New plasmalogen derivatives

Abstract

The present invention provides a novel compound which exhibits an excellent anti-inflammatory effect and which is formed from a compound represented by general formula (I), a racemate thereof, or salts of the compound and the racemate. (In general formula (I), X represents an oxygen atom, a nitrogen atom, or a sulfur atom; R1 represents an unsaturated aliphatic hydrocarbon group; R2 represents a saturated or unsaturated aliphatic hydrocarbon group; and R3 represents choline, ethanolamine, inositol, or serine.)

Description

[Technical field]

[0001] The present invention relates to novel compounds (plasmalogen derivatives) having a structure similar to that of plasmalogen. [Background technology]

[0002] Phospholipids are important components of biological membranes, and plasmalogens, which are ether phospholipids, account for approximately 18% of the phospholipids in mammalian biological membranes. Plasmalogens are known to be abundant in the brain, nervous system, cardiac muscle, skeletal muscle, white blood cells, and sperm.

[0003] Plasmalogens are bound to many polyunsaturated fatty acids such as docosahexaenoic acid and arachidonic acid, and therefore not only serve as reservoirs for secondary messengers of intercellular signals, such as prostaglandins and leukotrienes, which are produced from these polyunsaturated fatty acids, but also play important roles in cell fusion, ion transport, and other areas.

[0004] It has also become clear that plasmalogens themselves are involved in signal transduction via specific G protein-coupled receptors (GPCRs). For example, plasmalogens suppress neuronal cell death by enhancing the activity of protein kinases such as AKT and ERK in neurons (see Non-Patent Document 1), and the involvement of five specific GPCRs in the cell signal transduction mechanism has been revealed (see Non-Patent Document 2).

[0005] Furthermore, intraperitoneal injection of lipopolysaccharide (LPS) for 7 days in mice induced strong expression of IL-1β and TNF-α mRNA in the prefrontal cortex and hippocampus, expression of β-amyloid (Аβ1-16) positive neurons, and activation of glial cells. However, co-administration of plasmalogen intraperitoneally after LPS injection significantly attenuated glial cell activation accompanied by cytokine production and accumulation of Аβ protein. In addition, LPS reduced plasmalogen content in the prefrontal cortex and hippocampus, but the reduction was suppressed by co-administration of plasmalogen. In other words, plasmalogen is thought to have anti-neuroinflammatory effects and amyloid production preventive effects, and its application in the prevention or amelioration (treatment) of Alzheimer's disease has been suggested (see Non-Patent Document 3).

[0006] Plasmalogens are known to be decreased in neurological diseases such as dementia, Parkinson's disease, depression, and schizophrenia, as well as diabetes, metabolic syndrome, ischemic heart disease, various infectious diseases, and immune disorders. For example, in 1999, it was reported that ethanolamine-type plasmalogens were significantly reduced in the frontal lobe and hippocampus of brains with Alzheimer's disease (dead brains) (see Non-Patent Document 4), and in 2007, it was reported that plasmalogens were reduced in the serum of patients with Alzheimer's disease (see Non-Patent Document 5). It has also been reported that choline-type plasmalogens are decreased in patients with ischemic heart disease compared to normal controls (see Non-Patent Document 6).

[0007] Since it is believed that the prevention and improvement effects of these diseases can be expected by externally replenishing the decreased plasmalogen, attempts have been made to extract such plasmalogen from animal tissues. For example, Patent Document 1 proposes a method of extracting layer breast meat using ethanol as an extraction solvent and recovering the extract.

[0008] Furthermore, Patent Document 2 proposes a method in which bivalve shellfish such as scallops are extracted with a mixed solvent of a non-polar organic solvent and a branched alcohol, and then treated with phospholipase A1 (PLA1) to hydrolyze and remove contaminant diacyl glycerophospholipids. Furthermore, it has been reported that oral administration of plasmalogen extracted from the scallop strings in a randomized double-blind clinical trial targeting humans with mild Alzheimer's disease and mild cognitive impairment strongly suggested that cognitive function improved in mild Alzheimer's disease (see non-patent document 7).

[0009] On the other hand, as an example of the synthesis of plasmalogen derivatives, a novel plasmalogen precursor in which α-lipoic acid is bound to the sn-3 position of plasmalogen has been proposed for the purpose of preventing or ameliorating various diseases caused by plasmalogen deficiency by increasing the decreased plasmalogen level (see Patent Document 3). These derivatives have been shown to be effective in monkey models of Parkinson's disease (see Non-Patent Document 8). In addition, Patent Document 4 proposes that a derivative acetylated at the sn-1 position is a good carrier of docosahexaenoic acid, and it has been reported to be effective in an acute stroke model in rats (see Non-Patent Document 9). [Prior art documents] [Patent documents]

[0010] [Patent Document 1] Patent No. 5483846 [Patent Document 2] JP 2016-108466 A [Patent Document 3] International Publication No. 2010 / 071988 [Patent Document 4] International Publication No. 2013 / 037862 [Non-patent literature]

[0011] [Non-Patent Document 1] Md. Shamim Hossain et al, Plasmalogens rescue neuronal cell death through an activation of AKT and ERK survival signaling. PLoS ONE 8 (12): e83508, 2013 [Non-Patent Document 2] Md. Shamim Hossain et al, Neuronal Orphan G-Protein Coupled Receptor Proteins Mediate Plasmalogens-Induced Activation of ERK and Akt Signaling. PLoS ONE 11 (3):e0150846, 2016 [Non-Patent Document 3] M. Ifuku et al, Anti-inflammatory / anti-amyloidogenic effects of plasmalogens in lipopolysaccharide-induced neuroinflammation in adult mice. J of Neuroinflammation, 9:197, 2012 [Non-Patent Document 4] Z. Guan et al, Decrease and Structural Modifications of Phosphatidylethanolamine Plasmalogen in the Brain with Alzheimer Disease, J Neuropathol Exp Neurol , 58 (7), 7400-747, 1999 [Non-Patent Document 5] DB Goodenowe et al, Peripheral ethanolamine plasmalogen deficiency: a logical causative factor in Alzheimer's disease and dementia, J Lipid Res, 48, 2485-2498, 2007 [Non-Patent Document 6] Takeo Sanada et al., Serum Plasmalogen Levels in Ischemic Heart Disease, Atherosclerosis, 11, 535-539, 1983 [Non-Patent Document 7] T. Fujino et al, Efficacy and Blood Plasmalogen Changes by Oral Administration of Plasmalogen in Patients with Mild Alzheimer's Disease and Mild Cognitive Impairment: A Multicenter, Randomized, Double-blind, Placebo-controlled Trial, EBioMedicine, 17: 199-205, 2017 [Non-Patent Document 8] L. Gregoire et al, Plasmalogen precursor analog treatment reduces levodopa-induced dyskinesias in parkinsonian monkeys, Behav Brain Res, 286: 328-337, 2015 [Non-Patent Document 9] C. Fabien et al, Brain-Targeting Form of Docosahexaenoic Acid for Experimental Stroke Treatment: MRI Evaluation and Anti-Oxidant Impact, Current Neurovascular Res, 8 (2): 95-102, 2011 [Non-Patent Document 10] P. Wang et al, Improved Plasmalogen Synthesis Using Organobarium Intermediates, J. Org. Chem, 72: 5005-5007, 2007 Summary of the Invention [Problem to be solved by the invention]

[0012] The present invention aims to provide a novel compound which has a structure similar to that of plasmalogen and exhibits excellent anti-inflammatory activity. [Means for solving the problem]

[0013] While conducting research on plasmalogens and similar compounds, the inventors discovered that even if a vinyl ether bond is not formed at the sn-1 position, which is a major characteristic of plasmalogens, the presence of an unsaturated bond at the sn-1 position results in excellent anti-inflammatory effects, and thus completed the present invention.

[0014] That is, the present invention is as follows. [1] A compound represented by general formula (I), its racemate or a salt thereof.

[0015] [ka]

[0016] In the general formula (I), X represents an oxygen atom, a nitrogen atom, or a sulfur atom; R 1 represents an unsaturated aliphatic hydrocarbon group, R 2 represents a saturated or unsaturated aliphatic hydrocarbon group, R 3 represents choline, ethanolamine, inositol or serine.

[0017] [2] The compound according to the above [1], its racemate or a salt thereof, wherein X is an oxygen atom. [3] R 1The compound according to the above [1] or [2], a racemate thereof or a salt thereof, characterized in that it has at least one double bond. [4]R 1 The compound according to the above [3], its racemate or a salt thereof, characterized in that it has one double bond. [5]R 1 The compound according to the above [3] or [4], its racemic mixture, or a salt thereof, wherein the double bond is between the 1-position carbon and the 2-position carbon when the 1-position carbon is the carbon bonded to the carbon bonded to X.

