Phytosphingosine-3,4-cyclic phosphate compound and pharmaceutical composition containing same

Abstract

The present invention relates to a phytosphingosine-3,4-cyclic phosphate compound, which can increase the expression of SIRT1 and decrease the expression of amyloid and tau protein, as well as increase the expression of tyrosine hydroxylase and decrease the expression of α-synuclein, and can improve sepsis and acute respiratory distress syndrome induced by CLP surgery, and a pharmaceutical composition containing the same as an active ingredient.

Description

[Technical Field]

[0001] The present invention relates to a phytosphingosine-3,4-cyclic phosphate compound and a pharmaceutical composition containing the same, and more particularly to a phytosphingosine-3,4-cyclic phosphate compound that can increase the expression of SIRT1, reduce the expression of amyloid and tau protein, increase the expression of tyrosine hydroxylase, and reduce the expression of α-synuclein, as well as ameliorate sepsis and acute respiratory distress syndrome induced by CLP surgery, and a pharmaceutical composition containing the same as an active ingredient. [Background technology]

[0002] Degenerative brain diseases, as the name suggests, are degenerative diseases that occur in the brain as we age and are caused by the loss of nerve cells. Degenerative brain diseases include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease. Of these, Alzheimer's disease is the most common, followed by Parkinson's disease.

[0003] Alzheimer's disease is a major degenerative brain disease that causes senile dementia, and its main symptom is the progressive loss of memory and cognitive ability. Parkinson's disease manifests as symptoms such as tremor, bradykinesia, muscle rigidity, postural instability, and akinesia due to the death of dopamine-secreting neurons in the substantia nigra pars compacta (SNpc) of the midbrain and dopamine deficiency in the striatum.

[0004] It has been confirmed that abnormal protein aggregation occurs in both Alzheimer's disease and Parkinson's disease. In the case of Alzheimer's disease, abnormal aggregation of extracellular amyloid beta (Aβ) and intracellular tau is closely related to the pathophysiology of the disease, while in the case of Parkinson's disease, aggregation of α-synuclein is closely related to the pathophysiology of the disease.

[0005] In this regard, for example, Korean Patent Registration No. 10-1064258 discloses a composition containing an aryl ureidoacetate compound that prevents degeneration and damage to brain cells caused by amyloid beta, and Korean Patent Registration No. 10-1092620 discloses a composition containing a sesquiterpene compound as an active ingredient that inhibits the expression and transformation of alpha-synuclein, which causes damage and death of nerve cells, thereby preventing or treating degenerative brain diseases.

[0006] Recently, it has been discovered that the expression of SIRT1 (silent mating type information regulation 2 homolog; sirtuin 1) is reduced in such degenerative brain diseases, and as a result, SIRT1 activators have attracted attention as therapeutic agents for degenerative brain diseases.

[0007] Sepsis, on the other hand, is an inflammatory reaction caused by excessive activation of the body's immune system due to infection with pathogenic microorganisms, and in severe cases, can lead to shock and death in patients.

[0008] Specifically, sepsis is a systemic inflammatory response syndrome caused by infection, and it occurs acutely mainly in infants, the elderly, or surgical patients with weak immune systems. Most cases of sepsis are accompanied by a systemic inflammatory response, which can occur for a variety of reasons. Sepsis occurs when a systemic inflammatory response occurs due to infection with pathogenic microorganisms in the body. Sepsis is a common infectious disease that accounts for the majority of deaths in critically ill patients, accounting for approximately 25-30% of hospitalized patients, and is a dangerous condition with a mortality rate of 30-60%, but there is no effective treatment for this disease.

[0009] In the absence of effective therapeutic agents for sepsis, various attempts have been made to develop therapeutic agents, particularly those that suppress inflammation. However, many anti-inflammatory drugs have failed in clinical trials, and novel therapeutic strategies that do not suppress inflammation are needed.

[0010] Recently, it has been found that SIRT1 protein activity and synthesis are reduced in sepsis patients compared to normal controls, and that S1P (sphingosine-1-phosphate) content is also reduced (Critical care, 2015, 19, 372). Because S1P content tends to decrease rapidly in sepsis patients, attempts to treat sepsis have been made using S1P agonists to increase S1P content or inhibitors that inhibit the enzyme that degrades S1P (J. Pharmacol. Exp. Ther., 2015, 352, 61-66). Other attempts have also been made to increase the level and activity of SIRT1 protein, which is reduced in sepsis patients. The best methods to increase S1P content involve overproducing the gene that synthesizes S1P or knocking down or out the enzyme that degrades S1P. However, these methods involve the disadvantage of genetic manipulation, and S1P agonists have only limited therapeutic efficacy.

[0011] Furthermore, if sepsis becomes severe, it can lead to acute respiratory distress syndrome (ARDS).

[0012] Acute respiratory distress syndrome (ARS) is a group of symptoms that manifests as acute pulmonary edema, causing breathing difficulties that do not improve even with the supply of high-concentration oxygen. ARS can be caused by sepsis, trauma, massive blood transfusion, drugs, etc. ARS is a dangerous condition with a mortality rate of 40-50%, but there is no effective treatment for it. Summary of the Invention [Problem to be solved by the invention]

[0013] An object of the present invention is to provide a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof, which exhibits effects of preventing, treating, and ameliorating degenerative brain diseases, sepsis, and acute respiratory distress syndrome:

[0014] Another object of the present invention is to provide a pharmaceutical composition comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.

[0015] It is still another object of the present invention to provide a health functional food containing the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof. [Means for solving the problem]

[0016] One embodiment of the present invention relates to a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof:

[0017] [ka]

[0018] In the above formula, R is a hydrogen atom, a C1-C6 alkyl group, or an aryl group.

[0019] As used herein, a C1-C6 alkyl group refers to a linear or branched monovalent hydrocarbon group composed of 1 to 6 carbon atoms, including, but not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-pentyl, and n-hexyl.

[0020] As used herein, the term "aryl" includes both aromatic and heteroaromatic groups and their partially reduced derivatives. The aromatic group is a 5- to 15-membered monocyclic or fused ring, and the heteroaromatic group refers to an aromatic group containing one or more oxygen, sulfur, or nitrogen atoms. Representative examples of aryl groups include, but are not limited to, phenyl, naphthyl, pyridinyl, pyrimidinyl, furanyl, thiophenyl, indolyl, quinolinyl, imidazolinyl, oxazolyl, thiazolyl, and tetrahydronaphthyl.

[0021] In one embodiment of the present invention, R is a hydrogen atom or a C1-C6 alkyl group.

