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

Abstract

The present invention relates to a phytosphingosine-3, 4-cyclic phosphate compound and a pharmaceutical composition comprising the same as an active ingredient, in which the phytosphingosine-3, 4-cyclic phosphate compound is capable of increasing the expression of SIRT1 and reducing the expression of amyloid protein and tau protein, and in which the phytosphingosine-3, 4-cyclic phosphate compound is capable of increasing the expression of SIRT1 and reducing the expression of tau protein. The present invention relates to novel compounds and uses thereof, and also has the effects of increasing the expression of tyrosine hydroxylase and reducing the expression of alpha-synuclein, and further, ameliorates sepsis and acute respiratory distress syndrome induced by CLP surgical methods.

Description

Technical Field

[0001] The present disclosure relates to phytosphingosine-3,4-cyclic phosphate compounds and pharmaceutical compositions containing the same, and more particularly, to phytosphingosine-3,4-cyclic phosphate compounds capable of increasing SIRT1 expression, reducing amyloid and Tau protein expression, increasing tyrosine hydroxylase expression, reducing α-synuclein expression, and improving sepsis and acute respiratory distress syndrome induced by CLP surgery, and pharmaceutical compositions containing the same as an active ingredient. Background Art

[0002] Literally, neurodegenerative diseases are any degenerative diseases that occur in the brain as we age and are caused by the loss of nerve cells. Neurodegenerative 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), among which Alzheimer's disease is the most common, followed by Parkinson's disease.

[0003] Alzheimer's disease is the main neurodegenerative disease leading to dementia and is characterized by progressive memory and cognitive loss; while Parkinson's disease is caused by the death of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the lack of dopamine in the striatum, resulting in symptoms such as tremors, bradykinesia, muscle rigidity, postural instability, and akinesia.

[0004] Both Alzheimer's disease and Parkinson's disease have been identified as being characterized by abnormal aggregation of proteins. Abnormal aggregation of extracellular amyloid-β (Aβ) and intracellular Tau in Alzheimer's disease and α-synuclein in Parkinson's disease are closely related to the pathophysiology of these diseases.

[0005] For example, Korean Patent No. 10-1064258 discloses a composition for preventing brain cell degeneration and damage caused by β-amyloid, which contains an arylureidoacetate compound; and Korean Patent No. 10-1092620 discloses a composition for preventing or treating neurodegenerative diseases by inhibiting the expression and modification of α-synuclein that causes nerve cell damage and death, which contains a sesquiterpene compound as an active ingredient.

[0006] Recently, it has been reported that in these neurodegenerative diseases, the expression of SIRT1 (silent mating type information regulation 2 homolog; sirtuin 1) is reduced, and SIRT1 activators have become therapeutic agents for neurodegenerative diseases.

[0007] Meanwhile, sepsis is an inflammatory response induced by the over-activation of the body's immune system caused by pathogenic microbial infections. In severe cases, it leads to shock and death of the patient.

[0008] Specifically, sepsis is a systemic inflammatory response syndrome caused by infection and mainly occurs acutely in infants, the elderly, or surgical patients with weaker immunity. Sepsis is usually accompanied by a systemic inflammatory response, and the systemic inflammatory response can be caused by various reasons. The systemic inflammatory response caused by pathogenic microbial infections in the body is called sepsis. Sepsis is a disease that is the cause of most deaths in critically ill patients and is a common infectious disease accounting for about 25%-30% of inpatients. Sepsis is a dangerous condition with a mortality rate of 30%-60% but there is no effective treatment.

[0009] Due to the lack of effective sepsis therapeutics, various attempts are being made to develop therapeutics. In particular, many attempts have been made as a method of suppressing inflammation, but various anti-inflammatory drugs often fail in clinical practice, so new treatment strategies rather than methods of suppressing inflammation are needed.

[0010] Recently, it has been found that the activity and synthesis of SIRT1 protein and the content of sphingosine-1-phosphate (S1P) are decreased in sepsis patients compared with the normal group (Critical care, 2015, 19, 372). The content of S1P often drops sharply in sepsis patients, so attempts are being made to treat sepsis by increasing the S1P content using S1P agonists or by using inhibitors that inhibit the enzymes degrading S1P (J Pharmacol Exp Ther., 2015, 352, 61-66), and attempts are being made to increase the decreased SIRT1 protein content and activity in sepsis patients. The best way to increase the S1P content is to overproduce the gene synthesizing S1P or to knockdown or knockout the enzymes degrading S1P, but this method is inconvenient due to gene manipulation, and the method using S1P agonists has shown little therapeutic benefit.

[0011] In addition, severe sepsis may lead to acute respiratory distress syndrome (ARDS).

[0012] ARDS is a syndrome characterized by acute-onset pulmonary edema, which causes dyspnea that cannot be improved under high levels of oxygen. In addition to sepsis, this syndrome can also be caused by trauma, massive blood transfusion, and drugs. ARDS is a dangerous condition with a mortality rate of 40%-50% but there is no effective treatment. SUMMARY OF THE INVENTION

[0013] [Technical Problem]

[0014] The object of the present disclosure is to provide a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof, which exhibits preventive, therapeutic, and ameliorating effects on neurodegenerative diseases, sepsis, and acute respiratory distress syndrome.

[0015] Another object of the present invention is to provide a pharmaceutical composition comprising the compound represented by the above formula (1) or a pharmaceutically acceptable salt thereof.

[0016] Another object of the present invention is to provide a dietary supplement comprising the compound represented by the above formula (1) or a pharmaceutically acceptable salt thereof.

[0017] [Technical Solution]

[0018] One aspect of the present invention relates to a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[0019]

[0020] Wherein,

[0021] R is hydrogen, C1-C6 alkyl, or aryl.

[0022] As used herein, the term "C1-C6 alkyl" means a straight-chain or branched-chain monovalent hydrocarbon having 1 to 6 carbon atoms, and examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-propyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, and the like.

[0023] As used herein, the term "aryl" includes aromatic and heteroaromatic groups and their partially reduced derivatives. The aromatic group is of the monocyclic or fused-ring type composed of 5 to 15 carbon atoms, and the heteroaromatic group is an aromatic group containing one or more of oxygen, sulfur, or nitrogen. Representative examples of aryl include, but are not limited to, phenyl, naphthyl, pyridyl, pyrimidinyl, furyl, thienyl, indolyl, quinolinyl, imidazolinyl, oxazolyl, thiazolyl, tetrahydronaphthyl, and the like.