[0018] [6] A composition comprising the compound according to any one of the above [1] to [5], its racemate or a salt thereof as an active ingredient. [7] The composition described in [6] above, characterized in that it has an anti-inflammatory effect. [8] The composition according to [6] or [7] above, which is used for preventing or improving a neuroinflammatory disease. [9] The composition described in [8], wherein the brain neuroinflammatory disease is at least one disease selected from dementia, Parkinson's disease, depression and schizophrenia.

[10] The composition described in [6] or [7] above, which is used for preventing or ameliorating Rett syndrome.

[11] The composition according to any one of [6] to [9] above, which is a pharmaceutical composition. Effect of the Invention

[0019] The novel compound of the present invention exhibits excellent anti-inflammatory activity. [Brief description of the drawings]

[0020] [Figure 1] 1 shows the results (semi-quantitative PCR) of measuring the expression level of LPS-induced inflammatory cytokine IL-1β (relative expression level to the control) in microglial cells (BV2) of mice administered with the compound of the present invention (KIT-008). [Diagram 2]FIG. 1 shows the results of measurement (Western blotting) of the intranuclear expression level of p65 (relative expression level to the control) induced by LPS in microglial cells (BV2) of mice administered with the compounds of the present invention (KIT-008 and KIT-020). [Diagram 3] FIG. 1 shows the results of measurement (Western blotting) of the intranuclear expression level of p65 (relative expression level to the control) induced by LPS in microglial cells (MG6) of mice administered with the compounds of the present invention (KIT-019 and KIT-020). [Figure 4] 1 shows the results of measuring the expression level (relative expression level to the control) of LPS-induced inflammatory cytokine IL-1β in microglial cells (MG6) of mice administered with the compounds of the present invention (KIT-019 and KIT-020) (ELISA method). [Diagram 5] FIG. 1 shows the survival rate of MeCP2-deficient mice administered with the compound of the present invention (KIT-008). [Figure 6] 1 shows the results of evaluation by scoring of RTT-like symptoms in MeCP2-deficient mice administered with the compounds of the present invention (KIT-008, KIT-019, KIT-020). [Figure 7] FIG. 1 shows changes in body weight of MeCP2-deficient mice administered with the compounds of the present invention (KIT-008, KIT-019, KIT-020). [Figure 8] FIG. 1 shows changes in body weight of wild-type mice administered with the compounds of the present invention (KIT-008, KIT-019, KIT-020). [Figure 9] 13 is a micrograph showing impaired lysosomal acidification in neuronal Neuro2a cells induced by culture supernatant of LPS-treated BV2 microglia. [Figure 10] 1 shows micrographs showing that administration of the compounds of the present invention (KIT-008, KIT-019, KIT-020) suppressed impaired lysosomal acidification induced in neuronal Neuro2a cells. [Figure 11]1 is a micrograph showing that administration of the compound of the present invention (KIT-008) suppressed accumulation of amyloid beta (Aβ) in lysosomes induced in neuronal Neuro2a cells. [Figure 12] FIG. 1 shows the results of a water maze test in mice with LPS-induced memory impairment administered with the compounds of the present invention (KIT-008, KIT-020). [Figure 13] 1 is a micrograph showing that administration of the compounds of the present invention (KIT-008, KIT-020) inhibited LPS-induced amyloid beta (Aβ) deposition in mouse hippocampal tissue. [Figure 14] FIG. 1 shows that administration of the compounds of the present invention (KIT-008, KIT-020) inhibited LPS-induced amyloid beta (Aβ) deposition in mouse hippocampal tissue, and is a graph showing the average number of deposited amyloid beta. [Figure 15] 1 is a micrograph showing that the administration of the compounds of the present invention (KIT-008, KIT-020) suppressed the increase in the expression of inducible nitric oxide synthase (iNOS) in mouse hippocampal tissue. [Figure 16] FIG. 1 shows that administration of the compounds of the present invention (KIT-008, KIT-020) inhibited increased expression of inducible nitric oxide synthase (iNOS) in mouse hippocampal tissue, and is a graph showing the average number of iNOS-positive neurons. [Figure 17] FIG. 1 is a micrograph showing that administration of the compounds of the present invention (KIT-008, KIT-020) inhibited the LPS-induced increase in amoeboid microglia (Iba1 positive cells) and amoeboid astrocytes (GFAP positive cells) in mouse hippocampal tissue. [Figure 18]FIG. 1 shows that administration of the compounds of the present invention (KIT-008, KIT-020) suppressed LPS-induced increases in amoeboid microglia (Iba1 positive cells) and amoeboid astrocytes (GFAP positive cells), as well as increases in astrocytes (GFAP positive cells) in mouse hippocampal tissue. The upper graph shows the proportions of amoeboid microglia and amoeboid astrocytes, and the lower graph shows the average number of astrocytes (GFAP positive cells). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] [New compounds of the present invention] The novel compound of the present invention is a compound represented by the following general formula (I), its racemate, or a salt thereof. The carbon atom of the glycerol skeleton in general formula (I) may have a substituent. Examples of the substituent include an alkyl group having 1 to 4 carbon atoms and an alkoxy group having 1 to 4 carbon atoms.

[0022] [ka]

[0023] In formula (I), X represents an oxygen atom, a nitrogen atom or a sulfur atom, and is preferably an oxygen atom.

[0024] R 1 R represents an unsaturated aliphatic hydrocarbon group, preferably having at least one double bond. 1 The position of the double bond in may be any position, but when the carbon bonded to the carbon adjacent to X is the 1st carbon, it is preferable that the double bond is at least between the 1st carbon and the 2nd carbon.

[0025] Also, R 1 may be linear or branched, but is preferably linear. The number of carbon atoms is preferably 4 to 50, more preferably 8 to 30, further preferably 10 to 25, and particularly preferably 15 to 20. 1may have a substituent, and examples of the substituent include an alkoxy group having 1 to 4 carbon atoms.

[0026] R 2 R represents a saturated or unsaturated aliphatic hydrocarbon group, and an unsaturated aliphatic hydrocarbon group is preferred. 2 The unsaturated aliphatic hydrocarbon group preferably has at least one double bond, and may have two or more double bonds.

[0027] R 2 may be linear or branched, but is preferably linear. The number of carbon atoms is preferably 1 to 50, more preferably 8 to 30, further preferably 10 to 25, and particularly preferably 15 to 20. 2 may have a substituent, and examples of the substituent include an alkoxy group having 1 to 4 carbon atoms. Specifically, R 2 is R 2 In the case of COOH, those representing ω-3 fatty acids, ω-6 fatty acids, ω-7 fatty acids, ω-9 fatty acids, and ω-10 fatty acids are preferred, those representing ω-3 fatty acids, ω-6 fatty acids, and ω-9 fatty acids are more preferred, and those representing ω-6 fatty acids are particularly preferred.

[0028] R 3 represents choline, ethanolamine, inositol or serine, which may have a substituent, and examples of the substituent include an alkyl group having 1 to 4 carbon atoms and an alkoxy group having 1 to 4 carbon atoms.

[0029] The novel compound of the present invention has an anti-inflammatory effect and is effective in preventing or ameliorating (treating) Rett syndrome.

[0030] [Composition of the present invention] The composition of the present invention is characterized by containing the novel compound of the present invention represented by the above general formula (I), its racemate or a salt thereof as an active ingredient. The composition of the present invention may contain other pharma- ceutically acceptable ingredients.

[0031] Since the composition of the present invention has an anti-inflammatory effect, it can be used to prevent or improve (treat) inflammatory diseases, and is particularly effective in preventing or improving (treating) neuroinflammatory diseases. Specifically, it can be suitably used to prevent or improve (treat) neuroinflammatory diseases such as dementia, Parkinson's disease, depression, and schizophrenia, and is particularly effective in Alzheimer's dementia. It is also effective in improving brain function.

[0032] In addition, the composition of the present invention is effective in preventing or ameliorating (treating) Rett syndrome, a progressive neurodevelopmental disorder caused by a mutation in the MECP2 gene on the X chromosome.

[0033] The composition of the present invention can be used as a medicine or health food. Also, the composition of the present invention can be used as an oral or parenteral agent.

[0034] When used as an oral preparation, its form may be, for example, a tablet, capsule, powder, granule, liquid, particle, rod, plate, block, solid, round, paste, cream, caplet, gel, chewable, stick, etc. Examples of parenteral preparations include external preparations, injections, and infusions.

[0035] [Method of producing the novel compound of the present invention] The novel compound of the present invention is, for example, (R)-(2,2-dimethyl-1,3-dioxolan-4-yl)methanol represented by the following structural formula: 1 ~R 3 It should be noted that the compound represented by the following structural formula may be protected with a protecting group other than acetonide.

