KR102116166B1 - Nlrp3 inhibitor - Google Patents

Abstract

The present invention is 2-cyclohexyl-diamino-6 inhibitor as NLRP3-6,7-dihydro -5 H - benzo [1,3] oxa thiol-4-one (2-cyclohexylimino-6-methyl -6, 7-dihydro-5 H -benzo [1,3] oxathiol-4-one) relates to the use of 2-cyclohexylimino-6-methyl-6,7-dihydro- of Formula 1 according to the present invention 5 H -benzo [1,3] oxathiol-4-one inhibits its ATPase activity by alkylating NLRP3 and then ubiquitinates it, thereby inhibiting binding and migration with ASC, thereby inhibiting the NLRP3 inflammation control complex (inflammasome). Since it was confirmed to suppress activation, and it was confirmed that the therapeutic effect on peritonitis caused by mice was excellent, it can be used for the purpose of suppressing the NLRP3 alkylating agent and the NLRP3 or NLRP3 inflammatory regulatory complex, and preventing or treating inflammatory diseases It can also be useful as a medical composition.

Description

NLRP3 inhibitor {NLRP3 INHIBITOR}

The present invention is 2-cyclohexyl-diamino-6 inhibitor as NLRP3-6,7-dihydro -5 H - benzo [1,3] oxa thiol-4-one (2-cyclohexylimino-6-methyl -6, 7-dihydro-5 H- benzo [1,3] oxathiol-4-one).

The innate immune response is the first line of defense against microorganisms that are regulated by innate immune cells, such as macrophages and dendritic cells. When infected with microorganisms, these innate immune cells activate an immune response to resist infectious microorganisms. Innate immune cells include Toll-like receptors, NOD-like receptors, and RIG-I Recognize microorganisms through pattern recognition receptors (PRRs), such as RIG-I-like receptors and C-type lectin receptors. These pattern recognition receptors recognize microorganisms by conserving structures that exist conservatively between microbial species, such as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Identify and activate immune modulators to eliminate microbes, pathogens, and infectious cells accordingly. Among them, NOD-like receptors (NLRs) are intracellular immune receptor families with more than 20 family members in humans, and NLR family members are leucine-rich repeats (LRRs) and NATCH nucleotide-binding omain. It is known to have at least two or more common features such as (NBD). Among them, the LRR domain is known to recognize proto-associated molecular patterns (PAMPs) and play an important role in protein-to-protein interaction. The NOD-like receptor family members have the same common characteristics as above, but it is known that there is a difference in the N-terminal domain, and based on the difference, the NOD-like receptor family members are subdivided. Of these, NLRP is the largest group of NLR families with Nterminal pyrin domain (PYD), NOD1 (also known as nucleotide-binding ligomerization domain-containing 1), NOD2 and CARD domain-containing 4 NLRC4 (also known as IPAF) All known) have an N-terminal caspase recruitment domain.

On the other hand, family members having NLRP1, NLRP3 and NLRC4 among NOD-like receptor families are known to be able to form multi-molecular complexes by various active factors, thereby activating immune caspases. -1 regulates the cytokine secretion of the IL-1 family. NOD-like receptor multi-molecular complexes are also called inflammation control complexes (inflammasomes), which are key components for the immune surveillance system in cytosols. Inflammatory regulatory complexes are multiprotein complexes that activate caspase-1, a proteolytic enzyme, and mature pro-inflammatory cytokines such as IL-1β (interleukin-1β) and IL-18. Along with this activation reaction, the inflammatory regulatory complex is self-oligolysed and recruiting pro-caspase-1 through direct cross-linking of the NOD-like receptor and the CARDs domain of pro-caspase-1. However, since many NOD-like receptors do not have a CARDs domain, they are assisted by an apoptotic speck protein containing a caspase recruitment domain (ASC) containing CARDs or PYD domains, that is, the PYD domain of ASC. Interacts with the NOD-like receptor, and the CARD domain of ASC interacts with the CARD domain of pro-caspase-1. Caspase-1 is also known as an IL-1-converting enzyme, which forms an inflammatory regulatory complex, and then pro-caspase-1 self-cuts to form active caspase-1, and active caspase-1 is pro- This is because IL-1β matures into IL-1β and induces caspase-1 dependent cell death, known as pyroptosis. Active IL-1β matured by caspase-1 possesses potential pro-inflammatory activity, and inhibition of IL-1β is atherosclerosis regulated by IL-1β, insulin resistance, type 2 diabetes, rheumatism It is known to reduce autoimmune diseases such as sex arthritis and gout. Therefore, the rapid outflow of IL-1β can lead to fairly detrimental results, so the secretion of IL-1β will have to be very tightly controlled.

