CN118178362A - Application of 2,2'-(2-methylenepropane-1,3-diyl)bis(4-isopropylphenol) in the preparation of NLRP3 inflammasome inhibitors - Google Patents

Abstract

The present invention provides the use of 2,2'-(2-methylenepropane-1,3-diyl)bis(4-isopropylphenol) in the preparation of NLRP3 inflammasome inhibitors. The present invention first investigates the effect of the compound on the activation of macrophage NLRP3 inflammasome in vitro, and the results show that the compound can effectively inhibit the maturation and secretion of caspase-1 and IL-1β, and inhibit the activation of NLRP3 inflammasome in vitro. At the same time, the compound can also inhibit the activation of NLRP3 inflammasome in LPS-induced ALI mice in vivo, reduce the release of inflammatory factors, reduce lung tissue damage, and alleviate inflammatory response. The present invention provides a novel propylene bisphenol compound that can be used as an inhibitor of NLRP3 inflammasome, providing a new strategy for the treatment of diseases associated with uncontrolled inflammatory response, such as ALI and sepsis.

Description

Application of 2,2'-(2-methylenepropane-1,3-diyl)bis(4-isopropylphenol) in the preparation of NLRP3 inflammasome inhibitors

Technical Field

The present invention belongs to the field of biomedicine and relates to the application of 2,2′-(2-methylenepropane-1,3-diyl)bis(4-isopropylphenol) in the preparation of NLRP3 inflammasome inhibitors.

Background technique

NLRP3 (Nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3) inflammasome is an intracellular multiprotein complex that plays an important role in the host's immune response to microbial infection and cell damage. When cells are injured, infected, stressed or otherwise stimulated, NLRP3 aggregates to form inflammasomes and triggers a series of inflammatory responses. NLRP3 inflammasomes are composed of apoptosis-associated speck-like protein containing a CARD (ASC), NLRP3 and pro-caspase-1. Through the interaction between the PYD and CARD domains, they promote the cleavage activation of caspase-1 and the maturation of IL-1β and IL-18. NLRP3 inflammasomes play an important role in a variety of diseases, including lung injury, inflammatory bowel inflammation, gout, atherosclerosis, cardiovascular disease, diabetes, rheumatoid arthritis and neurodegenerative diseases. Therefore, controlling the activation of NLRP3 inflammasomes may help alleviate inflammatory responses and provide new ideas for the treatment of related diseases.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is closely related to uncontrolled inflammatory response and lung tissue vulnerability. During ALI, neutrophils accumulate in the lungs, exacerbating lung epithelial cell death, damaging alveolar structure, causing pulmonary capillary membrane damage and increased permeability, leading to plasma protein infiltration to form pulmonary edema and protein membrane, which may develop into fibrosis. Alveolar protein accumulation impairs surfactant function and exacerbates cell damage. Neutrophils attach to damaged endothelium and enter the alveoli. The degree of inflammation in ALI is closely related to the release of proinflammatory factors such as IL-6, IL-18 and IL-1β, and the activation of alveolar macrophages. Therefore, inhibiting NLRP3 inflammasome activation and then inhibiting uncontrolled inflammatory response can alleviate the disease and is crucial to promoting inflammation relief and tissue repair.

In summary, finding reagents that can inhibit NLRP3 inflammasome activation with excellent effects is of great significance for reducing inflammation and alleviating inflammation-related diseases.

Purpose of the Invention

The first object of the present invention is to provide an application of 2,2′-(2-methylenepropane-1,3-diyl)bis(4-isopropylphenol) in the preparation of NLRP3 inflammasome inhibitors in view of the deficiencies of the prior art. 2,2′-(2-methylenepropane-1,3-diyl)bis(4-isopropylphenol) is named MLS-03, and its structural formula is shown as follows:

Preferably, 2,2′-(2-methylenepropane-1,3-diyl)bis(4-isopropylphenol) inhibits the activation of NLRP3 inflammasome and reduces the expression levels of inflammatory factors IL-6, TNF-α, IL-8 and IL-1β.

