Steroidal compounds, methods of making and using the same

Abstract

The application discloses a kind of steroid compounds, with following structural formula I: the compound can slow down the progress of pulmonary fibrosis by inhibiting fibroblast proliferation, regulating extracellular matrix production degradation and inhibiting the effect of inflammation.

Description

Technical Field

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

[0002] Pulmonary fibrosis is a chronic, progressive lung disease often associated with destruction of alveolar structure and severe impairment of respiratory function. The disease is characterized by abnormal proliferation of fibroblasts and myofibroblasts in the lungs, and abnormal deposition of extracellular matrix (ECM), ultimately leading to decreased lung function and, in severe cases, even death. Idiopathic pulmonary fibrosis (IPF) is the most common type of pulmonary fibrosis with unknown etiology, and carries an extremely poor prognosis, with an average survival of only 2 to 4 years.

[0003] Currently, clinical drug options for the treatment of pulmonary fibrosis are limited. Approved anti-fibrotic drugs such as pirfenidone and nintedanib can slow disease progression, but their mechanisms of action are not fully understood and are often associated with significant side effects, such as hepatotoxicity and gastrointestinal reactions, limiting their widespread application. Therefore, developing anti-pulmonary fibrosis drugs with clear efficacy and minimal side effects has become a key research challenge. Summary of the Invention

[0004] To address the above-mentioned issues, the present invention aims to provide a steroidal compound, preparation method, and use thereof. This compound can slow the progression of pulmonary fibrosis by inhibiting the excessive proliferation of fibroblasts and suppressing inflammation. Furthermore, this compound has minimal 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 (CGMCC 1.12425) on a non-resistant LB solid plate. 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 reaches 0.6-0.8;

[0012] S3: Diosgenin was added to the culture medium at a final concentration of 0.3 mg / mL and the fermentation culture was continued for 3 days;

[0013] S4: Centrifuge the fermentation broth to separate the precipitate and supernatant. Resuspend the fermentation broth precipitate in methanol and disrupt the suspension using an ultrasonic disruptor for 30 minutes. After disruption, centrifuge and collect the supernatant, rotary evaporate to dryness, and then extract with an equal volume of a mixed solution of water and ethyl acetate, collecting the ethyl acetate layer. Extract the fermentation broth supernatant with an equal volume of ethyl acetate, collect the ethyl acetate layer, and combine it with the ethyl acetate layer from the fermentation broth precipitate, rotary evaporate to dryness, and obtain a crude extract.

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

[0015] S6: The preliminary isolated product was further purified and the target compound was separated using a semi-preparative liquid chromatography apparatus with a mobile phase of 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile solution at a flow rate of 1 mL / min and a sample injection volume of 200 μL. The effluent within 10-12 minutes was collected and rotary evaporated to dryness 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, operates 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 BEHC18OBD™ preparative column (10×150 mm, 5 μm).

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

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

[0021] As mentioned above, the drug is in an oral dosage form.

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

[0023] This invention discloses a novel compound that exhibits anti-pulmonary fibrosis activity in both in vitro and in vivo animal models. Unlike existing drugs, this compound can slow the progression of pulmonary fibrosis by inhibiting fibroblast proliferation, regulating the production and degradation of extracellular matrix, and suppressing inflammation.

[0024] Furthermore, the compounds disclosed in this invention exhibit high bioavailability and low toxicity in animal models, making them suitable for oral administration. Compared to existing anti-fibrotic drugs, the compounds of this invention not only have significant efficacy but also exhibit fewer side effects and a strong safety profile, suggesting promising 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 nuclear magnetic resonance 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 1 H NMR spectrum;

[0027] Figure 2 The nuclear magnetic resonance 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 13 C NMR spectrum;

[0028] Figure 3 The FT-IR spectrum 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 is shown;

[0029] Figure 4This is a 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 demonstrated 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; Figure 6 (A) is a pathological section of the 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 biosafety 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 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 examples and drawings. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and are not intended to limit the present invention.

[0034] Example 1

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

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

[0037] 1. Streak and activate Rhodococcus etherivorans on non-resistant LB solid medium and incubate at 30°C for three days. Then, use a toothpick to pick a single colony from the solid plate 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 2% of the inoculum into a 250 mL baffled shake flask containing 50 mL of non-resistant LB liquid medium. Incubate until the OD 600 When the optical density (OD) was 0.6-0.8, diosgenin previously dissolved in anhydrous ethanol was added to achieve a final concentration of 0.3 mg / mL of diosgenin, and the culture was continued for 3 days.

