CN1891687B - Inflammation factor receptor antagonist active compound derived from microorganism and application thereof - Google Patents

Abstract

This invention relates to rivalry active compound of inflammatory factor receptors origin from microorganisms, which especially refers to the rivalry active compound or acceptable derivates of tumour necrosis factor receptor (TNFR) and interleukin-6 receptor (IL-6). The invention also relates to the method for synthesizing the medicine for the disease treatment which is caused by excessive volume of ligand of natural tumor necrosis factor receptor (TNFR) and interleukin-6 receptor (IL-6R). In addition, the invention also relates to the medicine containing the described compounds.

Description

Inflammation factor receptor antagonist active compound derived from microorganism and application thereof

field of invention

The present invention relates to an inflammatory factor receptor antagonistic compound derived from microorganisms, especially an antagonistic compound of tumor necrosis factor receptor (TNFR) and interleukin-6 receptor (IL-6R) or a pharmaceutically acceptable derivative thereof. The present invention also relates to the use of the above-mentioned compound for preparing a medicine for treating diseases related to excessive ligands of natural tumor necrosis factor receptor (TNFR) and interleukin-6 receptor (IL-6R). Furthermore, the present invention also relates to pharmaceutical compositions containing said compounds.

Background of the invention

Inflammation is the body's response to various injuries, and cytokines play an important role in the aggravation, repair and chronication of inflammatory responses. Tumor necrosis factor (TNF) and interleukin-6 (IL-6) are known to be important mediators of inflammatory responses.

Tumor necrosis factor exerts its biological activity by binding to receptors on the cell surface and transmitting information into cells through the signal transduction system. Tumor necrosis factor has two receptors, whose genes have been isolated and purified, and their structures have been elucidated. The molecular weights of these two receptor proteins are known to be 55Kd and 75kD, respectively, and are called P55R (TNFR I) and P75R (TNFR II), respectively. The receptor fragments of TNFR on the cell surface hydrolyzed by protease exist in various human body fluids, that is, soluble TNF receptors, which correspond to the membrane surface, and those derived from TNFR I are called sTNFR I, and those derived from TNFR II are called for sTNFR II. sTNFR has a high affinity to TNF, once combined with TNF, it blocks the combination of TNF and TNFR, thereby indirectly inhibiting the activity of TNF. The role of sTNFR has two aspects: at high concentration, sTNFR can antagonize the action of TNF; at low concentration, it has a stabilizing effect on the structure of TNF.

Interleukin-6 (IL-6) is a multifunctional cytokine that participates in biological processes such as immune regulation, hematopoiesis, and acute phase response, and plays a certain role in the endocrine and nervous systems. Dysregulation of IL-6 expression is associated with many human diseases, such as infection, tumor, autoimmune disease, arthritis and aging process. IL-6 acts on target cells through a two-chain receptor system composed of two membrane proteins, the ligand-binding chain IL-6R and the non-ligand-binding chain gp130 as a signal transducer. IL-6R consists of an Ig-like region, a ligand-binding region, a transmembrane region, and an intracellular region. It is a transmembrane glycoprotein with two naturally occurring forms, namely: the membrane-bound receptor gp80 and the soluble receptor sIL-6R, both of which play an important role in the signaling process of IL-6.

In view of the fact that cytokine receptors are involved in various physiological and/or pathological processes of the body, they are important targets for drug action. In recent years, with the development of molecular biology in the field of pharmacology, especially the development of the Human Genome Project, new receptors and subtypes for cytokines are constantly being discovered, and the functions of the receptors (subtypes) and The role in disease development is gradually being elucidated. This drives drug research workers to start from the search for corresponding receptor antagonists or agonists, on the one hand through the receptor antagonists or agonists to study the biological functions of these messenger substances, on the other hand can also be specific New drugs that regulate body functions are obtained from receptor antagonists or agonists.