[0022] As used herein, pharmaceutically acceptable salts include both non-toxic inorganic and organic acid salts, and may include, for example, hydrochloride, sulfate, nitrate, phosphate, acetate, adipate, aspartate, benzoate, benzenesulfonate, citrate, camphorate, camphorsulfonate, diphosphate, ethanesulfonate, fumarate, glutamate, maleate, lactate, methanesulfonate, succinate, tartrate, picrate, tosylate, and the like, and may particularly be the hydrochloride salt of the compound represented by Chemical Formula 1, which is represented by Chemical Formula 1a below.

[0023] [ka]

[0024] On the other hand, one embodiment of the present invention is (i) protecting the amine group of a compound represented by the following Chemical Formula 2 to obtain a compound represented by the following Chemical Formula 3: (ii) protecting the primary hydroxy group of the compound represented by the following chemical formula 3 to obtain a compound represented by the following chemical formula 4: (iii) reacting a compound represented by the following chemical formula 4 with phosphoryl chloride (POCl3), or phosphoryl chloride (POCl3) and a compound represented by the following chemical formula 5, or a compound represented by the following chemical formula 6, to obtain a compound represented by the following chemical formula 7: (iv) selectively deprotecting the hydroxy-protecting group of the compound represented by the following formula 7 to obtain a compound represented by the following formula 8; and (v) A method for producing a compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof, comprising the step of deprotecting an amine protecting group of a compound represented by Chemical Formula 8:

[0025] [ka]

[0026] [ka]

[0027] [ka]

[0028] [ka]

[0029] [ka]

[0030] [ka]

[0031] [ka]

[0032] [ka]

[0033] In the above formula, PG1 is an amine protecting group, PG2 is a hydroxy protecting group, R' is a C1-C6 alkyl or aryl group; R is a hydrogen atom, a C1-C6 alkyl group, or an aryl group.

[0034] In one embodiment of the present invention, the amine protecting group may be, but is not limited to, t-butyloxycarbonyl, benzyloxycarbonyl, and the like.

[0035] In one embodiment of the present invention, the hydroxy protecting group may be, but is not limited to, t-butyldimethylsilyl, pivaloyl, and the like.

[0036] The production method of the present invention will be explained in more detail below with reference to the following reaction scheme 1.

[0037] The method described in the following reaction scheme 1 is merely an example of a method that is typically used, and the reaction reagents, reaction conditions, etc. may be appropriately changed depending on the case.

[0038] [ka]

[0039] Step 1: Synthesis of the compound represented by formula 3 The compound represented by Chemical Formula 3 can be prepared by protecting the amine group of the compound represented by Chemical Formula 2.

[0040] The protection may be carried out using di-t-butyl dicarbonate (Boc2O), benzyl chloroformate, and the like.

[0041] Step 2: Synthesis of the compound represented by formula 4 The compound represented by Chemical Formula 4 can be prepared by protecting the primary hydroxy group of the compound represented by Chemical Formula 3.

[0042] The protection may be carried out using t-butyldimethylsilyl chloride, pivaloyl chloride, or the like in the presence of a catalyst and a base.

[0043] In this case, 4-dimethylaminopyridine or the like may be used as the catalyst, and triethylamine, imidazole or the like may be used as the base.

[0044] Step 3: Synthesis of the compound represented by formula 7 The compound of formula 7 can be prepared by reacting the compound of formula 4 with phosphoryl chloride (POCl3), or phosphoryl chloride (POCl3) and the compound of formula 5, or the compound of formula 6.

[0045] The reaction may be carried out in the presence of a base, and in this case, pyridine or the like may be used as the base.

[0046] Step 4: Synthesis of the compound represented by formula 8 The compound represented by Chemical Formula 8 can be prepared by selectively deprotecting the hydroxy protecting group of the compound represented by Chemical Formula 7.

[0047] The deprotection may be carried out in the presence of a base and HF.

[0048] The base may be pyridine or the like.

[0049] Step 5: Preparation of the compound represented by formula 1 The compound represented by Chemical Formula 1 can be prepared by deprotecting the amine protecting group of the compound represented by Chemical Formula 8.

[0050] The deprotection may be carried out using an acid.

[0051] In this case, the acid may be an inorganic acid or an organic acid, for example, hydrochloric acid.

[0052] By carrying out deprotection using an acid as described above, an acid addition reaction can be carried out simultaneously with the deprotection, and the compound represented by Chemical Formula 1 can be obtained in the form of an acid addition salt.

[0053] The compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof according to the present invention can increase the expression of SIRT1 and restore the reduced intestinal length in an animal model of degenerative brain disease (see Test Examples 1 and 2).

[0054] In addition, the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof according to the present invention can reduce the expression of amyloid and tau proteins, which are biomarkers for Alzheimer's disease (see Test Example 3).

[0055] Furthermore, the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof according to the present invention exhibits neuronal proliferation efficacy in a Parkinson's disease cell model and can promote the differentiation of totipotent stem cells into dopamine-secreting neurons. It can also restore the expression of TH that is reduced by rotenone, and conversely, reduce the expression of α-synuclein that is increased by rotenone (see Test Examples 4 to 6).

[0056] In addition, the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof according to the present invention can ameliorate sepsis and acute respiratory distress syndrome (ARDS) induced by CLP surgery (see Test Examples 7 and 8).

[0057] Furthermore, the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof according to the present invention can improve sepsis and acute respiratory distress syndrome through inflammation suppression and vascular barrier strengthening (see Test Examples 9 and 10).

[0058] Therefore, one embodiment of the present invention relates to a pharmaceutical composition for preventing or treating degenerative brain diseases, comprising a compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[0059] Specifically, the degenerative brain disease may be Alzheimer's disease or Parkinson's disease.

[0060] Another embodiment of the present invention relates to a pharmaceutical composition for preventing or treating sepsis or acute respiratory distress syndrome, comprising the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[0061] The pharmaceutical composition of the present invention may be administered orally (e.g., by ingestion or inhalation) or parenterally (e.g., by injection, deposition, implant, or suppository), and the injection may be, for example, intravenous, subcutaneous, intramuscular, or intraperitoneal. Depending on the route of administration, the pharmaceutical composition of the present invention may be formulated into tablets, capsules, granules, fine subtilis, powder, sublingual tablets, suppositories, ointments, injections, emulsions, suspensions, syrups, sprays, etc. The various forms of the pharmaceutical composition of the present invention can be prepared by known techniques using pharmaceutically acceptable carriers commonly used for each dosage form. Examples of pharmaceutically acceptable carriers include excipients, binders, disintegrating agents, lubricants, preservatives, antioxidants, isotonic agents, buffers, coating agents, sweeteners, solubilizers, bases, dispersing agents, wetting agents, suspending agents, stabilizers, coloring agents, and the like.

[0062] The pharmaceutical composition according to the present invention contains the compound of the present invention or a pharmaceutically acceptable salt thereof in an amount ranging from about 0.001 to 95% by weight, depending on the form of the drug.

