CN116768956A

CN116768956A - RDH10 agonist

Info

Publication number: CN116768956A
Application number: CN202310247087.1A
Authority: CN
Inventors: 叶敏; 匡易; 乔雪; 苏惠飞
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2022-03-17
Filing date: 2023-03-15
Publication date: 2023-09-19

Abstract

The present invention relates to the compound 25S-anti C and its use for the preparation of RDH10 agonists. The compound has remarkable RDH10 agonism, can remarkably prevent or reduce lipid deposition, and can be used for preventing and treating lipid metabolism abnormality or diseases related to lipid metabolism abnormality.

Description

RDH10 agonist

Technical Field

The present invention relates to compound 25S-Antcin C or a salt thereof and its use for the preparation of an RDH10 agonist.

Background

Retinol dehydrogenase 10 (RDH 10) is a subtype of retinol dehydrogenase, belonging to the short chain dehydrogenase/reductase (SDR) superfamily, consisting of 341 amino acids, expressed in various tissues and organs such as retina, kidney, liver, small intestine, placenta, lung, heart and skeletal muscle. RDH10 has been shown to exhibit the highest affinity for retinol in SDR enzymes, catalyzing the oxidation of all-trans and 11-cis retinol to the corresponding retinoid, which is the first step in the metabolism of retinol to retinoic acid (Beliaeva OV, et al, J Biol Chem 283,29,20299-20308); RDH10 also plays an important role in the metabolic homeostasis of all-trans retinoic acid. Reduced levels of all-trans retinoic acid synthesis in RDH10 heterozygous mice have been reported in the literature to lead to lipid deposition (Yang D, et al, diabetes 2018,67,662-673).

Disclosure of Invention

The inventors of the present invention found that a compound of formula (I) (also referred to herein as "25S-Antcin C", "25S-AC" or "ACs") has a remarkable RDH10 agonism and is capable of remarkably preventing or reducing lipid deposition, and is useful for the prevention and treatment of lipid metabolism disorder or diseases associated with lipid metabolism disorder, thereby completing the present invention.

In particular, the present invention relates to the following.

One aspect of the present invention relates to a compound of formula (I):

another aspect of the invention relates to the use of a compound of the foregoing or a salt thereof for the preparation of an RDH10 agonist.

Another aspect of the invention relates to the use of a compound of the foregoing, or a salt thereof, in the manufacture of a medicament for preventing or reducing lipid deposition, and/or for treating or preventing a disease treatable by RDH10 agonism, such as lipid metabolism disorder (e.g. hyperlipidemia, cholesterol deposition, retinal lipidemia) or a disease associated with lipid metabolism disorder (e.g. cardiovascular disease associated with lipid metabolism disorder such as hypertension, atherosclerosis, or kidney disease associated with lipid metabolism disorder).

In a specific embodiment, the "disease treatable by RDH10 agonism" does not include obesity, diabetes or nonalcoholic steatohepatitis.

An advantage of the present invention is the provision of the aforementioned compounds of formula (I) which have a remarkable RDH10 agonistic activity and are markedly superior to their enantiomer 25R-Antcin C, for use in the preparation of a therapeutic or prophylactic agent for RDH10 agonists or related diseases.

Drawings

Fig. 1:25S-Antcin C-CO-NH-PEG-biotin molecular probe synthesis reaction formula.

Fig. 2:25S-Antcin C-CO-NH-PEG-biotin ¹ H-NMR spectrum (pyridine-d) ₅ ，400MHz)。

Fig. 3:25S-Antcin C-CO-NH-PEG-biotin ¹³ C-NMR spectrum (pyridine-d) ₅ ，100MHz)。

Fig. 4: molecular probe protein fishing results.

Fig. 5:25S-Antcin C acts directly on target RDH10 (note: in graphs B and C, the top-to-bottom arrows indicate the concentrations corresponding to the top-to-bottom curves, respectively).

Fig. 6: RDH10siRNA cell knockdown experimental results.

Fig. 7: results of 25S-Antcin C and 25R-Antcin C in RDH10siRNA cell knockdown experiments were compared.

Detailed description of the preferred embodiments

The definition of "compound of formula (I)" as referred to in the present specification includes salts thereof, preferably pharmaceutically acceptable salts (preferably pharmaceutically acceptable salts) of the compound with an acid or a base.

