Novel compounds and compositions containing them as active ingredients for the prevention, improvement, or treatment of bacterial inflammation and inflammasome-mediated diseases.

Abstract

The present application relates to a compound of Chemical Formula 1, its isomer, or a pharma- ceutically acceptable salt thereof, and its use in the prevention, amelioration, and / or treatment of inflammasome-mediated diseases, and / or diseases caused by or infected with Gram-negative bacteria, which is safe without causing cytotoxicity, can effectively inhibit inflammasomes, and has excellent preventive, ameliorative, and / or therapeutic effects against the above diseases.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority rights under Republic of Korea Patent Application No. 10-2022-0022320 dated February 21, 2022, Republic of Korea Patent Application No. 10-2022-0140639 dated October 27, 2022, Republic of Korea Patent Application No. 10-2022-0146145 dated November 4, 2022, and Republic of Korea Patent Application No. 10-2023-0022804 dated February 21, 2023, and all content disclosed in the literature of said Republic of Korea Patent Applications is incorporated herein by reference. [Background technology]

[0002] Gram-negative bacteria are known to cause periodontal disease, urethral infections, cystitis, pyelonephritis, abdominal infections, and pneumonia, and they have a high pathogenicity that affects the human body, often leading to secondary infections such as hospital-acquired infections and opportunistic infections. If bacteria that infect a local site are not properly controlled in the early stages, the pathogenicity of the bacteria increases, and eventually they can enter the bloodstream and lead to a systemic inflammatory response (sepsis), which can result in organ failure and even death from shock.

[0003] Gram-negative bacteria have an outer membrane that Gram-positive bacteria lack, making drug penetration difficult. Furthermore, the bacterial membrane, developed through quorum sensing in Gram-negative bacteria, requires high concentrations of antibiotics, resulting in a short duration of effectiveness and potentially leading to antibiotic abuse and resistance. However, by using quorum sensing inhibitors to suppress bacterial membrane formation in Gram-negative bacteria and control their pathogenicity, it is possible to fundamentally prevent the progression to related diseases (see Figures 9 and 10).

[0004] In particular, when quorum sensing inhibitors are administered together with antibiotics, they can counteract the bacterial film effect, increasing the effectiveness of the antibiotic, resulting in lower effective concentrations, longer effective periods, and ultimately enhancing the antibiotic effect while minimizing the induction of drug resistance.

[0005] There is a current need to develop new therapeutic agents targeting Gram-negative bacteria (such as Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Acinetobacter baumannii (A. baumannii), Porphyromonas gingivalis (P. gingivalis), etc.) that cause various inflammations such as sepsis of systemic inflammatory response syndrome, periodontitis, osteomyelitis, urinary tract infection, cystitis, abdominal infection, pneumonia, etc. <00000⑧3> On the other hand, the inflammasome inflammatory complex may be activated due to causes such as Gram-negative bacterial infection, etc., to induce an immune response. The over-activated NLRP3 inflammasome is known to cause various inflammasome-mediated diseases such as neurodegenerative diseases, autoinflammatory diseases, cancer, etc. Therefore, there is a need to develop a therapeutic agent that can effectively treat the said diseases by suppressing the NLRP3 inflammasome.

Summary of the Invention

[0007] Problems to be Solved by the Invention One example of the present application provides a compound of novel Chemical Formula 1, its isomers, or pharmaceutically acceptable salts thereof.

[0008]

Chemical Formula

[0009] <000⑩0>Another example provides a composition (pharmaceutical composition, food composition, feed composition, etc.) for the prevention, improvement, and / or treatment of inflammasome-mediated diseases and / or infections of Gram-negative bacteria or diseases induced by Gram-negative bacteria, for neuroprotection, and / or for neuronal regeneration (or generation), containing one or more selected from the group consisting of a compound of Chemical Formula 1, its isomers, and pharmaceutically acceptable salts thereof as an active ingredient.

[0010] Other examples include applications for using one or more compounds selected from the group consisting of compounds of chemical formula 1, their isomers, and pharmaceutically acceptable salts thereof for the prevention, improvement, and / or treatment, neuroprotection, and / or neuronal regeneration (or generation) of inflammasome-mediated diseases and / or diseases caused by or infection with Gram-negative bacteria.

[0011] Other examples include applications for use in the manufacture of compositions (pharmaceutical compositions, food compositions, feed compositions, etc.) for the prevention, improvement, and / or treatment, neuroprotection, and / or regeneration (or generation) of inflammasome-mediated diseases and / or diseases caused by or infected with Gram-negative bacteria, selected from the group consisting of compounds of chemical formula 1, their isomers, and pharmaceutically acceptable salts thereof.

[0012] Other examples include providing a method for preventing, improving and / or treating inflammasome-mediated diseases and / or diseases induced by or caused by Gram-negative bacteria, a method for neuronal protection, and / or a method for neuronal regeneration or generation, comprising the step of administering one or more effective amounts selected from the group consisting of compounds of chemical formula 1, their isomers, and pharmaceutically acceptable salts thereof, to a subject (individual) requiring prevention, improvement, and / or treatment of inflammasome-mediated diseases and / or infection with or caused by Gram-negative bacteria, a method for neuronal protection, and / or a method for neuronal regeneration or generation.

[0013] Another example provides an antibiotic containing a compound of chemical formula 1. The antibiotic may exhibit antiviral activity against Gram-negative bacteria.

[0014] The antibiotics provided herein (first antibiotic), when used in combination with other antibiotics (second antibiotic), have the advantages of increasing bacterial susceptibility to antibiotics, enhancing the bactericidal effect of the second antibiotic due to the bacteriostatic effect of the first antibiotic itself, and enabling excellent antibiotic efficacy even at low concentrations of the second antibiotic. This reduces side effects such as toxicity (e.g., nephrotoxicity, hepatotoxicity) caused by the use of high concentrations of antibiotics. Furthermore, it can suppress the emergence of antibiotic-resistant bacteria through the use of antibiotics with other mechanisms of action.

[0015] Therefore, one example is, (a) compound, and (b) Second antibiotic The present invention provides a combination antibiotic that includes [the specified ingredient].

[0016] Another example is the provision of a feed additive containing an antibiotic or a combination antibiotic as an active ingredient.

[0017] Another example is a disinfectant containing an antibiotic or a combination antibiotic as an active ingredient.

[0018] Another example provides a disinfection method that includes the step of applying an antibiotic or a combination antibiotic to an object requiring disinfection.

[0019] Another example is a cleaning agent containing an antibiotic or a combination antibiotic as an active ingredient.

[0020] Another example provides a cleaning method that includes the step of applying an antibiotic or concomitant antibiotic to an object that needs cleaning.

[0021] Means for solving the problem One example of this application provides a compound of the following chemical formula 1, its isomers, or pharmaceutically acceptable salts thereof.

[0022] [ka] [Brief explanation of the drawing]

[0023] [Figure 1] Figures 1 and 2 show graphs of the degree of biofilm formation (O.D 590nm) of P. gingivalis after treating the culture medium of P. gingivalis with the compounds from Examples 1-1 to 1-3 at different concentrations (2 μM, 0.2 μM, 0.02 μM, and 0.002 μM). "NT" represents the group with no treatment, "DPD" represents the group in which P. gingivalis was cultured after treating the culture medium with the compound DPD, "CT" represents the group in which P. gingivalis was cultured after treating the culture medium with PBS instead of DPD, and DPD+Example 1-1, DPD+Example 1-2, and DPD+Example 1-3 represent the groups in which DPD and the example compounds were treated together. [Figure 2] Same as above [Figure 3A] Figures 3A to 3E show the results of treating the compounds from Examples 1-2 and the comparative examples with P. gingivalis culture media at different concentrations (2 μM, 0.2 μM, 0.02 μM, and 0.002 μM), respectively, and then observing the degree of biofilm formation (O.D 590 nm) of P. gingivalis, as shown in graphs. [Figure 3B] Same as above [Figure 3C] Same as above [Figure 3D] Same as above [Figure 3E] Same as above [Figure 4A] Figures 4A to 4D are graphs showing the stability of the compounds from Examples 1-2 and the comparative examples in human plasma and rat plasma. [Figure 4B] Same as above [Figure 4C] Same as above [Figure 4D] Same as above [Figure 5A]Figures 5A to 5C show graphs illustrating the expression levels of three gingipain proteins ((lysine-gingipain (Kgp), arginine-gingipain A (RgpA), and arginine-gingipain B (RgpB))), which are major pathogenic factors and proteolytic enzymes produced by Porphyromonas gingivalis, after treating the culture medium with the compounds from Examples 1-1 to 1-3, respectively. The 16s rRNA gene was used to normalize the expression levels of the gingipain genes. "Control" is the group with no treatment, "DPD" is the group in which Porphyromonas gingivalis was cultured after treating the culture medium with the compound DPD, and "DPD + Example 1-1" and "DPD + Example 1-2" are groups in which Porphyromonas gingivalis was treated with both DPD and the example compounds. [Figure 5B] Same as above [Figure 5C] Same as above [Figure 6] Figure 6 is a graph showing the presence or absence of cytotoxicity when mouse bone marrow-derived macrophages were treated with the compounds from Examples 1-2 at different concentrations (0.5 μM to 16 μM). "Con" represents the control group, which received no treatment. [Figure 7A] Figures 7A to 7D show that treating Gram-negative bacteria with the compounds in the examples significantly suppressed Escherichia coli biofilm compared to the positive control group (CT) that was not treated with the compounds. (Figures 7A and 7B: Results for Escherichia coli (E. coli), Figure 7C: Results for Pseudomonas aeruginosa (P. aeruginosa), Figure 7D: Results for Acinetobacter baumannii) [Figure 7B] Same as above [Figure 7C] Same as above [Figure 7D] Same as above [Figure 8] Figure 8 shows that when E. coli was treated with the compound from the example and the antibiotic, the minimum growth inhibitory concentration of the antibiotic was significantly reduced compared to when the antibiotic was treated alone. [Figure 9] Figures 9 and 10 show schematic diagrams illustrating how the compounds of the present invention suppress biofilms of Gram-negative bacteria. [Figure 10] Same as above [Figure 11] Figure 11 shows that nerve cells were damaged by inflammation induced by a P. gingivalis infection stimulus, but were protected by treatment with the compounds in the examples. [Figure 12] Figures 12 and 13 are graphs showing that the expression of inflammatory cytokine genes increased in nerve cells due to P. gingivalis infection, but that neuroinflammation was alleviated by treatment with the compounds described in the examples. [Figure 13] Same as above [Figure 14] Figure 14 is a graph showing that the compounds in the examples do not exhibit toxicity to nerve cells. [Figure 15] Figure 15 is a graph showing the plasma concentration over time after intravenous injection of the compound in the example into male rats and collection of blood samples. [Figure 16] Figure 16 is a graph showing the brain concentration over time after intravenous injection of the compound in the example into male rats and collection of brain samples. It demonstrates that the compound in the example travels through the bloodstream, crosses the hematocratic barrier, and reaches the brain, thus acting on brain lesions in the target area to treat neurodegenerative diseases. [Figure 17] Figure 17 is a graph showing that when mouse bone marrow-derived macrophages were treated with the compounds from the examples, changes in the production of inflammatory cytokines (IL-1β) in the macrophages were measured, and the inflammatory response induced in the macrophages was suppressed by the compounds from the examples. [Figure 18] Figure 18 is a graph showing that the compounds in the examples do not exhibit cytotoxicity in mouse bone marrow-derived macrophages. [Figure 19] Figure 19 is a graph showing that the compounds in the examples do not exhibit cytotoxicity in human mononuclear cell lines. [Modes for carrying out the invention]

[0024] Definition of Terms The terms used in this specification are briefly explained below.

[0025] As used herein, the term "pharmaceutically acceptable salt" means a salt form of a compound that does not impair the biological activity and properties of the compound without inducing serious irritation to the organism to which the compound is administered. In the present invention, this can refer collectively to any salt that possesses equivalent biological efficacy and properties of the compound of chemical formula 1 and is preferable from the viewpoint of pharmaceutically, biological, or other properties. A pharmaceutically acceptable salt may be an acid addition salt formed by an acid that forms a nontoxic acid addition salt containing a pharmaceutically acceptable anion, such as an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid; an organic carboxylic acid such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid; or an acid addition salt formed by a sulfonic acid such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid. As a specific example, an acid addition salt of the compound of one embodiment can be obtained by reacting a compound in its free base form with a stoichiometric amount of a suitable acid. In this case, the reaction can proceed in water, an organic solvent, or a mixture thereof, and specifically in a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. In addition, each form of salt can be obtained by a conventional reaction obvious to those skilled in the art, depending on the pharmaceutically acceptable form of the salt. Furthermore, pharmaceutically acceptable salts may include alkali metal salts or alkaline earth metal salts formed by lithium, sodium, potassium, calcium, magnesium, etc.; amino acid salts such as lysine, arginine, and guanidine; or organic salts such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, diethanolamine, choline, and triethylamine.

[0026] As used herein, the term "isomer" means a compound or salt thereof that has the same chemical or molecular formula but is optically or sterically different. Such isomers, salts thereof, and racemic mixtures of isomers are also included in the scope of the present invention.

[0027] As used herein, the term "aryl" means a carbocyclic group having a shared pi-electron system and at least one ring (e.g., a phenyl group). This term includes monocyclic or fusion polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) groups.

[0028] As used herein, the term "heteroaryl" means a heterocyclic aryl group having a shared pi-electron system and at least one ring, and includes, but is not limited to, furan, thiophene, pyrrole, imidazole, oxazole, isoxazole, oxadiazole, tetrazole, thiazole, imidazole, pyrazole, isothiazole, triazole, thiadiazole, pyridine, pyridazine, pyrimidine, pyrazine, and triazine.

[0029] Aryl and / or heteroaryls are C 5-10 The aryl and / or heteroaryl compounds may be substituted or unsubstituted, and may have 5-10 carbon atoms (C5-C10), 5-9 carbon atoms (C5-C9), 5-8 carbon atoms (C5-C8), 5-7 carbon atoms (C5-C7), 5-6 carbon atoms (C5-C6), 6-10 carbon atoms (C6-C10), 6-9 carbon atoms (C6-C9), 6-8 carbon atoms (C6-C8), 6-7 carbon atoms (C6-C7), 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, or 10 carbon atoms.