[0038] 2. The fermented liquid was transferred to a 50 mL centrifuge tube and centrifuged at 4000 rpm for 10 minutes to separate the supernatant and the bacterial precipitation. The supernatant was extracted with equal volumes of ethyl acetate (40 mL) twice using a separating funnel and the ethyl acetate layers were combined. The bacterial precipitation was resuspended with 20 mL of methanol, and then the suspension was crushed for 30 minutes using an ultrasonic crusher. The supernatant was then centrifuged at 4000 rpm for 10 minutes to collect the supernatant methanol. The mixture was dried by rotary evaporation and dissolved in a mixed solution of equal volumes of ethyl acetate and water (each 25 mL). The extract was then extracted with a separating funnel using the ethyl acetate layer. Finally, the supernatant and the bacterial precipitation layers obtained were combined and dried by rotary evaporation to obtain 102.4 mg of crude extract.

[0039] 3. Separate by silica gel column chromatography. Dissolve the crude extract in 10 mL 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 mixture ratio of petroleum ether and ethyl acetate of 10:1 to separate the preliminary separation product. Further separation and purification are 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 1 mL / min, and the injection volume is 200 μL. Under the elution conditions, elution isocratic with 65% acetonitrile, and the target compound is collected within 10-12 minutes. After final concentration, 9.75 mg of pure target compound is obtained with a yield of 39%.

[0040] Example 2 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 was characterized:

[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 application is a yellow powder, and the optical rotation test shows a negative value: [a] D 25 = -14.5 (C = 0.15 mg / mL, MeOH); the compound was further analyzed by LC-MS-IT-TOF, and the ion peak 449.1857 m / z [M+Na] + signal and 425.2614 m / z [M-H] - signal, and its molecular formula was determined as C 27 H 38 O4. Infrared absorption spectrum Figure 3 ) showed hydroxyl (3356 cm -1 ), aromatic ring (1608 cm -1 , 1506 cm -1 , 1456 cm -1 ) and carbonyl (1707 cm -1 ) signals. Among them 1 H NMR spectrum Figure 1 ) and 13 C NMR spectrum Figure 2 ) data suggest that the benzene ring in the structure contains an 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 are four 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 is determined as (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. After comparison with Anychem, Chemicalbook, Scifinder and other databases, no report about this compound was found, indicating that this compound is a new compound.

[0042] The NMR data of compound (2R,3S,3aR,3bS,5'R,8aS)-7-(5-hydroxy-2-methylbenzyl)-3,3b,5'-trimethyldodecanohydrospiro[indene[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 (600 MHz, CDCl3)

[0044]

[0045] Example 3 Cytotoxicity Test of Compound (2R,3S,3aR,3bS,5'R,8aS)-7-(5-hydroxy-2-methylbenzyl)-3,3b,5'-trimethyldodecanohydrospiro[indene[2,1-b]furan-2,2'-pyran]-6(3H)-one

[0046] This experiment used the MTT assay to determine the activity of mouse lung fibroblasts. Culture conditions were set at 37°C in a 5% CO2 incubator using DMEM medium containing 10% fetal bovine serum (FBS). Mouse lung fibroblasts were seeded in 96-well cell culture plates with a 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 end of the culture, the culture medium was replaced, 10μL of MTT (12mM) was added to each well, and the cells were incubated in an incubator for another 4 hours. After the incubation was completed, 85μL of culture medium was discarded from each well, 50μL of DMSO was added to mix, and the cells were incubated in an incubator for 10 minutes. Subsequently, the absorbance of each well was measured using a microplate reader, 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'-trimethyldodecanohydrospiro[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'-trimethyldodecano[indene[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 and weighing about 22g, were used in the experiment and were raised in a specific pathogen-free (SPF) environment. The mice were randomly divided into a control group, a model group, a compound of the present invention group 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 levels of biomarkers MMP-7, KL-6, SP-A and SP-D in the serum were detected. The significant increase in the levels of biomarkers indicated that the model was successfully established. While the model was being constructed, mice in the compound of the present invention group 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 were as shown in FIG. Figure 6 (A) Mouse lung tissue was homogenized and centrifuged at 4000 rpm for 5 minutes to collect the supernatant. ELISA kits were used to detect the levels of IL-6 and TNF-α. The results are shown in Figure 6 (B) and Figure 6 (C).

[0052] Figure 6 The results shown in (A) indicate that the compound of the present invention can effectively slow down the progression of bleomycin-induced pulmonary fibrosis, and its effect is significantly better than that of pirfenidone. Figure 6 (B) and Figure 6 The results of (C) further indicate that the compound of the present invention also exhibits 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'-trimethyldodecano[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 orbits, 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 showed that during pirfenidone treatment, the weight of mice decreased and then increased, while serum AST and ALT levels increased significantly, suggesting that pirfenidone has significant hepatotoxicity and systemic toxicity. However, during treatment with the compound of the present invention, the weight of mice steadily increased, serum AST and ALT levels remained stable, and no significant toxic side effects were observed, demonstrating the good biosafety of the compound of the present invention.

[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 slowing down pulmonary fibrosis, characterized in that: The invention comprises 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: Rhodococcus etherivorans ( Rhodococcus aetherivorans CGMCC 1.12425) and then added with diosgenin for fermentation, centrifugation, extraction and purification.

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