The compounds of the invention may be administered to a subject, e.g., a human patient, using any convenient means capable of producing the desired result, e.g., the compounds may be formulated into pharmaceutical compositions as described previously, and/or into known or newly developed dosage forms (e.g., tablets, capsules, injections, etc.). The compounds of the present invention may be used in combination with other therapeutic agents, and when used in combination, the compounds or extracts of the present invention may be formulated into the same dosage form as the other therapeutic agents, or separately formulated into separate dosage forms. One or more pharmaceutically acceptable carriers or excipients may be added to the aforementioned pharmaceutical compositions or dosage forms, including but not limited to diluents, fillers, binders, wetting agents, disintegrants, lubricants, and the like.

Specific embodiments of the invention will be illustrated by the following examples, but it will be appreciated that the examples are not intended to limit the scope of the invention. The starting materials, reagents, etc. used in the examples are all those known to those skilled in the art and are available commercially or by literature methods; the test or characterization methods used are also well known to those skilled in the art.

Example 1:25S-Antcin Synthesis and structure identification of C-CO-NH-PEG-biotin (ACS-biotin) molecular probe Fixing device

Experimental materials used in this example include: 25S-Antcin C (isolated from Antrodia camphorate by chiral resolution), and (R) -and (S) -1- (9-anthryl) -2, 2-trifluoroethanol (Sigma-Aldrich, USA), et3N (triethylamine, carbofuran, beijing), DMAP (4- (dimethylamino) pyridine, carbofuran, beijing), EDCI (1-Ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride, 1-Ethyl- (3-dimethylmineopyl) carbodiimide hydrochloride, shanghai), HOBt (1-Hydroxybenzotriazole, shanghai), DIPEA (N, N-Diisopropylethylamine, N, N-diisopropylopylamines, an Naiji, beijing), DMF (N, N-Dimethylformamide, an Naiji, beijing), biotin-PEG ₂ -NH ₂ (N- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -5- ((3 aS,4S,6 aR) -2-oxahexahydro-1H-thieno [3, 4-d)]Imidazol-4-yl) pentanamide, after the medicine, shanghai); dichloromethane (carbofuran, beijing), ammonium chloride (Tongguang, beijing), sodium sulfate (Leyan, beijing), colorSpectrum methanol (thermo fisher, beijing), methanol (guang, beijing), preparation of grade acetonitrile (belowe, beijing), trifluoroacetic acid (An Naiji, beijing).

Firstly, 25kg of dried dish cultivated Antrodia camphorata is weighed and crushed. Adding 10 times of 95% ethanol, heating and refluxing for 2-3 h, and suction filtering. The residue was repeatedly extracted with 95% ethanol for 5 times. Mixing the extractive solutions, concentrating under reduced pressure, and recovering solvent to obtain total extract, which is ethanol extract of Antrodia Camphorata. About 4.8kg of total extract can be obtained, and the yield reaches 19.2%. 1.2kg of the ethanol extract was dissolved in 50% ethanol, and the solution was applied to 9.6kg of a macroporous adsorbent resin open column (AB-8) in 4 times, and eluted with a gradient of ethanol-water (50:50, 70:30, 85:15, 95:5) as a mobile phase, and combined into 6 fractions (A-F) according to TLC and HPLC analysis results. Fraction D (90.5 g) was dissolved in an appropriate amount of methanol, sonicated, and filtered to obtain filtrate DA (12.6 g) and solid DB (70.8 g). The filtrate DA was separated by a silica gel column using methylene chloride-methanol (10:1-1:1, v/v) as eluent and combined into 4 fractions DAA to DAD by TLC and HPLC detection. Fraction DAB (2.6 g) was separated by LH-20 gel chromatography to give 2 fractions (DABA and DABB), wherein fraction DABA (309.1 mg) was subjected to semi-preparative liquid chromatography (acetonitrile-water, 65:36, v/v) to give compound 25R-Antcin C (50.3 mg) and 25S-Antcin C (60.4 mg).

