Carbamoyl lipids with cyclic groups on the side chain, lipid nanoparticles thereof, and pharmaceutical compositions thereof

Abstract

The inventors have discovered carbamoyl lipids with cyclic groups on their side chains capable of forming lipid nanoparticles, and have elucidated that lipid nanoparticles composed of carbamoyl lipids with cyclic groups on their side chains can express proteins in astrocytes or hepatocytes. Furthermore, lipid nanoparticles composed of carbamoyl lipids with cyclic groups on their side chains encapsulating nucleic acids are expected to be a component of pharmaceutical compositions useful for the prevention and / or treatment of astrocyte-related diseases.

Description

Technical Field

[0001] The present invention provides cationic lipids (hereinafter also referred to as compounds or salts thereof) useful as constituent components of lipid nanoparticles, lipid nanoparticles of the cationic lipids, lipid nanoparticles containing nucleic acids (hereinafter also referred to as nucleic acid lipid nanoparticles), and pharmaceutical compositions containing nucleic acid lipid nanoparticles. Background Technology

[0002] Because nucleic acids such as DNA and mRNA are easily broken down in living organisms, lipid nanoparticles are used for these nucleic acids as pharmaceuticals. Lipid nanoparticles are used as carriers in drug delivery systems (DDS). Examples of applications of lipid nanoparticles include COVID-19 vaccines and cancer vaccines, which are either already on the market or undergoing clinical development. Various lipids are used as cationic lipids, which are one of the constituent components of these nucleic acid lipid nanoparticles.

[0003] It is believed that cationic lipids (also known as ionized lipids) form lipid nanoparticles and encapsulate anionic DNA, mRNA, etc. The most suitable cationic lipids are capable of encapsulating nucleic acids such as DNA and mRNA. Furthermore, it is believed that cationic lipids can deliver nucleic acids such as DNA and mRNA to cells and tissues, enabling the production and function of desired proteins (Patent Document 1).

[0004] As cationic lipids for use in lipid nanoparticles, lipids represented by the following general formula are described (Patent Document 1).

[0005]

[0006] (Please refer to Patent Document 1 for the symbols used in the formula.)

[0007] As cationic lipids for use in lipid nanoparticles, lipids represented by the following general formula are described (Patent Document 2).

[0008]

[0009] (Please refer to Patent Document 2 for the symbols used in the formula.)

[0010] There are technologies that induce the differentiation of somatic cells into target cells without relying on pluripotent stem cells such as iPS cells. If disease-related proteins can be produced in specific cells, tissues, or organs, they could potentially become effective therapeutic agents.

[0011] Astrocytes are among the most abundant cells in the central nervous system. Under normal conditions, they play an important role in the stability of the blood-brain barrier and the formation of synapses in nerve cells, and are crucial for supporting normal brain function (Frontiers in Cellular Neuroscience, 2022, vol.16, p.850866. Toxicologic Pathology, 2011, vol.39(1), p.115-123).

[0012] However, when the brain is impaired, astrocytes become activated and accumulate at the site of the impairment, forming glial proliferation. Glial proliferation is considered a factor that inhibits neurogenesis and has been reported in a wide range of central nervous system diseases, including neurodegenerative diseases such as Alzheimer's disease (Neuron, 2014, vol. 81, pp. 229-248).

[0013] Therefore, if an effective drug can be developed that can be taken up and act on by astrocytes, it may be possible to provide new treatments for a wide range of central nervous system diseases.

[0014] Existing technical documents

[0015] Patent documents

[0016] Patent Document 1: International Publication No. 2013 / 185116

[0017] Patent Document 2: International Publication No. 2023 / 091490 Summary of the Invention

[0018] The problem that the invention aims to solve

[0019] The main objective of this invention is to provide novel cationic lipids that can be constituent components of lipid nanoparticles, and to create lipid nanoparticles for the uptake of nucleic acids into cells and the expression of proteins. Furthermore, the objective of this invention is to provide lipid nanoparticles encapsulating nucleic acids (e.g., mRNA) useful for the prevention or treatment of various diseases, and pharmaceutical compositions containing the same.

[0020] In addition, the subject of this invention is to provide a pharmaceutical composition containing lipid nanoparticles containing nucleic acids (e.g., mRNA) that are useful for the prevention and / or treatment of astrocyte-related diseases.

[0021] Furthermore, the subject of this invention is to provide lipid nanoparticles containing nucleic acids (e.g., mRNA) capable of being delivered to astrocytes and / or hepatocytes, and pharmaceutical compositions containing the same.

[0022] Methods for solving problems

[0023] The inventors conducted in-depth research and discovered the compound of formula (I) of this invention or its salt, and found that using it as a cationic lipid can produce novel and useful lipid nanoparticles. Furthermore, by studying the proportions of the constituent components of the lipid nanoparticles (cationic lipids, neutral lipids, and PEGylated lipids, etc.), suitable compositional ratios were discovered. This invention can provide such cationic lipids, lipid nanoparticles, nucleic acid lipid nanoparticles, and pharmaceutical compositions containing them. This invention can provide lipid nanoparticles containing encapsulated nucleic acids (e.g., mRNA) capable of delivery to astrocytes and / or hepatocytes, and pharmaceutical compositions containing them.

[0024] That is, the present invention relates to the following (1) to (16).

[0025] (This invention)

[0026] (1) The compound of formula (I) or its salt.

[0027]

[0028] (in the formula, L 1 and L 2 Whether they are the same or different, they are -CH2-, -CH2CH2- or bonds. L 3 For key or C 1-10 Alkylene M is -CH2- or does not exist. n is 1 or 2, When M is -CH2-, n is 1. E 1 and E 2 Same or different, -C(=O)O- -OC(=O)- -OC(=O)O- -C(=O)- OR key, This indicates that at this location, R 1 or R 2 bonding, Among them, E 1 and E 2 Either of them is -C(=O)O- or -OC(=O)- , R 1 and R 2 Same or different, for -CH(-R) x )R y -CH2CH(-R)x )R y -CH2CH2CH(-R) x )R y -CH2CH(-OR) x OR y -CH2CH2CH(-OR) x OR y -CH2-(C 5-15 alkyl), -CH2-(C 5-20 alkenyl), -N(-R) x )R y -NR y (-C(=O)R x ) or -NR y C(=O)CH(-R x )R Z , Among them, R 1 and R 2 Any one of them is -N(-R) x )R y At that time, -E 1 -R 1 and -E 2 -R 2 Either of them is -OC (=O) -N (-R) x )R y or -C(=O)-N(-R) x )R y , R 1 and R 2 Either of them is -NR y (-C(=O)R x ) or -NR y C(=O)CH(-R x )R Z At that time, it should be with R 1 bonded E 1 Or with R 2 bonded E 2 As key, and, E 1 and E 2 All are -OC (=O)- At that time, R 1 and R 2 Same or different, -CH2CH2CH(-OR) x OR y or -CH2-(C 5-20 alkenyl), R x Ry and R Z Same or different, C 5-15 alkyl, R 3 To select groups from the group consisting of free formulas (a) to (i),

[0029] R a C 1-6 alkyl, R b -CH2-C 1-6 Alkyl or -C(=O)CH2N(CH3)2, R c and R d The same or different, is -CH3, -CH2CH3 or -CH2CH2OH, or, R c When it is H, R d It is -CH2C(=O)NH2, L cd It can be -CH2-, -CH2CH2-, -CH2CH2CH2-, or -CH2CH(CH3)-. R e It is H or OH. R f For H, R g C 1-6 alkyl, Among them, R f and R g They can form pyrrolidine rings together with the carbon and nitrogen atoms they are bonded to. R h C 1-6 alkyl, R i C 1-6 Alkyl or -CH2CH2OH, R j and R k Same or different, C 1-6 alkyl, R l and R m Same or different, C 1-6 alkyl, (s and t may be the same or different, and the value is 1 or 2.) (1a-1-1) According to the compound or salt thereof described in (1), wherein R 3 It can be any one of equations (a) to (d) and (f) to (i).

[0030] (1a-1-2) According to the compound or salt thereof described in (1), wherein R 3 Let it be equation (e).

[0031] (1a-2) The compound or its salt according to (1) to (1a-1-2), wherein, -E 1 -R 1 -C(=O)O-CH2CH(-R) x )R y -C(=O)O-CH2CH2CH(-R) x )R y -OC(=O)-C3H6-(C 1-6 (alkylene)-CH=CH-CH=CH-(C 1-6 Alkyl), -OC(=O)-N(-R) x )R y -OC(=O)O-CH(-R) x )R y -OC(=O)O-CH2CH(-R) x )R y -NR y (-C(=O)R x ) or -NR y C(=O)CH(-R x )R Z , -E 2 -R 2 -C(=O)O-CH2CH(-R) x )R y -C(=O)O-CH2CH2CH(-R) x )R y -C(=O)O-C3H6-(C 1-6 (alkylene)-CH=CH-CH=CH-(C 1-6 Alkyl group), -OC(=O)-CH2CH(-R) x )R y -OC(=O)-CH2CH2CH(-R) x )R y -OC(=O)-CH2CH2CH(-OR) x OR y -OC(=O)-C3H6-(C 1-6 (alkylene)-CH=CH-CH=CH-(C 1-6 Alkyl) or -OC(=O)O-CH2CH(-R x )R y .