[0024] In one embodiment of the present invention, R is hydrogen or C1-C6 alkyl.

[0025] The pharmaceutically acceptable salts of the present invention may include non-toxic inorganic acid salts and organic acid salts, and examples thereof include hydrochloride, sulfate, nitrate, phosphate, acetate, adipate, aspartate, benzoate, benzenesulfonate, citrate, camphorate, camphorsulfonate, diphosphate, ethanesulfonate, fumarate, glutamate, malate, lactate, mesylate, succinate, tartrate, picrate, toluenesulfonate, and the like. In particular, it may be the hydrochloride salt of the compound represented by formula (1), represented by the following formula (1a).

[0026]

[0027] On the other hand, the present invention provides a method for preparing a compound represented by formula (1) or a pharmaceutically acceptable salt thereof, which comprises:

[0028] (i) protecting the amino group of the compound represented by the following formula (2) to obtain a compound represented by the following formula (3);

[0029] (ii) protecting the primary alcohol hydroxyl group of the compound represented by the following formula (3) to obtain a compound represented by the following formula (4);

[0030] (iii) reacting the compound represented by the following formula (4) with phosphoryl chloride (POCl3), or with phosphoryl chloride (POCl3) and a compound represented by the following formula (5), or with a compound represented by the following formula (6) to obtain a compound represented by the following formula (7);

[0031] (iv) selectively deprotecting the hydroxyl protecting group of the compound represented by the following formula (7) to obtain a compound represented by the following formula (8); and

[0032] (v) deprotecting the amine protecting group of the compound represented by the following formula (8).

[0033]

[0034]

[0035] R′-OH (5)

[0036]

[0037] Wherein,

[0038] PG1 is an amine protecting group;

[0039] PG2 is a hydroxyl protecting group;

[0040] R' is a C1-C6 alkyl or aryl; and

[0041] R is hydrogen, a C1-C6 alkyl or aryl.

[0042] In one embodiment of the present invention, the amine protecting group can be, but is not limited to, tert-butoxycarbonyl, benzyloxycarbonyl, etc.

[0043] In one embodiment of the present invention, the hydroxyl protecting group can be, but is not limited to, tert-butyldimethylsilyl, pivaloyl, etc.

[0044] In the following, the preparation method of the present invention will be described in more detail with reference to the following reaction scheme (1). The method described in the following reaction scheme (1) is an example of a representative method, and reaction reagents, reaction conditions, etc. can be changed at any time according to circumstances.

[0045] [Reaction Scheme 1]

[0046]

[0047] Step 1: Synthesis of the compound represented by formula (3)

[0048] The compound represented by formula (3) can be prepared by protecting the amino group of the compound represented by formula (2).

[0049] The protection can be carried out using di-tert-butyl dicarbonate (Boc2O), benzyl chloroformate, etc.

[0050] Step 2: Synthesis of the compound represented by formula (4)

[0051] The compound represented by formula (4) can be prepared by protecting the primary alcohol hydroxyl group of the compound represented by formula (3).

[0052] The protection can be carried out using tert-butyldimethylchlorosilane, pivaloyl chloride, etc. in the presence of a catalyst and a base.

[0053] In this case, 4-dimethylaminopyridine, etc. can be used as the catalyst, and triethylamine, imidazole, etc. can be used as the base.

[0054] Step 3: Synthesis of the compound represented by formula (7)

[0055] The compound represented by formula (7) can be prepared by reacting the compound represented by formula (4) with phosphoryl chloride (POCl3), or with phosphoryl chloride (POCl3) and the compound represented by formula (5), or with the compound represented by formula (6).

[0056] The reaction can be carried out in the presence of a base, and the base can be pyridine, etc.

[0057] Step 4: Synthesis of the compound represented by formula (8)

[0058] The compound represented by formula (8) can be prepared by selectively deprotecting the hydroxyl protecting group of the compound represented by formula (7).

[0059] The deprotection can be carried out in the presence of a base and HF.

[0060] As the base, pyridine, etc. can be used.

[0061] Step 5: Synthesis of the compound represented by formula (1)

[0062] The compound represented by formula (1) can be prepared by deprotecting the amine protecting group of the compound represented by formula (8).

[0063] The deprotection can be carried out using an acid.

[0064] In this case, the acid can be an inorganic acid or an organic acid, such as hydrochloric acid.

[0065] By carrying out the deprotection using an acid as described above, an acid addition reaction can also be carried out together with the deprotection, so that the compound represented by formula (1) can be obtained in the form of an acid addition salt.

[0066] The compound represented by formula (1) according to the present invention or a pharmaceutically acceptable salt thereof can increase the expression of SIRT1 and can restore the shortened intestinal length in an animal model of neurodegenerative disease (see Experimental Examples 1 and 2).

[0067] Furthermore, the compound represented by formula (1) according to the present invention or a pharmaceutically acceptable salt thereof can reduce the expression of amyloid protein and Tau protein, which are biomarkers of Alzheimer's disease (see Experimental Example 3).

[0068] Furthermore, the compound represented by formula (1) according to the present invention or a pharmaceutically acceptable salt thereof can exhibit a neuronal proliferation effect in a Parkinson's disease cell model, promote the differentiation of pluripotent stem cells into dopaminergic neurons, and restore the TH expression reduced by rotenone, and in turn reduce the α-synuclein expression increased by rotenone (see Experimental Examples 4 to 6).

[0069] Furthermore, the compound represented by formula (1) according to the present invention or a pharmaceutically acceptable salt thereof can improve sepsis and acute respiratory distress syndrome (ARDS) induced by CLP surgery (see Experimental Examples 7 and 8).

[0070] Furthermore, the compound represented by formula (1) according to the present invention or a pharmaceutically acceptable salt thereof can improve sepsis and acute respiratory distress syndrome by inhibiting inflammation and enhancing the vascular barrier (see Experimental Examples 9 and 10).

[0071] Therefore, another aspect of the present invention relates to a pharmaceutical composition for preventing or treating neurodegenerative diseases, the pharmaceutical composition comprising the compound represented by formula (1) or a pharmaceutically acceptable salt thereof.