[0036] [ka]

[0037] Here, R 1Compounds containing halogenated unsaturated aliphatic hydrocarbons (ACR 1 (A; halogen)), for example, -R 1 But (-C=CR 1’ ), halogenated unsaturated aliphatic hydrocarbons (ACC=CR 1’ ) can be prepared as follows:

[0038] As a starting material, for example, an alcohol containing a triple bond, such as propargyl alcohol represented by the following chemical formula, is used.

[0039] [ka]

[0040] First, a protecting group (Z 1 ) will be introduced.

[0041] [ka]

[0042] Subsequently, the protected triple bond-containing alcohol is reacted with a halogenated aliphatic hydrocarbon to give the desired aliphatic group (R 1’ In other words, in this process, the halogenated aliphatic hydrocarbon having the desired carbon chain is reacted to produce the final halogenated unsaturated aliphatic hydrocarbon (ACC=CR 1’ ) carbon chain (R 1’ ) can be the objective.

[0043] [ka]

[0044] Next, the protecting group (Z 1 ) is deprotected.

[0045] [ka]

[0046] Next, alcohols containing cis double bonds (HO-CC=CR 1’ )

[0047] [ka]

[0048] Finally, halogen is introduced to form halogenated unsaturated aliphatic hydrocarbons (ACC=CR 1’ (A; halogen)).

[0049] [ka]

[0050] Here, examples of the halogen (A) include chlorine, bromine, and iodine. When introducing chlorine as a halogen, it can be introduced using PCl3, SOCl2, (COCl)2, a combination of CCl4 and PPh3, a combination of N-chlorosuccinimide (NCS) and PPh3, etc. When introducing bromine as a halogen, it can be introduced using PBr 3、 It can be introduced using a combination of CBr4 and PPh3, a combination of N-bromosuccinimide (NBS) and PPh3, a combination of imidazole, PPh3 and Br2, etc. When iodine is introduced as a halogen, it can be introduced using a combination of imidazole, PPh3 and I2, a combination of N-iodosuccinimide (NIS) and PPh3, etc.

[0051] The halogenated unsaturated aliphatic hydrocarbons (ACC=CR 1’ (ACR 1 )) is used to prepare the novel compounds of the present invention.

[0052] First, this halogenated unsaturated aliphatic hydrocarbon (ACR 1) with (R)-(2,2-dimethyl-1,3-dioxolan-4-yl)methanol to obtain the following compound.

[0053] [ka]

[0054] The acetonide of the above compound is then deprotected to give the following compound.

[0055] [ka]

[0056] Next, protecting groups (Z, Z') are introduced to the two OH groups.

[0057] [ka]

[0058] Thereafter, the protecting group (Z) is selectively deprotected to give an OH group.

[0059] [ka]

[0060] Next, the phosphate group and R 3 The following compound is obtained by introducing a substituent containing a phosphate group and R 3 is preferably protected by a protecting group.

[0061] [ka]

[0062] Subsequently, the protecting group (Z') is deprotected to give an OH group.

[0063] [ka]

[0064] Thereafter, the deprotected OH group is condensed with R 2 COOH.

[0065]

Chemical formula

[0066] Finally, the phosphate group and the protecting group of R 3Z are deprotected, etc. to obtain the compound of the present invention below.

[0067]

Chemical formula

Example

[0068] The compounds KIT-008, KIT-019, and KIT-020 of the present invention represented by the following structural formula were produced. The outline is shown below.

[0069]

Chemical formula

[0070] The following is a specific description. <Production of KIT-008> [Step 1: Protection of the OH group of propargyl alcohol]

[0071]

Chemical formula

[0072] To a solution of propargyl alcohol (5.6 g, 100 mmol) in ethyl acetate (100 mL) was added p-toluenesulfonic acid monohydrate (190 mg, 1.0 mmol) at room temperature. To this was slowly added 3,4-dihydro-2H-pyran (10 mL, 110 mmol) at 0°C. After stirring the solution at 0°C for 5 minutes, the reaction mixture was allowed to warm to room temperature. After stirring at room temperature for 40 minutes, saturated NaHCO3 was added to the reaction mixture, which was then extracted twice with ethyl acetate. The combined ethyl acetate layers were washed with water and saturated brine, then dried over Na2SO4, and the solvent was removed under reduced pressure. The residue was purified by vacuum distillation (boiling point 76°C / 10 mmHg) to give THP-protected propargyl alcohol as a yellow liquid (11.9 g, 85%).

[0073] 1 H NMR (500 MHz, CDCl3) δ 4.83 (1H, d, J = 3.4 Hz), 4.30 (1H, dd, J = 2.4, 15.7 Hz), 4.24 (1H, dd, J = 2.4, 15.7 Hz), 3.84 (1H, ddd, J = 2.8, 9.0, 11.5 Hz), 3.84 (1H, ddt, J d = 1.5, 11.1 Hz, J t = 4.3 Hz), 2.42 (1H, t, J = 2.4 Hz), 1.89-1.79 (1H, m), 1.78-1.71 (1H, m), 1.67-1.50 (4H, m).

[0074] [Step 2: Introduction of Aliphatic Group]

[0075] [ka]

[0076] A solution of THP-protected propargyl alcohol (4.6 g, 33 mmol) in tetrafuran (122 mL) was added at -78 °C. nA hexane solution of BuLi (1.57 mol / L) (21 mL, 33 mmol) was added dropwise. At 0°C, 1-bromopentadecane (8.7 g, 30 mmol) was slowly added, and the mixture was warmed to room temperature and then heated at 50°C for 21 hours. The mixture was cooled to room temperature, saturated aqueous NH4Cl was added, and the mixture was extracted twice with Et2O. The Et2O layers were combined, washed with saturated brine, dried over Na2SO4, and filtered through a pad of silica gel. The filtrate was concentrated under reduced pressure. The residue was dissolved in methanol (73 mL) and diluted with DOWEX TM 50WX8 (strong acid ion exchange resin) (994 mg) was added and stirred at 40° C. for 3 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was dissolved in petroleum ether at 30-40° C. and the resulting solution was cooled to room temperature. The solution was cooled to −78° C. and the resulting precipitate was collected by filtration to give the desired alcohol as a white solid (6.1 g, 77%).

[0077] 1 H NMR (500 MHz, CDCl3) δ 4.25 (2H, t, J = 2.1 Hz), 2.21 (2H, tt, J = 2.1, 7.1 Hz), 1.60 (1H, brs), 1.50 (2H, tt, J = 7.4, 7.4 Hz), 1.40-1.33 (2H, m), 1.33-1.21 (22H, m), 0.88 (3H, t, J = 7.0 Hz).

[0078] [Step 3: Conversion to cis-allyl alcohol]

[0079] [ka]

[0080] A solution of octadec-2-yn-1-ol (266 mg, 1.0 mmol) in tetrahydrofuran (4 mL) was added to a solution of LiAlH4 (42 mg, 1.1 mmol) in tetrahydrofuran (6 mL) at 0°C, and the mixture was warmed to room temperature and stirred for 19 hours. A saturated aqueous solution of Rochelle salt was added to the reaction mixture at 0°C, and the mixture was stirred for 1 hour and then filtered. The filtrate was extracted twice with ethyl acetate. The ethyl acetate layers were combined, washed with water and saturated saline, dried over Na2SO4, and the solvent was distilled off under reduced pressure. The residue was purified by column chromatography to obtain the desired (E)-octadec-2-en-1-ol (244 mg, 91%) as a white solid.

[0081] 1 H NMR (500 MHz, CDCl3) δ 5.73-5.60 (2H, m), 4.11-4.07 (2H, m), 2.06-2.01 (2H, m), 1.40-1.34 (2H, m), 1.32-1.21 (24H, m), 0.88 (3H, t, J = 7.0 Hz).

[0082] [Step 4: Halogenation of alcohol]

[0083] [ka]

[0084] PBr3 was added to a solution of (E)-octadec-2-en-1-ol in ethyl acetate at 0°C, and the mixture was stirred for 30 minutes. Ice water was added, and the mixture was extracted twice with ethyl acetate. The ethyl acetate layers were combined, washed with saturated saline, dried over Na2SO4, and the solvent was removed under reduced pressure. The residue was purified by column chromatography to obtain the desired (E)-1-bromooctadec-2-ene as a colorless liquid.

[0085] 1 H NMR (500 MHz, CDCl3) δ 5.78 (1H, dt, J d = 15.1 Hz, J t= 6.6 Hz), 5.68 (1H, dtt, J d = 15.1 Hz, J t = 1.2, 7.5 Hz), 3.95 (2H, dd, J = 0.6, 7.5 Hz), 2.05 (2H, dt, J d = 7.0 Hz, J t = 7.0 Hz), 1.40-1.34 (2H, m), 1.32-1.21 (24H, m), 0.88 (3H, t, J = 7.0 Hz).

