Benzofuran coumarin compound as well as preparation method and application thereof

Abstract

The invention relates to the field of pharmacy, in particular to a benzofuran coumarin compound as well as a preparation method and application thereof. The benzofuran coumarin compound is an optical isomer, a crystal and a pharmaceutically available salt as shown in the following structural formula: # imgabs0 #, wherein R1 is selected from hydrogen and C1-C6 alkoxy. The benzofuran coumarin compound disclosed by the invention has anti-hepatitis activity and a potential liver protection effect, and a new preparation method of direct C3 alkylation of the benzofuran coumarin compound is provided.

Description

Technical Field

[0001] The present invention relates to the field of pharmacy, and particularly relates to a benzofuran coumarin compound, a preparation method thereof, and an application thereof. Background Art

[0002] There are many literature reports on the direct C3-alkylation of coumarin with alkyl radicals through a radical substitution process. The precursors for the generation of alkyl radicals include (cyclo)alkyl ethers, alkylbenzenes, aliphatic aldehydes, alkyl hydrazines, N-hydroxyphthalimide esters, 4-alkyl-1,4-dihydropyridines, hypervalent iodine(III) reagents, cyclobutanone oxime esters, alkyl boronic acids, and alkyl diacyl peroxides, etc. Among them, N-hydroxyphthalimide esters have high efficiency in generating alkyl radicals. Examples of relevant literature are as follows:

[0003]

[0004] It can be seen that although there are many preparation methods for the C3-alkylation of coumarin provided in the prior art, according to the current results, the C3-alkylation methods in the literature cannot be used for the C3-alkylation of coumarin with a hydroxyl group on the benzene ring, and no target product is obtained by attempting the above methods. There is currently no literature report on the C3-alkylation of coumarin with a hydroxyl group on the benzene ring. Summary of the Invention

[0005] The purpose of the present invention is to provide a new benzofuran coumarin compound, a preparation method thereof, and an application thereof in the preparation of anti-hepatitis drugs.

[0006] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A benzofuran coumarin compound, which is an optical isomer, crystal, or pharmaceutically acceptable salt represented by the following structural formula:

[0008]

[0009] Wherein R1 is selected from hydrogen and C1-C6 alkoxy groups.

[0010] In a preferred embodiment, R1 is selected from hydrogen and C1-C3 alkoxy groups.

[0011] In a preferred embodiment, the benzofuran coumarin compound has the following structural formula:

[0012]

[0013] Based on the same inventive concept, the present invention also protects an extraction method of the benzofuran coumarin compound, comprising the following steps:

[0014] S1. Crush the thick stem part of rue and perform ultrasonic extraction to obtain an aqueous extract; concentrate the aqueous extract under reduced pressure and pass it through macroporous resin to obtain three components, Fr.A to Fr.C;

[0015] S2. Further subject Fr.C to silica gel column chromatography, elute with a gradient of dichloromethane - methanol system, and combine similar components to obtain 12 fractions, Fr.C-Ⅰ to Fr.C-Ⅻ;

[0016] S3. Subject Fr.C-Ⅲ to column chromatography, elute, and combine similar components to obtain 10 components, Fr.C-Ⅲ-1 to Fr.C-Ⅲ-10; separate Fr.C-Ⅲ-7 by thin layer preparative plate to obtain 3 components, Fr.C-Ⅲ-7-1 to Fr.C-Ⅲ-7-3; purify Fr.C-Ⅲ-7-2 by semi-preparative high performance liquid chromatography to obtain compound 2*, and then resolve compound 2* through a chiral column to obtain compound (+)-2* and compound (–)-2*; subject Fr.C-Ⅴ to column chromatography, elute, and combine similar components to obtain 3 components, Fr.C-Ⅴ-1 to Fr.C-Ⅴ-3; purify Fr.C-Ⅴ-3 by semi-preparative high performance liquid chromatography once to obtain 9 components, Fr.C-Ⅴ-3-1 to Fr.C-Ⅴ-3-9; purify Fr.C-Ⅴ-3-5 by semi-preparative high performance liquid chromatography twice to obtain compound 1*, and then resolve compound 1* through a chiral column to obtain compound (+)-1* and compound (–)-1*;

[0017] The structural formula of compound (+)-1* is The structural formula of compound (-)-1* is The structural formula of compound (+)-2* is The structural formula of compound (-)-2* is

[0018] In one preferred embodiment, in step S1, the number of ultrasonic extractions is 2 - 4 times, the extraction time for each time is 1 - 2 h; the liquid - solid ratio for each time is 1:1 - 2, and the filtrates are combined to obtain an aqueous extract.

[0019] In one preferred embodiment, in step S1, the macroporous resin is D101 macroporous resin.

[0020] In one preferred embodiment, in step S1, during the process of passing through the macroporous resin, elute successively with water, 50% ethanol, and 90% ethanol until the color of the solution does not change significantly, and three components, Fr.A to Fr.C, are obtained.

[0021] The component eluted by water is Fr.A, the component eluted by 50% ethanol is B, and the component eluted by 90% ethanol is C.

[0022] In one preferred embodiment, in step S2, the reagents used for gradient elution are dichloromethane, a solution of dichloromethane and methanol with a volume ratio of 100:1, dichloromethane, a solution of dichloromethane and methanol with a volume ratio of 50:1, dichloromethane, a solution of dichloromethane and methanol with a volume ratio of 30:1, dichloromethane, a solution of dichloromethane and methanol with a volume ratio of 20:1, dichloromethane, a solution of dichloromethane and methanol with a volume ratio of 10:1, a solution of dichloromethane and methanol with a volume ratio of 1:1, and methanol in sequence.

[0023] In one preferred embodiment, in step S2, an aqueous formic acid solution with a volume concentration of one-thousandth is added to the reagents used for gradient elution.

[0024] In one preferred embodiment, in step S3, in the elution solution for column chromatography of Fr.C-Ⅲ, the volume ratio of petroleum ether, dichloromethane, and methanol is 4 - 6:4 - 6:1.