[0032] (1a-2-1) According to the compound or salt thereof described in (1a-2), wherein R 3 It can be any one of equations (a) to (d) and (f) to (i).

[0033] (1a-2-2) According to the compound or its salt as described in (1a-2), wherein R 3 Let it be equation (e).

[0034] (1a-3) A compound of formula (I) or a salt thereof according to (1) to (1a-2-2), wherein, E 1 and E 2 Same or different, -C(=O)O- -OC(=O)- or-OC(=O)O- , This indicates that at this location, R 1 or R 2 bonding, Among them, E 1 and E 2 Either of them is -C(=O)O- , R 1 and R 2 Same or different, -CH2CH(-R) x )R y -CH2CH(-OR) x OR y -CH2CH2CH(-OR) x OR y -CH2-(C 5-15 alkyl) or -CH2-(C 5-20 alkenyl), R x and R y Same or different, C 5-15 alkyl, R 3 To select groups from the group consisting of free formulas (a) to (h),

[0035] R c and R d All are -CH3, -CH2CH3, or -CH2CH2OH, or, R c When it is H, R d It is -CH2C(=O)NH2, L cdIt is -CH2- or -CH2CH2-. Among them, R c R d When all are -CH3, -CH2CH3 or -CH2CH2OH, L cd They are -CH2-, -CH2CH2-, or -CH2CH2-, respectively.

[0036] (1a-3-1) The compound or its salt according to (1a-3), wherein R 3 For example, (a)~(d) or (f)~(h).

[0037] (1a-3-2) The compound or its salt according to (1a-3), wherein R 3 Let it be equation (e).

[0038] (1a-4) The compound or its salt according to (1a-3) to (1a-3-2), wherein, -E 1 -R 1 -C(=O)O-CH2CH(-R) x )R y -OC(=O)-C3H6-(C 1-6 (alkylene)-CH=CH-CH=CH-(C 1-6 Alkyl) or -OC(=O)O-CH2CH(-R x )R y , -E 2 -R 2 -C(=O)O-CH2CH(-R) x )R y -C(=O)O-C3H6-(C 1-6 (alkylene)-CH=CH-CH=CH-(C 1-6 Alkyl) or -OC(=O)-C3H6-(C 1-6 (alkylene)-CH=CH-CH=CH-(C 1-6 alkyl).

[0039] (1a-4-1) The compound or its salt according to (1a-4), wherein R 3 For example, (a)~(d) or (f)~(h).

[0040] (1a-4-2) The compound or its salt according to (1a-4), wherein R 3 Let it be equation (e).

[0041] (1a-5) The compound or its salt according to (1a-2), wherein, L1 As key, L 2 For -CH2- or bond, L 3 C 1-6 Alkylene -E 1 -R 1 -C(=O)O-CH2CH(-R) x )R y -C(=O)O-CH2CH2CH(-R x )R y -OC(=O)O-CH(-R) x )R y or -OC(=O)O-CH2CH(-R) x )R y , -E 2 -R 2 -C(=O)O-CH2CH(-R) x )R y -C(=O)O-CH2CH2CH(-R) x )R y -OC(=O)-CH2CH2CH(-R) x )R y Or -OC(=O)-CH2CH2CH(-OR) x OR y , R x and R y Same or different, C 5-15 alkyl, R 3 The groups selected are those from the group consisting of free formulas (d), (e), (g), or (h).

[0042] R c and R d Whether they are the same or different, they are -CH3, -CH2CH3, or -CH2CH2OH. L cd It can be -CH2-, -CH2CH2-, or -CH2CH2CH2-. R e For H, R f For H, R g C 1-6 alkyl, R i C 1-6 alkyl, R j and R k Same or different, C 1-6 alkyl, t is 1.

[0043] (1a-5-1) The compound or its salt according to (1a-5), wherein R 3 For example, (d), (h), or (g).

[0044] (1a-5-2) The compound or its salt according to (1a-5), wherein R 3 Let it be equation (e).

[0045] (1a-6) The compound or its salt according to (1a-5) to (1a-5-2), wherein, L 3 It is a C6 alkylene group. -E 1 -R 1 -C(=O)O-CH2CH(-R) x )R y or -OC(=O)O-CH2CH(-R) x )R y , -E 2 -R 2 -C(=O)O-CH2CH(-R) x )R y , R c and R d Whether they are the same or different, they are -CH3, -CH2CH3, or -CH2CH2OH. L cd It can be -CH2-, -CH2CH2-, or -CH2CH2CH2-. Among them, R c R d When all are -CH3, -CH2CH3 or -CH2CH2OH, L cd They are -CH2-, -CH2CH2-, or -CH2CH2-, respectively.

[0046] (1b-1) The compound or its salt according to (1), wherein -E 1 -R 1 or -E 2 -R 2 Either of them is -C(=O)O-CH2CH(-R) x )R y The other is -C(=O)O-CH2CH(-R) x )Ry -OC(=O)O-CH2CH(-R) x )R y -OC(=O)-CH2CH(-OR) x OR y -OC(=O)-CH2CH2CH(-OR) x OR y or -OC(=O)-CH2-(C 5-15 (alkenyl).

[0047] (1b-2) The compound or its salt according to (1), wherein R 3 The groups selected are those composed of free formulas (d), (e), (g), and (h).

[0048]

[0049] (1b-3) The compound or its salt according to (1b-2), wherein R c and R d Both are -CH2CH3, L cd For -CH2CH2-, R e For H, R f For H, R g C 1-6 Alkyl, R i =-CH3, t=1, R j and R k It is -CH2CH3.

[0050] (1b-4) The compound or its salt according to (1b-3), wherein R 3 For formula (e). According to the compound or its salt as described in (1b-3), wherein R 3 For example, (d) or (e).

[0051] (1b-5) The compound or its salt according to (1b-4), wherein R g It is -CH3.

[0052] (1b-6) The compound or its salt according to (1b-3), wherein R 3 Let it be the formula (h).

[0053] (2) The compound or a salt thereof according to (1), wherein the compound is selected from the group consisting of: 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester; 2-Nonylundecyl octanoate of 8-[(1-methyl-L-prolyl)(3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}bicyclo[1.1.1]pentan-1-yl)amino]octanoate; 8-{(N,N-diethyl-β-alanyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester; 8-{(1,4-diethyl-1,4-diazacycloheptane-6-carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester; 8-{[(4-methylpiperazin-1-yl)acetyl][(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester; and (1S,4r)-4-[{8-[(2-heptylnonyl)oxy]-8-oxooctyl}(1-methyl-L-prolyl)amino]cyclohexane-1-carboxylic acid 2-heptylnonyl ester.

[0054] (2-1) A compound or a salt thereof, wherein the compound is: 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester; 2-Nonylundecyl octanoate of 8-[(1-methyl-L-prolyl)(3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}bicyclo[1.1.1]pentan-1-yl)amino]octanoate; 8-{(1-ethyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester; 8-{(N,N-diethyl-β-alanyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester; 8-{(1,4-diethyl-1,4-diazacycloheptane-6-carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester; 8-{[(4-methylpiperazin-1-yl)acetyl][(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester; 8-[(1-methyl-L-prolyl){(1r,3S)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]octanoic acid 2-nonylundecyl ester; (1S,4r)-4-[{8-[(2-heptaylnonyl)oxy]-8-oxooctyl}(1-methyl-L-prolyl)amino]cyclohexane-1-carboxylic acid 2-heptaylnonyl ester; 8-{(1-ethyl-D-prolyl)[(1r,3R)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester; 8-{[(1r,3S)-3-{2-[(3-decyltridecyl)oxy]-2-oxoethyl}cyclobutyl](1-methyl-L-prolyl)amino}octanoic acid 3-decyltridecyl ester; 4,4-Bis(octyloxy)butyric acid 2-{(1-ethyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}ethyl ester; or 4-Octylododecanoic acid 4-[(1-ethyl-L-prolyl){(1r,3S)-3-[({[(2-nonylundecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]butyl ester.

[0055] (2-2) A compound or a salt thereof, wherein the compound is: 8-{(1-ethyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester; or 8-{(N,N-diethyl-β-alanyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonyl undecyl ester.

[0056] (3) A lipid nanoparticle containing the compound described in (1) or a salt thereof.

[0057] (4) A lipid nanoparticle containing the compound described in (1) or a salt thereof, neutral lipids and PEGylated lipids.

[0058] (5) The lipid nanoparticles according to (4) contain nucleic acids.

[0059] (6) The lipid nanoparticles according to (5), wherein the nucleic acid is mRNA.

[0060] (7) The lipid nanoparticles according to any one of (4) to (6), wherein, Neutral lipids are phospholipids and sterols. Phospholipids include DPPC, DSPC, SOPC, DoPhPE, DOPS, or DHSM. Steroids include cholesterol, 7α-hydroxycholesterol, or β-sitosterol. The PEGylated lipids are DMG-PEG2000, PEG monostearate, or C8 PEG2000 ceramide.

[0061] (8) The lipid nanoparticles according to any one of (5) to (7), wherein the lipid nanoparticles are capable of expressing proteins in astrocytes.