[0063] The specific dosage of the pharmaceutical composition of the present invention may vary depending on the species, body weight, sex, severity of the disease, and the doctor's judgment of the mammal (including human) being treated. Preferably, for oral administration, 0.01 to 50 mg of the active ingredient is administered per kg of body weight per day, and for parenteral administration, 0.001 to 10 mg of the active ingredient is administered per kg of body weight per day. The total daily dosage may be administered once or in divided doses depending on the severity of the disease, the doctor's judgment, etc.

[0064] One embodiment of the present invention relates to a health functional food for preventing or ameliorating degenerative brain diseases, comprising the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[0065] Specifically, the degenerative brain disease may be Alzheimer's disease or Parkinson's disease.

[0066] There are no particular limitations on the type of health functional food according to the present invention, and it may be in the form of an oral formulation such as powder, granules, tablets, capsules, suspensions, emulsions, or syrups, or it may be added to common foods such as candy, sweets, gum, ice cream, noodles, bread, or beverages.

[0067] The health functional food of the present invention can be manufactured by using fillers, extenders, binders, wetting agents, disintegrants, sweeteners, flavorings, preservatives, surfactants, lubricants, excipients, etc. in a conventional manner depending on the form.

[0068] The content of the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof in the production of the health functional food varies depending on the form of the health functional food, but is approximately 0.001 to 10% by weight, preferably 0.01 to 5% by weight. [Effects of the Invention]

[0069] The compounds of the present invention can increase the expression of SIRT1 and restore reduced intestinal length in animal models of degenerative brain disease. Furthermore, the compounds of the present invention reduce the expression of amyloid and tau proteins, which are biomarkers for Alzheimer's disease, and exhibit neuronal proliferation effects in Parkinson's disease cell models, promoting the differentiation of totipotent stem cells into dopamine-secreting neurons. They also restore the expression of TH reduced by rotenone and, conversely, reduce the expression of α-synuclein increased by rotenone. Furthermore, the compounds of the present invention can ameliorate sepsis and acute respiratory distress syndrome induced by CLP treatment.

[0070] Therefore, the compounds according to the present invention can be effectively used in compositions for preventing, treating and ameliorating degenerative brain diseases, sepsis and acute respiratory distress syndrome. [Brief explanation of the drawings]

[0071] [Figure 1]1 shows the results of Western blot analysis measuring changes in SIRT1 expression when HUVEC cells are treated with a compound represented by Chemical Formula 1a-1. [Figure 2] 1 shows the results of examining changes in intestinal length when a compound represented by chemical formula 1a-1 is treated in an AD-induced animal model. [Figure 3] 1 shows the results of changes in the expression of mAβ and Tau, which are AD biomarkers, observed by fluorescence microscopy when an AD-induced animal model was treated with the compound represented by chemical formula 1a-1. [Figure 4] 1 shows the results of evaluating the efficacy of treating a compound represented by chemical formula 1a-1 on the proliferation of nerve cells in a cell model of Parkinson's disease. [Figure 5] 1 shows the results of evaluating the efficacy of treating a compound represented by Chemical Formula 1a-1 with a compound for promoting differentiation from totipotent stem cells into dopamine-secreting neurons. [Figure 6] 1 shows the results of Western blot analysis measuring changes in expression of tyrosine hydroxylase and α-synuclein when treated with a compound represented by Chemical Formula 1a-1, and a graph illustrating the results. [Figure 7] 1 is a graph showing the effect on mouse survival rate when the compound represented by Formula 1a-1 was administered at 100 ng / mouse (5 μg / kg) 6 and 16 hours after CLP treatment. [Figure 8] 1 is a graph showing the effect of fingolimod (FTY720) administered at 100 ng / mouse (5 μg / kg) 6 and 16 hours after CLP treatment on mouse survival rate. [Figure 9] Figure 9A is a photograph of lung tissue observed under an optical microscope when treated with the compound represented by chemical formula 1a-1 after CLP treatment, Figure 9B is a graph showing the results of the lung injury score, in which lung tissue damage was evaluated by histological scoring, and Figure 9C is a graph showing the wet-dry weight ratio of the excised lungs. [Figure 10]1 is a graph showing the effect of treatment with a compound represented by Chemical Formula 1a-1 after CLP treatment on the secretion of inflammatory cytokines TNF-α (A) and IL-6 (B). [Figure 11] Figure 11A is a graph showing the effect on the permeability of the MYSEC cell line when treated with LPS in the presence of a compound represented by Chemical Formula 1a-1, and Figures 11B and 11C are a photograph and a graph, respectively, showing the effect on the permeability of mouse organs when treated with a compound represented by Chemical Formula 1a-1 after CLP treatment. DETAILED DESCRIPTION OF THE INVENTION

[0072] The present invention will be described in more detail below with reference to examples. These examples are merely for the purpose of illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not limited to these examples.

[0073] Example 1: Preparation of (4S,5R)-4-((S)-1-amino-2-hydroxyethyl)-2-hydroxy-5-tetradecyl-1,3,2-dioxaphospholane 2-oxide hydrochloride (1a-1)

[0074] [ka]

[0075] Example 1-1: Preparation of t-butyl ((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)carbamate (3-1)

[0076] [ka]

[0077] 100 g (314.951 mmol) of DS-phytosphingosine (2) was dissolved in 1000 mL of tetrahydrofuran, and then 82.5 g (377.941 mmol) of di-t-butyl dicarbonate (BocO) was added and stirred for 18 hours. After the reaction was completed, the insoluble material was filtered through filter paper and concentrated under reduced pressure. 800 mL of hexane was added to the concentrated oil phase and stirred. The resulting crystals were filtered and dried under vacuum to yield 118.36 g (90%) of the title compound.

[0078] 1 H NMR (DMSO-d6): δ 0.81-0.85(3H, t), 1.21-1.30(24H, m), 1.35-1.40(9H, m), 1.45-1.53(2H, m), 3.31-3.41(2H, m), 3.46-3.54(2H, m), 4.40-4.42(1H, m) ES-MS m / z: 418.44 [M+H] + Example 1-2: Preparation of t-butyl ((2S,3S,4R)-1-((t-butyldimethylsilyl)oxy)-3,4-dihydroxyoctadecan-2-yl)carbamate (4-1)

[0079] [ka]

[0080] 5 g (11.973 mmol) of t-butyl ((2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl)carbamate (3-1) prepared in Example 1-1 was dissolved in 50 mL of dichloromethane, and then 2 g (13.170 mmol) of t-butyldimethylsilyl chloride, 0.73 g (5.986 mmol) of 4-dimethylaminopyridine, and 1.85 mL (13.170 mmol) of triethylamine were added and stirred for 16 hours. After the reaction was completed, 30 mL of water was added. The separated organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The resulting material was purified using column chromatography to obtain 6.33 g (99%) of the title compound.