[0030] As used herein, the term "alkyl" means an aliphatic hydrocarbon group. The alkyl group may be a "saturated alkyl" group that does not contain any alkene or alkyne groups, or an "unsaturated alkyl" group that contains at least one alkene or alkyne group. The "alkene" group means a group consisting of at least one carbon-carbon double bond, and the "alkyne" group means a group consisting of at least one carbon-carbon triple bond. For example, "alkyl" may be a saturated or unsaturated alkyl group having a linear, branched, or cyclic structure. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, hexyl, ethenyl, propenyl, and butenyl. For example, C1-C4-alkyl groups have 1 to 4 carbon atoms in the alkyl chain and are selected from groups consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl.

[0031] In this specification, alkyl groups are defined as C 1-10 The alkyl group may be a linear or branched alkyl group, and the alkoxy group is C 1-10 It may be a linear or branched alkoxy group. 1-10 A linear or branched alkyl group, or C 1-10The linear or branched alkoxy groups can each be independently of the number of carbon atoms: 1-10 (C1-C10), 1-9 (C1-C9), 1-8 (C1-C8), 1-7 (C1-C7), 1-6 (C1-C6), 1-5 (C1-C5), 1-4 (C1-C4), 1-3 (C1-C3), 1-2 (C1-C2), 1 (C1), 2-10 (C2-C10), 2-9 (C2-C9), 2-8 carbons (C2-C8), 2-7 carbons (C2-C7), 2-6 carbons (C2-C6), 2-5 carbons (C2-C5), 2-4 carbons (C2-C4), 2-3 carbons (C2-C3), 2 carbons (C2), carbon Number of 3 to 10 carbons (C3-C10), 3 to 9 carbons (C3-C9), 3 to 8 carbons (C3-C8), 3 to 7 carbons (C3-C7), 3 to 6 carbons (C3-C6), 3 to 5 carbons (C3-C5), 3 to 4 carbons (C3-C4) ), 3 carbons (C3), 4-10 carbons (C4-C10), 4-9 carbons (C4-C9), 4-8 carbons (C4-C8), 4-7 carbons (C4-C7), 4-6 carbons (C4-C6), 4-5 carbons (C4-C5) ), 4 carbons (C4), 5-10 carbons (C5-C10), 5-9 carbons (C5-C9), 5-8 carbons (C5-C8), 5-7 carbons (C5-C7), 5-6 carbons (C5-C6), 5 carbons (C5), 6 carbons It may be up to 10 carbon atoms (C6-C10), 6-9 carbon atoms (C6-C9), 6-8 carbon atoms (C6-C8), 6-7 carbon atoms (C6-C7), 6 carbon atoms (C6), 7-10 carbon atoms (C7-C10), 7-9 carbon atoms (C7-C9), 7-8 carbon atoms (C7-C8), 7 carbon atoms (C7), 8-10 carbon atoms (C8-10), 8-9 carbon atoms (C8-C9), 9 carbon atoms (C9), 9-10 carbon atoms (C9-C10), or 10 carbon atoms (C10).

[0032] As used herein, the term "halo" or "halogen" may refer to a fluoro group (-F), a chloro group (-Cl), a bromo group (-Br), or an iodine group (-I).

[0033] As used herein, the term "heterocycle" refers to a group in which the ring carbon is replaced by oxygen, nitrogen, sulfur, etc., and which may optionally contain a double bond. Examples of heterocycles include, but are not limited to, pyrroline, pyrrolidine, tetrahydrofuran, imidazoline, imidazolidine, pyrazoline, pyrazolidine, pyran, piperidine, piperazine, morpholine, and thiomorpholine.

[0034] "Each hydrogen atom can be substituted independently" means that if there are two or more substituted hydrogen atoms, each hydrogen atom may be substituted with the same or different substituents.

[0035] In this specification, “prevention” means all actions that suppress or delay the onset of a disease by administration of the composition as an example; “treatment” means all actions that result in an improvement or favorable modification of the symptoms of an individual suspected of having the disease or who has developed the disease by administration of the composition as an example; and “improvement” can mean all actions that result in at least a reduction in the degree of symptoms, for example, of parameters related to the state in which the disease is treated by administration of the composition as an example. The disease may be an inflammasome-mediated disease.

[0036] As used herein, the term "pharmaceutical effective amount" means the amount of active ingredient that produces the desired pharmaceutical effect, and may, in some cases, mean the concentration or dosage of the active ingredient in the pharmaceutical composition required to produce the desired pharmaceutical effect.

[0037] Terms other than those mentioned above can be interpreted as having the meanings that are ordinarily understood by those skilled in the art in which this invention pertains.

[0038] Compounds of chemical formula 1 One example of this application provides a compound of the following chemical formula 1, its isomers, or pharmaceutically acceptable salts thereof.

[0039] [ka]

[0040] In Chemical Formula 1, [Chem.] may be a substituted or unsubstituted C 3-7 cycloalkyl, a substituted or unsubstituted C 3-7 cycloalkenyl containing one or more double bonds, a substituted or unsubstituted C 3-7 heterocycloalkyl containing one or more heteroatoms selected from the group consisting of N, O, and S, a substituted or unsubstituted C 3-7 heterocycloalkenyl containing one or more double bonds and one or more heteroatoms selected from the group consisting of N, O, and S, a substituted or unsubstituted C 6-10 aryl, or a substituted or unsubstituted C 5-10 heteroaryl containing one or more heteroatoms selected from the group consisting of N, O, and S. In one specific example, [Chem.] may be a substituted or unsubstituted C 6-10 aryl.

[0041] The substituted cycloalkyl, substituted cycloalkenyl, substituted heterocycloalkyl, substituted heterocycloalkenyl, substituted aryl, or substituted heteroaryl may be substituted with one or more (as used herein, "one or more" includes the case of being substituted with two or more of the same substituents: for example, the case of being substituted with two OHs, etc.) substituents selected from the group consisting of hydroxy (-OH), halogen (-F, -Br, -Cl, or -I), cyano (-CN), nitro (-NO2), amino (-NH), substituted or unsubstituted C 1-10 linear or branched alkyl, and substituted or unsubstituted C 1-10 linear or branched alkoxy.

[0042] Substituted alkyl or substituted alkoxy groups include hydroxy(-OH), halogen, cyano(-CN), nitro(-NO2), amino(-NH), and C. 1-5 Linear or branched alkyl groups, and C 1-5 It may be substituted with one or more substituents selected from the group consisting of linear or branched alkoxy chains.

[0043] R3 is either substituted or unsubstituted C 1-10 Linear or branched alkyl, substituted or unsubstituted C 1-10 Linear or branched alkoxys, substituted or unsubstituted C 3-7 A substituted or unsubstituted C containing one or more heteroatoms selected from the group consisting of cycloalkyl, N, O, and S 3-7 heterocycloalkyl, substituted or unsubstituted C 6-10 A substituted or unsubstituted C containing an aryl or one or more heteroatoms selected from the group consisting of N, O, and S. 5-10 It may also be a heteroaryl. In one specific example, R3 is a substituted or unsubstituted C 6-10 It could also be an arrow.

[0044] Substituted alkyl groups include hydroxy(-OH), halogen, cyano(-CN), nitro(-NO2), amino(-NH), and C. 1-5 Linear or branched alkyl groups, and C 1-5 It is substituted with one or more substituents selected from the group consisting of linear or branched alkoxys, or substituted or unsubstituted C 6-10 A substituted or unsubstituted C containing an aryl or one or more heteroatoms selected from the group consisting of N, O, and S. 5-10 It may be substituted with a heteroaryl compound.

[0045] Substituted alkoxy, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, or substituted heteroaryl are hydroxy(-OH), halogen, cyano(-CN), nitro(-NO2), amino(-NH), C 1-5Linear or branched alkyl and C 1-5 It may be substituted with one or more substituents selected from the group consisting of linear or branched alkoxy chains.

[0046] R1 and R2 are each (or all) hydrogen (H), halogen (-F, -Br, -Cl, or -I), substituted or unsubstituted carbon. 1-10 Linear or branched alkyl, substituted or unsubstituted C 1-10 Linear or branched alkoxys, substituted or unsubstituted C 6-10 A substituted or unsubstituted C containing an aryl or one or more heteroatoms selected from the group consisting of N, O, and S. 5-10 It may also be a heteroaryl. In one specific example, R1 and R2 are each independently substituted or unsubstituted C 6-10 A substituted or unsubstituted C containing an aryl or one or more heteroatoms selected from the group consisting of N, O, and S. 5-10 It may also be a heteroaryl. In one specific example, the substituted C 6-10 aryl or replaced C 5-10 The heteroaryl group may be substituted with one or more substituents selected from the group consisting of methyl, ethyl, methoxy, and ethoxy groups.

[0047] Substituted alkyl groups include hydroxy(-OH), halogen, cyano(-CN), nitro(-NO2), amino(-NH), and C. 1-5 Linear or branched alkyl groups, and C 1-5 It is substituted with one or more substituents selected from the group consisting of linear or branched alkoxys, or substituted or unsubstituted C 6-10 A substituted or unsubstituted C containing an aryl or one or more heteroatoms selected from the group consisting of N, O, and S. 5-10 It may be substituted with a heteroaryl compound.

[0048] Substituted alkoxys, substituted aryls, or substituted heteroaryls include hydroxy(-OH), halogens, cyano(-CN), nitro(-NO2), amino(-NH), and C. 1-5 Linear or branched alkyl and C 1-5 It may be substituted with one or more substituents selected from the group consisting of linear or branched alkoxy chains.

[0049] In one example, the compound of chemical formula 1 may be one or more compounds selected from the group consisting of the following compounds: 1) 3-(diphenylmethylene)-2-methylisoindolin-1-one, 2) 3-(di-p-tolylmethylene)-2-methylisoindolin-1-one, and 3) 3-(bis(4-methoxyphenyl)methylene)-2-methylisoindolin-1-one Other examples of this application provide the compound of the following chemical formula 1A, its isomers, or pharmaceutically acceptable salts thereof.

[0050] [ka]

[0051] In the compound of chemical formula 1A, [ka] R1, R2, and R3 are as previously described. However, the compound of chemical formula 1A does not necessarily have to contain the following compounds: 3-(diphenylmethylene)-2-methylisoindolin-1-one (CAS No. 92172-54-8) In one example, the compound of chemical formula 1A may be one or more compounds selected from the group consisting of the following compounds: 1) 3-(di-p-tolylmethylene)-2-methylisoindolin-1-one, and 2) 3-(bis(4-methoxyphenyl)methylene)-2-methylisoindolin-1-one).

[0052] Isomers of a compound of chemical formula 1 or chemical formula 1A (hereinafter, unless otherwise specified, compounds of chemical formula 1 or chemical formula 1A will be referred to collectively as compounds of chemical formula 1) may be optical isomers, stereoisomers, or mixtures of isomers (racemic mixtures) of the compound of chemical formula 1. In one embodiment of the compound of chemical formula 1, each substituent is understood to be able to attach to the chiral center of a carbon atom. Any chiral carbon atom on the compound of one embodiment can exist in any form of (R)-, (S)-, or (R,S)- coordination, preferably in the respective separated forms of (R)- or (S)- coordination. Furthermore, the compound of one embodiment can exist in any form of any possible isomer or mixture thereof, for example, in any form of pure geometric isomers, partial stereoisomers, optical isomers, racemic mixtures, or mixtures thereof. Additionally, if the compound of one embodiment has a double bond, each substituent attached to the double bond may be in an E or Z sequence.

[0053] Inflammasome-mediated diseases As used herein, the term "inflammasome" refers to a caspase-1-activating complex protein complex that may include: 1) a sensor protein, NLR (nucleotide-binding oligomerization domain and leucine-rich repeat-containing receptor) protein; 2) an adapter protein, ASC (adaptor protein apoptosis-associated spec-like protein containing a caspase-recruitment domain); and 3) an effector protein, a caspase precursor (pro-caspase-1). IL-1β, an inflammatory cytokine, is formed by a multiprotein polymer called the inflammasome and can mediate pyroptosis, a type of inflammatory cell death. When Toll-like receptors (TLRs) are activated by pathogen-associated molecular pattern molecules (PAMPs), which are substances present in pathogens, or damage-associated molecular pattern molecules (DAMPs), which are produced by dead cells, the expression of IL-1β precursor (pro-IL-1β) increases. Subsequently, the inflammasome protein complex degrades the IL-1β precursor while activating caspase-1, thereby producing activated IL-1β.

[0054] Inflammasomes can be selected from, but are not limited to, the group consisting of NLRP3 (NOD-like receptor family, pyrin domain-containing 3) inflammasomes, AIM2 (Absent in melanoma 2) inflammasomes, and NLRC4 (NLR family CARD domain-containing protein 4) inflammasomes. NLRP3, AIM2, or NLRC4 inflammasomes include, but are not limited to, nucleic acids, polynucleotides, oligonucleotides, sense and antisense polynucleotide chains, complementary sequences, peptides, polypeptides, proteins, homologous and / or heterologous molecules, isoforms, precursors, mutants, variants, derivatives, splice variants, alleles, different species, and their active fragments.

[0055] When inflammasomes are excessively activated, inflammasome-mediated diseases may occur. Inflammasome activation may, but is not limited to, be induced by Gram-negative bacterial infection or Gram-negative bacteria. For example, the compounds of this application can effectively regulate the secretion of the inflammatory cytokine IL-1β by suppressing inflammasome activation (e.g., by suppressing the production of the inflammatory cytokine IL-1β), and can exhibit excellent effects in preventing, improving, or treating inflammasome-mediated diseases.

[0056] Inflammasome-mediated diseases can refer to all types of diseases that occur when inflammasomes are abnormally and excessively activated.

[0057] In one example, the inflammasome-mediated disease may be a periodontal disease.

[0058] In one example, an inflammasome-mediated disorder may be a neurodegenerative disorder.

