Steroid compound as well as preparation method and application thereof

Abstract

The invention discloses a steroid compound, which has the following structural formula I: # imgabs0. The steroid compound can retard the pulmonary fibrosis progress by inhibiting fibroblast proliferation, regulating the generation and degradation of extracellular matrix and inhibiting inflammation.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical chemistry, and in particular to a steroid compound and a preparation method and application thereof. Background Art

[0002] Pulmonary fibrosis is a chronic, progressive lung disease that is usually accompanied by destruction of alveolar structure and severe impairment of respiratory function. The main characteristics of this disease are abnormal proliferation of fibroblasts and myofibroblasts in the lungs and abnormal deposition of extracellular matrix (ECM), which ultimately lead to decreased lung function and may even be fatal in severe cases. Idiopathic pulmonary fibrosis (IPF) is the most common type of pulmonary fibrosis with unknown etiology, and has a very poor prognosis, with an average survival of only 2 to 4 years.

[0003] Currently, there are limited drug options for the treatment of pulmonary fibrosis in clinical practice. Approved anti-fibrotic drugs such as pirfenidone and nintedanib can slow the progression of the disease, but their mechanism of action is not completely clear and is usually accompanied by significant side effects, such as hepatotoxicity and gastrointestinal reactions, which limits their widespread use. Therefore, the development of anti-pulmonary fibrosis drugs with clear efficacy and low side effects has become a key problem in current research. Summary of the invention

[0004] To solve the above problems, the present invention aims to provide a steroid compound, a preparation method and an application thereof, which can slow down the progression of pulmonary fibrosis by inhibiting the excessive proliferation of fibroblasts and inhibiting inflammation. The compound has low toxicity and side effects, high bioavailability and is suitable for the development of anti-pulmonary fibrosis drugs.

[0005] The present invention is achieved through the following technical solutions:

[0006] The present invention discloses a novel steroid compound having the following structural formula I:

[0007]

[0008] The structure of the compound is 1 H NMR and 13 C NMR analysis confirmed that it was (2R,3S,3aR,3bS,5'R,8aS)-7-(5-hydroxy-2-methylbenzyl)-3,3b,5'-trimethyldodecahydrospiro[indeno[2,1-b]furan-2,2'-pyran]-6(3H)-one.

[0009] The preparation method of the compound of the present invention comprises the following steps:

[0010] S1: Streak and activate Rhodococcus aetherivorans (Rhodococcus aetherivorans CGMCC 1.12425) on a non-resistant LB solid plate medium. This Rhodococcus aetherivorans was purchased from the China General Microbiological Culture Collection Center;

[0011] S2: Cultivate the activated Rhodococcus etherivorans in non-resistant LB liquid medium at 30°C and 220 rpm until the OD value is 0.6-0.8;

[0012] S3: Add diosgenin with a final concentration of 0.3 mg / mL to the culture medium and continue fermentation for 3 days;

[0013] S4: centrifuge the fermentation broth to separate the precipitate and the supernatant. After the fermentation broth precipitate is resuspended with methanol, the suspension is crushed with an ultrasonic crusher for 30 minutes. After crushing, the supernatant is collected by centrifugation and rotary evaporated to dryness, and then extracted with an equal volume of water and ethyl acetate mixed solution, and the ethyl acetate layer is collected; the fermentation broth supernatant is extracted with an equal volume of ethyl acetate, the ethyl acetate layer is collected, and combined with the ethyl acetate layer of the fermentation broth precipitate, and the crude extract is obtained by rotary evaporation and drying;

[0014] S5: Separate the crude extract by silica gel column chromatography, use 100-200 mesh silica gel to mix the sample, and 200-300 mesh silica gel to load the column. After loading the sample, use petroleum ether-ethyl acetate with a volume ratio of 10:1 as an eluent to elute to obtain a preliminary separated product;

[0015] S6: The preliminary isolated product is further purified, and the target compound is separated using a semi-preparative liquid chromatograph. The mobile phase is 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile solution. The flow rate is 1 mL / min, and the sample injection volume is 200 μL. The effluent within 10-12 minutes is collected and rotary evaporated to dry to obtain the novel steroid compound.