[0081] 1 H NMR (DMSO-d6): δ 0.81-0.85(12H, m), 1.13-1.27(24H, m), 1.34-1.39(9H, m), 1.45-1.51(2H, m), 3.23-3.41(2H, m), 3.61-3.64(1H, m), 3.74-3.77(1H, dd), 3.98-4.03(1H, m) Example 1-3: Preparation of t-butyl ((1S)-2-((t-butyldimethylsilyl)oxy)-1-((4S,5R)-2-hydroxy-2-oxide-5-tetradecyl-1,3,2-dioxaphospholan-4-yl)ethyl)carbamate (7-1)

[0082] [ka]

[0083] 9.3 g (17.472 mmol) of t-butyl ((2S,3S,4R)-1-((t-butyldimethylsilyl)oxy)-3,4-dihydroxyoctadecan-2-yl)carbamate (4-1) prepared in Example 1-2 was dissolved in 80 mL of dichloromethane and cooled to 0°C. 8.5 mL (104.832 mmol) of pyridine was added, and 2.1 mL (22.714 mmol) of phosphoryl trichloride diluted with 20 mL of dichloromethane was slowly added dropwise. The mixture was stirred at 20-25°C for 3 hours. Upon completion of the reaction, 50 mL of water was added to extract the compound. The separated organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain 10.3 g (100%) of the title compound.

[0084] 1 H NMR (DMSO-d6): δ 0.88-0.90(12H, m), 1.27-1.33(24H, m), 1.42-1.45(9H, m), 1.55-1.63(2H, m), 3.46-3.54(2H, m), 3.66-3.74(1H, m), 4.41-4.51(2H, m) ES-MS m / z: 594.62 [M+H]+ Example 1-4: Preparation of t-butyl ((1S)-2-hydroxy-1-((4S,5R)-2-hydroxy-2-oxide-5-tetradecyl-1,3,2-dioxaphospholan-4-yl)ethyl)carbamate (8-1)

[0085] [ka]

[0086] 10.3 g (17.472 mmol) of t-butyl ((1S)-2-((t-butyldimethylsilyl)oxy)-1-((4S,5R)-2-hydroxy-2-oxide-5-tetradecyl-1,3,2-dioxaphospholan-4-yl)ethyl)carbamate (7-1) prepared in Example 1-3 was dissolved in 100 mL of tetrahydrofuran, and then 1.8 mL (69.888 mmol) of hydrogen fluoride pyridine was added and stirred at 20-25°C for 16 hours. After the reaction was completed, 7.4 g (69.888 mmol) of sodium carbonate was added and stirred for 3 hours. 50 mL of 1N hydrochloric acid was added to the reaction solution, and then 100 mL of ethyl acetate was added for extraction. The separated organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain 6 g (71.6%) of the title compound.

[0087] 1 H NMR (DMSO-d6): δ 0.84-0.87(3H, t), 1.24-1.29(24H, m), 1.38-1.43(9H, m), 1.45-1.55(2H, m), 3.36-3.62(2H, m), 3.67-3.74(1H, m), 4.37-4.45(2H, m) ES-MS m / z: 480.44 [M+H] + Example 1-5: Preparation of (4S,5R)-4-((S)-1-amino-2-hydroxyethyl)-2-hydroxy-5-tetradecyl-1,3,2-dioxaphospholane 2-oxide hydrochloride (1a-1)

[0088] [ka]

[0089] 6 g (12.511 mmol) of t-butyl ((1S)-2-hydroxy-1-((4S,5R)-2-hydroxy-2-oxide-5-tetradecyl-1,3,2-dioxaphospholan-4-yl)ethyl)carbamate (8-1) prepared in Example 1-4 was dissolved in 120 mL of ethyl acetate, and then 31 mL of 4 M hydrochloride (in dioxane, 125.107 mmol) was added and stirred at 20-25°C for 18 hours. After completion of the reaction, the reaction solution was concentrated. 120 mL of acetonitrile was added to the concentrated residue to crystallize it, and the mixture was stirred for 3 hours. The resulting crystals were filtered and dried under vacuum to obtain 4.5 g (86%) of the title compound.

[0090] 1 H NMR (DMSO-d6): δ 0.84-0.87(3H, t), 1.16-1.24(24H, m), 1.44-1.68(2H, m), 3.65-3.81(2H, m), 3.93-4.00(1H, m), 4.43-4.48(2H, m) 31 P NMR (DMSO-d): δ -8.03 ppm 13 C NMR (DMSO-d6): δ 14.40(1C, s), 22.55(1C, s), 24.92(1C, s), 29.16(1C, s), 29.48-29.64(9C, s), 31.75(1C, s), 46.70(1C, s), 64.93(1C, s), 69.96(1C, s), 80.94(1C, s) ES-MS m / z: 380.39 [M+H] + Example 2: Preparation of (4S,5R)-4-((S)-1-amino-2-hydroxyethyl)-2-methoxy-5-tetradecyl-1,3,2-dioxaphospholane-2-oxide hydrochloride (1a-2)

[0091] [ka]

[0092] Example 2-1: Preparation of t-butyl ((1S)-2-((t-butyldimethylsilyl)oxy)-1-((4S,5R)-2-methoxy-2-oxide-5-tetradecyl-1,3,2-dioxaphospholan-4-yl)ethyl)carbamate (7-2)

[0093] [ka]

[0094] 10 g (18.801 mmol) of t-butyl ((2S,3S,4R)-1-((t-butyldimethylsilyl)oxy)-3,4-dihydroxyoctadecan-2-yl)carbamate (4-1) prepared in Example 1-2 was dissolved in 100 mL of dichloromethane and cooled to 0°C. 6.1 mL (75.205 mmol) of pyridine was added, and 2.4 mL (24.442 mmol) of methyl phosphorodichloridate diluted with 20 mL of dichloromethane was slowly added dropwise. The mixture was stirred at 20-25°C for 3 hours until the reaction was complete, after which the compound was concentrated. 100 mL of ethyl acetate (EA) was added to the concentrated compound and stirred, and the resulting crystals were filtered. The filtrate was concentrated under reduced pressure to obtain 4.8 g (48%) of the title compound.