[0025] In one preferred embodiment, in step S3, in the separation solvent used for separating Fr.C-Ⅲ-7 by thin-layer preparative plate, the volume ratio of petroleum ether to ethyl acetate is 3 - 5:1.

[0026] In one preferred embodiment, in step S3, the process for purifying Fr.C-Ⅲ-7-2 by semi-preparative high performance liquid chromatography is as follows: the chromatographic column is a Ph column, the mobile phase is an acetonitrile aqueous solution containing 0.1% fluoroacetic acid, the volume of acetonitrile and water is 36 - 40:62, and the flow rate is 2.0 - 3.0 ml / min.

[0027] In one preferred embodiment, in step S3, the process for resolving compound 2* by a chiral column is as follows: the resolving solvent is a mixed solution of n-hexane and isopropanol with a volume ratio of 88 - 92:10, and the flow rate is 0.7 - 0.9 ml / min.

[0028] In one preferred embodiment, in step S3, in the elution solution for column chromatography of Fr.C-Ⅴ, the volume ratio of petroleum ether, dichloromethane, and methanol is 4 - 6:4 - 6:1.

[0029] In one preferred embodiment, in step S3, the process for Fr.C-Ⅴ-3 by one-time semi-preparative high performance liquid chromatography is as follows: the chromatographic column is a Ph column, the mobile phase is a methanol aqueous solution containing 0.1% fluoroacetic acid, the volume of methanol and water is 50 - 60:45, and the flow rate is 2.0 - 3.0 ml / min.

[0030] In one preferred embodiment, in step S3, the process of purifying Fr.C-V-3-5 by secondary semi-preparative high performance liquid chromatography is as follows: the chromatographic column is a Ph column, the mobile phase is an acetonitrile aqueous solution containing 0.1% fluoroacetic acid, the volume ratio of acetonitrile to water is 36 - 40:62, and the flow rate is 2.0 - 3.0 ml / min.

[0031] In one preferred embodiment, in step S3, the process of resolving compound 1* by a chiral column is as follows: the resolving solvent is a mixed solution of n-hexane and isopropanol with a volume ratio of 88 - 92:10, and the flow rate is 0.7 - 0.9 ml / min.

[0032] Based on the same inventive concept, the present invention also protects the synthetic route of the benzofuran coumarin compounds as shown below:

[0033]

[0034] Based on the same inventive concept, the present invention also protects the preparation method of the benzofuran coumarin compounds, which includes the following steps:

[0035] A: Mix 2,2-dimethylcyclopropanecarboxylic acid, N-hydroxyphthalimide and a condensing agent evenly and stir to obtain an intermediate;

[0036] B: Mix the ketone, the intermediate and the catalyst under the protection of an inert gas, and stir under LED light irradiation until the reaction is complete to obtain the benzofuran coumarin compounds;

[0037] The catalyst includes N,N-diisopropylethylamine and also includes a base catalyst;

[0038] The base catalyst is any one or two of potassium ethylxanthate or sodium sulfide;

[0039] The wavelength of the LED lamp is 300 - 500 nM;

[0040] The molar ratio of 2,2-dimethylcyclopropanecarboxylic acid, N-hydroxyphthalimide (NHPI), and the condensing agent is 1:0.8 - 2:0.8 - 2; the molar ratio of the ketone, the intermediate, the base catalyst and N,N-diisopropylethylamine is 1:0.8 - 2:0.05 - 1:0.05 - 1.

[0041] In one preferred embodiment, in step A, an organic solvent is further included, and the organic solvent is selected from one or a combination of more of dichloromethane, chloroform, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, toluene, chlorobenzene, xylene, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, and preferably dichloromethane.

[0042] In one preferred embodiment, in step A, the condensing agent is selected from 4-N,N-dimethylpyridine (DMAP), 4-pyrrolidinopyridine (4-PPY), diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), preferably one or a combination of more of diisopropylcarbodiimide (DIC) and 4-N,N-dimethylpyridine (DMAP).

[0043] In one preferred embodiment, in step B, the ketone is umbelliferone or 7-hydroxy-6-methoxy-2H-benzopyran-2-one.

[0044] In one preferred embodiment, in step B, an organic solvent is further included, and the organic solvent is selected from one or a combination of more of dichloromethane, chlorobenzene, xylene, 1,4-dioxane, acetonitrile, water, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, preferably dimethyl sulfoxide.

[0045] In one preferred embodiment, in step B, the wavelength of the LED lamp is 300 - 500 nM, and the inert gas is argon or nitrogen.

[0046] In one preferred embodiment, the molar ratio of the ketone, intermediate, base catalyst, and N,N-diisopropylethylamine is 1:0.8 - 2:0.05 - 0.2:0.05 - 0.2.

[0047] In one preferred embodiment, in step A, the reaction time is 0.5 - 12 hours.

[0048] In one preferred embodiment, in step B, the reaction time is 1 - 48 hours.

[0049] In one preferred embodiment, an inorganic base is further included in step B, and the inorganic base is Li2CO3.

[0050] In one preferred embodiment, the molar ratio of the ketone to the inorganic base is 1:0.8 - 2.

[0051] In one preferred embodiment, work-up is included in both step A and step B.

[0052] In the above preparation method, the work-up is a suitable work-up that those skilled in the art can perform according to the reaction mechanism and reaction conditions of this step. This work-up operation aims to remove impurities and solvents and to a certain extent ensure the obtainment of a pure target product.

[0053] In one preferred embodiment, the post-treatment of step A includes the following steps:

[0054] Concentrate, add diethyl ether and mix evenly, then filter, take the filtrate, and dry under reduced pressure.

[0055] In one preferred embodiment, the post-treatment of step B includes the following steps:

[0056] Extract, wash with water, dry and concentrate, and purify by column chromatography.

