Benzofuran derivatives and uses thereof

Technical Field

The invention relates to the field of organic chemistry, in particular to furan compounds, in particular to a benzofuran derivatives.

Background Art

Lespedeza to leguminoseae lespedeza the lespedeza virgata Lespedeza   virgata (Thurb) DC of the grass, widely distributed all regions throughout the country. The lespedeza the medicinal value of "herbal providing famine relief " the, "classification herbal medicine ", "herbal findings" there are records in the, Patent ye Jigen obtain Yuanbao, taste micro painstakingly , mild nature, has strong muscle profit kidney , healing, the efficacy of the lung effects, in civil widely used, the conventional hubei civil for the treatment of nephritis crude drugs, used for the treatment of chronic glomerulonephritis, diseases such as pyelonephritis. Chinese nephritis to the four piece to effect chronic glomerulonephritis, small side effect, have been written in the state of the basic medical insurance a similar drug contents, in view of the precise taste piece the curative effect of four nephritis, the researchers is widely used in research with the positive control drug.

Since the last century 30 so far at the end of the 1990s, many scholars at home and abroad the lespedeza of the chemical components of a substantial amount of research, the alkaloids have been identified, flavone, sterol a plurality of chemical components, such as, the renal function is not fully have a curative effect, but have not yet found significant components of the special activity. "Studies   on   the   cnstituents   of   Lespedeza   cyrtobotrya   MIQ   II   Thestructures   of   HaginniC, HaginiD,   LespedeolC and" a text discloses a benzofuran compound, the compound has the following molecular structure:

Wherein R is H or OCOCH₃ (Toshio   miyase, Akira   Ueno, Tadataka   Noro, and   SeigoFukushima.Shisuoka   College   of   Pharmacy   2-2-1, Oshika, sjosuoka, 422, Japan). When the R is H, the compound formula C₁₄ H₁₀ O₄, named as 2-(2 the [...] , 4 the [...] -dihydroxy-phenyl)-5-hydroxy-benzofuran, is light brown needle, melting point of: the 207-208 [...] , ferric trichloride experiment and diazo experimental positive, the compound is from the installation lespedeza (Lespedeza   cyrtobotrya   M_1Q.) xylem methanol extract obtained by separation of the ethyl acetate portion, (Phytoalexin) a plant in sozin of; when the R OCOCH₃ time, the molecular formula is C₂₀ H₁₆ O₇, its melting point is 154-156 the [...] , can be through the 2-(2 the [...] , 4 the [...] -dihydroxy-phenyl)-5-hydroxy-benzofuran by the acetic anhydride reaction and pyridine, by acetylation.

the country knows the bureau in 9 January 2008 discloses an invention Patent application (CN101100463 public number), the application discloses a structure shown in the formula of the benzofuran derivatives, the benzofuran derivative is separated from the lespedeza thin stem , molecular formula is C₁₇ H₁₆ O₆, molecular weight is 316, normal temperature and normal pressure to yellow-green ruleless powder, with for treatment of nephritis and peroxidaton, can be used for the treatment of nephritis.

Content of the invention

The purpose of this invention is to provide a memory with a medical value of the benzofuran derivatives.

The purpose of this invention to realize the above-mentioned technical scheme is:

A benzofuran derivatives, its molecular structure is shown in formula I:

The benzofuran derivatives of the invention for the first-time separation thin stem lespedeza [Lespedeza   virgata (Thurb) DC], molecular formula is C₁₈ H₁₆ O₇, molecular weight is 344, to dark green under normal temperature and pressure ruleless powder.