[0095] 1 H NMR (DMSO-d6): δ 0.87-0.91(12H, m), 1.25-1.30(24H, m), 1.43-1.46(9H, m), 1.55-1.63(2H, m), 3.44-3.52(2H, m), 3.62(3H, s) 3.68-3.75(1H, m), 4.43-4.52(2H, m) ES-MS m / z: 608.85 [M+H] + Example 2-1-1: Preparation of t-butyl ((1S)-2-((t-butyldimethylsilyl)oxy)-1-((4S,5R)-2-methoxy-2-oxide-5-tetradecyl-1,3,2-dioxaphospholan-4-yl)ethyl)carbamate (7-2)

[0096] [ka]

[0097] 10 g (18.801 mmol) of t-butyl ((2S,3S,4R)-1-((t-butyldimethylsilyl)oxy)-3,4-dihydroxyoctadecan-2-yl)carbamate (4-1) prepared in Example 1-2 was dissolved in 100 mL of dichloromethane and cooled to 0°C. 6.1 mL (75.205 mmol) of pyridine was added, and 2.3 mL (24.442 mmol) of phosphoryl trichloride diluted with 20 mL of dichloromethane was slowly added dropwise. The mixture was stirred at 20-25°C for 3 hours, and upon completion of the reaction, 10 mL of methanol was added. After 2 hours, the mixture was concentrated, and 100 mL of ethyl acetate (EA) was added and stirred. The resulting crystals were filtered. The filtrate was concentrated under reduced pressure to obtain 5.7 g (55%) of the title compound.

[0098] 1 H NMR (DMSO-d6): δ 0.89-0.92(12H, m), 1.26-1.30(24H, m), 1.43-1.49(9H, m), 1.53-1.62(2H, m), 3.45-3.53(2H, m), 3.64(3H, s) 3.69-3.76(1H, m), 4.41-4.52(2H, m) ES-MS m / z: 608.85 [M+H] + Example 2-2: Preparation of t-butyl ((1S)-2-hydroxy-1-((4S,5R)-2-methoxy-2-oxide-5-tetradecyl-1,3,2-dioxaphospholan-4-yl)ethyl)carbamate (8-2)

[0099] [ka]

[0100] 4.8 g (7.896 mmol) of t-butyl ((1S)-2-((t-butyldimethylsilyl)oxy)-1-((4S,5R)-2-methoxy-2-oxide-5-tetradecyl-1,3,2-dioxaphospholan-4-yl)ethyl)carbamate (7-2) prepared in Example 2-1 was dissolved in 50 mL of tetrahydrofuran, and then 0.84 mL (31.584 mmol) of hydrogen fluoride pyridine was added and stirred at 20-25°C for 16 hours. After the reaction was completed, 3.3 g (31.584 mmol) of sodium carbonate was added and stirred for 3 hours. 50 mL of 1N hydrochloric acid was added to the reaction solution, and then 100 mL of ethyl acetate was added for extraction. The separated organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain 2.5 g (64%) of the title compound.

[0101] 1 H NMR (DMSO-d6): δ 0.84-0.86(3H, t), 1.22-1.30(24H, m), 1.37-1.43(9H, m), 1.46-1.55(2H, m), 3.36-3.60(2H, m), 3.61(3H, s), 3.67-3.71(1H, m), 4.38-4.47(2H, m) ES-MS m / z: 494.12 [M+H] + Example 2-3: Preparation of (4S,5R)-4-((S)-1-amino-2-hydroxyethyl)-2-methoxy-5-tetradecyl-1,3,2-dioxaphospholane 2-oxide hydrochloride (1a-2)

[0102] [ka]

[0103] 2.5 g (5.064 mmol) of t-butyl ((1S)-2-hydroxy-1-((4S,5R)-2-methoxy-2-oxide-5-tetradecyl-1,3,2-dioxaphospholan-4-yl)ethyl)carbamate (7-2) prepared in Example 2-2 was dissolved in 50 mL of ethyl acetate, and then 12.7 mL of 4 M hydrochloride (in dioxane, 50.647 mmol) was added and stirred at 20-25°C for 18 hours. After completion of the reaction, the reaction solution was concentrated. 100 mL of acetonitrile was added to the concentrated residue to crystallize it, and the mixture was stirred for 3 hours. The resulting crystals were filtered and dried under vacuum to obtain 1.9 g (90%) of the title compound.

[0104] 1 H NMR (DMSO-d6): δ 0.85-0.87(3H, t), 1.15-1.25(24H, m), 1.43-1.67(2H, m), 3.61(3H, s), 3.66-3.81(2H, m), 3.95-4.01(1H, m), 4.43-4.46(2H, m) 31 P NMR (DMSO-d): δ -8.01 ppm 13 C NMR (DMSO-d6): δ 14.40(1C, s), 22.55(1C, s), 24.92(1C, s), 29.16(1C, s), 29.48-29.64(9C, s), 31.75(1C, s), 46.70(1C, s), 55.3(1C, s), 64.93(1C, s), 69.96(1C, s), 80.94(1C, s) ES-MS m / z: 393.56 [M+H] + Test Example 1: Analysis of effects on SIRT1 expression To confirm the increase in SIRT1 expression in human umbilical vein endothelial cells (HUVECs) by the compound represented by Formula 1a-1 prepared in Example 1, 5 x 10 HUVEC cells were cultured in a 60 mm cell culture dish using ECBM2 medium (Promocell).5 The cells were seeded with 1 / 4 ml of PBS and allowed to stabilize for 12 hours in an incubator where the temperature and CO2 were maintained at 37°C and 5%, respectively.

[0105] Then, the cells were treated with the compound represented by Formula 1a-1 prepared in Example 1 at 10 nM, 100 nM, and 1000 nM, respectively, and then cultured for another 24 hours. SIRT1 expression was then confirmed by Western blot.

[0106] The results are shown in Figure 1.

[0107] From Figure 1, it can be seen that when HUVEC cells are treated with the compound represented by Chemical Formula 1a-1, the expression of SIRT1 is increased even at a very low concentration of 10 nM. In particular, the compound represented by Chemical Formula 1a-1 increased the expression of SIRT1 by 50% (EC 50 ) was found to be approximately 10 nM.

[0108] On the other hand, when mouse vascular cells were treated with the compound represented by chemical formula 1a-1, maximum expression was observed at 100 ng / mL.

[0109] Resveratrol, a well-known SIRT1 activator, showed EC 50 is known to be approximately 36 μM. Therefore, it can be confirmed that the compound represented by Chemical Formula 1a-1 increases the expression of SIRT1 at a concentration several thousand times lower than that of resveratrol.

[0110] Test Example 2: Analysis of intestinal length changes in AD-induced animal models In degenerative brain diseases (AD and PD), the length of the intestine tends to decrease as the disease progresses, so we confirmed the change in intestinal length in an AD-induced animal model.

[0111] APP / PS / Tau transgenic mice were used as an AD-induced animal model. 12-week-old transgenic mice were intraperitoneally administered 1 mg / kg of the compound represented by formula 1a-1 once every three days for two weeks, and the changes in intestinal length of the mice were measured one and four weeks later.

[0112] The results are shown in Figure 2.