[0057] Based on the same inventive concept, the present invention also protects another synthetic route of the benzofuran coumarin compounds, as shown below:

[0058]

[0059] Based on the same inventive concept, the present invention also protects another preparation method of the benzofuran coumarin compounds, including the following steps:

[0060] C. Add 2,2-dimethylcyclopropanecarboxylic acid, N-hydroxyphthalimide (NHPI) and N,N'-diisopropylcarbodiimide (DIC) into a reaction vessel and stir for reaction;

[0061] D. Add a ketone, sodium sulfide or potassium ethyl xanthate, N,N-diisopropylethylamine and lithium carbonate under the protection of an inert gas, and stir under LED light irradiation until the reaction is complete to obtain the benzofuran coumarin compounds.

[0062] In one preferred embodiment, in step C, an organic solvent is further included, and the organic solvent is selected from one or more of dichloromethane, chlorobenzene, xylene, 1,4-dioxane, acetonitrile, water, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, preferably dimethyl sulfoxide.

[0063] In one preferred embodiment, in step D, the ketone is umbelliferone or 7-hydroxy-6-methoxy-2H-benzopyran-2-one.

[0064] In one preferred embodiment, in step D, the wavelength of the LED lamp is 300-500 nM, and the inert gas is argon or nitrogen.

[0065] In one preferred embodiment, the molar ratio of the ketone, 2,2-dimethylcyclopropanecarboxylic acid, N-hydroxyphthalimide (NHPI), diisopropylcarbodiimide (DIC), sodium sulfide or potassium ethyl xanthate, N,N-diisopropylethylamine, and lithium carbonate is 1:1 to 5.0:1 to 5.0:1 to 5.0:0.1 to 1.0:0.1 to 1.0:1.0 to 5.0.

[0066] In one preferred embodiment, in step C, the reaction time is 0.5 to 12 hours.

[0067] In one preferred embodiment, in step D, the reaction time is 1 to 48 hours.

[0068] In one preferred embodiment, in step D, post-treatment is further included.

[0069] In the above preparation method, the post-treatment is a suitable post-treatment that those skilled in the art can perform according to the reaction mechanism and reaction conditions of this step. This post-treatment operation aims to remove impurities and solvents and, to a certain extent, ensure the obtainment of a pure target product.

[0070] In one preferred embodiment, the post-treatment of step D includes the following steps:

[0071] Extraction, washing with water, drying and concentration, and purification by column chromatography.

[0072] Moreover, it is worth supplementing that the above preparation method essentially adopts the "one-pot" method.

[0073] Based on the same inventive concept, the present invention also protects the use of the benzofuran coumarin compounds in the preparation of anti-hepatitis drugs.

[0074] In one preferred embodiment, the drug can be used directly or in the form of a pharmaceutical composition. In one preferred embodiment, the drug contains at least one of the benzofuran coumarin compounds as an active ingredient, combined with one or several pharmaceutically acceptable carriers and excipients.

[0075] In one preferred embodiment, the pharmaceutically acceptable carriers and excipients can be one or more solid, semi-solid, and liquid diluents, fillers, and pharmaceutical aids.

[0076] The pharmaceutical composition of the present invention is prepared into various dosage forms by methods recognized in the pharmaceutical field, such as liquid preparations (suspensions, syrups, oral liquids, or injections, etc.), solid preparations (tablets, capsules, or granules, etc.), sprays, etc. The above drugs can be administered through routes such as oral, sublingual, or injection (intravenous injection, intramuscular injection, or subcutaneous injection, etc.).

[0077] Through the preliminary evaluation of anti-hepatitis activity, the research results of the present invention show that the benzofuran coumarin compounds have anti-hepatitis effects.

[0078] The present invention has first discovered a benzofuran coumarin compound from the aqueous extract of Rutaceae. Through research, it is found that the compound has anti-hepatitis activity and potential liver protection effect, and a new preparation method for the direct C3 alkylation of the benzofuran coumarin compound is provided. The reaction reagents used in the preparation method of the present invention are significantly different from those in the prior art. Based on multiple exploratory experiments, it is found that the target product cannot be obtained by the preparation method in the prior art. According to a large number of exploratory experiments, a suitable catalyst is creatively added to obtain the target product. In addition, by exploring new reaction conditions completely different from those in the prior art, the target product is also obtained.

[0079] The beneficial effects of the present invention are as follows: The present invention has first extracted and isolated the benzofuran coumarin compound from the medicinal plant of Rutaceae. Through research, it is found that the compound has anti-hepatitis effect, and a direct and simple natural extraction and preparation process and the first chemical synthesis preparation route are provided. The preparation cost is low, the operation is simple, most of the organic solvents have low toxicity and can be recycled, and the treatment efficiency is high, which not only increases the benefit but also avoids pollution. Description of the Drawings

[0080] Figure 1 It is the HRESI-MS diagram of compound 1*;

[0081] Figure 2 It is the 1H NMR spectrum diagram of compound 1*;

[0082] Figure 3 It is the 13C NMR spectrum diagram of compound 1*;

[0083] Figure 4 It is the HRESI-MS diagram of compound 2*;

[0084] Figure 5 It is the 1H NMR spectrum diagram of compound 2*;

[0085] Figure 6 It is the 13C NMR spectrum diagram of compound 2*;

[0086] Figure 7 It is the determination of the safe concentration of the compound on HepG2 cells;

[0087] Figure 8 It is the effect of the compound in low, medium and high dose groups on ALT of HepG2 cells;

[0088] Figure 9 It is the effect of the compound in low, medium and high dose groups on AST of HepG2 cells;

[0089] Figure 10 It is the effect of the compound in low, medium and high dose groups on TNF-α of HepG2 cells;

[0090] Figure 11 Effects of the compounds in the low, medium, and high dose groups on IL-4 in HepG2 cells;

[0091] Figure 12 Effects of the compounds in the low, medium, and high dose groups on IL-10 in HepG2 cells. Specific implementation manners

[0092] The present invention is not limited to the following specific implementation manners. Those of ordinary skill in the art can implement the present invention in many other specific implementation manners according to the content disclosed in the present invention. Or, any simple changes or modifications made by adopting the design structure and idea of the present invention fall within the protection scope of the present invention. It should be noted that, without conflict, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0093] Example 1