[0062] (9) The lipid nanoparticles according to any one of (5) to (8), wherein the nucleic acid is an mRNA useful for the prevention and / or treatment of astrocyte-related diseases.

[0063] (10) The lipid nanoparticles according to any one of (5) to (9), wherein, The nucleic acid is mRNA encoding the NeuroD1 protein. The compound of formula (I) or its salt is 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonyl undecyl ester or its salt, Neutral lipids are DSPC and cholesterol. The PEGylated lipid was DMG-PEG2000.

[0064] (11) The lipid nanoparticles according to any one of (5) to (10), wherein the nucleic acid is mRNA encoding the NeuroD1 protein containing a base sequence encoding a protein consisting of the amino acid sequence shown in Serial No. 2.

[0065] (12) The lipid nanoparticles according to any one of (3) to (11), wherein, based on the total amount of lipid nanoparticles, the compound described in (1) or its salt is contained in a composition ratio of 20.0 to 80.0 mol%, neutral lipids are contained in a composition ratio of 18.5 to 78.5 mol%, and PEGylated lipids are contained in a composition ratio of 0.5 to 2.5 mol%.

[0066] (13) The lipid nanoparticles according to any one of (3) to (11), wherein, based on the total amount of lipid nanoparticles, the compound described in (1) or its salt is contained in a composition ratio of 30.0 to 60.0 mol%, neutral lipids are contained in a composition ratio of 38.5 to 68.5 mol%, and PEGylated lipids are contained in a composition ratio of 0.5 to 2.0 mol%.

[0067] (14) A pharmaceutical composition comprising any one of (5) to (13) lipid nanoparticles.

[0068] (15) A pharmaceutical composition comprising any one of (5) to (13) lipid nanoparticles and one or more pharmaceutically acceptable pharmaceutical additives.

[0069] (16) The pharmaceutical composition according to (15) is a pharmaceutical composition for the prevention and / or treatment of astrocyte-related diseases.

[0070] In addition, the present invention relates to a pharmaceutical composition for the prevention and / or treatment of astrocyte-related diseases, comprising lipid nanoparticles comprising any one of (5) to (13) of a compound of formula (I) or a salt thereof.

[0071] It should be noted that the pharmaceutical composition includes a preventive and / or therapeutic agent for astrocyte-related diseases, which contains lipid nanoparticles comprising any one of the compounds of formula (I) or salts thereof, in formulas (5) to (13).

[0072] In addition, the present invention also relates to: The use of lipid nanoparticles comprising any one of the compounds of formula (I) or salts thereof (5) to (13) in the manufacture of pharmaceutical compositions for the prevention and / or treatment of astrocyte-related diseases; Lipid nanoparticles comprising any one of (5) to (13) of a compound of formula (I) or a salt thereof for use in the prevention and / or treatment of astrocyte-related diseases; The use of lipid nanoparticles comprising any one of formula (I) or a salt thereof (5) to (13) in the prevention and / or treatment of astrocyte-related diseases; and A method for the prevention and / or treatment of astrocyte-related diseases, including the step of administering an effective amount of any one of the compounds of formula (I) or salts thereof (5) to a subject.

[0073] In addition, the present invention relates to a method for delivering nucleic acids (e.g., nucleic acids useful for the prevention and / or treatment of astrocyte-related diseases) to cells in a living organism, particularly astrocytes, by using nucleic acid lipid nanoparticles comprising a compound of formula (I) or its salts thereof.

[0074] This invention relates to lipid nanoparticles containing nucleic acids (e.g., mRNA) capable of being delivered to astrocytes or hepatocytes, and pharmaceutical compositions containing the same.

[0075] "Astrocyte-related diseases" are diseases associated with astrocytes, including cerebral infarction, cerebral hemorrhage, traumatic brain injury, neurodegenerative diseases, amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, Alexander's disease, multiple sclerosis, spinal cord injury, and neuromyelitis optica. In other words, these include cerebral hemorrhage, traumatic brain injury, neurodegenerative diseases, amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, Alexander's disease, multiple sclerosis, and neuromyelitis optica.

[0076] Here, regarding cerebral infarction, one definition is perforator infarction; another is cerebral infarction with brain damage in the perforator's innervation area; yet another is subacute cerebral infarction; yet another is cerebral infarction from subacute to chronic phase; yet another is chronic cerebral infarction; yet another is cerebral infarction presenting with severe motor dysfunction; yet another is cerebral infarction with a modified Rankin Scale (mRS) score of 2 or higher; yet another is cerebral infarction with an mRS score of 4 or higher; yet another is cerebral infarction combining these; and yet another is perforator infarction, cerebral infarction with brain damage in the perforator's innervation area, subacute to chronic phase cerebral infarction, and cerebral infarction presenting with severe motor dysfunction. Cerebral infarction can be divided into the following stages. The "acute phase" refers to the period within 72 hours of the onset of cerebral infarction, during which symptoms may worsen. The "subacute phase" refers to the period from 72 hours to less than one month after the onset of cerebral infarction. The "subacute to chronic phase" refers to the period after 72 hours of the onset of cerebral infarction. The "chronic phase" refers to the period after one month of the onset of cerebral infarction.

[0077] Here, regarding "spinal cord injury," it is categorized as follows: subacute to chronic phase spinal cord injury; chronic phase spinal cord injury; spinal cord injury with limb paralysis; spinal cord injury with upper arm motor dysfunction; spinal cord injury including cervical spinal cord injury; and spinal cord injury caused by compression. Spinal cord injury can be divided into the following stages: "Acute phase" refers to the period from the onset of the spinal cord injury to less than two weeks. "Subacute phase" (also known as the recovery phase) refers to the period from two weeks to less than four weeks after the spinal cord injury. "Chronic phase" refers to the period after four weeks. Regarding "subacute to chronic phase," it is defined as the period after two weeks of spinal cord injury; as the period after four weeks of spinal cord injury; as the period after three months of spinal cord injury; and as the period after six months of spinal cord injury.

[0078] This specification includes the disclosures of Japanese Patent Application No. 2023-221885 and Japanese Patent Application No. 2023-220683, filed on December 27, 2023, which form the basis of the priority claim of this application.

[0079] Invention Effects

[0080] Furthermore, the present invention is not limited to the above-described manner, but also includes a manner in which the contents described in the detailed invention specification are appropriately combined.

[0081] Lipids, as compounds of formula (I) or their salts, can be used as components of lipid nanoparticles to create lipid nanoparticles. The resulting lipid nanoparticles enable target cells, such as astrocytes, to take up nucleic acids and express proteins.

[0082] Lipid nanoparticles enable the uptake of nucleic acids useful for the prevention and / or treatment of astrocyte-related diseases into target cells and the expression of proteins. That is, pharmaceutical compositions useful for the prevention and / or treatment of astrocyte-related diseases can be provided. The pharmaceutical compositions of the present invention containing nucleic acid lipid nanoparticles can be used as preventive and / or therapeutic agents for astrocyte-related diseases. Attached Figure Description

[0083] Figure 1 This diagram shows the diurnal variation of motor dysfunction following endothelin-1 treatment (induced by cerebral ischemia) in the NeuroD1 (ND1) nucleic acid lipid nanoparticle group (ND1 mRNA) and the control group (Control mRNA) described in Example 4, evaluated using the modified Rankin Scale (mRS). The horizontal axis represents the number of days after endothelin-1 injection, and the vertical axis represents the mRS value (maximum value 6). White circles represent the mean mRS at each evaluation time point for the ND1 nucleic acid lipid nanoparticle group. It should be noted that mRS was evaluated every 2 weeks after day 28 post-endothelin-1 injection; however, the results on day 28 represent the mean of the results evaluated on day 28 or 29. Error bars represent the standard error (SEM). Black circles represent the mean mRS at each evaluation time point for the control group. It should be noted that the results on day 28 represent the mean of the results evaluated on day 28 or 29. Nucleic acid lipid nanoparticles were administered on day 21 post-endothelin-1 treatment. Detailed Implementation

[0084] The present invention will now be described in detail.

[0085] In this specification, unless otherwise specified, the following terms have the following meanings. The definitions below are intended to clarify the terms, not to limit them. When a term is not specifically defined herein, it is used in the sense that is commonly understood by those skilled in the art. Unless otherwise specified, the same symbol in one chemical formula in this specification has the same meaning when used in other chemical formulas.

[0086] "Alkyl" refers to a straight-chain or branched alkyl group. C 1-6 Alkyl groups refer to alkyl groups having 1 to 6 carbon atoms. (C) 5-10 Alkyl groups refer to alkyl groups with 5 to 10 carbon atoms. As a convention, alkyl groups are C14 and C24. 1-6 Alkyl group. In one manner, an alkyl group is C10. 5-10 Alkyl group. In one manner, an alkyl group is C10. 5-15 Alkyl group. In one manner, an alkyl group is C10. 6-10 Alkyl group. In one manner, an alkyl group is C10. 7-9 Alkyl group. In one manner, an alkyl group is C10. 7-8 Alkyl group. In one manner, an alkyl group is C10. 8-9 Alkyl. As a form, the alkyl group is a C7 alkyl group. As a form, the alkyl group is a C7 alkyl group. 6-9 Alkyl. As a form, the alkyl group is C6 alkyl. As a form, the alkyl group is C8 alkyl. As a form, the alkyl group is C9 alkyl. As a form, the alkyl group is n-hexyl. As a form, the alkyl group is n-heptyl. As a form, the alkyl group is n-octyl. As a form, the alkyl group is n-nonyl.