[0113] From Figure 2, it can be seen that the intestinal length of the mice administered with the compound represented by Chemical Formula 1a-1 was increased compared to the AD-induced mice in the control group (Control) that were not administered with the compound represented by Chemical Formula 1a-1.

[0114] In particular, when the intestinal length was measured 1 week (3W) and 4 weeks (6W) after administration of the compound represented by Chemical Formula 1a-1, it was confirmed that the intestinal length after 4 weeks was longer than the intestinal length after 1 week.

[0115] Test Example 3: Drug efficacy analysis in AD-induced animal models To confirm the therapeutic effect of Alzheimer's disease (AD), we analyzed the effects on amyloid beta monomer (mAβ) and tau protein, which are biomarkers of AD, in an AD-induced animal model.

[0116] As an animal model for AD induction, we used APP / PS / Tau transgenic mice.

[0117] Twelve-week-old transgenic mice were intraperitoneally administered the compound represented by Formula 1a-1 at a concentration of 1 mg / kg once every three days for two weeks, and the expression of mAβ and Tau proteins in the mouse brains was measured one and four weeks later. After anesthetizing the mice, the cervical vertebrae were incised using a dissection tool, and a portion of the back of the head was incised to remove both parts of the skull. The brains were then washed with PBS (phosphate buffered saline) and fixed in 4% paraformaldehyde (Sigma, St. Louis, MO, USA) for 12 hours at 4°C. The brains were cut into 10 μm-thick sections using a vibratome, and the hippocampal and cortical sections were collected and placed in 24-well plates. PBS was added to the wells containing the fixed tissue sections, and fluorescent nanoparticles (QD565, red fluorescence) conjugated with anti-amyloid antibody and anti-tau antibody (Santa Cruz, USA) were added at 2 μM each and incubated for 12 hours at 4° C. The presence or absence of mAβ and tau expression was confirmed using a fluorescence microscope.

[0118] The results are shown in Figure 3.

[0119] 3 shows that mice not administered the compound represented by formula 1a-1 exhibited very strong red fluorescence for mAβ and Tau, whereas the group administered the compound represented by formula 1a-1 exhibited faint red fluorescence for mAβ and Tau at 1 and 4 weeks after drug administration. Therefore, it can be seen that administration of the compound represented by formula 1a-1 significantly reduced the expression of amyloid and Tau proteins, which are biomarkers for Alzheimer's disease. These results indicate that the compound represented by formula 1a-1 can be used as a therapeutic agent for Alzheimer's disease.

[0120] Test Example 4: Analysis of effects on proliferation of human nerve cells The SH-SY5Y cell line, derived from human neuroblastoma and exhibiting properties similar to dopamine neurons, was used as a cell model for Parkinson's disease (PD) to evaluate the efficacy of neuronal proliferation.

[0121] The cells were seeded onto a T75 plate using a medium containing DMEM / F12 (Hyclone), 10% FBS (Hyclone), and 1% penicillin-streptomycin (Hyclone). The medium was changed every two days. Subculture was performed when the cell density reached 80-90%.

[0122] Thereafter, to confirm the effect of proliferation of nerve cells, the subcultured SH-SY5Y cells were plated in 96 wells at 5 × 10 3 Cells were seeded at 1000 cells / well. After one day, the medium was replaced with one containing 5% FBS and treated with the compound represented by formula 1a-1 at two-fold dilutions from 500 nM to 15.6 nM. The cells were then cultured for five days. The medium was replaced with 10% FBS containing the drug every two days. The 10% FBS culture medium served as a control. After five days, the amount of live cells was measured at 450 nm absorbance using EZ-cytox (DoGen) to compare cell viability.

[0123] The results are shown in Figure 4.

[0124] From FIG. 4, it can be seen that as the concentration of the compound represented by Chemical Formula 1a-1 increases, the proliferation of nerve cells increases, and the maximum proliferation effect is observed at around 100 nM.

[0125] Test Example 5: Analysis of effects on differentiation into nerve cells Totipotent stem cells were seeded at 5,000 cells / microwell onto AggreWell plates (stemcell) and cultured in E6 medium (GIBCO) for 5 days to induce embryonic body (EB) formation. For neural rosette formation, 30 EBs were seeded onto 35 mm dishes and cultured in N2bF medium for 10 days. The 35 mm dishes were coated with 15 μg / ml poly-L-ornithine (Sigma) and 1 μg / ml fibronectin (Sigma). N2bF medium consisted of DMEM / F12 (GIBCO), 1x N2 supplement (Stemcell), 10% NEAA (GIBCO), 55 μM beta-mercaptoethanol (GIBCO), and 20 ng / ml bFGF (Peprotech). For the formation of spherical neural masses (SNMs), rosette clumps were fragmented and cultured in N2bF medium in Petri dishes. SNMs were not larger than 500 μM and were cultured in fragments for 3–4 passages. After SNM subculture, clumps were finely fragmented using a tungsten mesh (Nilaco) and then attached to PLO / FN-coated dishes for the neural progenitor cell (NPC) culture stage. They were then cultured in N2bF medium. To differentiate NPCs into dopaminergic neurons, the NPCs were cultured for 2 days and then replaced with ITS-B27 medium (DMEM / F12 (GIBCO)), 1.5 mg / ml D-(+)-glucose (Sigma), 5 μg / ml insulin (Wako), 50 μg / ml transferrin (Wako), 30 nM selenite (Sigma), 2.5% GlutaMAX (GIBCO), and 1x B27 (GIBCO).After 4 days of NPC culture, Sonic Hedgehog (200 ng / ml) and FGF8 (100 ng / ml) (Peprotech) were added to the ITS-B27 medium. After 6 days of NPC culture, Sonic Hedgehog (200 ng / ml), GDNF (20 ng / ml), BDNF (20 ng / ml) (Peprotech), ascorbic acid (200 μM), and cAMP (500 μM) (Sigma) were added to the ITS-B27 medium and cultured for approximately 5 weeks. Compound 1a-1 was added after 2 days of NPC culture. Differentiation was induced without treatment with compound 1a-1 as a differentiated control (differentiated CTL). For comparison, undifferentiated control (undifferentiated CTL) cells were also treated without treatment with compound 1a-1.

[0126] The expression levels of tyrosine hydroxylase (TH) and dopamine transporter (DAT) in cells treated with or without the compound represented by formula 1a-1 were compared to evaluate the effect of the compound represented by formula 1a-1 on differentiation into neurons.

[0127] The results are shown in Figure 5.

