Compound as inhibitor against necroptosis

Abstract

The present invention relates to a novel use of oxidized apomorphine for preventing or treating diseases accompanied by or related to necroptosis. The present invention relates to a pharmaceutical composition comprising oxidized apomorphine or a pharmaceutically acceptable salt thereof for preventing or treating diseases accompanied by or related to necroptosis.

Description

Compounds as inhibitors of programmed cell necrosis

[0001] The present invention relates to apomorphine and its use for treating diseases related to necroptosis, and more particularly, to a pharmaceutical composition for preventing or treating diseases related to necroptosis, comprising apomorphine or a pharmaceutically acceptable salt thereof as an active ingredient, and a method for producing the same.

[0002] Programmed necrosis (necroptosis) is a type of programmed necrotic cell death associated with organ development, tissue homeostasis, inflammation, and disease pathogenesis. When stimulated by necrosis-inducing agents, cytoplasmic receptor-interacting protein kinase 1 (RIP1), RIP3, and MLKL are sequentially activated, ultimately resulting in the formation of oligomers of phosphorylated mixed lineage kinase domain-like pseudokinase (MLKL) that translocate to the cell membrane, mediating ion influx or disrupting the cell membrane, thereby inducing cell necrosis.

[0003] The first known necroptosis inhibitor was Nec-1, which inhibits RIP1 activity. However, Nec-1 is often used by researchers to distinguish between necroptosis and apoptosis in cell experiments, but it was found that it does not have substrate specificity sufficient to be used for therapeutic purposes. In addition, since RIP1 is involved in not only necroptosis but also apoptosis and NF-kB activity, its efficacy as a necroptosis inhibitor is reported to be limited.

[0004] Furthermore, RIP3 is a protein specifically involved in necroptosis, making it a good target for therapeutic development. However, considering that RIP3 without kinase function has recently been reported to induce apoptosis, RIP3 inhibitors may also induce other unwanted forms of cell death, which may result in serious drug side effects.

[0005] Due to the problems with these factors in the signaling pathway of programmed cell necrosis, MLKL has recently been studied as the optimal target for specifically inhibiting necroptosis. Furthermore, recent studies have demonstrated that MLKL is involved in various inflammatory diseases, including acute pancreatitis, multiple sclerosis, inflammatory bowel disease, and allergic colitis, leading to its investigation as a potential therapeutic target for inflammatory diseases.

[0006] Currently, two MLKL inhibitors are known: necrosulfonamide (NSA) and GW806742X. However, NSA's development as a drug was discontinued due to its moderate potency and narrow structure-activity-relationship (SAR) profile. Furthermore, GW806742X, which targets the pseudokinase domain of MLKL, also binds to RIP1 and RIP3, resulting in poor targeting specificity for MLKL.

[0007] Considering the problems of MLKL inhibitors reported to date, there is a need to develop new and potent MLKL inhibitors that can specifically inhibit only programmed cell necrosis.

[0008] The present invention aims to provide a pharmaceutical composition having a function of inhibiting cell-programmed necrosis.

[0009] The present invention aims to provide a pharmaceutical composition for preventing or treating a disease accompanied by or related to programmed cell necrosis.

[0010] The present invention aims to provide a composition for organ preservation that can improve the organ preservation period by suppressing organ damage or cell necrosis.

[0011] The present invention provides a pharmaceutical composition having the function of inhibiting programmed cell necrosis, comprising a compound of the following chemical formula 1, or a pharmaceutically acceptable salt thereof:

[0012] <Chemical Formula 1>

[0013]

[0014] The present invention also provides a pharmaceutical composition for preventing or treating a disease accompanied by or related to programmed cell necrosis, comprising a compound of the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0015] The present invention provides a method for treating a disease accompanied by or related to programmed cell necrosis, comprising administering to a subject in need thereof a compound of the above chemical formula 1, or a pharmaceutically acceptable salt thereof.

[0016] The present invention provides the use of a compound of formula 1, or a pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for the prevention or treatment of a disease accompanied by or related to programmed cell necrosis.

[0017] The present invention also provides a composition for long-term preservation or a long-term preservation solution comprising the compound of the above chemical formula 1 or a pharmaceutically acceptable salt thereof.

[0018] The present invention provides a use of a compound of formula 1, or a pharmaceutically acceptable salt thereof, for preparing a long-term preservation solution.

[0019] The present invention provides a method for preserving an organ, comprising a step of exposing the organ to a preservative solution containing the compound of the above chemical formula 1, or a pharmaceutically acceptable salt thereof.

[0020] By providing a pharmaceutical composition comprising the compound of the present invention as an active ingredient, the composition can be usefully utilized for the prevention and treatment of various diseases that are accompanied by or can be caused by apoptosis.

[0021] Figure 1 shows the structure (A) of oxoapomorphine (Ox-APO), a compound of the present invention manufactured through oxidation of apomorphine (APO), and the results of real-time measurement of the oxidation level of apomorphine to oxoapomorphine (B).

[0022] Figure 2 shows the results of confirming whether MLKL oligomerization and phosphorylation were inhibited by apomorphine (APO) and oxoapomorphine (Ox-APO).

[0023] Figure 3 shows the results of confirming whether MLKL oligomerization and phosphorylation were inhibited according to the concentration of apomorphine (APO) and oxoapomorphine (Ox-APO).

[0024] Figure 4 shows the results of SPR analysis performed to measure the affinity of APO and Ox-APO for MLKL, respectively.

