Method for preparing benzopyran compound and application thereof in treating pulmonary fibrosis

Abstract

The present invention opens to the public a method to prepare a benzopyran compound and its use for treating pulmonary fibrosis. A benzopyran compound has a structure (I): ##STR00001##
in which: R1 represents hydrogen, C1-C4 alkyl, or various remaining amino acid moieties after removal of an amino group; R2 represents hydrogen, C1-C4 alkyl, or various remaining amino acid moieties after removal of an amino group; R3 represents hydrogen, or C1-C4 alkyl; and n is any integer of 1-4, wherein the benzopyran compound is derived from a broth of Streptomyces xiamenensis CGMCC No. 5675 by extraction, separation and purification. The derivatives of xiamenmycin made from the present invention have a higher bioactivity to suppress the proliferation of normal human lung fibroblast, and medicinal products containing the same are useful in the treatment of pulmonary fibrosis.

Claims

1. A preparation method of medicines for treating pulmonary fibrosis, comprising steps of: A) inoculating and culturing Streptomyces xiamenensis strain DSM 41903 in a liquid medium, then extracting and concentrating a broth for a crude extract; wherein extraction of the broth is as follows: the broth is centrifuged to separate a supernatant and a residue; then the supernatant is extracted with ethyl acetate and the residue with a solvent mixture; finally, the two extractions from all above are combined; and a solvent mixture described above is a mixture of ethyl acetate/methanol/acetic acid 80:15:5, v:v:v; B) subjecting the crude extract to a column chromatography on silica gel, eluting by CHCl.sub.3:MeOH, 100:1, with a rate of 15 second/drop and 200 ml per elution fraction; C) subjecting the fraction eluted by CHCl.sub.3:MeOH 15:1, v:v from the step B to a column chromatography on Sephadex LH-20 with a rate of 70-90 second/drop and purifying to obtain a benzopyran compound; wherein the fraction is collected and guided by HPLC fingerprints to find peaks containing a target UV profile max 206, 260 nm, then a combination is purified to obtain the benzopyran compound; wherein purification is as follows, the eluted fraction is collected and purified by an HPLC with a mobile phase of acetonitrile/water and by a rate of 45:55 acetonitrile/water, v:v to 55:45 acetonitrile/water, v:v within 35 minutes; and D) applying a therapeutically effective amount of the benzopyran compound to the medicines; wherein the benzopyran compound is shown in a formula (II): ##STR00005##

2. The preparation method as claimed in claim 1 for preparing the medicines for treating the pulmonary fibrosis, comprising treatments of acute respiratory distress syndrome, acute interstitial pneumonia or chronic acute worsening disease of idiopathic pulmonary fibrosis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) By reading and referring to the detailed description made by the following figures to the non-restrictive embodiment example, other characteristics, purposes and advantages of the present invention will become more apparent:

(2) FIG. 1 is a structure of a benzopyran compoundxiamenmycin C of the present invention;

(3) FIG. 2 is a quantification schematic diagram by a CCK-8 assay in an experiment for the benzopyran compoundxiamenmycin C to inhibit human lung fibroblast proliferation.

(4) FIG. 3 is a quantification schematic diagram by the CCK-8 assay in the experiment for xiamenmycin to suppress the human lung fibroblast proliferation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(5) In the following paragraphs, the present invention is described in detail with figures and examples which will assist the technical people in this field to understand the present invention. However, these examples as follows are unable to cover all the present invention. So it should be pointed out that any evolve and improvement made by the skilled person always belong to the scope of the patent protection asked for.

Example 1. Extraction and Purification

(6) S. xiamenensis M6 (i.e. Streptomyces xiamenensis CGMCC No. 5675) is cultured in a liquid medium (30 liters) for 7 days, then a broth is harvested and centrifuged to separate supernatant and mycelium. The supernatant is partitioned for 3 times by ethyl acetate and a residue is extracted for 3 times by a comparable amount of ethyl acetate/methanol/acetic acid (80:15:5, v:v:v) with 12 hours each time. Total supernatants were combined and concentrated to afford a crude extract (13.4 g). The crude extract is subjected to a column chromatography on silica gel (10 g, 200-300 meshes, produced by Qingdao Ocean Chemical Group Company) with 50 g silica gel, and eluted by CHCl.sub.3:MeOH (gradient from chloroform:methanol, 100:1, 70:1, 60:1, 50:1, 30:1, 15:1, 10:1, 5:1, 2:1, 1:1 to MeOH) with a rate of 15 second/drop and 150-100 ml/bottle. Then each fraction (200 ml) eluted by CHCl.sub.3:MeOH (15:1, v:v) is subjected to a column chromatography on Sephadex LH-20 with the rate of 70-90 second/drop and eluted by MeOH, then the combination above is isolated by a HPLC (semi-preparative column, C18) with a mobile phase of acetonitrile/water and by a rate of 45:55 (acetonitrile/water, v:v) to 55:45 (acetonitrile/water, v:v) within 35 minutes to obtain a benzopyran compound 1. Next, rest fractions eluted by a solvent mixture of chloroform/methanol, especially eluting ratio near CHCl.sub.3:MeOH (15:1, v:v) are also collected. The rest fractions are combined guiding by HPLC fingerprints to find peaks containing a target UV profile (max 206, 260 nm) and then the combination is purified by the HPLC (semi-preparative column, C18) with the eluent of acetonitrile/water and the gradient change from acetonitrile:water (45:55, v:v) to acetonitrile:water (55:45, v:v) to obtain a purified benzopyran compound 1, i.e. xiamenmycin C (1.5 mg).