[0086] [Step 5: Introduction of an aliphatic group into the Sn-1 position of (R)-(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (hereinafter referred to as substrate)]

[0087] [ka]

[0088] t (R)-(2,2-dimethyl-1,3-dioxolan-4-yl)methanol (1.30 g, 9.8 mmol) was added to a tetrahydrofuran solution (50 mL) of BuOK (1.65 g, 14.7 mmol) at 0°C, and the mixture was stirred for 1 hour. To this, a tetrahydrofuran solution (50 mL) of (E)-1-bromooctadec-2-ene (4.86 g, 14.7 mmol) prepared above was added, heated to 40°C, and stirred overnight. To this, a phosphate buffer solution (pH 7) was added, and the mixture was extracted twice with Et2O. The Et2O layers were combined, washed with water and saturated saline, dried over Na2SO4, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography to give the desired (R,E)-2,2-dimethyl-4-((octadec-2-en-1-yloxy)methyl)-1,3-dioxolane as a colorless liquid (3.62 g, 97%).

[0089] 1 H NMR (500 MHz, CDCl3) δ 5.69 (1H, dt, J d= 15.3 Hz, J t = 6.7 Hz), 5.53 (1H, dtt, J d = 15.3 Hz, J t = 1.4, 6.3 Hz), 4.28 (1H, tt, J = 6.0, 6.0 Hz), 4.06 (1H, dd, J = 6.4, 8.2 Hz), 4.01-3.93 (2H, m), 3.72 (1H, dd, J = 6.4, 8.3 Hz),3.50 (1H, dd, J = 5.9, 9.8 Hz), 3.41 (1H, dd, J = 5.6, 9.8 Hz), 2.03 (2H, dt, J d = 6.9 Hz, J t = 6.9 Hz), 1.43 (3H, s), 1.39-1.33 (2H, m), 1.36 (3H, s), 1.32-1.21 (24H, m), 0.88 (3H, t, J = 7.0 Hz).

[0090] [Step 6: Deprotection of the substrate]

[0091] [ka]

[0092] To a solution of (R,E)-2,2-dimethyl-4-((octadec-2-en-1-yloxy)methyl)-1,3-dioxolane (6.91 g, 18.0 mmol) in ethanol (60 mL)-water (7.5 mL) was added a solution of tosylic acid monohydrate (0.69 g, 3.6 mmol) in ethanol (15 mL) at 0°C. The mixture was heated to 70°C and stirred for 6 h, then cooled to 0°C and neutralized with triethylamine. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography to give the desired (S,E)-3-(octadec-2-en-1-yloxy)propane-1,2-diol as a white solid (5.9 g, 95%).

[0093] 1H NMR (500 MHz, CDCl3) δ 5.70 (1H, dt, J d = 15.3 Hz, J t = 6.8 Hz), 5.53 (1H, dtt, J d = 15.3 Hz, J t = 1.4, 6.3 Hz), 3.96 (2H, d, J = 6.2 Hz), 3.90-3.85 (1H, m), 3.72 (1H, ddd, J = 4.0, 7.0, 11.2 Hz), 3.65 (1H, dt, J d = 11.0 Hz, J t = 5.4 Hz), 3.54 (1H, dd, J = 3.8, 9.7 Hz), 3.49 (1H, dd, J = 6.3, 9.7 Hz), 2.56 (1H, d, J = 5.0 Hz), 2.04 (2H, dt, J d = 6.8 Hz, J t = 6.9 Hz), 1.40-1.34 (2H, m), 1.32-1.21 (24H, m), 0.88 (3H, t, J = 7.0 Hz).

[0094] [Project 7: Protection of the OH group at the Sn-3 position of the matrix]

[0095]

change

[0096] Pivaloyl chloride (0.19 mL, 1.52 mmol) was added to a dichloromethane solution (13 mL) of (S,E)-3-(octadec-2-en-1-yloxy)propane-1,2-diol (436 mg, 1.27 mmol) and pyridine (0.31 mL, 3.81 mmol) at 0°C. The reaction mixture was warmed to room temperature and stirred for 8 hours. Phosphate buffer (pH 7) was added and the mixture was extracted twice with Et2O. The Et2O layers were combined, washed with water and saturated saline, dried over Na2SO4, and the solvent was removed under reduced pressure. The residue was purified by column chromatography to give the desired (R,E)-2-hydroxy-3-(octadec-2-en-1-yloxy)propyl pivalate as a colorless liquid (440 mg, 81%).

[0097] 1 H NMR (500 MHz, CDCl3) δ 5.70 (1H, dt, J d = 15.3 Hz, J t = 6.7 Hz), 5.53 (1H, dtt, J d = 15.3 Hz, J t = 1.4, 6.3 Hz), 4.16 (1H, dd, J = 4.8, 11.4 Hz), 4.13 (1H, dd, J = 5.5, 11.3 Hz), 4.03-3.98 (1H, m), 3.96 (2H, d, J = 6.3 Hz), 3.49 (1H, dd, J = 4.3, 9.7 Hz), 3.43 (1H, dd, J = 6.3, 9.7 Hz), 2.46 (1H, d, J = 4.3 Hz), 2.04 (2H, dt, J d = 6.9 Hz, J t = 6.9 Hz), 1.40-1.34 (2H, m), 1.32-1.22 (24H, m), 1.22 (9H, s), 0.88 (3H, t, J = 7.0 Hz).

[0098] [Step 8: Protection of the OH group at the Sn-2 position of the substrate]

[0099] [ka]

[0100] To a DMF solution (9 mL) of tert-butyldimethylchlorosilane (R,E)-2-hydroxy-3-(octadec-2-en-1-yloxy)propyl pivalate (774 mg, 1.81 mmol) and imidazole (493 mg, 7.24 mmol), a DMF solution (9 mL) of tert-butyldimethylchlorosilane (546 mg, 3.62 mmol) was added at 0°C, and the mixture was warmed to room temperature and stirred for 20 hours. Phosphate buffer (pH 7) was added to the mixture, and the mixture was extracted twice with Et2O. The Et2O layers were combined, washed with water and saturated saline, dried over Na2SO4, and the solvent was removed under reduced pressure. The residue was purified by column chromatography to give the desired (R,E)-2-((tert-butyldimethylsilyl)oxy)-3-(octadec-2-en-1-yloxy)propyl pivalate as a colorless liquid (921 mg, 94%).

[0101] 1 H NMR (500 MHz, CDCl3) δ 5.68 (1H, dt, J d = 15.3 Hz, J t = 6.7 Hz), 5.51 (1H, dt, J d = 15.3 Hz, J t = 6.2 Hz), 4.19-4.14 (1H, m), 4.02-3.95 (2H, m), 3.93 (2H, dd, J = 0.6, 6.2 Hz), 3.49 (2H, dd, J = 4.3, 9.7 Hz), 3.43-3.35 (2H, m), 2.03 (2H, dt, J d = 6.9 Hz, J t = 7.0 Hz), 1.40-1.32 (2H, m), 1.32-1.22 (24H, m), 1.20 (9H, s), 0.88 (3H, t, J = 6.9 Hz), 0.88 (9H, s), 0.09 (6H, s).

[0102] [Step 9: Deprotection of the Sn-3 position of the substrate]

[0103] [ka]

[0104] A 1.014 mol / L toluene solution of diisobutylaluminum hydride (5 mL, 5.07 mmol) was added to a dichloromethane solution (60 mL) of (R,E)-2-((tert-butyldimethylsilyl)oxy)-3-(octadec-2-en-1-yloxy)propyl pivalate (915 mg, 1.69 mmol) at -78°C, and the mixture was stirred for 3 hours. A 30% aqueous solution of Rochelle salt was added to the mixture, and the mixture was stirred for 1 hour, filtered through Celite, and extracted twice with ethyl acetate. The ethyl acetate layers were combined, washed with water and saturated saline, dried over Na2SO4, and the solvent was removed under reduced pressure. The residue was purified by column chromatography to obtain the desired (S,E)-2-((tert-butyldimethylsilyl)oxy)-3-(octadec-2-en-1-yloxy)propan-1-ol as a colorless liquid (756 mg, 98%).

[0105] 1 H NMR (500 MHz, CDCl3) δ 5.68 (1H, dt, J d = 15.3 Hz, J t = 6.8 Hz), 5.51 (1H, dt, J d = 15.3 Hz, J t = 6.2 Hz), 3.92 (2H, d, J = 6.2 Hz), 3.91-3.87 (1H, m), 3.65 (1H, dt, J d = 11.1 Hz, J t = 4.7 Hz), 3.58 (1H, ddd, J = 4.5, 7.1, 11.3 Hz), 3.41 (2H, d, J = 6.2 Hz), 2.12-2.08 (1H, m), 2.03 (2H, dt, J d = 7.0 Hz, Jt = 7.0 Hz), 1.40-1.33 (2H, m), 1.32-1.21 (24H, m), 0.90 (9H, s), 0.88 (3H, t, J = 7.1 Hz), 0.10 (3H, s), 0.09 (3H, s).