[0025] Figure 5 shows the results of analyzing the improvement effects of APO and Ox-APO in the DSS-induced mouse colitis model. (5a) Experimental method and schedule, (5b) Histopathological evaluation results by tissue staining (Scale bar, 100 μm. Mean ± SD (n = 5)), (5c) Histological score evaluation results ( * p < 0.05, *** p < 0.001, One-way ANOVA), and (5d) disease activity index (DAI) of colitis was evaluated (mean ± SD (n = 5). * p < 0.05, *** p < 0.001, t-test.), (5e) Colon length evaluation results (Mean ± SD (n = 5). * p < 775 0.05, ** p < 0.01,*** p < 0.001, One-way ANOVA).

[0026] Figure 6 shows the results of mutation experiments and SPR analysis to analyze the interactions between Nt-hMLKL and APO and Ox-APO.

[0027] Figure 7 shows the results of analyzing the liver damage protective effects of APO and Ox-APO in an APAP-induced liver damage mouse model. (7a) Experimental method and schedule, (7b) liver tissue color changes in the groups treated with Re-APO and Re-APO at a concentration of 15 mg / kg, (7c) liver tissue H&E staining results, (7d) IHC analysis results, and (7e) serum ALT and AST levels.

[0028] Figure 8 shows the results of confirming whether the p-MLKL oligomerization inhibition effect is observed even at low temperature (4°C) to confirm the possibility of using Ox-APO as a preservative in long-term transplantation.

[0029] The present invention provides a pharmaceutical composition for preventing or treating a disease accompanied by or related to programmed cell necrosis, comprising a compound of the following chemical formula 1, or a pharmaceutically acceptable salt thereof:

[0030] <Chemical Formula 1>

[0031]

[0032] The compound represented by chemical formula 1 of the present invention can be prepared by oxidizing apomorphine in the form of apomorphine, as shown in FIG. 1.

[0033] The term "programmed necrosis" as used herein refers to programmed cell death. While distinct from necrosis and apoptosis, it can refer to a form of necrosis that shares the same morphology as both of these cell death types. Programmed necrosis is known to be induced by substances that induce necrosis and is mediated by the sequential signaling of RIP1, RIP3, and MLKL within the cytoplasm. The final step, MLKL, oligomerizes upon phosphorylation and translocates to the cell membrane, forming pores in the membrane and causing cell necrosis. For further details, see Sun L, et al. Trends Biochem Sci. 2014 Dec;39(12):587-93. doi: 10.1016 / j.tibs.2014.10.003. PMID: 25455759.

[0034] The present inventors confirmed that the compound specifically inhibits the formation of pores in the cell membrane by inhibiting the oligomerization of MLKL (mixed lineage kinase domain like pseudokinase), a terminal mediator of programmed cell necrosis, and confirmed that the compound has superior MLKL specificity and ability to inhibit programmed cell necrosis compared to existing MLKL inhibitors.

[0035] Accordingly, the present invention provides a novel use of the compound for the prevention or treatment of diseases accompanied by or related to programmed cell necrosis.

[0036] In addition, the present invention provides a method for preventing or treating a disease accompanied by or related to programmed cell necrosis, comprising a step of administering the compound or a pharmaceutical composition comprising the compound to a subject in need thereof.

[0037] Diseases accompanying or associated with programmed cell necrosis of the present invention include ischemic, neurodegenerative, and inflammatory diseases. Specifically, the diseases related to the above-mentioned programmed cell necrosis include toxic epidermal necrolysis (TEN), Stevens-Johnson syndrome (SJS), burns, frostbite, erythema multiforme, skin diseases caused by bacterial or viral infections, pityriasis versicolor, lupus erythematosus, lichen planus, drug rash, cutaneous vasculitis, stroke, autoimmune disease, Inflammatory Bowel Disease (IBD), retinal degeneration, sepsis, chronic obstructive pulmonary disease, acute respiratory distress disorder, transfusion-related acute lung injury, transplant rejection, atherosclerosis, aortic aneurysm, myocardial infarction, terminal ileitis, acute kidney injury, kidney These include renal ischemia reperfusion injury, liver injury, steatohepatitis, pancreatitis, bone marrow failure, and cell death during organ transplantation.

[0038] [ PMC free article ] [ PubMed ] 1. Khoury MK, Gupta K, Franco SR, Liu B. Necroptosis in the Pathophysiology of Disease. Am J Pathol. 2020 Feb;190(2):272-2 doi: 10.1016 / j.ajpath.2019.10.012.2. Epub 2019 Nov 26. PMID: 31783008; PMCID: PMC6983729;

[0039] 2. Ware LB. Transfusion-induced lung endothelial injury: a DAMP death? Am J Respir Crit Care Med. 2014 Dec 15;190(12):1331-2. doi: 10.1164 / rccm.201411-2047ED. PMID: 25496097.;

[0040] [ PubMed ] 3. Leeper NJ. The role of necroptosis in atherosclerotic disease. JACC Basic Transl Sci. 2016 Oct;1(6):548-550. doi: 10.1016 / j.jacbts.2016.08.002. PMID: 28480338; PMCID: PMC5419689.;

[0041] 4. Gunther C, Martini E, Wittkopf N, Amann K, Weigmann B, Neumann H, Waldner MJ, Hedrick SM, Tenzer S, Neurath MF, Becker C. Caspase-8 regulates TNF-α-induced epithelial necroptosis and terminal ileitis. Nature. 2011 Sep 14;477(7364):335-9. doi: 10.1038 / nature10400. PMID: 21921917; PMCID: PMC3373730.;

[0042] 5. Jun W, Benjanuwattra J, Chattipakorn SC, Chattipakorn N. Necroptosis in renal ischemia / reperfusion injury: A major mode of cell death? Arch Biochem Biophys. 2020 Aug 15;689:108433. doi: 10.1016 / j.abb.2020.108433. Epub 2020 May 26. PMID: 32470461.;

[0043] 6. Gautheron J, Vucur M, Luedde T. Necroptosis in Nonalcoholic Steatohepatitis. Cell Mol Gastroenterol Hepatol. 2015 Mar 12;1(3):264-265. doi: 10.1016 / j.jcmgh.2015.02.001. PMID: 28210679; PMCID: PMC5301189.;

[0044] 7. Roderick JE, Hermance N, Zelic M, Simmons MJ, Polykratis A, Pasparakis M, Kelliher MA. Hematopoietic RIPK1 deficiency results in bone marrow failure caused by apoptosis and RIPK3-mediated necroptosis. Proc Natl Acad Sci U S A. 2014 Oct 7;111(40):14436-41. doi: 10.1073 / pnas.1409389111. Epub 2014 Sep 22. PMID: 25246544; PMCID: PMC4209989.