[0087] "Alkylene" refers to a straight-chain or branched alkylene group. C 5-10 Alkylene refers to an alkylene group having 5 to 10 carbon atoms. As a convention, an alkylene group is C1. 1-10 Alkylene. In one manner, alkylene is C10. 1-6 Alkylene. In one manner, alkylene is C10. 5-8 Alkylene. In one manner, alkylene is C10. 7-9 Alkylene. In one manner, alkylene is C10. 6-8 Alkylene. As one embodiment, the alkylene is a C6 alkylene. As one embodiment, the alkylene is hexanediyl, heptanediyl, octanediyl, or nonanediyl. As one embodiment, the alkylene is heptanediyl, octanediyl, or nonanediyl. As one embodiment, the alkylene is heptane-1,7-diyl, octane-1,8-diyl, or nonane-1,9-diyl. As one embodiment, the alkylene is n-hexane-1,6-diyl.

[0088] "Alkenyl" refers to a straight-chain or branched alkenyl group, C 5-20Alkenyl groups refer to straight-chain or branched alkenyl groups with 5 to 20 carbon atoms, such as vinyl, propenyl, butenyl, pentenyl, 1-methylvinyl, 1-methyl-2-propenyl, 1,3-butadienyl, 1,3-pentadienyl, etc. As a convention, the alkenyl group is -C. 5-10 Alkylene -CH=CH-CH=CH-C 5-10 Alkyl. In one form, it is a straight-chain alkenyl group. In another form, the alkenyl group is -C8 alkylene-(CH=CH-CH2)2-C4 alkyl, -C8 alkylene-(CH=CH-CH2)2-C6 alkyl, -C7 alkylene-CH=CH-C8 alkyl, -C7 alkylene-(CH=CH-CH2)2-C4 alkyl, -C7 alkylene-(CH=CH-CH2)3-C4 alkyl, -C3 alkylene-(CH=CH-CH2)4-C4 alkyl, -C3 alkylene-(CH=CH-CH2)5-C1 alkyl, or -C2 alkylene-(CH=CH-CH2)6-C1 alkyl. In one embodiment, the alkenyl group is -C8 alkylene-(CH=CH-CH2)2-C4 alkyl (wherein, the double bonds are all Z-bonded), -C8 alkylene-(CH=CH-CH2)2-C6 alkyl (wherein, the double bonds are all Z-bonded), -C7 alkylene-CH=CH-C8 alkyl (wherein, the double bonds are all Z-bonded), -C7 alkylene-(CH=CH-CH2)2-C4 alkyl (wherein, the double bonds are all Z-bonded), -C7 alkylene-(CH=CH-CH2)3-C4 alkyl (wherein, the double bonds are all Z-bonded), -C3 alkylene-(CH=CH-CH2)4-C4 alkyl (wherein, the double bonds are all Z-bonded), -C3 alkylene-(CH=CH-CH2)5-C1 alkyl (wherein, the double bonds are all Z-bonded), or -C2 alkylene-(CH=CH-CH2)6-C1 alkyl (wherein, the double bonds are all Z-bonded). As one embodiment, the alkenyl group is -C7 alkylene-(CH=CH-CH2)2-C4 alkyl (wherein, all double bonds are Z-form). As another embodiment, the alkenyl group is -C6 alkylene-(CH=CH-CH2)2-C4 alkyl (wherein, all double bonds are Z-form). As another embodiment, the alkenyl group is -C8 alkylene-(CH=CH-CH2)2-C4 alkyl (wherein, all double bonds are Z-form). As yet another embodiment, it is "-CH2-(C 5-20 "Alkenyl)" is -C3H6-(C 1-6 (alkylene)-CH=CH-CH2-CH=CH-(C 1-6 alkyl).

[0089] "Halogens" refer to F, Cl, Br, and I.

[0090] "Lipid nanoparticles" refer to nanoparticles whose main component is lipid. Typically, lipid nanoparticles contain cationic lipids, neutral lipids, and PEGylated lipids. As another type, they are nucleic acid lipid nanoparticles that also encapsulate nucleic acids. Additionally, "nucleic acid lipid nanoparticles" refer to lipid nanoparticles that encapsulate nucleic acids useful for the prevention and / or treatment of diseases; as another type, they are lipid nanoparticles that encapsulate nucleic acids useful for the prevention and / or treatment of astrocyte-related diseases; and as yet another type, they are lipid nanoparticles that encapsulate mRNA.

[0091] "Particle size" refers to the particle size of lipid nanoparticles. For dispersions in a static state, it is determined using a particle size analyzer (Zetasizer Nano ZSP or Ultra, Malvern Panalytical) employing Dynamic Light Scattering (DLS) to measure the diameter as hydrodynamic diameter (Dh). This is based on ISO 22412. The "particle size" of lipid nanoparticles is calculated as the Z-mean particle size. Regarding the particle size of lipid nanoparticles, one method is 10 nm to 1000 nm; another is 30 nm to 500 nm; another is 30 nm to 250 nm; another is 60 nm to 180 nm; another is 70 nm to 110 nm; another is 70 nm to 100 nm; another is 80 nm to 100 nm; and another is 90 nm to 100 nm.

[0092] "Cationic lipids" refer to compounds or salts thereof that are capable of carrying intramolecular cations (positive charges) in response to pH and have fatty acid chains. One example is a compound or salt thereof of formula (I).

[0093] "Neutral lipids" refers to "phospholipids" and "sterols". In one sense, neutral lipids are phospholipids and sterols; in another sense, neutral lipids are phospholipids; and in yet another sense, neutral lipids are sterols.

[0094] Phospholipids are lipids containing phosphate ester groups. Phospholipids are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), or sphingomyelin (SM), and combinations thereof.

[0095] Phosphatidylcholine (PC) includes 1,2-disorcinyl-sn-glycerol-3-phosphate choline (DEPC), 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-diundecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), or 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate choline (SOPC), etc.

[0096] Phosphatidylethanolamine (PE) includes 1,2-disorcinyl-sn-glycerol-3-phosphate ethanolamine (DEPE), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine (DMPE), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (DPPE), 1,2-di-O-phytanoyl-sn-glycerol-3-phosphate ethanolamine (DoPhPE; CAS150135-14-1), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine (DSPE), or 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine (POPE), etc.

[0097] Phosphatidylglycerol (PG) is available in sodium salts of various types, including 1,2-dimyristoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) (DMPG), 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) (DOPG), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) (DPPG), and 1,2-distearatel-sn-glycerol-3-phosphate-rac-(1-glycerol) (DSPG).

[0098] Phosphatidylserine (PS) is phosphatidylserine (PS) or 1,2-dioleoyl-sn-glycerol-3-phosphate-L-serine (DOPS), etc.

[0099] As a sphingomyelin (SM), it is sphingomyelin (SM) or dihydrosphingomyelin (DHSM), etc. As another method, the phospholipid is DPPC, DSPC, SOPC, DOPE, DoPhPE, DOPS, or DHSM. As yet another method, the phospholipid is DSPC. As a method where no phospholipid is used, it is DOTAP or DOTMA.

[0100] "Sterol" refers to one of the steroid alcohols, a compound with a hydroxyl group on ring A of the steroid skeleton. In one sense, sterols are cholesterol and corticosteroids. In another sense, sterols include cholesterol, 7α-hydroxycholesterol, campesterol, ergosterol, fecosterol, campesterol, sitosterol, β-sitosterol, stigmasterol, tomatine, tomatine, ursolic acid, or mixtures thereof. Additionally, it can be a combination of two or more. In one sense, sterols are cholesterol, 7α-hydroxycholesterol, campesterol, or β-sitosterol. In another sense, sterol is cholesterol.

[0101] "PEGylated lipids" refer to lipids containing polyethylene glycol (PEG). PEGylated lipids are lipids modified with polyethylene glycol. PEGylated lipids include PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, PEG-modified phosphatidic acids, PEG-modified phosphatidylethanolamines, and mixtures thereof. As an example, PEGylated lipids include 1,2-dilauroyl-sn-glycerol-3-phosphate ethanolamine-PEG (DLPE-PEG), 1,2-dimyristoyl-rac-glycerol-3-methoxy-PEG (DMG-PEG2000, also known as DMG-PEG), 1,2-dimyristoyl-sn-glycerol-3-phosphate ethanolamine-PEG (DMPE-PEG), and 1,2-dipalmitoyl-rac-glycerol-3-methoxy- PEG (DPG-PEG), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine-PEG (DPPC-PEG), 1,2-distearyl-rac-glycerol-3-PEG (DSG-PEG), 1,2-distearyl-sn-glycerol-3-phosphoethanolamine-PEG (DSPE-PEG), PEG monostearate or N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]} (C8 PEG2000 ceramide).

[0102] As one method, the PEGylated lipid is DMG-PEG2000, PEG monostearate, or N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]} (C8 PEG2000 ceramide), and as another method, the PEGylated lipid is DMG-PEG2000.

