylene groups, 6 methine groups and 8 quaternary carbon groups are included. According to the nuclear magnetic resonance dataδ _H 7.21 (1H, d, J=2.1 Hz, H-8), 7.17 (1H, dd, J=8.2, 2.2 Hz, H-12)，6.78（1H，d, J=8.2 Hz, H-11), 6.65 (1H, d, J=1.8 Hz, H-6), 6.63 (1H, d, J=1.8 Hz, H-2), 3.99 (1H, dd, J=10.7, 5.8 Hz, H-4＇), 3.91 (1H, dd, J=10.7, 6.6 Hz, H-4＇), 3.87 (3H, s, 3-OMe), 2.68 (1H, m, H-1＇), 2.62 (1H, m, H-1＇), 2.02 (3H, s, 4＇- OAc), 1.81 (1H, m, H-3＇), 1.71 (1H, m, H-2＇), 1.47 (1H, m, H-2＇), 0.99 (3H, d, J=6.7 Hz, H-5＇) ; δ _C134.2 (C-1), 103.1 (C-2), 149.9 (C-3), 134.1 (C-4), 146.6 (C-5), 108.2 (C-6), 134.1 (C-7), 129.2 (C-8), 130.1 (C-9), 155.3 (C-10), 116.2 (C-11), 126.1 (C-12), 28.7 (C-1＇), 34.7 (C-2＇), 33.6 (C-3＇), 70.6 (C-4＇), 17.3 (C-5＇), 56.8 (3-OMe), 173.6, 21.0 (4＇-OAc)]The compound is preliminarily presumed to be a biphenyl compound containing acetoxy isoamyl. H-2 and H-6 with C-7 andthe HMBC correlation of H-8 and H-12 with C-1 further confirms the biphenyl backbone. In thatδ _CThe 3 signals at 134.1 (C-4), 146.6 (C-5), 155.3 (C-10) indicate the presence of three hydroxyl groups on the two phenyl rings due to H-1' being on the two phenyl ringsδ _H2.68, 2.62 and 129.2 (C-8), 130.1 (C-9), 155.3 (C-10) showed HMBC correlation, H-2' and C-9 showed HMBC correlation, so that acetoxy isoamyl group was determined at C-9,δ _H3.87 and C-3 (δ _C149.9) showed that the methoxy group was attached at C-3 in the molecule, and thus the structure of the compound was determined.

The third purpose of the invention is realized by the application of the isopentyl substituted biphenyl compound in preparing the anti-rotavirus medicaments. By an anti-rotavirus activity experiment, ribavirin is selected as a control, and the compound has a TI value of more than 10 to rotavirus and 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 the NMR spectrum of the compound (C:¹³C NMR）；

FIG. 2 shows the NMR spectrum of the compound (A)¹H NMR）；

Figure 3 is the primary HMBC (→) correlation of the compound.

Detailed Description

The invention is further illustrated by the following examples and figures, but is not limited in any way thereto, and any variations or modifications based on the teachings of the invention are within the scope of the invention.

The isopentane-substituted biphenyl compound is prepared 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-pressure liquid chromatography separation, and the molecular formula of the compound is C₂₀H₂₄O₆Having the following structural formula:

the invention relates to a preparation method of isopentyl-substituted biphenyl, which takes dry branches, leaves or fruits of arbors of Guttiferae as raw materials and is obtained by extract extraction, organic solvent extraction, silica gel column chromatography and high-pressure liquid chromatography separation, and specifically comprises the following steps:

Further, the organic solvent in the step A is 70-100% of acetone, ethanol or methanol.

Further, the organic solvent in the step B is ethyl acetate, chloroform, diethyl ether, petroleum ether or benzene.

Further, the mixed organic solvent in the step C is chloroform-acetone, chloroform-methanol, petroleum ether-acetone, dichloromethane-ethyl acetate or petroleum ether-ethyl acetate.

Further, the volume ratio of the mixed organic solvent in the step C is 1:0, 4:1, 2:1, 1:1 and 0: 1.