[0045] 8. A., Nakatake, S., Notomi, S., et al. (2014). Programmed necrosis, not apoptosis, is a key mediator of cell loss and DAMP-mediated inflammation in dsRNA-induced retinal degeneration. Cell Death Differ 21, 270-277. 10.1038 / cdd.2013.109.

[0046] 9. Lau A., Wang S., Jiang J., Haig A., Pavlosky A., Linkermann A., Zhang Z.-X., Jevnikar AM RIPK3-Mediated Necroptosis Promotes Donor Kidney Inflammatory Injury and Reduces Allograft Survival. Am. J.Transplant. 2013;13:2805-2818.

[0047] 10. Zhao Y, Main K, Aujla T, Keshavjee S, Liu M. Necroptosis in Organ Transplantation: Mechanisms and Potential Therapeutic Targets. Cells. 2023 Sep 17;12(18):2296.

[0048] The inflammatory bowel disease may include Crohn's disease or ulcerative colitis. The autoimmune disease may include psoriasis or rheumatoid arthritis.

[0049] In a specific embodiment of the present invention, when the compound of the present invention was administered to a mouse model of inflammatory bowel disease, the therapeutic effect was confirmed by confirming that damage to the mouse mucosal barrier and DAI score were reduced.

[0050] The pharmaceutically acceptable salts of the present invention can be prepared by a method conventional in the art, for example, salts of inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sodium hydrogen sulfate, phosphoric acid, nitric acid, carbonic acid, etc.; salts of organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, succinic acid, benzoic acid, citric acid, maleic acid, malonic acid, tartaric acid, gluconic acid, lactic acid, gestic acid, fumaric acid, lactobionic acid, salicylic acid, trifluoroacetic acid, or acetylsalicylic acid (aspirin); salts of amino acids such as glycine, alanine, vanillin, isoleucine, serine, cysteine, cystine, aspartic acid, glutamine, lysine, arginine, tyrosine, proline, etc.; salts of sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, etc. Includes metal salts resulting from reactions with alkali metals such as sodium and potassium, or salts with ammonium ions. Pharmaceutically acceptable salts are not particularly limited as long as they can be used in pharmaceuticals.

[0051] The pharmaceutical composition of the present invention may include, in addition to the active ingredient, one or more pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" refers to a known pharmaceutical excipient that is useful in formulating the administered compound and is substantially non-toxic and non-sensitizing. The precise proportions of such excipients are determined by the solubility and chemical properties of the active compound, the selected route of administration, and standard pharmaceutical practice.

[0052] The pharmaceutical composition of the present invention can be formulated into a form suitable for a desired administration method using additives such as suitable and physiologically acceptable excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, glidants, and flavoring agents.

[0053] The above pharmaceutical composition may be formulated in the form of, but is not limited to, tablets, capsules, pills, granules, powders, injections, films or liquids.

[0054] The formulation of the pharmaceutical composition and the pharmaceutically acceptable carrier can be appropriately selected according to techniques known in the art, and reference can be made, for example, to the following references: [Urquhart et al., Lancet, 16:367, 1980]; [Lieberman et al., PHARMACEUTICAL DOSAGE FORMS-DISPERSE SYSTEMS, 2nd ed., vol. 3, 1998]; [Ansel et al., PHARMACEUTICAL DOSAGE FORMS & DRUG DELIVERY SYSTEMS, 7th ed., 2000]; [Martindale, THE EXTRA PHARMACOPEIA, 31st ed.]; [Remington's PHARMACEUTICAL SCIENCES, 16th-20th editions]; [THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Goodman and Gilman, eds., 9th ed., 1996]; [Wilson and Gisvolds' TEXTBOOK OF ORGANIC MEDICINAL AND PHARMACEUTICAL CHEMISTRY, Delgado and Remers, eds., 10th ed., 1998]. Principles of formulating pharmaceutical compositions are also described, for example, in the following references: [Platt, Clin Lab Med, 7:289-99, 1987]; [Aulton, PHARMACEUTICS: THE SCIENCE OF DOSAGE FORM DESIGN, Churchill Livingstone, NY, 1988]; See [EXTEMPORANEOUS ORAL LIQUID DOSAGE PREPARATIONS, CSHP, 1998], ["Drug Dosage," J Kans Med Soc, 70(1):30-32, 1969], etc.

[0055] Although not limited thereto, in one specific embodiment, the pharmaceutical composition of the present invention may be formulated as an injection. The injection may be formulated as an intravenous injection or a subcutaneous injection. In the case of a parenteral injection, the composition may include components included in a general injection composition. For example, the injection composition may include a liquid carrier such as sterile water, water for injection, or physiological saline. In addition, the composition may further include one or more components selected from the group consisting of amino acids, sugars, lipids, vitamins, electrolytes, pH adjusters, stabilizers, osmotic pressure adjusters, and solubilizers.