[0106] [Step 10: Removal of a protected phosphate group and R 3 Introduction of

[0107] [ka]

[0108] To a toluene solution (8 mL) of (S,E)-2-((tert-butyldimethylsilyl)oxy)-3-(octadec-2-en-1-yloxy)propan-1-ol (722 mg, 1.58 mmol), t A 0.1 mol / L hexane solution of BuOLi (16 mL, 1.60 mmol) was added at 0°C, and the mixture was stirred for 1 hour. A toluene solution (8 mL) of tert-butyl (2-((tert-butoxy(2,2,2-trifluoroethoxy)phosphoryl)oxy)ethyl)carbamate (899 mg, 2.37 mmol) was added to the mixture at 0°C, and the mixture was warmed to room temperature and stirred for 24 hours. A phosphate buffer solution (pH 7) was added to the mixture, and the mixture was extracted twice with ethyl acetate. The ethyl acetate layers were combined, washed with water and saturated saline, dried over Na2SO4, and the solvent was distilled off under reduced pressure. The residue was purified by column chromatography to give the desired tert-butyl (2-((tert-butoxy((R)-2-((tert-butyldimethylsilyl)oxy)-3-(((E)-octadec-2-en-1-yl)oxy)propoxy)phosphoryl)oxy)ethyl)carbamate as a colorless liquid (1.035 g, 89%).

[0109] 1H NMR (500 MHz, CDCl3) δ 5.71-5.64 (1H, m), 5.55-5.47 (1H, m), 5.15 (1H, brs), 4.09-4.02 (3H, m), 4.00-3.88 (4H, m), 3.44-3.35 (4H, m), 2.03 (2H, dt, J d = 6.5 Hz, J t = 6.6 Hz), 1.50 (9H, s), 1.44 (9H, s), 1.39-1.33 (2H, m), 1.32-1.22 (24H, m), 0.89 (9H, s), 0.88 (3H, t, J = 7.1 Hz), 0.094+0.091+0.086+0.064+0.060 (6H, five singlet signals).

[0110] [Step 11: Deprotection of OH group at Sn-2 position of substrate]

[0111] [ka]

[0112] To a solution (14 mL) of tert-butyl (2-((tert-butoxy((R)-2-((tert-butyldimethylsilyl)oxy)-3-(((E)-octadec-2-en-1-yl)oxy)propoxy)phosphoryl)oxy)ethyl)carbamate (1.00 g, 1.36 mmol) in tetrahydrofuran, triethylamine trihydrofluoride (1.7 mL, 13.6 mmol) was added at room temperature, and the mixture was heated to 40°C and stirred for 10 hours. A saturated aqueous solution of sodium bicarbonate was added to the mixture, and the mixture was extracted twice with ethyl acetate. The ethyl acetate layers were combined, washed with saturated saline, dried over Na2SO4, and the solvent was removed under reduced pressure. The residue was purified by column chromatography to give the desired tert-butyl (2-((tert-butoxy((R)-2-hydroxy-3-(((E)-octadec-2-en-1-yl)oxy)propoxy)phosphoryl)oxy)ethyl)carbamate as a colorless liquid (0.535 g, 65%).

[0113] 1 H NMR (500 MHz, CDCl3) δ 5.73-5.66 (1H, m), 5.55-5.48 (1H, m), 5.15 (1H, brs), 4.15-3.98 (5H, m), 3.96 (2H, dd, J = 0.8, 6.3 Hz), 3.48 (2H, d, J = 5.3 Hz), 3.43-3.38 (2H, m), 3.10 (1H, brs), 2.03 (2H, dt, J d = 7.1 Hz, J t = 7.1 Hz), 1.51 (9H, s), 1.44 (9H, s), 1.39-1.33 (2H, m), 1.32-1.22 (24H, m), 0.88 (3H, t, J = 7.0 Hz).

[0114] [Project 12: Aliphatic group (R) at the Sn-2 position of the matrix 2 ) import]

[0115]

change

[0116] To a dichloromethane solution (0.5 mL) of tert-butyl (2-((tert-butoxy((R)-2-hydroxy-3-(((E)-octadec-2-en-1-yl)oxy)propoxy)phosphoryl)oxy)ethyl)carbamate (131.6 mg, 0.21 mmol), a dichloromethane solution (0.5 mL) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (81.8 mg, 0.42 mmol), dimethylaminopyridine (26.7 mg, 0.21 mmol) and docosahexaenoic acid (93.4 mg, 0.28 mmol) was added at room temperature, and the mixture was stirred for 5 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography to give the desired (2R)-1-((tert-butoxy(2-((tert-butoxycarbonyl)amino)ethoxy)phosphoryl)oxy)-3-(((E)-octadec-2-en-1-yl)oxy)propan-2-yl (4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoate as a colorless liquid (0.535 g, 65%).

[0117] 1H NMR (500 MHz, CDCl3) δ 5.72-5.65 (1H, m), 5.53-5.46 (1H, m), 5.43-5.28 (12H, m), 5.19-5.12 (2H, m), 4.25-4.10 (2H, m), 4.08-4.02 (2H, m), 3.97-3.90 (2H, m), 3.59 (2H, d, J = 5.2 Hz, one diastereomer), 3.55 (2H, d, J = 5.2 Hz, the other diastereomer), 3.42-3.37 (2H, m), 2.87-2.80 (10H, m), 2.41-2.39 (4H, m), 2.11-2.00 (4H, m), 1.50 (9H, s, the other diastereomer), 1.49 (9H, s, one diastereomer), 1.44 (9H, s), 1.39-1.33 (2H, m), 1.32-1.22 (24H, m), 0.97 (3H, t, J = 7.5 Hz), 0.89 (3H, t, J = 7.0 Hz).

[0118] [Project 13: Deprotection and びアミンization of the Sn-3 position of the matrix]

[0119]

change

[0120] (2R)-1-((tert-Butoxy(2-((tert-butoxycarbonyl)amino)ethoxy)phosphoryl)oxy)-3-(((E)-octadec-2-en-1-yl)oxy)propan-2-yl (4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoate (29.1 mg, 0.031 mmol) in dichloromethane (0.3 mL) was treated with 0.3 mL of trifluoroacetic acid at room temperature and stirred for 2 h. The reaction mixture was concentrated under reduced pressure without further treatment to afford the title 2-((((R)-2-(((4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoyl)oxy)-3-(((E)-octadec-2-en-1-yl)oxy)propoxy)(hydroxy)phosphoryl)oxy)ethane-1-ammonium 2,2,2-trifluoroacetate (KIT-008) as a colorless liquid (27.4 mg, quant).

[0121] 1 H NMR (500 MHz, CDCl3) δ 8.09 (3H, brs), 5.67 (1H, dt, J d = 15.1 Hz, J t = 6.9 Hz), 5.48 (1H, dt, J d = 15.0 Hz, J t = 6.5 Hz), 5.43-5.28 (12H, m), 5.18-5.12 (1H, m), 4.19-3.88 (6H, m), 3.53 (2H, brd, J = 4.2 Hz), 3.21 (2H, brs), 2,87-2.79 (10H, m), 2.42-2.34 (4H, m), 2.11-1.99 (4H, m), 1.39-1.22 (26H, m), 0.97 (3H, t, J = 7.6 Hz), 0.88 (3H, t, J = 7.0 Hz).

[0122] <Production of KIT-019> Steps 1 to 11 are the same as those in the above "Production of KIT-008".

[0123] [Step 12: Adding an aliphatic group (R 2 ) Introduction

[0124] [ka]

[0125] To a dichloromethane solution (0.5 mL) of tert-butyl (2-((tert-butoxy((R)-2-hydroxy-3-(((E)-octadec-2-en-1-yl)oxy)propoxy)phosphoryl)oxy)ethyl)carbamate (61.0 mg, 0.10 mmol), a dichloromethane solution (0.5 mL) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (38.0 mg, 0.20 mmol), dimethylaminopyridine (12.0 mg, 0.10 mmol) and oleic acid (34.0 mg, 0.12 mmol) was added at room temperature, and the mixture was stirred for 17 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography to give the desired (2R)-1-((tert-butoxy(2-((tert-butoxycarbonyl)amino)ethoxy)phosphoryl)oxy)-3-(((E)-octadec-2-en-1-yl)oxy)propan-2-yl oleate as a colorless liquid (49.0 mg, 55%).