The second object of the present invention is to provide an NLRP3 inflammasome inhibitor, comprising 2,2′-(2-methylenepropane-1,3-diyl)bis(4-isopropylphenol).

The third object of the present invention is to provide the use of 2,2′-(2-methylenepropane-1,3-diyl)bis(4-isopropylphenol) in the preparation of drugs for preventing or treating NLRP3 inflammasome-related diseases.

The fourth object of the present invention is to provide a pharmaceutical composition, the active ingredient of which includes 2,2′-(2-methylenepropane-1,3-diyl)bis(4-isopropylphenol).

For the above-mentioned pharmaceutical composition, MLS-03, as an active ingredient, can be introduced into the body by oral administration, injection, spray, nasal drops, eye drops, penetration, absorption, physical or chemical mediation methods, such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue, or introduced into the body after mixing or encapsulating with other substances.

In the present invention, the pharmaceutical composition for preventing and/or treating diseases mediated by NLRP3 inflammasome may contain only MLS-03 (as an active ingredient), or may be prepared by combining MLS-03 with one or more pharmaceutically acceptable carriers that are pharmaceutically permitted; these carriers include diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. The drug may be an oral agent or a non-oral agent.

In the present invention, the pharmaceutical composition adopts any pharmaceutically acceptable dosage form; when preparing capsules, tablets, pills and powders suitable for oral administration, sucrose, lactose, corn starch, microcrystalline cellulose, carboxymethyl cellulose and the like can be used as carriers or excipients.

In the present invention, preventing and/or treating a disease mediated by NLRP3 inflammasome refers to preventing the occurrence of the disease, alleviating symptoms that have occurred, delaying disease progression, or reversing the pathological process.

In the present invention, MLS-03 can inhibit the production of IL-1β, a product of NLRP3 inflammasome activation, in macrophages treated with LPS and ATP/Nigericin, inhibit the shearing of NLRP3 inflammasome activation marker caspase-1/pro-cysteine protease-1, and inhibit the release of lactate dehydrogenase caused by NLRP3 inflammasome activation, thereby preventing and/or treating the pathological conditions of diseases mediated by NLRP3 inflammasomes such as ALI/ARDS. The NLRP3 inflammasome-related diseases include, but are not limited to, acute lung injury or acute respiratory distress syndrome. Other diseases include infectious inflammatory diseases and autoimmune diseases, including but not limited to: inflammatory bowel disease, sepsis, peritonitis, gouty arthritis, and multiple sclerosis.

Compared with the prior art, the beneficial effects of the present invention are at least:

The present invention provides the use of 2,2′-(2-methylenepropane-1,3-diyl)bis(4-isopropylphenol) in the preparation of NLRP3 inflammasome inhibitors, and the compound is a symmetrical polyphenol compound containing an isopropyl group. The present invention expands the application field of the compound, verifies that the compound can effectively inhibit the maturation and secretion of caspase-1 and IL-1β, and inhibit the activation of NLRP3 inflammasome in vitro; at the same time, the compound can also inhibit the activation of NLRP3 inflammasome in LPS-induced ALI mice in vivo, reduce the release of inflammatory factors, reduce lung tissue damage, and alleviate inflammatory response. The present invention provides a new strategy for the treatment of diseases associated with uncontrolled inflammatory response, such as ALI and sepsis.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a synthetic route of MLS-03, wherein compound 1 is 4-4-isopropylphenol; compound 2 is 4,4′-((2-methylenepropane-1,3-diyl)bis(oxy))bis(isopropylbenzene); and compound 3 is 2,2′-(2-methylenepropane-1,3-diyl)bis(4-isopropylphenol).

Figure 2 shows that MLS-03 can alleviate acute lung injury induced by LPS in mice, where A: Representative images of H&E stained sections of lung tissues of mice in different groups (100×, 200×), showing pathological damage of lung tissues (scale length is 100μM); B: Protein concentration in bronchoalveolar lavage fluid; C: MPO activity in bronchoalveolar lavage fluid. *P<0.05, **P<0.01.