And further, the high performance liquid chromatography separation and purification in the step E takes 50-70% of methanol as a mobile phase, a reversed-phase preparation column with the flow rate of 2-5 ml/min, the thickness of 9.4 x 250 mm and the thickness of 5 mu m as a stationary phase, an ultraviolet detector detects the wavelength of 254nm, the sample injection is carried out for 45-60 mu L each time, chromatographic peaks of 7-25 min are collected, and the isopentyl substituted biphenyl compounds are obtained by evaporation after multiple accumulation.

The isopentyl substituted biphenyl compound is applied to preparation of rotavirus resisting medicines.

The gambogic plant of the invention is not limited by regions and varieties, and can be realized.

Example 1

Taking 3.5 kg of dried branches, leaves and/or fruits of arbor of Guttiferae, coarsely pulverizing to 20 mesh, ultrasonically extracting with 70% acetone for 30min for 4 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 260g of extract a; adding 260g of water into the extract a, extracting for 5 times by using ethyl acetate with the same volume as the water, combining extract phases, and concentrating under reduced pressure to obtain 154g of extract b; 1500g of 200-plus-300-mesh silica gel is used for filling a column, 200g of methanol is added into the extract b for dissolving, 160g of 100-plus-200-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-acetone 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 eluent, concentrating, monitoring by TLC, mixing the same parts to obtain 9 parts, and eluting with petroleum ether-acetone mixed organic solvent at volume ratio of 7:3 (c) 13 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 60-70%, taking 65% methanol as a mobile phase, taking an Altima C18 reversed-phase preparation column with the flow rate of 3ml/min and the thickness of 9.4 x 250 mm and the thickness of 5 mu m as a stationary phase, taking an ultraviolet detector with the detection wavelength of 254nm, feeding 50 mu L of sample each time, collecting chromatographic peaks for 12 min, accumulating for multiple times, and evaporating to dryness to obtain the isopentyl substituted biphenyl compound.

Example 2

Collecting dried branches, leaves and/or fruits of arbor of Guttiferae 3.2kg, coarse pulverizing to 20 mesh, ultrasonic extracting with 10% water-containing ethanol for 3 times, each for 20min, and mixing extractive solutions; filtering the extractive solution, and concentrating under reduced pressure to 1/3; standing, filtering out precipitates, and concentrating to obtain 360g of extract a; adding 360g of water into the extract a, extracting for 3 times by using ethyl acetate with the same volume as the water, combining extraction phases, and concentrating under reduced pressure to obtain 120g of extract b; filling 1200g of 200-mesh silica gel into the column, adding 240g of methanol into the extract b for dissolution, then adding 120g of 100-mesh 200-mesh silica gel for sample mixing, and filling 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, 3:2, 1:1, 1:2, and 0:1, collecting gradient eluate, concentrating, monitoring by TLC, and mixing the same fractions; the volume ratio of chloroform-acetone mixed organic solvent eluent c is 46 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 50-100%, 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 60-70%, then taking 60% methanol as a mobile phase, taking an Altima C18 reversed-phase preparation column with the flow rate of 3ml/min, the flow rate of 2-5 ml/min, the flow rate of 9.4 x 250 mm and the size of 5 mu m as a stationary phase, taking an ultraviolet detector with the detection wavelength of 254nm, feeding 50 mu L of sample each time, collecting a chromatographic peak for 20min, accumulating for multiple times, and evaporating to dryness to obtain the isopentyl substituted biphenyl compound.