As a precedent patent related to the treatment of inflammatory diseases, the U.S. registered extract (No. 10-1732365) for reducing the production of inflammatory mediators by regulating the activity of the inflammatory control complex activity, inflammasome mediated inflammatory disease of green pepper extract Republic of Korea Patent Registration (No. 10-1637476) for the composition for improvement has been reported.

The present inventors have found that 2-cyclohexyl-6-a mino-6,7-dihydro -5 H - [1,3] oxazol-4-thiol of NLRP3 (NACHT, LRR and PYD domains -containing protein 3 ) The present invention was completed by confirming the inhibitory effect on the inflammatory regulatory complex (inflammasome) and confirming the inhibitory mechanism therefor.

In the present invention, 2-cyclohexyl-6-butylimino-6,7-dihydro -5 H - benzo [1,3] NLRP3 active inflammation or NLRP3 control complex (inflammasome) activity comprising a thiol-oxa-4-one It is an object to provide a reagent composition for inhibition.

In the present invention, 2-cyclohexyl-6-butylimino-6,7-dihydro -5 H - is an object of the present invention to provide a NLRP3 alkylating agent containing the benzo [1,3] oxa thiol-4-one .

In the present invention, 2-cyclohexyl-6-butylimino-6,7-dihydro -5 H - [1,3], including the step of processing the oxa thiol-4-one in vitro (in It is an object to provide a method for inhibiting NLRP3 activity or NLRP3 inflammatory regulatory complex activity in vitro.

In the present invention, 2-cyclohexyl-6-butylimino-6,7-dihydro -5 H - [1,3] for the prevention or treatment of inflammatory diseases containing oxa thiol-4-one as an active ingredient It is an object to provide a pharmaceutical composition.

In addition, in the present invention, 2-cyclohexyl-6-butylimino-6,7-dihydro -5 H - benzo [1, 3] a food composition for the prevention or amelioration of inflammatory disorders, containing a thiol-oxa-4-one It aims to provide.

In order to achieve the above object, the present inventors have found that 2-cyclohexyl-6-a mino-6,7-dihydro -5 H - [1,3] oxazol-4-thiol (or BOT-4-one ) And the effect on NLRP3 activity and NLRP3 inflammatory regulatory complex (inflammasome) and specific action mechanism were confirmed.

2-cyclohexyl-6-a mino-6,7-dihydro -5 H of the formula (I) according to the present invention benzo [1,3] oxa thiol-4-one inhibits the ATPase activity thereof, by alkylating the NLRP3 And increasing ubiquitination, thereby inhibiting the movement and binding of ASC, thereby inhibiting the activation of the NLRP3 inflammatory regulatory complex (inflammasome), and confirming that the therapeutic effect on peritonitis caused by the mouse is excellent. The NLRP3 alkylating agent and the NLRP3 ATPase can be used for suppressing the NLRP3 inflammatory regulatory complex through inhibitory activity, and it can also be usefully used as a pharmaceutical composition for preventing or treating inflammatory diseases.

Figure 1 is 2-cyclohexyl-6-a mino-6,7-dihydro -5 H - is a check the cytotoxicity of the concentrations BMDMs of benzo [1,3] oxa thiol-4-one:
Sup: Cell supernatant.
Figure 2 is a 2-cyclohexyl-6-butylimino-6,7-dihydro -5 H - is the benzo [1, 3] oxazol-thiol is also confirming the NLRP3 inflammation control complex activity inhibitory effect of the 4-one:
BOT: 2- cyclohexyl-6-butylimino-6,7-dihydro -5 H - benzo [1,3] oxa thiol-4-one;
Nig: nigericin; And
Sil: Silica crystal.
3 is a 2-cyclohexyl-6-butylimino-6,7-dihydro -5 H - is the benzo [1, 3] oxazol-thiol is also confirming the NLRP3 ASC oligomerization mediated inhibitory effect of 4-one:
Sup: cell supernatant;
Pel: pellets (crosslinked cytoplasmic pellets); And
Lys: cell debris.
Figure 4 is 2-cyclohexyl-6-a mino-6,7-dihydro -5 H - movement in the benzo [1,3] oxa thiol-4-one of NLRP3 and ASC of Triton X-100 insoluble fraction This is a diagram confirming the inhibitory effect:
Sup: cell supernatant (soluble fraction of Triton X-100);
Pel: pellet (insoluble fraction of Triton X-100); And
Lys: Cell lysate (soluble fraction of Triton X-100).
5 is a 2-cyclohexyl-6-butylimino-6,7-dihydro -5 H - is the benzo [1, 3] oxazol-thiol is also confirming the NLRP3 ATPase activity and inhibit the alkylation of 4-one:
Cys: L-cysteine; And
Bay: Bay11-7082.
Figure 6 is a 2-cyclohexyl-6-diamino-6,7-dihydro -5 H - [1,3] oxazol-thiol movement of ASC NLRP3 oligomerization and / ASC via the NLRP3 alkylated-4-one It is also confirmed to suppress:
Cys: L-cysteine;
Sup: cell supernatant (soluble fraction of Triton X-100);
Pel: pellet (insoluble fraction of Triton X-100); And
Lys: Cell lysate (soluble fraction of Triton X-100).
7 is a 2-cyclohexyl-6-butylimino-6,7-dihydro -5 H - induced ubiquitination of NLRP3 through benzo [1, 3] oxazol-4-thiol, followed by alkylation of the NLRP3 It is the figure which confirmed that it suppresses the binding of NLRP3 / ASC.
Figure 8 is 2-cyclohexyl-6-butylimino-6,7-dihydro -5 H - [1,3] is also confirming the MSU- peritonitis induced relaxation effect in the in vivo oxa thiol-4-one .