[0094] Extraction, separation, and structural identification of furocoumarin compounds

[0095] A total of 10 Kg of dried above-ground parts of rue were used. The thick stem parts were crushed using a pulverizer and ultrasonically extracted twice with pure water at room temperature (8 L of pure water each time for 1 h). The two filtrates were combined to obtain an aqueous extract of dried rue. The aqueous extract of rue was concentrated under reduced pressure to 1823.7 g and roughly separated by macroporous resin (D101, about 2 Kg). Elution was carried out successively with water, 50% ethanol, and 90% ethanol until the color of the solution did not change significantly, obtaining three fractions (Fr.A - Fr.C) with volumes of 5 L, 4 L, and 5 L respectively. The fraction eluted with water was Fr.A, the fraction eluted with 50% ethanol was B, and the fraction eluted with 90% ethanol was C. Among them, the fraction Fr.C was concentrated under reduced pressure to obtain 14.3 g of an extract. Fr.C was further separated by silica gel column chromatography. Through thin-layer chromatography plate spotting analysis, finally, gradient separation by silica gel column chromatography was carried out using a dichloromethane - methanol system. The elution gradients were successively 100% dichloromethane, dichloromethane:methanol (volume ratio, 100:1, 50:1, 30:1, 20:1, 10:1, and 1:1), and 100% methanol. One-thousandth of an aqueous formic acid solution was added to the eluent. The fractions were collected and spotted on the plate for color development. Similar components were combined to obtain 12 fractions (Fr.C-I - Fr.C-XII).

[0096] Fr.C-Ⅲ was chromatographed again on a Sephadex LH-20 column with elution conditions of petroleum ether:dichloromethane:methanol = 5:5:1. The fractions from the gel column chromatography were combined after developing and visualizing on a thin layer plate, yielding 10 components (Fr.C-Ⅲ-1 to Fr.C-Ⅲ-10). Fr.C-Ⅲ-7 was separated on a preparative thin layer plate (petroleum ether:ethyl acetate volume ratio 4:1), giving 3 components (Fr.C-Ⅲ-7-1 to Fr.C-Ⅲ-7-3). Among them, Fr.C-Ⅲ-7-2 was purified by semi-preparative high performance liquid chromatography (Ph column, ACN-H2O-0.1% FA, v / v, 38:62, 2.0 ml / min) to obtain compound 2* (4.3 mg, tR = 32.3 min). Compound 2* was further resolved by a chiral column (n-Hexane-Isopropyl alcohol, v / v, 90:10, 0.7 ml / min) to obtain four optically pure compounds and compound (+)-2* (1.7 mg) and (–)-2 (1.9 mg).

[0097] Fr.C-Ⅴ was chromatographed again on a Sephadex LH-20 column with elution conditions of petroleum ether:dichloromethane:methanol = 5:5:1. The fractions from the gel column chromatography were combined after developing and visualizing on a thin layer plate, yielding 10 components, and were further combined by liquid phase analysis to finally obtain 3 components (Fr.C-Ⅴ-1 to Fr.C-Ⅴ-3). Fr.C-Ⅴ-3 was roughly separated by the first semi-preparative high performance liquid chromatography (Ph column, MeOH-H2O-0.1% FA, v / v, 55:45, 2.0 ml / min) to obtain 9 components (Fr.C-Ⅴ-3-1 to Fr.C-Ⅴ-3-9). Fr.C-Ⅴ-3-5 was continuously purified by semi-preparative high performance liquid chromatography (Ph column, ACN-H2O-0.1% FA, v / v, 38:62, 2.0 ml / min) to obtain compound 1* (4.6 mg, tR = 34.0 min). Compound 1* was further resolved by a chiral column (n-Hexane-Isopropyl alcohol, v / v, 90:10, 0.7 ml / min) to obtain four optically pure compounds and (+)-1* (2.2 mg) and (–)-1 (1.9 mg).

[0098] The structures of the compounds were identified as follows:

[0099] Compound (+)-1* is a white amorphous powder, and its chemical structural formula is:

[0100]

[0101] The spectral data are as follows: ESI-MSm / z 231.1016[M+H] + (calcd for C 14H 15 O3, 231.1016). +103.8 (c 0.025, MeOH); UV (MeOH) λ max (log ε) = 330 (1.81) nm; CD (MeOH) λ max = 211 (mdge – 5.298), 229 (mdge + 2.178), 254 (mdge + 1.438), 32 (mdge – 2.251) nm; IR (KBr) ν max 3416, 3285, 2925, 2854, 1599, 1260, 1192, 1120, 862 cm -1 .

[0102] 1 H NMR (500 MHz, MeOD) δ 7.48 (s, 1H), 7.40 (d, J = 8.5 Hz, 1H), 6.77 (dd, J = 8.5, 2.3 Hz, 1H), 6.71 (d, J = 2.1 Hz, 1H), 1.73 (t, J = 7.1 Hz, 1H), 1.27 (s, 3H), 0.87 (s, 3H), 0.81 (dt, J = 8.0, 5.1 Hz, 2H). 13 C NMR (100 MHz, MeOD) δ 165.3, 162.0, 155.8, 140.6, 129.9, 124.9, 114.3, 113.5, 103.0, 27.0, 26.6, 20.4, 19.7, 18.1.

[0103] The compound (-)-1* is a white amorphous powder, and its chemical structural formula is:

[0104]

[0105] The spectral data are as follows: –90.0 (c 0.036, MeOH); The UV and IR spectra of (-)-1* show characteristic absorptions similar to those of (+)-1*. CD (MeOH) λ max = 211 (mdge + 4.251), 228 (mdge – 2.538), 252 (mdge – 1.176), 328 (mdge + 1.985) nm; The 1H NMR and 13C NMR spectra are consistent with those of (+)-1*.

[0106] The HRESI-MS of compound 1* is as Figure 1 shown, and the 1H NMR spectrum of compound 1* is as Figure 2 shown, and the 13C NMR spectrum of compound 1* is as Figure 3 shown.