The benzofuran derivatives of the invention from the lespedeza thin stem can be obtained by separation in, the specific method of:

(1) the drying of grass after crushing the lespedeza thin stem , using 95% ethanol percolation extraction, reclaiming and extracting solution of ethanol to obtain the extract, the extract with the petroleum ether extraction, takes the aqueous phase , extraction using chloroform, takes the aqueous phase , then using the ethyl acetate extraction, takes the aqueous phase , finally, the n-butanol extraction, saw vertically from butanol portion;

(2) the positive butanol portion by forward silica gel column, the ratio of the volume in the 98 [...] 2, the 95 [...] 5 and 9 the [...] 1 a mixture of chloroform and methanol gradient elution, chloroform and methanol volume ratio of the collection of the 9 [...] 1 the eluant, then the liver ODS, sequentially with the concentration of 30%, 50%, 60% of the methanol gradient elution, collecting 50% methanol eluant, then Sephadex   LH-20 column chromatography, using pure methanol to elute, collect eluant recycling solvent after the liver, the volume ratio of the 6 [...] 4 eluting methanol-water mixtures, the chromatography conditions for the eluant chloroform: methanol =9   [...] 1, iodine color thin-layer chromatography detection, taking thin-layer after analyzing, iodine the eluant of the color is blue-black color, after removing methanol to obtain greenish black crystalline.

The benzofuran derivatives of the invention with for treatment of nephritis and removing free radicals, can be used for preparing a medicament for the treatment of nephritis, the medicament the compounds of the formula I in a pharmaceutically acceptable adjuvant, may be injection, oral tablet, capsule, and the like.

In order to better understand the invention, will be through the in vitro cell experiment to prove the technical effect of the present invention.

A, body external renal tubular epithelial cell experiment:

1, material and method

Normal kidney tubule cell strain (HK-2) bought from Wuhan China typical culture collection center. HK-2 for the 37 thaw [...] warm water, 1000r/min centrifugal 5 min, discard the upper layer of frozen stock solution, plus 10% FBS   DMEM-F₁₂ culture solution, 37      5% CO₂ static culture under the conditions of, conventional liquid exchange, cell fusion 80% when above, 0.25% trypsin digestion, passage, inverted microscope every day under observation of cell morphology, taking 4-6 generation cells are used for the experiment.

2, drug intervention and experiment packet

Blank control: 10% FBS   DMEM/F12 culture solution;

Model set (TGF-β₁) group: 10% FBS   DMEM/F12 culture solution +TGF-β₁ (8ng/ml);

benzene that Pulley group : Benazepril (10 mg/L) +TGF-β₁ (8ng/ml);

Group DPI (DPI, 10     mol/L) +TGF-β₁ (8ng/ml);

The invention relates to a high-dose group: the invention benzofuran derivatives (100     g/ml) +TGF-β₁ (8ng/ml);

Dose group in the present invention: the invention benzofuran derivatives (50     g/ml) +TGF-β₁ (8ng/ml);

Low dosage group of the present invention: the invention benzofuran derivatives (25     g/ml) +TGF-β₁ (8ng/ml)

3, confocol microscope semiquantify determining the intracellular ROS

Method for measuring intracellular ROS method of   Mochizuki reference T (T   Mochizuki, Furuta   S, Mitsushita   J, et   al.Inhibition   of   NADPH   oxidase   4   activates   apoptosis   Via   the   AK7/apoptosis   signal-regulating   Kinase1   pathway   in   pancreatic   cancer   PANC-1cells .Oncogene, 2006, 25, 3699-3707). Fluorescent probe, dichlorofluorescein diacetate ester H₂-DCFDA is esterase hydrolysis in vivo, forming non-fluorescent also prototype dichlorofluorescein DCFH, the latter to reactive oxygen-sensitive, can be rapidly oxidized so as to have a high fluorescence material oxidized DCF. Using confocal microscope may be of the direct observation of the generated active oxygen in the cell, the fluorescence intensity reflects the degree of oxidative stress. Taking 3rd generation HK-2 cell for the six holes tested in the plate culture to 80% or more after fusion growth, deprivation of serum 24 hours after synchronization, in addition the blank set according to the packet given DPI and NADPH oxidase inhibitors (1 subsidence mol/L) intervention 24 hours later, for the each group of cells 10     mol/LH₂-DCFDA for the 37 lucifuge [...] incubate 30 minutes, three times with wash buffer solution D-Hanks, laser confocal microscope observation, the excitation wavelength of 488 nm, emission wavelength 510 nm, camera. Measuring each group of fluorescent intensity value, and in order to control the intensity of the fluorescent baseline, calculating the relative fluorescent intensity value of each experimental group.