[0126] 1 H NMR (500 MHz, CDCl3) δ 5.68 (1H, dt, J d = 15.2 Hz, J t = 6.8 Hz), 5.49 (1H, dtt, J d = 15.3 Hz, J t= 1.3, 6.3 Hz), 5.38-5.30 (2H, m), 5.19-5.12 (2H, m), 4.21-4.15 (1H, m), 4.14-4.09 (1H, m), 4.08-4.01 (2H, m), 3.97-3.90 (2H, m), 3.55 (2H, d, J = 5.2 Hz), 3.42-3.37 (2H, m), 2.33 (2H, t, J = 7.6 Hz), 2.06-1.98 (6H, m), 1.65-1.58 (2H, m), 1.502 (9H, s, one diastereomer), 1.495 (9H, s, the other diastereomer), 1.44 (9H, s), 1.38-1.23 (46H, m), 0.88 (6H, t, J = 6.9 Hz).

[0127] [Step 13: Deprotection and amination of the Sn-3 position of the substrate]

[0128] [ka]

[0129] To a dichloromethane solution (0.55 mL) of (2R)-1-((tert-butoxy(2-((tert-butoxycarbonyl)amino)ethoxy)phosphoryl)oxy)-3-(((E)-octadec-2-en-1-yl)oxy)propan-2-yl oleate (46.0 mg, 0.055 mmol), 0.55 mL of trifluoroacetic acid was added at room temperature and stirred for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain the desired 2-((hydroxy((R)-3-3-(((E)-octadec-2-en-1-yl)oxy)-2-(oleoyloxy)propyl)phosphoryl)oxy)ethane-1-ammonium 2,2,2-trifluoroacetate (KIT-019) as a colorless liquid (46.2 mg, quant).

[0130] 11H NMR (500 MHz, CDCl3) δ 7.88 (3H, brs), 5.68 (1H, dt, J d = 15.2 Hz, J t = 6.7 Hz), 5.48 (1H, dt, J d = 15.0 Hz, J t = 6.5 Hz), 5.38 - 5.30 (2H, m), 5.19 - 5.12 (1H, m), 4.22 - 3.97 (4H, m), 3.97 - 3.89 (2H, m), 3.53 (2H, brd, J = 4.1 Hz), 2.32 (2H, t, J = 7.6 Hz), 2.05 - 1.97 (6H, m), 1.61 - 1.55 (2H, m), 1.37 - 1.21 (m, 46H), 0.88 (6H, t, J = 6.9 Hz).

[0131] <Production of KIT-020> Steps 1 to 11 are the same as those in the above "Production of KIT-008".

[0132] [Step 12: Introduction of an aliphatic group (R 2 ) at the Sn-2 position of the substrate]

[0133] [Chemical formula]

[0134] To a dichloromethane solution (0.5 mL) of tert-butyl (2-((tert-butoxy((R)-2-hydroxy-3-(((E)-octadec-2-en-1-yl)oxy)propoxy)phosphoryl)oxy)ethyl)carbamate (61.0 mg, 0.10 mmol), a dichloromethane solution (0.5 mL) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (38.0 mg, 0.20 mmol), dimethylaminopyridine (12.0 mg, 0.10 mmol) and arachidonic acid (37.0 mg, 0.12 mmol) was added at room temperature, and the mixture was stirred for 16 hours. The reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography to give the desired (2R)-1-((tert-butoxy(2-((tert-butoxycarbonyl)amino)ethoxy)phosphoryl)oxy)-3-(((E)-octadec-2-en-1-yl)oxy)propan-2-yl(5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoate as a colorless liquid (33.8 mg, 38%).

[0135] 1 H NMR (500 MHz, CDCl3) δ 5.68 (1H, dt, J d = 15.3 Hz, J t = 6.8 Hz), 5.49 (1H, dtt, J d = 15.3 Hz, J t = 1.3, 6.3 Hz), 5.43-5.30 (8H, m), 5.19-5.12 (2H, m), 4.21-4.16 (1H, m), 4.15-4.09 (1H, m), 4.08-4.01 (2H, m), 3.97-3.89 (2H, m), 3.55 (2H, d, J = 5.2 Hz), 3.42-3.37 (2H, m), 2.86-2.78 (6H, m), 2.36 (2H, t, J = 7.6 Hz), 2.11 (2H, dt, J d = 7.0 Hz, J t = 6.9 Hz), 2.06 (2H, dt, J d= 7.1 Hz, J t = 7.1 Hz), 2.03 (2H, dt, J d = 7.2 Hz, J t = 7.2 Hz), 1.75-1.67 (2H, m), 1.50 (9H, s, one diastereomer), 1.49 (9H, s, the other diastereomer), 1.44 (9H, s), 1.39-1.22 (32H, m), 0.89 (3H, t, J = 6.9 Hz), 0.88 (3H, t, J = 6.9 Hz).

[0136] [Step 13: Deprotection and amination of the Sn-3 position of the substrate]

[0137] [ka]

[0138] To a dichloromethane solution (0.36 mL) of (2R)-1-((tert-butoxy(2-((tert-butoxycarbonyl)amino)ethoxy)phosphoryl)oxy)-3-(((E)-octadec-2-en-1-yl)oxy)propan-2-yl (5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoate (32.5 mg, 0.036 mmol), 0.36 mL of trifluoroacetic acid was added at room temperature, and the mixture was stirred for 2 hours. The reaction mixture was concentrated under reduced pressure to give the desired 2-((hydroxy((R)-2-(((5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoyl)oxy)-3-(((E)-octadec-2-en-1-yl)oxy)propoxy)phosphoryl)oxy)ethane-1-ammonium 2,2,2-trifluoroacetate (KIT-020) as a colorless liquid (31.3 mg, quant).

[0139] 1 H NMR (500 MHz, CDCl3) δ 7.91 (3H, brs), 5.68 (1H, dt, J d= 15.2 Hz, J t = 6.8 Hz), 5.48 (1H, dt, J d = 14.7 Hz, J t = 6.6 Hz), 5.43-5.30 (8H, m), 5.19-5.14 (1H, m), 4.22-4.12 (2H, m), 4.12-3.97 (2H, m), 3.96-3.89 (2H, m), 3.53 (2H, d, J = 4.7 Hz), 3.30-3.21 (2H, m), 2.85-2.78 (6H, m), 2.35 (2H, t, J = 7.7 Hz), 2.10 (2H, dt, J d = 7.2 Hz, J t = 7.2 Hz), 2.05 (2H, dt, J d = 7.5 Hz, J t = 7.5 Hz), 2.02 (2H, dt, J d = 7.4 Hz, J t = 7.4 Hz), 1.72-1.64 (2H, m), 1.38-1.22 (32H, m), 0.89 (3H, t, J = 6.9 Hz), 0.88 (3H, t, J = 7.5 Hz). EXAMPLES

[0140] The effects of the above-prepared compounds of the present invention, KIT-008, KIT-019, and KIT-020, were confirmed.

[0141] [Test 1: Evaluation of the IL-1β reducing effect (anti-inflammatory effect) of the compound KIT-008 of the present invention] Mouse microglial cells (BV2) were cultured for 12 hours in DMEM medium containing 2% FBS (fetal bovine serum) with and without the addition of the compound KIT-008 of the present invention (5 μg / ml). After that, the cells were treated with LPS (1 μg / ml) for 2 hours, and intracellular RNA was extracted using an RNA extraction kit (TRI Reagent, Molecular Research Center Inc.). The expression of the inflammatory cytokine IL-1β and the RNA expression of the Gapdh gene as a control were detected by semi-quantitative PCR (TaKaRa PrimeScript 1st strand cDNA synthesis Kit, TAKARA BIOTECHNOLOGY Co., LTD.).

[0142] The results are shown in Figure 1. As shown in FIG. 1, the expression of LPS-induced inflammatory cytokine IL-1β was reduced in microglial cells (BV2) cultured in a medium supplemented with KIT-008.

[0143] [Test 2: Evaluation of the p65 nuclear accumulation inhibitory effect (anti-inflammatory effect) of the compounds of the present invention, KIT-008 and KIT-020] Mouse microglial cells (BV2) were cultured for 12 hours in DMEM medium containing 2% FBS (fetal bovine serum) with or without the addition of the compounds of the present invention, KIT-008 and KIT-020 (5 μg / ml).The cells were then treated with LPS (1 μg / ml) for 2 hours, and proteins in the cell nuclei were extracted.The expression of p65 (NFkB) protein was detected by Western blotting using NF-κB p65 (Cell Signaling Technology, clone name D14E12) as the primary antibody and Anti-Rabbit IgG HRP-linked antibody (Cell Signaling Technology) as the secondary antibody.

[0144] The results are shown in Figure 2. As shown in Figure 2, LPS-induced nuclear accumulation of p65 was suppressed in microglial cells (BV2) cultured in a medium containing KIT-008 and KIT-020. In particular, it was revealed that the use of KIT-020 significantly suppressed the nuclear accumulation of p65.