[0128] Figure 5A shows the results of cell fluorescent immunostaining, confirming that treatment with the compound represented by Chemical Formula 1a-1 increases TH expression. In particular, TH expression, which does not increase until differentiation is induced for at least five weeks, is elevated after just three weeks of treatment with the compound represented by Chemical Formula 1a-1. Figure 5B shows the results of Western blot analysis of samples from five weeks of differentiation. Treatment with the compound represented by Chemical Formula 1a-1 confirms increased TH expression and DAT glycosylation. This demonstrates that the compound represented by Chemical Formula 1a-1 can promote the differentiation of totipotent stem cells into dopamine-secreting neurons.

[0129] Test Example 6: Analysis of effects on neuron-specific protein expression Western blot was performed to analyze the effect on the expression of tyrosine hydroxylase (TH) and α-synuclein, which are neuron-specific proteins.

[0130] To induce differentiation of SH-SY5Y cells into TH-secreting neurons, the cells were treated with retinoic acid and 12-O-tetradecanoylphorbol-13-acetate (TPA) for 6 days.

[0131] Differentiated dopamine-secreting neurons were lysed in PRO-PREP (Intronbio) cell lysis solution supplemented with a protease inhibitor cocktail (Gendepot) and then electrophoresed at 20 μg / well on a 10% SDS polyacrylamide gel. The transfer was performed using a PVDF (Merck Millipore) membrane, and the gel was incubated for over 2 hours with tyrosine hydroxylase antibody, α-synuclein antibody (Cell Signaling), and β-actin antibody (Santa Cruz). Protein expression levels were confirmed by developing the gel with ECL substrate (Biorad) and then graphed using the Image J program.

[0132] The results are shown in Figure 6.

[0133] Specifically, FIG. 6A shows the results of Western blot analysis measuring changes in the expression of tyrosine hydroxylase and α-synuclein upon treatment with a compound represented by Chemical Formula 1a-1, and FIGS. 6B and 6C are graphs showing the results of Western blot analysis measuring changes in the expression of tyrosine hydroxylase and α-synuclein, respectively.

[0134] Figure 6 shows that TH expression was significantly increased in differentiated dopamine-secreting neurons (differentiated CTL) compared to undifferentiated neurons (SH-SY5Y cell line, undifferentiated CTL). However, when differentiated dopamine-secreting neurons were treated with rotenone (RT), TH expression decreased and α-synuclein expression increased. However, when treated with the compound represented by Chemical Formula 1a-1, the TH expression decreased by rotenone was restored, and conversely, the α-synuclein expression increased by rotenone was decreased. In particular, it can be seen that the compound represented by Chemical Formula 1a-1 has a superior effect compared to resveratrol, which was used as a control drug.

[0135] Rotenone is known to be a drug that induces Parkinson's disease, and therefore, the above results indicate that the compound represented by formula 1a-1 can be used as a therapeutic agent for Parkinson's disease.

[0136] Test Example 7: Analysis of the effect on sepsis induced by CLP treatment Seven- to eight-week-old C57BL / 6 mice were purchased from JA BIO (Suwon, Gyeonggi-do), and sepsis was induced by cecal ligation and puncture (CLP).

[0137] Specifically, to induce sepsis, mice were anesthetized with 150 mg / kg ketamine and 17.5 mg / kg rompun. A 1-2 cm abdominal incision was then made to expose the cecum. The cecum was ligated distal to the ileocecal valve with 6-0 silk suture and punctured with an 18-gauge needle. The cecum was gently squeezed to expel a 1-2 cm stool mass, and then returned to the abdominal cavity. The abdomen was then closed, and pre-warmed saline (2.5 mL / 100 g body weight) was injected intraperitoneally into the mice immediately after the procedure. Six and 16 hours after CLP, the compound represented by formula 1a-1 was dissolved in phosphate buffered saline (PBS) and intravenously injected at 100 ng / mouse (5 μg / kg), and survival rates were assessed for 10 days. For comparison, the survival rate of mice treated with PBS alone after CLP was investigated as a sham control group.

[0138] The results are shown in Figure 7.

[0139] 7, it can be seen that all animals in the drug-negative control group, which received only PBS, died within 48 hours, demonstrating that the CLP treatment conditions provided a highly effective severe sepsis model.

[0140] In this severe sepsis model, the experimental group in which the compound represented by formula 1a-1 was administered 6 and 16 hours after CLP treatment showed a survival rate of over 80% for 10 days. No deaths occurred in the experimental group even after 10 days. This demonstrates that the compound represented by formula 1a-1 is effective in treating severe sepsis.

[0141] For comparison, the survival rate of a control group was investigated under the same conditions, but administered fingolimod (FTY720), a known S1P modulator, instead of the compound represented by chemical formula 1a-1.

[0142] The results are shown in Figure 8.

[0143] From FIG. 8, it can be seen that FTY720 does not show any effect of improving survival rate.

[0144] Test Example 8: Analysis of the effect on acute respiratory distress syndrome induced by CLP treatment CLP increases inflammation and disrupts the vascular barrier, causing pulmonary edema and resulting in acute respiratory distress syndrome (ARDS). To confirm the effect on ARDS, mice were treated with CLP in the same manner as in Test Example 7, and then treated with the compound represented by Formula 1a-1 6 and 16 hours later. At 18 hours, the lungs were excised and subjected to histological analysis to determine the wet-to-dry weight ratio of the lungs. For comparison, mice not treated with CLP were used as a control.

[0145] For histological analysis, the excised lungs were fixed in 10% paraformaldehyde, infiltrated with paraffin, and sectioned into 5-μm-thick tissues. The tissues were then deparaffinized with xylene. The sections were mounted on slides, stained with H&E, and observed under a light microscope. Furthermore, lung tissue damage was assessed histologically, and a lung injury score (LIS) was calculated for each group.

[0146] To determine the wet-dry weight ratio, lung organs were excised 18 hours after CLP treatment and the wet weight was quickly measured. After drying in a 60°C oven for 48 hours, the dry weight was measured and the wet-dry weight ratio was calculated. The wet-dry weight ratio is used as an index of organ edema formation.

[0147] The results of the histological analysis and wet-dry weight ratio measurements are shown in FIG.

[0148] Figure 9A is a photograph of lung tissue observed under an optical microscope when treated with the compound represented by chemical formula 1a-1 after CLP treatment, Figure 9B is a graph showing the results of the lung injury score, in which lung tissue damage was evaluated by histological scoring, and Figure 9C is a graph showing the wet-dry weight ratio of the excised lungs.

[0149] Figure 9A confirms that lung tissue is damaged after CLP treatment, but that treatment with the compound represented by chemical formula 1a-1 restores the lung tissue to nearly normal. Figure 9B also confirms that the lung injury score significantly increases after CLP treatment, but that treatment with the compound represented by chemical formula 1a-1 restores the lung injury score to nearly normal. Figure 9C also confirms that inflammation and vascular barrier breakdown after CLP treatment cause water accumulation in the lung tissue, resulting in pulmonary edema, which increases the wet-to-dry lung weight ratio. Treatment with the compound represented by chemical formula 1a-1 restores the lung wet-to-dry lung weight ratio to normal.