[0059] In one example, an inflammasome-mediated disease may be an inflammatory disease. An inflammatory disease may be an inflammasome-mediated inflammatory disease.

[0060] In one example, the inflammasome-mediated disease may be cancer.

[0061] In one example, an inflammasome-mediated disease may be a disease caused by or infected with Gram-negative bacteria.

[0062] In one example, an inflammasome-mediated disease may be, but is not limited to, one or more selected from the group consisting of periodontal disease, neurodegenerative disease, inflammatory diseases (e.g., inflammasome-mediated inflammatory diseases), cancer, Gram-negative bacterial infection or disease induced by Gram-negative bacteria, metabolic disease, sepsis, septic shock, inflammatory bowel disease, osteoarthritis, hyperimmunoglobulin D syndrome, and cryopyrin-associated periodic syndromes.

[0063] The compounds of this application may exhibit antibiotic effects against Gram-negative bacteria, specifically, inhibitory activity against the pathogenicity of Porphyromonas bacteria, as described below, and may have preventive, ameliorative, and / or therapeutic activity for periodontal disease.

[0064] The activity of inhibiting the pathogenicity of Gram-negative bacteria may be, but is not limited to, inhibiting quorum sensing in Gram-negative bacteria and / or suppressing the expression of pathogenic factors in Gram-negative bacteria (e.g., biofilm of Gram-negative bacteria, or gingipain in the case of Porphyromonas bacteria) (e.g., repression of mRNA transcription or repression of protein translation). Gingipain may be, but is not limited to, one or more selected from the group consisting of lysine-gingipain (Kgp), arginine-gingipain A (RgpA), arginine-gingipain B (RgpB), etc.

[0065] By suppressing the pathogenicity of Gram-negative bacteria, preventive, ameliorative, and / or therapeutic effects on periodontal disease can be achieved. In other words, the prevention, ameliorative, and / or therapeutic effects of periodontal disease may be achieved, but are not limited to, suppressing the pathogenicity of Gram-negative bacteria, for example, by suppressing quorum sensing of Porphyromonas bacteria, and / or by suppressing the expression of pathogenic factors of Porphyromonas bacteria (e.g., gingipain, biofilm, etc.) (e.g., by suppressing mRNA transcription or protein translation).

[0066] Periodontal disease may be one or more diseases selected from periodontitis, gingivitis, etc.

[0067] The compounds of this application are characterized by having activity to suppress the expression of inflammatory proteins (e.g., inflammatory cytokines, etc.), inflammation and death of nerve cells (e.g., neuroinflammation), and / or suppression of nerve cell death and / or inflammation caused by Porphyromonas gingivalis in patients with neurodegenerative diseases.

[0068] As used herein, neurodegenerative diseases may be selected from all diseases that exhibit abnormalities in motor control ability, cognitive function, perceptual function, sensory function, and autonomic nervous system function due to the death, reduction, dysfunction, or loss of nerve cells. For example, neurodegenerative diseases may be one or more selected from the group consisting of dementia (e.g., Alzheimer's disease, AD, vascular dementia, mixed dementia, Lewy body dementia, frontotemporal dementia, etc.), Parkinson's disease, PD, Huntington's disease, amyotrophic lateral sclerosis (Lou Gehrig's disease), tremor, Taranto's disease, multiple sclerosis, motor neuron disease, spinal muscular atrophy, Creutzfeldt-Jakob disease, Pick's disease, prion disease, spinocerebellar degeneration, etc.

[0069] Neurodegenerative diseases may be caused by, but are not limited to, infection with, Gram-negative bacteria, such as Porphyromonas species, or induced by Porphyromonas species. The Porphyromonas species may be one or more selected from the group consisting of Porphyromonas gingivalis, Porphyromonas endodontalis, and others.

[0070] Nerve cells may also be central nervous system cells, such as brain (nerve) cells.

[0071] Inflammatory proteins (e.g., inflammatory cytokines) may include, but are not limited to, IL-6, IL-8, IL-18, TNF-α, IL-1, etc.

[0072] Inflammatory diseases can refer to abnormal inflammatory diseases that occur when inflammasomes are abnormally and excessively activated, and can include neuroinflammatory diseases, autoinflammatory diseases, and autoimmune diseases.

[0073] Neuroinflammatory diseases can refer to diseases caused by damage to nerve tissue due to an inflammatory response, and may include, but are not limited to, one or more diseases selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, multiple sclerosis, amyotrophic lateral sclerosis, inflammatory spinal cord injury, diffuse Lewy body disease, leukoencephalitis, temporal lobe epilepsy, and inflammatory spinal cord injury.

[0074] Autoinflammatory diseases may include, but are not limited to, one or more of the following: Muckle-Wells syndrome (MWS), latent autoimmune diabetes mellitus (LADA) in adults, familial cold autoinflammatory syndrome (FCAS), cryopyrin-associated periodic syndromes (CAPS), neonatal-onset multiorgan inflammatory syndrome (NOMID), chronic infantile neurocutaneous arthritis (CINCA) syndrome, familial Mediterranean fever (FMF), pediatric arthritis (e.g., systemic idiopathic pediatric arthritis (SJIA)), pediatric rheumatoid arthritis (e.g., systemic idiopathic pediatric arthritis), and gout.

[0075] Autoimmune diseases may include, but are not limited to, one or more conditions selected from the group consisting of rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), atopic dermatitis (AD), and psoriasis.

[0076] Furthermore, inflammasome activation is known to be involved in the pathogenesis of many cancers, including colorectal cancer, acute myeloid leukemia, adrenocortical carcinoma, Kaposi's sarcoma, lymphoma, anal cancer, appendiceal cancer, teratomatoid rhabdoid tumor, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain cancer, breast cancer, bronchial tumor, carcinoid tumor, cardiac tumor, cervical cancer, chondroma, chronic lymphocytic leukemia, chronic myeloproliferative neoplasm, colon cancer, colorectal cancer, craniopharyngioma, endometrial cancer, ependymal cell tumor, esophageal cancer, sensory neuroblastoma, Ewing's sarcoma, eye cancer, and egg cancer. One or more cancers selected from the group consisting of ductal cancer, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal matrix tumor, germ cell tumor, hairy cell leukemia, head and neck cancer, heart cancer, liver cancer, hypopharyngeal cancer, pancreatic cancer, kidney cancer, laryngeal cancer, chronic myeloid leukemia, lip cancer, oral cancer, lung cancer, melanoma, Merkel cell carcinoma, mesothelioma, oral cancer, osteosarcoma, ovarian cancer, penile cancer, pharyngeal cancer, prostate cancer, rectal cancer, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, testicular cancer, throat cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, and vulvar cancer may be selected, but are not limited to these.

[0077] The compounds described in this application can effectively prevent, improve, or treat cancer by inhibiting inflammasomes.

[0078] Metabolic diseases are a general term for diseases caused by disorders of metabolism of substances in the body, and may include, but are not limited to, one or more diseases selected from the group consisting of obesity, hyperlipidemia, hypercholesterolemia, arteriosclerosis, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH).

[0079] Antibiotics for Gram-negative bacteria and Gram-negative bacterial infections or diseases induced by Gram-negative bacteria Another example provides an antibiotic containing a compound.

[0080] Other examples include applications for using the compound for antibiotics against Gram-negative bacteria (e.g., inhibiting the growth of Gram-negative bacteria, bacteriostatic effects on Gram-negative bacteria, bactericidal effects on Gram-negative bacteria, and control of Gram-negative bacteria).

[0081] Another example provides a method for killing Gram-negative bacteria (or a method for bacteriostasis, control, or growth inhibition) which includes the step of applying (or administering) an effective amount of a compound to a target requiring antibiotic (or bacteriostasis, bactericidal, or control of Gram-negative bacteria) treatment.

[0082] Another example provides an application for using the compound in the manufacture of antibiotics.

[0083] A bacteriostatic effect can mean that an antibiotic reversibly binds to bacteria and inhibits their growth, but does not kill them. A bactericidal effect can mean that an antibiotic irreversibly binds to bacteria and kills them, going beyond simply inhibiting their growth. The distinction between bacteriostatic and bactericidal effects is not absolute, and depending on the concentration of the antibiotic administered, it can function as either a bacteriostatic or bactericidal agent.

[0084] The compound of chemical formula 1 may have (i) activity to inhibit the virulence of Gram-negative bacteria, (ii) antibiotic effect against Gram-negative bacteria (e.g., bacteriostatic effect and / or bactericidal effect), and / or (iii) preventive, ameliorative, and / or therapeutic activity against infection by Gram-negative bacteria or diseases induced by Gram-negative bacteria.

[0085] The inhibitory activity against the virulence of Gram-negative bacteria, or the antibiotic effect against Gram-negative bacteria, may be, but is not limited to, the suppression of quorum sensing in Gram-negative bacteria and / or the suppression of the expression of virulence factors (e.g., biofilms, extracellular polysaccharides (EPSs), toxins, etc.) in Gram-negative bacteria (e.g., suppression of mRNA transcription or suppression of protein translation).

[0086] The prevention, improvement, and / or treatment of infections or diseases induced by Gram-negative bacteria can be achieved by suppressing the virulence of Gram-negative bacteria or by exerting an antibiotic effect against Gram-negative bacteria. In other words, the prevention, improvement, and / or treatment of infections or diseases induced by Gram-negative bacteria are achieved by, but are not limited to, suppressing the virulence of Gram-negative bacteria or exerting an antibiotic effect against Gram-negative bacteria, for example, by suppressing quorum sensing in Gram-negative bacteria and / or by suppressing the generation and / or development of virulence factors (e.g., biofilms, extracellular polysaccharides (EPSs), toxins, etc.) in Gram-negative bacteria (e.g., repression of mRNA transcription or repression of protein translation).

[0087] The antibiotic may be one that has an antibiotic effect against Gram-negative bacteria. The Gram-negative bacteria may be one or more species (for example, one, two, three, four, or five species) selected from the group consisting of Escherichia, Pseudomonas, Acinetobacter, Enterobacter, Klebsiella, Porphyromonas, Fusobacteria, Tannerella, etc. For example, a bacterium of the genus Escherichia may be Escherichia coli, a bacterium of the genus Pseudomonas may be Pseudomonas aeruginosa, a bacterium of the genus Acinetobacter may be Acinetobacter baumannii, a bacterium of the genus Enterobacter may be Enterobacter aerogenes (also known as Klebsiella aerogenes) or Enterobacter cloacae, a bacterium of the genus Klebsiella may be Klebsiella pneumoniae, and a bacterium of the genus Porphyromonas may be Porphyromonas gingivalis or Porphyromonas endodonthalis It may be endodontalis, the genus Fusobacteria may be Fusobacterium nucleatum, and the genus Tannerella may be Tannerella forsythia, but is not limited to these.

[0088] Gram-negative bacterial infections or diseases induced by Gram-negative bacteria can be selected from all diseases caused by Gram-negative bacterial infections, and may include, but are not limited to, one or more diseases selected from the group consisting of, for example, enteritis, Crohn's disease, ulcerative colitis, bacterial dysentery, urethral infections, skin infections, bacteremia, sepsis, skin infections, bedsores, pneumonia, endocarditis, meningitis, otitis externa, otitis media, keratitis, osteomyelitis, peritonitis, cystic fibrosis, and periodontal diseases (e.g., gingivitis, periodontitis, etc.).

[0089] Gram-negative bacterial infections or diseases induced by Gram-negative bacteria can be selected from, but are not limited to, the group consisting of diseases induced by Escherichia bacteria such as enteritis, Crohn's disease, ulcerative colitis, bacterial dysentery, urethral infections, skin infections, bacteremia, and sepsis; diseases induced by Pseudomonas bacteria such as skin infections, bedsores, pneumonia, bacteremia, sepsis, endocarditis, meningitis, otitis externa, otitis media, keratitis, osteomyelitis, enteritis, peritonitis, and cystic fibrosis; diseases induced by Acinetobacter bacteria such as skin infections, pneumonia, bacteremia, and sepsis; and diseases induced by Porphyromonas, Fusobacterium, or Tannerella bacteria such as gingivitis and periodontitis.

[0090] The antibiotics provided herein can be used in combination with other antibiotics (secondary antibiotics) in addition to the compounds described above.

[0091] Therefore, another embodiment provides a combination antibiotic comprising an antibiotic and a second antibiotic.

[0092] The concomitant antibiotic may also have an antibiotic effect against Gram-negative bacteria. Gram-negative bacteria are as explained earlier.

[0093] The second antibiotic may be one or more commonly used antibiotics, for example, antibiotics that have antiviral activity against Gram-negative bacteria. The second antibiotic may be one or more selected from the group consisting of beta-lactam antibiotics, bacterial cell membrane permeability inhibitors, bacterial ribosome inhibitors, bacterial nucleic acid synthesis inhibitors, and bacterial folic acid synthesis inhibitors.

[0094] In one specific example, the second antibiotic is a beta-lactam antibiotic. One or more bacterial cell membrane permeability inhibitors selected from the group consisting of polymicin, amphotericin B, ketoconazole, fluconazole, and itraconazole. One or more bacterial ribosome inhibitors selected from the group consisting of aminoglycosides, tetracyclines, macrolides, lincosamides, chloramphenicol, etc. One or more bacterial nucleic acid synthesis inhibitors selected from the group consisting of quinolones, fluoroquinolones, rifampicin, and the like, and One or more bacterial folate synthesis inhibitors selected from the group consisting of sulfonamides, trimethoprims, etc. It may be one or more selected from the group consisting of such as, but is not limited to, these.

[0095] Beta-lactam antibiotics include one or more penicillin antibiotics selected from the group consisting of penicillin G, methicillin, oxacillin, flucloxacillin, amoxicillin, ampicillin, ampicillin / sulbactam, piperacillin, piperacillin / tazobactam, ticarcillin, azlocillin, carbenicillin, and amoxicillin / clavulanic acid. One or more cephalosporin antibiotics selected from the group consisting of cefazolin, cephalexin, cephalothin, ceftezol, cefazedone, cefaclor, cefamandole, cefmetazole, cefotiam, cefuroxime, cefotaxime, ceftriaxone, ceftazidime, and cefepime. Monobactam antibiotics such as aztreonam, or The antibiotic may be one or more carbapenem antibiotics selected from the group consisting of imipenem, imipenem / cilastatin, faropenem, meropenem, doripenem, and artapenem, but is not limited to these.