[0145] [Test 3: Evaluation of the p65 nuclear accumulation inhibitory effect (anti-inflammatory effect) of the compounds of the present invention, KIT-019 and KIT-020] Mouse microglial cells (MG6) were cultured for 24 hours in DMEM medium containing 2% FBS (fetal bovine serum) with or without the addition of the compounds of the present invention, KIT-019 and KIT-020 (5 μg / ml).The cells were then treated with LPS (1 μg / ml) for 8 hours, and proteins in the cell nuclei were extracted.The expression of p65 (NFkB) protein was detected by Western blotting using NF-κB p65 (Cell Signaling Technology, clone name D14E12) as the primary antibody and Anti-Rabbit IgG HRP-linked antibody (Cell Signaling Technology) as the secondary antibody.

[0146] The results are shown in Figure 3. As shown in FIG. 3, in microglial cells (MG6) cultured in a medium containing KIT-019 and KIT-020, LPS-induced intranuclear accumulation of p65 was significantly suppressed.

[0147] [Test 4: Evaluation of the IL-1β reducing effect (anti-inflammatory effect) of the compounds of the present invention, KIT-019 and KIT-020] 5000 mouse microglial cells (MG6) were cultured in a 96-well dish containing 100 μl of DMEM medium containing 2% FBS (fetal bovine serum). Then, 5 μg / ml of the compounds of the present invention KIT-019 and KIT-020 were added and cultured for 24 hours. After that, the cells were treated with LPS (1 μg / ml) for 8 hours. The culture medium was collected, and the secreted IL-1β protein was measured by ELISA using DuoSet Mouse IL-1β (R&D Systems).

[0148] The results are shown in Figure 4. As shown in FIG. 4, in microglial cells (MG6) to which KIT-019 and KIT-020 were added, the expression of LPS-induced inflammatory cytokine IL-1β protein was significantly reduced.

[0149] From the above, it is apparent that the compound of the present invention exhibits excellent anti-inflammatory activity. EXAMPLES

[0150] Rett syndrome (RTT) is a progressive neurodevelopmental disorder caused by a mutation in the MeCP2 gene located on the X chromosome. It mainly affects girls, who grow normally for about six months to one year after birth, but then show various neurological symptoms, including autistic tendencies. However, the details of the molecular mechanism by which MeCP2 dysfunction causes developmental disorders such as RTT remain unclear. In this example, the effect of the compound of the present invention on mouse growth was investigated using MeCP2-deficient mice that exhibit a phenotype similar to that of RTT.

[0151] [Test 1: Evaluation of impact on lifespan] For the experiment, 4-week-old male MeCP2-deficient mice were used. The MeCP2-deficient mice were divided into two groups: a KIT-008-administered group (KO-KIT-008, n=13) and a control group (KO-All, n=28). The weights of the mice in the administration group were measured in advance, and the compound KIT-008 was suspended in water by ultrasonic treatment so that the mice ingested KIT-008 at 20 ng / g body weight. The mice were administered the compound in drinking water twice a week, and survival was verified. The control group was given only water in the same manner.

[0152] The results are shown in Figure 5. As shown in Figure 5, MeCP2-deficient mice administered with the compound KIT-008 of the present invention had a higher survival rate than MeCP2-deficient mice not administered with the compound, and the number of days until the survival rate reached 50% was 64.5 days for the control group mice, whereas the average survival time for the KO-KIT-008-administered group mice was 78 days.

[0153] From the above, it was demonstrated that the life span of mice administered with the compound of the present invention was extended.

[0154] [Test 2: Evaluation of RTT-like scoring] The RTT-like symptoms of MeCP2-deficient mice administered the compound of the present invention were evaluated by scoring. Four-week-old male MeCP2-deficient mice were used in the experiment. MeCP2-deficient mice were divided into four groups: KIT-020 administration group (KO-KIT-020, n = 15), KIT-019 administration group (KO-KIT-019, n = 16), KIT-008 administration group (KO-KIT-008, n = 13), and control group (KO-2021, n = 15). The compound was dissolved in water to 20 ng / g body weight in the same manner as above and administered to the administration group via drinking water. The control group was given only water in the same manner. Scoring was performed once a week from 5 weeks of age on the following items 1 to 6.

[0155] 1.Mobility The mouse was gently placed on the platform and observed. 0 points: same as wild type. 1 point: Less movement than wild type (longer freezing period when first placed on the platform, longer time spent still). Score 2: No spontaneous movement since being placed on the platform, but may move in response to gentle stimulation or food placed nearby. 2. Gait 0 points: same as wild type. Score 1: Hind legs spread wider than in wild type when walking or running, with reduced pelvic elevation. Waddling gait. Duck gait. Score 2: More pronounced abnormalities. Legs sway when lifted, walking backwards, hind legs lifting in unison.

[0156] 3. Hindlimb clasping Observe the mouse when it is hung by the base of its tail. 0 points: Legs spread outward. 1 point: Pulling hind legs in opposite directions or pulling one leg towards the body. 2 points: Feet are drawn tightly together, either touching or touching each other. 4. Tremor Observe the mouse standing on a flat palm. 0 points: No shaking. 1 point: Intermittent, mild shaking. 2 points: Continuous shaking or intermittent violent shaking.

[0157] 5. Breathing Flank movement is observed while the animal is stationary. 0: Normal breathing. Score 1: Periods of regular breathing are interspersed with short periods of faster breathing or breathing cessation. 2 points: Very irregular breathing. Gasping or shortness of breath. 6. General condition Observations were based on general indicators of well-being such as coat condition, eyes and posture. 0 points: Clean, shiny coat, good eyes, normal posture. Score 1: Blank eyes, dull coat / no grooming, slightly hunched posture. 2 points: Eyes closed or narrowed, piloerection, hunched posture.

[0158] The results are shown in Figure 6. As shown in Figure 6, it was confirmed that the RTT-like score of mice was reduced by using the compound of the present invention. Therefore, it was revealed that the RTT-like symptoms were improved by administering the compound of the present invention.

[0159] [Test 3: Evaluation of weight change] The body weight of MeCP2-deficient mice administered the compound of the present invention was measured every week, and the transition of body weight was evaluated. For the experiment, 4-week-old male MeCP2-deficient mice were used. MeCP2-deficient mice were divided into four groups: KIT-020 administration group (KO-KIT-020, n = 16), KIT-019 administration group (KO-KIT-019, n = 16), KIT-008 administration group (KO-KIT-008, n = 12), and control group (KO-2021, n = 21). For the administration group, the compound was dissolved in water to 20 ng / g body weight in the same manner as above, and administered in drinking water. In addition, only water was given to the control group in the same manner.

[0160] The results are shown in Figure 7. The changes in body weight are shown relative to the initial body weight. As shown in FIG. 7, no statistical difference was found in the weight transition between the mice administered with the compound of the present invention and the control group, the KO mice (t-test).

[0161] [Test 4: Toxicity evaluation] The toxicity of the compound of the present invention to mice was evaluated based on the change in body weight of wild-type mice administered with the compound of the present invention.

[0162] The compound of the present invention was administered in drinking water at 20 ng / g body weight to 4-week-old male wild-type mice in the same manner as in Test 3. The wild-type control group was given only water.

[0163] The results are shown in Figure 8. As shown in FIG. 8, it was confirmed that the mice administered with the compound of the present invention showed a weight gain similar to that of the control group. Therefore, the compounds of the present invention are believed to be non-toxic. EXAMPLES

[0164] Lysosomes, organelles that contain enzymes that work under acidic conditions, are involved in the decomposition, removal, and recycling of metabolic waste, proteins, and nucleic acids. However, when nerve cells are damaged by Alzheimer's disease, the acidity level in the lysosomes decreases, and the lysosomes become enlarged as they combine with autophagic vacuoles filled with waste products that could not be completely decomposed. These autophagic vacuoles also contain early amyloid beta (Aβ), and before amyloid beta accumulates, nerve cells are damaged due to impaired acidity in the lysosomes of nerve cells (Ju-Hyun Lee et al. (2022) Faulty autolysosome acidification in Alzheimer's disease mouse models induces autophagic build-up of Aβ in neurons, yielding senile plaques. Nat. Neurosci. 25: 688-701).

[0165] [Test 1] In this study, the effect of compounds of the invention on lysosomal acidity disorders was investigated.

[0166] [Preliminary study: Establishment of an experimental system for impairing lysosomal acidity] In mouse neuronal cells Neuro2a (N2a) cultured in standard DMEM medium, lysosomes were labeled with LysoPrime Green (Dojindo), a fluorescent dye that is highly specific to lysosomes, an acidic organelle, and resistant to pH changes. Next, N2a cells were cultured in the culture supernatant of mouse glial cells BV2 treated with endotoxin lipopolysaccharide (LPS) (1 μg / ml, 18 h) or untreated BV2 culture supernatant, and the LysoTracker, which targets acidic organelles, was expressed. TM Labeling was performed with Red DND-99 (Thermo Fisher Scientific).