Example 3

Collecting dried branch, leaf and/or fruit of arbor of Guttiferae 6.5kg, coarse pulverizing to 30 mesh, ultrasonic extracting with 20% methanol for 3 times, each for 20min, and mixing extractive solutions; filtering the extractive solution, and concentrating under reduced pressure to 1/2; standing, filtering out precipitate, and concentrating to obtain 675g extract a; adding 700g 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 342g of extract b; 3400g of 200-plus-300-mesh silica gel is used for filling a column, 900g of methanol is added into the extract b for dissolving, 360g of 100-plus-200-mesh silica gel is added for sample mixing, and the mixture is loaded on the column after the sample mixing; gradient eluting with dichloromethane-ethyl acetate 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, collecting gradient eluate, concentrating, monitoring by TLC, and mixing the same fractions; the volume ratio of dichloromethane-ethyl acetate 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; and (2) taking an eluent obtained by eluting with a methanol aqueous solution with the volume content of 60-70%, taking 68% methanol as a mobile phase, taking an Altima C18 reversed-phase preparation column with the flow rate of 3ml/min and the thickness of 9.4 x 250 mm and the thickness of 5 mu m as a stationary phase, taking an ultraviolet detector with the detection wavelength of 254nm, feeding 50 mu L of sample each time, collecting chromatographic peaks for 10min, accumulating for multiple times, and evaporating to dryness to obtain the isopentyl substituted biphenyl compound.

Example 4

Collecting dried branch, leaf and/or fruit of arbor of Guttiferae 5.9kg, coarse pulverizing to 40 mesh, extracting with 90% ethanol for 3 times, and mixing extractive solutions; filtering the extractive solution, and concentrating under reduced pressure to 1/4; standing, filtering out precipitate, and concentrating to obtain extract a of 760 g; adding 800g 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 305g of extract b; loading 3000g of 200-mesh silica gel into a column, adding 300g of methanol into the extract b for dissolution, then adding 300g of 100-mesh 200-mesh silica gel for sample mixing, and loading the sample 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, 7:3, 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-acetone mixed organic solvent with the volume ratio of 8:2 is 43 g; 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 (2) taking an eluent obtained by eluting with 70-80% methanol aqueous solution by volume, taking 70% methanol as a mobile phase, taking a 9.4 x 250 mm 5 mu m Altima C18 reversed-phase preparation column as a stationary phase at a flow rate of 2ml/min, collecting chromatographic peaks for 8min after an ultraviolet detector detects that the wavelength is 254nm, and evaporating to dryness after multiple accumulation to obtain the isopentyl-substituted biphenyl compound.

Example 5

Collecting dried branches, leaves and/or fruits of arbor of Guttiferae 5.6 kg, coarse pulverizing to 20 mesh, ultrasonic extracting with 80% methanol for 3 times, each time for 30min, and mixing extractive solutions; filtering the extractive solution, and concentrating under reduced pressure to 1/2; standing, filtering out precipitates, and concentrating to obtain 430g of extract a; adding 800g of water into the extract a, extracting for 4 times by using acetone with the same volume as the water, combining extract phases, and concentrating under reduced pressure to obtain 260g of extract b; 2200g of 200-mesh silica gel 300 meshes is used for filling the column, 420g of ethyl acetate is added into the extract b for dissolving, 400g of 100-mesh silica gel 200 meshes 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; 26g of eluent c of petroleum ether-ethyl acetate mixed organic solvent with the volume ratio of 9: 1; 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 a methanol aqueous solution with the volume content of 50-70%, taking 58% methanol as a mobile phase, taking an Altima C18 reversed-phase preparation column with the flow rate of 3ml/min and the size of 9.4 x 250 mm and the size of 5 mu m as a stationary phase, taking an ultraviolet detector to detect the wavelength of 254nm, collecting a chromatographic peak for 17min, accumulating for multiple times, and evaporating to dryness to obtain the isopentyl-substituted biphenyl compound.

Example 6

The compound prepared in example 1 was taken as a pale yellow gum; the determination method comprises the following steps: nuclear magnetic resonance, in combination with other spectroscopic techniques, was used to identify structures.

(1) Ultraviolet spectrum (the solvent is methanol),λ _max（log ε）：570 (1.71), 270 (3.43), 208 (3.89) nm；

(2) infrared spectrum (Potassium bromide tablet) v_max 3425, 2934, 2958, 1711, 1610, 1501, 1462, 1453, 1367, 1254, 1201, 1124, 1092, 1036, 986 cm^-1；

(3) HRESIMS shows that the peak of the excimer of the compound ism/z 360.1565 [M]⁺(calculated 360.1573), combined¹³C and¹h NMR spectrum (FIG. 1 and FIG. 2, the data of carbon spectrum and hydrogen spectrum are shown in Table 1) shows that the molecular formula is C₂₀H₂₄O₆。¹H NMR(400 MHz,CD₃OD) and¹³C NMR(100 MHz,CD₃OD) data, see table 1.

