Triphenylphosphonium Cation-Conjugated Thiadiazole Derivatives as Mitochondrial Reactive Oxygen Inhibitors

Abstract

The novel triphenylphosphonium cationic-bound thiadiazole derivative according to the present invention or a pharmaceutically acceptable salt thereof exhibits inhibition of reactive oxygen species within mitochondria and inhibition of Rubicon-p22Phox protein interaction activity, and thus a pharmaceutical composition comprising said derivative can be usefully utilized for the prevention and treatment of inflammatory diseases.

Description

Technology Field

[0001] The present invention relates to a novel triphenylphosphonium cationic-linked thiadiazole derivative having mitochondrial reactive oxygen species inhibitory activity and the use thereof. Background Technology

[0002] Oxygen-derived free radicals, known as reactive oxygen species (ROS), are small, highly active molecules such as superoxide and hydroxide. The concentration of ROS plays a significant role in both physiological and pathological cellular responses. ROS production is strictly regulated by specific cellular subdivisions and complex cellular signaling and negative feedback mechanisms. However, under certain conditions, excessive ROS generation induces oxidative stress, damaging systems within the host cell. Consequently, this leads to various diseases, including inflammatory bowel disease (IBD), sepsis, atherosclerosis, heart failure, cancer, aging, and neurodegeneration.

[0003] Mitochondria are membrane-bound organelles essential for maintaining cellular energy and performing various metabolic functions through oxidative phosphorylation. Mitochondria are a major source of reactive oxygen species (ROS). Furthermore, mitochondrial ROS (mtROS) play a key role in regulating inflammatory signaling. However, the overproduction of ROS can cause oxidative damage to cellular components such as proteins, membranes, and DNA, leading to mitochondrial dysfunction. Mitochondrial dysfunction is a major underlying cause of the pathogenesis of various diseases, including metabolic disorders, age-related diseases, and chronic inflammatory conditions. Increasing research has demonstrated that disrupted gut function may occur in response to mitochondrial dysregulation. In particular, elevated mtROS levels have been observed primarily in patients with IBD. Given the importance of mitochondrial function in the gut immune system, targeting the regulation of mtROS could be a promising therapeutic strategy for improving the pathogenesis of colitis.

[0004] Rubicon is a protein involved in cellular autophagy and is known to play a crucial role in various intracellular processes. It primarily acts as an inhibitor of autophagy and regulates the activation phase of the process. Autophagy refers to the cellular process of removing damaged organelles or unnecessary substances and recycling them; Rubicon regulates the formation and maturation stages of autophagosomes, which are part of this process. Rubicon interacts with another protein called p22phox to increase the production of reactive oxygen species (ROS) in the cytoplasm, and as a component of NADPH oxidase, it is involved in the production of ROS. ROS are associated with oxidative stress within cells, and their excessive production can cause cell damage. Since Rubicon is involved not only in regulating autophagy but also in ROS production, it can be viewed as a protein that significantly influences intracellular oxidative stress and inflammatory responses.

[0005] Meanwhile, IBD is an idiopathic, recurrent, and chronic inflammatory disease of the intestinal and colonic regions affecting all ages, accompanied by symptoms such as diarrhea, bloody stools, abdominal pain, and weight loss. Although the precise pathophysiology of IBD is not fully understood, it is known to be caused by a complex interplay of factors, including genetic background, environmental factors such as diet, lifestyle, and smoking, as well as disorders of gut microbiota and immune regulation. IBD is classified into two main types: Crohn's disease (which can affect discontinuous sections of the gastrointestinal tract from the mouth to the anus) and ulcerative colitis (which can affect only the large intestine). Currently, while there are various types of treatments to manage the severity of IBD (e.g., anti-inflammatory drugs, 5-aminosalicylates and mesalazine, immunosuppressants, corticosteroids, biological and biosimilar drugs, antibodies, anti-cytokines and anti-adhesion molecules, fecal microbiota transplantation), there is no definitive cure, and a significant proportion of patients do not respond to treatment or lose their response to it.

[0006] Therefore, there is an urgent need for an alternative treatment for inflammatory diseases that is highly effective, accurate, and safe for long-term treatment. Prior art literature

[0007] (Patent Document 0001) KR 10-2023-0061243 A(Patent Document 0002) KR 10-1840674 B1

[0008] Identification of highly potent and selective inhibitor, TIPTP, of the p22phox-Rubicon axis as a therapeutic agent for rheumatoid arthritis, Sci Rep. 2020 Mar 12;10(1):4570.doi: 10.1038 / s41598-020-61630-x.Mito-TIPTP Increases Mitochondrial Function by Repressing the Rubicon-p22phox Interaction in Colitis-Induced Mice, Antioxidants (Basel). 2021 Dec 6;10(12):1954.doi: 10.3390 / antiox10121954. The problem to be solved

[0009] The present invention was devised to solve the aforementioned problems, and the inventors have made diligent research efforts to find a novel substance with potential for development as a treatment for inflammatory diseases such as inflammatory bowel disease and sepsis.

[0010] As a result, a novel triphenylphosphonium cation-bound thiadiazole derivative exhibiting inhibitory activity against reactive oxygen species within mitochondria and inhibition of Rubicon-p22Phox protein interactions was identified, and the present invention was completed based on this.

[0011] Accordingly, the object of the present invention is to provide a novel triphenylphosphonium cation-bound thiadiazole derivative or a pharmaceutically acceptable salt thereof that exhibits activity of inhibiting reactive oxygen species within mitochondria and inhibiting Rubicon-p22Phox protein interactions.

[0012] Another objective of the present invention is to provide a method for preparing a novel triphenylphosphonium cation-bound thiadiazole derivative that exhibits inhibitory activity against reactive oxygen species within mitochondria and inhibitory activity against Rubicon-p22Phox protein interactions.

[0013] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising the triphenylphosphonium cationic-bound thiadiazole derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

[0014] Another object of the present invention is to provide a method for treating an inflammatory disease, comprising the step of administering the triphenylphosphonium cationic-bound thiadiazole derivative or a pharmaceutically acceptable salt thereof to an individual or subject in need thereof.

[0015] Another object of the present invention is to provide the triphenylphosphonium cationic-bound thiadiazole derivative or a pharmaceutically acceptable salt thereof for use in the treatment of inflammatory diseases.

[0016] Another object of the present invention is to provide a use of the triphenylphosphonium cationic-bound thiadiazole derivative or a pharmaceutically acceptable salt thereof for use in the manufacture of a drug for treating inflammatory diseases.

[0017] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0018] To achieve the above-mentioned objective of the present invention, a compound of Formula 1 or a pharmaceutically acceptable salt thereof is provided.

[0019] [Chemical Formula 1]

[0020]

[0021] In the above chemical formula 1,

[0022] R1 is And,

[0023] R2 is a haloalkyl or And,

[0024] R3 is And,

[0025] X1 is an NH or nitrogen atom, and

[0026] X2 is an NH or oxygen atom, and

[0027] n is 1 to 10.

[0028] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising a derivative of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0029] As one embodiment of the present invention, the composition can inhibit reactive oxygen species within mitochondria and inhibit Rubicon-p22Phox protein interaction activity. Effects of the invention

[0030] The novel triphenylphosphonium cationic-bound thiadiazole derivative according to the present invention or a pharmaceutically acceptable salt thereof exhibits inhibition of reactive oxygen species within mitochondria and inhibition of Rubicon-p22Phox protein interaction activity, and thus a pharmaceutical composition comprising said derivative can be usefully utilized for the prevention and treatment of inflammatory diseases. Brief explanation of the drawing

[0031] Figure 1 is a graph showing the results of evaluating the active oxygen inhibitory efficacy of a compound according to the present invention. Figure 2 is a figure showing the results of evaluating the efficacy of a compound according to the present invention in inhibiting Rubicon-p22phox interactions. Specific details for implementing the invention

[0032] The present invention will be described in detail below.

[0033] The present invention provides a compound of the following formula 1 or a pharmaceutically acceptable salt thereof.

[0034] [Chemical Formula 1]

[0035]

[0036] In the above chemical formula 1,

[0037] R1 is And,

[0038] R2 is a haloalkyl or And,

[0039] R3 is And,

[0040] X1 is an NH or nitrogen atom, and

[0041] X2 is an NH or oxygen atom, and

[0042] n is 1 to 10.

[0043] Here, “haloalkyl” refers to a compound in which one or more hydrogen atoms of an alkyl group (a hydrocarbon chain consisting only of carbon and hydrogen) are substituted with halogen atoms (e.g., fluorine, chlorine, bromine, iodine, etc.). Haloalkyls have a structure in which one or more hydrogen atoms in an alkyl chain are replaced by halogen atoms (e.g., methyl chloride (CH3Cl), bromoethane (C2H5Br), 1-chloropropane (C3H7Cl)), and haloalkyls are formed by halogen atoms bonding to carbons of the alkyl chain, and halogens can be bonded at various positions as long as the attachment does not violate atomic requirements. Additionally, haloalkyls may include one or more non-hydrogen substituents.

[0044] As another embodiment of the present invention, the compound of Formula 1 is,

[0045] ( R )-2-(3-(4,5,6,7-tetrahydro-2 H -Indazole-2-il)Phenoxy)-N -(5-trifluoromethyl)-1,3,4-thiadiazole-2-yl)propanamide;

[0046] ( R )-triphenyl(10-(2-((2-(3-(4,5,6,7-tetrahydro-2 H -Indazole-2-yl)phenoxy)propanoyl)imino)-5-(trifluoromethyl)-1,3,4-thiadiazole-3(2 H )-1)decyl)phosphonium bromide;

[0047] (10-Oxo-10-((2-(3-((( R )-1-oxo-1-((5-(trifluoromethyl)-1,3,4-thiadiazole-2-yl)amino)propane-2-yl)oxy)phenyl)-4,5,6,7-tetrahydro-2 H -Indazole-5-yl)methoxy)decyl)triphenylphosphonium bromide;

[0048] (10-Oxo-10-((2-(3-((( R )-1-oxo-1-((5-(trifluoromethyl)-1,3,4-thiadiazole-2-yl)amino)propane-2-yl)oxy)phenyl)-4,5,6,7-tetrahydro-2 H -Indazole-5-yl)amino)decyl)triphenylphosphonium bromide;

[0049] ( R )-(10-oxo-10-((5-(2-(3-(4,5,6,7-tetrahydro-2 H -Indazole-2-yl)phenoxy)propaneamido)-1,3,4-thiadiazole-2-yl)methoxy)decyl)triphenylphosphonium bromide; or

[0050] ( R )-(10-oxo-10-(((5-(2-(3-(4,5,6,7-tetrahydro-2 H -Indazole-2-yl)phenoxy)propaneamido)-1,3,4-thiadiazole-2-yl)methyl)amino)decyl)triphaylphosphonium bromide; may be

[0051] In one embodiment of the present invention, the compound of Formula 1 may be a compound having the structure of Formula 2 below.