4, chemical detection HK-2FN immune cell expression

The HK-2 made suspension, by 1 × 10⁶/L inoculating to the pre-placed with 6 × 22 mm of the cover glass in the six-hole plate, set CO₂ an incubator, culture 1 day, single-layer grown to be close to the cells, according to the section 2 after the packet processing section according to the following method to measure HK-2FN expression:

A. Experimental procedure:

(1) culture for the six holes tested in the plate the cells fixed with cold acetone for 20 minutes

(2) adding of each hole 50     l   3% H₂ O₂ incubate 10 min, remove the activity of the endogenous catalase

(3) PBS washing three times, every 3 min

(4) with a normal fluid rabbit blood serum drop which the 50  l, room temperature incubation 15 min, is dumped

(5) in each experimental group adds by drops one anti- the 50  l, the controls dropwise PBS50  l, the 4 [...] incubation sleepovers

(6) PBS washing three times, every 3 min

(7) the dropping IgG goat anti-mouse horse-radish enzyme mark the   50  l, 37 the incubating [...] 15 min

(8) three times with PBS, each 3 min

(9) anti- adds by drops two the 50  l, 37 the incubating [...] 1h

(10) wash PBS three times, every 3 min

(11) the color solution DAB 10 min, under a microscope, the degree master dyeing

(12) the tap water washing

(13) hematoxiline duplicate dyes usually 15 seconds, hydrochloric acid alcohol differentiation

(14) water flushing 15 min

(15) dehydration, transparent, sealed sheet, microscopic examination

B. Result judgment: to cell slurry for positive cells appear brown particles, and imaging system using optical microscope analysis (olympus), with 40 times FN objective lens measuring the cell immune response percentage of positive cells of, counting of not less than 200 cell, calculate the number of cells positive for FN total cell number of the percentage value.

5, results

(1) confocol microscope semiquantify determining the intracellular ROS: confocol microsopy, HK-2 normal group can be observed in cells of a relatively weak green fluorescent ROS, TGF-β₁ stimulate HK-2 cells 24 hours later, compared with the normal group can be a significant increase in the generation of ROS in the cell (P=0.000); DPI (10 subsidence mol/L) and acts on the cells of different concentration Lespedezavirgatol for medicine group 24 hours later, and Control group, can be significantly reversed by TGF-β₁ the increase of the induced ROS, there has been a significant statistical significance of differences (P=0.000), as shown in table 1 is shown.

Table 1 compound of the present invention, to confocol microscope each group of semi-quantitative detection of the level of ROS in the cell (X±S)

Notes: F=797.333, P=0.000^{black triangular}: P <0.05,^▲▲: P <0.0   1VS   Normal;

*: P <0.05, **: P <0.01   Control   VS.

(2) detection of immune cell chemical HK-2 cell FN expression:

Normal control group HK-2 cells very little in the expression of FN, model group adds the TGF-β₁   8ng/ml   24h rear, HK-2 cytolymph FN expression in a strong and positive in, compared with the normal group (P=0.000) there is significant difference, following intervention Lespedezavirgatol, with the increase of the density of the (25, 50,100 μg/ml), express a partially FN is inhibited, compared with the model group, (P=0.000) there is significant difference. Positive control drug Benazepril also make similar outcome.

Table 2 experiment each group of immunohistochemistical FN (X±S) expression

F=153.404, P=0.000^{black triangular}: P <0.05,^▲▲: P <0.01 relative to the blank-control group;

*P <0.05, **: P <0.01 with respect to the model set.