[0016] Preferably, the fermentation broth and the precipitation suspension are centrifuged at 4000 rpm for 10 min.

[0017] Preferably, the ultrasonic crusher has an operating power of 200W, works for 5s, and rests for 5s;

[0018] Preferably, the semi-preparative liquid chromatograph is composed of a Waters 1525 pump and a Waters The detector consisted of a Waters 2998 PDA with a BEHC18OBDTM preparative column (10×150 mm, 5 μm).

[0019] Use of the above-mentioned compound in preparing drugs for resisting pulmonary fibrosis or slowing down pulmonary fibrosis.

[0020] A drug for preventing or slowing down pulmonary fibrosis, comprising the above-mentioned compound.

[0021] The drug as mentioned above is in the oral dosage form.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0023] The present invention discloses a novel compound, which exhibits anti-pulmonary fibrosis activity in both in vitro experiments and in vivo animal models. Unlike existing drugs, the compound can slow down the progression of pulmonary fibrosis by inhibiting fibroblast proliferation, regulating the generation and degradation of extracellular matrix and inhibiting inflammation.

[0024] In addition, the compounds disclosed in the present invention show high bioavailability and low toxicity in animal models and are suitable for oral administration. Compared with existing anti-fibrosis drugs, the compounds of the present invention not only have obvious efficacy, but also have low side effects and strong safety, and have good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The drawings described herein are used to provide a further understanding of the embodiments of the present invention, constitute a part of this application, and do not constitute a limitation of the embodiments of the present invention. In the drawings:

[0026] Figure 1 The NMR of (2R,3S,3aR,3bS,5'R,8aS)-7-(5-hydroxy-2-methylbenzyl)-3,3b,5'-trimethyldodecanoic acid spiro[indeno[2,1-b]furan-2,2'-pyran]-6(3H)-one 1 H NMR spectra;

[0027] Figure 2 The NMR of (2R,3S,3aR,3bS,5'R,8aS)-7-(5-hydroxy-2-methylbenzyl)-3,3b,5'-trimethyldodecanoic acid spiro[indeno[2,1-b]furan-2,2'-pyran]-6(3H)-one 13 C NMR spectra;

[0028] Figure 3 is the FT-IR spectrum of (2R,3S,3aR,3bS,5'R,8aS)-7-(5-hydroxy-2-methylbenzyl)-3,3b,5'-trimethyldodecahydrospiro[inden[2,1-b]furan-2,2'-pyran]-6(3H)-one;

[0029] Figure 4The high resolution mass spectrum HR-ESI-MS chart of (2R,3S,3aR,3bS,5'R,8aS)-7-(5-hydroxy-2-methylbenzyl)-3,3b,5'-trimethyldodecahydrospiro[indeno[2,1-b]furan-2,2'-pyran]-6(3H)-one;

[0030] Figure 5 demonstrated the toxic effects of (2R,3S,3aR,3bS,5'R,8aS)-7-(5-hydroxy-2-methylbenzyl)-3,3b,5'-trimethyldodecahydrospiro[indeno[2,1-b]furan-2,2'-pyran]-6(3H)-one on mouse lung fibroblasts;

[0031] Figure 6 The effects of (2R,3S,3aR,3bS,5'R,8aS)-7-(5-hydroxy-2-methylbenzyl)-3,3b,5'-trimethyldodecahydrospiro[indeno[2,1-b]furan-2,2'-pyran]-6(3H)-one administration on pulmonary fibrosis were demonstrated; Figure 6 (A) is a pathological section of lung tissue of mice in the control group, model group, compound of the present invention group and pirfenidone group; Figure 6 (B) is the level of IL-6 (interleukin-6) in lung tissue of the control group, model group, compound of the present invention group and pirfenidone group; Figure 6 (C) is the level of TNF-α (tumor necrosis factor-α) in lung tissue of the control group, model group, compound of the present invention group and pirfenidone group;