[0052] [Chemical Formula 2]

[0053]

[0054] In the above chemical formula 2,

[0055] R1 is And,

[0056] R2 is a haloalkyl or And,

[0057] X1 is an NH or nitrogen atom, and

[0058] X2 is an NH or oxygen atom, and

[0059] n is 1 to 10.

[0060] As another embodiment of the present invention, the compound of Formula 2 is,

[0061] ( R )-triphenyl(10-(2-((2-(3-(4,5,6,7-tetrahydro-2 H -Indazole-2-yl)phenoxy)propanoyl)imino)-5-(trifluoromethyl)-1,3,4-thiadiazole-3(2 H It may be )-1)decyl)phosphonium bromide.

[0062] As another embodiment of the present invention, the compound of Formula 1 may be a compound having the structure of Formula 3 below.

[0063] [Chemical Formula 3]

[0064]

[0065] In the above chemical formula 3,

[0066] R2 is a haloalkyl or And,

[0067] R3 is And,

[0068] X1 is an NH or nitrogen atom, and

[0069] X2 is an NH or oxygen atom, and

[0070] n is 1 to 10.

[0071] As another embodiment of the present invention, the compound of Formula 3 is,

[0072] (10-Oxo-10-((2-(3-((( R )-1-oxo-1-((5-(trifluoromethyl)-1,3,4-thiadiazole-2-yl)amino)propane-2-yl)oxy)phenyl)-4,5,6,7-tetrahydro-2 H -Indazole-5-yl)methoxy)decyl)triphenylphosphonium bromide; or

[0073] (10-Oxo-10-((2-(3-((( R )-1-oxo-1-((5-(trifluoromethyl)-1,3,4-thiadiazole-2-yl)amino)propane-2-yl)oxy)phenyl)-4,5,6,7-tetrahydro-2 H -Indazole-5-yl)amino)decyl)triphenylphosphonium bromide; may be

[0074] As another embodiment of the present invention, the compound of Formula 1 may be a compound having the structure of Formula 4 below.

[0075] [Chemical Formula 4]

[0076]

[0077] In the above chemical formula 4,

[0078] R1 is And,

[0079] R3 is And,

[0080] X1 is an NH or nitrogen atom, and

[0081] X2 is an NH or oxygen atom, and

[0082] n is 1 to 10.

[0083] As another embodiment of the present invention, the compound of Formula 4 is,

[0084] ( R)-(10-oxo-10-((5-(2-(3-(4,5,6,7-tetrahydro-2 H -Indazole-2-yl)phenoxy)propaneamido)-1,3,4-thiadiazole-2-yl)methoxy)decyl)triphenylphosphonium bromide; or

[0085] ( R )-(10-oxo-10-(((5-(2-(3-(4,5,6,7-tetrahydro-2 H -Indazole-2-yl)phenoxy)propaneamido)-1,3,4-thiadiazole-2-yl)methyl)amino)decyl)triphaylphosphonium bromide; may be

[0086] The present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising as an active ingredient a compound of Formulas 1 to 4, a derivative thereof, or a pharmaceutically acceptable salt thereof.

[0087] As another embodiment of the present invention, the inflammatory disease may be one or more selected from the group consisting of inflammatory bowel disease, sepsis, septic shock, peritonitis, nephritis, acute bronchitis, chronic bronchitis, osteoarthritis, intestinal spondylitis, chronic obstructive pulmonary disease, rheumatoid arthritis, acute lung injury, and bronchopulmonary dysplasia, but is not limited thereto.

[0088] As another embodiment of the present invention, the composition may inhibit reactive oxygen species within mitochondria or inhibit Rubicon-p22Phox protein interactions.

[0089] Meanwhile, the compound of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful.

[0090] The term “salt” as used in the present invention may be an acid addition salt formed by a pharmaceutically acceptable free acid. The acid addition salt is obtained from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromide, hydroiodide, nitrous acid, or phosphoric acid, and non-toxic organic acids such as aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates and alkandioates, aromatic acids, and aliphatic and aromatic sulfonic acids. These pharmaceutically non-toxic salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphate chloride, bromides, iodides, fluorides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caprates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, malieates, butyne-1,4-dioate, hexane-1,6-dioate, benzoates, chlorobenzoates, methyl benzoates, dinitrobenzoates, hydroxybenzoates, and methoxybenzoates. Includes phthalates, terephthalates, benzenesulfonates, toluenesulfonates, chlorobenzenesulfonates, xylenesulfonates, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, or mandelate.

[0091] The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving a compound in an excess amount of aqueous acid solution and precipitating the salt using a water-miscible organic solvent, for example, methanol, ethanol, acetone, or acetonitrile. Alternatively, it can be prepared by drying the mixture after evaporating the solvent or excess acid, or by suction filtration of the precipitated salt.

[0092] In addition, pharmaceutically acceptable metal salts can be produced using a base. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess amount of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, it is pharmaceutically suitable to produce sodium, potassium, or calcium salts as the metal salts. The corresponding silver salt is obtained by reacting the alkali metal or alkaline earth metal salt with a suitable base salt (e.g., silver nitrate).

[0093] In addition, the compounds of the present invention include not only pharmaceutically acceptable salts but also all salts, isomers, hydrates, and solvates that can be prepared by conventional methods.

[0094] As can be seen in the following examples, the compound of Formula 1 can be used as an intramitochondrial reactive oxygen species inhibitor or a Rubicon-p22Phox protein interaction inhibitor, and as described in the background of the invention, it is a widely known fact to those skilled in the art that intramitochondrial reactive oxygen species inhibitors or Rubicon-p22Phox protein interaction inhibitors can be used for the treatment of inflammatory diseases.

[0095] The present invention provides a pharmaceutical composition for the prevention or treatment of an inflammatory disease comprising a compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, the use of the compound of Formula 1 or a pharmaceutically acceptable salt thereof for the treatment of said disease, and a method for treating said disease comprising administering a therapeutically effective amount of the compound of Formula 1 or a pharmaceutically acceptable salt thereof to a subject.

[0096] As used in the present invention, the term "prevention" refers to any act of suppressing an inflammatory disease or delaying its onset by administering a pharmaceutical composition according to the present invention.

[0097] As used in the present invention, the term "treatment" refers to any act in which symptoms of an inflammatory disease are improved or beneficially altered by the administration of a pharmaceutical composition according to the present invention.

[0098] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient. In this case, the pharmaceutically acceptable carrier is one that is commonly used in formulations and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, in addition to the above components, it may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc.

[0099] The pharmaceutical composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage may be appropriately selected by those skilled in the art, although it depends on the patient's condition and body weight, the severity of the disease, the form of the drug, the route of administration, and the time.

[0100] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by taking all of the above-mentioned factors into account, and this can be easily determined by a person skilled in the art.

[0101] Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, gender, condition, body weight, absorption rate, inactivation rate, and excretion rate of the active ingredient in the body, the type of disease, and concomitant drugs. Generally, 0.0001 to 1000 mg, preferably 0.001 to 500 mg per kg of body weight, may be administered daily or every other day, or divided into 1 to 3 doses per day. However, since the dosage may increase or decrease depending on the route of administration, severity of obesity, gender, body weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0102] In the present invention, "individual" refers to a subject requiring treatment for a disease, and more specifically, to mammals such as humans or non-human primates, mice, dogs, cats, horses, and cattle.

[0104] Hereinafter, preferred manufacturing examples and embodiments are presented to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the present invention, and the content of the present invention is not limited by the following manufacturing examples and embodiments.

[0106] [Synthesis of Mito-TIPTP-1]

[0107] <Preparation Example 1> 2-(3-methoxyphenyl)-4,5,6,7-tetrahydro-2 H -Indazole (Compound 1)

[0108]

[0109] 4,5,6,7-tetrahydro-2 H To a solution of indazole (1.00 g, 8.2 mmol) dissolved in DMF (11 ml), 3-iodoanisole (1.3 ml, 9.8 mmol), trans-1,2-cyclohexanediamine (197 μL, 1.64 mmol), K2CO3 (2.38 g, 17.2 mmol), and CuI (80 mg, 0.40 mmol) were added, and the mixture was stirred at 130 °C for 15 hours. After the reaction was complete, water was added, the organic layer was extracted using EtOAc, and washed with water and saturated brine. The organic layer was dried by adding anhydrous MgSO4, and the solvent was removed under reduced pressure. The mixture was purified by silica gel column chromatography (Hexane / EtOAc = 10:1) to obtain a pure colorless oil target compound. 1 It was obtained with a yield of 38%.

[0110] 1 ¹H NMR (400 MHz, CDCl₃) δ 7.59 (s, 1H), 7.30 (t, J = 8.1 Hz, 1H), 7.24 (s, 1H), 7.16 (dd, J = 8.0, 1.1 Hz, 1H), 6.77 (dd, J = 8.2, 2.4 Hz, 1H), 3.85 (s, 3H), 2.79 (t, J= 5.9 Hz, 2H), 2.62 (t, J = 5.9 Hz, 2H), 1.87-1.75 (m, 4H).

[0112] <Preparation Example 2> 3-(4,5,6,7-tetrahydro-2 H -Indazole-2-yl)phenol (compound 2)

[0113]

[0114] compound 1 712 mg (3.12 mmol) was dissolved in 31 ml of DCM and cooled to 0 °C. Then, 6.2 ml of BBr3 (1.0 M DCM) was slowly added at 0 °C, followed by stirring at 25 °C for 18 hours. After the reaction was complete, water was added, the organic layer was extracted using DCM, and washed with water and saturated brine. The organic layer was dried with anhydrous MgSO4, and the solvent was removed under reduced pressure to obtain the target compound as a pure white solid. 2 It was obtained with an 83% yield.

[0115] 1 ¹H NMR (400 MHz, CDCl₃) δ 7.52 (s, 1H), 7.30 (t, J = 2.2 Hz, 1H), 7.22 (t, J = 8.1 Hz, 1H), 7.02-6.99 (m, 1H), 6.72-6.69 (m, 1H), 2.78 (t, J = 6.2 Hz, 2H), 2.60 (t, J = 6.2 Hz, 2H), 1.85-1.75 (m, 4H). 13 13C NMR (100 MHz, CDCl3) δ 157.7, 151.5, 140.9, 130.4, 130.4, 125.1, 118.3, 113.9, 110.6, 107.4, 23.3, 23.3, 23.2, 20.6.