Hereinafter, the present invention will be described in detail. However, the present invention can be implemented by being modified in various forms, and is not limited to the embodiments described herein.

In one aspect, the present invention is 2-cyclohexyl-6,7-dihydro-6-butylimino of the following general formula (1) -5 H - [1,3] oxa thiol-4-one (2-cyclohexylimino-6 -methyl-6,7-dihydro-5 H -benzo [1,3] oxathiol-4-one, also referred to herein as BOT-4-one) relates to a reagent composition for inhibiting NLRP3 activity :

In one embodiment, the NLRP3 activity may be the ATPase activity of NLRP3, ubiquitination of NLRP3 can inhibit the activity of NLRP3, and then the binding of NLRP3 and ASC can also be inhibited.

In one aspect, the present invention is 2-cyclohexyl-6-a mino-6,7-dihydro -5 H of the formula (1) benzo [1,3] NLRP3 inflammation control complex including a oxazole-4-thiol (inflammasome) Reagent composition for inhibiting activity .

In one embodiment, the present invention is 2-cyclohexyl-6-diamino-6,7-dihydro -5 H - benzo [1,3] oxa thiol-4-one is in the ASC and ASC oligomerization and NLRP3 By inhibiting the migration to the Triton X-100 insoluble fraction, and inhibiting the binding of NLRP3 and the ASC (apoptotic speck protein containing a caspase recruitment domain), it is possible to inhibit the activity of the NLRP3 inflammatory regulatory complex.

In one aspect, the present invention is a 2-cyclohexyl-6-methyl-6,7-diamino of formula dihydro -5 H - [1,3] on the NLRP3 alkylating agent containing the thiol-oxa-4-one will be.

In one implementation, the 2-cyclohexyl-6-a mino-6,7-dihydro -5 H of the present invention benzo [1,3] oxa thiol-4-one is NLRP3 of ATPase activity by alkylating the NLRP3 And inhibits the activity of NLRP3 by ubiquitination, thereby inhibiting the binding and migration of ASC oligomerization and NLRP3 / ASC, thereby inhibiting the NLRP3 inflammatory regulatory complex, resulting in maturation and interleukin-1 beta maturation and Secretion can be suppressed.

In one embodiment, the 2-cyclohexyl-6 of the present invention diamino-6,7-dihydro -5 H - a mechanism for controlling the benzo [1,3] oxazole The thiol NLRP3 activity of the 4-one Since it was confirmed by alkylation, it can be used as an NLRP3 alkylating agent.

In one aspect, the present invention is a 2-cyclohexyl-6-methyl-6,7-diamino of formula dihydro -5 H - comprising the step of treating the benzo [1,3] oxa thiol-4-one It relates to a method for inhibiting NLRP3 inflammation control complex activity in vitro.

In one embodiment, the present invention is 2-cyclohexyl-6-diamino-6,7-dihydro -5 H - of benzo [1, 3] oxazol-4-thiol NLRP3 inflammation control complex on the in vitro Since it was confirmed to inhibit activation, it can be used to inhibit the activity of the NLRP3 inflammatory regulatory complex in vitro.

In one aspect, the present invention is a 2-cyclohexyl-6-methyl-6,7-diamino of formula dihydro -5 H - comprising the step of treating the benzo [1,3] oxa thiol-4-one , It relates to a method for inhibiting NLRP3 activity in vitro.

In one embodiment, the 2-cyclohexyl-6 of the present invention diamino-6,7-dihydro -5 H - NLRP3 of inhibiting activity on the benzo [l, 3] The in vitro oxa thiol-4-one Since it was confirmed that it can be used to suppress NLRP3 activity in vitro.

In one aspect the present invention is a 2-cyclohexyl-6-methyl-6,7-diamino of formula dihydro -5 H - benzo [1,3] oxa thiol-4-one (2-cyclohexylimino-6-methyl -6,7-dihydro-5 H -benzo [ 1,3] oxathiol-4-one) the present invention relates to inflammatory disease prevention or treatment a pharmaceutical composition containing as an active ingredient.