[0056] The pharmaceutical composition of the present invention may be a subcutaneous injection, and in this case, in addition to the compound, it may include sodium metabisulfite, benzyl alcohol, hydrochloric acid, sodium hydroxide, etc. as additives.

[0057] In one specific embodiment, the pharmaceutical composition of the present invention can be formulated as a sublingual tablet or film. When the composition is formulated as a tablet, it may further comprise one or more ingredients selected from the group consisting of excipients, binders, disintegrants, lubricants, solvents, antioxidants, emulsifiers, thickeners, anticaking agents, flavoring agents, coloring agents, sweeteners, pH adjusters, sustained-release agents, stabilizers, and coating agents. In one embodiment of the present invention, the additives may include, but are not limited to, sodium edetate, ascorbic acid, magnesium stearate, hypromellose, citric acid, microcrystalline cellulose, colloidal silicon dioxide, cool mint orange (WONF WL-28499), red iron oxide (E172), acesulfame potassium, and mannitol.

[0058] When the composition is formulated as a film, it may further include a binder, a stabilizer, an antioxidant, a chelating agent, a thickener, a coating agent, a flavoring agent, a sweetener, and a pH adjuster. The additives may include, but are not limited to, sodium metabisulfite, disodium EDTA, dihydrate, glycerol, glyceryl monostearate, hydroxyethyl cellulose, hypromellose, maltodextrin, (-)-menthol, pyridoxine hydrochloride, sodium hydroxide, sucralose, and white ink.

[0059] The preferred dosage of the compound may vary depending on the condition and weight of the subject, the type and severity of the disease, the drug form, the route and duration of administration, and may be appropriately selected by those skilled in the art. According to one embodiment of the present invention, although not limited thereto, the daily dosage may be 0.01 to 200 mg / kg, specifically 0.1 to 200 mg / kg, and more specifically 0.1 to 100 mg / kg. Administration may be administered once a day or divided into several doses, and the scope of the present invention is not limited thereby.

[0060] In the present invention, the term "subject" means a subject requiring prevention or treatment of a disease, and more specifically, may mean a mammal such as a human or non-human primate, mouse, dog, cat, horse, or cow requiring prevention or treatment of a disease accompanied by or related to programmed cell necrosis.

[0061] As described above, the present inventors confirmed that the compound effectively inhibits necroptosis by inhibiting oligomerization of MLKL (mixed lineage kinase domain-like pseudokinase).

[0062] Accordingly, the present invention provides, as another embodiment, a composition for long-term preservation or a long-term preservation solution comprising the compound.

[0063] The present invention also provides a method for preserving an organ, comprising exposing the organ to a composition or preservative solution containing the compound, or a use of the compound for the preparation of such an organ preservative solution.

[0064] The term "organ" used herein merely specifically describes the object to which the composition of the present invention is applied. The composition of the present invention can be applied not only to organs but also to the preservation of parts of organs, tissues, and portions of tissues. Therefore, the term "organ" in this specification should be interpreted as encompassing organs, parts of organs, tissues, and portions of tissues.

[0065] During organ transplantation, organs separated from the body undergo cellular damage and necrosis. To delay this process, the organ is typically preserved in a low-temperature (4°C) environment in a preservative solution and then reperfused. Representative preservative solutions currently in use include Collins' solution, Euro-Collins' solution, HTK (Histidine-Tryptophan-Ketoglutarate) solution, and Celsior's solution. Of these, UW solution, developed at the University of Wisconsin, is the most widely used.

[0066] According to a specific embodiment of the present invention, the compound of the present invention exhibits an excellent effect of inhibiting cell-programmed necrosis, and therefore can be utilized as a main or auxiliary component of a preservative solution for inhibiting or delaying damage to isolated organs, such as organs for transplantation.

[0067] In particular, in one embodiment, when the compound of the present invention was treated under a low-temperature (4°C) environment, it was confirmed that it effectively inhibited the formation of octamers and tetramers of p-MLKL, thereby inhibiting cell-programmed necrosis, as shown in FIG. 8. These results clearly support the possibility that the compound of the present invention can be used as an effective ingredient in long-term preservation solutions.

[0068] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms. These embodiments are provided solely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention. The present invention is defined solely by the scope of the claims.

[0069]

[0070] [Example]

[0071] Experimental method

[0072] 1-1. Cell culture and reagents

[0073] THP-1-HMGB1-Lucia stably expressing HMGB1::Lucia-luciferase fusion protein (HMGB1-luciferase) for screening tests TMCells (InvivoGen, CA, USA) were maintained in complete medium supplemented with 25 mM HEPES, 100 μg / mL Zeocin (ant-z-1; InvivoGen), and Mycozap (VZA-2031; Lonza, Swiss) for selection at 37°C in 5% CO2. THP-1 (TIB-202; ATCC, Manassas, VA, USA) were used for programmed necrosis pathway studies and were maintained in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin (complete medium) at 37°C in 5% CO2. THP-1 cells were treated in complete medium containing 500 nM PMA (phorbol 12-myristate 13-acetate) for 3 h to induce cell maturation and attachment.