[0096] The second antibiotic can exhibit improved antiviral activity when used in combination with beta-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam, or with cilastatin.

[0097] In one example, the second antibiotic can be used in combination with substances such as clavulanic acid, sulbactam, tazobactam, and cilastatin. For example, it can be used in combination with amoxicillin / clavulanic acid, ampicillin / sulbactam, piperacillin / tazobactam, and imipenem / cilastatin. Acids such as sulbactam, tazobactam, and cilastatin are found in weight-based ratios of 1:10~0.1, 1:9~0.1, 1:8~0.1, 1:7~0.1, 1:6~0.1, 1:5~0.1, 1:4~0.1, 1:3~0.1, 1:2~0.1, 1:10~0.2, 1:9~0.2, 1:8~0.2, 1:7~0.2, 1:6~0.2, 1:5~0.2, and 1:4~0.2. It can be used in a variety of ratios, such as 1:3~0.2, 1:2~0.2, 1:10~0.5, 1:9~0.5, 1:8~0.5, 1:7~0.5, 1:6~0.5, 1:5~0.5, 1:4~0.5, 1:3~0.5, 1:2~0.5, 1:10~0.75, 1:9~0.75, 1:8~0.75, 1:7~0.75, 1:6~0.75, 1:5~0.75, 1:4~0.75, 1:3~0.75, 1:2~0.75, 1:2, and 1:0.5.

[0098] Furthermore, two or more secondary antibiotics can be used in combination. When two or more secondary antibiotics are used in combination, the weight ratios of each secondary antibiotic are as follows: 1:10~0.1, 1:9~0.1, 1:8~0.1, 1:7~0.1, 1:6~0.1, 1:5~0.1, 1:4~0.1, 1:3~0.1, 1:2~0.1, 1:10~0.2, 1:9~0.2, 1:8~0.2, 1:7~0.2, 1:6~0.2, 1:5~0.2, 1:4 It can be used in a variety of ratios, such as ~0.2, 1:3~0.2, 1:2~0.2, 1:10~0.5, 1:9~0.5, 1:8~0.5, 1:7~0.5, 1:6~0.5, 1:5~0.5, 1:4~0.5, 1:3~0.5, 1:2~0.5, 1:10~0.75, 1:9~0.75, 1:8~0.75, 1:7~0.75, 1:6~0.75, 1:5~0.75, 1:4~0.75, 1:3~0.75, 1:2~0.75, 1:2, and 1:0.5.

[0099] Thus, combining an antibiotic (first-line antibiotic) with another antibiotic (second-line antibiotic) offers several advantages: it increases bacterial susceptibility to the antibiotic, enhances the bactericidal effect of the second antibiotic due to the bacteriostatic action of the first antibiotic itself, and allows the second antibiotic to exert excellent antibiotic efficacy even at low concentrations. Alternatively, the first antibiotic's effect of inhibiting quorum sensing and / or biofilm formation and / or weakening the virulence of Gram-negative bacteria allows the second antibiotic, which is effective against Gram-negative bacteria, to exert excellent antibiotic efficacy even at low concentrations when used in combination with the first and second antibiotics. This reduces side effects such as toxicity (e.g., nephrotoxicity, hepatotoxicity) associated with the use of high concentrations of antibiotics. Furthermore, it can suppress the emergence of antibiotic-resistant bacteria through the combined use of antibiotics with other mechanisms of action.

[0100] In this specification, the term "antibiotic" encompasses all forms of formulations that have growth inhibitory activity (e.g., quorum sensing inhibition, biofilm formation inhibition, and / or virulence attenuation) and / or killing activity against Gram-negative bacteria, and is interchangeable with antimicrobial agents, preservatives, bactericides, etc., unless otherwise specified.

[0101] Pharmaceutical composition Other embodiments provide pharmaceutical compositions for the prevention or treatment of inflammasome-mediated diseases and / or diseases caused by or infected with Gram-negative bacteria, comprising a compound, its isomers, or pharmaceutically acceptable salts thereof as an active ingredient.

[0102] Other embodiments provide applications for the prevention, improvement, or treatment of one or more inflammasome-mediated diseases and / or diseases caused by or infected with Gram-negative bacteria, selected from the group consisting of compounds of chemical formula 1, their isomers, and pharmaceutically acceptable salts thereof.

[0103] Other embodiments provide compositions for the prevention, improvement, or treatment of inflammasome-mediated diseases and / or diseases caused by or infected with Gram-negative bacteria, comprising as an active ingredient one or more selected from the group consisting of compounds of chemical formula 1, isomers thereof, and pharmaceutically acceptable salts thereof.

[0104] Another embodiment provides a method for preventing, improving, or treating inflammasome-mediated diseases and / or diseases induced by Gram-negative bacteria, comprising the step of administering one or more effective amounts selected from the group consisting of the compound of chemical formula 1, its isomers, and pharmaceutically acceptable salts thereof, to a subject in need of prevention, improvement, or treatment of inflammasome-mediated diseases and / or infection with Gram-negative bacteria or diseases induced by Gram-negative bacteria. The method may additionally include a step of identifying a subject in need of prevention, improvement, and / or treatment of inflammasome-mediated diseases and / or infection with Gram-negative bacteria or diseases induced by Gram-negative bacteria, prior to the administration step.

[0105] Other embodiments provide applications for use in the manufacture of compositions for the prevention, improvement, or treatment of inflammasome-mediated diseases and / or diseases caused by infection with or induced by Gram-negative bacteria, selected from the group consisting of compounds of chemical formula 1, their isomers, and pharmaceutically acceptable salts thereof.

[0106] Other examples include pharmaceutical compositions for neuroprotection and / or for neuronal regeneration or generation, each containing a compound.

[0107] Other examples include providing a method for neuroprotection and / or neuronal regeneration or generation, comprising the step of administering a compound to a subject requiring protection of nerve cells and / or regeneration (or generation) of nerve cells.

[0108] Other embodiments provide applications for use in the manufacture of pharmaceutical compositions for neuroprotection and / or neuronal regeneration (or generation) of the compound.

[0109] Hereinafter, unless otherwise specified, the compound of chemical formula 1 as an active ingredient in a pharmaceutical composition encompasses all isomers of the compound and pharmaceutically acceptable salts of the compound or its isomers, and all of these compounds, isomers, and salts should be interpreted as being included within the scope of this application. However, for convenience, unless otherwise mentioned herein, one or more compounds selected from the group consisting of the compound of chemical formula 1 as an active ingredient in a pharmaceutical composition, its isomers, and pharmaceutically acceptable salts thereof will be simply referred to as "the compound of chemical formula 1."

[0110] Inflammasome-mediated diseases are as explained earlier.

[0111] The target of the pharmaceutical composition may be one or more mammals selected from humans, primates such as monkeys, rodents such as mice and rats, livestock such as dogs, cats, pigs, cattle, horses, sheep, and goats, and poultry such as chickens, ducks, geese, pheasants, quail, and turkeys, or cells, tissues, or cultures thereof derived from these. For example, the target of administration may be a person who needs prevention, improvement, and / or treatment of inflammasome-mediated diseases and / or infection with or diseases induced by Gram-negative bacteria, and may be, for example, a human.

[0112] In one example, the target of administration can be selected from among humans with neurodegenerative diseases, primates such as monkeys, mammals including rats and mice, or cells (brain cells) or tissues (brain tissue) isolated from mammals, or cultures thereof. For example, it can be selected from among humans with neurodegenerative diseases, or brain cells, brain tissue, or cultures thereof isolated from them.

[0113] A pharmaceutical composition containing one or more compounds selected from the group consisting of compounds of chemical formula 1, their isomers, and pharmaceutically acceptable salts thereof as an active ingredient can be formulated and used in the form of a conventional pharmaceutical preparation. For example, a pharmaceutical preparation may be manufactured in various formulations for oral or parenteral administration, and the form of the preparation can be determined in various ways depending on the method of use, method of administration, purpose of administration, etc. When manufactured in various formulations for oral or parenteral administration, the preparation can be formulated using one or more substances selected from the group consisting of commonly used fillers, bulking agents, binders, wetting agents, disintegrants, diluents such as surfactants, excipients, etc.

[0114] When a pharmaceutical composition is administered orally, proteins or peptides are digested; therefore, oral compositions must be formulated in a way that coats the active agent or protects it from digestion in the stomach. Solid formulations for oral administration may include tablets, pills, powders, granules, capsules, etc., and such solid formulations can be prepared by mixing the active ingredient with at least one excipient, such as one or more selected from the group consisting of starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stylate talc may also be used. Liquid formulations for oral administration may include suspensions, oral solutions, emulsions, syrups, etc. When formulating into liquid formulations, commonly used simple diluents such as water and / or liquid paraffin may be used, and optionally, one or more other diverse excipients, such as humectants, sweeteners, fragrances, and preservatives, may be additionally included. Furthermore, the composition may be administered by any device capable of delivering the active substance to target cells.

[0115] When a pharmaceutical composition is administered parenterally, the parenteral administration may be carried out by any convenient route, such as intravenous administration (e.g., arterial administration, intravenous administration), injection or insertion into the patient's lesion site (e.g., gums), intramuscular administration, subcutaneous administration, intraperitoneal administration, intranasal administration, transdermal administration, endothelial administration, local administration, intrapulmonary administration, rectal administration, intravenous infusion, subcutaneous infusion, intramuscular infusion, or intraperitoneal infusion. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents for the manufacture of non-aqueous solutions, or suspension solvents for the manufacture of suspensions, can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suitable bases for suppositories include witepsol, macrogol, tween 61, cocoa butter, lauric butter, and glyceroloselatin. When administered intranasally, the pharmaceutical composition can be diluted and administered by nasal spray using a sprayer or spray system to allow absorption into the nasal cavity. For nasal spraying, aerosol formulations or other respiratory dosage forms can be used.

[0116] The amount of active ingredients contained in the pharmaceutical composition is, on a weight basis of the total pharmaceutical composition, 0.01% to 99.9% by weight, 0.01% to 90% by weight, 0.01% to 80% by weight, 0.01% to 70% by weight, 0.01% to 60% by weight, 0.01% to 50% by weight, 0.01% to 40% by weight, 0.01% to 30% by weight, 1% to 99.9% by weight, 1% to 90% by weight, 1% to 80% by weight, 1% to 70% by weight, 1% to 60% by weight, 1% to 50% by weight, 1% to 40% by weight, and 1% to 30% by weight. The amount may be 5% to 99.9% by weight, 5% to 90% by weight, 5% to 80% by weight, 5% to 70% by weight, 5% to 60% by weight, 5% to 50% by weight, 5% to 40% by weight, 5% to 30% by weight, 10% to 99.9% by weight, 10% to 90% by weight, 10% to 80% by weight, 10% to 70% by weight, 10% to 60% by weight, 10% to 50% by weight, 10% to 40% by weight, or 10% to 30% by weight, but is not limited thereto and can be appropriately adjusted depending on the form of the formulation, method of administration, purpose of administration, etc. In addition, the pharmaceutical composition may contain a pharmaceutically acceptable carrier in addition to the active ingredient. A pharmaceutically acceptable carrier can mean a carrier that is commonly used in the formulation of drugs including proteins, nucleic acids, or cells, and does not irritate living organisms or inhibit the biological activity and / or properties of the active ingredient. In one example, the carrier may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc., but is not limited thereto. The pharmaceutical composition may additionally contain one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., which are commonly used in the manufacture of pharmaceutical compositions.

[0117] The pharmaceutically effective amount of a pharmaceutical composition can be formulated in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, medical condition, food intake, administration time, administration interval, administration route, excretion rate, and response sensitivity. It can be administered or taken once to several times a day, but is not limited to this, and can be administered in a variety of dosages and methods. In one example, the single dose of the pharmaceutical composition is 0.0001-5000 mg / kg, 0.0001-4000 mg / kg, 0.0001-3000 mg / kg, 0.0001-2500 mg / kg, 0.0001-2000 mg / kg, 0.0001-1500 mg / kg, 0.0001-1000 mg / kg, 0.0001-500 mg / kg, 0.0001-100 mg / kg, 0.0001-50 mg / kg, 0.0001-10 mg / kg, 0.0001-5 mg / kg, 0.0001~1mg / kg, 0.001~5000mg / kg, 0.001~4000mg / kg, 0.001~3000mg / kg, 0.001~2500mg / kg, 0.001~2000mg / kg, 0.001~1500mg / kg, 0.001~1000mg / kg, 0.001~500mg / kg, 0.001~10mg / kg, 0.001~5mg / kg, 0.001~1mg / kg 0.01~5000mg / kg, 0.01~4000mg / kg, 0.01~3000mg / kg, 0.01~2500mg / kg, 0.01~2000mg / kg, 0.01~1500mg / kg, 0.01~1000mg / kg, 0 .01~500mg / kg, 0.01~100mg / kg, 0.01~50mg / kg, 0.01~10mg / kg, 0.01~5mg / kg, 0.01~1mg / kg, 0.1~5000mg / kg, 0.1~4000mg / kg, 0 .1~3000mg / kg, 0.1~2500mg / kg, 0.1~2000mg / kg, 0.1~1500mg / kg, 0.1~1000mg / kg, 0.1~500mg / kg, 0.1~100mg / kg, 0.1~50mg / kg , 0.1~10mg / kg, 0.1~5mg / kg, 0.1~1mg / kg, 1~5000mg / kg, 1~4000mg / kg, 1~3000mg / kg, 1~2500mg / kg, 1~2000mg / kg, 1~1500mg / kg,1~1000mg / kg、1~500mg / kg、1~100mg / kg、1~50mg / kg、1~10mg / kg、1~5mg / kg、5~5000mg / kg、5~4000mg / kg、5~3000mg / kg、5~2500mg / kg、5~2000mg / kg、5~1500mg / kg、5~1000mg / kg、5~500mg / kg、5~100mg / kg、5~50mg / kg、5~10mg / kg、10~5000mg / kg、10~4000mg / kg、10~3000mg / kg、10~2500mg / kg、10~2000mg / kg、10~1500mg / kg、10~1000mg / kg、10~500mg / kg、10~100mg / kg、10~50mg / kg、20~5000mg / kg、20~4000mg / kg、20~3000mg / kg、20~2500mg / kg、20~2000mg / kg、20~1500mg / kg、20~1000mg / kg、20~500mg / kg、20~100mg / kg、20~50mg / kg、50~5000mg / kg、50~4000mg / kg、50~3000mg / kg、50~2500mg / kg、50~2000mg / kg、50~1500mg / kg、50~1000mg / kg、50~500mg / kg、50~100mg / kg、100~5000mg / kg、100~4000mg / kg、100~3000mg / kg、100~2500mg / kg、100~2000mg / kg、100~1500mg / kg、100~1000mg / kg、100~500mg / kg、200~5000mg / kg、200~4000mg / kg、200~3000mg / kg、200~2500mg / kg、200~2000mg / kg、200~1500mg / kg、200~1000mg / kg、200~500mg / kg、500~5000mg / kg、500~4000mg / kg、500~3000mg / kg、500~2500mg / kg、500~2000mg / kg、500~1500mg / kg、500~1000mg / kg、1500~5000mg / kg、1500~4000mg / kg、1500~3000mg / kg、1500~2500mg / kg、1500~2000mg / kg、2000~5000mg / kg、2000~4000mg / kg、2000~3000mg / kg、Alternatively, it may be 2000-2500 mg / kg, but is not limited to this range.