[0117] <Preparation Example 3> Methyl ( R )-2-(3-(4,5,6,7-tetrahydro-2 H -Indazole-2-yl)phenoxy)propanoate (Compound 3)

[0118]

[0119] compound 2 496 mg (2.31 mmol) was dissolved in 11.6 ml of DMF, followed by the addition of methyl (S)-(-)-2-chloropropionate (0.50 ml, 4.62 mmol) and K2CO3 (639 mg, 4.62 mmol), and the mixture was stirred at 80 °C for 6 hours. After the reaction was complete, water was added, the organic layer was extracted using EtOAc, and the mixture was washed with water and saturated brine. The organic layer was dried by adding anhydrous MgSO4, and the solvent was removed under reduced pressure. The mixture was purified by silica gel column chromatography (Hexane / EtOAc = 3:1) to obtain a pure, colorless oil target compound. 3 It was obtained with a yield of 56%.

[0120] 1 ¹H NMR (400 MHz, CDCl₃) δ 7.57 (s, 1H), 7.30-7.20 (m, 3H), 6.72-6.69 (m, 1H), 4.88 (q, J = 6.8 Hz, 1H), 3.75 (s, 3H), 2.78 (t, J = 6.2 Hz, 2H), 2.62 (t, J = 6.2 Hz, 2H), 1.88-1.74 (m, 4H), 1.63 (d, J = 6.8 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 172.4, 158.4, 151.3, 141.6, 130.2, 123.8, 118.4, 112.0, 111.8, 106.0, 72.7, 52.4, 23.5, 23.4, 23.4, 20.7, 18.5. [α] D 21.0 =+11.0(c=0.5 in CH2Cl2)

[0122] <Preparation Example 4> ( R )-2-(3-(4,5,6,7-tetrahydro-2H-indazole-2-yl)phenoxy)propanoic acid (compound 4)

[0123]

[0124] compound 3 390 mg (1.30 mmol) was dissolved in a mixture of MeOH (6.5 ml) and H2O (2.2 ml), cooled to 0 °C, and then LiOH (37 mg, 1.56 mmol) was added and stirred at 25 °C for 2 hours. After the reaction was complete, 1 N HCl was slowly added until the pH reached 3, and the organic layer was extracted using DCM. The organic layer was dried by adding anhydrous MgSO4, and the solvent was removed under reduced pressure to obtain the target compound as a white solid. 4 It was obtained with a 90% yield.

[0125] 1 ¹H NMR (400 MHz, MeOD) δ 7.82 (s, 1H), 7.34 (t, J = 8.5 Hz, 1H), 7.22-7.20 (m, 1H), 6.80 (m, 1H), 4.89-4.86 (m, 1H), 2.72 (t, J = 6.0 Hz, 2H), 2.63(t, J = 6.2 Hz, 2H), 1.89-1.75 (m, 4H), 1.60 (d, J = 6.8 Hz, 3H). [α] D 22.0 =+16.4 (c=0.5 in CH2Cl2)

[0127] <Preparation Example 5> Ethyl ( R )-5-(2-(3-(4,5,6,7-tetrahydro-2 H -Indazole-2-yl)phenoxy)propanoamido)-1,3,4-thiadiazole-2-carboxylate (Compound 5)

[0128]

[0129] compound 4 After dissolving (143 mg, 0.499 mmol) in DMF (5.0 ml), N , N- Diisopropylethylamine (442 μL, 2.54 mmol), HBTU (378 mg, 0.998 mmol), and ethyl 5-amino-1,3,4-thiadiazole-2-carboxylate (130 mg, 0.749 mmol) were added, and the mixture was stirred at 80 °C for 5 hours. After the reaction was complete, water was added, the organic layer was extracted using EtOAc, and washed with water and saturated brine. The organic layer was dried by adding anhydrous MgSO4, and the solvent was removed under reduced pressure. The mixture was purified by silica gel column chromatography (Hexane / EtOAc = 1:1) to obtain the target compound as a pure orange solid. 5 It was obtained with a yield of 73%.

[0130] 1 ¹H NMR (400 MHz, CDCl₃) δ 10.31 (broad, 1H), 7.60 (s, 1H), 7.37-7.32 (m, 2H), 7.27-7.25 (m, 1H), 6.80 (dd, J = 8.1, 1.8 Hz, 1H), 5.15 (q, J = 6.8 Hz, 1H), 4.53 (q, J = 7.1 Hz 2H), 2.77 (t, J = 6.3 Hz, 2H), 2.62 (t, J = 6.0 Hz, 2H), 1.88-1.76 (m, 4H), 1.70 (d, J = 6.8 Hz, 3H), 1.46 (t, J = 7.1 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 170.4, 161.3, 159.1, 156.9, 155.7, 151.7, 141.7, 130.7, 124.0, 118.8, 112.7, 112.5, 106.6, 74.3, 63.1, 23.4, 23.3, 20.6, 18.2, 14.2. [α] D 23.8=-17.0(c=0.5 in CH2Cl2)

[0132] <Preparation Example 6> ( R) - N -(5-(hydroxymethyl)-1,3,4-thiadiazole-2-yl)-2-(3-(4,5,6,7-tetrahydro-2 H -Indazole-2-yl)phenoxy)propenamide (Compound 6)

[0133]

[0134] compound 5 160 mg (0.362 mmol) was dissolved in 3.6 ml of THF, and 0.54 ml of LiBH4 (2.0 M THF) was slowly added at 0 °C, followed by stirring at 25 °C for 12 hours. After the reaction was complete, the mixture was cooled to 0 °C, and then H2O (25 μL), 15% NaOH aqueous solution (25 μL), and water (75 μL) were added in sequence, followed by stirring at 25 °C for 20 minutes. Subsequently, the mixture was filtered under reduced pressure using Celite 545 and methanol to remove the solvent. The mixture was purified by silica gel column chromatography (Hexane / EtOAc = 1:3) to obtain the target compound as a pure white solid. 6 It was obtained with a yield of 36%.

[0135] 1 ¹H NMR (400 MHz, CDCl₃) δ 7.52 (s, 1H), 7.27-7.25 (m, 1H), 7.23 (s, 1H), 7.13 (d, J = 7.6 Hz, 1H), 6.72 (d, J = 8.1 Hz, 1H), 5.11 (q, J = 6.5 Hz, 1H), 4.93 (s, 2H), 2.74 (t, J = 6.1 Hz, 2H), 2.58 (t, J = 5.9 Hz, 2H), 1.86-1.71 (m, 4H), 1.67 (d, J = 6.7 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ170.4, 166.8, 159.6, 157.4, 151.9, 141.5, 130.4, 124.5, 118.7, 112.5, 112.4, 107.6, 74.5, 59.4, 29.8, 23.4, 23.3, 20.6, 18.3. LRMS (ESI) m / z: [M + H] + calcd for C 19 H 20 N5O3S 400.1; found 400.1.[α] D 26.0 =-6.0 (c=0.5 in CH2Cl2)

[0137] <Preparation Example 7> ( R )- N -(3-(10-bromodecyl)-5-(hydroxymethyl)-1,3,4-thiadiazole-2(3 H )-ylidene)-2-(3-(4,5,6,7-tetrahydro-2H-indazole-2-yl)phenoxy)propenamide (Compound 7)

[0138]

[0139] compound 6 (52 mg, 0.130 mmol) was dissolved in DMF (2.6 ml), and then NaH (5.2 mg, 0.130 mmol, 60% dispersion in mineral oil) and 1,10-dibromodecane (15.7 μL, 0.070 mmol) were added at 0 °C, followed by stirring at 25 °C for 4 hours. After the reaction was complete, water was added, the organic layer was extracted using DCM, and washed with water and saturated brine. The organic layer was dried by adding anhydrous MgSO4, and the solvent was removed under reduced pressure. The mixture was purified by silica gel column chromatography (Hexane / EtOAc = 2:1) to obtain the target compound in a pure colorless oil. 7 It was obtained with a 25% yield.

[0140] 1 ¹H NMR (400 MHz, CDCl₃) δ 7.54 (s, 1H), 7.23-7.16 (m, 3H), 6.74 (dd, J= 10.1, 1.4 Hz, 1H), 5.05 (q, J = 6.7 Hz, 1H), 4.72 (m, 2H), 4.34 (dt, J = 13.2, 7.2 Hz, 2H), 3.41 (t, J = 6.8 Hz, 2H), 2.75 (t, J = 6.4 Hz, 2H), 2.60 (t, J = 6.3 Hz, 2H), 1.87-1.70 (m, 8H), 1.65 (d, J = 6.7 Hz, 3H), 1.42-1.36 (m, 2H), 1.25 (m, 10H). 13 C NMR (100 MHz, CDCl3) δ 181.4, 166.2, 160.6, 159.1, 151.2, 141.3, 130.1, 124.1, 118.3, 112.4, 111.3, 106.0, 76.0, 59.8, 51.0, 34.1, 32.9, 29.46, 29.43, 29.1, 28.8, 28.4, 28.2, 26.5, 23.5, 23.4, 20.7, 19.2. LRMS (ESI) m / z: [M+H] + calcd for C 29 H 40 BrN5O3S 618.2; found 618.3. [α] D 23.7 =+5.2 (c=0.5 in CH2Cl 2)

[0142] <Preparation Example 8> ( R )-(10-(5-(hydroxymethyl)-2-((2-(3-(4,5,6,7-tetrahydro-2 H -Indazole-2-yl)phenoxy)propanoyl)imino)1,3,4-thiadiazole-3(2 H -yl)-decyl)triphenylphosphonium bromide (Compound 8, Mito-TIPTP-1)

[0143]

[0144] 화합물 7(20 mg, 0.0323 mmol) and PPh3 (7.0 mg, 0.0258 mmol) were dissolved in ACN (0.32 ml) and refluxed at 85 °C for 12 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the mixture n - The target compound is a white solid after washing with hexane and Et2O. 8, Mito-TIPTP-1 It was obtained with a yield of 67%.