[0072] Specifically, the neurodegenerative disease can be Alzheimer's disease, Parkinson's disease, etc.

[0073] In addition, another aspect of the present invention relates to a pharmaceutical composition for preventing or treating sepsis or acute respiratory distress syndrome, the pharmaceutical composition comprising a compound represented by formula (1) or a pharmaceutically acceptable salt thereof.

[0074] The pharmaceutical composition according to the present invention can be administered orally (e.g., by ingestion or inhalation) or parenterally (e.g., by injection, deposition, implantation, suppository), and the injection can be, for example, intravenous, subcutaneous, intramuscular or intraperitoneal injection. Depending on the route of administration, the pharmaceutical composition according to the present invention can be formulated as tablets, capsules, granules, fine subtilae, dispersions, sublingual tablets, suppositories, ointments, injections, emulsions, suspensions, syrups, sprays, etc. The pharmaceutical composition according to the present invention in the various forms described above can be prepared by methods known in the art using pharmaceutically acceptable carriers conventionally used for each formulation. Examples of pharmaceutically acceptable carriers include excipients, binders, disintegrants, lubricants, preservatives, antioxidants, isotonic agents, buffers, coating agents, sweeteners, solubilizers, bases, dispersants, wetting agents, suspending agents, stabilizers, colorants, etc.

[0075] Depending on the form of the drug, the pharmaceutical composition according to the present invention contains about 0.001 to 95% by weight of the compound of the present invention or a pharmaceutically acceptable salt thereof.

[0076] The specific dose of the pharmaceutical composition of the present invention can vary depending on the type of mammal (including humans) being treated, body weight, gender, severity of the disease and the judgment of the physician. Preferably, in the case of oral administration, 0.01 to 50 mg of the active ingredient is administered per kilogram of body weight per day, and in the case of parenteral administration, 0.001 to 10 mg of the active ingredient is administered per kilogram of body weight per day. Depending on the severity of the disease, the judgment of the physician, etc., the total daily dose can be administered in one or multiple doses.

[0077] Another aspect of the present invention relates to a dietary supplement for preventing or improving neurodegenerative diseases, the dietary supplement comprising a compound represented by formula (1) above or a pharmaceutically acceptable salt thereof.

[0078] Specifically, the neurodegenerative disease can be Alzheimer's disease, Parkinson's disease, etc.

[0079] There is no particular limitation on the type of the dietary supplement according to the present invention, and it can be in the form of an oral preparation, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, or added to common foods, such as candies, confectionery, chewing gums, ice creams, noodles, breads, beverages, etc.

[0080] The dietary supplement of the present invention can be prepared according to its form by conventional methods, and when appropriate, fillers, extenders, binders, wetting agents, disintegrants, sweeteners, flavoring agents, preservatives, surfactants, lubricants, excipients, etc. are used.

[0081] In the preparation of the dietary supplement described above, the content of the compound represented by formula (1) or its pharmaceutically acceptable salt depends on the form of the dietary supplement, but is about 0.001 to 10% by weight, preferably 0.01 to 5% by weight.

[0082] Beneficial effects

[0083] The compound according to the present invention can increase the expression of SIRT1 and restore the shortened intestinal length in an animal model of neurodegenerative disease. In addition, the compound according to the present invention can reduce the expression of amyloid and Tau proteins, which are biomarkers of Alzheimer's disease, show the proliferation effect of nerve cells in a cell model of Parkinson's disease, promote the differentiation of pluripotent stem cells into dopaminergic neurons, and restore the TH expression reduced by rotenone, and in turn reduce the α-synuclein expression increased by rotenone. In addition, the compound according to the present invention can improve sepsis and acute respiratory distress syndrome induced by CLP treatment.

[0084] Therefore, the compound according to the present invention can be effectively used in a composition to prevent, treat and improve neurodegenerative diseases, sepsis and acute respiratory distress syndrome. Brief description of the drawings

[0085] Figure 1 The Western blot results showing the change in SIRT1 expression after treatment with the compound of formula (1a-1) in HUVEC cells are shown.

[0086] Figure 2 The results showing the change in intestinal length after treatment with the compound of formula (1a-1) in an AD-induced animal model are shown.

[0087] Figure 3 The fluorescence microscopy results showing the change in the expression of AD biomarkers mAβ and Tau after treatment with the compound of formula (1a-1) in an AD-induced animal model are shown.

[0088] Figure 4 The results showing the evaluation of the nerve cell proliferation effect after treatment with the compound of formula (1a-1) in a cell model of Parkinson's disease are shown.

[0089] Figure 5 The results showing the evaluation of the effect of promoting the differentiation of pluripotent stem cells into dopaminergic neurons after treatment with the compound of formula (1a-1) are shown.

[0090] Figure 6 The Western blot results showing the changes in tyrosine hydroxylase and α-synuclein expression after treatment with the compound of formula (1a-1) and the figures describing them.

[0091] Figure 7 The figure showing the effect on the survival rate of mice when 100 ng / mouse (5 μg / kg) of the compound of formula (1a-1) was administered 6 and 16 hours after CLP treatment.

[0092] Figure 8 The figure showing the effect on the survival rate of mice when 100 ng / mouse (5 μg / kg) of fingolimod (FTY720) was administered 6 and 16 hours after CLP treatment.

[0093] Figure 9 A is a photograph of lung tissue observed under an optical microscope when treated with the compound of formula (1a-1) after CLP treatment, Figure 9 B is a figure showing the results of lung injury scoring, which uses histological scoring to evaluate lung tissue injury, and Figure 9 C is a figure showing the ratio of the wet weight to the dry weight of the harvested lungs.

[0094] Figure 10 The figure showing the effect of treatment with the compound of formula (1a-1) after CLP treatment on the secretion of inflammatory cytokines TNF-α (A) and IL-6 (B).

[0095] Figure 11 A is a figure showing the effect on the permeability of the MYSEC cell line after treatment with LPS in the presence of the compound of formula (1a-1), and Figure 11 B and 11C are respectively a photograph and a figure showing the effect on the permeability of mouse organs after treatment with the compound of formula (1a-1) after CLP treatment. Detailed Description of the Invention

[0096] Hereinafter, the present invention will be further illustrated by the following examples. These examples are only intended to illustrate the present invention, and those skilled in the art should understand that the scope of the present invention is not limited to these examples.