[0074] 1-2. Induction of programmed cell necrosis and luciferase assay

[0075] THP-1-HMGB1-Lucia TMCells were pretreated with the pan-caspase inhibitor Z-VAD (tlrl-vad; InvivoGen) and each chemical for 1 h, and then TNF-α (TNF0501; NKMAXBio, Seongnam, Korea) and the Smac mimetic BV6 (B1332-5; Biovision, San Francisco, CA, USA) (abbreviated as TBZ) were added and incubated for the indicated times for the screening of programmed necrosis of cells. Necrostatin-1 (NEC-1, 1846; Biovision), a RIP1 inhibitor, and necrosulfonamide (NSA, 432531-71-0; Sigma-Aldrich, St. Louis, MO, USA), a MLKL inhibitor, were used as control chemicals. Cell supernatants were then collected in 96-well plates with clear bottoms and white walls, and HMGB1 levels were measured using a luciferase assay. For this purpose, the Luciferase assay kit (E1500; Promega, Madison, WI, USA) was used according to the manufacturer's instructions and measured with a luminometer (Centro XS3 LB 960; Berthold Technologies, Bad Wildbad, Germany). The inhibition of programmed necrosis (%) was calculated as follows: (sample treatment data - untreated medium data) x 100 / (TBZ treatment data - untreated medium data). Apomorphine (APO) (STK088477, Vitas-M Laboratory, Hong Kong), (6aS)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol, was purchased, aliquoted in DMSO, and stored at -70°C. APO has autooxidative properties and was prepared by dilution using DPBS containing 5 mM 1,4-Dithiothreitol (DTT) and used immediately. Oxidized APO (Oxoapomorphine, Ox-APO) was prepared by incubating APO in DPBS at 25°C in the presence of light and air for 24 hours, and then adding 0.It was manufactured by removing sediment through filtration using 2㎛.

[0076] 1-3. Propidium iodide (PI) uptake test

[0077] HMGB1-luciferase can be actively secreted regardless of necrotic pore formation in the cell membrane. To identify candidate compounds that inhibit pore formation in the cell membrane by preventing MLKL oligomerization, a PI uptake test was performed. This allowed the exclusion of candidates that inhibit HMGB1 secretion without being associated with the programmed necrotic pathway.

[0078] For analysis, 2 x 10 THP-1 cells were cultured 5 Cells were plated in 96-well black / clear bottom plates at 10 μg / well and treated with candidate compounds for 8 h to induce programmed cell death. PI (556463, BD biosciences, San Jose, CA, USA) was added to a final concentration of 50 μg / mL, and cells were washed for 10 min. Fluorescence was measured in real time using a Varioskan Flash 3001 plate reader (Thermo Fisher Scientific, MA, USA) by excitation at 530 nm and emission at 617 nm. Data acquisition was performed at 37°C.

[0079] 1-4. Western blot analysis

[0080] THP-1 cells were treated with candidate compounds and harvested by centrifugation at 1,500 × g for 5 min at 4°C. The harvested cells were lysed using 1X RIPA buffer (R4100-010, GenDEPOT Inc., Katy, TX, USA) containing 1x protease inhibitor (P3100-001; GenDEPOT) and phosphatase inhibitor (1862495, Thermo Fisher Scientific). Whole cell lysates (WCL) were then centrifuged at 20,000 × g for 30 min at 4°C to remove debris, and protein concentrations were measured using a BCA protein assay kit (23225, Thermo Fisher Scientific). WCL was subjected to non-reducing or reducing 8-12% gel SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat milk in TBS containing 1% Tween 20 (TR1027-500-00; Biosesang, Seongnam, South Korea) and probed with antibodies specific for each molecule. Antibodies against MLKL (14993S), phospho-MLKL (Ser358) (91689S), phospho-RIP1 (Ser166) (65746S), phospho-RIP3 (Ser227) (93654), and LDH (2012S) were purchased from Cell Signaling Technology (Danvers, MA, USA), and antibodies against GAPDH (LF-PA0018; Abfrontier, Seoul, Korea), RIP1 (ab72139; Abcam, Cambridge, UK), RIP3 (sc-135170; Santa Cruz Biotechnology, Dallas, TX, USA), HMGB1 (ab78923; Abcam), and LAMP1 (ab24170; Abcam) were used.

[0081] 1-5. Cell membrane fractionation

[0082] To examine whether MLKL oligomers are present in the plasma membrane fraction, plasma membrane fractionation was performed. Briefly, THP-1 cells were harvested and resuspended in 20 mM TBS (pH 7.4) containing 10 mM KCl, 1 mM MgCl2, 1x protease inhibitor (P3100-001; GenDEPOT Inc.), and phosphatase inhibitor (78420; Thermo Fisher Scientific, Massachusetts, USA). The cell suspension was kept on ice for 30 min, passed through a 22-gauge needle 30 times, and centrifuged at 500 × g for 15 min. The resulting supernatant was centrifuged at 20,000 × g for 15 min, fractionated into cytosol, and stored. The pellet was resuspended in lysis buffer, centrifuged at 20,000 × g for 15 min, fractionated into crude membrane, and stored.

[0083] 1-6. Confocal microscopy

[0084] THP-1 cells were seeded on cell culture-treated glass chamber slides and treated with TBZ and candidate compounds that inhibit programmed necrosis for 8 hours. Cells were washed with DPBS and fixed in 4% paraformaldehyde for 10 minutes. After washing three times, cells were incubated in 0.25% Triton X-100 for 10 minutes, blocked in 5% BSA-containing PBS for 30 minutes, and stained with MLKL antibody. Nuclei were stained with DAPI (00-4959-52, Invitrogen, Waltham, MA, USA). Image analysis was performed using a confocal microscope (FV1000, Olympus, Tokyo, Japan). Signal intensities were measured using FluoView FV1000 software.