[0145] 1 ¹H NMR (400 MHz, CDCl₃) δ 7.82-7.65 (m, 15H), 7.48 (s, 1H), 7.16-7.12 (m, 3H), 6.67-6.64 (m, 1H), 5.02 (q, J = 6.7 Hz, 1H), 4.89-4.80 (m, 2H), 4.35-4.27 (m, 2H), 3.68-3.53 (m, 2H), 2.71 (t, J = 6.2 Hz, 2H), 2.57 (t, J = 6.0 Hz, 2H), 1.86-1.70 (m, 8H), 1.61 (d, J = 6.7 Hz, 3H), 1.22-1.11 (m, 3H), 1.03 (m, 9H). 13 13C NMR (100 MHz, CDCl3) δ 180.5, 166.4, 162.5, 159.2, 151.1, 141.4, 135.05, 135.02, 133.7, 133.6, 130.6, 130.4, 130.0, 123.8, 118.9, 118.2, 118.0, 112.1, 111.0, 106.0, 76.2, 59.3, 50.6, 30.5, 30.4, 28.5, 28.4, 27.9, 27.4, 25.7, 23.6, 23.5, 23.4, 22.7, 22.6, 22.5, 22.0, 20.7, 19.3. HRMS (ESI) m / z: [M-Br] + calcd for C 47 H 55 N5O3PS +800.3757; found800.3764. [α] D 22.2 =+5.8 (c=0.5 in CH2Cl2)

[0147] [Synthesis of TIPTP]

[0148] <Preparation Example 9> ( R )-2-(3-(4,5,6,7-tetrahydro-2 H -Indazole-2-il)Phenoxy)- N -(5-trifluoromethyl)-1,3,4-thiadiazole-2-yl)propanamide (Compound 9, TIPTP)

[0149]

[0150] compound 4 After dissolving (0.300 mmol, 1 equiv) in DMF (8.6 ml) N , N - Diisopropylethylamine (1.53 mmol, 5.1 equiv), HBTU (0.600 mmol, 2 equiv), and 2-amino-5-trifluoromethyl-1,3,4-thiadiazole (0.300 mmol, 1 equiv) were added, and the mixture was stirred at 70 °C for 4 hours. After the reaction was complete, water was added, the organic layer was extracted using EtOAc, washed with water and saturated brine, dried with anhydrous MgSO4, and the solvent was removed under reduced pressure. The mixture was purified by silica gel column chromatography (Hexane / EtOAc = 2:1) to obtain the target compound as a pure white solid. 9, TIPTP It was obtained with a 65% yield.

[0151] 1 ¹H NMR (400 MHz, CDCl₃) δ 10.27 (s, broad, 1H), 7.61 (s, 1H), 7.40 (t, J = 2.2 Hz, 1H), 7.37 (t, J = 8.1 Hz, 1H), 7.27-7.26 (m, 1H), 6.81 (dd, J = 7.9, 2.1 Hz, 1H), 5.17 (q,J = 6.7 Hz, 1H), 2.78 (t, J = 6.7 Hz, 2H), 2.63 (t, J = 6.3 Hz, 2H), 1.87-1.76 (m, 4H), 1.71 (d, J = 6.8 Hz, 3H). 13 C NMR (100 MHz, CDCl3) δ 170.5, 160.1, 156.9, 151.7, 141.6, 130.9, 124.2, 121.1, 119.0, 118.4, 113.1, 112.7, 106.7, 74.4, 29.8, 23.4, 23.3, 20.7, 18.3. LRMS (ESI) m / z: [M + H] + calcd for C 19 H 18 F3N5O2S 438.1; found 438.1. [α] D 22.0 =-37.0 (c=0.5 in CH2Cl2)

[0153] [Synthesis of Mito-TIPTP-2]

[0154] <Preparation Example 10> ( R )- N -(3-(10-bromodecyl)-5-(trifluoromethyl)-1,3,4-thiadiazole-2(3 H )-ylidene)-2-(3-(4,5,6,7-tetrahydro-2 H -Indazole-2-yl)phenoxy)propenamide (Compound 10)

[0155]

[0156] Compound 9 (92 mg, 0.210 mmol) was dissolved in DMF (4.2 ml), and then LiOH (6 mg, 0.252 mmol) and 1,10-dibromodecane (71 μL, 0.315 mmol) were added at 0 °C, followed by stirring at 25 °C for 12 hours. After the reaction was complete, water was added, the organic layer was extracted using EtOAc, and washed with water and saturated brine. The organic layer was dried by adding anhydrous MgSO4, and the solvent was removed under reduced pressure. The mixture was purified by silica gel column chromatography (Hexane / EtOAc = 8:1) to obtain the target compound 10 as a pure colorless oil with a yield of 56%.

[0157] 1 ¹H NMR (400 MHz, CDCl₃) δ 7.55 (s, 1H), 7.25-7.17 (m, 3H), 6.72-6.70 (m, 1H), 5.11 (q, J = 6.8 Hz, 1H), 4.43 (dt, J = 13.4, 6.7 Hz, 2H), 3.41 (t, J = 6.8 Hz, 2H), 2.76 (t, J = 6.4 Hz, 2H), 2.60 (t, J = 6.3 Hz, 2H), 1.86-1.74 (m, 8H), 1.67 (d, J = 6.7 Hz, 3H), 1.44-1.36 (m, 2H), 1.25 (m, 10H). 13 13C NMR (100 MHz, CDCl3) δ182.0, 165.4, 159.0, 151.2, 146.4, 145.9, 141.4, 130.1, 123.9, 120.5, 118.3, 117.8, 112.3, 111.3, 106.0, 75.9, 51.9, 34.0, 32.8, 29.4, 29.3, 29.0, 28.8, 28.2, 28.1, 26.4, 23.5, 23.4, 20.7, 19.0. LRMS (ESI) m / z: [M+H] + calcd for C 29 H 37 BrF3N5O2S 656.1; found 656.3.

[0159] <Preparation Example 11> ( R )-triphenyl(10-(2-((2-(3-(4,5,6,7-tetrahydro-2 H -Indazole-2-yl)phenoxy)propanoyl)imino)-5-(trifluoromethyl)-1,3,4-thiadiazole-3(2 H )-1)decyl)phosphonium bromide (Compound 11, Mito-TIPIP-2)

[0160]

[0161] compound 10 (77 mg, 0.117 mmol) and PPh3 (31 mg, 0.117 mmol) were dissolved in ACN (1.2 ml) and refluxed at 85 °C for 12 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the mixture n - The target compound is a white solid after washing with hexane and Et2O. 11, Mito-TIPTP-2 It was obtained with a 55% yield.

[0162] 1 ¹H NMR (400 MHz, CDCl₃) δ 7.88-7.83 (m, 6H), 7.79-7.75 (m, 3H), 7.71-7.66 (m, 6H), 7.57 (s, 1H), 7.24-7.17 (m, 3H), 6.72-6.69 (m, 1H), 5.11 (q, J = 6.7 Hz, 1H), 4.41 (dt, J = 13.4, 6.7 Hz, 2H), 3.90-3.81 (m, 2H), 2.74 (t, J= 6.1 Hz, 2H), 2.60 (t, J = 6.0 Hz, 2H), 1.86-1.71 (m, 8H), 1.66 (d, J = 6.7 Hz, 3H), 1.22 (m, 12H). 13 C NMR (100 MHz, CDCl3) δ 182.0, 165.3, 159.0, 151.1, 141.4, 135.05, 135.02, 133.8, 133.7, 130.6, 130.4, 130.1, 124.0, 119.0, 118.3, 118.1, 112.3, 111.3, 106.0, 75.9, 51.9, 30.6, 30.4, 29.3, 29.2, 28.9, 28.1, 26.3, 23.5, 23.4, 23.1, 22.87, 22.83, 22.6, 20.7, 19.0. HRMS (ESI) m / z: [M-Br] + calcd for C 47 H 52 F3N5O2PS + 838.3525; found 838.3533. [α] D 23.2 =+5.8 (c=0.5 in CH2Cl2)

[0164] [Synthesis of Mito-TIPTP-3]

[0165] <Preparation Example 12> Ethyl 4,5,6,7-tetrohydro-2H-indazole-5-carboxylate (Compounds 12, 13)

[0166]

[0167] A solution of ethyl 4-oxocyclohexane carboxylate (2.0 g, 11.75 mmol) dissolved in THF (38.2 ml) was mixed at 0 °C with sodium hydride (564 mg, 14.1 mmol, 60% dispersion in mineral oil), ethyl formate (1.84 ml, 22.91 mmol), and EtOH (1.0 ml), and stirred at 25 °C for 6 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the mixture was filtered under reduced pressure with Et2O. Subsequently, the solvent was removed under reduced pressure to obtain Compound 12 in the form of a sodium salt, which was then subjected to the following reaction without a separate purification process. The obtained compound 12 [The substance] was dissolved in water (51 ml), semicarbazide hydrochloride (1.71 g, 15.3 mmol) was added, and the mixture was stirred at 105 °C for 4 hours. The organic layer was extracted using DCM after the reaction was terminated and washed with water and saturated brine. The organic layer was dried by adding anhydrous MgSO4, and the solvent was removed under reduced pressure. The mixture was purified by silica gel column chromatography (DCM / MeOH = 8:1) to obtain the target compound of the pure yellow oil. 13 It was obtained with a yield of 38%.

[0168] 1 ¹H NMR (400 MHz, CDCl₃) δ 7.33 (s, 1H), 4.19 (q, J = 7.1 Hz, 2H), 2.91-2.64 (m, 5H), 2.28-2.21 (m, 1H), 1.96-1.87 (m, 1H), 1.29 (t, J = 7.1 Hz, 3H). (Two-step reaction)

[0170] <Preparation Example 13> (4,5,6,7-tetrahydro-2H-indazole-5-yl)methanol (Compound 14)

[0171]

[0172] compound 13(875 mg, 4.51 mmol) was dissolved in THF (45 ml), and LiAlH4 (2.7 ml, 1.0 M in THF) was added at 0 °C, followed by stirring at 25 °C for 4 hours. After the reaction was complete, the mixture was cooled to 0 °C, and H2O (0.1 ml), 15% NaOH aqueous solution (0.1 ml), and H2O (0.3 ml) were added sequentially, followed by stirring at 25 °C for 20 minutes. Subsequently, the mixture was filtered under reduced pressure using Celite 545 and methanol to remove the solvent. The mixture was purified by silica gel column chromatography (DCM / MeOH = 1:3) to separate the target compound of the colorless oil. 14 It was obtained with a yield of 92%.

[0173] 1 ¹H NMR (400 MHz, MeOD) δ 7.26 (s, 1H), 3.54 (d, J = 6.5 Hz, 2H), 2.80-2.55 (m, 3H), 2.22-2.14 (m, 1H), 2.08-2.01 (m, 1H), 1.91-1.81 (m, 1H), 1.53-1.41 (m, 1H).

[0175] <Preparation Example 14> 5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5,6,7-tetrahydro-2 H -Indazole (Compound 15)

[0176]

[0177] After dissolving Compound 14 (630 mg, 4.14 mmol) in DMF (21 ml), imidazole (547 mg, 8.28 mmol) and TBSCl (1.25 g, 8.28 mmol) were added at 0 °C, followed by 3 hours at 25 °C. The mixture was stirred. After the reaction was complete, water was added, the organic layer was extracted using DCM, and washed with water and saturated brine. The obtained organic layer was dried by adding anhydrous MgSO4, and the solvent was removed under reduced pressure. The mixture was purified by silica gel column chromatography (Hexane / EtOAc = 2:1) to obtain the target compound 15 as a pure white solid in an 81% yield.