In one embodiment, the 2-cyclohexyl-6 of the present invention diamino-6,7-dihydro -5 H - have any one or more of the following properties [1,3] oxa thiol-4-one Can:

1) inhibition of NLRP3 activity;

2) inhibition of NLRP3 inflammatory regulatory complex activity;

3) NLRP3 and ASC binding and migration inhibition; And

4) IL-1β maturation and secretion inhibition.

In one embodiment, the inflammatory disease may be an inflammatory disease-mediated inflammatory disease.

In one embodiment, the inflammatory disease is peritonitis, spondylitis, inflammatory pain, urethritis, cystitis, sepsis, dermatitis, periodontitis, gingivitis, diabetes, asthma, tuberculosis, bronchitis, allergic and non-allergic rhinitis, chronic and acute rhinitis, Chronic and acute gastritis or enteritis, ulcerative gastritis, acute and chronic nephritis, acute and chronic hepatitis, chronic obstructive pulmonary disease, pulmonary fibrosis, irritable bowel syndrome, inflammatory pain, migraine, headache, back pain, fibromyalgia, fascia disease, virus Infections (e.g., type C infection), bacterial infections, fungal infections, burns, wounds caused by surgical or dental surgery, prostaglandin E hypersyndrome, atherosclerosis, gout, arthritis, rheumatoid arthritis, ankylosing spondylitis, Hodgkin's disease, pancreatitis, conjunctivitis, keratitis, scleritis, uveitis, eczema, and multiple sclerosis, more preferably peritonitis.

In one implementation, the 2-cyclohexyl-6-a mino-6,7-dihydro -5 H of the present invention benzo [1,3] oxa thiol-4-one can be included in more than 3μM.

The compositions of the present invention is 2-cyclohexyl-6-diamino-6,7-dihydro -5 H - benzo [1,3] oxa thiol-4-one, as well as this same or other active ingredients having a similar function By further containing, or by further containing another active ingredient having a different function from the above components, it can be prepared as a pharmaceutical composition for the prevention or treatment of inflammatory diseases.

In the present invention, the term "prevention" means the present invention according to the 2-cyclohexyl-6-diamino-6,7-dihydro -5 H -, including benzo [1,3] oxa thiol-4-one, or it Any action that inhibits or delays the development, spread and recurrence of inflammation by administration of the composition.

The term "treatment" is 2-cyclohexyl-6-a mino-6,7-dihydro -5 H used in the present invention benzo [1,3] oxa thiol-4-one or the dose of the composition comprising the same By means all actions to improve or beneficially improve the symptoms of inflammatory diseases or complications. If the person having ordinary knowledge in the technical field to which the present invention belongs, can refer to the data presented by the Korean Medical Association and the like to know the exact criteria of the disease in which the composition of the present invention is effective, and to determine the degree of improvement, improvement and treatment. will be.

The therapeutically effective amount of the composition of the present invention may vary depending on several factors, for example, the method of administration, the target site, the condition of the patient, and the like. Therefore, when used in the human body, the dosage should be determined in an appropriate amount in consideration of safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. Such considerations to be taken into account when determining an effective amount include, for example, Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; And E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term “pharmaceutically effective amount” used in the present invention means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and does not cause side effects, and an effective dose level of the patient Well known in the medical field, factors including inflammatory disease type, severity, drug activity, sensitivity to the drug, administration method, administration time, administration route and discharge rate, duration of treatment, combination or concurrent drug use and other medical fields It can be determined by factors. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, which can be easily determined by those skilled in the art.

In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group comprising oral dosage forms, external preparations, suppositories, sterile injectable solutions and sprays.

The compositions of the present invention may also include carriers, diluents, excipients, or combinations of two or more of those commonly used in biological agents. The pharmaceutically acceptable carrier is not particularly limited as long as the composition is suitable for in vivo delivery, for example, Merck Index, 13th ed., Merck & Co. Inc. The compound, saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be used in combination, and other antioxidants, buffers, bacteriostatic agents, etc. may be used as required. Conventional additives can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into main-use formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component using methods appropriate in the art or using methods disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

The composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions. The composition of the present invention, based on the total weight of the composition, comprises 0.0001 to 10% by weight of the protein, preferably 0.001 to 1% by weight.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, wherein pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, Lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opadry, sodium starch glycolate, carnauba lead, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, stearic acid Calcium, white sugar, dextrose, sorbitol and talc can be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

The composition of the present invention may be administered orally (for example, intravenously, subcutaneously, intraperitoneally, or topically) or orally, depending on the desired method, and the dosage is the patient's weight, age, sex, health status, The range varies depending on the diet, administration time, administration method, excretion rate and disease severity. The daily dosage of the composition according to the present invention is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, and more preferably, divided and administered once to several times a day.

