Marine fungus-derived sesquiterpenoid talaroterpene D, preparation method thereof, and application thereof in the preparation of RORα agonists

Abstract

The present invention discloses talaroterpene D, a sesquiterpene derived from a marine fungus, its preparation method, and its use in the preparation of RORα agonists. The structural formula of talaroterpene D is shown in Formula I. The present invention isolated talaroterpene D, a novel sesquiterpene derived from the fermentation culture of the marine fungus Talaromyces sp. SCSIO 41412, which stimulates the transcriptional activity of RORα and regulates downstream genes such as BMAL1. This compound, or a pharmaceutically acceptable salt thereof, can be used to prepare agonists for retinoic acid-related orphan receptor α.

Description

Technical field:

[0001] The present invention belongs to the technical field of natural product applications, and particularly relates to a sesquiterpene talaroterpene D derived from marine fungi, a preparation method thereof, and an application thereof in the preparation of a RORα agonist. Background technology:

[0002] The nuclear receptor retinoic acid-related orphan receptor α (RORα, NR1F1) is a member of the steroid / thyroid hormone receptor transcription factor superfamily. Its protein can bind to hormone response elements upstream of several genes as a monomer or as a homodimer to enhance the expression of these genes. It is widely expressed in tissues such as the liver, lung, skin, kidney, and adipose tissue, and has been shown to contribute to the transcriptional regulation of certain genes involved in circadian rhythms. It also controls multiple target genes related to lipid metabolism and inflammation regulation. In patients with fatty liver disease, including NASH, hepatic expression of RORα is significantly reduced. RORα has anti-lipogenic and anti-oxidative stress functions in the liver, thereby preventing the development of NASH. Treatment with activating RORα ligands can improve the symptoms of NASH and insulin resistance. RORα also plays a role in the dynamic switching of M1 / M2 polarization in macrophages. Therefore, RORα is expected to serve as a drug target for the treatment of fatty liver disease, inflammation, type 2 diabetes, and other diseases. Summary of the invention:

[0003] The first object of the present invention is to provide a sesquiterpene talaroterpene D which has the ability to stimulate the transcriptional activity of RORα, thereby causing downstream genes such as BAML1 to be regulated accordingly. The sesquiterpene talaroterpene D is a novel compound that has not been reported.

[0004] The marine fungus-derived sesquiterpene talaroterpene D of the present invention, or a pharmaceutically acceptable salt thereof, has a structural formula as shown in Formula I:

[0005]

[0006] Formula I.

[0007] The present invention found that talaroterpene D, a sesquiterpene derived from marine fungi, can significantly upregulate the RORα target gene BMAL1 ( Figure 5 b) confirmed that talaroterpene D is a RORα agonist. Therefore, talaroterpene D activates RORα transcriptional activity by binding to it, leading to the corresponding regulation of downstream genes such as BMAL1.

[0008] Therefore, the second object of the present invention is to provide the use of the above-mentioned sesquiterpene talaroterpene D, or a pharmaceutically acceptable salt thereof, in the preparation of a RORα agonist.

[0009] Preferably, the sesquiterpene talaroterpene D, or a pharmaceutically acceptable salt thereof, is used in the preparation of a medicament for treating fatty liver disease, inflammation, and type II diabetes.

[0010] The third object of the present invention is to provide a RORα agonist comprising talaroterpene D, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0011] The fourth object of the present invention is to provide a method for preparing sesquiterpene talaroterpene D, which is isolated from the fermentation culture of Talaromyces p. SCSIO 41412.

[0012] Preferably, the method specifically comprises the following steps:

[0013] The fermentation product of Talaromyces sp.SCSIO 41412 was prepared. The fermentation product was extracted with ethyl acetate. The ethyl acetate extract was concentrated to remove ethyl acetate to obtain an extract. The extract was chromatographed on a silica gel column with gradient elution of dichloromethane / petroleum ether (0:1, 1:1, 1:0, v / v) and methanol / dichloromethane (1:99, 2:98, 3:97, 5:95, 10:90, 20:80, 50:50, v / v) to sequentially obtain 10 elution fractions Fr.1–Fr.10. Fr.4 eluted with methanol:dichloromethane 1:99 was eluted with methanol / water (volume fraction 5% to 100%) on a medium-pressure ODS column to obtain 14 subfractions. Fr.4-3 eluted with methanol / water 15% was purified multiple times to obtain the monomer compound talaroterpene D.