Mode of execution

Example 1: from thin stem lespedeza extracting and separating the compounds of the present invention.

Lespedeza thin stem the drying of the grass 40 kg, crushed, with 95% ethanol percolation extraction, reclaiming and extracting solution of ethanol to obtain the extract, the extract with the petroleum ether extraction, takes the aqueous phase , extraction using chloroform, takes the aqueous phase , then using the ethyl acetate extraction, takes the aqueous phase , finally, the n-butanol extraction, the butanol part, by forward silica gel column, the ratio of the volume in the 98 [...] 2, the 95 [...] 5, the 9 [...] 1 mixture of chloroform and methanol gradient elution, chloroform and methanol volume ratio of the collection of the 9 [...] 1 the eluant, through the ODS reverse column chromatography, are sequentially with the concentration of 30%, 50%, 60% of the methanol gradient elution, collecting 50% methanol eluant, through Sephadex   LH-20 column chromatography, eluted with pure methanol, collecting solution recovery solvent, the liver, methanol-water (the 6 [...] 4) elution, the eluent is chromatography conditions for chloroform: methanol =9   [...] 1, iodine color thin-layer chromatography, thin-layer after analyzing, iodine color to blue-black color of the eluant, remove the solvent to obtain dark green crystalline 0.78g (0.02g/kg).

Identifying: molecular formula C₁₈ H₁₆ O₇, ruleless dark green powder, HR-ESI-MS: 344.0433 (M)⁺.¹ H-NMR,¹³ C-NMR detected data in table 2.

Table 2¹ H-NMR (400MHz)¹³ C-NMR (100MHz) (DMSO-d₆) Spectral   date

Example 2:

The example 1 of the compound, the method for preparing medical injection of the injection liquid: the compound 1000 mg dissolved in 1000 ml in physiological saline, heating solution, after mixing in the bottle is filled, sealed, sterilized into a parenteral solution of 2 mg/2 ml. Vigiv used for intravenous injection (iv.), adult 6 mg/day, equivalent to 3 supporting; children often consumption: by weight 0.05 mg/kg/day.

Example 3:

The example 1 of the compound, the method for preparing medical injection of the injection liquid. The compound 2000 mg dissolved in 1000 ml in physiological saline, heating solution, after mixing in the bottle is filled, sealed, disinfection is made of a 4 mg/2 ml injection. Vigiv used for intravenous injection (iv.), adult 6 mg/day, equal to 1.5 a; common for infant consumption: by weight 0.05 mg/kg/day.

Example 4:

The example 1 of the compound, the process for the preparation of pharmaceutical tablet made into tablets. The compound 1600 mg, plus starch 13440 mg, plus acetyllactosyl 8960 mg mixed evenly, tablets heavy to 150 mg tablet, each containing vigiv 10 mg. Usage: oral. Adult normal consumption: a 20 mg (2 particles), once every six hours; children often consumption: a weight 0.5 mg/kg, once every six hours.

Example 5:

The example 1 by using the compound of preparation method of tablet made into tablets, the compound 3200 mg, plus starch 17280 mg, plus acetyllactosyl 11520 mg mixed evenly, heavy sheet is 200 mg tablet, each containing vigiv 20 mg. Usage: oral. Adult normal consumption: a 20 mg (1 granulata), once every six hours; children often consumption: a weight 0.5 mg/kg, once every six hours.

Example 6:

The example 1 by using the compound of preparation method of the capsule made into capsule. The compound 3200 mg, plus starch 11520 mg, plus acetyllactosyl 17280 mg is mixed to make particles, filling into each grain to the 200 mg capsule, each capsule containing vigiv 20 mg. Usage: oral. Adult normal consumption: a 20 mg (1 granulata), once every six hours; children often consumption: a weight 0.5 mg/kg, once every six hours.