Liquid preparations for oral administration of the compositions of the present invention include suspending agents, intravenous solutions, emulsions, syrups, etc., and various excipients, such as wetting agents, sweeteners, flavoring agents, preservatives, in addition to water and liquid paraffin, which are commonly used simple diluents. Etc. may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like.

In one aspect, the present invention is 2-cyclohexyl-6-a mino-6,7-dihydro -5 H - [1,3] for the prevention or amelioration of inflammatory disorders, containing a thiol-oxa-4-one It relates to a food composition.

When the composition of the present invention is used as a food composition, the above extract can be added as it is or used with other foods or food ingredients, and can be suitably used according to a conventional method. The composition may include a food additive that is food-acceptable in addition to the active ingredient, and the mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health, or therapeutic treatment).

The term "food supplement additive" used in the present invention means a component that can be supplementally added to food, and is added to prepare a health functional food of each formulation, and can be appropriately selected and used by those skilled in the art. Examples of food supplements include flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonic acid used in carbonated beverages, and the like, but the types of food supplements of the present invention are not limited by the examples.

The food composition of the present invention may include a health functional food. The term "health functional food" used in the present invention refers to food prepared and processed in the form of tablets, capsules, powders, granules, liquids and pills using ingredients or ingredients having useful functional properties for the human body. Here, the term 'functional' refers to obtaining a useful effect for health use, such as regulating nutrients or physiological effects on the structure and function of the human body. The health functional food of the present invention can be manufactured by a method conventionally used in the general technical field, and may be prepared by adding raw materials and components conventionally added in the general technical field. In addition, the formulation of the health functional food can also be prepared without limitation as long as the formulation is recognized as a health functional food. The food composition of the present invention can be prepared in various types of formulations, and has the advantage of not having side effects that may occur when taking the drug for a long time using food as a raw material, unlike general drugs, and has excellent portability, and the present invention Can be consumed as a supplement to enhance the effectiveness of anticancer drugs.

In addition, there is no limitation on the type of healthy foods to which the composition of the present invention can be used. In addition, the composition comprising the extract of the extract of the present invention as an active ingredient may be prepared by mixing a known additive with other suitable auxiliary ingredients that may be contained in a health functional food according to the choice of a person skilled in the art. Examples of foods that can be added are meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, dairy products including gums, ice creams, various soups, beverages, teas, drinks, alcoholic beverages, and There are vitamin complexes and the like, and can be prepared by adding the extract according to the present invention as a main ingredient, and adding it to juice, tea, jelly, and juice.

The present invention will be described in more detail through the following examples. However, the following examples are only intended to materialize the contents of the present invention, and the present invention is not limited thereto.

Example 1 2-cyclohexylimino-6-methyl-6,7-dihydro-5 H -Benzo [1,3] oxathiol-4-one (2-cyclohexylimino-6-methyl-6,7-dihydro-5 H -Cytotoxicity confirmation of benzo [1,3] oxathiol-4-one, BOT-4-one)

[Formula 1]

In order to determine the toxicity and the effective concentration of the BOT-4-one (2- cyclohexylimino-6-methyl-6,7-dihydro-5 H -benzo [1,3] oxathiol-4-one) of the formula (1), mouse Cell survival analysis was performed using bone-marrow derived macrophages (BMDMs). Specifically, the femur and tibia of 8-week-old C57BL / 6 mice were washed with sterile PBS to obtain bone marrow cells. Then, bone marrow cells were cultured for 7 days in RPMI 1640 medium (Gibco, Grand Island, NY, USA) containing 10% FBS, 30% L929 cell-conditioned medium (LCM) and 1% penicillin-streptomycin. (BMDM). Differentiated cells are dispensed into non-tissue culture-treated Petri dishes (SPL Life Science Co., Gyeonggi-do, Republic of Korea) and incubated at 37 ° C. and 5% CO ₂ conditions. Cultured in Prepared BMDMs were treated with BOT-4-one (Seoul National University of College of Medicine, South Korea) at 0, 1.5626, 3.125 and 6.25 mM, respectively, and cell viability was confirmed by MTT analysis.

As a result, it was found that the BOT-4-one concentration up to 3 μM had no effect on the cell viability of BMDMs (FIG. 1).