[0167] As a result, as shown in Figure 9, targeting of LysoTracker Red to the lysosomes of N2a cells cultured with the culture supernatant of LPS-treated BV2 cells was significantly suppressed, and impaired lysosomal acidity was observed, indicating that inflammation-induced glial cell-derived factors in the culture medium impaired the acidification of lysosomes in neurons.

[0168] [Test: Confirmation of the effect of the compound of the present invention on lysosomal acidity disorders] Using the above experimental system, the effect of the compound of the present invention on lysosomal acidification disorders was confirmed.

[0169] KIT-008, KIT-019, and KIT-020 of the present invention were added to the medium of N2a cells and cultured for 24 hours, and then the cells were cultured for another 24 hours in the presence of each compound in the culture supernatant of BV2 cells administered LPS. As a result, as shown in Figure 10, KIT-008, KIT-019, and KIT-020 of the present invention improved the acidification disorder of lysosomes in nerve cells caused by glial cell-derived factors. Among them, the effect of KIT-008 was the highest.

[0170] [Test 2] In this study, we investigated whether the compounds of the present invention actually inhibit amyloid beta (Aβ) accumulation in N2a cells.

[0171] A plasmid carrying cDNA encoding CFPAPPsw (CFPAPPsw, which fuses the fluorescent protein CFP to the N-terminus of amyloid precursor protein (APPsw) with a Swedish amino acid substitution that is susceptible to cleavage by BACE) was expressed in N2a cells, and the localization of amyloid beta (Aβ) in untreated mouse glial cell BV2 culture supernatant (-LPS), culture supernatant of BV2 treated with LPS (LPS-treated BV2-derived medium), and N2a cells administered KIT-008 was examined using the antibody 6E10 (BioLegend), which recognizes the Aβ portion.

[0172] As a result, as shown in Figure 11, Aβ was not localized in lysosomes in normal medium, but was detected in lysosomes in N2a cells in which lysosomal acidification was impaired due to glial cell-derived factors caused by inflammation. This accumulation of Aβ in lysosomes was eliminated by pretreatment with KIT-008. In other words, it is believed that the accumulation of Aβ in lysosomes due to impaired lysosomal acidity, which is seen in the early stage of Alzheimer's disease, was suppressed by improving the impaired lysosomal acidity with KIT-008.

[0173] The compound of the present invention ameliorates disorders of lysosomal acidity and inhibits accumulation of amyloid beta (Aβ) in lysosomes, and is therefore expected to be effective in preventing and improving Alzheimer's disease. EXAMPLES

[0174] The effects of the compounds of the present invention on LPS-induced memory impairment in mice were investigated. Specifically, 8-week-old male c57BL6J mice (5 mice per group, n=5) were given the compounds of the present invention (KIT-008, KIT-020) at a dose of 10 mg / 50 kg / day for 4 weeks, and then treated with LPS (200 mg / kg / day) for 7 days while also drinking the compounds. Then, water maze tests were performed for 2 days (3 trials per day).

[0175] The results are shown in Figure 12. Escape latency indicates the time required to reach the escape platform (reach time). Comparison between groups was performed using ANOVA and Bonferroni's post-hoc test. The LPS group showed a decrease in memory (increase in reach time) compared to the control group (P<0.01). The KIT-008 group and the KIT-020 group showed an improvement in memory compared to the LPS group in Trial 6 (P<0.05 for each). Therefore, the compound of the present invention is effective in improving brain function (cognitive function). EXAMPLES

[0176] The effect of the compounds of the present invention on LPS-induced amyloid beta (Aβ) deposition in mouse brain tissue (hippocampal tissue) was investigated. Specifically, 8-week-old male c57BL6J mice were given the compounds of the present invention (KIT-008, KIT-020) at a dose of 10 mg / 50 kg / day for 4 weeks, and then treated with LPS (200 mg / kg / day) for 10 days while also drinking the compounds. The hippocampal tissues of the mice were subjected to immunohistochemistry using anti-amyloid beta antibodies (a cocktail of two antibodies, 6E10 (BioLegend) and 82E1 (Immuno-Biological Laboratories Co, Ltd)). DAPI was used to stain the cell nuclei.

[0177] The results are shown in Figures 13 and 14 (graphs). The scale bar in Figure 13 is 100 μm, and the arrow indicates the deposited amyloid beta. The data in Figure 14 represent the average number of deposited amyloid beta in each group (3 mice, n=3). Amyloid beta deposition was observed in the LPS group compared to the control group, but amyloid beta deposition by LPS was suppressed in the KIT-008 group and the KIT-020 group. EXAMPLES

[0178] The effect of the compound of the present invention on the induction of iNOS expression was investigated using the increase in expression of inducible nitric oxide synthase (iNOS) caused by intracerebral inflammation in mouse brain tissue (hippocampal tissue). Specifically, 8-week-old male c57BL6J mice were given the compounds of the present invention (KIT-008, KIT-020) at a dose of 10 mg / 50 kg / day for 4 weeks, and then treated with LPS (200 mg / kg / day) for 10 days while also drinking the compounds. Mouse brain tissues were subjected to immunohistochemistry (IHC) studies using anti-NeuN antibody (neuron marker, Millipore MAB377) and anti-iNOS antibody (inflammatory marker, Invitrogen PA1-036). DAPI was used to stain cell nuclei.

[0179] The results are shown in Figures 15 and 16 (graphs). The scale bar in Figure 15 is 50 µm, and the arrows indicate iNOS-positive neurons. The data in Figure 16 represent the average values ​​for each group (3 mice, n = 3). The LPS group showed an increased number of iNOS-positive neurons compared to the control group, but the increase in iNOS-positive neurons due to LPS was suppressed in the KIT-008 and KIT-020 groups. EXAMPLES

[0180] The effect of the compound of the present invention on the increase in LPS-induced amoeboid microglia (Iba1 positive cells) and amoeboid astrocytes (GFAP positive cells) in mouse brain tissue (hippocampal tissue) was investigated. The amoeboid represents inflammation in microglia and astrocytes. The effect of the compound of the present invention on the increase in LPS-induced astrocytes (GFAP positive cells) in mouse brain tissue (hippocampal tissue) was also investigated.

[0181] Specifically, 8-week-old male c57BL6J mice were given the compounds of the present invention (KIT-008, KIT-020) at a dose of 10 mg / 50 kg / day for 4 weeks, and then treated with LPS (200 mg / kg / day) for 10 days while also drinking the compounds. Mouse brain tissues were subjected to immunohistochemistry studies using Iba1 (microglia marker, Fujifilm 019-19741) and GFAP (astrocyte marker, Sigma C9205). DAPI was used to stain cell nuclei.

[0182] The results are shown in Figures 17 and 18 (graphs). The scale bar in Figure 17 is 100 μm. The data in Figure 18 show the percentage of amoeboid microglia and amoeboid astrocytes (upper row) and the number of astrocytes (GFAP-positive cells) (lower row) in each group (3 mice, n=3). The LPS group showed an increase in amoeboid microglia (Iba1-positive cells) and amoeboid astrocytes (GFAP-positive cells) compared to the control group. In addition, astrocytes (GFAP-positive cells) increased. However, in the KIT-008 and KIT-020 groups, the increase in amoeboid microglia (Iba1-positive cells), amoeboid astrocytes (GFAP-positive cells), and astrocytes (GFAP-positive cells) due to LPS was suppressed. [Industrial Applicability]

[0183] The novel compound of the present invention can be used as a pharmaceutical composition and is therefore industrially useful.

Claims

1. A compound represented by the general formula (I), a racemate thereof or a salt thereof. 【Chemistry 1】 In the general formula (I), X represents an oxygen atom, a nitrogen atom, or a sulfur atom; R １ represents an unsaturated aliphatic hydrocarbon group having one double bond, and the double bond is present between the carbon atom bonded to the carbon bonded to X and the carbon atom bonded to X, where the carbon atom bonded to the carbon atom bonded to X is the carbon atom bonded to X, and R ２ represents a saturated or unsaturated aliphatic hydrocarbon group, R ３ represents choline, ethanolamine, inositol or serine.

2. 2. The compound according to claim 1, its racemate or a salt thereof, wherein X is an oxygen atom.

3. A composition comprising the compound according to claim 1, its racemate or a salt thereof as an active ingredient.

4. The composition according to claim 3, characterized in that it has an anti-inflammatory effect.

5. 5. The composition according to claim 3 or 4, characterized in that it is used for preventing or improving a neuroinflammatory disease.

6. The composition according to claim 5, characterized in that the brain neuroinflammatory disease is at least one disease selected from dementia, Parkinson's disease, depression and schizophrenia.

7. 5. The composition according to claim 3 or 4, which is for preventing or ameliorating Rett syndrome.

8. 5. The composition according to claim 3 or 4, which is a pharmaceutical composition.

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