[0107] Compound (+)-2* is a pale yellow amorphous powder, and its chemical structural formula is:

[0108]

[0109] The spectral data are as follows: ESI-MS m / z 261.1125 [M+H] + (calcd for C 15 H 17 O4, 261.1122). +60.5 (c 0.096, MeOH); UV (MeOH) λ max (logε) = 345 (1.78) nm; CD (MeOH) λ max = 219 (mdge –4.391), 244 (mdge –0.812), 262 (mdge +1.861), 339 (mdge –1.190) nm; IR (KBr) ν max 2922.3, 2851.5, 1709.5, 1583.0, 1274.8, 1149.9, 1036.2 cm -1 ; 1 1H NMR (400 MHz, MeOD) δ 7.50 (s, 1H), 7.08 (s, 1H), 6.77 (s, 1H), 3.91 (s, 3H), 1.74 (ddd, J = 7.6, 6.2, 1.0 Hz, 1H), 1.27 (s, 3H), 0.87 (s, 3H), 0.81 (dt, J = 8.1, 5.0 Hz, 2H). 13 13C NMR (100 MHz, MeOD) δ 165.6, 151.7, 149.9, 147.0, 140.7, 125.2, 112.9, 109.5, 103.6, 56.8, 27.0, 26.7, 20.4, 19.7, 18.2.

[0110] Compound (–)-2* is a pale yellow amorphous powder, and its chemical structural formula is:

[0111]

[0112] The spectral data are as follows: ESI-MS m / z 261.1125 [M+H] + (calcd for C 15 H 17 O4, 261.1122). –51.0 (c 0.094, MeOH); The UV and IR spectra of (–)-2* showed characteristic absorptions similar to those of compound (+)-2**. CD (MeOH) λmax = 218 (mdge + 4.496), 244 (mdge + 0.875), 263 (mdge – 1.866), 338 (mdge + 1.206) nm; The 1H-NMR and 13C-NMR spectra were consistent with those of (+)-1*.

[0113] The HRESI-MS of compound 2* was as Figure 4 shown. The 1H-NMR spectrum of compound 2* was as Figure 5 shown. The 13C-NMR spectrum of compound 2* was as Figure 6 shown.

[0114] Example 2

[0115] The synthesis of compound 1 included two steps. One was the synthesis of the intermediate as follows:

[0116] Synthetic route of Alkyl-C(O)ONPhth:

[0117]

[0118] Add dichloromethane (8 ml), 2,2-dimethylcyclopropanecarboxylic acid (1 g), N-hydroxyphthalimide (1.4 g), 4-dimethylaminopyridine (DMAP, 107.0 mg) and N,N'-diisopropylcarbodiimide (DIC, 1.7 g) into the reaction flask, and stir at room temperature for 3 hours until the reaction is complete. Concentrate under reduced pressure to remove dichloromethane, add ether (10 mL), filter to obtain the filtrate, and rotary evaporate to obtain white solid Alkyl-C(O)ONPhth.

[0119] The other was the synthesis of compound 1:

[0120]

[0121] Add umbelliferone, Alkyl-C(O)ONPhth and other reaction reagents into the reaction vessel under the protection of N2 gas, continue to stir and react under the reaction conditions until the reaction is complete, extract with ethyl acetate, wash with water, dry and concentrate, and then purify by normal-phase silica gel column chromatography to obtain benzofuran coumarin compound 1* (81 mg).

[0122] According to this synthetic route, multiple schemes were optimized, and the conditions and yields of each scheme are shown in the following table:

[0123] Table 1 Conditions and yields for the synthesis of compound 1

[0124]

[0125]

[0126] Example 3

[0127] The synthesis route of Compound 1 proceeds according to the following formula:

[0128]

[0129] 2,2-Dimethylcyclopropanecarboxylic acid, N-hydroxyphthalimide (NHPI), an organic solvent, and N,N'-diisopropylcarbodiimide (DIC) are added to a reaction vessel and stirred for reaction; under the protection of an inert gas, a ketone, sodium sulfide, N,N-diisopropylethylamine, and lithium carbonate are added, and the mixture is stirred under LED light irradiation until the reaction is complete to obtain the benzofuran coumarin compound.

[0130] According to this synthesis route, multiple schemes were optimized, and the conditions and yields of each scheme are shown in the following table:

[0131] Table 2 Conditions and Yields for the Synthesis of Compound 1

[0132]

[0133] Explanation of the abbreviations in the table:

[0134] NHPI: N-Hydroxyphthalimide;

[0135] DIC: N,N'-Diisopropylcarbodiimide;

[0136] DMF: Dimethylformamide;

[0137] DIPEA: N,N-Diisopropylethylamine;

[0138] ]]DMSO: Dimethyl sulfoxide;

[0139] DCC: N,N'-Dicyclohexylcarbodiimide;

[0140] DMAP: 4-Dimethylaminopyridine;

[0141] DABCO: Triethylenediamine;

[0142] DMA: Dimethylacetamide;

[0143] TMEDA: Tetramethylethylenediamine;

[0144] DIPEA: N,N-Diisopropylethylamine;

[0145] Example 4

[0146] Synthesis of Compound 2

[0147] 7-Hydroxy-6-methoxy-2H-chromen-2-one (20 mg), Alkyl-C(O)ONPhth (26 mg), potassium ethyl xanthate (1.6 mg), N,N-diisopropylethylamine (1.3 mg) and dimethyl sulfoxide (0.25 mL) were added to a reaction vessel under N2 gas protection, and the reaction was continued to stir under 365 nm LED light irradiation for 12 - 48 h until the reaction was complete. After extraction with ethyl acetate, washing with water, drying and concentration, the benzofuran coumarin compound 2* (6.8 mg) was obtained by purification on a normal phase silica gel column, with a yield of 26%.