[0178] 1 ¹H NMR (400 MHz, CDCl₃) δ 7.30 (s, 1H), 3.61-3.54 (m, 2H), 2.82-2.57 (m, 3H), 2.30-2.23-2.15 (m, 1H), 2.05-1.97 (m, 1H), 1.96-1.85 (m, 1H), 1.54-1.42 (m, 1H), 0.90 (s, 9H), 0.05 (s, 6H). 13 13C NMR (100 MHz, CDCl3) δ 143.8, 132.1, 115.1, 67.4, 37.8, 26.2, 26.0, 23.5, 21.4, 18.4, -5.21.

[0180] <Preparation Example 15> Methyl ( R )-2-(3-iodophenoxy)propanoate (Compound 16)

[0181]

[0182] Methyl (S)-(-)-2-chloropropaonate (0.98 ml, 9.10 mmol) and K2CO3 (1.26 g, 9.10 mmol) were added to a solution of 3-iodophenol (1.0 g, 4.55 mmol) dissolved in DMF (23 ml), and the mixture was stirred at 80 °C for 4 hours. After the reaction was complete, water was added, the organic layer was extracted using EtOAc, and washed with water and saturated brine. The organic layer was dried by adding anhydrous MgSO4, and the solvent was removed under reduced pressure. The mixture was purified by silica gel column chromatography (Hexane / EtOAc = 8:1) to obtain a pure colorless oil target compound. 16 It was obtained with a yield of 74%.

[0183] 1 ¹H NMR (400 MHz, CDCl₃) δ 7.32-7.29 (m, 1H), 7.24-7.23 (m, 1H), 7.00 (t, J = 8.1 Hz, 1H), 6.83-6.80 (m, 1H), 4.75 (q, J = 6.8 Hz, 1H), 3.76 (s, 3H), 1.61 (d, J = 6.8 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 172.1, 158.0, 130.9, 130.8, 124.6, 114.4, 94.4, 72.6, 52.4, 18.5. [α] D 23.4 =+31.0 (c=0.5 in CH2Cl2)

[0185] <Preparation Example 16> ( R )-2-(3-iodophenoxy)propanoic acid (compound 17)

[0186]

[0187] compound 161.04 g (3.38 mmol) was dissolved in a mixture of MeOH (25.4 ml) and H2O (8.5 ml), cooled to 0 °C, and then LiOH (122 mg, 5.07 mmol) was added and stirred at 25 °C for 1 hour. After the reaction was complete, 1 N HCl was slowly added until the pH reached 3, and the organic layer was extracted using DCM. The organic layer was dried by adding anhydrous MgSO4, and the solvent was removed under reduced pressure to obtain the target compound as a white solid. 17 It was obtained with a yield of 92%.

[0188] 1 ¹H NMR (400 MHz, CDCl₃) δ 7.35-7.32 (m, 1H), 7.27-7.26 (m, 1H), 7.02-6.98 (t, J = 8.1 Hz, 1H), 6.87-6.84 (m, 1H), 4.80 (q, J = 6.8 Hz, 1H), 1.66 (d, J = 6.8 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 177.7, 157.8, 131.2, 131.0, 124.8, 114.4, 94.5, 72.1, 18.4. [α] D 22.7 =+7.0 (c=0.5 in CH2Cl2)

[0190] <Preparation Example 17> ( R )-2-(3-iodophenoxy)- N -(5-(trifluoromethyl)-1,3,4-thiadiazole-2-yl)propenamide (compound 18)

[0191]

[0192] compound 17 After dissolving (911 mg, 3.11 mmol) in DMF (31 ml), N , NDiisopropylethylamine (2.72 ml, 15.6 mmol), HBTU (1.41 g, 3.74 mmol), and 2-amino-5-trifluoromethyl-1,3,4-thiadiazole (632 mg, 3.74 mmol) were added, and the mixture was stirred at 70 °C for 4 hours. After the reaction was complete, water was added, the organic layer was extracted using EtOAc, and washed with water and saturated brine. The organic layer was dried by adding anhydrous MgSO4, and the solvent was removed under reduced pressure. The mixture was purified by silica gel column chromatography (Hexane / EtOAc = 5:1) to obtain the target compound as a pure white solid. 18 It was obtained with an 89% yield.

[0193] 1 ¹H NMR (400 MHz, CDCl₃) δ 11.26 (broad, 1H), 7.38 (d, J = 7.8 Hz, 1H), 7.29-7.28 (m, 1H), 7.03 (t, J = 8.1 Hz, 1H), 6.91-6.89 (m, 1H), 5.08 (q, J = 6.8 Hz, 1H), 1.70 (d, J = 6.8 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 170.7, 161.1, 156.6, 153.4, 153.0, 132.1, 131.2, 125.4, 121.0, 118.3, 115.2, 94.6, 74.4, 18.2. [α] D 23.8 =-12.6(c=0.5 in CH2Cl2)

[0195] <Preparation Example 18> (2 R )-2-(3-(5-(((tert-butyldimethylsilyl)oxy)methyl)-4,5,6,7-tetrahydro-2 H -Indazole-2-il)Phenoxy)- N -(5-(trifluoromethyl)-1,3,4-thiadiazole-2-yl)propenamide (compound 19)

[0196]

[0197] compound15 Trans-1,2-cyclohexanediamine (46 μL, 0.384 mmol) dissolved in DMF (2.74 ml) containing (511 mg, 1.918 mmol) and Compound 18 (850 mg, 1.918 mmol), K2CO3 (530 mg, 3.836 mmol), and CuI (18 mg, 0.096 mmol) were added, and the mixture was stirred at 130 °C for 18 hours. After the reaction was complete, water was added, the organic layer was extracted using EtOAc, and washed with water and saturated brine. The organic layer was dried by adding anhydrous MgSO4, and the solvent was removed under reduced pressure. The mixture was purified by silica gel column chromatography (Hexane / DCM / EtOAc = 4:0.5:1) to obtain the target compound as a pure white solid. 19 It was obtained with a yield of 16%.

[0198] 1 ¹H NMR (400 MHz, CDCl₃) δ 10.79 (broad, 1H), 7.60 (s, 1H), 7.40-7.24 (m, 3H), 6.79 (d, J = 7.9 Hz, 1H), 5.15 (m, 1H), 3.60 (d, J = 6.3 Hz, 2H), 2.92-2.64 (m, 3H), 2.31-2.22 (m, 1H), 2.07-2.00 (m, 1H), 1.96-1.86 (m, 1H), 1.70 (d, J = 6.7, 3H), 1.55-1.45 (m, 1H), 0.91 (s, 9H), 0.06 (s, 6H).

[0200] <Preparation Example 19> (2 R )-2-(3-(5-(hydroxymethyl)-4,5,6,7-tetrahydro-2 H -Indazole-2-il)Phenoxy)- N -(5-(trifluoromethyl)-1,3,4-thiadiazole-2-yl)propenamide (compound 20)

[0201]

[0202] compound 19(127 mg, 0.218 mmol) was dissolved in THF (1.1 ml), followed by the addition of TBAF (0.44 ml, 0.436 mmol) and stirring at 25 °C for 15 hours. After the reaction was complete, water was added, the organic layer was extracted using DCM, and washed with water and saturated brine. The organic layer was dried by adding anhydrous MgSO4, and the solvent was removed under reduced pressure. The mixture was purified by silica gel column chromatography (Hexane / EtOAc = 1:3) to obtain the target compound as a pure white solid. 20 It was obtained with a yield of 64%.

[0203] 1 ¹H NMR (400 MHz, CDCl₃) δ 10.94 (broad, 1H), 7.58 (s, 1H), 7.34-7.29 (m, 2H), 7.25-7.21 (m, 1H), 6.78-6.76 (m, 1H), 5.16 (q, J = 6.7 Hz, 1H), 3.66 (d, J = 6.4 Hz, 2H), 2.92-2.63 (m, 3H), 2.35-2.27 (m, 1H), 2.10-2.01 (m, 1H), 1.99-1.89 (m, 1H), 1.81-1.73 (m, 1H), 1.70 (t, J = 6.8 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 170.7, 160.5, 157.0, 151.6, 141.8, 130.8, 124.1, 121.1, 118.4, 118.0, 112.67, 112.64, 106.7, 74.4, 67.2, 37.6, 26.3, 23.7, 22.5, 18.3. LRMS (ESI) m / z: [M+H] + calcd for C 20 H 20 F3N5O3S 468.1;found 468.1. [α] D 23.1=-14.2(c=0.5 in CH2Cl2)

[0205] <Preparation Example 20> (2-(3-((( R )-1-oxo-1-((5-(trifluoromethyl)-1,3,4-thiadiazole-2-yl)amino)propane-2-yl)oxy)phenyl)-4,5,6,7-tetrahydro-2 H -Indazole-5-yl)methyl 10-bromodecanoate (Compound 21)

[0206]

[0207] Thionyl chloride (19 μL, 0.265 mmol) was added to 10-bromodecanosic acid (33.4 mg, 0.133 mmol) and stirred at 25 °C for 18 hours. Subsequently, the mixture was subjected to reduced pressure to obtain 10-bromodecanoylic chloride, which was used in the next step without a separate purification process. Compound 20 31 mg (0.066 mmol) was dissolved in 0.33 ml of DCM. Then, at 0 °C, 10-bromodecanoylic chloride dissolved in 0.33 ml of DCM was slowly added, followed by the addition of 10 μL (0.080 mmol) of triethylamine, and the mixture was stirred at 0 °C for 2 hours. After the reaction was complete, water was added, the organic layer was extracted using DCM, and the mixture was washed with water and saturated brine. The organic layer was dried by adding anhydrous MgSO4, and the solvent was removed under reduced pressure. The mixture was purified by silica gel column chromatography (Hexane / EtOAc = 2:1) to obtain the target compound as a pure white solid. 21 It was obtained with a yield of 52%. (Two-step reaction)

[0208] 1 ¹H NMR (400 MHz, CDCl₃) δ 10.59 (broad, 1H), 7.68 (s, 1H), 7.56 (s, 1H), 7.40 (t, J = 8.2 Hz, 1H), 7.22 (d, J = 7.9 Hz, 1H), 6.91 (dd, J = 8.2, 2.0 Hz, 1H), 5.31 (q, J = 6.7 Hz, 1H), 4.11 (d,J = 6.4 Hz, 2H), 3.41 (t, J = 6.8 Hz, 2H), 3.08-2.98 (m, 1H), 2.85-2.75 (m, 2H), 2.36-2.31 (m, 1H), 2.35 (t, J = 7.5 Hz, 2H), 2.17-2.06 (m, 2H), 1.88-1.82 (m, 2H), 1.72 (d, J = 6.7 Hz, 3H) 1.66-1.61 (m, 2H), 1.44-1.39 (m, 2H), 1.30 (m, 10H). LRMS (ESI) m / z: [M+H] + calcd for C 30 H 37 BrF3N5O4S 700.1; found 700.3.