Example 2. Confirmation of inhibition of NLRP3 inflammation control complex (inflammasome) of BOT-4-one

To confirm the effect of BOT-4-one on NLRP3 inflammatory regulatory complex activation, LPS pretreated BMDMs were treated with canonical or noncanonical NLRP3 inflammatory regulatory complex activators and BOT-4-one. The effect of existence was confirmed. Specifically, 100 ml / mL of LPS was pretreated for 4 hours as an activator of the NLRP3 inflammatory regulatory complex normal to BMDMs, replaced with Opti-MEM medium, and treated with BOT-4-one and zVAD (20 μM) 1 Incubated for hours. It was then stimulated with ATP (5 mM) or nigericin (10 μM) for 1 hour, or with silica crystals (150 μg / mL) for 3 hours. In addition, Pam3CSK4 (200 ng / mL) as an activator of the irregular NLRP3 inflammatory regulatory complex was pre-treated for 3 hours, replaced with Opti-MEM medium, and treated with BOT-4-one and KCl (150 mM) 1 Incubated for hours. Thereafter, LPS ( E.coli 011: B4) (70 μg / mL) was transfected with Lipofectamine 2000 (Invitrogen), and stimulated by incubation for 3 hours. Then, Western blot analysis (Western blot chemiluminescence reagent kit, Pierce Chemical, Rockford, IL, USA), ELISA (ELISA kit, R & D Systems, Minneapolis, MN, USA) and LDH analysis were performed using the crushed cells. The effect of BOT-4-one on the activation of the NLRP3 inflammatory regulatory complex was confirmed.

As a result, it was found that BOT-4-one inhibits IL-1β secretion and caspase-1 cleavage induced by activation of the NLRP3 inflammatory regulatory complex in a concentration-dependent manner (FIGS. 2A-C). In addition, inhibition of IL-1β secretion of BOT-4-one was found to be about 1.28 μM (ATP), 0.67 μM (nigericin) and 0.59 μM (silica crystals) with IC ₅₀ values (FIG. 2D). In addition, BOT-4-one also inhibited the release of lactate dehydrogenase (LDH) caused by the NLRP3 inflammatory regulatory complex (Figure 2E). In addition, BOT-4-one also inhibited IL-1β secretion and caspase-1 cleavage by the non-regular NLRP3 inflammatory regulatory complex (Figure 2F). Accordingly, it was found that the BOT-4-one of the present invention inhibits the activation of the NLRP3 inflammatory regulatory complex in both normal and non-normal conditions according to the above results.

Example 3. Confirmation of blocking NLRP3-mediated ASC oligomerization of BOT-4-one

To confirm the effect of BOT-4-one on inflammatory regulatory complex-mediated ASC oligomer formation, ASC oligomer cross-linking was confirmed by immunofluorescence imaging and Western blot. Specifically, as in Example 2, BPS-4-one or KCl was treated with LPS pre-treated BMDMs for 1 hour, and after activation of the NLRP3 inflammatory regulatory complex with ATP, nigericin, or silica crystals, cells were A0. It was crushed with a buffer (0.5% Triton X100, 20 mM HEPES-KOH, pH 7.5, 150 mM KCl and complete protease and phosphatase inhibitor cocktail) and a G26 syringe. The pellet obtained by centrifuging the cell lysate at 4 ° C. at 6000 rpm for 10 minutes was resuspended in PBS, and then crosslinked with DSS (disuccinimidyl suberate) (2 mM) (Thermo Scientific-Pierce, Rockford, IL, USA). The cross-linked pellet was centrifuged at 13000 rpm for 15 minutes to mix with the SDS sample buffer to prepare a Western blot sample. IL-1β for supernatant of cell debris, presence of oligomers, dimers and monomers of ASC for pellet debris, pro-IL-1β, NLRP3, ASC, pro-caspase-1 and β-actin for whole cell debris Confirmed the existence of. In addition, for ASC spot staining, NLRP3 inflammatory regulatory complex-activated BMDMs were fixed with 4% paraformaldehyde, permeabilized with acetone, and blocked with 10% horse serum. Cells were then stained with ASC antibody (Santa Cruz) and Cy3-bound anti-rabbit antibody (Jackson ImmunoResearch Lab). Nuclei were observed by staining with DAPI (Sigma-Aldrich).

As a result, BOT-4-one significantly inhibited the formation of ASC spots stimulated by nigericin in LPS-pretreated BMDMs (FIG. 3A). In addition, BOT-4-one inhibited ASC oligomerization in cytoplasmic pellets cross-linked by nigericin, ATP, and silica crystals without concentration change of the NLRP3 inflammatory regulatory complex and BOT-4-one concentration-dependently. (Figure 3B). That is, it can be seen that BOT-4-one modulates NLRP3-dependent ASC spot formation.