[0014] The purification was performed by semi-preparative HPLC using a YMC-Pack ODS-A column (5 μm, 10×250 mm). Fr.4-3 was separated by semi-preparative HPLC using 50% CH3CN / H2O at 3 mL / min to obtain Fr.4-3-3 with a retention time of 9.7 min. The residue was then purified again by semi-preparative HPLC using a mobile phase of 68% CH3OH / H2O at 2.5 mL / min to obtain talaroterpene D with a retention time of 15.5 min.

[0015] The method for preparing the fermentation product of Talaromyces sp. SCSIO 41412 comprises the following steps: inoculating the strain Talaromyces sp. SCSIO 41412 into 200 mL of seed culture medium, culturing the product in a shaking incubator at 28° C. and 180 rpm for 3 days to obtain a seed culture, then using the seed culture to inoculate large-scale fermentation, preparing a rice culture medium in an Erlenmeyer flask, and statically fermenting the product at 26° C. for 28 days to obtain a rice fermentation product. The seed culture medium comprises 15 g of malt extract and 10 g of sea salt per liter of water, and the rice culture medium comprises 200 g of rice and 2% sea salt by mass per 230 mL of water.

[0016] A fifth object of the present invention is to provide the use of Talaromyces sp. SCSIO 41412 in the preparation of sesquiterpene talaroterpene D.

[0017] The present invention isolates sesquiterpenes from the fermentation culture of Talaromyces sp. SCSIO 41412, which can stimulate the transcriptional activity of RORα and cause downstream genes such as BMAL1 to be regulated accordingly. The sesquiterpenes can be used as RORα agonists.

[0018] Talaromyces sp.SCSIO 41412 was deposited on January 5, 2024 in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, and its deposit number is GDMCCNO: 64247. Description of the drawings:

[0019] Figure 1 The chemical structure of talaroterpene D and important two-dimensional NMR related information (COSY and HMBC) are shown.

[0020] Figure 2 This is an important two-dimensional nuclear magnetic resonance (NOESY) image of talaroterpene D.

[0021] Figure 3 The calculated and measured ECD diagrams of talaroterpene D.

[0022] Figure 4Molecular docking analysis of talaroterpene D and the RORα ligand binding domain. (a) Three-dimensional structure of the RORα ligand binding pocket of talaroterpene D; (b) Two-dimensional structure of the predicted binding mode of talaroterpene D and RORα ligand. Computer analysis results show that talaroterpene D binds well to RORα.

[0023] Figure 5 Figure 2. RORα and BMAL1 mRNA levels in HepG2 cells after co-culture with talaroterpene D. Results are presented as mean ± SEM (n = 3, ns = not significant, **p < 0.01). These results demonstrate that talaroterpene D significantly upregulates the RORα target gene BMAL1, confirming that talaroterpene D is a RORα agonist. Specific implementation method:

[0024] In order to make the purpose, technical solution and beneficial technical effects of the present invention clearer, the present invention is further described in detail below with reference to the embodiments. It should be understood that the embodiments described in this specification are only for the purpose of explaining the present invention and are not intended to limit the present invention. The parameters, proportions, etc. of the embodiments can be selected according to local conditions without substantial impact on the results.

[0025] Example 1 Fermentation of the fungus Talaromyces sp. SCSIO 41412 and isolation of the sesquiterpene talaroterpene D

[0026] Each component of seed culture medium (15g malt extract, 10g sea salt) is dissolved in 1L distilled water, be sub-packed in 1000mL triangular flask (300mL / bottle), put into 121 ℃ of sterilizing 30min of autoclave after the cotton plug sealing bandage.After treating that sterilized seed culture medium cools off, from agar plate, picking fungus Penicillium sp.SCSIO 41412 mycelium is inoculated in the bottle, and 28 ℃ of following shaking tables (180rpm) are cultivated 3 days, obtain seed culture.Use this seed culture to inoculate large-scale fermentation then, use Erlenmeyer flask configuration rice culture medium (200g rice, 2% sea salt, 230mL water), through autoclaving, amount to 47 bottles.26 ℃ of following static fermentations 28 days, obtain rice fermentation thing 326.1g.

[0027] The rice fermentation product was extracted three times with ethyl acetate. The ethyl acetate extracts were concentrated to remove the ethyl acetate, resulting in an extract. The extract was chromatographed on a silica gel column using a gradient elution of dichloromethane / petroleum ether (0:1, 1:1, 1:0, v / v) and methanol / dichloromethane (1:99, 2:98, 3:97, 5:95, 10:90, 20:80, 50:50, v / v). Ten elution fractions, Fr.1–Fr.10, were obtained sequentially. Fr.4 (methanol:dichloromethane 1:99) was eluted on a medium-pressure ODS column using methanol / water (5%–100%) to yield 14 subfractions. Fractions Fr.4-3, eluting at 15% methanol / water, were collected. Fraction Fr.4-3 was separated by semi-preparative HPLC using a YMC-Pack ODS-A column (5μm, 10×250mm) (eluent 50% CH3CN / H2O, flow rate 3mL / min) to obtain Fr.4-3-3 (retention time 9.7min). It was then purified again by semi-preparative HPLC using a YMC-Pack ODS-A column (5μm, 10×250mm) with a mobile phase of 68% CH3OH / H2O by volume at a flow rate of 2.5mL / min to obtain talaroterpene D at a retention time of 15.5min.