[0210] <Preparation Example 21> (10-oxo-10-((2-(3-((( R )-1-oxo-1-((5-(trifluoromethyl)-1,3,4-thiadiazole-2-yl)amino)propane-2-yl)oxy)phenyl)-4,5,6,7-tetrahydro-2 H -Indazole-5-yl)methoxy)decyl)triphenylphosphonium bromide (Compound 22, Mito-TIPTP-3)

[0211]

[0212] compound 21 (28 mg, 0.0400 mmol) and PPh3 (10.5 mg, 0.0400 mmol) were dissolved in ACN (0.40 ml) and stirred at 85 °C for 24 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (DCM / MeOH = 15:1) to obtain the colorless solid target compound. 22, Mito-TIPTP-3 It was obtained with a yield of 55%.

[0213] 1 ¹H NMR (400 MHz, MeOD) δ 7.89-7.70 (m, 16H), 7.31-7.27 (m, 2H), 7.24-7.21 (m, 1H), 6.87-6.84 (m, 1H), 5.11 (q, J= 6.7 Hz, 1H), 4.14-4.04 (m, 2H), 3.36-3.28 (m, overlapped with solvent peak, 2H), 2.85-2.74 (m, 2H), 2.70-2.60 (m, 1H), 2.35 (t, J = 7.2 Hz, 2H), 2.13-1.99 (m, 2H), 1.66 (d, J = 6.7 Hz, 3H), 1.62-1.54 (m, 4H), 1.52-1.43 (m, 2H), 1.28 (m, 10 H). 13 C NMR (100 MHz, MeOD) δ 175.4, 174.5, 164.7, 159.5, 152.6, 152.1, 142.6, 136.26, 136.23, 134.8, 134.7, 131.6, 131.5, 131.4, 126.4, 122.9, 120.3, 120.2, 119.5, 118.5, 113.8, 113.0, 107.4, 75.6, 69.0, 35.7, 35.0, 31.5, 31.4, 30.1, 30.06, 30.00, 29.7, 27.3, 25.9, 24.5, 23.48, 23.44, 23.1, 22.8, 22.3, 18.9. HRMS (ESI) m / z: [M-Br] + calcd for C 48 H 52 F3N5O4PS + 882.3424; found 882.3432. [α] D 23.9 =+19.0 (c=0.5 in CH2Cl2)

[0215] [Synthesis of Mito-TIPTP-4]

[0216] <Preparation Example 22> Tert-butyl (4,5,6,7-tetrahydro-2 H -Indazole-5-yl)carbamate (Compound 24)

[0217]

[0218] tertA solution of β-butyl (4-oxocyclohexyl) carbamate (1.0 g, 4.68 mmol) dissolved in THF (23.4 ml) was mixed with sodium hydride (225 mg, 5.62 mmol, 60% dispersion in mineral oil), ethyl formate (1.13 ml, 14.04 mmol), and EtOH (1.0 ml) at 0 °C, and stirred at 25 °C for 6 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the mixture was filtered under reduced pressure with Et2O. Subsequently, the solvent was removed under reduced pressure to obtain the compound in the form of a sodium salt. 23 The compound was obtained, and the following reaction was performed without a separate purification process. 23 ... was dissolved in water (23.4 ml), semicarbazide hydrochloride (627 mg, 5.62 mmol) was added, and the mixture was stirred at 105 °C for 4 hours. The organic layer was extracted using DCM after reaction termination and washed with water and saturated brine. The organic layer was dried by adding anhydrous MgSO4, and the solvent was removed under reduced pressure. The mixture was separated by silica gel column chromatography ( n -Hexane / EtOAc = 1:3) Purified to obtain the target compound as a pure white solid 24 It was obtained with a yield of 26%. (Two-step reaction)

[0219] 1 ¹H NMR (400 MHz, CDCl₃) δ 7.32 (s, 1H), 4.60 (s, broad, 1H), 3.98 (s, broad, 1H), 2.94-2.77 (m, 3H), 2.44-2.39 (m, 1H), 2.09-2.01 (m, 1H), 1.92-1.81 (m, 1H), 1.45 (s, 9H).

[0221] <Preparation Example 23> Tert-butyl(2-(3-(((R)-1-oxo-1-((5-(trifluoromethyl)-1,3,4-thiadiazole-2-yl)amino)propane-2-yl)oxy)phenyl)-4,5,6,7-tetrahydro-2H-indazole-5-yl)carbamate (Compound 25)

[0222]

[0223] compound 24To a solution of (293 mg, 1.235 mmol) and Compound 18 (657 mg, 1.482 mmol) dissolved in DMF (1.76 ml), trans-1,2-cyclohexanediamine (30 μL, 0.247 mmol), K2CO3 (341 mg, 2.47 mmol), and CuI (24 mg, 0.124 mmol) were added, and the mixture was stirred at 130 °C for 18 hours. After the reaction was complete, water was added, the organic layer was extracted using EtOAc, and washed with water and saturated brine. The organic layer was dried by adding anhydrous MgSO4, and the solvent was removed under reduced pressure. The mixture was purified by silica gel column chromatography (Hexane / DCM / EtOAc = 3:0.5:2) to obtain the pure yellow solid target compound. 25 It was obtained with a yield of 21%.

[0224] 1 ¹H NMR (400 MHz, CDCl₃) δ 10.33 (broad, 1H), 7.64 (s, 1H), 7.40 (s, 1H), 7.39 (t, J = 8.2 Hz, 1H), 7.27-7.23 (m, 1H), 6.84 (d, J = 8.2 Hz, 1H), 5.17-5.12 (m, 1H), 4.63-4.61 (m, 1H), 4.01 (s, broad, 1H), 3.03-2.83 (m, 3H), 2.54-2.45 (m, 1H), 2.12-2.05 (m, 1H), 1.94-1.87 (m, 1H), 1.71 (d, J = 6.8 Hz, 3H), 1.45 (s, 9H). 13 13C NMR (100 MHz, CDCl3) δ170.7, 160.7, 157.0, 155.4, 150.1, 141.7, 130.7, 130.4, 124.5, 121.0, 118.3, 116.5, 112.88, 112.84, 112.66, 106.8, 74.3, 46.5, 29.7, 29.0, 28.5, 27.7, 20.9, 18.2. LRMS (ESI) m / z: [M+H] + calcd for C 24 H 27 F3N6O4S553.1; found 553.2. [α] D 21.5 =-10.0(c=0.5 in CH2Cl2)

[0226] <Preparation Example 24> (2 R )-2-(3-(5-amino-4,5,6,7-tetrahydro-2 H -Indazole-2-il)Phenoxy)- N -(5-(trifluoromethyl)-1,3,4-thiadiazole-2-yl)propenamide (compound 26)

[0227]

[0228] compound 25 60 mg (0.127 mmol) was dissolved in 1.3 ml of DCM, followed by the addition of 0.49 ml of TFA (6.35 mmol) and stirring at 25 °C for 20 minutes. After the reaction was complete, saturated NaHCO3 solution was added to alkalize the mixture to pH 10; the organic layer was then extracted using DCM and washed with water and saturated brine. The organic layer was dried with anhydrous MgSO4, and the solvent was removed under reduced pressure to obtain the target compound as a white solid. 26 It was obtained with a yield of 57%.

[0229] 1 ¹H NMR (400 MHz, MeOD) δ 7.82-7.80 (m, 1H), 7.31 (t, J= 8.1 Hz, 1H), 7.26-7.21 (m, 1H), 7.19-7.15 (m, 1H), 6.86-6.84 (m, 1H), 4.97-4.91 (m, 1H), 3.54-3.48 (m, 1H), 3.09-2.96 (m, 1H), 2.91-2.73 (m, 2H), 2.63-2.55 (m, 1H), 2.25-2.16 (m, 1H), 1.97-1.88 (m, 1H), 1.64 (d, J = 6.6 Hz, 3H). LRMS (ESI) m / z: [M+H] + calcd for C 19 H 19 F3N6O2S 453.1; found 453.1.

[0231] <Preparation Example 25> 10-Bromo- N -(2-(3-((( R )-1-oxo-1-((5-(trifluoromethyl)-1,3,4-thiadiazole-2-yl)amino)propane-2-yl)oxy)phenyl)-4,5,6,7-tetrahydro-2 H -Indazole-5-yl)decanamide (Compound 27)

[0232]

[0233] Thionyl chloride (3 μL, 0.034 mmol) was added to 10-bromodecanosic acid (4.2 mg, 0.017 mmol) and stirred at 25 °C for 18 hours. Subsequently, the mixture was subjected to reduced pressure to obtain 10-bromodecanoylic chloride, which was used in the next step without a separate purification process. Compound 267 mg (0.017 mmol) was dissolved in 0.09 ml of THF, and 10-bromodecanoylic chloride dissolved in 0.08 ml of THF was slowly added at 0 °C. Then, 3 μL (0.021 mmol) of triethylamine was added, and the mixture was stirred at 0 °C for 2 hours. After the reaction was complete, water was added, the organic layer was extracted using DCM, and washed with water and saturated brine. The organic layer was dried by adding anhydrous MgSO4, and the solvent was removed under reduced pressure. The mixture was purified by silica gel column chromatography (DCM / MeOH = 20:1) to obtain the target compound as a pure white solid. 27 It was obtained with a yield of 54%. (Two-step reaction)

[0234] 1 ¹H NMR (400 MHz, CDCl₃) δ 10.51 (s, broad, 1H), 7.63 (s, 1H), 7.38-7.32 (m, 2H), 7.27-7.23 (m, 1H), 6.82-6.80 (m, 1H), 5.51 (d, J = 7.9 Hz, 1H), 5.15-5.10 (m, 1H), 4.39-4.28 (m, 1H), 3.41 (t, J = 6.8 Hz, 2H), 3.04-2.97 (m, 1H), 2.89-2.82 (m, 2H), 2.53-2.46 (m, 1H), 2.17 (t, J = 7.6 Hz, 2H), 2.07-2.02 (m, 1H), 1.98-1.89 (m, 1H), 1.71 (d, J = 6.8 Hz, 3H) 1.65-1.58 (m, 2H), 1.44-1.37 (m, 2H), 1.28 (m, 10H). 13 13C NMR (100 MHz, CDCl3) δ173.0, 170.7, 160.5, 156.9, 149.8, 141.2, 130.8, 130.5, 124.8, 120.9, 118.2, 116.3, 112.9, 112.7, 112.5, 106.6, 106.5, 74.0, 44.9, 37.0, 34.4, 32.8, 29.8, 29.34, 29.30, 28.7, 28.4, 28.1, 27.3, 25.8, 20.7, 18.3. LRMS (ESI) m / z: [M+H] + calcd for C 29 H 36 BrF3N6O3S 685.1; found 685.2.