Characterization data for Antcin C (25 r,25 s) were: ¹ h NMR (400 MHz, pyridine-d) ₅ )δ:1.25,2.90(2H,m,H-1),2.38(2H,m,H-2),4.53(1H,t,J＝8.6Hz,H-7),2.47(1H,d,J＝13.8Hz,H-12a),3.00(1H,d,J＝13.8Hz,H-12b),0.90(3H,s,H-18),1.61(3H,s,H-19),0.92(3H,d,J＝5.1Hz,H-21),1.53(3H,d,J＝7.0Hz,H-27),5.10(1H,s,H-28a),5.25(1H,s,H-28b),1.13(3H,d,J＝6.5Hz,H-29)。 ¹³ C NMR (100 MHz, pyridine-d) ₅ )δ:36.6(C-1),38.5(C-2),211.8(C-3),44.5(C-4),49.0(C-5),33.9(C-6),69.7(C-7),141.3(C-8),156.2(C-9),37.8(C-10),201.7(C-11),58.9(C-12),48.3(C-13),54.0(C-14),25.8(C-15),28.6(C-16),55.1(C-17),12.9(C-18),18.1(C-19),36.5(C-20),19.0(C-21),34.8(C-22),32.3(C-23),150.3(C-24),46.9(C-25),176.8(C-26),17.4(C-27),110.9(C-28),12.3(C-29)。

To determine the configuration of C at position 25, mosher ester reactions were performed using (R) -and (S) -1- (9-anthryl) -2, 2-trifluoroethanol reagents. 25S-antcin C (14.67, about 0.031 mmol), (R) -1- (9-anthryl) -2, 2-trifluoroethanol (8.56 mg, about 0.031 mmol), EDCI (17.83 mg, about 0.093 mmol), et3N (8.7 uL, about 0.031 mmol) and DMAP (5.74 mg, about 0.047 mmol) were first weighed out and dissolved in 1mL of deuterated chloroform, sonicated for 20min and allowed to stand for 1 day. After the reaction is completed, the mixture is evaporated to dryness under reduced pressure, extracted by chloroform-water, and the organic layer is evaporated to dryness. Similarly, the 25R-ester of 25S-Antcin C, the 25R/S ester of 25R-Antcin C was obtained by the same procedure. By comparison of the esters obtained by the reaction ¹ The H-NMR data in turn infer the steric configuration of the carbon at position 25 of the compound.

And comparing the nuclear magnetic data of the 25R/S-antcin C with a product obtained after the mosher reaction, thereby determining the 25-carbon steric configuration of the 25R/S-antcin C obtained by separation. Specific hydrogen spectrum data are shown in table 1.

TABLE 1 Mosher ester partial Hydrogen Spectrum data for 25R/S-antcin C (400M, pyridine-d ₅ ，δin ppm，J in Hz)

a, (R) -1- (9-anthryl) -2, 2-trifluoroethanol ester; b, (S) -1- (9-anthryl) -2, 2-trifluoroethanol ester.

Next, 25S-Antcin C (20.00 mg,0.0425 mmol), EDCI (10.61 mg,0.5525 mmol), HOBt (7.46 mg,0.05525 mmol) and DIPEA (47.28 uL,0.2677 mmol) were dissolved in anhydrous DMF (1 mL) and Biotin-PEG was added thereto according to the chemical reaction shown in FIG. 1 ₂ -NH ₂ (19.07 mg,0.051 mmol) in anhydrous DMF (0.5 mL). The reaction was stirred at room temperature for 10h. The reaction system was diluted with dichloromethane, washed with saturated ammonium chloride solution, water, saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and redissolved with 1-2mL of chromatographic methanol.

Semi-preparative liquid chromatography YMC Pack ODS-A column (10X 250mm,5 mm), chromatographic conditions: 0-70min,37% B (B is preparation grade acetonitrile, A is 0.03% trifluoroacetic acid water); detection wavelength: 254nm, flow rate: 2mL/min to obtain the compound 25S-Antcin C-CO-NH-PEG ₂ Biotin (19.33 mg, 65% yield, white solid), andthe structure was determined by NMR. The nuclear magnetic spectrum is shown in figures 2-3.