It is known that human sTNFR and sIL-6R can be used as antagonists of natural TNFR and IL-6R for the treatment of diseases related to the excess of their corresponding ligands, including rheumatoid arthritis, septic shock, acute and chronic myeloid failure Blood diseases, atherosclerosis, autoimmune diseases, transplant rejection, etc. Therefore, antagonists of TNFR and IL-6R from different sources have attracted widespread attention.

In order to overcome the defects in the prior art of using animal tissue to extract receptors to screen drugs, for example, the content of receptors on tissue cells is very small (each cell has 10-1000 binding sites); directly use animal tissue or tissue uniform The specificity of screening receptor antagonists or agonists is not high, the inventors screened samples derived from microorganisms by utilizing the TNFR and IL-6R antagonist screening models established by the corresponding recombinant receptors respectively, and obtained Compounds of the invention.

Contents of the invention

In order to obtain new TNFR and IL-6R antagonists, the inventors screened a large number of metabolites derived from microorganisms by utilizing the TNFR and IL-6R antagonist screening models established by the corresponding recombinant receptors, and obtained by further separation and purification A new microbial source of inflammatory factor receptor antagonist activity compounds.

In one aspect of the present invention, a microbial-derived inflammatory factor receptor antagonist activity compound, or a pharmaceutically acceptable derivative thereof, has a structure shown in formula I through a TNFR receptor antagonist screening model:

The name of the compound is N-(3-acetylthio-2-methyl-1-carbonylpropyl)-proline, hereinafter also referred to as 1487B, which has TNFR receptor antagonistic activity.

In the present invention, the pharmaceutically acceptable derivatives of the compounds include salts, esters or esters of the above compounds.

In yet another aspect, the present invention also relates to a microorganism producing the compound 1487B. Specifically, in order to meet the patent examination requirements, the inventor deposited the fungus (Fungus) 1487 on March 14, 2005 in the General Microbiology Center of the China Microbiological Strain Management Committee (CGMCC, Beijing, No. 13, North Zhongguancun, Haidian District, 100080), deposit number CGMCC 1327.

Another aspect of the present invention also relates to a method for preparing the compound 1487B, comprising the following steps: cultivating the microorganism CGMCC1327 under conditions suitable for the production of the compound 1487B, and recovering the compound 1487B from the culture solution.

In another aspect of the present invention, a microbial-derived inflammatory factor receptor antagonist activity compound, or a pharmaceutically acceptable derivative thereof, has a structure shown in formula II through the IL-6R receptor antagonist screening model:

The name of the compound is 2-(N-n-heptyl)imino-3-oxo-4a-methyl-5-oxo-7-(2-hydroxyethyl)-8-(E-isopropene base)-2,3,-dihydronaphtho[2,3-b]1,4-dioxane, hereinafter also referred to as 2460A, has IL-6R receptor antagonistic activity.

In the present invention, the pharmaceutically acceptable derivatives of the compounds include salts, esters or esters of the above compounds.

In yet another aspect, the present invention also relates to a microorganism producing the compound 2460A. Specifically, in order to meet the patent examination requirements, the inventor deposited the fungus (Fungus) 2460 on March 14, 2005 in the General Microbiology Center of the China Microbiological Strain Management Committee (CGMCC, Beijing, No. 13, North Zhongguancun, Haidian District, 100080), deposit number CGMCC1328.

Another aspect of the present invention also relates to a method for preparing the compound 2460A, comprising the following steps: cultivating the microorganism CGMCC1328 under conditions suitable for the production of the compound 2460A, and recovering the compound 2460A from the culture solution.

Another aspect of the present invention relates to a pharmaceutical composition, which contains the compound described in the above formula I and/or formula II2 or a pharmaceutically acceptable derivative thereof, and a pharmaceutically acceptable carrier.