Of the compounds of Table 1¹H and¹³c NMR data (400/100 MHz, CD)₃OD)

HRESIMS shows that the peak of the excimer ion is the peak of the excimer ionm/z 360.1565 [M]⁺(calculated 360.1573), combined¹³C NMR spectrum to confirm the molecular formula as C₂₀H₂₄O₆The unsaturation degree was 9. The infrared spectrum showed hydroxyl groups (3425 cm)^-1) Carbonyl group (1711 cm)^-1) The absorption peak of (1). In that¹³In C-NMR, 4' -OAc: (δ _C173.6,21.0) also confirmed the presence of carbonyl groups. Of the compound¹H-and¹³C-NMR data is classified and shows that the carbon number of the carbon is 20, wherein 3 methyl groups, 3 methylene groups, 6 methine groups and 8 quaternary carbon groups are included. From nuclear magnetic resonance data [δ _H 7.21 (1H, d, J=2.1 Hz, H-8), 7.17 (1H, dd, J=8.2, 2.2 Hz, H-12)，6.78（1H，d, J=8.2 Hz, H-11), 6.65 (1H, d, J=1.8 Hz, H-6), 6.63 (1H, d, J=1.8 Hz, H-2), 3.99 (1H, dd, J=10.7, 5.8 Hz, H-4＇), 3.91 (1H, dd, J=10.7, 6.6 Hz, H-4＇), 3.87 (3H, s, 3-OMe), 2.68 (1H, m, H-1＇), 2.62 (1H, m, H-1＇), 2.02 (3H, s, 4＇- OAc), 1.81 (1H, m, H-3＇), 1.71 (1H, m, H-2＇), 1.47 (1H, m, H-2＇), 0.99 (3H, d, J=6.7 Hz, H-5＇); δ _C134.2 (C-1), 103.1 (C-2), 149.9 (C-3), 134.1 (C-4), 146.6 (C-5), 108.2 (C-6), 134.1 (C-7), 129.2 (C-8), 130.1 (C-9), 155.3 (C-10), 116.2 (C-11), 126.1 (C-12), 28.7 (C-1＇), 34.7 (C-2＇), 33.6 (C-3＇), 70.6 (C-4＇), 17.3 (C-5＇), 56.8 (3-OMe), 173.6, 21.0 (4＇-OAc)]The compound is preliminarily presumed to be a biphenyl compound containing acetoxy isoamyl. The HMBC correlation of H-2 and H-6 with C-7 and H-8 and H-12 with C-1 further confirms the biphenyl backbone. In thatδ _CThe 3 signals at 134.1 (C-4), 146.6 (C-5), 155.3 (C-10) indicate the presence of three hydroxyl groups on the two phenyl rings due to H-1' being on the two phenyl ringsδ _H2.68, 2.62 and 129.2 (C-8), 130.1 (C-9), 155.3 (C-10) showed HMBC correlation, H-2' and C-9 showed HMBC correlation, so that acetoxy isoamyl group was determined at C-9,δ _H3.87 and C-3 (δ _C149.9) showed that the methoxy group was attached at C-3 in the molecule, and thus the structure of the compound was determined.

Example 7

The compound prepared in example 2 was taken as a pale 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 C₂₀H₂₄O₆. The compound prepared in example 2 was confirmed to be the isopentenyl-substituted biphenyl compound.

Example 8

The compound prepared in example 3 was taken as a pale yellow gum; the structure determination was carried out as in example 6, with the results: the structure is the same as that of the embodiment6, molecular formula C₂₀H₂₄O₆. The compound prepared in example 3 was confirmed to be the isopentenyl-substituted biphenyl compound.