[0118] Food composition Other examples include food compositions containing compounds for the prevention and / or improvement of inflammasome-mediated diseases and / or diseases induced by or infection with Gram-negative bacteria, for neuroprotection, and / or neuronal regeneration (or generation). The food compositions may also be functional foods.

[0119] Inflammasome-mediated diseases, and / or diseases caused by or infected with Gram-negative bacteria, are as previously described.

[0120] An example of a food composition can include meats, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, energy drinks, alcoholic beverages, vitamin complexes, health functional foods, and health foods, encompassing all foods in the ordinary sense.

[0121] Functional foods are the same term as foods for special health use (FoSHU), and refer to foods with high medical and therapeutic effects that are processed to efficiently exhibit biological regulatory functions in addition to nutritional supply. Here, "function" means obtaining effects useful for health purposes, such as regulating nutrients or physiological effects on the structure and function of the human body. The food described in this application can be manufactured by methods commonly used in the industry, and can be manufactured by adding raw materials and components that are commonly added in the industry. Furthermore, there are no restrictions on the dosage form of the food, as long as it is a dosage form recognized as a food. The food composition described in this application can be manufactured in a variety of dosage forms, and unlike general drugs, it has the advantage of not having side effects that can occur when drugs are taken long-term, as it is made from food as a raw material, and is highly portable. The food described in this application can be taken as an adjunct to enhance the effect of preventing or improving inflammasome-mediated inflammatory diseases.

[0122] Health foods refer to foods that have a more active effect on maintaining or improving health compared to general foods, while health supplement foods refer to foods intended to supplement health. In some cases, the terms health functional foods, health foods, and health supplement foods can be used interchangeably.

[0123] Specifically, health functional foods are foods that are manufactured by adding a composition (as exemplified) to food ingredients such as beverages, teas, spices, gums, and confectionery, or by encapsulating, powdering, or suspending them. When consumed, they are said to have a specific health effect, but unlike general medicines, they have the advantage of not having the side effects that can occur with long-term use of medicines, as they are made from food ingredients.

[0124] Because the food composition described in this example can be consumed on a daily basis, it is expected to have excellent preventive or ameliorative effects on inflammasome-mediated inflammatory diseases, making it extremely useful.

[0125] Food compositions may additionally contain physiologically acceptable carriers, but the type of carrier is not particularly limited, and any carrier commonly used in the art may be used. Furthermore, food compositions may contain additional components commonly used in food compositions to improve odor, taste, appearance, etc. For example, they may contain vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, and pantothenic acid. They may also contain minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr). In addition, they may contain amino acids such as lysine, tryptophan, cysteine, and valine.

[0126] Furthermore, food compositions may contain food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder and high-grade bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), colorants (tar dyes, etc.), color fixatives (sodium nitrite, sodium acetate, etc.), bleaching agents (sodium sulfite), seasonings (MSG, monosodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, potassium bitartrate, etc.), fortifiers, emulsifiers, thickeners, coating agents, gum bases, foam inhibitors, solvents, and improvers. Additives are selected according to the type of food and can be used in appropriate amounts.

[0127] The example composition can be added as is or used in combination with other foods or food ingredients, and can be used appropriately by conventional methods. The amount of active ingredient mixed can be suitably determined depending on the purpose of use (prevention, health, or therapeutic treatment). In general, when manufacturing food or beverages, the food composition of this application can be added to the food or beverage in an amount of 50 parts by weight or less, specifically 20 parts by weight or less. However, for long-term intake for health and hygiene purposes, the active ingredient can be used in amounts below this range, as there are no safety concerns.

[0128] One example of a food composition is a health beverage composition, in which case it can contain various flavorings or natural carbohydrates as additional ingredients, as in a regular beverage. The aforementioned natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; or sugar alcohols such as xylitol, sorbitol, and erythritol. Sweeteners that can be used include natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame.

[0129] In addition, the health beverage composition may contain a variety of nutrients, vitamins, electrolytes, flavorings, colorings, pectin, pectin salts, alginic acid, alginic acid salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonation agents. Furthermore, it may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, or vegetable beverages. Such components can be used individually or in combination. While the proportion of such additives is not particularly important, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of this application.

[0130] A food composition, as an example, can be included in various weight percentages if it can demonstrate an effect in preventing or improving inflammasome-mediated inflammatory diseases.

[0131] Feed compositions, etc. Other examples include providing feed additives (feed additive compositions) containing compounds, antibiotics, or concomitant antibiotics as active ingredients for the prevention and / or improvement of inflammasome-mediated diseases and / or diseases caused by or infection with Gram-negative bacteria, for neuroprotection, and / or neuronal regeneration (or generation).

[0132] Inflammasome-mediated diseases, and / or diseases caused by or infected with Gram-negative bacteria, are as previously described.

[0133] Another example is providing feed containing feed additives.

[0134] Feed can be manufactured by separately preparing antibiotics or concomitant antibiotics as feed additives and mixing them into the feed, or by directly adding them during the feed manufacturing process.

[0135] The compounds, antibiotics, or concomitant antibiotics in the feed may be in liquid or dry form, for example, in dry powder form. The antibiotics may be present in amounts of 0.005–10% by weight, 0.05–10% by weight, 0.1–10% by weight, 0.005–5% by weight, 0.05–5% by weight, 0.1–5% by weight, 0.005–2% by weight, 0.05–2% by weight, or 0.1–2% by weight of the total weight of the feed, but are not limited to these amounts. In addition to the compounds, antibiotics, or concomitant antibiotics, the feed may also contain common additives that improve the shelf life of the feed.

[0136] In this specification, feeds to which compounds, antibiotics, or concomitant antibiotics may be added may be selected from, but are not limited to, commercially available feeds, or from the group consisting of grains, root fruits, food processing by-products, algae, fiber, pharmaceutical by-products, milk fats, starches, melons, grain by-products, proteins, inorganic substances, oils and fats, mineral substances, single-cell proteins, zooplankton, and leftover food and beverages.

[0137] Other examples include providing food additives or drinking water additives containing compounds, antibiotics, or combination antibiotics as active ingredients. By mixing the compounds, antibiotics, or combination antibiotics with drinking water and supplying it, the number of Gram-negative bacteria in the drinking water can be reduced. Gram-negative bacteria are as previously described.

[0138] Other examples include providing disinfectants containing compounds, antibiotics, or combination antibiotics as active ingredients. Other examples include providing disinfection methods that include the step of applying a compound, antibiotic, or combination antibiotic to an object requiring disinfection. Disinfectants are a general term for formulations for preventing pathogenic bacterial infections and can be used as general household disinfectants, disinfectants for food and cooking areas and equipment, disinfectants for buildings such as poultry farms and livestock barns, livestock bodies, drinking water, straw, egg sacks, transport vehicles, tableware, and various other breeding supplies.

[0139] Other examples include providing a cleaning agent containing a compound, antibiotic, or combination antibiotic as an active ingredient. Other examples include providing a cleaning method comprising the step of applying a compound, antibiotic, or combination antibiotic to an object requiring cleaning. Since the antibiotic has an antibiotic effect against Gram-negative bacteria, it is applicable to cleaning the skin surface or body parts of individuals that are exposed to or may be exposed to Gram-negative bacteria. Gram-negative bacteria are as previously described.

[0140] Manufacturing method In another example, a method for producing a compound of chemical formula 1 is provided.

[0141] In one example, the manufacturing method may include a step of introducing phenylboronic acid or a derivative thereof (for example, phenylboronic acid (Cas No. 98-80-6), 4-methylphenylboronic acid (or p-Tolylboronic acid, Cas No. 5720-05-8), or 4-methoxyphenylboronic acid ((4-methoxyphenyl)boronic acid, Cas No. 5720-07-0)) into the compound of chemical formula 4 to produce the compound of chemical formula 1.

[0142] [ka]

[0143] In the compound of chemical formula 1, [ka] When R3 is an unsubstituted C6 aryl group and R3 is a C1 alkyl (methyl group), the step of producing the compound of chemical formula 1 may be carried out by reacting the compound of chemical formula 4 with phenylboronic acid (Cas No. 98-80-6), 4-methylphenylboronic acid (or p-Tolylboronic acid, Cas No. 5720-05-8), or 4-methoxyphenylboronic acid ((4-methoxyphenyl)boronic acid, Cas No. 5720-07-0).

[0144] Anyone with ordinary skill in the art to which this invention pertains can produce compounds based on the structure of chemical formula 1 by various methods, and all such methods should be interpreted as falling within the scope of the invention. In other words, compounds of chemical formula 1 can be produced within the scope of this invention by arbitrarily combining various synthesis methods described herein or listed in the prior art. Therefore, the production methods according to the present invention are not limited to those presented below.

[0145] Effects of the invention This application relates to the compound of chemical formula 1, its isomers, or pharmaceutically acceptable salts thereof, and their use in preventing, improving, and / or treating inflammasome-mediated diseases and / or infections with or diseases induced by Gram-negative bacteria, wherein they are safe without exhibiting cytotoxicity, can effectively suppress inflammasomes, and have excellent preventive, improving, and / or therapeutic effects against diseases. [Examples]

[0146] The present invention will be described more specifically below with reference to the following examples. However, these are merely illustrative examples of the present invention, and the scope of the present invention is not limited by these examples.

[0147] Example 1. Preparation of the compound Example 1-1. Preparation of 3-(diphenylmethylene)-2-methylisoindorin-1-one The compound of Example 1-1 (3-(diphenylmethylene)-2-methylisoindolin-1-one (CAS No. 92172-54-8)) was prepared by the following process.

[0148] Step 1: Manufacturing of Compound A [ka]

[0149] 10 mL of THF (Cas No. 109-99-9, sigma aldrich) containing Zn dust (Cas No. 7440-66-6, sigma aldrich) (490 mg, 7.50 mmol) was cooled to 0°C under Ar (Cas No. 7440-37-1, Songguan special gas) with triisopropyl phosphite (P(i-PrO)3, Triisopropylphosphite, Cas No. 116-17-6, sigma aldrich) (2.7 ml, 11.00 mmol). A solution of CBr4 (Cas No. 558-13-4, TCI) (2.49 g, 7.50 mmol) in the THF was slowly added to the P(i-PrO)3 ​​solution, and the reaction mixture was stirred until the color changed to yellow. Next, a solution of N-methylphthalimide anhydride (Cas No. 550-44-7, sigma aldrich) (806 mg, 5.00 mmol) in THF was added. The reaction mixture was stirred at room temperature for 12 hours. After quenching with the addition of sat.aq NaHCO3 solution (Cas No. 144-55-8, Samchun Chemicals) (2.5 mL), the phases were separated, and the crude product was extracted from the aqueous layer with ether. The residue was dissolved in ether and filtered through a Celite pad. The resulting filtrate was concentrated by rotary evaporation and purified by column chromatography (50% DCM (dichloromethane) in hexane) to obtain compound A.

[0150] Step 2: Preparation of the compound of Example 1-1 Under argon air, compound A (158 mg, 0.5 mmol) prepared in step 1, Na2CO3 (212 mg, 2 mmol), Pd(OAC)2 (Cas No. 3375-31-3, TCI) (22 mg, 0.1 mmol), PPh3 (Cas No. 603-35-0, TCI) (52 mg, 0.2 mmol), H2O (2 ml), and THF (8 ml) were added to a Schlenk tube. Phenylboronic acid (Cas No. 98-80-6, TCI) (1.2 mmol) was added to the stirred mixture, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was extracted with DCM (Cas No. 75-09-2, sigma aldrich) (10 mL), washed with brine, dried over MgSO4 (Cas No. 10034-99-8, Samchun Chemical), and filtered. The crude product was purified by flash chromatography on silica gel to obtain the compound of Example 1-1.

[0151] [ka]

[0152] Examples 1-2. Preparation of 3-(di-p-tolylmethylene)-2-methylisoindorin-1-one The compounds of Example 1-2 (3-(di-p-tolylmethylene)-2-methylisoindolin-1-one) were prepared by the following steps.

[0153] Step 1: Manufacturing of Compound A Compound A was prepared in the same manner as in step 1 of Example 1-1.

[0154] Step 2: Preparation of the compounds of Examples 1-2 Under argon air, compound A (158 mg, 0.5 mmol) prepared in step 1, Na2CO3 (212 mg, 2 mmol), Pd(OAC)2 (22 mg, 0.1 mmol), PPh3 (52 mg, 0.2 mmol), H2O (2 ml), and THF (8 ml) were added to a Schlenk tube. 4-Methylphenylboronic acid (or p-Tolylboronic acid, Cas No. 5720-05-8, TCI) (1.2 mmol) was added to the stirred mixture, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was extracted with DCM (10 mL), washed with brine, dried over MgSO4, and filtered. The crude product was purified by flash chromatography on silica gel to obtain the compounds of Examples 1-2.