[0103] "Inclusion rate" refers to the amount of nucleic acid incorporated into the lipid nanoparticles relative to the total amount of nucleic acid present in the lipid nanoparticle dispersion. For example, if 98 mg of nucleic acid out of a total of 100 mg of nucleic acid is incorporated into the lipid nanoparticles, the inclusion rate can be expressed as 98%. In this specification, "inclusion" means completely or substantially contained within, or contained in. For nucleic acid lipid nanoparticles such as mRNA, the inclusion rate of the nucleic acid can be determined by the methods described in detail below. Values ​​for the inclusion rate of the nucleic acid include, for example, 70% or more, 80% or more, 90% or more, 93% or more, and 95% or more.

[0104] The "N / P ratio" refers to the value obtained by dividing the molar number of amino groups (N) of the cationic lipids in nucleic acid lipid nanoparticles by the molar number of phosphate groups (P) of the RNA. In this specification, for amino groups, the N / P ratio is converted to nitrogen atoms with a calculated ACD / pKa GALAS value greater than 6 using ACD / Percepta (ACD / LabsRelease 2019.2.2 or 2023.1.1, registered trademark, Advanced Chemistry Development, Inc.). One method is an N / P ratio of 1 to 12; another method is an N / P ratio of 6 to 12; yet another method is an N / P ratio of 6 or 12; and a third method is an N / P ratio of 6.

[0105] "Nucleic acid" refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Examples of nucleic acids include messenger RNA (mRNA), microRNA (miRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), ribozymes, and antisense oligonucleotides. As a general term, mRNA can be listed. As a general term, siRNA can be listed. As a general term, nucleic acids can include Ascl1 mRNA (mRNA encoding the Ascl1 protein is also called Ascl mRNA, and hereinafter, mRNA encoding xxx protein will also be called xxx mRNA), Dlx2 mRNA, NeuroD1 mRNA (mRNA encoding the NeuroD1 protein), Ngn2 mRNA, BDNF mRNA, NGF mRNA, or NT-3 mRNA, etc.

[0106] As one method, the nucleic acid is NeuroD1 mRNA. As one method, the nucleic acid is Ascl1 mRNA. As one method, the nucleic acid is NeuroD1 mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in Serial Number 2. As one method, the nucleic acid is NeuroD1 mRNA containing the base sequence shown in Serial Number 1. As one method, the nucleic acid is NeuroD1 mRNA containing the base sequence shown in Serial Number 3. As one method, the nucleic acid is NeuroD1 mRNA containing the base sequence shown in Serial Number 4. As one method, the nucleic acid is NeuroD1 mRNA containing the base sequence shown in Serial Number 5. As one method, the nucleic acid is mRNA encoding a NeuroD1 protein containing a sequence for capping. As one method, the nucleic acid is mRNA encoding a NeuroD1 protein with a 5' end sequence suitable for capping using CleanCap (registered trademark) or Reagent AG (TriLink BioTechnologies). As one method, the nucleic acid is mRNA encoding a NeuroD1 protein with a 5' end sequence of AGG. In one manner, the nucleic acid is an mRNA encoding the NeuroD1 protein with a 5' terminal sequence (positions 1 to 3 of sequence number 4). In another manner, the mRNA may include BDNF mRNA. In one manner, the lipid nanoparticles contain two or more nucleic acids; in another manner, the lipid nanoparticles contain only one nucleic acid. Based on the total weight of the lipid nanoparticles, the lipid nanoparticles containing nucleic acids contain, in one manner, 0.001–60% by weight of nucleic acid; in another manner, 0.1–40% by weight of nucleic acid; in another manner, 1.0–25% by weight of nucleic acid; and in another manner, 3.0–10% by weight of nucleic acid. The mRNA can be a natural nucleic acid or an artificial nucleic acid (e.g., a nucleic acid containing natural nucleic acid bases and / or artificial nucleic acid bases).

[0107] In this specification, "mRNA encoding xxx protein" refers to RNA containing a polynucleotide sequence encoding xxx protein and capable of being translated into xxx protein. In this specification, mRNA encoding xxx protein is also referred to as xxx mRNA. That is, it includes polynucleotides encoding xxx protein in an expressible state. As an example, the mRNA encoding xxx protein in this invention is a polynucleotide encoding xxx protein containing a functional sequence for expressing xxx protein (including, for example, CDS, 5'UTR, 3'UTR, 5' cap structure, and poly-A sequence, but not limited thereto).

[0108] In this specification, the representative DNA sequence of the polynucleotide is sometimes described as a sequence of bases containing "T". However, for example, in RNA (e.g., mRNA) containing a base sequence specified by a specific sequence number, where the specific sequence number represents the DNA sequence, the base sequence can be understood as an RNA sequence formed by replacing each "T" in the DNA sequence with "U". Unless otherwise specified in the sequence listing, the bases "adenine (A)", "thymine (T)", "guanine (G)", "cytosine (C)" and "uracil (U)" constituting the base sequence (e.g., the base sequences shown in sequence numbers 1 and 3), and the nucleosides and nucleotides containing them, can be either native or modified, and / or may have other modifications (methylation, etc.).

[0109] “mRNA” refers to messenger ribonucleic acid, which can be translated into the desired protein. Its structure contains a 5' cap, a 5' untranslated region (hereinafter also referred to as UTR), a coding region (CDS), a 3' UTR, and a polyA chain.

[0110] The "5' cap" refers to the modification structure visible at the 5' end, which is related to the stability of mature mRNA and translation initiation. It involves the modification of 7-methylguanosine (also known as m...) via triphosphates (also called ppp). 7 G) The structure where mRNA is bonded at 5' to 5' is called Cap-0. A structure where the 2' position of the ribose sugar at the first position of the mRNA nucleoside is further methylated based on the Cap-0 structure is called Cap-1; a structure where the 2' positions of the ribose sugars at the first and second positions of the nucleoside are further methylated is called Cap-2. Cap-0, Cap-1, and Cap-2 are sometimes referred to as mRNA nucleoside ... 7 GpppNp, m 7 GpppN1mp, m 7 GpppN1mpN2mp (N1 and N2 represent nucleosides, respectively. Where m represents 2'-O methyl) (Nature Reviews Molecular Cell Biology 2014, vol.15(5), p.313-326). As a mode, it is Cap-0, Cap-1, or Cap-2. As a mode, it is Cap-0. As a mode, it is Cap-1. As a mode, it is Cap-2.

[0111] "UTR" refers to untranslated regions, which contain both 5' and 3' UTRs. The 5' and 3' UTRs can originate from the gene to be expressed or from a different gene. A UTR can be naturally occurring or modified by inserting, deleting, substituting, and / or adding one or more nucleotides (e.g., 2, 3, 4, 5, or 6) to the naturally occurring UTR. In this specification, "UTR derived from…gene" includes not only naturally occurring UTRs but also such modified UTRs. A UTR can be formed by connecting multiple UTRs directly or through spacer sequences. The start codon and 5' UTR may contain a portion of the Kozak sequence (e.g., a 5' portion of the Kozak sequence beyond the start codon). Examples include: UTRs derived from globin genes; UTRs derived from α-globin genes; UTRs derived from human α-globin genes; UTRs derived from β-globin genes; and UTRs derived from human β-globin genes. In one manner, the 5'UTR and 3'UTR are derived from the 5'UTR and 3'UTR of the human α-globin gene.

[0112] "CDS" refers to the coding sequence, which is the DNA sequence region that is translated into a protein. Its codons can be optimized. The stop codon at the 3' end of the CDS can be any of the stop codons TAA, TGA, or TAG. Alternatively, two or more stop codons can be used consecutively (e.g., TAATGATAG).

[0113] "Modified nucleotides" refer to nucleotides that have undergone modifications. Examples include nucleotides modified by methylation, atomic exchange, double bond saturation, deamination, or the replacement of oxygen atoms with sulfur atoms. As a type, nucleotides with modified nucleic acid bases are also considered. As a type, nucleotides with modified ribose are also considered. As a type, nucleotides with modified phosphate groups are also considered. As a type, nucleotides containing 1-methyladenosine, pseudouridine, N1-methylpseuuridine (also known as 1-methylpseuuridine), dihydrouridine, 5-methoxycytidine, 5-methylcytidine, 7-methylguanosine, N6-methyladenosine, inosine, or thiouridine are also considered. As a type, nucleotides containing 1-methyladenosine are also considered. As a type, nucleotides containing modified uridine are also considered. As a type, nucleotides containing pseudouridine or N1-methylpseuuridine are also considered. As a type, nucleotides containing pseudouridine are also considered. As a type, nucleotides containing N1-methylpseuuridine are also considered. As a type, nucleotides containing dihydrouridine are also considered. One approach is to use a nucleotide containing inosine. Another approach is to use a nucleotide containing 4-thiouridine. It should be noted that, in this specification, the description of a modified nucleotide as "part or all" may also include cases where a portion of the specified modified nucleotide in the entire sequence is not substituted. The percentage of residues in the entire sequence that are substituted with a specific nucleotide for a modified nucleotide can be 1–100%, 1–90%, 1–80%, 1–70%, 1–60%, 1–50%, 1–40%, 1–30%, 1–20%, 1–10%, 1–5%, 75–99%, 50–75%, 25–50%, 10–25%, but is not limited to these. In one manner, the "part" of the proportion of the number of residues replaced with the modified nucleotide relative to the total number of residues can be, for example, 60–99%, 70–99%, 80–99%, 90–99%, 95–99%, 60–99.9%, 70–99.9%, 80–99.9%, 90–99.9%, or 95–99.9%, but is not limited thereto.