[0028] Example 2 Structural Identification of Talaroterpene D

[0029] The chemical structure of the sesquiterpene talaroterpene D isolated and obtained in Example 1 is as follows: Figure 1 The structure derivation process is as follows:

[0030] Talaroterpene D is a colorless oil. Its molecular formula was determined to be C by high resolution mass spectrometry (HRESIMS) data. 15 H 22 O2, unsaturation is 5. Analysis 1 The H NMR data (Table 1) show that the two alkenes [δ H 5.45 (1H, s, H-9), 5.00 (1H, d, J = 1.25 Hz, H2-13), 4.90 (1H, s, H2-13)], 4 methines [3.29 (1H, m, H-3), 0.96 (1H, m, H-4), 2.59 (1H, m, H-7), 3.97 (1H, m, H-8), two of which are connected to oxygen atoms], 4 methylenes [δ H2.22 (1H, m, H2-1), 2.06 (1H, dt, J = 14.0, 4.0 Hz, H2-1), 1.88 (1H, m, H2-2), 1.13 (1H, m, H2-2), 1.65 (1H, dd, J = 12.9, 5.0 Hz, H2-6), 1.06 (1H, m, H2-6), 4.35 (1H, d, J = 13.4 Hz, H2-12), 4.10 (1H, d, J = 13.4 Hz, H2-12)], and 2 methyl groups [0.90 (3H, s, H3-14), 0.93 (3H, d, J = 4.7 Hz, H3-15)]. 13 In addition to the carbon signals associated with the 12 hydrogen atoms mentioned above, the CNMR and HSQC data also showed three carbon signals including two olefin carbons [δ C 149.1(C-10), δ C 153.7(C-11)] and one quaternary carbon [δ C 38.3 (C-5)]. The above one-dimensional NMR data suggest that it is a sesquiterpenoid compound. 1 H- 1 H COSY spectrum ( Figure 1 )'s two spin systems H2-1 / H2-2 / H-3 / H-4 / H3-15 and H2-6 / H-7 / H2-8 / H-9, and the HMBC spectrum ( Figure 1 ) in the HMBC spectrum, the signals of H2-1 / C-5, C-9, C-10 and H3-14 / C-4, C-5, C-6 indicate that the compound is a sesquiterpene derivative with a 6 / 6 bicyclic skeleton, with two methyl groups attached to C-4 and C-5, respectively. The signals of H3-13 / C-12, C-11, C-7 and H2-12 / C-7, C-8, C-11 in the HMBC spectrum indicate that a furan ring is formed in the branched part. The relative configuration of the ring was determined by NOESY ( Figure 2 ) The related signals of H3-15 / H-3, H3-15 / H3-14, H3-14 / H-7, H3-14 / H-8 and H-7 / H-8 in the spectrum were determined to be rel-(3R,4R,5R,7S,8S). Based on the ECD curves of (3R,4R,5R,7S,8S) and (3R,4R,5R,7S,8R)( Figure 3 ), and the comparison found that the measured ECD was consistent with the calculated configuration (3R, 4R, 5R, 7S, 8S), proving that its absolute configuration is 3R, 4R, 5R, 7S, 8S, and it was named talaroterpene D. Its structural formula is shown in Formula I:

[0031]

[0032] Table 1. Talaroterpene D 1 H NMR (500 MHz) and 13 C NMR (125 MHz) data (deuterated DMSO)

[0033]

[0034]

[0035] Example 3 Determination of RORα Activity by Talaroterpene D

[0036] In order to search for potential RORα agonists, we screened multiple sesquiterpenoid natural products by molecular docking analysis using high-performance computers. The RORα ligand binding domain was retrieved from the available crystal structure (PDBID: 1N83) using the 2017-1 software and constructed according to the Protein Prepare Wizard workflow in the Maestro software package. The bonding site was selected using the Grid generator, and then the prepared ligand was flexibly docked to the receptor using Glide (XP mode) under default parameters. The molecular interaction between the RORα ligand binding domain and talaroterpene D was studied and analyzed. The results showed that talaroterpene D can bind to the RORα receptor binding domain ( Figure 4 ), with a binding free energy value (S value) of -8.613. In the two-dimensional binding model, hydrogen bonds were formed between the hydroxyl group of talaroterpene D and the tyrosine residue at position 380 on the RORα protein.