[0236] <Preparation Example 26> (10-oxo-10-((2-(3-((( R )-1-oxo-1-((5-(trifluoromethyl)-1,3,4-thiadiazole-2-yl)amino)propane-2-yl)oxy)phenyl)-4,5,6,7-tetrahydro-2 H -Indazole-5-yl)amino)decyl)triphenylphosphonium bromide (Compound 28, Mito-TIPTP-4)

[0237]

[0238] compound 27 (6 mg, 0.009 mmol) and PPh3 (3 mg, 0.011 mmol) were dissolved in ACN (0.1 ml) and refluxed at 85 °C for 24 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (DCM / MeOH = 10:1) to obtain the colorless solid target compound. 28, Mito-TIPTP-4 It was obtained with a yield of 47%.

[0239] 1 ¹H NMR (400 MHz, MeOD) δ 7.90-7.85 (m, 4H), 7.82-7.72 (m, 13H), 7.36-7.33 (m, 2H), 7.29-7.25 (m, 1H), 6.91 (dd, J = 8.0, 2.1 Hz, 1H), 5.20 (q, J = 6.7 Hz, 1H), 4.09 (m, 1H), 3.42-3.35 (m, 2H), 2.98-2.72 (m, 3H), 2.55-2.47 (m, 1H), 2.20 (t, J= 7.3 Hz, 2H), 2.05-1.99 (m, 1H), 1.88-1.81 (m, 1H), 1.69 (d, J = 6.7 Hz, 3H), 1.60-1.51 (m, 4H), 1.28 (m, 10H). 13 1C NMR (100 MHz, MeOD) δ 174.4, 172.2, 158.0, 149.8, 141.2, 134.89, 134.86, 133.4, 133.35, 133.31, 130.3, 130.1, 130.0, 125.4, 119.0, 118.1, 116.4, 112.6, 111.8, 106.2, 106.1, 74.0, 45.6, 35.6, 30.2, 30.0, 29.3, 28.7, 28.6, 28.4, 28.3, 26.2, 25.5, 22.1, 22.0, 21.5, 21.0, 20.8, 17.3. HRMS (ESI) m / z: [M-Br] + calcd for C 47 H 51 F3N6O3PS + 867.3427; found 867.3435. [α] D 22.3 =+7.25 (c=0.4 in CH2Cl2)

[0241] [Synthesis of Mito-TIPTP-5]

[0242] <Preparation Example 27> ( R )-(5-(2-(3-(4,5,6,7-tetrahydro-2 H -Indazole-2-yl)phenoxy)propaneamido)-1,3,4-thiadiazole-2-yl)methyl10-bromodecanoate (Compound 29)

[0243]

[0244] Thionyl chloride (19 μL, 0.265 mmol) was added to 10-bromodecanosic acid (33.4 mg, 0.133 mmol) and stirred at 25 °C for 18 hours. Subsequently, the mixture was subjected to reduced pressure to obtain 10-bromodecanoylic chloride, which was used in the next step without a separate purification process. Compound 6After dissolving (53 mg, 0.133 mmol) in DCM (0.34 mL), 10-bromodecanoylic chloride dissolved in DCM (0.33 mL) was slowly added at 0 °C, followed by the addition of triethylamine (22 μL, 0.160 mmol) and stirring at 0 °C for 2 hours. After the reaction was complete, water was added, the organic layer was extracted using DCM, and washed with water-supersaturated brine. The organic layer was dried by adding anhydrous MgSO4, and the solvent was removed under reduced pressure. The mixture was separated by silica gel column chromatography ( n -Hexane / EtOAc = 2:1) Purified to obtain the target compound as a pure white solid 29 It was obtained with a yield of 49%.

[0245] 1 ¹H NMR (400 MHz, CDCl₃) δ 10.27 (broad, 1H), 7.59 (s, 1H), 7.36-7.31 (m, 2H), 7.27-7.24 (m, 1H), 6.80-6.77 (m, 1H), 5.43 (s, 2H), 5.11 (q, J = 6.7 Hz, 1H), 3.41 (t, J = 6.9 Hz, 2H), 2.77 (t, J = 6.3 Hz, 2H), 2.62 (t, J = 6.1 Hz, 2H), 2.40 (t, J = 7.5 Hz, 2H), 1.87-1.75 (m, 6H), 1.69 (d, J = 6.7 Hz, 3H) 1.67-1.61 (m, 2H), 1.43-1.39 (m, 2H), 1.29 (m, 8H). 13 13C NMR (100 MHz, CDCl3) δ 178.4 ,173.2, 170.3, 160.6, 160.0, 157.1, 151.7, 141.8, 130.6, 123.9, 118.7, 112.6, 112.5, 106.6, 74.3, 60.1, 34.1, 33.9, 32.9, 29.3, 29.18, 29.12, 28.7, 28.2, 24.8, 23.5, 23.48, 23.46, 20.7, 18.3. LRMS (ESI) m / z: [M+H] + calcd for C 29 H 38 BrN5O4S 632.1; found 632.3.

[0247] <Preparation Example 28> ( R )-(10-oxo-10-((5-(2-(3-(4,5,6,7-tetrahydro-2 H -Indazole-2-yl)phenoxy)propaneamido)-1,3,4-thiadiazole-2-yl)methoxy)decyl)triphenylphosphonium bromide (Compound 30, Mito-TIPTP-5)

[0248]

[0249] compound 29 (10 mg, 0.0158 mmol) and PPh3 (4.1 mg, 0.0158 mmol) were dissolved in DMF (0.16 ml) and stirred at 85 °C for 24 hours. After the reaction was complete, the mixture was purified by silica gel column chromatography (DCM / MeOH = 15:1) to obtain the white solid target compound. 30, Mito-TIPTP-5 It was obtained with a 35% yield.

[0250] 1 ¹H NMR (400 MHz, MeOD) δ 7.89-7.84 (m, 3H), 7.81-7.71 (m, 13H), 7.34-7.30 (m, 2H), 7.25-7.23 (m, 1H), 6.85-6.83 (m, 1H), 5.40 (s, 2H), 5.12 (q, J = 6.7 Hz, 1H), 3.39-3.33 (m, 2H), 2.69 (t, J = 6.3 Hz, 2H), 2.62 (t, J = 6.1 Hz, 2H), 2.39 (t, J= 7.2 Hz, 2H), 1.87-1.74 (m, 4H), 1.66 (d, J = 6.7 Hz, 3H), 1.63-1.55 (m, 3H), 1.52-1.44 (m, 2H), 1.24 (m, 9H). 13 C NMR (100 MHz, MeOD) δ 174.6, 172.8, 162.0, 161.8, 159.4, 152.6, 142.7, 136.26, 136.23, 134.8, 134.7, 131.6, 131.5, 131.4, 126.1, 120.3, 119.6, 119.5, 113.5, 113.0, 107.4, 75.3, 60.7, 34.5, 31.4, 31.2, 29.83, 29.80, 29.7, 29.4, 25.6, 24.44, 24.42, 23.4, 23.3, 22.8, 22.3, 21.4, 18.8. HRMS (ESI) m / z: [M-Br] + calcd for C 47 H 53 N5O4PS + 814.3550; found 814.3555. [α] D 21.1 =-2.9(c=0.5 in CH2Cl2)

[0252] [Synthesis of Mito-TIPTP-6]

[0253] <Preparation Example 29> ( R )- N -(5-(azidomethyl)-1,3,4-thiadiazole-2-yl)-2-(3-(4,5,6,7-tetrahydro-2 H -Indazole-2-yl)phenoxy)propanamide (Compound 31)

[0254]

[0255] 화합물 6After dissolving (60.0 mg, 0.150 mmol) in THF (1.0 ml), diphenylphosphoryl azide (80.6 μL, 0.375 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (56.0 μL, 0.375 mmol) were added at 0 °C, followed by stirring at 25 °C for 23 hours. After the reaction was complete, water was added, the organic layer was extracted using EtOAc, and washed with water and saturated brine. The organic layer was dried by adding anhydrous MgSO4, and the solvent was removed under reduced pressure. The mixture was purified by silica gel column chromatography (Hexane / EtOAc = 1:1) to obtain the target compound as a pure white solid. 31 It was obtained with a yield of 54%.

[0256] ¹H NMR (400 MHz, CDCl3) δ 7.59 (s, 1H), 7.38 - 7.35 (m, 1H), 7.33 (t, J = 8.1 Hz, 1H), 7.26 - 7.23 (m, 1H), 6.79 (dd, J = 1.9, 8.7 Hz, 1H), 5.13 (q, J = 6.8 Hz, 1H), 4.73 (s, 2H), 2.77 (t, J = 6.3 Hz, 2H), 2.61 (t, J = 6.3 Hz, 2H), 1.90 - 1.83 (m, 2H), 1.82 - 1.75 (m, 2H), 1.70 (d, J = 6.4 Hz, 3H). 13 13C NMR (100 MHz, CDCl3) δ 170.41, 170.39, 160.4, 159.9, 157.2, 151.7, 141.8, 130.5, 123.8, 118.7, 112.47, 112.31, 106.7, 74.4, 48.7, 23.6, 23.44, 23.41, 20.7, 18.34, 18.32.

[0257] LRMS (ESI) m / z: [M+H]+ calcd for C19H20N8O2S 425.1 ; found 425.1

[0258] [α] D 26.0 = - 21.8 (c = 0.5 in CH2Cl2)

[0260] <Preparation Example 30> ( R )- N -(5-(aminomethyl)-1,3,4-thiadiazole-2-yl)-2-(3-(4,5,6,7-tetrahydro-2 H -Indazole-2-yl)phenoxy)propanamide (Compound 32)

[0261]

[0262] compound 31 Pd / C (6.6 mg) was added to a solution of (33.0 mg, 0.0777 mmol) dissolved in MeOH (1.9 ml), and the mixture was stirred for 1 hour at 25 °C in an H₂ environment. After the reaction was complete, the mixture was filtered under reduced pressure using Celite 545 and MeOH, and the yellow solid target compound was removed under reduced pressure. 32 It was obtained with a 97% yield.