[0148] Example 5

[0149] Synthesis of Compound 2

[0150] Dimethyl sulfoxide (4 ml), 2,2-dimethylcyclopropanecarboxylic acid (183 mg), N-hydroxyphthalimide (261 mg) and N,N'-diisopropylcarbodiimide (202 mg) were added to a reaction flask and stirred at room temperature for 1 - 6 h until the reaction was complete. Under N2 protection, umbelliferone (154 mg), N,N-diisopropylethylamine (20 mg) and sodium sulfide (12 mg) were added to the reaction solution, and the reaction was continued to stir under 365 nm LED light irradiation for 12 - 48 h until the reaction was complete. After extraction with ethyl acetate, washing with water, drying and concentration, the benzofuran coumarin compound 1* (12 mg) was obtained by purification on a normal phase silica gel column, with a yield of 11%.

[0151] Example 6

[0152] Evaluation of the anti-hepatitis activity of the compound to be tested

[0153] The liver plays a very important role in drug metabolism. Factors such as viral infection, alcohol, drugs and obesity can all induce liver diseases. Although the treatment methods for different types of liver diseases vary, controlling the inflammatory process of the liver is an essential means for the vast majority of liver disease treatments. ALT and AST are sensitive markers reflecting hepatocyte damage and are the most commonly used liver function test indicators. ALT is mainly distributed in the cytoplasm of hepatocytes, and AST is mainly distributed in the cytoplasm and mitochondria of hepatocytes. The increase in their levels can reveal the degree of liver damage. In this experiment, the safety concentration of the compound was detected by testing the cytotoxicity of the compound to HepG2 cells; subsequently, an inflammatory model of HepG2 cells was established by using LPS, and whether the compound had anti-hepatitis and inhibitory effects on liver damage to play a hepatoprotective role was judged by detecting alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the cells. LPS can strongly induce the body's inflammatory response, resulting in the large expression of inflammatory factors. Therefore, we tested whether the compound to be tested could reduce the pro-inflammatory factor TNF-α and increase the levels of anti-inflammatory factors IL-4 and IL-10 to explore its anti-inflammatory activity.

[0154] (1) Evaluation of the cytotoxicity of the compound to be tested on HepG2 cells

[0155] (1) Experimental procedure: Appropriate amounts of compounds (–)-1*, (+)-1*, (+)-2*, and (–)-2* were accurately weighed and dissolved in DMSO to prepare a 10 mmol / L stock solution for standby. HepG2 cells were cultured in MEM medium containing 10% FBS + 1% double antibiotics and cultured in an incubator at 37 °C, 5% CO2, and saturated humidity. When the cell growth state was good and the growth amount was about 80% - 90%, subculture was carried out, and the cells were subcultured 2 - 3 times. The cell suspension was added to a 96-well plate at 5×10 3 cells per well, 100 μL per well. At the same time, a blank group (Blank group) with only complete medium was set. All groups were cultured in an incubator at 37 °C, 5% CO2, and saturated humidity. Logarithmically growing cells were treated with the drug to be tested. Seven concentrations (0 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM) of each drug to be tested were set and cultured for 24 h. Then, the medium in each well was removed, and 100 μL of medium containing 10 μL of CCK-8 was added. Three replicates were set for each treatment. After continuing to incubate at 37 °C, 5% CO2 for 4 h, it was taken out and the absorbance (OD) value at 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the mean value was plotted.

[0156] (2) Experimental results: The cells were treated with different concentrations (10 μM - 100 μM) of the compound to be tested for 24 h, and the cell viability of each group was detected by the CCK-8 method. The cytotoxicity results of (–)-1*, (+)-1*, (+)-2*, and (–)-2* on HepG2 cells are as Figure 7 shown. It can be seen from the results that compared with the blank group (cell survival rate was 100%), the survival rates of the compounds to be tested at different concentrations were significantly higher than those of the blank group, and there was no obvious toxicity to the cells. According to the above experimental results, the low, medium, and high concentrations of the compound to be tested in subsequent experiments were set to 10 μM, 20 μM, and 40 μM.

[0157] (2) Effects of the compound to be tested on the production of ALT and AST in HepG2 cells induced by LPS

[0158] (1) Cell grouping: An inflammatory model of HepG2 cells was established by selecting LPS (100 ng / mL), and the success of model establishment was judged by detecting alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the cells. Using fraxetin as the positive drug and the LPS-induced HepG2 inflammation as the model, a normal group, a model group, a positive drug group, and experimental low, medium, and high-dose groups (LPS + L-compound, LPS + M-compound, and LPS + H-compound) were set up respectively. Cell administration: Administration was carried out when the cells were in the logarithmic growth phase. The cells in the normal group were not treated with LPS and the test drug;; The cells in the model group were treated with 100 ng / mL lipopolysaccharide; The positive group was treated with LPS and 20 μM fraxetin; The experimental groups were treated with the corresponding test compounds and LPS at low, medium, and high doses (10 μM, 20 μM, and 40 μM) respectively. All groups were continued to be treated in an incubator at 37 °C, 5% CO2, and saturated humidity for 24 h. Each treatment was set up with three parallel groups.

[0159] The structure of fraxetin is:

[0160] (2) Sample collection: Centrifuge at 1000 g for 15 min at 2-8 °C, and take the supernatant for immediate detection. The aliquoted samples need to be stored at -80 °C. The thawed samples need to be centrifuged again and then detected. The microplate method was used, and the operation was carried out strictly according to the detection methods of the ALT and AST kits. After adding the stop solution, gently shake the 96-well plate horizontally to mix it evenly, place it at room temperature for 15 min, and measure the OD value of each well with an enzyme-labeling instrument at a wavelength of 510 nm.

[0161] (3) Experimental results: ALT and AST are sensitive markers reflecting hepatocyte damage and are the most commonly used liver function detection indicators. ALT is mainly distributed in the cytoplasm of hepatocytes, and AST is mainly distributed in the cytoplasm and mitochondria of hepatocytes. The increase in its level can reveal the degree of liver damage. The low, medium, and high-dose groups of (-)-1*, (+)-1*, (+)-2*, and (-)-2* on the contents of ALT and AST in the supernatant of HepG2 cells are as Figures 8-9 shown. It can be seen from the results that compared with the normal group, the levels of ALT and AST in the model group were significantly increased (P < 0.01), indicating that the model establishment was successful. Compared with the model group, the levels of ALT and AST in the low, medium, and high-dose groups of (-)-1*, (+)-1*, (+)-2*, and (-)-2* all decreased to varying degrees (P < 0.05). Compared with fraxetin, the contents of ALT and AST in (-)-1*, (+)-1*, (+)-2*, and (-)-2* were significantly reduced, and they could better exert the activity of treating hepatitis.