[0037] To further investigate the effect of talaroterpene D on RORα, quantitative polymerase chain reaction (qPCR) was used to detect the effect of talaroterpene D on the mRNA levels of RORα and its target gene BMAL1 in cells. HepG2 cells were cultured in vitro, and talaroterpene D compound was added to the HepG2 cell culture medium to a final concentration of 20 μM, and the cells were cultured for 24 hours. Total RNA was extracted from talaroterpeneD-treated and untreated HepG2 cell lysates using the RNAprep Pure Tissue Kit (Tiangen Biotechnology Co., Ltd., China). qPCR was performed using the PrimeScript RT Kit (Takara, Japan) and SYBR Green PCR Premix (Promega, USA) according to the kit instructions. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control gene to evaluate the relative mRNA levels of RORα and BMAL1. The results showed that talaroterpene D could significantly upregulate the RORα target gene BMAL1 ( Figure 5 ), confirming that talaroterpene D is a RORα agonist. Therefore, the sesquiterpenoid talaroterpene D activates RORα transcriptional activity by binding to the RORα receptor, leading to corresponding regulation of downstream genes such as BMAL1.

Claims

1. Sesquiterpene talaroterpene D, or a pharmaceutically acceptable salt thereof, having the structural formula shown in Formula I: Formula I.

2. Use of the sesquiterpene talaroterpene D according to claim 1, or a pharmaceutically acceptable salt thereof, in the preparation of a RORα agonist.

3. The use according to claim 2, characterized in that The invention is used in the preparation of medicines for treating fatty liver disease, inflammation and type II diabetes.

4. A RORα agonist, characterized in that Comprising the sesquiterpene talaroterpene D according to claim 1, or a pharmaceutically acceptable salt thereof as an active ingredient.

5. A method for preparing the sesquiterpene talaroterpene D according to claim 1, characterized in that: It is from Talaromyces It was isolated from the fermentation culture of sp.SCSIO 41412.

6. The preparation method according to claim 5, characterized in that The following steps are involved: preparation Talaromyces The fermentation product of sp. SCSIO 41412 was extracted with ethyl acetate. The ethyl acetate extract was concentrated to remove ethyl acetate to obtain an extract. The extract was purified by silica gel column chromatography with a gradient elution of dichloromethane / petroleum ether (v / v) (0:1, 1:1, 1:0) and methanol / dichloromethane (1:99, 2:98, 3:97, 5:95, 10:90, 20:80, 50:50). Ten elution fractions, Fr.1–Fr.10, were sequentially obtained. Fr.4, eluted with methanol:dichloromethane (1:99), was purified by a medium-pressure ODS column with methanol / water (volume fraction) ranging from 5% to 100%, yielding 14 subfractions. Fr.4-3, eluted with methanol / water (15%), were purified multiple times to obtain the monomer compound talaroterpene D.

7. The preparation method according to claim 6, characterized in that The purification was semi-preparative HPLC purification using a YMC-Pack ODS-A column 5 μ m, 10 × 250 mm, Fr.4-3 was separated by semi-preparative HPLC with 50% CH3CN / H2O at 3 mL / min to obtain Fr.4-3-3 with a retention time of 9.7 min, and then purified again by semi-preparative HPLC with a mobile phase of 68% CH3OH / H2O at 2.5 mL / min to obtain talaroterpene D with a retention time of 15.5 min.

8. The preparation method according to claim 6, characterized in that The preparation Talaromyces The fermentation product of sp.SCSIO41412 is the Talaromyces sp. SCSIO 41412 was inoculated into 200 mL of seed culture medium and cultured in a shaker at 28°C, 180 rpm, for 3 days to obtain a seed culture. The seed culture was then used to inoculate a large-scale fermentation. A rice culture medium was prepared in an Erlenmeyer flask and statically fermented at 26°C for 28 days to obtain a rice fermentation product. The seed culture medium was prepared by adding 15 g of malt extract and 10 g of sea salt per liter of water. The rice culture medium was prepared by adding 200 g of rice and 2% sea salt per 230 mL of water.

9. Preparation of the sesquiterpene talaroterpene D according to claim 1 Talaromyces sp.SCSIO 41412, deposit number is GDMCC NO: 64247.

10. The method according to claim 9 Talaromyces Use of sp.SCSIO 41412 in the preparation of the sesquiterpene talaroterpene D described in claim 1.

Images