25S-Antcin C-CO-NH-PEG ₂ Biotin, yield: 55%,19.33mg. HRESIMS: M/z 827.49871 ([ M+H)] ⁺ ,C ₃₅ H ₅₃ O ₁₁ Calculated values: 826.49088). ¹ H NMR (400 MHz, pyridine-d) ₅ ):δ:2.90,1.45(2H,H-1),2.55,2.22(2H,H-2),2.45(1H,H-4),2.41(1H,H-5),2.18,1.58(2H,H-6),4.34(1H,H-7),2.46,2.75(2H,H-12),2.78(1H,H-14),2.14(2H,H-15),1.96(2H,H-16),1.46(1H,H-17),0.79(3H,H-18),1.47(3H,H-19),1.46(1H，H-20),0.95(3H,H-21),1.60,1.24(2H,H-22),2.12,1.99(2H,H-23),3.06(1H,H-25),1.25(3H,H-27),4.97,4.91(2H，H-28),1.01(3H,H-29),4.49(1H，H-2'),4.30(1H,H-3'),2.92,2.71(2H,H-4'),3.20(1H,H-5'),1.66(2H,H-6'),1.44(2H,H-7'),1.73(2H，H-8'),2.22(2H,H-9'),3.36(2H,H-11'),3.54(2H,H-12'),3.61(2H,H-13'),3.61(2H,H-14'),3.54(2H,H-15'),3.36(2H,H-16')。 ¹³ C NMR (100 MHz, pyridine-d) ₅ )δ:37.0(C-1),37.0(C-2),215.0(C-3),45.0(C-4),49.5(C-5),33.5(C-6),70.4(C-7),156.9(C-8),142.3(C-9),38.2(C-10),204.0(C-11),59.1(C-12),48.9(C-13),54.6(C-14),26.0(C-15),29.0(C-16),55.6(C-17),12.6(C-18),17.9(C-19),37.1(C-20),19.1(C-21),35.3(C-22),32.4(C-23),150.6(C-24),47.9(C-25),177.1(C-26),16.7(C-27),111.4(C-28),11.9(C-29),166.1(C-1'),61.6(C-2'),63.4(C-3'),41.1(C-4'),57.0(C-5'),26.9(C-6'),27.8(7'),29.5(C-8'),36.7(C-9'),176.1(C-10'),40.3(C-11'),71.3(C-12'),70.6(13’),70.6(C-14'),71.3(C-15'),40.4(C-16')。

Example 2: protein fishing finding 25S-Antcin C (ACS) binding target RDH10

Avidin microspheres (Sigma, usa), other reagents (beijing chemical factory, beijing).

Protein fishing experiment steps:

(1) Cutting about 50mg of liver tissue of a C57BL/6J mouse, adding 1mL of RIPA lysate, fully lysing proteins on ice, centrifuging at 12000rpm for 30min at 4 ℃, taking supernatant, measuring the protein concentration by a BCA method, and quantifying to 1mg/mL by PBS;

(2) Taking a new 1.5mL EP tube, dividing into a control group, a drug group and a competition inhibition group, respectively adding 100 mu L of PBS solution containing 40 mu M Biotin, 100 mu L of PBS solution containing 40 mu M ACS-Biotin (prepared in example 1), 100 mu L of PBS solution containing 40 mu M ACS-Biotin and 400 mu M ACS, adding 100 mu L of hepatic protein lysate (1 mg/mL) into each tube, and incubating for 2h at 4 ℃ in a shaking table;

(3) Taking 100 mu L of avidin microspheres, adding a new 1.5mL EP tube, centrifuging at 2700rpm for 1min, discarding the supernatant, adding 100 mu L of PBS, and washing three times;

(4) Adding each group of liquid in the step (2) into the washed avidin microspheres, incubating for 2 hours at 4 ℃ in a shaking table, centrifuging at 2700rpm for 1min, discarding the supernatant, and adding 100 mu L of PBS for washing three times;

(5) Adding 50 μl PBS, mixing, adding 5×SDS-PAGE sample buffer, boiling for 5min, centrifuging at 13000rpm for 5min, preserving supernatant at-20deg.C, and running SDS-Page;

(6) Coomassie blue staining, gel cutting detection proteomics.

As shown in FIG. 4A, the drug group showed a significantly deeper protein band between 40-50kDa compared to the control group and the competitive inhibition group, and there may be a highly bound target protein of compound 25S-Antcin C between this band. This protein band was excised and the sample was subjected to proteomic analysis. As shown in the proteomics results of FIG. 4B, log of the drug group compared with the control group ₂ (ACS-biotin/Biotin)>0 protein is maximally different from 342 proteins>6) Of which RDH10 is a molecule closely related to reducing lipid deposition. As shown in FIG. 4C, log of RDH10 protein was compared with the drug group and the competitive inhibition group ₂ (ACS-biotin/ACS-biotin+ACS)>0, indicating that the addition of 25S-Antcin C as a competitive inhibitor reduces binding of the molecular probe to RDH10 protein.

