Compound isarubrolone c and use thereof

Abstract

The present application relates to the compound Isarubrolone C and its application, specifically, the structure of Isarubrolone C is as shown in the following formula, the present application also relates to the application of the compound Isarubrolone C in the preparation of the drug for preventing and / or treating the liver cell or nerve cell damage caused by nitration stress.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biotechnology, specifically relating to the compound Isarubrolone C and its applications. Background Technology

[0002] Isarubrolone C is a secondary metabolite produced by *Streptomyces* CGMCC 15540 (identical to *Streptomyces* CPCC204095), which has a fused pentacyclic core structure consisting of 6-deoxyhexose, furan, cycloheptatrienone, cyclopentanone, and pyridine. This *Streptomyces* strain was deposited on April 10, 2018, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing, 100101, China. The taxonomic name of this strain is *Streptomyces* sp., and the accession number is CGMCC 15540.

[0003] Isarubrolone C has been found to possess biological activities such as activating autophagy, antiparasitic activity (Leishmania donovani), and antiviral activity. With ongoing research and exploration into the biological activities of Isarubrolone C, its potential pharmaceutical applications are being continuously discovered and explored.

[0004] Nitrosative stress is the excessive production of peroxynitrite (ONOO) anions during certain pathological processes. - Nitrification stress induces tyrosine nitration of intracellular protein molecules, leading to a cascade of reactions that cause cell damage or apoptosis. During nitrification stress, tyrosine residues in intracellular protein molecules are nitrated into 3-nitrotyrosine. If 3-nitrotyrosine is not cleared in time, it affects the normal biological function of protein molecules, causing toxic damage to cells and ultimately leading to apoptosis.

[0005] The liver is one of the most important organs in the human body, possessing complex physiological and biochemical functions, including the metabolism, detoxification, transformation, synthesis, and immunity of proteins, lipids, carbohydrates, hormones, and vitamins. Under pathophysiological conditions (such as in alcoholic liver disease), the liver exhibits enhanced synthesis of inducible nitric oxide synthase (iNOS) in cells, leading to a significant increase in nitric oxide (NO) levels. This nitric oxide combines with reactive oxygen species generated by oxidative stress to form peroxynitrite anions, resulting in hepatocyte damage.

[0006] The nervous system is the dominant system regulating human physiological functions and activities. The nerve tissues (cells) that make up the nervous system exist in various organs and tissues, including the brain, and are responsible for receiving, integrating, transmitting, and outputting information, as well as facilitating information exchange. In neurodegenerative diseases, nerve cells (neurons) can be damaged or die due to excessive nitrification stress (and oxidative stress).

[0007] Therefore, inhibiting the level of nitrification stress in liver cells and nerve cells can reduce the damage or apoptosis of liver cells and nerve cells caused by nitrification stress, thereby achieving the goal of protecting the normal physiological functions of the liver and nervous system.

[0008] Sodium nitroprusside (SNP) is a short-acting, rapid-release vasodilator used clinically. Its mechanism of action involves the intracellular generation of NO, which directly dilates the smooth muscle of arteries and veins. In basic research, sodium nitroprusside is often used as a NO donor, exhibiting a significant inhibitory effect on cell growth and proliferation. It is widely used in in vitro studies simulating cytotoxic damage under nitrification stress.

[0009] Natural products such as silibinin and artemisinin are known to have anti-nitrification stress-induced neuronal damage caused by sodium nitroprusside. Edaravone, a clinical drug used to treat acute cerebral infarction and cerebral edema and improve post-stroke neurological function, also possesses anti-nitrification stress-induced neuronal damage as a free radical scavenger. In studies antagonizing sodium nitroprusside-induced nitrification stress damage in human HepG2 liver cancer cells and rat pheochromocytoma cells PC12, isarubrolone C showed superior activity compared to these compounds or drugs.

[0010] Alcoholic liver disease and neurodegenerative diseases are both common clinical conditions; nitrification stress plays a significant role in the development and progression of these diseases; it is expected that drugs antagonizing nitrification stress can protect the liver or nervous system and slow down hepatocyte or nerve cell damage or apoptosis caused by nitrification stress. However, there are currently no approved drugs on the market for the prevention and / or treatment of hepatocyte or nerve cell damage caused by nitrification stress.

[0011] Based on this, the present invention is proposed. Summary of the Invention

[0012] This invention first relates to a secondary metabolite of Streptomyces, compound Isarubrolone C, whose chemical structure is shown in formula (1) below.

[0013]

[0014] Its structure is a fused pentacyclic ring, consisting of a 6-deoxyhexose ring, a furan ring, a cycloheptatrienone ring, a cyclopentanone ring, and a pyridine ring from left to right.

[0015] This invention also relates to a method for preparing the compound Isarubrolone C, characterized in that the method is as follows:

[0016] Method (1) Fermentation of Streptomyces CGMCC 15540 to isolate the compound from the fermentation culture, or

[0017] Method (2) uses Isatropolone C (proflavonolone C) as a substrate and reacts it with NH3 (ammonia) to obtain Isarubrolone C.

[0018]

[0019] The present invention also relates to the use of the compound Isarubrolone C in the preparation of formulations / medications for the treatment of hepatocyte and nerve cell damage caused by nitrification stress; preferably, the drug is used to treat alcoholic liver disease and / or neurodegenerative diseases.

[0020] The present invention also relates to pharmaceutical compositions comprising the compound Isarubrolone C; preferably, the pharmaceutical compositions further comprising necessary pharmaceutical excipients.