[0097] Example 1: Preparation of (4S,5R)-4-((S)-1-amino-2-hydroxyethyl)-2-hydroxy-5-tetradecyl-1,3,2-dioxaphosphinan-2-oxide hydrochloride (1a-1)

[0098]

[0099] Example 1-1: Preparation of tert-butyl ((2S,3S,4R)-1,3,4-trihydroxyoctadec-2-yl)carbamate (3-1)

[0100]

[0101] Dissolve 100 g (314.951 mmol) of DS-sphingosine (2) in 1000 mL of tetrahydrofuran, add 82.5 g (377.941 mmol) of di-tert-butyl dicarbonate (Boc2O) thereto, and stir the mixture for 18 hours. After completion of the reaction, filter the insoluble material through filter paper and concentrate under reduced pressure. Add 800 mL of hexane to the concentrated oil phase and stir, then filter the resulting crystals and dry them under vacuum to obtain 118.36 g (90%) of the title compound.

[0102] 1 1H 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)

[0103] ES-MS m / z: 418.44 [M + H] +

[0104] Example 1-2: Preparation of ((2S,3S,4R)-1-((tert-butyldimethylsilyl)oxy)-3,4-dihydroxyoctadec-2-yl)carbamic acid tert-butyl ester (4-1)

[0105]

[0106] Dissolve 5 g (11.973 mmol) of ((2S,3S,4R)-1,3,4-trihydroxyoctadec-2-yl)carbamic acid tert-butyl ester (3-1) obtained in Example 1-1 above in 50 mL of dichloromethane, add 2 g (13.170 mmol) of tert-butyldimethylchlorosilane, 0.73 g (5.986 mmol) of 4-dimethylaminopyridine, and 1.85 mL (13.170 mmol) of triethylamine thereto, and stir the mixture for 16 hours. After completion of the reaction, add 30 mL of water. Dry the separated organic layer over anhydrous magnesium sulfate and concentrate under reduced pressure. Purify the obtained material by column chromatography to obtain 6.33 g (99%) of the title compound.

[0107] 11H 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)

[0108] Examples 1 - 3: Preparation of tert-Butyl ((1S)-2-((tert-butyldimethylsilyl)oxy)-1-((4S,5R)-2-hydroxy-2-oxo-5-tetradecyl-1,3,2-dioxaphosphinan-4-yl)ethyl)carbamate (7 - 1)

[0109]

[0110] Dissolve 9.3 g (17.472 mmol) of tert-Butyl ((2S,3S,4R)-1-((tert-butyldimethylsilyl)oxy)-3,4-dihydroxyoctadec-2-yl)carbamate (4 - 1) obtained in Examples 1 - 2 above in 80 mL of dichloromethane, and then cool to 0 °C. After adding 8.5 mL (104.832 mmol) of pyridine, slowly add dropwise 2.1 mL (22.714 mmol) of phosphorus oxychloride diluted in 20 mL of dichloromethane. Stir the mixture at 20 - 25 °C for 3 hours, and after the reaction is complete, add 50 mL of water to extract the compound. Dry the separated organic layer over anhydrous magnesium sulfate and concentrate under reduced pressure to obtain 10.3 g (100%) of the title compound.

[0111] 1 1H 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)

[0112] ES-MS m / z: 594.62 [M + H] +

[0113] Example 1 - 4: Preparation of tert-Butyl ((1S)-2-hydroxy-1-((4S,5R)-2-hydroxy-2-oxo-5-tetradecyl-1,3,2-dioxaphosphinan-4-yl)ethyl)carbamate (8 - 1)

[0114]

[0115] 10.3 g (17.472 mmol) of tert-butyl ((1S)-2-((tert-butyldimethylsilyl)oxy)-1-((4S,5R)-2-hydroxy-2-oxo-5-tetradecyl-1,3,2-dioxaphosphinan-4-yl)ethyl)carbamate (7-1) obtained in Examples 1-3 above was dissolved in 100 mL of tetrahydrofuran, then 1.8 mL (69.888 mmol) of pyridinium hydrofluoride was added, and the mixture was stirred at 20 - 25 °C for 16 h. After completion of the reaction, 7.4 g (69.888 mmol) of sodium carbonate was added and stirred for 3 h. After adding 50 mL of 1 N hydrochloric acid to the reaction solution, 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.

[0116] 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)

[0117] ES-MS m / z: 480.44 [M+H] +

[0118] Example 1-5: Preparation of (4S,5R)-4-((S)-1-amino-2-hydroxyethyl)-2-hydroxy-5-tetradecyl-1,3,2-dioxaphosphinan-2-oxide hydrochloride (1a-1)

[0119]

[0120] 6 g (12.511 mmol) of tert-butyl ((1S)-2-hydroxy-1-((4S,5R)-2-hydroxy-2-oxo-5-tetradecyl-1,3,2-dioxaphosphinan-4-yl)ethyl)carbamate (8-1) obtained in Examples 1-4 above was dissolved in 120 mL of ethyl acetate, 31 mL of 4 M hydrochloride (in dioxane, 125.107 mmol) was added, and the mixture was stirred at 20 - 25 °C for 18 h. After completion of the reaction, the reaction solution was concentrated. After adding 120 mL of acetonitrile to the concentrated residue and crystallization, the mixture was stirred for 3 h. The resulting crystals were filtered and dried in vacuo to obtain 4.5 g (86%) of the title compound.

[0121] 11H 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)

[0122] 31 31P NMR (DMSO-d6): δ -8.03 ppm

[0123] 13 13C 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)

[0124] ES-MS m / z: 380.39 [M + H] +

[0125] 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)

[0126]

[0127] Example 2-1: Preparation of tert-butyl ((1S)-2-((tert-butyldimethylsilyl)oxy)-1-((4S,5R)-2-methoxy-2-oxo-5-tetradecyl-1,3,2-dioxaphospholan-4-yl)ethyl)carbamate (7-2)

[0128]

[0129] Dissolve 10 g (18.801 mmol) of tert-butyl ((2S,3S,4R)-1-((tert-butyldimethylsilyl)oxy)-3,4-dihydroxyoctadec-2-yl)carbamate (4-1) obtained in Example 1-2 above in 100 mL of dichloromethane, and then cool to 0 °C. After adding 6.1 mL (75.205 mmol) of pyridine, slowly add dropwise 2.4 mL (24.442 mmol) of methyl dichlorophosphate diluted in 20 mL of dichloromethane. Stir the mixture at 20 - 25 °C for 3 hours, and concentrate the compound after the reaction is complete. Add 100 mL of ethyl acetate (EA) to the concentrated compound, stir, and filter the resulting crystals. Concentrate the filtrate under reduced pressure to obtain 4.8 g (48%) of the title compound.