[0263] ¹H NMR (400 MHz, CDCl3) δ7.56 (s, 1H), 7.31 (t, J = 9.6 Hz, 2H), 7.22 (d, J = 8.0 Hz, 1H), 6.76 (d, J = 7.9 Hz, 1H), 5.09 (q, J = 6.5 Hz, 1H), 4.22 (s, 2H), 2.73 (t, J = 6.1 Hz, 2H), 2.73 (t, J = 6.1 Hz, 2H), 2.59 (t, J = 6.0 Hz, 2H), 2.59 (t, J = 6.0 Hz, 2H), 1.90 - 1.79 (m, 2H), 1.75 (m, 2H), 1.68 (d, J = 6.6 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 169.97, 168.7, 158.6, 157.1, 151.7, 141.9, 130.6, 123.7, 118.6, 112.40, 112.33, 106.5, 74.4, 41.3, 23.51, 23.39, 23.36, 20.6, 18.3. LRMS (ESI) m / z: [M]+ calcd for C19H22N6O2S 398.2 ; found 399.1. [α] D 26.0 = - 10.3 (c = 0.1 in CH2Cl2)

[0265] <Preparation Example 31> ( R )-10-Bromo- N -((5-(2-(3-(4,5,6,7-tetrahydro-2 H -Indazole-2-yl)phenoxy)propaneamido)-1,3,4-thiadiazole-2-yl)methyl)decanamide (Compound 33)

[0266]

[0267] Thionyl chloride (0.6 ml, 1.0 M in DCM) was added to 10-bromodecanosic acid (114 mg, 0.452 mmol) and stirred at 25 °C for 18 hours. Subsequently, the mixture was subjected to reduced pressure to obtain 10-bromodecanoylic chloride, which was used in the next step without a separate purification process. Compound 32After dissolving (90.0 mg, 0.226 mmol) in DCM (1.15 ml), 10-bromodecanoylic chloride dissolved in DCM (1.15 ml) was slowly added at 0 °C, followed by the addition of triethylamine (63 μL, 0.452 mmol) and stirring at 25 °C for 1 hour. After the reaction was complete, water was added, the organic layer was extracted using DCM, and washed with water and saturated brine. The organic layer was dried by adding anhydrous MgSO4, and the solvent was removed under reduced pressure. The mixture was separated by silica gel column chromatography ( n -Hexane / EtOAc=1:2) Purified to obtain the target compound as a pure white solid 33 It was obtained with a yield of 56%. (Two-step reaction)

[0268] ¹H NMR (400 MHz, MeOD) δ 7.83 (s, 1H), 7.35 (t, J = 8.3 Hz, 1H), 7.32 (t, J = 2.3 Hz, 1H), 7.27 (ddd, J = 1.0, 1.9, 8.1 Hz, 1H), 6.89 - 6.85 (m, 1H), 5.10 (q, J = 6.7 Hz, 1H), 4.65 (s, 2H), 3.40 (t, J = 6.8 Hz, 2H), 2.71 (t, J = 6.3 Hz, 2H), 2.65 - 2.60 (m, 2H), 2.24 (t, J = 7.3 Hz, 2H), 1.90 - 1.83 (m, 2H), 1.82 - 1.75 (m, 4H), 1.67 (d, J = 6.7 Hz, 3H), 1.63 (t, J = 7.1 Hz, 2H), 1.44 - 1.35 (m, 2H), 1.34 - 1.26 (m, 8H). 13 1C NMR (100 MHz, MeOD) δ175.3, 175.2, 171.1, 164.0, 159.6, 158.0, 151.3, 141.3, 130.2, 124.8, 118.3, 112.2, 111.8, 106.1, 74.0, 37.7, 35.3, 35.2, 33.1, 32.5, 29.0, 28.8, 28.7, 28.3, 27.7, 25.4, 23.1, 23.0, 22.9, 20.1, 17.4. LRMS (ESI) m / z: [M] + calcd for C 29 H 39 BrN6O3S 630.2; found 631.3. [α] D 26.0 = - 15.0 (c = 0.5 in CH2Cl2)

[0270] <Preparation Example 32> ( R )-(10-oxo-10-(((5-(2-(3-(4,5,6,7-tetrahydro-2 H -Indazole-2-yl)phenoxy)propaneamido)-1,3,4-thiadiazole-2-yl)methyl)amino)decyl)triphaylphosphonium bromide (Compound 34, Mito-TIPTP-6)

[0271]

[0272] compound 33 (30 mg, 0.0475 mmol) and PPh3 (12.5 mg, 0.0475 mmol) were dissolved in ACN (0.48 ml) and refluxed at 85 °C for 24 hours. After the reaction was complete, the mixture was purified by silica gel column chromatography (DCM / MeOH = 10:1) to obtain the white solid target compound. 34, Mito-TIPTP-6 It was obtained with a 30% yield.

[0273] ¹H NMR (400 MHz, MeOD) δ 7.91 - 7.83 (m, 3H), 7.82 - 7.71 (m, 13H), 7.33 - 7.27 (m, 2H), 7.24 - 7.22 (m, 1H), 6.83 (dd, J = 2.5, 7.8 Hz, 1H), 5.09 (q, J = 6.6 Hz, 1H), 4.63 (s, 2H), 3.42 - 3.33 (m, 2H), 2.67 (t, J= 6.1 Hz, 2H), 2.60 (t, J = 5.9 Hz, 2H), 2.21 (t, J = 8.0 Hz, 2H), 1.88 - 1.79 (m, 2H), 1.79 - 1.73 (m, 2H), 1.70 - 1.53 (m, 6H), 1.52 - 1.45 (m, 2H), 1.31 - 1.18 (m, 9H). 13 1C NMR (100 MHz, MeOD) δ 176.5, 172.7, 165.3, 161.3, 159.4, 152.7, 142.8, 136.27, 136.24, 134.86, 134.76, 131.64, 131.58, 131.5, 126.2, 120.4, 119.7, 119.6, 113.6, 113.0, 107.5, 75.4, 39.1, 36.6, 36.5, 31.5, 31.3, 29.88, 29.83, 29.80, 29.4, 26.5, 24.46, 24.43, 24.3, 23.40, 23.35, 22.9, 22.4, 21.5, 18.9. HRMS (ESI) m / z: [M-Br] + calcd for C 47 H 54 N6O3PS + 813.3710; found813.3716. [α] D 26.0 = - 23.8 (c = 0.1 in CH2Cl2)

[0275] <Example 1> Evaluation of the active oxygen inhibitory efficacy of a compound

[0276] The reactive oxygen species inhibitory efficacy was evaluated by treating LPS / ATP-primed BMDM cells with each of the Mito-TIPTP-1, Mito-TIPTP-2, Mito-TIPTP-3, Mito-TIPTP-4, Mito-TIPTP-5, and Mito-TIPTP-6 compounds.

[0277] Intracellular ROS levels were measured by flow cytometry using cells cultured in serum-free medium, and loaded with the redox-sensitive dyes 2 μM dihydroethidium (DHE, for O2; Calbiochem, San Diego, CA, USA) or 1 μM MitoSox Red (for mitochondrial ROS; Calbiochem, San Diego, CA, USA). 1 × 10^5 BMDMs were rapidly washed with a pulse spin and immediately collected for analysis on a FACSCalibur (BD Biosciences, San Jose, CA, USA). Data were derived using CellQuest software (BD Biosciences, San Jose, CA, USA).

[0278] As a result of the experiment, the Mito-TIPTP-6 compound showed the most superior reactive oxygen species inhibitory efficacy (Fig. 1).

[0280] <Example 2> Evaluation of Rubicon-p22phox Interaction Inhibitory Efficacy

[0281] The efficacy of inhibiting Rubicon-p22phox interactions was evaluated by treating LPS / ATP-primed BMDM cells with each of the Mito-TIPTP-5 and Mito-TIPTP-6 compounds. The efficacy was compared using Mito-TIPTP-1 as a control.

[0282] Mitochondria fraction-BMDMs prepared with LPS (100 ng / mL) were treated with Mito-TIPTP-1, Mito-TIPTP-5, and Mito-TIPTP-6 (0.1, 0.5, 1, 10, 100 nM) for 1 hour, then activated with ATP for 30 minutes, and immunoprecipitation (IP) was performed with α-Rubicon. Immunoblotting (IB) was performed using αp22phox, and WCLs (whole cell extracts) were immunoblotted using α-Rubicon, αp22phox, αCOX IV, and αActin.

[0283] Experimental results showed that the Mito-TIPTP-6 compound exhibited excellent inhibitory activity against Rubicon-p22phox interactions and showed efficacy in a concentration-dependent manner (Fig. 2).

Claims

Claim 1 Compound of Formula 1 below or a pharmaceutically acceptable salt thereof: [Formula 1] In the above chemical formula 1, R1 is and R2 is a haloalkyl or And, R3 is And, X1 is an NH or nitrogen atom, X2 is an NH or oxygen atom, and n is 1 to 10. Claim 2 In claim 1, the compound of Chemical Formula 1 is a compound having the structure of Chemical Formula 2 below: [Chemical Formula 2] In the above chemical formula 2, R1 is and R2 is a haloalkyl or And, X1 is an NH or nitrogen atom, X2 is an NH or oxygen atom, and n is 1 to 10. Claim 3 In claim 1, the compound of Chemical Formula 1 is a compound having the structure of the following Chemical Formula 3: [Chemical Formula 3] In the above chemical formula 3, R2 is a haloalkyl or And, R3 is And, X1 is an NH or nitrogen atom, X2 is an NH or oxygen atom, and n is 1 to 10. Claim 4 In claim 1, the compound of Chemical Formula 1 is a compound having the structure of Chemical Formula 4 below: In the above chemical formula 4, R1 is And, R3 is And, X1 is an NH or nitrogen atom, X2 is an NH or oxygen atom, and n is 1 to 10. Claim 5 A pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising as an active ingredient a compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof. Claim 6 A pharmaceutical composition according to claim 5, wherein the inflammatory disease is one or more selected from the group consisting of inflammatory bowel disease, sepsis, septic shock, peritonitis, nephritis, acute bronchitis, chronic bronchitis, osteoarthritis, intestinal spondylitis, chronic obstructive pulmonary disease, rheumatoid arthritis, acute lung injury, and bronchopulmonary dysplasia. Claim 7 A pharmaceutical composition according to claim 5, characterized in that the composition inhibits reactive oxygen species within mitochondria or inhibits Rubicon-p22Phox protein interactions.

Images