[0155] [ka]

[0156] Examples 1-3. Preparation of 3-(bis(4-methoxyphenyl)methylene)-2-methylisoindoline-1-one The compounds of Examples 1-3 (3-(bis(4-methoxyphenyl)methylene)-2-methylisoindolin-1-one) were prepared by the following steps.

[0157] Step 1: Manufacturing of Compound A Compound A was prepared in the same manner as in step 1 of Example 1-1.

[0158] Step 2: Preparation of the compounds of Examples 1-3 Under argon air, compound A (158 mg, 0.5 mmol) prepared in step 1, Na2CO3 (212 mg, 2 mmol), Pd(OAC)2 (22 mg, 0.1 mmol), PPh3 (52 mg, 0.2 mmol), H2O (2 ml), and THF (8 ml) were added to a Schlenk tube. 4-methoxyphenylboronic acid ((4-methoxyphenyl)boronic acid, Cas No. 5720-07-0, TCI) (1.2 mmol) was added to the stirred mixture, and the mixture was stirred at room temperature for 24 hours. The reaction mixture was extracted with DCM (10 mL), washed with brine, dried over MgSO4, and filtered. The crude product was purified by flash chromatography on silica gel to obtain the compounds of Examples 1-3.

[0159] [ka]

[0160] Manufacturing of the comparative compound DPD The compound DPD (4,5-dihydroxy-2,3-pentanedione, CAS NO. 142937-55-1) used in the experimental example below was prepared by following steps 1 to 5 below (see Molecules. 2018 Oct, 23(10):2545).

[0161] Step 1: 1.3 eq of 1-propynyl magnesium bromide (0.5 M in THF) was slowly added at 0°C to a THF solution of anhydrous (t-butyldimethylsililioxy)acetaldehyde (1.0 eq). The mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated by rotary evaporation, and then quenched by adding a cooled saturated NH4Cl solution. Next, the reaction mixture was extracted with Et2O, washed with brine, dried over MgSO4, and filtered. A yellow oily substance 1 was obtained.

[0162] Step 2: The obtained substance 1 (1.0 eq) was dissolved in MeOH, Dowex 50WX8 100-200 mesh (100 mg / 1 mL) was added, and the mixture was stirred at room temperature for 12 hours. The product was filtered to obtain substance 2.

[0163] Step 3: The obtained substance 2 (1.0 eq) was oxidized with cyclohexanone dimethyl ketal (3.0 eq) and catalyst p-TSA, and stirred at room temperature for 12 hours. The reaction solution was concentrated by rotary evaporation, then dissolved again in Et2O, washed with NaHCO3, dried over MgSO4, and filtered to obtain substance 3.

[0164] Step 4: The obtained substance 3 was placed in a 1:1:1 CHCl3 / ACN / H2O solution, and NaIO4 (4.4 eq) and RuO2_H2O (2.5% mol) were added. The mixture was stirred at room temperature for 12 hours. The reaction solution was concentrated by rotary evaporation, extracted with DCM, and dried over MgSO4 to obtain substance 4.

[0165] Step 5: The obtained substance 4 was placed in MeOH, Dowex 50WX8 resin was added, and the mixture was stirred at room temperature for 12 hours. After filtration, compound DPD was obtained.

[0166] Example 2. Inhibitory effect of P. gingivalis on biofilm formation. The inhibitory effect of the compounds in Examples 1-1 to 1-3 on biofilm formation by Porphyromonas gingivalis was confirmed.

[0167] The compounds from Examples 1-1 to 1-3 were used to treat culture media containing P. gingivalis (obtained from the Korea Oral Microbial Resource Center (KCOM)), and the degree of biofilm formation by P. gingivalis was confirmed.

[0168] Sterile coverslips (round, 12 mm radius) were placed one at a time at the bottom of each well of a cell culture plate (24-well plate) using tweezers. DPD (4,5-dihydroxy-2,3-pentadione, CAS NO. 142937-55-1), a precursor of the interbacterial signaling molecule AI-2 (Autoinducer-2), was added to the culture medium for P. gingivalis (BHI medium + supplement) to a concentration of 10 μM (BHI, a component of the medium, is Brain Heart Infusion Medium, and hemin (10 μg / ml) and vitamin K (0.2 μg / ml) were used as supplements). It is known that bacterial biofilm formation increases when bacteria use DPD for quorum sensing. The compounds from the examples (compounds from Examples 1-1 to 1-3) were each diluted in a culture medium containing DPD to prepare final concentrations of 0.002 μM, 0.02 μM, 0.2 μM, and 2 μM, respectively.

[0169] The prepared culture medium is dispensed into cell culture plates lined with glass slips, and bacteria are placed in 2 × 10⁶ cells. 8 Cells were inoculated to a concentration of cells / ml. For the positive control group (DPD), which was not treated with the compound in the example, DPD was added to the culture medium (BHI medium + supplement) before inoculating the same number of bacteria. For the negative control group (CT), which was not treated with either the compound or DPD in the example, the same amount of PBS (phosphate-buffered saline) was added to the culture medium (BHI medium + supplement) instead of DPD. The completed cell culture plates were maintained at 37°C under anaerobic conditions (10% H2, 10% CO2, and 80% N2) for 48 hours to culture the bacteria.

[0170] Biofilms formed on glass slips by bacteria cultured separately were stained with 1% crystal violet solution for 15 minutes, washed three times with PBS (phosphate-buffered saline) solution, and then destained with acetone-alcohol (20:80, vol / vol). 200 μl of the destained solution containing crystal violet was placed into each microplate, and the absorbance (OD) was measured using an absorbance microplate reader (Absorbance Microplatereader, Epoch2, Bio-Tek, USA). 590nm The following measurements were taken and compared and analyzed for each group. The results are shown in Figures 1 and 2.

[0171] As shown in Figures 1 and 2, no biofilm formation was observed in the culture medium without culturing Porphyromonas gingivalis (NT). Compared to the culture medium in which Porphyromonas gingivalis was cultured with PBS (CT), the culture medium in which Porphyromonas gingivalis was cultured with DPD (DPD) showed a significant increase in Porphyromonas gingivalis biofilm formation due to quorum sensing. In the culture media in which Porphyromonas gingivalis was cultured with DPD and the compound of the example together (DPD + Example 1-1, DPD + Example 1-2, DPD + Example 1-3), it was confirmed that the Porphyromonas gingivalis biofilm increased by DPD was suppressed in a concentration-dependent manner by the compound of the example.

[0172] Comparative experiment To demonstrate that the compounds in Examples 1-1 to 1-3 have superior effects compared to the comparative example compounds, the following experiment was conducted. The comparative example compound used was the compound ((Z)-3-(bromo(phenyl)methylene)isobenzofuran-1(3H)-one) from Example 1 of the Korean Patent Publication (KR10-2019-0130983A).

[0173] 1. Inhibitory effect of P. gingivalis on biofilm formation The inhibitory effect of the compounds from Examples 1-2 and the comparative examples on biofilm formation of Porphyromonas gingivalis was confirmed. The experimental procedure was carried out in the same manner as in Experimental Example 1, and the experimental results are shown in Figures 3A to 3E.

[0174] As can be seen from Figures 3A to 3E, no biofilm formation was observed in the culture medium without culturing Porphyromonas gingivalis (NT). When compared with the culture medium in which Porphyromonas gingivalis was cultured with PBS (CT), biofilm formation of Porphyromonas gingivalis was significantly increased in the culture medium in which Porphyromonas gingivalis was cultured with DPD (DPD) due to quorum sensing. In the culture medium in which Porphyromonas gingivalis was cultured with DPD and the compound together (DPD + Comparative Example, DPD + Examples 1-2), it was confirmed that the biofilm of Porphyromonas gingivalis increased by DPD was suppressed in a concentration-dependent manner by the compound. In a comparison of the biofilm formation inhibitory ability of Porphyromonas gingivalis at the same concentration, it was confirmed that the biofilm formation inhibitory ability of the compound in Examples 1-2 was better than that of the compound in the Comparative Example.

[0175] 2.Plasma stability The plasma stability of the compounds from Examples 1-2 and the comparative examples was compared in human and rat plasma.

[0176] 1) Sample preparation The compounds from Examples 1-2 and the comparative examples, along with the substances used as positive controls (eucatropin and loterpresnol), were dissolved in DMSO to prepare 10 mM stock solutions. These stock solutions were then diluted to produce 0.2 mM and 1 mM working solutions, which were used in the tests (70% acetonitrile was used as the solvent for the comparative examples, and 50% acetonitrile for the compounds from Examples 1-2).

[0177] 2) Test method The positive control group and the test substance standard solution were placed in tubes containing two types of plasma (human and rat) at concentrations of 1 μM and 5 μM, respectively. These tubes were then cultured at 37°C for a set period of time (0, 30, 60, 120, and 240 minutes) while being mixed by shaking. At each time interval, 100 μl of the mixed sample was dispensed into a cell culture plate (96-well plate) containing 400 μl of quench reagent to complete the test. After centrifugation (5000 × g, 10 minutes), 100 μl of the supernatant was removed and mixed with 200 μl of ultrapure water. The solution was then injected into an LC-MS / MS system to analyze the drug at each time point, thereby evaluating the plasma stability of the comparative example and the compounds of Examples 1-2.

[0178] 3) Test results The results showing stability in human plasma are shown in Figure 4A (compound from Example 1-2) and Figure 4B (compound from the comparative example), and the results showing stability in rat plasma are shown in Figure 4C (compound from Example 1-2) and Figure 4D (compound from the comparative example).

[0179] As can be seen from Figures 4A to 4D, the amount of the comparative compound remaining in human and rat plasma decreased over time (Figures 4B and 4D), while the compounds of Examples 1-2 were confirmed to remain stably in plasma (Figures 4A and 4C).

[0180] Example 3. Inhibitory effect of gingipain expression in P. gingivalis. The compounds in each example were treated with culture media containing Porphyromonas gingivalis, and the expression levels of three gingipain proteins (gingipain lysine-gingipain (Kgp), arginine-gingipain A (RgpA), and arginine-gingipain B (RgpB)), which are major pathogenic factors and proteolytic enzymes produced by Porphyromonas gingivalis, were confirmed.

[0181] After dispensing a culture medium containing either 10 μM of DPD (4,5-dihydroxy-2,3-pentanedione), a precursor of the interbacterial signaling molecule (Autoinducer-2), and 2 μM of the compound of the example, or a culture medium without the compound of the example, into a cell culture plate (48-well plate), the bacteria Porphyromonas gingivalis (2 × 10⁶) were added. 8 Cells (per ml) were inoculated into groups, and the culture media inoculated with bacteria were incubated at 37°C under anaerobic conditions (10% H2, 10% CO2, and 80% N2) for 48 hours.

[0182] After 48 hours, each culture medium was centrifuged at 13,000 rpm for 1 minute to remove the supernatant. Total RNA was extracted using the easy-BLUE™ Total RNA Extraction kit (Seongnam City, South Korea, iNtRON Biotechnology), and cDNA was synthesized. The expression of the gingipain gene was then confirmed using real-time PCR. The cDNA was synthesized using AccuPower® CycleScript RT PreMix dN6 (Daejeon Metropolitan City, South Korea, Bioneer), and Power SYBR TM Quantitative reverse transcription chain reaction (Real-time (quantitative) reverse transcription PCR, RT-qPCR) was performed using Green PCR Master Mix (Applied Biosystems, Foster City, California, USA).

[0183] The primer sequences used for amplification are shown in Table 1 below. The RT-qPCR amplification conditions involved enzyme activation at 95°C for 3 minutes, followed by denaturation at 95°C for 10 seconds, and primer annealing at 60°C for 30 seconds. This process was repeated 40 times, and the results were analyzed using the Bio-Rad CFX Connect Real-Time PCR Detection System (Hercules, California, USA, Bio-Rad). To normalize the expression level of the gingipain gene, the 16s rRNA gene was used, and the gene expression level was calculated using the Comparative Ct Method (ΔΔCt), which is shown in Figures 5A to 5C.

[0184] [Table 1]

[0185] As shown in Figures 5A to 5C, compared to the control group (no treatment), the group treated with DPD, a precursor of AI-2 (DPD), showed increased expression levels of all three gingipain genes (rgpA, rgpB, kgp) in the group treated with DPD, which recognizes quorum sensing signaling molecules.

[0186] Example 4. Cytotoxicity evaluation We evaluated whether the compounds in the examples exhibited cytotoxicity in mouse bone marrow-derived macrophages.

[0187] Bone marrow was isolated from the femur and tibia of 8-12 week old C57BL / 6J mice (source: Dehan Biolink) using a 26g syringe. The isolated bone marrow was differentiated into macrophages for 6 days in a 150π cell culture plate using a medium consisting of IMDM medium + 30% L929 cell supernatant + 10% fetal bovine serum f + 1% penicillin / streptomycin + 1% non-essential amino acids + 1% sodium pyruvate, and then used in experiments.

[0188] Differentiated macrophages are 1 × 10 6 Cells were seeded in 200 μl portions into 48-well plates at a concentration of cells / ml and stabilized at 37°C under 5% CO2 conditions for 12 hours. After removing the supernatant of the cultured cells, the compound from Example 2 was diluted to concentrations of 0.5, 1, 2, 4, 8, and 16 μM, and 200 μl of each was added to serum-free medium. The cells were then cultured at 37°C under 5% CO2 conditions for 24 hours.

[0189] Subsequently, the supernatant of the cultured cells was removed, and 200 μl of MTT solution at a concentration of 400 μg / ml was added. The cells were then reacted at 7°C under 5% CO2 conditions for 4 hours. After that, the reaction solution was removed, 200 μl of DMSO was added, and the absorbance at 570 nm was measured. At this time, the cell viability of the treated cells was calculated using the cell viability of untreated cells as the baseline (100%), and is shown in Figure 6.