Figure 3 shows that MLS-03 significantly reduced the expression levels of inflammatory factors IL-6, TNF-α, IL-8 and IL-1β in LPS-induced ALI mice, where A: the secretion level of IL-6 in serum; B: the secretion level of TNF-α in serum; C: the secretion level of IL-8 in serum; D: the secretion level of IL-1β in serum.

FIG4 shows that MLS-03 significantly inhibits the secretion of IL-1β in mouse bone marrow-derived macrophages (BMDM) and THP-1 cells induced by the combined action of LPS and ATP or nigericin (Nig), wherein A: BMDM; B: THP-1.

Figure 5 shows that MLS-03 significantly inhibits the activation of NLRP3 inflammasome in BMDM cells and THP-1 cells induced by the combined action of LPS and ATP or nigericin (Nig), which shows the protein levels of caspase-1 and IL-1β in the cell culture supernatant and the protein levels of NLRP3, pro-caspase-1, pro-IL-1β and ASC in the cell lysate, respectively, where A: BMDM; B: THP-1.

Detailed ways

The present invention is further described below in conjunction with the accompanying drawings and examples, verifying that MLS-03, i.e., 2,2′-(2-methylenepropane-1,3-diyl)bis(4-isopropylphenol), can inhibit the activation of NLRP3 inflammasomes and improve LPS-induced acute lung injury and inflammatory response in mice.

Experimental Materials:

Cells: Mouse peritoneal macrophages (primary extraction); THP-1 cells, incubated with 10 ng/mL Phorbol 12-myristate 13-acetate (PMA) for 16 h before drug addition experiments, and then differentiated into human macrophages.

Animals: C57BL/6 healthy male mice aged 4-6 weeks were provided by Shanghai Shrek Company; mice were fed with complete nutritional pellet feed provided by the Experimental Animal Center of Hangzhou Normal University.

Instruments: IL-6 and TNF-α ELISA detection kits were purchased from Invitrogen; IL-8, IL-1β ELISA detection kits were purchased from Shenzhen Xinbosheng Biotechnology Co., Ltd.

Reagents: MLS-03, independently synthesized, the synthesis route is shown in Figure 1, and the synthesis method refers to patent CN202110185671.X; LPS and PMA were provided by Sigma-Aldrich, DMEM high glucose medium was provided by BI; fetal bovine serum (FBS) was provided by BI; trypsin (0.25% Trypsin-EDTA) was provided by BI. BCA protein quantitative detection kit was provided by Kangwei Century Biological Company. All antibodies are shown in Table 1.

Table 1 Antibodies used in the experiments

Example 1: MLS-03 improves LPS-induced ALI

An ALI mouse model induced by intraperitoneal injection of LPS was established, and the protective or mitigating effects of MLS-03 on the disease were studied in the model. A variety of different indicators were used to determine whether MLS-03 could reduce the inflammatory response in mice.

C57BL/6 mice, male, 30 in total, were randomly divided into 3 groups, 10 in each group. Each group was: 1) control group, each mouse was injected with an equal volume of saline; 2) LPS group, each mouse was intraperitoneally injected with 1 mg/kg LPS dissolved in 200 μL saline; 3) LPS+MLS-03 group, each mouse was first injected with MLS-03 at a dose of 12 mg/kg, dissolved in 100 μL saline, and 1 hour later, each mouse was intraperitoneally injected with 1 mg/kg LPS dissolved in 200 μL saline; mice were killed 16 hours later.

Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital 50 mg/kg. Five mice were randomly selected from each group to remove their eyeballs for blood collection, and the serum was separated for ELISA detection of related cytokines; lung tissue was separated, and the lung lobe was fixed with 4% paraformaldehyde for H&E staining to detect pathological changes in lung tissue. Five mice were randomly selected from each group, and after being killed by cervical dislocation, bronchoalveolar lavage was performed with 0.9% sodium chloride injection, and bronchoalveolar lavage fluid (BALF) was collected for detection of protein concentration and MPO activity.