[0130] 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)

[0131] ES-MS m / z: 608.85 [M+H] +

[0132] Example 2-1-1: Preparation of tert-butyl ((1S)-2-((tert-butyldimethylsilyl)oxy)-1-((4S,5R)-2-methoxy-2-oxo-5-tetradecyl-1,3,2-dioxaphosphinan-4-yl)ethyl)carbamate (7-2)

[0133]

[0134] Dissolve 10 g (18.801 mmol) of tert-butyl ((2S,3S,4R)-1-((tert-butyldimethylsilyl)oxy)-3,4-dihydroxyoctadec-2-yl)carbamate (4-1) prepared in Example 1-2 above in 100 mL of dichloromethane, and then cool to 0 °C. After adding 6.1 mL (75.205 mmol) of pyridine, slowly add dropwise 2.3 mL (24.442 mmol) of phosphorus oxychloride diluted in 20 mL of dichloromethane. Stir the mixture at 20 - 25 °C for 3 hours, and add 10 mL of methanol after the reaction is complete. After 2 hours, concentrate the compound, add 100 mL of ethyl acetate (EA) and stir, and filter the resulting crystals. Concentrate the filtrate under reduced pressure to obtain 5.7 g (55%) of the title compound.

[0135] 1 1H 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)

[0136] ES-MS m / z: 608.85 [M + H] +

[0137] Example 2-2: Preparation of tert-butyl ((1S)-2-hydroxy-1-((4S,5R)-2-methoxy-2-oxo-5-tetradecyl-1,3,2-dioxaphosphinan-4-yl)ethyl)carbamate (8-2)

[0138]

[0139] Dissolve 4.8 g (7.896 mmol) of tert-butyl ((1S)-2-((tert-butyldimethylsilyl)oxy)-1-((4S,5R)-2-methoxy-2-oxo-5-tetradecyl-1,3,2-dioxaphosphinan-4-yl)ethyl)carbamate (7-2) obtained in Example 2-1 above in 50 mL of tetrahydrofuran, add 0.84 mL (31.584 mmol) of pyridinium fluoride, and stir the mixture at 20 - 25 °C for 16 hours. After completion of the reaction, add 3.3 g (31.584 mmol) of sodium carbonate and stir for 3 hours. After adding 50 mL of 1N hydrochloric acid to the reaction solution, add 100 mL of ethyl acetate for extraction. Dry the separated organic layer over anhydrous magnesium sulfate and concentrate it under reduced pressure to obtain 2.5 g (64%) of the title compound.

[0140] 1 1H 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)

[0141] ES-MS m / z: 494.12 [M + H] +

[0142] 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)

[0143]

[0144] Dissolve 2.5 g (5.064 mmol) of tert-butyl ((1S)-2-hydroxy-1-((4S,5R)-2-methoxy-2-oxo-5-tetradecyl-1,3,2-dioxaphospholan-4-yl)ethyl)carbamate (7-2) obtained in Example 2-2 above in 50 mL of ethyl acetate, then add 12.7 mL of 4 M hydrochloride (in dioxane, 50.647 mmol), and stir the mixture at 20 - 25 °C for 18 h. After completion of the reaction, concentrate the reaction solution. Add 100 mL of acetonitrile to the concentrated residue for crystallization, and stir the mixture for 3 h. Filter the resulting crystals and dry them under vacuum to obtain 1.9 g (90%) of the title compound.

[0145] 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)

[0146] 31 P NMR (DMSO-d6): δ -8.01 ppm

[0147] 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)

[0148] ES-MS m / z: 393.56 [M+H] +

[0149] Experimental Example 1: Analysis of the effect on SIRT1 expression

[0150] To determine the increase in SIRT1 expression in human umbilical vein endothelial cells (HUVECs) by the compound of formula (1a-1) prepared in Example 1 above, HUVECs were plated at 5×10 5 / 4 ml in a 60 mm cell culture dish using ECBM2 medium (Promocell) and stabilized for 12 hours in an incubator maintained at 37 °C and 5% CO2, respectively.

[0151] The cells were then treated with 10 nM, 100 nM, and 1000 nM of the compound of formula (1a-1) prepared in Example 1 above and incubated for an additional 24 hours, and then SIRT1 expression was determined by Western blotting.

[0152] The results are shown in Figure 1 In.

[0153] From Figure 1 It can be seen that treatment of HUVECs with the compound of formula (1a-1) increased SIRT1 expression even at a very low concentration of 10 nM. In particular, the compound of formula (1a-1) showed that a concentration of approximately 10 nM (EC 50 ) increased SIRT1 expression by 50%.

[0154] On the other hand, when mouse vascular cells were treated with the compound of formula (1a-1), it was shown that the maximum expression was exhibited at 100 ng / mL.

[0155] For resveratrol, which is well-known as a SIRT1 activator, the EC 50 is known to be approximately 36 μM. Therefore, it can be confirmed that the compound of formula (1a-1) increased SIRT1 expression at a concentration thousands of times lower than that of resveratrol.

[0156] Experimental Example 2: Analysis of changes in intestinal length in an Alzheimer's disease animal model

[0157] In neurodegenerative diseases (AD and PD), the intestinal length tends to shorten as the disease progresses, so we looked for changes in intestinal length in an AD-induced animal model.

[0158] APP / PS / Tau transgenic mice were used as an AD-induced animal model. The compound of formula (1a-1) was intraperitoneally administered to 12-week-old transgenic mice at a concentration of 1 mg / kg once every 3 days for 2 weeks, and the changes in the intestinal length of the mice were measured 1 week and 4 weeks later.

[0159] The results are shown in Figure 2 In.

[0160] Refer to Figure 2, it can be seen that the intestinal length increased in the group of mice treated with the compound of formula (1a-1) compared to the AD-induced mice in the control group without the compound of formula (1a-1).