[0190] As can be seen in Figure 6, the compounds in Examples 1-2 were confirmed not to show cytotoxicity in mouse bone marrow-derived macrophages at concentrations of 16 μM or less.

[0191] Example 5. Inhibitory effect on biofilm formation of Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), and Acinetobacter baumannii (A. baumannii) 1) Inhibitory effect on E. coli biofilm formation The inhibitory effect of the compounds in Examples 1-2 on biofilm formation in Escherichia coli (KCTC2571 (ATCC8739), obtained from the Korea Oral Microbial Resource Center (KCOM)) was confirmed.

[0192] A sterile coverslip (round, 12 mm radius) was placed on the bottom of each well of a cell culture plate (24-well plate) using tweezers. The compounds from Examples 1-2 were then diluted in the bacterial culture medium (LB medium) to achieve final concentrations of 0.006 μg / ml, 0.06 μg / ml, 0.6 μg / ml, 1.2 μg / ml, 2.4 μg / ml, and 4.8 μg / ml.

[0193] The prepared culture medium is dispensed into cell culture plates lined with glass slips, and E. coli is added in 2 × 10⁶ units. 7 Cells were inoculated to a concentration of cells / ml. In the case of the positive control group (CT), which was not treated with the compound, the same number of bacteria were inoculated into a compound-free medium (LB). In the case of the negative control group (NT), which was not treated with either the compound or the bacteria, only the medium (LB medium) was used for treatment. The completed cell culture plates were maintained at 37°C under anaerobic conditions (10% H2, 10% CO2, and 80% N2) for 48 hours to culture the bacteria.

[0194] Biofilms formed on glass slips by bacteria cultured separately were stained with 1% crystal violet solution for 15 minutes, washed three times with PBS (phosphate-buffered saline) solution, and then destained with acetone-alcohol (20:80, vol / vol). 200 μl of the destained solution containing crystal violet was placed in each 96-well microplate, and the absorbance (OD) was measured using an absorbance microplate reader (Absorbance Microplatereader, Epoch2, Bio-Tek, USA). 590nm The degree of biofilm formation was compared by measuring the biofilm formation rate, and the results are shown in Figures 7A and 7B.

[0195] 2) Inhibitory effect on biofilm formation of Pseudomonas aeruginosa and Acinetobacter bacteria The inhibitory effects of the compounds in Examples 1-2 on biofilm formation by Pseudomonas aeruginosa (obtained from the Korea Oral Microbial Resource Center (KCOM)) and Acinetobacter baumannii (obtained from the Korea Microbial Conservation Center (KCCM)) were confirmed.

[0196] Sterile coverslips (round, 12 mm radius) were placed one at a time at the bottom of each well of a cell culture plate (24-well plate) using tweezers. The compounds from Examples 1-2 were then diluted in bacterial culture media (Pseudomonas aeruginosa: LB medium, Acinetobacter: NB medium) to final concentrations of 0.006 μg / ml, 0.06 μg / ml, and 0.6 μg / ml, respectively.

[0197] The prepared culture medium is dispensed into cell culture plates lined with glass slips, and each bacterium is placed in 2 × 10⁶ cells. 7 Cells were inoculated to a concentration of cells / ml. In the case of the positive control group (CT), which was not treated with the compound, the same number of bacteria were inoculated into a compound-free medium (LB or NB). In the case of the negative control group (NT), which was not treated with either the compound or bacteria, only the medium (LB or NB) was treated. The completed cell culture plates were maintained at 37°C under anaerobic conditions (10% H2, 10% CO2, and 80% N2) for 48 hours to culture the bacteria.

[0198] Biofilms formed on glass slips by bacteria cultured separately were stained with 1% crystal violet solution for 15 minutes, washed three times with PBS (phosphate-buffered saline) solution, and then destained with acetone-alcohol (20:80, vol / vol). 200 μl of the destained solution containing crystal violet was placed in each 96-well microplate, and the absorbance (OD) was measured using an absorbance microplate reader (Absorbance Microplatereader, Epoch2, Bio-Tek, USA). 590nm The degree of biofilm formation was compared by measuring the biofilm formation rate, and the results are shown in Figures 7C and 7D.

[0199] As can be seen from Figures 7A to 7D, after 48 hours of culture, the biofilm of each bacterium (Escherichia coli, Pseudomonas aeruginosa, Acinetobacter) increased in the control group (CT), but it was confirmed that the biofilm was significantly suppressed in a concentration-dependent manner in the groups treated with the compounds of Example 1-2.

[0200] Example 6. Combination effect of antibiotics against Escherichia coli (E. coli) After dispensing 100 μl of E. coli culture medium (LB) into cell culture plates (96-well plates), the control group was sequentially diluted by adding three antibiotics (imipenem (IPM, Sigma-Aldrich), meropenem (MEM, Sigma-Aldrich), and ceftriaxone (CRO, Sigma-Aldrich)) to final concentrations ranging from 0.0312 μg / ml to 32 μg / ml. To confirm whether the efficacy of the antibiotics was increased by the compound treatment in Examples 1-2, the experimental group was mixed with each antibiotic dilution containing 1 μg / ml of the compound from Examples 1-2. The final volume for both the control and experimental groups was 200 μl, and 1 × 10⁶ E. coli cells were placed in each well. 5 After inoculation with cells / ml, the cells were cultured at 37°C for 24-48 hours. After culturing, the absorbance (OD) of each well was measured using an Absorbance MicroplateReader (Epoch2, Bio-Tek, USA). 600nm The minimum growth inhibition concentration was measured and compared between the control group and the experimental group, and the results are shown in Figure 8.

[0201] As can be seen in Figure 8, the minimum growth inhibitory concentrations of the three antibiotics against Escherichia coli (imipenem (IPM), meropenem (MEM), and ceftriaxone (CRO)) were reduced by 20% to 50% or more with the compounds in the examples. This confirms that the efficacy of existing antibiotics was improved when the compounds in the examples of the present invention were used in combination with the antibiotics compared to when the antibiotics were treated individually.

[0202] Example 7. Confirmation of inhibitory and protective effects on nerve cell inflammation. Place 3 x 10⁶ human neuroblastoma cells (SH-SY5Y cells) (source: Korea Cell Line Bank) into a cell culture plate (6-well plate). 6 After treatment with cells / wells, the cells were cultured for 24 hours at 37°C in a 5% CO2 incubator. For the nerve cells attached to the cell culture plates, the negative control group was treated with 3 ml of culture medium only, while the positive control group was inoculated with P. gingivalis (obtained from the Korea Oral Microbial Resource Center (KCOM)) at an infection ratio of 500 (MOI 500). To confirm the neuronal cell inflammation-reducing effect of the compounds in the examples, the experimental groups were inoculated with the same number of P. gingivalis bacteria, and then treated with the compounds from Examples 1-2 diluted in culture medium to 1 μg / ml (2.95 μM).

[0203] Each completed cell culture plate was cultured for 24 hours at 37°C in a 5% CO2 incubator. After the culture was complete, the morphological changes of nerve cells due to the inflammatory response were examined by light microscopy for each group, and the results are shown in Figure 11.

[0204] As can be seen in Figure 11, nerve cells in which inflammation was induced by a P. gingivalis infection stimulus showed cell damage, such as deformation of cell morphology. However, it was confirmed that nerve cells were protected by treatment with the compounds in the examples.

[0205] Real-time PCR analysis After optical microscopy analysis, the supernatant was removed from the cell culture plate, and the levels of inflammatory cytokines in nerve cells were analyzed separately for each group using real-time PCR analysis of the remaining cells. For each group, nerve cells were lysed by treating them with 1 ml of reagent from the easy-BLUE™ Total RNA Extraction kit (Seongnam, South Korea, iNtRON Biotechnology). Total RNA was then extracted from the lysate to synthesize cDNA, and the expression of inflammatory cytokine genes was confirmed using real-time PCR. cDNA was synthesized using AccuPower® CycleScript RT PreMix dT20 (Daejeon, South Korea, Bioneer) and Power SYBR TM Real-time (quantitative) reverse transcription PCR (RT-qPCR) was performed using Green PCR Master Mix (Applied Biosystems, Foster City, California, USA).

[0206] The primer sequences used are shown in Table 2 below. The amplification conditions involved enzyme activation at 95°C for 10 minutes, followed by denaturation at 95°C for 10 seconds, and primer annealing at 60°C for 30 seconds, repeated 40 times. The instrument used was the Bio-Rad CFX Connect Real-Time PCR Detection System (Bio-Rad, Hercules, California, USA). The relative expression levels of inflammatory cytokine genes were calculated using the Comparative Ct Method (ΔΔCt) based on the expression level of the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and the results are shown in Figure 12.

[0207] [Table 2]

[0208] ELISA analysis In the supernatant sample separated from the cell culture plate, the amount of inflammatory cytokines secreted from nerve cells was measured by the ELISA method. After coating the capture antibody at an appropriate concentration on a 96-well plate, culturing at 25 °C for 24 hours, it was washed three times with wash buffer. Blocking was carried out at 25 °C for 1 hour using 300 μl of Reagent Diluent buffer and washed three times with wash buffer. Then, 100 μl each of the test sample and the standard solution were treated and cultured for 2 hours. All experimental groups were identically treated in two wells (duplicates) per group, and during treatment, they were washed three times with wash buffer. After treating 100 μl each of the detection antibody at an appropriate concentration and culturing for 2 hours, it was washed three times with wash buffer. After treating 100 μl of streptavidin-HRP, light was blocked for 20 minutes, then cultured at 25 °C, and after the culture was completed, it was treated with 50 μl of stop solution, and then the absorbance (O.D 450nm ) was measured. The result value was calculated as the average of the measured values, and the calculated amount of inflammatory cytokines was shown in Fig. 13.

[0209] As can be confirmed from Figs. 12 and 13, it was confirmed that the gene expression and production of major inflammatory cytokines (IL-6, IL-8, TNF-α) in nerve cells were suppressed by the treatment with the compound of the example, and neuroinflammation was alleviated.

[0210] Example 8. Cytotoxicity evaluation To confirm the cell safety of the compound of the example, the cytotoxicity against the nerve cells used in the experiment was confirmed.

[0211] To evaluate whether the compound of the example shows toxicity to nerve cells, human neuroblastoma (SH-SY5Y, 3×10 5After dispensing into cells / wells, the compounds of Examples 1-2 were treated at concentrations of 2, 4, 8, 16, and 32 μM and cultured in a 37°C, 5% CO2 incubator for 24 hours. After 24 hours, 50 μl of CCK-8 (cell counting kit-8, Dojindo Molecular Technologies, Inc) solution was identically added to each well, and then re-cultured in a 37°C, 5% CO2 incubator for 1-3 hours. The cell viability was evaluated while measuring the absorbance (O.D 450nm ) at 1-hour intervals of culture. The cell viability of each experimental group was calculated with the cell survival rate of the control group (Control) not treated with the compound of the example as the 100% standard, and the results are shown in Figure 8.

[0212] As can be confirmed from Figure 14, the compounds of the examples showed no cytotoxicity at concentrations of 16 μM or less in neurons.

[0213] Example 9. Results of blood-brain barrier penetration test The compounds of Examples 1-2 were diluted to a concentration of 0.4 mg / ml in an excipient with a composition of 10% DMSO + 65% PEG400 + 25% physiological saline. After preparation, male SD rats (Zhejiang Vital River Laboratory Animal Technology Co., Ltd) were injected intravenously (IV) at 5 ml / kg each, and then blood samples were collected at 60 μl each at different times (0.25 h, 0.5 h, 1 h, 5 h, 10 h). The collected blood samples were centrifuged at 8000 rpm for 6 minutes at 4°C, and then the supernatant was removed. Plasma samples at different times were collected, stored at -50°C until analysis, and then used for analysis.

[0214] To analyze the concentrations of substances in plasma, 20 μl of each time-series sample, a standard sample, and a QC (quality control) sample were mixed with 60 μl of acetonitrile (ACN) (Cas No. 75-05-8) containing internal standards (tolbutamide (Cas No. 64-77-7) 200 ng / ml, propranolol (Cas No. 318-98-9) 50 ng / ml, and Dic (N,N'-diisopropylcarbodiimide) (Cas No. 693-13-0) 500 ng / ml). The mixture was vortexed for 1 minute, then centrifuged (13000 rpm, 4°C, 10 min), and the supernatant (50 μl) was separated and transferred to a 96-well plate containing purified water (150 μl), where it was shaken and mixed for 10 minutes. The final mixture (3 μl) was injected into an LC-MS / MS system, and the concentrations of compounds in the plasma samples at each time point were analyzed. The results are shown in Figure 15.

[0215] In the case of brain samples, heart perfusion was performed to obtain only the sample contained in the brain tissue, and then the brain tissue was separated. After adding 3 times (w / v) saline solution by weight to the separated brain tissue, a homogenized sample solution was prepared by grinding together. 20 μl each of brain samples, standard samples, and QC samples were mixed with 60 μl of acetonitrile (ACN) (Cas No. 75-05-8) containing internal standards (tolbutamide (Cas No. 64-77-7) 200 ng / ml, propranolol (Cas No. 318-98-9) 50 ng / ml, and Dic (N,N'-diisopropylcarbodiimide) (Cas No. 693-13-0) 500 ng / ml). The mixture was then vortexed for 1 minute, centrifuged (13000 rpm, 4°C, 10 min), and the supernatant (50 μl) was separated and transferred to a 96-well plate containing purified water (150 μl), where it was shaken and mixed for 10 minutes. The final mixture (3 μl) was injected into an LC-MS / MS system to analyze the concentration of compounds in the brain samples at each time point. The concentration of compounds in brain tissue was analyzed by converting the calculated sample solution concentration (ng / ml) to the concentration in brain tissue (ng / g), reflecting the dilution factor, and the results are shown in Figure 16.

[0216] As can be seen in Figures 15 and 16, when the compound of the example was administered intravenously to male SD rats at a dose of 2 mg / kg, it was confirmed that the compound of the example traveled through the bloodstream, crossed the hematocratic barrier, reached the brain, and remained in the brain for up to 10 hours after administration. This confirmed that the compound of the example can act on brain lesions at the target site and treat neurodegenerative diseases.