[0021] The beneficial effects of this invention are as follows:

[0022] (1) Isarubrolone C, with a fused pentacyclic core structure consisting of a 6-deoxyhexose ring, a furan ring, a cycloheptatrienone ring, a cyclopentanone ring and a pyridine ring, was confirmed;

[0023] (2) It was found that the compound Isarubrolone C has the effect of antagonizing the damage to hepatocytes and nerve cells caused by nitrification stress;

[0024] The application of Isarubrolone C or its pharmaceutically acceptable salts as active ingredients in pharmaceuticals or pharmaceutical compositions has the potential to treat or prevent diseases caused by nitrification stress-induced damage to hepatocytes and nerve cells. Attached Figure Description

[0025] Figure 1 The protective effect of Isarubrolone C against sodium nitroprusside-induced damage to HepG2 cells

[0026] Figure 2 The protective effect of Isarubrolone C against sodium nitroprusside-induced damage to PC12 cells Detailed Implementation

[0027] Example 1: Determination of the bioactivity of Isarubrolone C in antagonizing nitrification stress damage

[0028] 1. Experimental Principle

[0029] Sodium nitroprusside (SNP) is often used as a NO donor in basic research. It has a significant inhibitory effect on cell growth and proliferation and is widely used in in vitro studies simulating cell damage under nitrification stress.

[0030] In the study of antagonizing sodium nitroprusside-induced cellular nitrification stress injury (model cells were human hepatocellular carcinoma HepG2 cells and rat pheochromocytoma PC12 cells), edaravone, silibinin, and artemisinin, as reported in the literature, were selected as positive control drugs.

[0031] 2. Cells and reagents

[0032] Human hepatocellular carcinoma HepG2 cells and rat pheochromocytoma PC12 cells were purchased from ATCC.

[0033] DMEM high glucose medium, RPMI-1640 medium, fetal bovine serum, and heat-inactivated horse serum (HS) were purchased from Gibco, USA.

[0034] HepG2 cells were cultured in DMEM medium containing 10% FBS, and PC12 cells were cultured in 1640 medium containing 5% FBS and 10% HS. Both were incubated in a cell culture incubator at 37°C and 5% CO2.

[0035] The CCK-8 (Cell Counting Kit-8) was purchased from Nanjing Novizan Co., Ltd., and the Spark 20M microplate reader was purchased from TECAN, Switzerland.

[0036] The positive control drug edaravone was purchased from Sigma-Aldrich, USA, while silibinin and artemisinin were purchased from Shanghai Bichen Biochemical Technology Co., Ltd.

[0037] Isarubrolone C was obtained by the applicant through fermentation and isolation of *Streptomyces* CGMCC 15540 (CGMCC NO. 15540). The fermentation and isolation methods can be found in the applicant's prior patents (CN201810661996.9, CN201810660648.X). NMR data for Isarubrolone C (Pyridine-d5, 600MHz) are shown in Table 1. Alternatively, Isarubrolone C can also be prepared through in vitro chemical synthesis. The preparation method for the key substrate, Isatropolone C (proflavonolone C), can be found in the applicant's prior application CN 201810660648.X.

[0038] Table 1. NMR data of Isarubrolone C

[0039]

[0040] 3. Experimental Methods

[0041] SNP-damaged hepatocyte (HepG2) and neural cell (PC12) models were constructed respectively. Cells in the culture plate were randomly divided into control group, model group (1 mM SNP-damaged HepG2 cells; 0.3 mM SNP-damaged PC12 cells) and drug administration groups with different concentrations (n=5).

[0042] The treatment groups were incubated with Isarubrolone C (edaravone, silymarin, or artemisinin as positive control drugs) at final concentrations of 3.125, 6.25, 12.5, 25, 50, and 100 μM, respectively, while applying SNP-damaged HepG2 or PC12 cells; a blank group (a culture medium without cells and compounds) was also set up.

[0043] After 24 hours, the absorbance (OD) was measured at 450 nm using a microplate reader with CCK-8 reagent.

[0044] Formula for calculating cell viability: Cell viability (%) = [(OD)] 给药组 -OD 空白组 )] / [(OD 对照组 -OD 空白组 []×100%. EC was calculated based on the dose-response relationship between cell protection rate and the concentration of Isarubrolone C or the positive control compound. 50 Values ​​and plot EC 50 curve.

[0045] When the cell protection rate is less than 50%, the protective effect is shown by cell survival rate (bar chart).

[0046] The formula for calculating cell protection rate is: Cell protection rate (%) = [(OD)] 给药组 -OD 空白组 )-(OD 模型组 -OD 空白组 )] / [(OD 模型组 -OD 空白组 )]×100%.

[0047] 4. Experimental Results

[0048] The results are as follows Figure 1 and Figure 2 As shown,

[0049] (1) Compound Isarubrolone C showed good anti-nitrification stress effects in both the SNP-damaged HepG2 cell model and the SNP-damaged PC12 cell model. 50 The values ​​were 25.45±5.24 μM and 41.77±2.62 μM, respectively.

[0050] (2) Positive control drugs edaravone, silibinin, and artemisinin antagonized the nitrification stress damage induced by sodium nitroprusside in human hepatocellular carcinoma HepG2 cells and rat pheochromocytoma PC12 cells. Figure 1 and Figure 2 However, the activity cannot be calculated for EC. 50 .

[0051] Isarubrolone C exhibits superior antinitrification activity compared to the positive control. Therefore, Isarubrolone C can inhibit SNP-induced nitrification stress damage in hepatocytes and nerve cells, and its activity is superior to that of the positive controls edaravone, silymarin, and artemisinin.

[0052] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not intended to limit the scope of protection of the present invention.

Claims

1. Use of a compound represented by formula (1) Isarubrolone C in the manufacture of a medicament for the treatment of hepatocyte and neuronal cell damage caused by nitrosative stress. Formula (1).

2. Use according to claim 1, characterized in that, The medicament is for the treatment of alcoholic liver disease and / or neurodegenerative disease.

Images