Example 4. Confirmation of movement inhibition of NLRP3 and ASC by BOT-4-one

To confirm the effect of BOT-4-one on the migration of the inflammatory regulatory complex (inflammasome), the effect of redistribution into Triton X-100 soluble or insoluble fraction was confirmed. Specifically, as in Example 3, BPS-4-one or KCl was treated with LPS pre-treated BMDMs for 1 hour, and after activation of the NLRP3 inflammatory regulatory complex with ATP, nigericin, or silica crystals, the cells were TTNE. Crushed with buffer (1% Triton X-100, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA and complete protease and phosphatase inhibitor cocktail). The cell lysate was centrifuged at 6000 rpm for 15 minutes at 4 ° C., and then supernatant and pellets were used as soluble and insoluble fractions of Triton X-100, respectively. The presence of caspase-1, pro-IL-1β, NLRP3, ASC, pro-caspase-1 and β-actin were confirmed by Western blot analysis, respectively.

As a result, it was found that after activation of the NLRP3 inflammatory regulatory complex, BOT-4-one decreased not only the secretion of IL-1β and caspase-1 from the supernatant, but also the migration of NLRP3 and ASC to the insoluble fraction (pellet). (Figure 4). Through this, it can be seen that BOT-4-one specifically inhibits NLRP3 inflammatory regulatory complex activation by inhibiting migration of NLRP3 and ASC to the Triton X-100 insoluble fraction.

Example 5. Confirmation of the mechanism of inhibition of BOT-4-one's NLRP3 inflammation control complex activity

5-1. Inhibition of ATPase activity of NLRP3

To confirm the mechanism by which BOT-4-one inhibits the activation of NLRP3 inflammatory regulatory complex, the effect of NLRP3 on ATPase activity was confirmed. Specifically, human recombinant NLRP3 (BPS Bioscience, San Diego, CA, USA) was reacted with DMSO (control) or BOT-4-one at 37 ° C in a reaction buffer (20mM Tris HCl, pH 7.8, 133 mM NaCl, 20 mM MgCl ₂ , 3 mM KCl and 0.56 mM EDTA) for 20 minutes. Then, ATP (250 mM, ultra-pure ATP) was added to react at 37 ° C. for 40 minutes. ATP hydrolysis by NLRP3 was confirmed by detecting luminescent ADP in the cell supernatant using the ADP-Glo Kinase Assay (Promega, Madison, WI, USA).

As a result, it was found that BOT-4-one significantly inhibited the NLRP3 ATPase activity in a concentration-dependent manner (FIG. 5A).

5-2. NLRP3 alkylation

BOT-4-one or Bay11-7082 (Bay) (control) for 1 hour in LPS pretreated BMDMs, as in the above examples, to confirm that the inhibition of NLRP3 activity of BOT-4-one is due to NLRP3 alkylation During the treatment, the alkylation inhibitor L-cysteine was treated together. Thereafter, ATP, nigericin, or silica crystals were treated to activate the NLRP3 inflammatory regulatory complex. Thereafter, NLRP3-mediated LDH release from the cell supernatant was confirmed, and IL-1β and caspase-1 were confirmed by Western blot analysis.

As a result, BOT-4-one significantly inhibited NLRP3-mediated LDH release and maturation of IL-1β and caspase-1, and NLRP3-mediated LDH release and IL-1β and caspase-1 maturation during L-cysteine treatment It was confirmed that recovery was again dependent on the concentration of L-cysteine (FIGS. 5B and C). Through this, it was found that BOT-4-one inhibits ATPase activity of NLRP3 by alkylating NLRP3, and thereby inhibits NLRP3 inflammatory regulatory complex activity.

Example 6. ASC oligomerization and migration inhibition through NLRP3 alkylation

The effect of inhibiting ATPase activity of NLRP3 through alkylation of NLRP3 with BOT-4-one was to determine the effect of ASC oligomerization and migration of NLRP3 and ASC to Triton X-100 insoluble fraction. Specifically, while treating BOT-4-one with LPS pre-treated BMDMs for 1 hour, the alkylation inhibitor L-cysteine was treated together, and after activating the NLRP3 inflammation-regulating complex with ATP, nigericin, or silica crystals, Examples The oligomerization of ASC was confirmed by the same method as in 3. In addition, the movement of NLRP3 and ASC to Triton X-100 soluble or insoluble fraction was confirmed in the same manner as in Example 4.

As a result, maturation and ASC oligomerization of NLRP3-mediated IL-1β and caspase-1 inhibited by BOT-4-one treatment were recovered in a concentration-dependent manner by an alkylation inhibitor (FIG. 6A). In addition, BOT-4-one inhibited the migration of NLRP3 and ASC to the insoluble fraction, but was recovered by the alkylation inhibitor (Figure 6B), which appeared dependent on the concentration of L-cysteine (Figure 6C). Through this, it was found that BOT-4-one inhibits ASC oligomerization and migration of NLRP3 and ASC to the Triton X-100 insoluble fraction by alkylation of NLRP3.