(7) Physical and chemical properties of the compound 1: yellow amorphous powder; [].sup.26.sub.D +28.45 (c 0.0034, MeOH); UV (MeOH) .sub.max=206, 260 nm; CD (c 0.0024, MeOH) .sub.201 +12.3, .sub.202 +9.6, .sub.205 +8.0, .sub.207.4 +7.1, .sub.213.6 +0.14, .sub.217 1.2, .sub.245.2 +2.0, .sub.259.8 +3.14, .sub.283 +0.03; .sup.1H and .sup.13C NMR data, see Table 2; HRESIMS m/z 290.1768 [M+H].sup.+, (calcd for C.sub.17H.sub.24NO.sub.3, m/z 290.1756), 288.1610 [MH], (calcd for C.sub.17H.sub.22NO.sub.3, m/z 288.2073).

(8) TABLE-US-00001 TABLE 1 Compound 1 Location .sub.H (J in Hz) .sub.C, type HMBC 1 2 79.7, C 3 3.74, dd (7.4, 5.2) 66.3, CH 4a, 2, 9, 15 4 2.66, dd (17.3, 7.4) 31.3, CH.sub.2 8a, 5, 4a, 2, 3, 6 2.93, dd (17.3, 5.2) 4a 120.4, C 5 7.63, d (1.8) 130.2, CH 7, 8a, 4, 1 6 126.3, C 7 7.60, dd (8.4, 1.8) 127.4, CH 5, 8a, 1 8 6.74, d (8.4) 116.5, CH 4a, 8a, 6 8a 156.0, C 9 1.59, m 38.0, CH.sub.2 11, 12, 2, 3, 10 10 2.10, m 21.6, CH.sub.2 11, 12, 2, 9 11 5.10, t (7.3) 124.8, CH 13, 10, 14, 9 12 131.3, C 13 1.56, s 18.0, CH.sub.3 11, 12, 14 14 1.63, s 25.9, CH.sub.3 11, 12, 13 15 1.16, s 18.8, CH.sub.3 2, 3, 9 1 168.0, C 2 3 4 5 6

(9) Signals in Table 1 are based on spectrum analysis results of DEPT, .sup.1H-.sup.1H COSY, HMQC and HMBC. Multiplicity of a carbon signal is determined by using DEPT and represented by C (single peak), CH (double peak), CH.sub.2 (triple peak) and CH.sub.3 (quadruple peak) respectively.

Example 2. Inhibitory Effect of Compound 1 on WI26 Cells Proliferation and Activity

(10) Materials:

(11) Cells: Human lung fibroblasts, WI26 cell;

(12) Drug: Compound 1 (Xiamenmycin C), compound 2 (Xiamenmycin) obtained from the above example

(13) ##STR00004##
wherein: a group treated by the compound 1 is a drug treatment group 1 and a group treated by the compound 2 is a drug treatment group 2 for comparison with the compound 1.

(14) Methods: wherein WI26 cells at 70-80% confluent were trysinized by 0.25% trypsin solution and seeded in 96-well plates at an initial density of 110.sup.4 cells/mL. A medium was replaced 24 h later by the fresh with 15 g/ml compound 1 and 1/1000 DMSO (drug treatment group 1 and its control) or 30 g/ml compound 2 and 1/1000 DMSO (drug treatment group 2 and its control), then the medium was refreshed and cell viability was measured by using a CCK-8 method at day 0, 1, 2, 3, 4, 5, 6, respectively. Proliferation measurement was applied by adding 100 L complete medium and 10 L CCK-8 solutions to each well and incubating for 1 h. OD values of each well were measured at a primary wavelength =450 nm by using a Microplate Spectrophotometer. Data are shown as meansstandard deviation (SD) of three independent experiments, each performed in triplicate.

(15) Results of the experiment are shown as FIG. 2: the compound 1 has an inhibitory effect on WI26 cells proliferation. Inhibition of the WI26 cells exposed to 15 g/ml compound 1 for day one is 13.8% and day six is 38%. This illuminates that the benzopyran compound hereinbefore defined in the present invention is applicable for preparing drugs treating pulmonary fibrosis. Compared between these two drug treatment groups, an effective concentration of the compound 1 is only half while the inhibition increased near 10%. Therefore the compound 1 has priority in an aspect of the inhibitory effect on the WI26 cells proliferation and activation. Besides, both the compound 1 and the xiamenmycin have a characteristic of low toxicity to cell based on dynamics of the inhibitory effect in FIG. 2. So changing of a side chain of the compound 1 to a higher anti-proliferative effect and low toxicity is a technical difficulty in related field. According to the results above, the skilled person may appreciate that the modifications of the compound 1 (as claimed in claim 1), such as modification of amino acid moiety or increase the side chain, will inevitably bring with itself the considerable activity.

(16) Results of a treatment group 2 (30 g/ml compound 2 and 1/1000 DMSO) are shown as FIG. 3: the compound 2 has some effects on the WI26 cells proliferation and activation, but a inhibition rate for the day one is 10% and the day six is 28.5%.

(17) The above is a detailed description of the present invention. What needs to understand is that the present invention is not limited to the above specific embodiment. The technical people in this field can make various changes or modifications within the scope of claim and this will not influence the substantial contents of the present invention.