[0032] Figure 7 The biological safety of (2R,3S,3aR,3bS,5'R,8aS)-7-(5-hydroxy-2-methylbenzyl)-3,3b,5'-trimethyldodecanoic acid spiro[indeno[2,1-b]furan-2,2'-pyran]-6(3H)-one was demonstrated; Figure 7 (A) is a graph showing the changes in body weight of mice in the control group, model group, compound of the present invention group and pirfenidone group; Figure 7 (B) is a graph showing changes in the levels of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) in the serum of mice in the control group, model group, compound of the present invention group and pirfenidone group. DETAILED DESCRIPTION

[0033] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments and drawings. The exemplary implementation modes of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0034] Example 1

[0035] The methods are conventional methods unless otherwise specified. The raw materials can be obtained from public commercial channels unless otherwise specified.

[0036] The specific steps of synthesizing (2R,3S,3aR,3bS,5'R,8aS)-7-(5-hydroxy-2-methylbenzyl)-3,3b,5'-trimethyldodecanoic spiro[indene[2,1-b]furan-2,2'-pyran]-6(3H)-one by transforming diosgenin with Rhodococcus etherovorans are as follows:

[0037] 1. Activate Rhodococcus etherivorans by streaking on non-resistant LB solid medium and incubate at 30°C for three days. Then, pick a single colony on the solid plate with a toothpick and inoculate it into 5 mL of non-resistant LB liquid medium. Continue to incubate at 30°C until the bacterial suspension becomes turbid. Take the turbid bacterial suspension and inoculate it into a 250 mL baffled shake flask containing 50 mL of non-resistant LB liquid medium at a 2% inoculum. Incubate until OD 600 When the optical density (OD) was 0.6-0.8, diosgenin previously dissolved in anhydrous ethanol was added to make the final concentration of diosgenin reach 0.3 mg / mL, and the culture was continued for 3 days.

[0038] 2. Transfer the fermentation broth to a 50 mL centrifuge tube and centrifuge at 4000 rpm for 10 minutes to separate the supernatant and the bacterial precipitate. Use a separatory funnel to extract the supernatant twice with equal volumes (40 mL) of ethyl acetate, and combine the ethyl acetate layers. The bacterial precipitate was first resuspended with 20 mL of methanol, and then the suspension was crushed for 30 minutes using an ultrasonic crusher, and then centrifuged at 4000 rpm for 10 minutes to collect the supernatant methanol. After rotary evaporation and drying, it was dissolved in a mixed solution of equal volumes of ethyl acetate and water (25 mL each), and the ethyl acetate layer was extracted using a separatory funnel. Finally, the supernatant obtained by the treatment and the ethyl acetate layer of the bacterial precipitate were combined, rotary evaporated and dried to obtain 102.4 mg of crude extract.

[0039] 3. Separate by silica gel column chromatography. Dissolve the crude extract with 10mL of ethyl acetate, add 1.5 times the weight of 100-200 mesh silica gel to mix the sample, and use 200-300 mesh silica gel to pack the column. The eluent is composed of a mixing ratio of petroleum ether and ethyl acetate of 10:1 to separate the preliminary separation product. Further separation and purification is carried out by semi-preparative liquid chromatography, and the mobile phase is set to 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile solution, the flow rate is 1mL / min, and the injection volume is 200μL. Under the elution conditions, 65% acetonitrile isocratic elution is used, and the target compound is collected within 10-12 minutes. After final concentration, 9.75mg of the pure product of the target compound is obtained with a yield of 39%.