[0085] 1-7. DNA structures and recombinant proteins

[0086] Human MLKL (hMLKL) plasmid was purchased from OriGene (RC213152). Recombinant wild-type (Wt) hMLKL protein was purchased from Abcam (ab241453). N-terminal hMLKL (Nt-hMLKL, amino acids 2-154), Nt-hMLKL containing one residue mutation at C86A and F148A (Nt-hMLKL C86A and hMLKL F148A ) and triple mutation of C86A / L89A / D94A (Nt-MLKL C86A / L89A / D94A), and the quadruple mutant (Nt-MLKLR145A / R146A / F148A / M150A) of R145A / R146A / F148A / M150A were mutated using a Site-directed Mutagenesis Kit (EZ004S; Enzynomics, Daejeon, South Korea). Nt-hMLKL (Nt-hMLKLC86A / L89A / D94 / R145A / R146A / F148A / M150A), which contains both the triple and quadruple mutants (C86A / L89A / D94 / R145A / R146A / F148A / M150A), was synthesized and constructed by Bioneer (Daejeon, S, South Korea). These constructs were subcloned into pBT7-N-His and pET-15b plasmid vectors. E. coli BL21 cells transformed with the cloned expression vector were selected from LB agar plates and cultured in 2 L LB medium supplemented with 100 μg / mL ampicillin at 37°C and 200 rpm until the optical density at 600 nm (OD) reached 0.5–0.6. The culture was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (367-93-1; Sigma-Aldrich) at 30°C and 200 rpm for 18 h. Cells were harvested, resuspended in lysis buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1% Triton X-100, and 5% glycerol), and sonicated. Cell lysates were centrifuged at 15,000 rpm for 40 min at 4°C to remove cell debris. The clear supernatant was subjected to Ni-NTA affinity chromatography (175018169; QIAGEN, Hilden, Germany). Purified proteins were quantified using a Protein Assay Kit (23225, Thermo Fisher Scientific).

[0087] 1-8. Surface plasmon resonance (SPR) analysis

[0088] The interactions of Wt-hMLKL and Nt-hMLKL proteins with selected drug candidates, APO or Ox-APO, were detected by SPR analysis using a Biacore® T200 (GE Healthcare, Chicago, IL, USA). A 10 mM sodium acetate buffer (pH 4.0–6.0) was used for pH scouting. Each MLKL protein was immobilized on the surface of a CM5 sensor chip (GE Healthcare). The coupling process was performed based on the molecular weights of MLKL protein (MW ligand) and APO (MW analyte) to derive the R ligand values. APO was injected (at a flow rate of 10 μL / min) in 10 mM HEPES (pH 7.4) running buffer at 25°C. The sensorgrams were recorded and analyzed in real time using the control software of the BIAcore T200 system. Six concentrations of APO were used for binding to derive K values. All data were calculated using BIAevaluation software (GE Healthcare).

[0089] 1-9. NMR experiments

[0090] 13 C / 15 To obtain N-labeled Nt-hMLKL protein, transformed E. coli cells were cultured in a nitrogen- and carbon-only medium, respectively. 15 N]NH4Cl and [ 13Cells were grown in M9 minimal medium containing [C]glucose (Becton Dickinson & Co, Sparks, MD, USA). All labeled materials were purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Protein expression was induced in cells with isopropyl-β-D-thiogalactopyranoside (IPTG) and then harvested. Nt-hMLKL protein was purified using Ni-affinity column chromatography followed by TEV cleavage during dialysis. Further purification was performed using size-exclusion column chromatography using a Superdex-75 column (Cytiva Swedish AB, Uppsala, Sweden). The concentration of the purified Nt-hMLKL protein was determined by UV absorbance at 280 nm and stored at -70°C.

[0091] NMR experiments were performed at 25°C in NMR buffer (pH 7.0, 20 mM HEPES, 100 mM NaCl, 5% D2O) to assign backbone chemical shifts of the purified proteins using triple resonance NMR spectra. The chemical shift values ​​of Nt-hMLKL (BMRB code: 25135) were used as a reference. NMR data were processed using the NMRpipe program and analyzed using the NMRFAM-Sparky program. Chemical shift perturbation (CSP) experiments were used to monitor drug molecule binding, and chemical shift differences were calculated using a previously reported method. 1 H- 15 N heteronuclear single-quantum correlation spectra were recorded in the absence and presence of APO. The concentration of Ox-APO was estimated from peak integration of the 1D spectra. CSP data were visualized using the UCSF Chimera program (https: / www.cgl.ucsf.edu / chimera / ).

[0092] 1-10. DSS-induced colitis

[0093] The experiments were performed using 6- to 8-week-old C57BL / 6 male mice (Central Lab Animal Inc., Seoul, S. Korea) according to procedures approved by the Institutional Animal Care and Use Committee of Yonsei University Laboratory Animal Research Center (YLARC, 2018-0024). For DSS-induced colitis, the mice were administered 2.5% DSS (36-50 kDa, 9011-18-1, MP Biomedicals, Solon, OH, USA) in drinking water for 8 days. Body weight and disease activity index (DAI) scores were recorded daily. DAI was calculated based on body weight loss (0 = none, 1 = 1% - 5% body weight loss, 2 = 5% - 10% body weight loss, 3 = 10% - 15% body weight loss, 4 = 15% or more body weight loss), stool consistency / diarrhea (0 = normal, 2 = loose stool, 4 = watery diarrhea), and bleeding (0 = no bleeding, 2 = slight bleeding, 4 = severe bleeding). At the end of the experiment, mice were sacrificed and tissue samples were prepared.

[0094] 1-11. APAP-induced liver injury animal model

[0095] BALB / c male mice (Central Lab Animal Inc., SEOUL, S. Korea), 6–8 weeks of age, were used in accordance with procedures approved by the Institutional Animal Care and Use Committee of Yonsei University Laboratory Animal Research Center (YLARC, 2018-0024). After fasting for 12 h, the mice were administered intraperitoneally (i.p.) with acetaminophen (APAP; 400–500 mg / kg, 103-90-2, Sigma-Aldrich) to induce liver damage. Subsequently, 15 mg / kg of Ox-APO or Re-APO was injected i.p. once, and the mice were sacrificed 24 h later, and blood and liver tissues were used for analysis.