[0217] Example 10. Confirmation of anti-inflammatory effect in macrophages. To confirm the anti-inflammatory effect of the compounds in the examples, mouse bone marrow-derived macrophages were differentiated, and changes in the production of inflammatory cytokines (IL-1β) in the macrophages after treatment with the compounds were measured.

[0218] Bone marrow was isolated from the femur and tibia of 8-12 week old C57BL / 6J mice (source: Dehan Biolink) using a 26g syringe. The isolated bone marrow was differentiated into macrophages for 6 days in a 150pi cell culture plate using a medium consisting of IMDM medium + 30% L929 cell supernatant + 10% fetal bovine serum + 1% penicillin / streptomycin + 1% non-essential amino acids + 1% sodium pyruvate before being used in the experiment.

[0219] Differentiated macrophages are 1 × 10 6 The cells were dispensed into 48-well plates at a concentration of cells / ml in 200 μl each and stabilized at 37°C under 5% CO2 conditions for 12 hours. After removing the supernatant of the cultured cells, serum-free medium was primed with 180 μl of LPS at a concentration of 100 ng / ml for 5 hours. The compounds from Examples 1-2 were diluted to concentrations of 0.78125, 1.5625, 3.125, 6.25, 12.5, and 25 mg / ml and added in 20 μl each (effective concentrations of 0.078125, 0.15625, 0.3125, 0.625, 1.25, and 2.5 mg / ml). After 1 hour, ATP (20 μl added to a concentration of 10 mM, cultured for 30 minutes) was added according to the conditions. After collecting supernatant samples from cultured cells, the amount of IL-1β, a representative inflammatory cytokine released by NLRP3 inflammasome activity, was measured using the ELISA method and is shown in Figure 17.

[0220] As can be seen in Figure 17, we were able to confirm that the release of IL-1β, which was increased in macrophages by the stimulus, was suppressed in a concentration-dependent manner by the compounds of Example 1-2. This confirmed that the inflammatory response induced in macrophages was suppressed by the compounds of the examples.

[0221] Example 11. Cytotoxicity evaluation To confirm the cytotoxicity of the compounds in the examples, we evaluated whether they exhibited cytotoxicity in mouse bone marrow-derived macrophages and human mononuclear cell line (THP-1) (source: Korea Cell Line Bank (KCLB)).

[0222] In the case of macrophages, differentiated macrophages are 1 × 10⁶ 6 Cells / ml were seeded in 200 μl portions into 48-well plates and stabilized at 37°C under 5% CO2 conditions for 12 hours. After removing the supernatant of the cultured cells, the compounds from Examples 1-2 were diluted to concentrations of 0.5, 1, 2, 4, 8, and 16 μM, and 200 μl of each portion was treated in serum-free medium. The cells were then cultured at 37°C under 5% CO2 conditions for 24 hours.

[0223] Subsequently, the supernatant of the cultured cells was removed, and 200 μl of MTT solution at a concentration of 400 μg / ml was added. The cells were then reacted at 7°C under 5% CO2 conditions for 4 hours. After that, the reaction solution was removed, 200 μl of DMSO was added, and the absorbance at 570 nm was measured. At this time, the cell viability of the treated cells was calculated using the cell viability of untreated cells as the baseline (100%) and is shown in Figure 18.

[0224] Human mononuclear cell line (THP-1) was cultured in RPMI1640 medium (Hyclone, Waltham, Massachusetts, USA) supplemented with 10% fetal bovine serum (Hyclone, Waltham, Massachusetts, USA), 2.05 mM L-glutamic acid, and antibiotics (100 units / ml penicillin, 100 μg / ml streptomycin), and then 1 × 10⁶ cells were cultured. 5Cells were seeded in a 96-well microtiter plate at a cell / well concentration. The compounds from Example 1-2 were diluted to final concentrations of 0.002 μM, 0.02 μM, 0.2 μM, and 2 μM, and the cells in each well were treated with these solutions. The cells were then cultured for 24 hours. Subsequently, 10 μl of Cell Counting Kit-8 (CCK-8, Kumamoto Prefecture, Japan, DOJINDO) solution was applied to each well of the cultured cells. The absorbance at 450 nm was then measured at different time intervals (1h, 2h, 3h), and the absorbance data from the final time interval (3h) was used as the baseline to derive the results.

[0225] At this time, the cell viability of cells treated with the sample was calculated using the cell viability of untreated cells as the baseline of 100%, and the results are shown in Figure 19.

[0226] As can be seen from Figures 18 and 19, the compounds in Examples 1-2 did not show cytotoxicity in mouse bone marrow-derived macrophages at concentrations below 16 μM, and did not show cytotoxicity in the human mononuclear cell line (THP-1) at any concentration.

[0227] Example 12. Drug safety evaluation using the rat micronucleus test. To confirm the safety of the compound used in the experimental rat (purchased from ORIENTBIO INC., South Korea), general symptoms, weight changes, and the presence or absence of micronucleus induction (genotoxicity) were evaluated after administration of the substance to Sprague-Dawley rat bone marrow cells.

[0228] The subcutaneous area of ​​the SD rats was dehaired, and the compound of the example was administered using a disposable syringe at low (500 mg / kg / day), medium (1000 mg / kg / day), and high (2000 mg / kg / day) concentrations, divided into two doses (10 ml / kg / day each) subcutaneously to the left and right sides of the dorsal neck of the rat. General symptoms such as appearance, behavior, and excretions were observed for each individual rat twice a day during the administration period (immediately before and immediately after administration), and once a day during other periods. The results are shown in Table 3 below.

[0229] In addition, body weights were measured on the administration start date and the bone marrow collection date to confirm whether there were any changes due to substance administration, and the results are shown in Table 4 below.

[0230] At 24 hours after the second administration of the test substance, the femurs were excised for each group to collect bone marrow cells. 0.5 mL of 10% neutral formalin was added to the bone marrow cell suspension and fixed for 5 minutes, and then centrifuged for 5 minutes (4 °C, 1,000 rpm, Micro17TR, Republic of Korea, Hanil Science Industrial) to remove the supernatant. Thereafter, 0.3 mL of 10% neutral formalin was added to the precipitated bone marrow cells to suspend them, and then filtered through a cell strainer and transferred to a storage tube. After dropping the fixed bone marrow cell suspension onto a cover glass, a slide glass coated with 20 μL of 0.05% acridine orange was covered to prepare an observation slide, and the observation slide was observed with a microscope (600-fold magnification, BX51, Japan, Olympus). 4,000 polychromatic erythrocytes (PCE) per solid were observed, and the appearance rate (MNPCE / PCE) of micronucleated polychromatic erythrocytes (MNPCE) relative to polychromatic erythrocytes was determined for each individual, and the results are shown in Table 3 below.

[0231] In addition, it was confirmed from the observation results whether the ratio of polychromatic erythrocytes to the total number of erythrocytes in the test substance group (treatment with the compounds of Examples 1-2) appeared to be 20% or more of that in the negative control group. 500 total erythrocytes per individual were observed as an index of bone marrow cell growth inhibition, and the ratio of polychromatic erythrocytes to total erythrocytes (PCE / (PCE+NCE)) was determined, and the results are shown in Table 5 below. As the positive control group, cyclophosphamide (Cas No: 50-18-0) was administered at a dose of 20 mg / kg / day.

[0232]

Table 3

[0233] <000,0977>

Table 4

[0234] [Table 5]

[0235] As can be seen from Table 3, the experimental results showed no abnormal clinical signs in the rats of the test substance group (the group administered with the compounds of Example 1-2) at all doses of the compounds of Example 1-2 during the observation period.

[0236] Furthermore, as can be seen from Table 4, at all doses of the compounds in Examples 1-2, the test substance group did not show a statistically significant change in body weight compared to the negative control group.

[0237] Furthermore, as can be seen from Table 5, at all doses of the compounds in Examples 1-2, the test substance group did not show a statistically significant difference in the frequency of micronuclear polychromatic erythrocytes (MNPCE / PCE) among polychromatic erythrocytes compared to the negative control group, and the ratio of polychromatic erythrocytes in total erythrocytes [PCE / (PCE+NCE)] also did not show a statistically significant difference compared to the negative control group. In contrast, in the positive control group, although no statistically significant difference was observed in the ratio of polychromatic erythrocytes compared to the negative control group, a statistically significant increase in the frequency of micronuclear polychromatic erythrocytes among polychromatic erythrocytes was observed compared to the negative control group (p<0.01).

[0238] The results confirmed that the compounds used in the examples were safe and did not induce micronuclei in rat bone marrow cells.

[0239] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. In this regard, the embodiments described above should be understood to be illustrative and not restrictive in all respects. The scope of the present invention should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, which are described below in more detail than the above, and their equivalent concepts.

Claims

1. A pharmaceutical composition for the prevention or treatment of inflammasome-mediated diseases, comprising as an active ingredient one or more selected from the group consisting of 3-(di-p-tolylmethylene)-2-methylisoindolin-1-one and pharmaceutically acceptable salts thereof.

2. The pharmaceutical composition according to claim 1, wherein the inflammasome-mediated disease is one or more selected from the group consisting of periodontal disease, neurodegenerative disease, and inflammatory disease.

3. The pharmaceutical composition according to claim 2, wherein the periodontal disease is one or more selected from the group consisting of periodontitis and gingivitis.

4. The pharmaceutical composition according to claim 2, wherein the neurodegenerative disease is one or more selected from the group consisting of dementia, Parkinson's disease, tremor, Taranto's disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, motor neuron disease, spinal muscular atrophy, Creutzfeldt-Jakob disease, Pick's disease, prion disease, and spinocerebellar degeneration.

5. The aforementioned inflammatory diseases are One or more metabolic diseases selected from the group consisting of obesity, hyperlipidemia, hypercholesterolemia, arteriosclerosis, type 2 diabetes, non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH), One or more autoinflammatory diseases selected from the group consisting of Muckle-Wells syndrome (MWS), latent autoimmune diabetes mellitus (LADA), familial cold autoinflammatory syndrome (FCAS), cryopyrin-associated periodic syndromes (CAPS), neonatal-onset multiorgan inflammatory syndrome (NOMID), chronic infantile neurocutaneous arthritis (CINCA) syndrome, familial Mediterranean fever (FMF), pediatric arthritis, pediatric rheumatoid arthritis, and gout. Autoimmune diseases, one or more selected from the group consisting of rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), atopic dermatitis (AD), and psoriasis. Septic shock, inflammatory bowel disease, osteoarthritis, hyperimmunoglobulin D syndrome, and cryopyrin-associated periodic syndromes. The pharmaceutical composition according to claim 2, wherein one or more are selected from the group consisting of the following.

6. A food composition for the prevention or improvement of inflammasome-mediated diseases, comprising as an active ingredient one or more selected from the group consisting of 3-(di-p-tolylmethylene)-2-methylisoindolin-1-one and pharmaceutically acceptable salts thereof.

7. A feed composition for the prevention or improvement of inflammasome-mediated diseases, comprising as an active ingredient one or more selected from the group consisting of 3-(di-p-tolylmethylene)-2-methylisoindolin-1-one and pharmaceutically acceptable salts thereof.

8. A pharmaceutical composition for the prevention or treatment of Gram-negative bacterial infection or disease induced by Gram-negative bacteria, comprising as an active ingredient one or more selected from the group consisting of 3-(di-p-tolylmethylene)-2-methylisoindolin-1-one and pharmaceutically acceptable salts thereof.

9. The pharmaceutical composition according to claim 8, wherein the Gram-negative bacterial infection or disease induced by Gram-negative bacteria is one or more selected from the group consisting of enteritis, Crohn's disease, ulcerative colitis, bacterial dysentery, urethral infection, skin infection, bacteremia, sepsis, pneumonia, endocarditis, meningitis, otitis media, periodontitis, gingivitis, and keratitis.

10. A food composition for the prevention or improvement of Gram-negative bacterial infection or disease induced by Gram-negative bacteria, comprising as an active ingredient one or more selected from the group consisting of 3-(di-p-tolylmethylene)-2-methylisoindolin-1-one and pharmaceutically acceptable salts thereof.

11. A feed composition for the prevention or improvement of Gram-negative bacterial infection or disease induced by Gram-negative bacteria, comprising as an active ingredient one or more selected from the group consisting of 3-(di-p-tolylmethylene)-2-methylisoindolin-1-one and pharmaceutically acceptable salts thereof.

12. An antibiotic having antiviral activity against Gram-negative bacteria, comprising as an active ingredient one or more selected from the group consisting of 3-(di-p-tolylmethylene)-2-methylisoindolin-1-one and pharmaceutically acceptable salts thereof.

13. The antibiotic according to claim 12, wherein the Gram-negative bacteria is one or more selected from the group consisting of Escherichia, Pseudomonas, Acinetobacter, Enterobacter, Klebsiella, Porphyromonas, Fusobacteria, and Tannerella.

14. The antibiotic according to claim 12, One or more secondary antibiotics selected from the group consisting of beta-lactam antibiotics, bacterial cell membrane permeability inhibitors, bacterial ribosome inhibitors, bacterial nucleic acid synthesis inhibitors, and bacterial folic acid synthesis inhibitors. Antibiotics used in combination with [this].

15. A feed additive comprising the antibiotic described in claim 12.

16. A disinfectant comprising the antibiotic described in claim 12.

17. A cleaning agent comprising the antibiotic described in claim 12.

18. i) 3-(di-p-tolylmethylene)-2-methylisoindolin-1-one, or ii) 3-(bis(4-methoxyphenyl)methylene)-2-methylisoindolin-1-one A compound that is a compound, or a pharmaceutically acceptable salt thereof.

19. A method for producing the compound according to claim 18, comprising the step of introducing 4-methylphenylboronic acid (4-methylphenylboronic Acid, Cas No. 5720-05-8) or 4-methoxyphenylboronic acid ((4-methylphenyl)boronic acid, Cas No. 5720-07-0) into the compound of the following chemical formula 4. 【Chemistry 1】

Images