In another aspect of the present invention, the compounds of the above-mentioned formula I and/or formula II are used for the preparation of compounds related to the excess ligand of natural tumor necrosis factor receptor (TNFR) and interleukin-6 receptor (IL-6R). The use of drugs for diseases. In the present invention, the diseases include, but are not limited to, rheumatoid arthritis, septic shock, acute and chronic myeloid sepsis, atherosclerosis, autoimmune diseases, and transplant rejection.

Description of drawings

Figure 1: Figures 1a-c are the H-spectrum results of compound 2460A, respectively.

Figure 2: C-spectrum results of compound 2460A.

Figure 3: ESI mass spectrometry results of compound 2460A.

Figure 4: Infrared IR spectrum results of compound 2460.

Figure 5: High resolution FAB mass spectrometry results of compound 2460.

Figure 6: Figures 6a-d are the HMBC NMR two-dimensional spectrum results of compound 2460, respectively.

Figure 7: Figure 7a-b are the HSQC NMR two-dimensional spectrum results of compound 2460, respectively.

Figure 8: Figure 8a-c are the H-H COZY NMR two-dimensional spectrum results of compound 2460, respectively.

Figure 9: Figure 9a-b are the DEPT C-spectrum results of compound 2460, respectively.

Figure 10: Figures 10a-c are the C-spectrum results of compound 2460, respectively.

Figure 11: UV spectrum results of compound 2460.

The compounds of the present invention and their uses are further described below in conjunction with the examples.

The preparation and purification of the antagonistic active compound 2460 of embodiment 1 microbial source

After the fungus 2460 was inoculated, it was cultured on a shaker at 26°C for 96 hours. The obtained fermentation broth was filtered, and the filtrate was passed through X-5 macroporous adsorption resin, eluted with 30% acetone, then 80% acetone was eluted and collected, evaporated to dryness at room temperature until solid. The dry product was dissolved in 50% methanol, passed through a Sephadex LH-20 column, eluted with 50% methanol, detected by the IL-6R antagonist screening model established by the inventor, and the active components were collected and evaporated to dryness. The dry product was dissolved in 80% methanol, passed through an ODS column, eluted with 50% methanol, and the active components were collected.

After evaporating to dryness, the sample was dissolved in an organic solvent and freeze-dried to obtain a red solid, namely compound 2046A of the present invention. Through the UV, IR, NMR and MS analysis of this compound, its physical and chemical properties are as follows:

Molecular weight: 425

Solubility: Hardly soluble in water, easily soluble in methanol and chloroform.

Melting point: 84-87°C

Color: solid is red, methanol solution is bright red.

pH value: 5.3 (in saturated aqueous solution).

Stability: Both acid and alkali are unstable (pH about 2 or 12), and the solution color is yellow, which can improve its water solubility.

UV max: 500nm.

Preparation and purification of the antagonistic activity compound 1487B of embodiment 2 microbial source

After fungus 1487 was inoculated, it was cultured on a shaker at 26°C for 96 hours. The obtained fermentation broth was filtered, and the filtrate was passed through X-5 macroporous adsorption resin, eluted with 30% acetone, then 80% acetone was eluted and collected, evaporated to dryness at room temperature until solid. The dry product was dissolved in 50% methanol, passed through a Sephadex LH-20 column, eluted with 50% methanol, detected by the screening model for TNFR antagonists established by the inventors, and the active components were collected and evaporated to dryness. The dry product was dissolved in 80% methanol, passed through an ODS column, eluted with 50% methanol, and the active components were collected.

After evaporating to dryness, the sample was dissolved in an organic solvent and freeze-dried to obtain the compound 1487B of the present invention.

Example 3 In vitro prediction of intestinal absorption of 2460A by the model established by Caco-2 cells

Caco-2 cells were cultured on the semi-permeable membrane of the Transwell corning plate. After 3 days, the cells were covered with the semi-permeable membrane. Add 0.5ml of 2460A solution (concentration is 100μmol/l, HBSS buffer solution containing 20% ethanol) to the upper part of the semipermeable membrane, and add 1.5ml HBSS buffer solution containing 20% ethanol to the lower part of the semipermeable membrane. , 90, and 120 minutes respectively take 200 μl of the solution from the lower part, and replenish 200 μl of HBSS buffer solution containing 20% ethanol in time after taking each time.