[0114] "Poly-A chain" refers to a polyadenylated nucleotide chain that functions in stabilizing mRNA, extranuclear transport, and translation. It is an mRNA region containing multiple (not limited below, typically more than 10) consecutive adenosine monophosphates, located downstream of the 3' UTR, for example, immediately downstream of it (i.e., on the 3' side). It is also called a poly-A sequence. As one possibility, the poly-A chain length is 20–1000 bases. As another possibility, the poly-A chain length is 30–500 bases, 50–200 bases, 60–150 bases, 70–130 bases, 70–120 bases, 70–80 bases, 120 bases, or 79 bases. As one possibility, the poly-A chain length is 120 bases. As another possibility, the poly-A chain length is 79 bases. As one method, the polyA chain length is 30–500 bases (as one method, 40–200, 50–200, 50–150, 50–100, 50–90, 60–150, 60–100, 60–90, 70–130, 70–120, 70–100, 70–90, 70–85, 70–80, 75–130, 75–120, 75–100, 75–90 bases; as another method, 74–84, 75–85, 75–83, or 76–82 bases). As one method, the polyA chain length is 120 bases. As one method, the polyA chain length is 79 bases. As one method, the polyA chain length is 74–84 bases. In one approach, the length of the poly-A chain is 75–85 bases.

[0115] In this specification, the term "expression" of nucleic acids, i.e., nucleic acid sequences, refers to the translation of mRNA into polypeptides, the construction of proteins from polypeptides, and post-translational modifications of polypeptides / proteins. Furthermore, in this specification, the terms "expression" and "production" are used interchangeably for the expression of proteins.

[0116] "NeuroD1" refers to Neuronal differentiation 1, a basic helix-loop-helix (bHLH) transcription factor belonging to the NeuroD family. NeuroD1 protein activates the transcription of genes containing specific DNA sequences known as E-boxes (also known as transcription factor activity). NeuroD1 plays a central role in inducing differentiation from neural stem cells into neural cells. Glial cells can be classified into four cell types: astrocytes, oligodendrocytes, ependymal cells, and microglia. It is known that ectopic expression of NeuroD1 in glial cells such as NG2 cells and in astrocytes can convert glial cells into neurons (International Publication No. 2014 / 015261). In one embodiment, NeuroD1 is a protein with transcription factor activity consisting of an amino acid sequence that has more than 90% (e.g., more than 95%, more than 98%, more than 99%, more than 99.4%, or more than 99.5%) sequence identity relative to the amino acid sequence shown in Serial No. 2. In one manner, NeuroD1 is a protein having transcription factor activity and consisting of an amino acid sequence comprising 1 to 50 (e.g., 1 to 2, 1 to 3, 1 to 5, 1 to 7, 1 to 10, or 1 to 30) inserted, deleted, substituted, and / or added amino acids in the amino acid sequence shown in Serial Number 2. In another manner, NeuroD1 is a protein consisting of the amino acid sequence shown in Serial Number 2.

[0117] "Transcription factor activity" refers to the ability of a protein to activate the transcription of its associated gene (i.e., transcriptional activation capacity). Specifically, regarding whether a protein possesses transcription factor activity, for example, for recombinant vectors, it can be determined by comparing it with cells that have not been introduced with the vector and by whether the expression level of the associated gene increases or decreases. It should be noted that recombinant vectors can be created by integrating a polynucleotide encoding a protein into a vector with any promoter. The created recombinant vector can then be introduced into cells to induce protein expression. For example, if the expression of the associated gene increases in cells introduced with a vector containing a polynucleotide encoding NeuroD1, it can be determined that the NeuroD1 protein possesses transcription factor activity.

[0118] "Sequence identity" refers to the percentage (%) of identical residues between a baseline biological sequence (base sequence or amino acid sequence, etc.) and a target biological sequence (usually the percentage of identical residues relative to the full length of the target sequence). Sequence identity can be calculated, for example, using the EMBOSS Needle (Nucleic Acids Res., 2015; Vol.43:pW580-W584) with default parameters, as an identity value. These parameters are as follows (Gap Open Penalty = 10, Gap Extend Penalty = 0.5, Matrix = EBLOSUM62, End Gap Penalty = false).

[0119] "Perforator infarction" refers to a disease in which infarction occurs in the thalamus, caudate nucleus, putamen, globus pallidus, and / or internal capsule innervated by a perforator, resulting in functional impairment of the infarcted area. A "perforator" is a small artery branching from the middle cerebral artery, which forms the main artery of the brain, and innervates the basal ganglia / thalamus and internal capsule. It should be noted that "innervation" of a brain region by a blood vessel means that blood is supplied to that brain region through that vessel; the brain region is located within the vascular territory of that vessel.

[0120] "Perforator innervation area" refers to the area innervated by perforators, namely the basal ganglia / thalamus region and internal capsule.

[0121] "Cerebral infarction with brain damage in the perforator innervation area" refers to cerebral infarction where the brain damage caused by the infarction primarily occurs in the perforator innervation area. Cerebral infarction with brain damage in the perforator innervation area includes not only cerebral infarctions that cause brain damage in the perforator innervation area due to infarction occurring within it, but also cerebral infarctions that secondarily cause brain damage in the perforator innervation area, such as cases where cell necrosis caused by infarction occurring around the perforator innervation area extends to the perforator innervation area, resulting in functional impairment in that area. In this specification, brain damage refers to functional damage to the brain caused by cell necrosis resulting from infarction. Brain damage typically includes cell necrosis in the area with the brain damage. "Cerebral infarction with brain damage in the perforator innervation area" can be cerebral infarction with cell necrosis in the perforator innervation area.

[0122] The following illustrates one method of using a compound of formula (I) as a compound of the present invention, or a salt thereof, lipid nanoparticles containing the same, and a pharmaceutical composition. It should be noted that all methods can be freely combined, as long as any two or more combinations are not contradictory. Even if not specifically described, one or more methods may be combined with a particular method.

[0123] 1. Cationic lipids

[0124] The following illustrates one manner of a compound of formula (I) or a salt thereof as a compound of the present invention. The following describes the manner of describing the compound. "Compound or salt thereof" will be referred to as "compound".

[0125] (The present invention relates to cationic lipids)

[0126] (1)L 1 Compounds of formula (I) that are -CH2-. L 1 Compounds of formula (I) with bonds.

[0127] (2)L 2 Compounds of formula (I) that are -CH2-. L 2 Compounds of formula (I) with bonds. L 2 Compounds of formula (I) with -CH2- or a bond.

[0128] (3)L 3 C 6-9 Alkylene compounds of formula (I). L 3 Compounds of formula (I) that are C6 alkylene groups.

[0129] (4) Compounds of formula (I) where M is absent and n is 1 or 2. Compounds of formula (I) where M is absent and n is 1. Compounds of formula (I) where M is absent and n is 2. Compounds of formula (I) where M is -CH2- and n is 1. Compounds of formula (I) where M is -CH2- or absent and n is 1.

[0130] (5)E 1 -C(=O)O- or -OC(=O)- , This indicates that at this location, R 1 A bonded compound of formula (I). E 1 -OC(=O)- or-OC(=O)O- , This indicates that at this location, R 1 A bonded compound of formula (I). E 1 is -OC(=O)O- or -C(=O)O- , This indicates that at this location, R 1 A bonded compound of formula (I). E 1 -C(=O)O- , This indicates that at this location, R1 A bonded compound of formula (I). E 1 -OC(=O)- , This indicates that at this location, R 1 A bonded compound of formula (I). E 1 is -OC(=O)O- , This indicates that at this location, R 1 A bonded compound of formula (I). E 1 and E 2 Same or different and of type -C(=O)O- -OC(=O)- or-OC(=O)O- , This indicates that at this location, R 1 or R 2 Bonding, where E 1 and E 2 Either of them is -C(=O)O- Compounds of formula (I).

[0131] (6)E 2 -C(=O)O- or -OC(=O)- , This indicates that at this location, R 2 A bonded compound of formula (I). E 2 -OC(=O)- or-OC(=O)O- , This indicates that at this location, R 2 A bonded compound of formula (I). E 2 is -OC(=O)O- or -C(=O)O- , This indicates that at this location, R 2 A bonded compound of formula (I). E 2 -C(=O)O- , This indicates that at this location, R 2 A bonded compound of formula (I). E 2 -OC(=O)- , This indicates that at this location, R 2 A bonded compound of formula (I). E 2 is -OC(=O)O- , This indicates that at this location, R2 A bonded compound of formula (I).

[0132] (7)R 1 -CH2CH(-R) x )R y or -CH2-(C 5-15 Compounds of formula (I) with an alkenyl group. R 1 -CH2CH(-R) x )R y Compounds of formula (I). R 1 -CH2CH(-OR) x OR y Compounds of formula (I). R 1 -CH2-(C 5-15 Compounds of formula (I) with alkyl groups. R 1 -CH2-(C 5-15 Compounds of formula (I) with an alkenyl group. R 1 -CH2CH(-R) x )R y Compounds of formula (I). R 1 and R 2 Same or different and of the type -CH2CH(-R) x )R y -CH2CH(-OR) x OR y -CH2CH2CH(-OR) x OR y -CH2-(C 5-15 alkyl) or -CH2-(C 5-20 Compounds of formula (I) with an alkenyl group.