[0150] Therefore, it can be seen that the compound represented by Chemical Formula 1a-1 can treat ARDS.

[0151] Test Example 9: Analysis of the effect on inflammatory cytokines after CLP treatment After CLP treatment, the inflammatory cytokines TNF-α and IL-6 were analyzed by ELISA.

[0152] Specifically, after CLP, the mice were treated with the compound represented by formula 1a-1 6 and 16 hours later, and then blood was collected from the mice 18 hours later and left at room temperature for 1 hour. The blood was then centrifuged at 3000 rpm for 10 minutes to obtain serum, which was then analyzed for the amounts of TNF-α and IL-6.

[0153] The results are shown in Figure 10.

[0154] FIG. 10 is a graph showing the effect of treatment with the compound represented by Chemical Formula 1a-1 after CLP treatment on the secretion of inflammatory cytokines TNF-α (A) and IL-6 (B).

[0155] As can be seen from FIG. 10, the levels of inflammatory cytokines TNF-α and IL-6 were significantly increased in the CLP-treated control group, whereas the levels of inflammatory cytokines were significantly reduced when treated with the compound represented by chemical formula 1a-1.

[0156] Therefore, it can be seen that the compound represented by chemical formula 1a-1 has excellent ability to suppress inflammatory responses, which are important for the treatment of sepsis or ARDS.

[0157] Test Example 10: Analysis of effects on permeability Permeability was measured in vitro using cell culture and in vivo using Evans blue dye. To increase permeability, LPS was used in the in vitro method, and CLP treatment was used in the in vivo method.

[0158] To measure permeability in vitro, the murine yolk sac endothelial cell (MYSEC) cell line was used. 2.5 × 10 5 MYSEC cell line cells / well were placed in the top chamber of a transwell and treated with 300 ng of Gram-negative bacterial lipopolysaccharide (LPS) in the presence of compound 1a-1 for 24 hours. The medium in the top chamber was replaced with 300 μl of serum-free medium containing 5 μl of streptavidin-HRP. After 5 minutes, 20 μl of medium from the lower chamber was removed, and HRP activity was measured by absorbance at 450 nm.

[0159] To confirm the mechanism by which the compound represented by formula 1a-1 improves permeability, the same experiment was performed in MYSEC cell line after knocking down SIRT1 protein.

[0160] To measure permeability in vivo, CLP was performed in the same manner as in Test Example 7, and the compound of Formula 1a-1 was treated 6 and 16 hours later. At 18 hours, 200 μL of 0.5% Evans blue dye (EBD) solution was administered intravenously and allowed to circulate for 1 hour. The organs were then excised and the presence of the dye was confirmed visually. The organs were then homogenized in a homogenizer and incubated at 55°C for 48 hours to extract the dye, after which the absorbance at 620 nm was measured to confirm permeability.

[0161] The results are shown in Figure 11.

[0162] Figure 11A is a graph showing the effect on the permeability of the MYSEC cell line when treated with LPS in the presence of a compound represented by Chemical Formula 1a-1, and Figures 11B and 11C are a photograph and a graph, respectively, showing the effect on the permeability of mouse organs when treated with a compound represented by Chemical Formula 1a-1 after CLP treatment.

[0163] Figure 11A shows that treatment of the MYSEC cell line with LPS increases permeability, but treatment with the compound represented by Chemical Formula 1a-1 restores the increased permeability caused by LPS to normal. However, knockdown of SIRT1 protein in the MYSEC cell line does not reveal the effect of the compound represented by Chemical Formula 1a-1. This confirms that the compound represented by Chemical Formula 1a-1 increases SIRT1 expression and improves permeability.

[0164] From Figure 11B, it can be seen that when CLP is induced, the vascular barrier is destroyed and a large amount of EBD is distributed to various organs.

[0165] Furthermore, it can be seen from FIG. 11C that the amount of EBD in each organ increases when CLP is induced, but is restored to normal when treated with the compound represented by chemical formula 1a-1.

[0166] Therefore, it can be seen that the compound represented by chemical formula 1a-1 can improve sepsis and ARDS by strengthening the vascular barrier.

Claims

1. A compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: 【Chemistry 1】 In the above formula, R is a hydrogen atom, C １ -C ６ is an alkyl group or an aryl group of the formula:

2. R is a hydrogen atom or C １ -C ６ 2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R is an alkyl group represented by the formula:

3. 2. The compound according to claim 1, which is the hydrochloride salt of the compound represented by Chemical Formula 1, or a pharmaceutically acceptable salt thereof.

4. (i) protecting the amine group of a compound represented by the following Chemical Formula 2 to obtain a compound represented by the following Chemical Formula 3: (ii) protecting the primary hydroxy group of the compound represented by the following Chemical Formula 3 to obtain a compound represented by the following Chemical Formula 4: (iii) A compound represented by the following chemical formula 4 is reacted with phosphoryl chloride (POCl ３ ), or phosphoryl chloride (POCl ３ ) and a compound represented by the following Chemical Formula 5 or a compound represented by the following Chemical Formula 6 to obtain a compound represented by the following Chemical Formula 7: (iv) selectively deprotecting the hydroxy-protecting group of the compound represented by the following formula 7 to obtain a compound represented by the following formula 8: (v) A method for producing a compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof, comprising the step of deprotecting an amine protecting group of a compound represented by Chemical Formula 8: 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 【Transformation 5】 【Transformation 6】 【Transformation 7】 【Transformation 8】 【Chemistry 9】 In the above formula, PG １ is an amine protecting group, PG ２ is a hydroxy protecting group, R' is C １ -C ６ is an alkyl or aryl group of the formula R is a hydrogen atom, C １ -C ６ is an alkyl group or an aryl group of the formula:

5. PG １ The method according to claim 4, wherein is t-butyloxycarbonyl.

6. PG ２ The method according to claim 4, wherein is t-butyldimethylsilyl.

7. The method according to claim 4, wherein in step (iv), the deprotection is carried out in the presence of a base and HF.

8. The method according to claim 4 , wherein in step (v), the deprotection is carried out using an acid.

9. A pharmaceutical composition for preventing or treating a degenerative brain disease, comprising the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

10. The pharmaceutical composition according to claim 9, wherein the degenerative brain disease is Alzheimer's disease or Parkinson's disease.

11. A pharmaceutical composition for preventing or treating sepsis or acute respiratory distress syndrome, comprising the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

12. A health functional food for preventing or ameliorating degenerative brain diseases, comprising the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

13. The health functional food according to claim 12, wherein the degenerative brain disease is Alzheimer's disease or Parkinson's disease.

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