[0161] In particular, when the intestinal length was examined at 1 week (3W) and 4 weeks (6W) after administration of the compound of formula (1a-1), it was found that the intestinal length at 4 weeks was longer than that at 1 week.

[0162] Experimental Example 3: Analysis of the drug efficacy in an AD-induced animal model

[0163] To determine the therapeutic effect on Alzheimer's disease (AD), the effects of monomeric amyloid-β (monomeric Aβ, mAβ) and Tau protein, which are biomarkers of AD, were analyzed in an AD-induced animal model.

[0164] APP / PS / Tau transgenic mice were used as the AD-induced animal model. The compound of formula (1a-1) was intraperitoneally administered to 12-week-old transgenic mice at a concentration of 1 mg / kg once every 3 days for 2 weeks, and the expression of mAβ and Tau protein in the mouse brain was measured after 1 week and 4 weeks. After the mice were anesthetized, the neck region was incised with dissection tools, and then the occipital region was incised to remove both sides of the skull. The brain was washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (Sigma, St Louis, MO, USA) at 4 °C for 12 hours. They were cut into a thickness of about 10 μm using a vibratome, and hippocampal and cortical sections were collected and placed in 24-well plates. PBS was added to the wells containing the fixed tissue sections, and 2 μM each of fluorescent nanoparticles (QD565, red fluorescence) conjugated with anti-amyloid antibody and anti-Tau antibody (Santa Cruz, USA) was added, and the reaction was carried out at 4 °C for 12 hours. The expression of mAβ and Tau was confirmed by fluorescence microscopy.

[0165] The results are shown in Figure 3 in.

[0166] It can be seen from Figure 3 that the mice not treated with the compound of formula (1a-1) showed very strong red fluorescence for mAβ and Tau, while the mice treated with the compound of formula (1a-1) showed negligible red fluorescence for both mAβ and Tau at 1 week and 4 weeks after drug administration. Therefore, it can be seen that when the compound of formula (1a-1) is administered, the expression of amyloid and Tau proteins, which are biomarkers of Alzheimer's disease, is significantly reduced. These results indicate that the compound of formula (1a-1) can be used as a treatment for Alzheimer's disease.

[0167] Experimental Example 4: Analysis of the effect on human neuron proliferation

[0168] The SH-SY5Y cell line derived from human neuroblastoma exhibits characteristics similar to dopaminergic neurons and is used as a cell model for Parkinson's disease (PD) to evaluate the proliferation efficacy of neurons.

[0169] A medium containing DMEM / F12 (Hyclone), 10% FBS (Hyclone), and 1% penicillin-streptomycin (Hyclone) was used, and the medium was changed every two days after inoculation on a T75 plate. Subculture was performed when the cell density reached 80 - 90%.

[0170] Then, to determine the neuron proliferation effect, the subcultured SH-SY5Y cells were inoculated into a 96-well plate at a density of 5×10 3 cells / well. After 1 day, the medium was replaced with a 5% FBS ratio, and the cells were treated with the compound of formula (1a-1) at concentrations serially diluted 2-fold from 500 nM to 15.6 nM and cultured for 5 days. The medium was replaced with 10% FBS containing the drug every other day. The medium with 10% FBS was used as a control. After 5 days, the amount of live cells was measured at 450 nm absorbance using EZ-cytox (DoGen) to compare cell viability.

[0171] The results are shown in Figure 4 .

[0172] It can be seen from Figure 4 that the proliferation of nerve cells is enhanced with the increase in the concentration of the compound of formula (1a-1), and the maximum proliferation efficacy is shown around 100 nM.

[0173] Experimental Example 5: Analysis of the effect on differentiation into neurons

[0174] Pluripotent stem cells were seeded at 5,000 cells / microwell in an AggreWell plate (for stem cells) and cultured in E6 medium (GIBCO) for 5 days to induce embryoid body (EB) formation. For neural rosette formation, EBs were seeded in groups of 30 into 35 mm culture dishes and cultured in N2bF medium for 10 days. The 35 mm culture dishes were used after being coated with 15 μg / ml poly-L-ornithine (sigma) and 1 μg / ml fibronectin (sigma). The N2Bf medium consisted of DMEM / F12 (GIBCO), N2 supplement (for stem cells) 1×, 10% NEAA (GIBCO), 55 μM β-mercaptoethanol (GIBCO), 20 ng / ml bFGF (peprotech). For the formation of spherical neural mass (SNM), the rosette clusters were sliced and cultured in a culture dish with N2bF medium. The size of the SNM was not more than 500 μM, and it was sliced and cultured for 3 to 4 passages. After the passage culture of the SNM, the clusters were broken into small pieces using a tungsten mesh (Nilaco) for the neural progenitor cell (NPC) culture step, and then attached to a culture dish coated with PLO / FN and cultured in N2bF medium. To differentiate NPCs into dopaminergic neurons, the medium was replaced with ITS-B27 medium 2 days after NPC culture. The ITS-B27 medium consisted of 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 B27 (GIBCO) 1×. 4 days after NPC culture, 200 ng / ml Sonic Hedgehog and 100 ng / ml FGF8 (peprotech) were added to the ITS-B27 medium. 6 days after NPC culture, 200 ng / ml Sonic Hedgehog, 20 ng / ml GDNF, 20 ng / ml BDNF (peprotech), 200 μM ascorbic acid, and 500 μM cAMP (sigma) were added to the ITS-B27 medium, and cultured for about 5 weeks. Starting from the stage 2 days after NPC culture, treatment with the compound of formula (1a-1) was carried out. The case of induced differentiation without treatment with the compound (1a-1) was designated as the differentiation control group (differentiated CTL). In addition, for comparison, the case of not being treated with the compound of formula (1a-1) and not being induced to differentiate was used as the undifferentiated control group (undifferentiated CTL).

[0175] The effect of the compound of formula (1a-1) on differentiation into neurons was evaluated by comparing the expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT) according to the presence or absence of treatment with the compound of formula (1a-1).

[0176] The results are shown in Figure 5 .