The results in Figure 2 show that the H&E staining results of lung tissues showed that the alveolar structure of mice in the normal control group was intact, with no inflammatory cells and fluid exudation in the alveolar cavity, and no inflammatory cell infiltration and edema in the alveolar septum; the alveolar structure of mice in the LPS-induced ALI group was destroyed, with a large number of inflammatory cells and fluid exudation in the alveolar cavity, a large number of inflammatory cells infiltrated in the alveolar septum, and pulmonary interstitial edema; in the MLS-03 treatment group (LPS+MLS-03 group), the pathological phenomena such as bronchial wall thickening, inflammatory cell infiltration, alveolar cavity and vascular congestion induced by LPS were significantly alleviated (Figure 2A). In acute lung injury, the permeability of alveolar capillaries increases, the air-blood barrier is damaged, and a large amount of protein and other substances are exuded, thus leading to an increase in the protein content in BALF. In our experiment, it was found that compared with the normal control group, the protein content in BALF of mice in the LPS group was significantly increased, while the protein content in BALF of mice in the MLS-03 treatment group was significantly lower than that in the LPS group (Figure 2B). Myeloperoxidase (MPO) is abundant in neutrophils, so the MPO concentration in lung tissue can reflect the degree of neutrophil infiltration during acute lung injury. We observed that compared with the normal control group, the MPO activity of mice in the LPS group was significantly increased, while the MPO activity of mice in the MLS-03 treatment group was significantly decreased compared with the LPS group (Figure 2C).

The results in Figure 3 show that compared with the normal control group, the concentrations of proinflammatory cytokines IL-6, TNF-α, IL-1β and IL-8 in the serum of mice in the LPS-induced acute lung injury group (LPS group) were significantly increased; while the concentrations of proinflammatory cytokines IL-6, TNF-α, IL-1β and IL-8 in the serum of mice in the MLS-03 treatment group were significantly lower than those in the LPS group. The above results show that MLS-03 can significantly inhibit the production of inflammatory factors in LPS-induced ALI mice.

Embodiment 2:

MLS-03 inhibits the activation of NLRP3 inflammasome in macrophages: Macrophages differentiated from mouse primary macrophages BMDM and human monocytes THP-1 were stimulated with LPS and ATP/Nigericin/, and then treated with different concentrations of MLS-03 to observe and record the activation of NLRP3 inflammasome.

(1) Mouse primary bone marrow-derived macrophages (BMDM)

BMDM cells were divided into 8 groups: Group 1: blank control group, cells were treated with normal saline; Group 2: cells were treated with 100ng/mL MLS-03 for 6 hours; Group 3: cells were treated with 100ng/mL LPS for 4 hours, and then 5mMATP was added for 1 hour; Group 4: cells were treated with 50ng/mL MLS-03 for 1 hour, and then 100ng/mL LPS for 4 hours, and then 5mMATP was added for 1 hour; Group 5: cells were treated with 100ng/mL MLS-03 for 1 hour, and then 100ng/mL LPS for 4 hours, and then 5mMATP was added for 1 hour; Group 6: cells were treated with 100ng/mL LPS for 4 hours, and then 10μM Nigericin was added for 1 hour;

Group 7: After the cells were treated with 50ng/mL LMS-03 for 1 hour, 100ng/mL LPS was used for 4 hours, and then 10μM Nigericin was added for 1 hour; Group 8: After the cells were treated with 100ng/mL LMS-03 for 1 hour, 100ng/mL LPS was used for 4 hours, and then 10μM Nigericin was added for 1 hour.