[0133] (8)R 2 -CH2CH(-R) x )R y or -CH2-(C 5-15 Compounds of formula (I) with an alkenyl group. R 2 -CH2CH(-R) x )R y -CH2CH(-OR) x OR y -CH2-(C 5-15 alkyl) or -CH2-(C 5-15 Compounds of formula (I) with an alkenyl group. R 2 -CH2CH(-R) x )R y Compounds of formula (I). R 2 -CH2CH(-OR) xOR y Compounds of formula (I). R 2 -CH2-(C 5-15 Compounds of formula (I) with alkyl groups. R 2 -CH2-(C 5-15 Compounds of formula (I) with an alkenyl group.

[0134] (9)-E 1 -R 1 or -E 2 -R 2 Either of them is -C(=O)O-CH2CH(-R) x )R y Compounds of formula (I).

[0135] -E 1 -R 1 -C(=O)O-CH2CH(-R) x )R y -OC(=O)O-CH2CH(-R) x )R y -OC(=O)-CH2CH(-OR) x OR y or -OC(=O)-(C 5-15 Compounds of formula (I) with an alkenyl group. -E 1 -R 1 -C(=O)O-CH2CH(-R) x )R y Compounds of formula (I). -E 1 -R 1 -C(=O)O-CH2CH(-R) x )R y -C(=O)O-CH2CH2CH(-R) x )R y -OC(=O)-C3H6-(C 1-6 (alkylene)-CH=CH-CH=CH-(C 1-6 Alkyl), -OC(=O)-N(-R) x )R y -OC(=O)O-CH(-R) x )R y -OC(=O)O-CH2CH(-R) x )R y -NR y (-C(=O)R x ) or -NR y C(=O)CH(-R x )R ZCompounds of formula (I). -E 1 -R 1 -C(=O)O-CH2CH(-R) x )R y -OC(=O)-C3H6-(C 1-6 (alkylene)-CH=CH-CH=CH-(C 1-6 Alkyl) or -OC(=O)O-CH2CH(-R x )R y Compounds of formula (I). -E 1 -R 1 -C(=O)O-CH2CH(-R) x )R y -C(=O)O-CH2CH2CH(-R) x )R y -OC(=O)O-CH(-R) x )R y or -OC(=O)O-CH2CH(-R) x )R y Compounds of formula (I). -E 1 -R 1 -C(=O)O-CH2CH(-R) x )R y or -OC(=O)O-CH2CH(-R) x )R y Compounds of formula (I).

[0136] (10)-E 1 -R 1 or -E 2 -R 2 The other one is -C(=O)O-CH2CH(-R) x )R y -OC(=O)O-CH2CH(-R) x )R y -OC(=O)-CH2CH(-OR) x OR y or -OC(=O)-CH2-(C 5-15 Compounds of formula (I) with an alkenyl group. -E 2 -R 2 -C(=O)O-CH2CH(-R) x )R y -OC(=O)O-CH2CH(-R) x )R y -OC(=O)-CH2CH(-OR) x OR yor -OC(=O)-CH2-(C 5-15 Compounds of formula (I) with an alkenyl group. -E 2 -R 2 -C(=O)O-CH2CH(-R) x )R y Compounds of formula (I). -E 2 -R 2 -C(=O)O-CH2CH(-R) x )R y -C(=O)O-CH2CH2CH(-R) x )R y -C(=O)O-C3H6-(C 1-6 (alkylene)-CH=CH-CH=CH-(C 1-6 Alkyl group), -OC(=O)-CH2CH(-R) x )R y -OC(=O)-CH2CH2CH(-R) x )R y -OC(=O)-CH2CH2CH(-OR) x OR y -OC(=O)-C3H6-(C 1-6 (alkylene)-CH=CH-CH=CH-(C 1-6 Alkyl) or -OC(=O)O-CH2CH(-R x )R y Compounds of formula (I). -E 2 -R 2 -C(=O)O-CH2CH(-R) x )R y -C(=O)O-C3H6-(C 1-6 (alkylene)-CH=CH-CH=CH-(C 1-6 Alkyl) or -OC(=O)-C3H6-(C 1-6 (alkylene)-CH=CH-CH=CH-(C 1-6 Compounds of formula (I) with alkyl groups. -E 2 -R 2 -C(=O)O-CH2CH(-R) x )R y -C(=O)O-CH2CH2CH(-R) x )R y -OC(=O)-CH2CH2CH(-R) x )R y Or -OC(=O)-CH2CH2CH(-OR) x ORy Compounds of formula (I). -E 2 -R 2 -C(=O)O-CH2CH(-R) x )R y Compounds of formula (I).

[0137] (11)R x C 5-10 Alkyl compounds of formula (I). R x C 7-9 Alkyl compounds of formula (I). R x Compounds of formula (I) that are C7 alkyl groups. R x It is a C8 alkyl compound of formula (I). R x It is a C9 alkyl compound of formula (I). R x Compounds of formula (I) that are C8 alkyl or C9 alkyl. R x and R y Same or different and is C 5-15 Alkyl compounds of formula (I).

[0138] (12)R y C 5-10 Alkyl compounds of formula (I). R y C 7-9 Alkyl compounds of formula (I). R y Compounds of formula (I) that are C7 alkyl groups. R y It is a C8 alkyl compound of formula (I). R y Compounds of formula (I) that are C9 alkyl groups.

[0139] (13)R 3 The compound of formula (I) is selected from the group consisting of formulas (d), (e), (g) and (h) below.

[0140]

[0141] R 3 For compounds of formula (I) selected from the group consisting of groups of formulas (e), (g), and (h). R 3 R is a compound of formula (I) of formula (d). 3 R is a compound of formula (I) of formula (e). 3 R is a compound of formula (I) of formula (g). 3 R is a compound of formula (I) of formula (h). 3 A compound of formula (I) consisting of groups selected from the group consisting of the following formulas (d), (e), (g) and (h).

[0142]

[0143] R 3 For compounds of formula (I) selected from the group consisting of groups of formulas (e), (g), and (h). R 3 It is a compound of formula (I) of formula (d).

[0144] (14)R a Compounds of formula (I) with -CH3.

[0145] (15)R b -CH2-C 1-6 Alkyl compounds of formula (I). R b The compound of formula (I) is -C(=O)CH2N(CH3)2.

[0146] (16)R c and R d All are -CH3, L cd The compound of formula (I) is -CH2-. R c and R d Both are -CH2CH3, L cd Compounds of formula (I) that are -CH2CH2-. R c and R d Both are -CH2CH2OH, L cd Compounds of formula (I) that are -CH2CH2-. R c For H, R d The compound of formula (I) is -CH2C(=O)NH2. R c and R d All are -CH3, -CH2CH3 or -CH2CH2OH, or R c When R is H d -CH2C(=O)NH2, L cd It is -CH2- or -CH2CH2-, where R c R d When all are -CH3, -CH2CH3 or -CH2CH2OH, L cd Compounds of formula (I) that are -CH2-, -CH2CH2- or -CH2CH2- respectively.

[0147] (17)R e The compound of formula (I) is H. R e Compounds of formula (I) with OH.

[0148] (18)R f Compounds of formula (I) with H.

[0149] (19)Rg The compound of formula (I) is -CH3. R f and R g Compounds of formula (I) that form a pyrrolidine ring together with the carbon and nitrogen atoms they are bonded to. R g C 1-6 Alkyl compounds of formula (I).

[0150] (20)R h Compounds of formula (I) with -CH2CH3.

[0151] (21)R i The compound of formula (I) is -CH3. R i C 1-6 Alkyl compounds of formula (I).

[0152] (22)R j R k All are compounds of formula (I) with -CH2CH3. R j and R k Same or different and is C 1-6 Alkyl compounds of formula (I).

[0153] (23) Compounds of formula (I) with s = 1. Compounds of formula (I) with s = 2.

[0154] (24) Compounds of formula (I) with t = 1.

[0155] (25) Compounds of formula (I) that are not contradictory combinations of the groups described in (1) to (24) above.

[0156] As examples of combinations of the above methods, the following compounds or their salts can be specifically listed.

[0157] L 1 For key, L 2 -CH2-, L 3 It is a C6 alkylene group, M is absent, n is 1, and E is... 1 -C(=O)O- E 2 -C(=O)O- R 1 -CH2CH(-R) x )R y R 2 -CH2CH(-R) x )R y R x and R y It is a C8 alkyl or C9 alkyl, R 3 For equation (e), R eFor H, R f For H, R g The compound of formula (I) is CH3.

[0158] L 1 For key, L 2 -CH2-, L 3 It is a C6 alkylene group, M is absent, n is 1, and -E 1 -R 1 -C(=O)O-CH2CH(-R) x )R y Among them, R x and R y C8 alkyl, -E 2 -R 2 -C(=O)O-CH2CH(-R) x )R y Among them, R x and R y C9 alkyl, R 3 For equation (e), R e For H, R f For H, R g The compound of formula (I) is CH3.

[0159] As one example of a specific compound of the present invention, the following compounds or their salts may be listed.