[0162] (III) Effects of the test compounds on LPS-induced production of inflammatory factors IL-4, IL-10, and TNF-α in HepG2 cells

[0163] (1) Experimental procedure: Cell grouping, modeling, drug administration and sample collection are the same as above. Strictly follow the instructions of the ELISA detection kit to determine the content of IL-4, IL-10 and TNF-α in the supernatant of each group of HepG2 cells. 1) Equilibrate the reagents at room temperature (18-25℃) for at least 30 minutes, prepare the standard and working solution according to the description of the ELISA kit, and set aside. 2) Sample addition: Set up standard wells and test sample wells respectively, add 100μL of standard or test sample to each well, shake gently to mix, cover with plate sticker, and incubate at 37℃ for 2h. 3) Discard the liquid in the well and spin dry, wash the plate 4 times, soak for 30s each time, 200μL per well, and spin dry. 4) Add 100 μL of detection antibody working solution to each well, cover with a new plate sticker, and incubate at 37°C for 1 hour (IL-4 and TNF-α); add 100 μL of detection antibody-HRP enzyme working solution to each well, cover with a new plate sticker, and incubate at 37°C for 40 minutes (IL-10). 5) Discard the liquid in the wells and spin dry. Wash the plate four times. Soak for 30 seconds each time, 200 μL per well, and spin dry. 6) Add 100 μL of avidin working solution to each well, cover with a new plate sticker, and incubate for 40 minutes. 7) Spin dry and wash the plate four times. Soak for 30 seconds each time, 200 μL per well. 8) Color development: Add 100 μL of substrate solution to each well in sequence and develop in the dark for approximately 20 minutes. 9) Stop: Add 100 μL of stop solution to each well in sequence to stop the reaction. 10) Detection: Measure the OD value of each well at 450 nm within 5 minutes after stopping the reaction.

[0164] (2) Experimental results: The contents of pro-inflammatory factor TNF-α and anti-inflammatory factors IL-4 and IL-10 in the supernatant of HepG2 cells in the (–)-1*, (+)-1*, (+)-2* and (–)-2* low, medium and high experimental groups were as follows: Figures 10-12As shown. Compared with TNF-α, IL-4, and IL-10 in the normal group, there were significant differences in TNF-α, IL-4, and IL-10 in the model group (P<0.01). Compared with the model group, except for TNF-α (83.33 pg / mL, P>0.05), IL-4 (11.00 pg / mL, P<0.05), and IL-10 (8.25 pg / mL, P>0.05) in the LPS+L-(–)-1* group, the TNF-α levels in other experimental groups were significantly decreased (P<0.01), and the IL-4 and IL-10 levels were significantly increased (P<0.01). The TNF-α levels in the low, medium, and high dose groups of (–)-1*, (+)-1*, (+)-2*, and (–)-2* all showed a decreasing trend with the increase of the administration dose; the IL-4 and IL-10 levels in the low, medium, and high dose groups of (–)-1*, (+)-1*, (+)-2*, and (–)-2* showed an increasing trend with the increase of the administration dose. Compared with Fraxetin, the TNF-α levels of (–)-1*, (+)-1*, (+)-2*, and (–)-2* were significantly decreased and the L-4 and IL-10 levels were significantly increased, which could better exert the activity of treating hepatitis.

[0165] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to enumerate all the implementation manners here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. A benzofuran coumarin compound, characterized in that, It is an optical isomer, crystal, pharmaceutically acceptable salt represented by the following structural formula: Wherein R1 is selected from hydrogen and C1-C6 alkoxy.

2. The benzofuran coumarin compound according to claim 1, wherein The benzofuran coumarin compound has the following structural formula:

3. The extraction method of the benzofuran coumarin compound according to claim 1 or 2, characterized in that It includes the following steps: S1. Crush the thick stem part of rue, extract by ultrasound to obtain an aqueous extract; concentrate the aqueous extract under reduced pressure and pass it through a macroporous resin to obtain three components Fr.A to Fr.C; S2. Fr.C is further subjected to silica gel column chromatography, eluted with a gradient of dichloromethane-methanol system, and similar components are combined to obtain 12 fractions Fr.C-I to Fr.C-XII; S3. Fr.C-III is subjected to column chromatography, eluted, and similar components are combined to obtain 10 components Fr.C-III-1 to Fr.C-III-10; Fr.C-III-7 is separated by a preparative thin layer plate to obtain 3 components Fr.C-III-7-1 to Fr.C-III-7-3; Compound 2* is obtained by semi-preparative high performance liquid purification of Fr.C-III-7-2, and compound 2* is further resolved by a chiral column to obtain compound (+)-2* and compound (–)-2*; Fr.C-V is subjected to column chromatography, eluted, and similar components are combined to obtain 3 components Fr.C-V-1 to Fr.C-V-3; Fr.C-V-3 is purified by semi-preparative high performance liquid once to obtain 9 components Fr.C-V-3-1 to Fr.C-V-3-9; Compound 1* is obtained by secondary semi-preparative high performance liquid purification of Fr.C-V-3-5, and compound 1* is further resolved by a chiral column to obtain compound (+)-1* and compound (–)-1*; The structural formula of compound (+)-1* is The structural formula of compound (-)-1* is The structural formula of compound (+)-2* is The structural formula of compound (-)-2* is 4. The extraction method according to claim 3, characterized in that, In step S1, the number of ultrasonic extractions is 2-4 times, and the extraction time for each time is 1-2 h; the liquid-solid ratio for each time is 1:1-2, and the filtrates are combined to obtain an aqueous extract; in step S1, during the process of passing through the macroporous resin, water, 50% ethanol, and 90% ethanol are used for elution in sequence, until the color of the solution does not change significantly, and three components Fr.A to Fr.C are obtained.