Example 9

The compound prepared in example 4 was taken as a pale 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 C₂₀H₂₄O₆. The compound prepared in example 4 was confirmed to be the isopentenyl-substituted biphenyl compound.

Example 10

The compound prepared in example 5 was taken as a pale 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 C₂₀H₂₄O₆. The compound prepared in example 5 was confirmed to be the isopentenyl-substituted biphenyl compound.

Example 11

Any one of the isopentyl substituted biphenyl compounds prepared in the embodiments 1-5 is used for carrying out an anti-rotavirus activity detection test, and the test conditions are as follows:

cell lines: rhesus monkey kidney cell line (MA-104).

Experiment design: incubating MA-104 cells with compounds with different concentrations for 72 hr, repeating experiment for each cell for 2 times, processing data with the results of 3 experiments, evaluating the inhibition degree of the compounds on cell proliferation by improved MTT method, calculating inhibition rate, and calculating IC by Logit method according to the inhibition rate₅₀In vitro antiviral 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.

(a) Compound cytotoxicity assays

Dissolving the compound with dimethyl sulfoxide (DMSO), sterilizing in microwave for l0min, preparing into lmg/ml mother solution with MEM, and diluting with MEM solution to desired concentration. 96-well cell culture plate, add l x l0⁵Mal04 cell suspension at a concentration of 100 mlul/well, 37 ℃ and 5% CO₂Culturing in incubator for 24h, and adding l mg/ml, 0.2 mg/ml, and 40 mg/ml onto the well-grown monolayer cells respectivelyug/ml、8 ug/ml、1.25 uCompound 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.

(b) Preventive effect of Compound on viral infection

At a cell 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、75 ug/ml、50 ug/ml、25 ug/ml、l ug/ml of the compound was preincubated with l.5 h cells at 37 ℃ and washed with PBS and 100 wells of 100TCID50/ml rotavirusul adsorption for lh, discard, add MEM Medium 100uThe cells were maintained at the same concentration in the same well, and incubated at 37 ℃ and 5% CO2 for observation of cytopathic effect every day. After 48h, the virus inhibition rate is detected by an MTT method.

(c) Therapeutic effect of compounds on viral infections

At a cell concentration of 10⁴L00 per well/mlul inoculating cells in 96-well plate, culturing for 24 hr until the cells grow into monolayer and grow well, and first 100 wells with 100TCID50/ml rotavirusul adsorbing for lh and discarding, then adding the above-mentioned compounds of different concentrations, 100ul/well, culturing and detecting as above. Each experiment was set with a virus control group (group C) and a normal cell control group (group N).

(d) Cell viability assay

Adding 5mg/ml Methyl Thiazolyl Tetrazolium (MTT) 20 into cells cultured for 48h by MTT methodul, continuing the cultivation3-4 h, discarding the supernatant, adding DMSO in 100 wellsul, immediately after shaking to completely dissolve the crystals in the wells at 490luThe absorbance A value was measured at n wavelength.

Cell viability = mean a value of drug group/a value of cell control group x100%

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 ] x100%

Therapeutic Index (TI) = half toxic concentration (CC)₅₀) Half maximal Inhibitory Concentration (IC)₅₀)

(e) Results of the experiment

The experimental results show that: by the activity experiment of anti-rotavirus, ribavirin is selected as a control, and Lanconiphynyl A is used for CC of rotavirus₅₀And EC₅₀Values of 305.3 and 17.3, respectivelyμmol/L, shown in Table 2, has better anti-rotavirus activity.

Anti-rotavirus activity of the compounds of table 2

No.	CC₅₀ (µM)	EC₅₀ (µM)	TI
				Said compounds	305.3+2.0	17.3+0.9	17.65
Ribavirin	263.2+1.9	13.3+0.7	19.8

^aAll data are expressed as mean ± SD (standard deviation); n = 3

TI therapeutic index, CC₅₀/EC₅₀。

Claims

1. An application of isopentyl substituted biphenyl compounds in preparing anti-rotavirus medicaments is characterized in that the isopentyl substituted biphenyl compounds have the following structural formula:

。