[0096] Liver tissues were perfused with DPBS, fixed with 4% paraformaldehyde (PFA; PC2031-050-00, Biosesang), and embedded in paraffin to prepare tissue slides. Tissue sections were analyzed for the location of p-MLKL by immunohistochemistry (IHC) using an anti-p-MLKL antibody (MA5-32752, Thermo Fisher Scientific). In addition, histopathological analysis was performed using hematoxylin (1.05175; Sigma-Aldrich) and eosin (17372-87-1; Sigma-Aldrich) staining, and serum ALT (Alanine Aminotransferase; 3250, FUJIFILM) and AST (Aspartate Aminotransferase; 3150, FUJIFILM) levels were measured to assess the degree of liver damage.

[0097] 1-12. Tissue sample preparation and histological evaluation

[0098] The colons of each mouse were removed, measured in length, dissected, fixed in Bouin's fixative (50% ethanol / 5% acetic acid in distilled water), and embedded in paraffin. Tissue sections were stained with hematoxylin (1.05175; Sigma-Aldrich) and eosin (17372-87-1; Sigma-Aldrich). Histological evaluation of colitis lesions along the entire colon length was performed according to the following scoring system: degree of crypt structural damage (0–3), degree of inflammatory cell infiltration (0–3), and submucosal edema (0–3). For periodic acid-Schiff (PAS) staining, deparaffinized and rehydrated tissue sections were treated with 0.5% periodic acid (10450-60-9; Junsei Chemical Co., Ltd., Tokyo, Japan) for 5 min, then treated with Schiff reagent (3952016; Sigma-Aldrich) for 10 min at room temperature, and then washed with water for 7 min. The sections were washed with TBS for 1 min, dehydrated with ethyl alcohol, and coverslipped.

[0099] 1-13. Statistical Analysis

[0100] Analysis of experimental data was performed using GraphPad Prism ((GraphPad Software Inc., San Diego, CA, USA). Results are expressed as mean, SD, SEM as indicated in individual figure legends. A value of p < 0.05 was considered statistically significant.

[0101]

[0102] Manufacturing Example 1: Manufacturing of the compound of the present invention

[0103] Oxidized APO (Oxoapomorphine, Ox-APO) was prepared by incubating apomorphine in DPBS at 25°C in the presence of light and air for 24 hours, and then removing the precipitate through filtration using 0.2 μm.

[0104] APO was found to spontaneously auto-oxidize in solution over time in the presence of light and air to form green-colored oxoapomorphine (Ox-APO) (Fig. 1A).

[0105] Oxidative instability of APO is a major barrier to developing therapeutic APO in aqueous solutions. When freshly dissolved APO was incubated at 25°C for 36 h, real-time 1D 1H-NMR spectra revealed that the level of APO oxidation increased with increasing retention time relative to the normalized concentration, reaching saturation after 24 h (Fig. 1B).

[0106]

[0107] Experimental Example 1: Confirmation of Ox-APO's ability to inhibit MLKL oligomerization.

[0108] To observe the effect of Ox-APO on MLKL oligomerization, APO was cultured in aqueous solution for 24 h and treated with cells. Western blot analysis results confirmed that Ox-APO strongly inhibited the formation of MLKL tetramers and octamers (Fig. 2). Furthermore, while APO showed an effect at concentrations above 20 μM, Ox-APO inhibited the tetramerization and octamerization of p-MLKL in a concentration-dependent manner (Figs. 3A and 3B). However, Ox-APO had little effect on the phosphorylation of MLKL (Fig. 3A).

[0109] We performed SPR analysis to measure the affinity binding of APO and Ox-APO to MLKL. Wt-hMLKL protein contains an N-terminal domain (Nt-hMLKL, amino acids 2-154, MW: 23 kDa) involved in oligomerization and a C-terminal domain (Ct-hMLKL, amino acids 155-471, MW: 31 kDa) involved in phosphorylation. CM5 chips were coated with endotoxin-free wild-type human MLKL protein (Wt-hMLKL) and the N-terminal domain (Nt-hMLKL). The K values ​​of APO and Ox-APO for Wt-hMLKL protein were 2.442 × 10, respectively. -7 M and 4.998x10 -9 It was measured as M. Ox-APO was confirmed to bind to Wt-hMLKL 48.9 times more potently than APO (Fig. 4). Ox-APO (1.312x10) for Nt-hMLKL involved in oligomerization -6 The combination of M) is APO(1.14x10 -3 It was confirmed that the binding of M) was 1000 times higher (Fig. 4).

[0110]

[0111] Experimental Example 2: Confirmation of improvement in DSS-induced colitis by Ox-APO.

[0112] To observe the inhibitory effect of APO on colitis symptoms associated with programmed cell necrosis, an in vivo experiment was conducted using a mouse DSS-induced colitis model. C57BL / 6 mice were treated with 2.5% DSS in drinking water for 8 days, and APO was injected intraperitoneally (ip) daily (Fig. 5A). NEC-1 and DPBS served as positive and negative controls, respectively. Body weight and DAI scores were recorded daily, and colon samples were prepared at the end of the experiment.

[0113] As a result, H&E and PAS staining analyses showed that the 15 mg / kg Ox-APO treatment group showed better histological scores than the 15 mg / kg APO treatment group (Fig. 5B). The average histological scores of 1.5 and 15 mg / kg Ox-APO and 15 mg / kg APO were 3.0, 1.2, and 2.6, respectively (Fig. 5C). In addition, the DAI score and colon length also showed that Ox-APO was more effective than APO at the same concentration (Figs. 5D and 5E). The effect of 1.5 mg / kg Ox-APO treatment was similar to that of 15 mg / kg APO treatment. In conclusion, it was confirmed that Ox-APO could improve the symptoms of DSS-induced mouse colitis better than APO.