[0040] Example 2 Characterization of the compound (2R, 3S, 3aR, 3bS, 5'R, 8aS)-7-(5-hydroxy-2-methylbenzyl)-3,3b,5'-trimethyldodecanoic acid spiro[indene[2,1-b]furan-2,2'-pyran]-6(3H)-one:

[0041] The compound (2R, 3S, 3aR, 3bS, 5'R, 8aS)-7-(5-hydroxy-2-methylbenzyl)-3, 3b, 5'-trimethyl dodecahydrospiro[indene[2, 1-b]furan-2, 2'-pyran]-6(3H)-one disclosed in the present invention is a yellow powder, and the optical rotation is negative after detection: [α] D 25 =-14.5 (C = 0.15 mg / mL, MeOH); the compound was further analyzed using LC-MS-IT-TOF, and the ion peak of the compound was detected at 449.1857 m / z [M+Na] + Signal and 425.2614m / z[MH] - signal, and its molecular formula was determined to be C 27 H 38 O4. Infrared absorption spectrum ( Figure 3 ) appears in the hydroxyl group (3356cm -1 ), aromatic ring (1608cm -1 、1506cm -1 、1456cm -1 ) and carbonyl (1707cm -1 ) signal. 1 H NMR spectrum ( Figure 1 )and 13 C NMR spectrum ( Figure 2 ) data suggested that the structure contained a benzene ring of ABX spin coupling system [δ6.97 (1H, d, 8.5 Hz, H-1), 6.58 (1H, dd, 2.0 Hz, 8.5 Hz, H-2), 6.64 (1H, d, 2.0 Hz, H-4)]; there were 4 methyl signals [2.22 (H3-18), 1.08 (H3-19), δ0.98 (H3-21) and 0.79 (H3-27)]; and carbonyl signal [δC 212.4 (C-9)]. Therefore, the structure of the compound was determined to be (2R, 3S, 3aR, 3bS, 5'R, 8aS)-7-(5-hydroxy-2-methylbenzyl)-3, 3b, 5'-trimethyldodecahydrospiro[inden[2,1-b]furan-2,2'-pyran]-6(3H)-one. After comparing with databases such as Anychem, Chemicalbook, and Scifinder, no reports on this compound were found, indicating that this compound is a new compound.

[0042] The NMR data of the compound (2R,3S,3aR,3bS,5'R,8aS)-7-(5-hydroxy-2-methylbenzyl)-3,3b,5'-trimethyldodecanoic spiro[inden[2,1-b]furan-2,2'-pyran]-6(3H)-one are shown in Table 1 below (see Figure 1 and Figure 2 ):

[0043] Table 1 Compounds of the present invention 1 H and 13 C NMR data (600MHz,CDCl3)

[0044]

[0045] Example 3 Cytotoxicity test of compound (2R,3S,3aR,3bS,5'R,8aS)-7-(5-hydroxy-2-methylbenzyl)-3,3b,5'-trimethyldodecanoic acid spiro[inden[2,1-b]furan-2,2'-pyran]-6(3H)-one

[0046] In this experiment, the MTT method was used to determine the activity of mouse lung fibroblasts. The culture conditions were set at 37°C and 5% CO2 in an incubator, and DMEM medium containing 10% fetal bovine serum (FBS) was used. Mouse lung fibroblasts were inoculated in a 96-well cell culture plate, with a liquid volume of 100 μL per well, containing 2×10 4 cells. After 24 hours of culture, the experimental group added different concentrations (400μM, 200μM, 100μM, 50μM, 25μM, 12.5μM, 6.25μM, 3.125μM, 1.5625μM and 0μM) of the compound of the present invention, and the other group added different concentrations (same concentration range) of pirfenidone and continued to culture for 48 hours. After the culture was completed, the culture medium was replaced, 10μL of MTT (12mM) was added to each well, and incubated in the incubator for 4 hours. After the incubation was completed, 85μL of culture medium was discarded from each well, 50μL of DMSO was added to mix, and then incubated in the incubator for 10 minutes. Subsequently, the absorbance of each well was measured using an ELISA instrument, and the detection wavelength was set to 540nm to calculate the growth inhibition rate of the compound on mouse lung fibroblasts. The experimental results are shown in Table 2, where pirfenidone was used as a positive control.