Example 3: western Blot, SPR, CETSA verifies that RDH10 is a 25S-Antcin C direct target

Experimental materials used in this example include: RIPA lysate, BSA protein detection kit (bi yun, shanghai), RDH10 antibody, GAPDH antibody (Bioss, beijing), goat anti-mouse secondary antibody, goat anti-rabbit secondary antibody (Bai Aoyi j, beijing).

Western Blot experiment procedure:

(1) Tissue material selection: about 100mg of liver tissue is sheared from the same part of the liver of each mouse, and the mice are washed by precooled PBS and put into a precooled 1.5mL EP tube;

(2) Preparing protein lysate: RIPA+phosphatase inhibitor A (1:50) +phosphatase inhibitor B (1:50) +protease inhibitor (1:100) +0.1M EDTA (1:100) +100mM PMSF (1:100)

(3) Protein extraction: adding 1mL of precooled protein lysate into liver tissue, grinding for 30s by a sample grinder, and placing on ice;

(4) Centrifuging at 13000rpm at 4deg.C for 30min, transferring supernatant to a new pre-cooled 1.5mL EP tube, detecting protein concentration by BCA method, and diluting PBS to 2mg/mL;

(5) Adding 5 XSDS-PAGE loading buffer, mixing, boiling for 5min to denature protein, and performing SDS-PAGE protein electrophoresis;

(6) Gel transfer printing, namely, film transfer printing for 1h at a constant current of 200 mA;

(7) Closing: cutting the transferred PVDF film according to the molecular weight of the target protein and the position corresponding to the marker, completely immersing the PVDF film in a sealing liquid, and sealing for 1h at room temperature;

(8) Incubation resistance: adding the corresponding primary antibody diluted by 5% BSA-TBST, and incubating for 12-14h at 4 ℃;

(9) Secondary antibody incubation: after the primary antibody incubation is finished, washing the PVDF membrane by TBST for 3 times for 10min each time, adding a corresponding secondary antibody diluted by 5% BSA-TBST, incubating for 1-4h at 4 ℃, and after the secondary antibody incubation is finished, washing by TBST for 3 times for 15min each time;

(10) Exposure: ECL color developing agent is prepared according to the ratio of the solution A to the solution B1:1, and uniformly dripped on the PVDF film, and the color is developed by a chemiluminescent imaging system.

Surface Plasmon Resonance (SPR) experimental procedure:

RDH10 protein was immobilized on the sensor chip CM5 using a Biacore T200 plasmon surface resonance apparatus by a primary amine coupling reaction, the pH of the RDH10 protein was 4.5, and the final immobilization concentration was 50. Mu.g/mL. The running buffer was 150. Mu.M NaCl, 2mM MgCl ₂ 50mM Tris buffer, 0.05% Tween-20, 5% DMSO. In the binding assay, different concentration gradients (25, 12.5, 6.25, 3.12, 1.56, 0.78. Mu.M) of 25S-A were usedThe ntcin C was dissolved in running buffer at a flow rate of 30mL/min, a contact time of 60s and a dissociation time of 60s. The data are analyzed by Biacore software, and the affinity constant K is calculated by kinetic analysis _D Values.

Cell thermal transition analysis (CETSA) experimental procedure:

(1) Mouse liver total protein (500. Mu.g/mL) was incubated with 25S-Antcin C (100. Mu.M) or equivalent DMSO, respectively, for 2h at room temperature;

(2) After incubation, 25S-Antcin C treatment group and DMSO control group were respectively dispensed into 13 PCR tubes, 30. Mu.L each, and placed on ice;

(3) On a PCR instrument, setting a gradient heating program of CETSA, setting 12 temperature points (37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81 ℃), heating 13 samples of a 25S-Antcin C treatment group and a DMSO control group at the 13 temperature points for 3min respectively, immediately taking out and incubating at room temperature for 3min, and immediately placing on ice for rapid cooling;

(4) The sample was transferred to a 1.5mL EP tube and centrifuged at 15000rpm at 4℃for 40min;

(5) The supernatant was transferred to a new EP tube, added with 5 XSDS-PAGE loading buffer, mixed and boiled for 5min to denature the protein, and Western Blot detection was performed.

To confirm the proteomic analysis results, western Bolt was performed after performing the protein fishing in the steps (1) to (5) of example 2, and the methods of control group, drug group and competitive inhibition group analysis were the same as those of example 2. As shown in FIG. 5A, the experimental group showed a distinct RDH10 protein band, whereas neither the control group nor the competitive inhibition group showed a distinct RDH10 protein band, which further demonstrates proteomic analysis.