[0114]

[0115] Experimental Example 3: Analysis of MLKL binding ability of Ox-APO

[0116] Using mutational studies and SPR analysis of hMLKL, we validated the covalent binding of Ox-APO to Cys86 and adjacent residues (Leu89, Ala91, Asp94, Arg145, Arg146, Phe148, and Met150) using a 3D docking model. We observed that Ox-APO binding to hMLKL was reduced when necrosulfonamide (NSA), an MLKL inhibitor that covalently targets Cys86, was used ( Fig. 6A ).

[0117] We performed triple mutations (C86A, L89A, D94A) in three residues within α4 of Nt-hMLKL for SPR analysis. As a result, both Ox-APO and APO were mutated Nt-hMLKL. C86A / L89A / D94A showed that it does not bind to Ox-APO. A quadruple mutant (hMLKLR145A / R146A / F148A / M150A) containing R145A, R146A, F148A, and M150A in α6 of hMLKL showed some binding to Ox-APO, but not to APO (Fig. 6B-C). Collectively, these results confirm that Cys86, Phe148, and their neighboring residues play a crucial role in mediating the interaction between Ox-APO and hMLKL.

[0118] Experimental Example 4: Effect of Ox-APO in an APAP-induced liver injury model

[0119] The in vivo efficacy of Ox-APO was confirmed in an APAP-induced liver injury model using BALB / c mice. In the APAP-treated group, liver tissue darkened, but in the groups treated with Ox-APO and Re-APO at a concentration of 15 mg / kg, the liver tissue color was similar to that of the normal group treated with DPBS alone (Fig. 7B). H&E staining revealed extensive necrotic areas around the central vein in the APAP-treated group, whereas almost no necrotic areas were observed in the Ox-APO and Re-APO-treated groups (Fig. 7C). IHC analysis confirmed that the membrane localization of p-MLKL was significantly increased by APAP administration, but was significantly suppressed by Ox-APO and Re-APO treatment (Fig. 7D). In addition, serum ALT and AST levels were restored to normal levels by Ox-APO and Re-APO treatment (Fig. 7E).

[0120]

[0121] Experimental Example 5: Confirmation of the effect of Ox-APO on p-MLKL multimerization inhibition by cold shock.

[0122] THP-1 cells were pretreated with the pan-caspase inhibitor Z-VAD (tlrl-vad; InvivoGen) and each chemical for 1 h, and then TNF-α (TNF0501; NKMAXBio, Seongnam, Korea) and Smac mimetic BV6 (B1332-5; Biovision, San Francisco, CA, USA) (abbreviated as TBZ) and 20 μM Ox-APO were added and incubated for 8 h at 37°C plus 4°C for cold shock. The cells were then harvested using centrifugation at 1,500 × g for 5 min at 4°C and probed with antibodies specific for each molecule by Western blot analysis. WCL was subjected to non-reducing or reducing 8-12% gel SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat milk in TBS containing 1% Tween 20 (TR1027-500-00; Biosesang, Seongnam, South Korea) and probed with antibodies specific for each molecule. The antibody against phospho-MLKL (Ser358) (91689S) was purchased from Cell Signaling Technology (Danvers, MA, USA), and the antibody against GAPDH (LF-PA0018; Abfrontier, Seoul, South Korea) was used.

[0123] In this experiment and the previous experimental example, it was confirmed that the multimerization of p-MLKL that appears at 37°C is inhibited by Ox-APO, and this was confirmed at 4°C to confirm its possibility as a preservative in organ transplantation. As a result, in both the group treated with TBZ and the group untreated at 4°C in THP-1 cells, the octamer band of p-MLKL increased, whereas both the octamer and tetramer bands were confirmed to decrease in the group treated with Ox-APO (Fig. 8). These results clearly support the possibility that the compound of the present invention can be used as an effective ingredient in an organ preservation solution by effectively inhibiting the formation of octamer and tetramer of p-MLKL even in a low-temperature (4°C) environment and thereby inhibiting programmed cell necrosis.

Claims

1. A compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof, Toxic epidermal necrolysis (TEN), Stevens-Johnson syndrome (SJS), burns, frostbite, erythema multiforme, skin diseases caused by bacterial or viral infections, pityriasis versicolor, lupus erythematosus, lichen planus, drug rash, cutaneous vasculitis, stroke, autoimmune disease, inflammatory bowel disease (IBD), retinal degeneration, sepsis, chronic obstructive pulmonary disease, acute respiratory distress disorder, transfusion-related acute lung injury, transplant rejection, atherosclerosis, aortic aneurysm, myocardial infarction, terminal ileitis, acute kidney injury, renal ischemia-reperfusion injury A pharmaceutical composition for the prevention or treatment of diseases accompanied by or related to necroptosis selected from ischemia reperfusion injury, liver injury, steatohepatitis, pancreatitis, and bone marrow failure, and cell death during organ transplantation. <Chemical Formula 1> 2. In paragraph 1, A pharmaceutical composition characterized in that the compound or a pharmaceutically acceptable salt thereof binds to MLKL (mixed lineage kinase domain like pseudokinase) and inhibits oligomerization.

3. In paragraph 1, A pharmaceutical composition wherein the disease accompanied by the above cell necrosis is inflammatory bowel disease.

4. In paragraph 3, A pharmaceutical composition, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

5. In paragraph 1, The above pharmaceutical composition is a pharmaceutical composition in the form of an injection.

6. A pharmaceutical composition according to claim 5, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

7. A composition for long-term preservation comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: <Chemical Formula 1> 8. A long-term preservation solution containing a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: <Chemical Formula 1>

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