[0047] Table 2 Inhibitory effect of compound (2R,3S,3aR,3bS,5'R,8aS)-7-(5-hydroxy-2-methylbenzyl)-3,3b,5'-trimethyldodecanoic spiro[inden[2,1-b]furan-2,2'-pyran]-6(3H)-one on the growth of mouse lung fibroblasts (n=5)

[0048]

[0049] The experimental results are expressed as mean ± standard deviation (SD), and the sample size is n = 5. The results show that the compound of the present invention has a better inhibitory effect on the growth of mouse lung fibroblasts than pirfenidone.

[0050] Example 3 Effects of the compound (2R,3S,3aR,3bS,5'R,8aS)-7-(5-hydroxy-2-methylbenzyl)-3,3b,5'-trimethyldodecanoic acid spiro[inden[2,1-b]furan-2,2'-pyran]-6(3H)-one on bleomycin-induced pulmonary fibrosis in mice

[0051] Twelve healthy male C57BL / 6J mice, 6 weeks old, weighing about 22g, were used in the experiment and were kept in an environment free of specific pathogens (SPF). The mice were randomly divided into a control group, a model group, a compound group of the present invention, and a pirfenidone group. The model was induced by tracheal instillation of bleomycin hydrochloride injection (0.51mg / kg). After induction, orbital blood samples were taken from the mice, the serum was separated, and the contents of biomarkers MMP-7, KL-6, SP-A and SP-D in the serum were detected. The significant increase in the content of biomarkers indicated that the model was successfully established. While the model was being constructed, mice in the compound group of the present invention and the pirfenidone group were gavage-administered, once every other day, for a total of 5 times, and the mice were killed after being raised for 22 days. Subsequently, the lung tissues of the mice were removed for HE staining, and the results are as follows Figure 6 (A) As shown. Mouse lung tissue was homogenized and centrifuged at 4000 rpm for 5 minutes to collect the supernatant. The levels of IL-6 and TNF-α were detected using ELISA kits. The results are shown in Figure 6 (B) and Figure 6 (C).

[0052] Figure 6 The results shown in (A) indicate that the compounds of the present invention can effectively slow down the progression of bleomycin-induced pulmonary fibrosis, and their effects are significantly better than those of pirfenidone. Figure 6 (B) and Figure 6 The results of (C) further show that the compounds of the present invention also exhibit superior effects in reducing lung inflammation, which is better than pirfenidone.

[0053] Example 4 Biosafety Evaluation of Compound (2R,3S,3aR,3bS,5'R,8aS)-7-(5-hydroxy-2-methylbenzyl)-3,3b,5'-trimethyldodecanoic acid spiro[indene[2,1-b]furan-2,2'-pyran]-6(3H)-one

[0054] After the mice were administered, the body weights of the mice in the control group, model group, compound of the present invention group and pirfenidone group were monitored and weighed every other day. Figure 7 (A) As shown. Subsequently, blood was collected from the mouse eye sockets, and the serum was separated and the levels of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) in the serum were detected. The experiment was carried out according to the operating instructions of the ALT and AST kits. The results are shown in Figure 7 (B) shown.

[0055] The experimental results show that during the treatment with pirfenidone, the weight of mice first decreased and then increased, and the levels of AST and ALT in serum increased significantly, indicating that pirfenidone has non-negligible hepatotoxicity and systemic toxicity. During the treatment with the compound of the present invention, the weight of mice increased steadily, the levels of AST and ALT in serum remained stable, and no obvious toxic side effects were observed, indicating that the compound of the present invention has good biosafety.

[0056] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A steroid compound, the structural formula of which is shown in I:

2. Use of the compound according to claim 1 in the preparation of drugs for resisting or slowing down pulmonary fibrosis.

3. A drug for preventing or alleviating pulmonary fibrosis, characterized in that: Comprising the steroid compound as claimed in claim 1.

4. The drug according to claim 3, characterized in that The dosage form is oral.

5. The method for preparing a steroid compound according to claim 1, characterized in that: The product is obtained by culturing Rhodococcus etheriferus, adding diosgenin, fermenting, centrifuging, extracting and purifying.

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