[0177] Figure 5 A shows the results of immunofluorescence staining of cells, confirming an increase in the expression of TH after treatment with the compound of formula (1a-1). In particular, the expression of TH increased after more than 5 weeks of differentiation, which had already increased 3 weeks after treatment with the compound of formula (1a-1). Figure 5 B shows the results of Western blotting of samples differentiated for 5 weeks. It can be seen that after treatment with the compound of formula (1a-1), the expression of TH increased and the glycosylation of DAT increased. Therefore, it can be seen that the compound of formula (1a-1) can promote the differentiation of pluripotent stem cells into dopaminergic neurons.

[0178] Experimental Example 6: Analysis of the effect on the expression of neuron-specific proteins

[0179] To analyze the effect on the expression of tyrosine hydroxylase (TH) and α-synuclein as neuron-specific proteins, Western blotting was performed.

[0180] The SH-SY5Y cell line was treated with retinoic acid and 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce differentiation into TH-secreting neurons for 6 days.

[0181] The differentiated dopaminergic neurons were lysed in PRO-PREP (Intronbio) (a cell lysis solution containing a protease inhibitor mixture (Gendepot)) and electrophoresed in a 10% SDS polyacrylamide gel at 20 μg / well. Transfer was performed using PVDF (Merck millipore) as the membrane, and tyrosine hydroxylase antibody, α-synuclein antibody (Cell signaling) and β-actin antibody (Santa Cruz) were used for reaction for more than 2 hours. Protein expression was detected by staining with an ECL substrate (Biorad) for development, and graphs were made using the image J program.

[0182] The results are shown in Figure 6 .

[0183] Specifically, Figure 6 A shows the Western blotting results measuring the changes in the expression of tyrosine hydroxylase and α-synuclein after treatment with the compound of formula (1a-1), and Figure 6B and 6C are graphs depicting the results of Western blotting for measuring changes in tyrosine hydroxylase and α-synuclein expression, respectively.

[0184] As can be seen from Figure 6 compared with the undifferentiated neuron SH-SY5Y cell line (undifferentiated CTL), TH expression was significantly increased in differentiated neurons (differentiated CTL), but when differentiated dopaminergic neurons were treated with rotenone (RT), TH expression decreased and α-synuclein expression increased. However, after treatment with the compound of formula (1a-1), the TH expression decreased due to rotenone was restored, and conversely, the α-synuclein expression increased due to rotenone decreased. In particular, the compound of formula (1a-1) was more effective than resveratrol used as a control drug.

[0185] Rotenone is known as a drug that induces Parkinson's disease. Therefore, the above results show that the compound of formula (1a-1) can be used for the treatment of Parkinson's disease.

[0186] Experimental Example 7: Effect on sepsis induced by CLP treatment

[0187] C57BL / 6 mice at 7-8 weeks of age were purchased from Jabio (Suwon, Gyeonggi-do) and sepsis was induced by cecal ligation and puncture (CLP).

[0188] Specifically, after anesthesia with 150 mg / kg ketamine and 17.5 mg / kg xylazine, a 1-2 cm incision was made in the abdomen to expose the cecum to induce sepsis. The cecum was ligated with 6-0 silk suture at the distal end of the ileocecal valve and punctured with an 18-gauge needle. The punctured cecum was gently squeezed to push out 1-2 cm of feces, and then the cecum was retracted into the abdominal cavity. Then the abdomen was closed, and pre-warmed saline (2.5 mL / 100 g body weight) was injected into the abdominal cavity of the mice immediately after the surgery. At 6 hours and 16 hours after CLP, the compound of formula (1a-1) was dissolved in phosphate buffered saline (PBS) and intravenously injected at 100 ng / mouse (5 μg / kg), and survival was examined for 10 days. For comparison, as a surgical negative control (sham control), survival was examined in mice that did not receive CLP treatment but underwent the same open surgery. In addition, as a drug negative control, the survival rate in mice that received only PBS after CLP treatment was examined.

[0189] The results are shown in Figure 7 .

[0190] As can be seen from Figure 7It can be seen that all individuals in the drug-negative control group that only received PBS died within 48 hours. Therefore, it can be seen that the above-mentioned CLP treatment condition is a very high-level severe sepsis model.

[0191] In this severe sepsis model, the experimental groups treated with the compound of formula (1a-1) 6 hours and 16 hours after CLP treatment showed more than 80% survival for 10 days. There were no deaths in the experimental groups after 10 days. Therefore, it can be seen that the compound of formula (1a-1) is effective in treating severe sepsis.

[0192] For comparison, we also examined the survival of the control group treated with the known S1P modulator fingolimod (FTY720) instead of the compound of formula (1a-1) under the same conditions.

[0193] The results showed in Figure 8 in.

[0194] From Figure 8 it can be seen that FTY720 did not improve survival.

[0195] Experimental Example 8: Analysis of the effect on acute respiratory distress syndrome induced by CLP treatment

[0196] CL induces pulmonary edema due to increased inflammation and disruption of the vascular barrier, leading to acute respiratory distress syndrome (ARDS). To determine the effect on ARDS, in the same manner as in Experimental Example 7 above, the compound of formula (1a-1) was used for treatment 6 hours and 16 hours after CLP treatment. Mice lungs were harvested at 18 hours for histological analysis, and the ratio of wet weight to dry weight of the lungs was obtained. For comparison, mice not receiving CLP treatment served as the control group.

[0197] For histological analysis, the harvested lungs were fixed in 10% paraformaldehyde, infiltrated with paraffin, cut into 5-μm thick tissues, and then dewaxed with xylene. The sectioned tissues were mounted on glass slides, stained with H&E, and observed under an optical microscope. In addition, histological scoring of the lung injury score (LIS) was also used to evaluate lung tissue injury for each group.

[0198] To calculate the ratio of wet weight to dry weight, lung organs were harvested 18 hours after CLP treatment, and the wet weight was measured immediately after harvest, and the dry weight was measured after drying in an oven at 60 °C for 48 hours to calculate the ratio of wet weight to dry weight. The ratio of wet weight to dry weight was used as an index of organ edema formation.

[0199] The results of histological analysis and measurement of the ratio of wet weight to dry weight are shown in Figure 9 in.

[0200] Figure 9 A is a photograph of lung tissue observed under an optical microscope when treated with the compound of formula (1a-1) after CLP treatment, Figure 9 B is a graph showing the results of lung injury scores, which uses histological scores to evaluate lung tissue injury, and Figure 9 C is a graph showing the ratio of the wet weight to the dry weight of the harvested lungs.