(2) Macrophages differentiated from THP-1 cell line

THP-1 cell line was induced by PMA (10ng/mL) for 16 hours, differentiated into macrophages after adherence, and then divided into 8 groups: Group 1: blank control group, cells were treated with normal saline; Group 2: cells were treated with 100ng/mL MLS-03 for 6 hours; Group 3: cells were treated with 100ng/mL LPS for 4 hours, and then 5mM ATP was added for 1 hour; Group 4: cells were treated with 50ng/mL MLS-03 for 1 hour, 100ng/mL LPS for 4 hours, and then 5mM ATP was added for 1 hour; Group 5: cells were treated with 100ng/mL MLS-03 for 1 hour, 100ng/mL LPS for 4 hours, and then 5mM ATP was added for 1 hour; Group 6: cells were treated with 100ng/mL LPS for 4 hours, and then 10μM Nigericin was added for 1 hour; Group 7:

After the cells were treated with 50ng/mL MLS-03 for 1 hour, 100ng/mL LPS was used for 4 hours, and then 10μM Nigericin was added for 1 hour; the fifth group: the cells were treated with 100ng/mL MLS-03 for 1 hour, 100ng/mL LPS was used for 4 hours, and then 10μM Nigericin was added for 1 hour.

The culture supernatants of the BMDM cells and THP-1 cells in the above groups were collected, and the secretion level of IFN-β in the cell culture supernatants was detected by ELISA. The experimental results are shown in FIG4 .

After lysing the BMDM cells and THP-1 cells in the above groups, the cell proteins were collected for Western-blot detection of the levels of NLRP3, pro-caspase-1, pro-IL-1β and ASC in the cells, with β-actin as an internal reference. At the same time, the supernatants of the above groups of cells were collected, and the proteins were extracted and Western-blot was performed to detect the levels of mature caspase-1 and IL-1β in the cell supernatants. The experimental results are shown in Figure 5.

The results of Figures 4 and 5 show that MLS-03 can significantly inhibit the activation of inflammasomes in BMDM and THP-1 cells stained under LPS+ATP stimulation or LPS+Nigericin stimulation, and reduce the secretion levels of caspase-1 and IL-1β in the supernatant.

In summary, the present invention confirms that MLS-03 can inhibit the activation of NLRP3 inflammasome, thereby reducing the inflammatory response and exerting a protective effect on acute lung injury in mice. The present invention belongs to the field of medical technology and has broad application prospects.

Although the embodiments described above have been described in detail, these embodiments are only used to illustrate the specific manner of the technical solution of the present invention and are not intended to limit it. Without departing from the technical principles of the present invention, the embodiments may be modified, replaced or improved to expand their scope of application. These modifications, replacements or improvements should be regarded as equivalent implementations within the scope of protection of the present invention and should be included in the scope of protection of the claims of the present invention to ensure comprehensive protection of the present invention.

Claims (8)

1. The use of 2,2'-(2-methylenepropane-1,3-diyl)bis(4-isopropylphenol) in the preparation of NLRP3 inflammasome inhibitors, characterized in that the structural formula of 2,2'-(2-methylenepropane-1,3-diyl)bis(4-isopropylphenol) is as shown below: 2. The use according to claim 1, characterized in that 2,2'-(2-methylenepropane-1,3-diyl)bis(4-isopropylphenol) inhibits the activation of NLRP3 inflammasome and reduces the expression levels of inflammatory factors IL-6, TNF-α, IL-8 and IL-1β. 3. A NLRP3 inflammasome inhibitor, characterized in that it comprises 2,2'-(2-methylenepropane-1,3-diyl)bis(4-isopropylphenol). 4. Application of 2,2'-(2-methylenepropane-1,3-diyl)bis(4-isopropylphenol) in the preparation of drugs for preventing or treating NLRP3 inflammasome-related diseases. 5. The use according to claim 4, characterized in that the NLRP3 inflammasome-related diseases include acute lung injury or acute respiratory distress syndrome. 6. A pharmaceutical composition, characterized in that the active ingredient of the pharmaceutical composition includes 2,2'-(2-methylenepropane-1,3-diyl)bis(4-isopropylphenol). 7. The pharmaceutical composition according to claim 6, characterized in that it further comprises a pharmaceutically acceptable carrier or excipient. 8. The pharmaceutical composition according to claim 6, characterized in that the pharmaceutical composition adopts any pharmaceutically acceptable dosage form.