As shown in the SPR results of FIGS. 5B and 5C, RDH10 has a strong binding capacity with 25S-Antcin C, K _D 8.31. Mu.M, while RDH10 has very weak binding ability to 25R-Antcin C, K _D 71.38. Mu.M.

As shown in FIG. 5D, after incubation of the total mouse liver protein with 25S-Antcin C (100. Mu.M), the CETSA curves of the 25S-Antcin C group drift, especially between 53-69℃and, compared to the DMSO group, were heated at different temperatures (37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81 ℃), andthe undeposited RDH10 protein increased significantly after 25S-Antcin C incubation. On the other hand, as shown in FIG. 5E, the isothermal dose-response curves (ITDRF) were obtained by incubating 25S-Antcin C (0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500, 1000. Mu.M) at different concentrations while keeping the temperature at 61℃unchanged _CETSA ) RDH10 protein stability increased with increasing concentration of compound 25S-Antcin C at 61℃and tended to stabilize after 100. Mu.M. CETSA curve and ITDRF _CETSA The results indicate that compound 25S-Antcin C can bind to RDH10 protein, resulting in increased thermostability of RDH10 protein.

Example 4: cell siRNA experiments

The 1640 medium, KREBS buffer, penicillin-streptomycin cell culture diabodies used in this example were purchased from Michaelis technology Co., ltd., china, beijing. Priority bovine serum was purchased from Gibco (New York, USA). EGTA and collagenase IV were purchased from Huazhonghaiwei Gene technologies Inc. (China, beijing). CaCl (CaCl) ₂ Heparin, rat tail collagen type i, acetic acid, palmitic acid, oleic acid were purchased from beijing solebao technologies limited (china, beijing). Opti-MEM medium was purchased from Gibco (New York, USA). Mouse RDH10siRNA (sc-76377), human RDH10siRNA (sc-76376), control siRNA (sc-37007, commonly used in humans and mice) were purchased from Santa Cruz (Dallas, USA). Lipofectamine ^TM RNAiMAX transfection reagent was purchased from Invitrogen (Calsbard, U.S.A.). L02 human hepatocytes were purchased from beijing synergetic cell bank.

The primary cells of the mice were isolated as follows:

(1) Preparation of reagents

Collagen I solution: preparing 0.02N acetic acid by deionized water, performing sterile filtration, and diluting the collagen I to 50 mug/mL;

perfusion liquid: KREBS buffer with 0.1mM EGTA, sterile filtered, and pre-heated at 37deg.C;

digestive juice: containing 2.7mM CaCl ₂ 0.05% of KREBS buffer solution of collagenase IV, sterile filtering and preheating at 37 ℃;

heparin solution: deionized water is used for preparing 2mg/mL heparin solution, and sterile filtration is carried out.

(2) Collagen paving: adding 2mL of collagen I solution into each hole of a 6-hole plate, standing for 0.5-1h, sucking out the collagen solution, and irradiating for 5h by ultraviolet;

(3) After the mice are anesthetized and sterilized by alcohol, separating the inferior vena cava, tying a dummy knot, inserting a vein retention needle below the dummy knot, and fixing the dummy knot;

(4) Injecting 1mL heparin into the vein of the mouse through the retaining needle, preparing to inject perfusion liquid, and simultaneously shearing the vein open, and perfusing for 3-5min;

(5) Injecting digestive juice for 3-6min until liver digests, and removing gallbladder;

(6) Picking up the liver, placing the liver in a culture dish on ice, transferring the culture dish to a sterile operation table, repeatedly and gently blowing the liver by using a 1640 culture medium precooled at 4 ℃, filtering the liver by a 400-mesh screen, taking 20 mu L of cells, adding 2 mu L of trypan blue for dyeing, and observing the survival condition of the cells under a microscope;

(7) Transferring the liver cells into a 50mL centrifuge tube, centrifuging for 2min at 50 Xg, discarding the supernatant, adding 25mL 1640 culture medium, centrifuging again, and repeating for 3 times;

(8) 25mL of 1640 medium containing 10% serum was used to resuspend cells, counted and counted at 5X 10 ⁵ The cells/holes are spread on a 6-hole plate, and the liquid is changed after 6 hours.