The concentration of 2460A in the liquid was detected by HPLC, which were marked as A0, A30, A60, A90, and A120 respectively, and AT was the residual liquid concentration.

Add 1.5ml 2460A solution to the lower part of the semipermeable membrane, add 0.5ml HBSS buffer solution containing 20% ethanol to the upper part of the semipermeable membrane, add 1.5ml HBSS buffer solution containing 20% ethanol to the lower part of the semipermeable membrane, at 0, 30, 60 , 90, and 120 minutes respectively take 400 μl of the solution from the lower part, and replenish 400 μl of HBSS buffer solution containing 20% ethanol in time after taking each time.

The concentration of 2460A in the liquid was detected by HPLC, which were marked as B0, B30, B60, B90, and B120 respectively, and BT was the residual liquid concentration.

The test results are shown in Table 1

Table 1

A0 A30 A60 A90 A120 AT B0 B30 B60 B90 B120 BT Concentration μmol/l 0 0 0 1.4 1.3 23.4 0 2.9 5.1 3.7 3.8 42.7

It can be seen from Table 1 that compound 2460A can penetrate from the cavity (upper part of the semipermeable membrane) to the blood (lower part of the semipermeable membrane) after 90 minutes. After 30 minutes, compound 2460A was able to permeate from the blood (lower part of the semipermeable membrane) into the lumen (upper part of the semipermeable membrane). Moreover, the penetration ability of the latter is stronger than that of the former.

Example 4 Inhibitory Effect of Compound 2460A on Human Myeloma Cell RPMI8226 in Vitro

Human myeloma RPMI8226 cells were cultured in RPMI1640 medium (product of Hyclone Company) supplemented with 10% fetal bovine serum at 37° C. in a 5% CO ₂ incubator, passaged once every 3-4 days, and grown to logarithmic phase.

The compound 2460A of the present invention was dissolved in a small amount of 100% ethanol, and the final concentration of ethanol was not more than 0.1%. Make sure to dissolve completely. The drugs were diluted with complete medium to the following concentrations: 25, 10, 5, 1, 0.5, 0.1, 0.01*10 ^-6 Mol/L.

The cells cultured to the logarithmic growth phase as above were collected to prepare a cell suspension with a concentration of 5.6*10 ⁵ /ml. Take two 96-well cell culture plates and add 50 μl of cell suspension to each well. A control group without drugs was set up. In the dosing group, 6 wells were set up in parallel according to different concentrations; cultured for 48 and 72 hours, respectively. 4 hours before the end of the culture, 20 μl of MTT was added to each well; 4 hours later, the liquid in each well was removed by centrifugation, and 200 μl of dimethyl sulfoxide was added to each well; 30 minutes later, an automatic microplate reader was used to measure the OD value of each well at 630 nm. Experiments were repeated three times.

Results: Compound 2460A had a significant inhibitory effect on human myeloma cell RPMI8226 at a concentration greater than 1*10 ^-6 Mol/L at 24 hours and 72 hours, up to 63%.

Claims (5)

1. A microbial-derived inflammatory factor receptor antagonistic active compound, which has a structure shown in formula II: It is an antagonistically active compound of the interleukin-6 receptor. 2. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier. 3. Use of the compound of claim 1 for the preparation of a medicament for treating diseases associated with excess ligands of natural tumor necrosis factor receptor TNFR and interleukin-6 receptor IL-6R. 4. The use according to claim 3, wherein said diseases include septic shock, acute and chronic myeloid sepsis, atherosclerosis, autoimmune diseases. 5. The use according to claim 4, wherein said autoimmune disease is rheumatoid arthritis, transplant rejection. the

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