5. The extraction method according to claim 3, characterized in that, In step S2, the reagents used for gradient elution are dichloromethane, dichloromethane, a solution of dichloromethane and methanol in a volume ratio of 100:1, dichloromethane, a solution of dichloromethane and methanol in a volume ratio of 50:1, dichloromethane, a solution of dichloromethane and methanol in a volume ratio of 30:1, dichloromethane, a solution of dichloromethane and methanol in a volume ratio of 20:1, dichloromethane, a solution of dichloromethane and methanol in a volume ratio of 10:1, dichloromethane, a solution of dichloromethane and methanol in a volume ratio of 1:1, and methanol in sequence.

6. The extraction method according to claim 3, characterized in that In step S3, in the elution solution for Fr.C-III column chromatography, the volume ratio of petroleum ether, dichloromethane, and methanol is 4-6:4-6:1; in the separation solvent for Fr.C-III-7 separation by preparative thin layer plate, the volume ratio of petroleum ether to ethyl acetate is 3-5:1; The process for purifying Fr.C-Ⅲ-7-2 by semi-preparative high performance liquid chromatography is as follows: the chromatographic column is a Ph column, the mobile phase is an acetonitrile aqueous solution containing 0.1% fluoroacetic acid, the volume ratio of acetonitrile to water is 36 - 40:62, and the flow rate is 2.0 - 3.0 ml / min; the process for resolving compound 2* by a chiral column is as follows: the resolving solvent is a mixed solution of n-hexane and isopropanol with a volume ratio of 88 - 92:10, and the flow rate is 0.7 - 0.9 ml / min; in step S3, in the eluting solution for Fr.C-Ⅴ by column chromatography, the volume ratio of petroleum ether, dichloromethane, and methanol is 4 - 6:4 - 6:1; the process for Fr.C-Ⅴ-3 by one-time semi-preparative high performance liquid chromatography is as follows: the chromatographic column is a Ph column, the mobile phase is a methanol aqueous solution containing 0.1% fluoroacetic acid, the volume ratio of methanol to water is 50 - 60:45, and the flow rate is 2.0 - 3.0 ml / min; the process for purifying Fr.C-Ⅴ-3-5 by two-time semi-preparative high performance liquid chromatography is as follows: the chromatographic column is a Ph column, the mobile phase is an acetonitrile aqueous solution containing 0.1% fluoroacetic acid, the volume ratio of acetonitrile to water is 36 - 40:62, and the flow rate is 2.0 - 3.0 ml / min; the process for resolving compound 1* by a chiral column is as follows: the resolving solvent is a mixed solution of n-hexane and isopropanol with a volume ratio of 88 - 92:10, and the flow rate is 0.7 - 0.9 ml / min.

7. The preparation method of the benzofuran coumarin compound according to claim 1 or 2, characterized in that, It includes the following steps: A: Mix 2,2-dimethylcyclopropanecarboxylic acid, N-hydroxyphthalimide, and a condensing agent evenly, and stir to obtain an intermediate; B: Mix a ketone, the intermediate, and a catalyst under the protection of an inert gas, and stir under LED light irradiation until the reaction is complete to obtain the benzofuran coumarin compound; The catalyst includes N,N-diisopropylethylamine and also includes a basic catalyst; The basic catalyst is any one or two of potassium ethylxanthate or sodium sulfide; The wavelength of the LED lamp is 300 - 500 nM; The molar ratio of 2,2-dimethylcyclopropanecarboxylic acid, N-hydroxyphthalimide (NHPI), and the condensing agent is 1:0.8 - 2:0.8 - 2; the molar ratio of the ketone, the intermediate, the basic catalyst, and N,N-diisopropylethylamine is 1:0.8 - 2:0.05 - 1:0.05 - 1.

8. The preparation method according to claim 7, characterized in that, In the step A, an organic solvent is further included. The organic solvent is selected from one or a combination of more than one of dichloromethane, chloroform, acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, toluene, chlorobenzene, xylene, 1,4-dioxane, N,N-dimethylformamide, N,N-dimethylacetamide, and dimethyl sulfoxide; the condensing agent is selected from 4-N,N-dimethylpyridine (DMAP), 4-pyrrolidinylpyridine (4-PPY), diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), preferably a combination of one or more of diisopropylcarbodiimide (DIC) and 4-N,N-dimethylpyridine (DMAP); in the step B, the ketone is umbelliferone or 7-hydroxy-6-methoxy-2H-benzopyran-2-one; the inert gas is argon or nitrogen; the molar ratio of the ketone, the intermediate, the base catalyst, and N,N-diisopropylethylamine is 1:0.8-2:0.05-0.2:0.05-0.

2.

9. The preparation method of the benzofuran coumarin compound according to claim 1 or 2, characterized in that, The method includes the following steps: C. Add 2,2-dimethylcyclopropanecarboxylic acid, N-hydroxyphthalimide (NHPI), and N,N'-diisopropylcarbodiimide (DIC) into a reaction vessel and stir for reaction. D. Under the protection of an inert gas, add the ketone, sodium sulfide or potassium ethylxanthate, N,N-diisopropylethylamine, and lithium carbonate, and stir under LED light irradiation until the reaction is complete to obtain the benzofuran coumarin compound. Preferably, the molar ratio of the ketone, 2,2-dimethylcyclopropanecarboxylic acid, N-hydroxyphthalimide (NHPI), diisopropylcarbodiimide (DIC), sodium sulfide or potassium ethylxanthate, N,N-diisopropylethylamine, and lithium carbonate is 1:1-5.0:1-5.0:1-5.0:0.1-1.0:0.1-1.0:1.0-5.

0.

10. Use of the benzofuran coumarin compound according to claim 1 or 2 in the preparation of a drug for treating hepatitis.

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