Next, cellular siRNA experiments were performed on the mouse primary hepatocytes and L02 hepatocytes isolated as described previously, respectively. The experimental procedure was as follows:

(1) The cells (mouse primary hepatocytes or human L02 hepatocytes) were cultured at 5X 10 ⁵ The individual/wells were plated in 6-well plates;

(2) Cell density reaches 60%, and is divided into a control siRNA group, a control siRNA plus administration group, an RDH10siRNA group and an RDH10siRNA plus administration group;

(3) Preparing a transfection A solution: mu.L of Opti-MEM medium was added to 1.5mL of RNase-free EP tube, 30pmol of human or mouse RDH10siRNA, or 30pmol of control siRNA was added, and gently mixed;

(4) Preparing a transfection B solution: mu.L of Opti-MEM medium was added to 1.5mL of RNase-free EP tube, and 5. Mu.L of Lipofectamine was added ^TM The RNAiMAX transfection reagent is gently mixed;

(5) Preparing transfection reagent: adding the solution A into the solution B, reversing the solution A upside down, mixing the solution A and the solution B evenly, and standing the solution A for 15 to 20 minutes at room temperature;

(6) Cell suction and discarding culture medium, wherein the control siRNA+ administration group and the RDH10 siRNA+ administration group are replaced by complete 1640 culture medium without double antibodies containing palmitic acid-oleic acid and 25S-Antcin C medicine, the control siRNA group and the RDH10siRNA group are replaced by complete 1640 culture medium without double antibodies containing palmitic acid-oleic acid and equivalent DMSO, the control siRNA group and the control siRNA+ administration group are uniformly dripped with a transfection reagent containing control siRNA, and the control siRNA+ administration group and the RDH10 siRNA+ administration group are uniformly dripped with a transfection reagent containing RDH10 siRNA;

(7) Culturing for 24h, and carrying out subsequent detection. The specific detection method comprises the following steps: after 24h incubation, the medium was discarded, washed with PBS and 4% paraformaldehyde fixed for 10min at room temperature; absorbing 4% paraformaldehyde and incubating with 60% isopropanol for 10min; dyeing with oil red O for 20-30min; rapidly passing the mixture through the mixture for a plurality of times by using 60% isopropanol; and (5) cleaning with deionized water, and sealing the tablet with glycerol.

As shown in fig. 6A, L02 human hepatocytes and mouse primary hepatocytes knocked down RDH10, respectively, and Western Blot showed that RDH10siRNA group RDH10 protein expression was significantly reduced compared to control siRNA group, indicating successful gene knockdown. As shown in FIG. 6B, oleic acid-palmitic acid induced lipid deposition by cells, in both L02 human hepatocytes and mouse primary hepatocytes, the control siRNA group had significantly reduced lipid droplets after 25S-Antcin C (20. Mu.M) treatment, indicating that 25S-Antcin C had significant lipid lowering activity. However, compared to the control siRNA, the activity of 25S-Antcin C on improving lipid deposition of RDH10siRNA group was almost completely lost, and the lipid size and the number were almost equivalent to RDH10siRNA group. This result indicates that RDH10 knockdown resulted in a decrease in lipid lowering activity of 25S-Antcin C, and therefore RDH10 was the target of 25S-Antcin C.

In addition, a comparative experiment was performed on 25S-Antcin C and its diastereoisomer 25R-Antcin C using mouse primary cells and L02 human hepatocytes, which were not knocked down by RDH10, under the same experimental conditions. The results showed that both 25S-Antcin C (10, 20. Mu.M) and 25R-Antcin C (20. Mu.M) exhibited lipid lowering activity, were able to reduce palmitic acid-oleic acid induced lipid deposition, and that the effect of 25S-Antcin C was significantly superior to 25R-Antcin C, as shown in FIG. 7.

Claims

1. A compound of formula (I):

2. use of a compound according to claim 1 or a salt thereof in the preparation of an RDH10 agonist.

3. Use of a compound or salt thereof according to claim 1 in the manufacture of a medicament for the treatment or prophylaxis of a disease which can be treated by RDH10 agonism, such as lipid metabolism disorders (e.g. hyperlipidemia, cholesterol deposition, retinal lipidemia) or diseases associated with lipid metabolism disorders (e.g. cardiovascular diseases associated with lipid metabolism disorders such as hypertension, atherosclerosis, or renal diseases associated with lipid metabolism disorders).

4. The use according to claim 3, wherein the disease treatable by RDH10 agonism does not include obesity, diabetes or nonalcoholic steatohepatitis.