[0201] As Figure 9 can be seen from A, after CLP treatment, the lung tissue was damaged, and when treated with the compound of formula (1a-1), the lung tissue almost returned to normal. In addition, Figure 9 B shows that after CLP treatment, the lung injury score increased significantly, but treatment with the compound of formula (1a-1) almost restored the lung injury score to normal. In addition, Figure 9 C shows that after CLP treatment, due to inflammation and disruption of the vascular barrier, pulmonary edema formed due to water accumulation in the lung tissue, and the ratio of the wet weight to the dry weight of the lung increased, but treatment with the compound of formula (1a-1) restored the ratio of the wet weight to the dry weight of the lung to normal.

[0202] Therefore, it can be seen that the compound of formula (1a-1) can treat ARDS.

[0203] Experimental Example 9: Analysis of the effect on inflammatory cytokines after CLP treatment

[0204] After CLP treatment, the inflammatory cytokines TNF-α and IL-6 were analyzed by ELISA.

[0205] Specifically, after treatment with the compound of formula (1a-1) at 6 hours and 16 hours after CLP, mouse blood was collected at 18 hours, allowed to stand at room temperature for 1 hour, and then centrifuged at 3000 rpm for 10 minutes to obtain serum for analyzing the amounts of TNF-α and IL-6.

[0206] The results are shown in Figure 10 .

[0207] Figure 10 is a graph showing the effect of treatment with the compound of formula (1a-1) on the secretion of inflammatory cytokines TNF-α (A) and IL-6 (B) after CLP treatment.

[0208] As Figure 10 can be seen, in the control group treated with CLP, the amounts of the inflammatory cytokines TNF-α and IL-6 increased significantly, but treatment with the compound of formula (1a-1) significantly reduced the amounts of the inflammatory cytokines.

[0209] Thus, it can be seen that the compound of formula (1a-1) has excellent ability to inhibit the inflammatory response, which is important in the treatment of sepsis or ARDS.

[0210] Experimental Example 10: Analysis of the effect on permeability

[0211] Permeability was measured by an in vitro method using cell culture and an in vivo method 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.

[0212] To measure permeability by the in vitro method, the MYSEC (mouse yolk sac endothelial cell) cell line was used. The MYSEC cell line was placed in the upper chamber of a trans well at 2.5×10 5 cells / well and treated with 300 ng lipopolysaccharide (LPS) from Gram-negative bacteria for 24 hours in the presence of the compound of formula (1a-1). The medium in the upper chamber was replaced with 300 μl serum-free medium containing 5 μl streptavidin-HRP. After 5 minutes, 20 μl of the medium in the lower chamber was taken and the HRP activity was measured as the absorbance at 450 nm.

[0213] To determine the mechanism by which the compound of formula (1a-1) improves permeability, the same experiment was conducted by knocking down the SIRT1 protein in the MYSEC cell line.

[0214] To measure permeability by the in vivo method, in the same manner as in Experimental Example 7 above, the compound of formula (1a-1) was administered 6 hours and 16 hours after CLP treatment, and 200 μl of a 0.5% Evans blue dye (EBD) solution was intravenously administered at 18 hours, circulated for 1 hour, and the organs were harvested to visually confirm the presence of the dye. Then the organs were homogenized in a homogenizer at 55 °C for 48 hours to extract the dye, and permeability was determined by measuring the absorbance at 620 nm.

[0215] The results are shown in Figure 11 .

[0216] Figure 11 A is a graph showing the effect on the permeability of the MYSEC cell line after treatment with LPS in the presence of the compound of formula (1a-1), and Figure 11 B and 11C are a photograph and a graph, respectively, showing the effect on the permeability of mouse organs after treatment with the compound of formula (1a-1) after CLP treatment.

[0217] From Figure 11It can be seen that treating the MYSEC cell line with LPS increases permeability, but treating with the compound of formula (1a-1) restores the LPS-induced increased permeability to normal. However, knockdown of SIRT1 protein in the MYSEC cell line did not show the effect of the compound of formula (1a-1). Therefore, it can be confirmed that the compound of formula (1a-1) improves permeability by increasing SIRT1 expression.

[0218] From Figure 11 It can be seen from B that inducing CLP leads to the breakdown of the vascular barrier and a large amount of EBD is distributed in various organs.

[0219] In addition, in Figure 11 It can be seen from C that inducing CLP increases the amount of EBD in each organ, but after treatment with the compound of formula (1a-1), it returns to normal.

[0220] Therefore, it can be seen that the compound of formula (1a-1) can improve sepsis and ARDS by strengthening the vascular barrier.

Claims

1. A compound of formula (1) or a pharmaceutically acceptable salt thereof: Wherein, R is hydrogen, C1-C6 alkyl or aryl.

2. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R is hydrogen or C1-C6 alkyl.

3. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the pharmaceutically acceptable salt is hydrochloride.

4. A method for preparing a compound of formula (1) or a pharmaceutically acceptable salt thereof, the method comprising: (i) Protecting the amino group of the compound of formula (2) to obtain a compound of formula (3); (ii) Protecting the primary alcohol hydroxyl group of the compound of formula (3) to obtain a compound of formula (4); (iii) Reacting the compound of formula (4) with phosphoryl chloride (POCl3), or with phosphoryl chloride (POCl3) and a compound of formula (5), or with a compound of formula (6) to obtain a compound of formula (7); (iv) Selectively deprotecting the hydroxyl protecting group of the compound of formula (7) to obtain a compound of formula (8); and (v) Deprotecting the amine protecting group of the compound of formula (8): R'-OH (5) Wherein, PG1 is an amine protecting group; PG2 is a hydroxyl protecting group; R' is C1-C6 alkyl or aryl; and R is hydrogen, C1-C6 alkyl or aryl.

5. The method according to claim 4, wherein PG1 is tert-butoxycarbonyl.

6. The method according to claim 4, wherein PG2 is tert-butyldimethylsilyl.

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

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

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

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

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

12. A dietary supplement for preventing or improving neurodegenerative diseases, comprising the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3.

13. The dietary supplement according to claim 12, wherein the neurodegenerative disease is Alzheimer's disease or Parkinson's disease.

Images