Example 7. Confirmation of NLRP3 ubiquitination

To confirm the ubiquitination of NLRP3 by BOT-4-one, BMDMs pretreated with LPS were treated with L-cysteine (Cys) for 15 minutes, and then BOT-4-one was treated for 1 hour. Subsequently, BMDMs were crushed with TINE buffer, and the cell lysate and NLRP3 antibody bound to beads were incubated at 4 overnight. After washing the beads, eluting with an elution buffer (0.1 M Tris-HCl, pH 6.8, 0.4% sodium dodecyl sulfate, 12% glycerol, 0.286 M β-mercaptoethanol and 0.32% bromophenol blue) confirmed ubiquitination of NLRP3. In addition, in order to confirm the interaction between NLRP3 and ASC, BOT-4-one was treated with L-cysteine in BMDMs pre-treated with LPS for 1 hour, and then NLRP3 inflammation control complex with ATP, nigericin, or silica crystals. Activation was induced. Then, the cells were crushed with IP buffer (1% NP-40, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate and complete protease and phosphatase inhibitor cocktail), and ultrasonicated at 20 amp for 5 seconds. Shredded. After washing the cell debris, the ASC antibody (Santa Cruz) was incubated overnight at 4 ° C., and immunoprecipitates were Western-blotted with the NLRP3 antibody to confirm the interaction between NLRP3 and ASC.

As a result, NLRP3 ubiquitination was induced by BOT-4-one treatment, which was shown to be inhibited by an alkylation inhibitor (FIG. 7A). Thus, it can be seen that the alkylation of NLRP3 by BOT-4-one and the inhibition of ATPase activity induce ubiquitination of NLRP3. In addition, BOT-4-one was shown to inhibit the binding of NLRP4 and ASC, which was inhibited by alkylation inhibitors (FIG. 7B). Through this, it can be seen that BOT-4-one induces ubiquitination of NLRP3 through alkylation of NLRP3 and then also inhibits NLRP3 / ASC binding.

Example 8. Effect of BOT-4-one on peritonitis relief

In order to confirm the effect of BOT-4-one on NLRP3 inflammatory regulatory complex-related diseases, peritoneitis was induced in mice with MSU (Monosodiumurate) and its effect was confirmed. Specifically, BOT-4-one (10 mg / kg) was intraperitoneally administered 2 and 12 hours before MSU treatment. MSU (50 mg / kg) or PBS (control) was then intraperitoneally administered to the mice. After 6 hours, the mice were sacrificed, from which peritoneal cells were obtained. Thereafter, the cells were stained with F4 / 80 and Ly-6G antibody (eBioscience), counted by flow cytometry (BD San Diego, CA, USA), and the number of neutrophils was obtained through Equation 1 below. Did. In addition, the concentration of IL-1β in the supernatant of the abdominal cavity was measured by ELISA.

As a result, BOT-4-one significantly reduced the number of MSU-mediated total cells and the number of Ly-6G ⁺ / F480 ⁻ neutrophils (FIGS. 8A and B). In addition, MSU-induced IL-1β production was reduced (Figure 8C). Through this, it was found that BOT-4-one relieves MSU-induced peritonitis by inhibiting NLRP3 inflammatory regulatory complex-mediated IL-1β production.

Claims (12)

To the 2-cyclohexyl-6-methyl-6,7-diamino of formula dihydro -5 H - benzo [1,3] oxa thiol-4-one (2-cyclohexylimino-6-methyl -6,7- dihydro-5 H -benzo [1,3] oxathiol-4-one) a reagent composition for NLRP3 inhibition, including:
[Formula 1]

. The reagent composition for inhibiting NLRP3 activity according to claim 1, which inhibits ATPase activity of NLRP3. The reagent composition for inhibiting NLRP3 activity according to claim 1, wherein NLRP3 is inhibited by ubiquitination. To 2-cyclohexyl-6-a mino-6,7-dihydro -5 H of the formula (1) benzo [1,3] for NLRP3 inflammation control complex (inflammasome) inhibition, including oxa thiol-4-one Reagent composition:
[Formula 1]

. According to claim 4, NLRP3 and inhibiting the binding of ASC (apoptotic speck protein containing a caspase recruitment domain), NLRP3 reagent composition for inhibiting the activity of the inflammatory inflammatory regulatory complex. NLRP3 reagent composition for the alkylation, including benzo [1,3] oxa thiol-4-one - To a 2-cyclohexyl-6-methyl-6,7-diamino of formula dihydro -5 H:
[Formula 1]

. To the 2-cyclohexyl-6-methyl-6,7-diamino of formula dihydro -5 H - comprising the step of treating the benzo [1,3] oxa thiol-4-one, in vitro (in vitro ) How to inhibit the NLRP3 inflammation control complex activity:
[Formula 1]

. To formula (I) 2-cyclohexyl-6-a mino-6,7-dihydro -5 H of benzo [1,3] NLRP3 activity in, in vitro comprising the step of processing the oxa thiol-4-one To suppress:
[Formula 1]

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