[0160] A compound or a salt thereof, wherein the compound is: 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester, 8-[(1-methyl-L-prolyl)(3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}bicyclo[1.1.1]pentan-1-yl)amino]octanoic acid 2-nonylundecyl ester, 8-{(1-ethyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonyl undecyl ester, 8-{(N,N-diethyl-β-alanyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester, 8-{(1,4-diethyl-1,4-diazacycloheptane-6-carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester, 8-{[(4-methylpiperazin-1-yl)acetyl][(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester, 8-[(1-methyl-L-prolyl){(1r,3S)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]octanoic acid 2-nonylundecyl ester, (1S,4r)-4-[{8-[(2-heptylonyl)oxy]-8-oxooctyl}(1-methyl-L-prolyl)amino]cyclohexane-1-carboxylic acid 2-heptylonyl ester, 8-{(1-ethyl-D-prolyl)[(1r,3R)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester, 8-{[(1r,3S)-3-{2-[(3-decyltridecyl)oxy]-2-oxoethyl}cyclobutyl](1-methyl-L-prolyl)amino}octanoic acid 3-decyltridecyl ester, 4,4-Bis(octyloxy)butyric acid 2-{(1-ethyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}ethyl ester, or 4-Octylododecanoic acid 4-[(1-ethyl-L-prolyl){(1r,3S)-3-[({[(2-nonylundecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]butyl ester.

[0161] As one example of a specific compound of the present invention, the following compounds or their salts may be listed.

[0162] A compound or a salt thereof, wherein the compound is: 8-{(1-ethyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonyl undecyl ester, or 8-{(N,N-diethyl-β-alanyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonyl undecyl ester.

[0163] In this specification, sometimes only one isomer of a compound of formula (I) or a salt thereof is described, but the invention also includes other isomers, substances obtained by isomer separation, or mixtures thereof.

[0164] Compounds of formula (I) may exist as tautomers and geometric isomers depending on the type of substituents. In this specification, compounds of formula (I) are sometimes described in only one isomer form, but the invention also includes other isomers, substances obtained by isomer separation, or mixtures thereof. In this specification, when stated as "both double bonds are Z-forms," ​​it refers to the (Z) form, i.e., the cis form. Conversely, when stated as "both double bonds are E-forms," ​​it refers to the (E) form, i.e., the trans form.

[0165] Additionally, compounds of formula (I) or their salts sometimes possess a chiral center or axial chirality, and enantiomers (optical isomers) may exist based on this. Compounds of formula (I) or their salts also include any of the isolated (R) bodies, (S) bodies, and other enantiomers, or mixtures thereof (including racemic or non-racemic mixtures). In one manner, the enantiomer is "stereochemically pure." "Stereochemically pure" means a purity that can be recognized by a person skilled in the art as substantially stereochemically pure. As an example, the enantiomer is, for instance, a compound having a stereochemical purity of 90% ee (enantiomeric excess) or more, 95% ee or more, 98% ee or more, or 99% ee or more.

[0166] Furthermore, the salts of compounds of formula (I) refer to pharmaceutically acceptable salts, and depending on the type of substituent, sometimes form acid addition salts. Specifically, examples include: acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; and acid addition salts with organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, mandelic acid, tartaric acid, dibenzoyl tartaric acid, dibenzoyl tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, aspartic acid, and glutamic acid.

[0167] Furthermore, the present invention also includes various hydrates, solvates and polymorphs of compounds of formula (I) or their salts.

[0168] Furthermore, this invention includes pharmaceutically acceptable compounds of formula (I) or salts thereof labeled with one or more radioactive or non-radioactive isotopes. Examples of suitable isotopes used in the isotopic labeling of compounds of this invention include hydrogen (…). 2 H and 3 H, etc.), carbon ( 11 C 13 C and 14 C, etc.), nitrogen ( 13 N and 15 N, etc.), oxygen ( 15 O、 17 O and 18 O, etc.), fluorine (18 F, etc.), chlorine ( 36 Cl, etc.), iodine ( 123 I and 125 I, etc.), phosphorus ( 32 P, etc.), sulfur ( 35 Isotopes of tritium (e.g., S). The isotopically labeled compounds of this invention can be used in studies such as tissue distribution research of drugs and / or matrices. For example, tritium ( 3 H), carbon-14 ( 14 Radioactive isotopes such as C can be used for this purpose due to their ease of labeling and simple detection. Substitution into heavier isotopes, such as hydrogen substitution into deuterium (… 2 H), sometimes has therapeutic advantages due to improved metabolic stability (e.g., increased half-life in vivo, reduced dosage, and fewer drug interactions). Substitution into positron-emitting isotopes ( 11 C 18 F, 15 O and 13 (e.g., N) can be used in positron emission tomography (PET) tests to test matrix acceptor occupancy. The isotopically labeled compounds of the present invention can generally be manufactured by existing methods known to those skilled in the art, or by using suitable isotopically labeled reagents instead of unlabeled reagents, through the same manufacturing methods as in the examples or manufacturing examples.

[0169] 2. Lipid nanoparticles

[0170] The following illustrates one method of lipid nanoparticles containing a compound of formula (I) or a salt thereof of the present invention. It should be noted that "a compound of formula (I) or a salt thereof" in the following method includes the "compound of formula (I) or a salt thereof" described above in the (method of cationic lipids of the present invention) method.

[0171] (The lipid nanoparticle method of this invention)

[0172] (1) Lipid nanoparticles containing a compound of formula (I) or a salt thereof.

[0173] (2) Lipid nanoparticles containing a compound of formula (I) or its salt, neutral lipids and PEGylated lipids.

[0174] (3) Lipid nanoparticles described in (2) containing nucleic acids.

[0175] (4) The lipid nanoparticles described in (2) to (3) are composed of phospholipids and sterols as neutral lipids. The lipid nanoparticles described in (2) to (3) are composed of DSPC and cholesterol as neutral lipids.

[0176] (4a) The phospholipids are lipid nanoparticles described in (4) of DSPC.

[0177] (4b) Sterol is cholesterol. (4) The lipid nanoparticles described above.

[0178] (5) The PEGylated lipid is the lipid nanoparticle described in (3) of DMG-PEG2000.

[0179] (6) Based on the total amount of lipid nanoparticles, the composition ratio of each component is shown in (6a) to (6e) as mol% of lipid nanoparticles.

[0180] (6a) The lipid nanoparticles of (3) having a composition ratio of 20.0 to 80.0 mol% of the compound of formula (I) or its salt, based on the total amount of lipid nanoparticles. Lipid nanoparticles containing the compound of formula (I) or its salt, having a composition ratio of 30.0 to 60.0 mol% of the compound of formula (I) or its salt, based on the total amount of lipid nanoparticles.

[0181] (6b) The lipid nanoparticles of (3) having a neutral lipid composition of 18.5 to 78.5 mol% based on the total amount of lipid nanoparticles. The lipid nanoparticles of (3) having a neutral lipid composition of 38.5 to 68.5 mol% based on the total amount of lipid nanoparticles.

[0182] (6c) The lipid nanoparticles of (6b) having a phospholipid composition ratio of 4.0 to 18.1 mol% based on the total amount of lipid nanoparticles. The lipid nanoparticles of (6b) having a phospholipid composition ratio of 5.0 to 18.1 mol% based on the total amount of lipid nanoparticles.

[0183] (6d) The lipid nanoparticles of (6b) having a sterol content of 14.5 to 62.3 mol% based on the total amount of lipid nanoparticles. The lipid nanoparticles of (6b) having a sterol content of 25.5 to 54.4 mol% based on the total amount of lipid nanoparticles.

[0184] (6e) The lipid nanoparticles of (3) having a PEGylated lipid composition ratio of 0.5 to 2.5 mol% based on the total amount of lipid nanoparticles. The lipid nanoparticles of (3) having a PEGylated lipid composition ratio of 0.5 to 2.0 mol% based on the total amount of lipid nanoparticles.

[0185] (7) Lipid nanoparticles of (3) wherein the nucleic acid is mRNA. Lipid nanoparticles of (3) wherein the nucleic acid is useful for the prevention and / or treatment of astrocyte-related diseases. Lipid nanoparticles of (3) wherein the nucleic acid is mRNA useful for the prevention and / or treatment of astrocyte-related diseases. Lipid nanoparticles of (3) wherein the nucleic acid is Ascl1 mRNA, Dlx2 mRNA, NeuroD1 mRNA, Ngn2 mRNA, BDNF mRNA, NGF mRNA or NT-3 mRNA. Lipid nanoparticles of (3) wherein the nucleic acid is BDNF mRNA or NeuroD1 mRNA. Lipid nanoparticles of (3) wherein the nucleic acid is NeuroD1 mRNA.

[0186] (8) The nucleic acid is the lipid nanoparticle described in (7) containing a NeuroD1 mRNA that encodes a protein with a base sequence that has a sequence identity of 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) with respect to the amino acid sequence shown in Serial No. 2. The nucleic acid is the lipid nanoparticle described in (7) containing a NeuroD1 mRNA that has a sequence identity of 70% or more (e.g., 80% or more, 90% or more, 95% or more) with respect to the base sequence shown in Serial No. 1 or 3 and encodes the amino acid sequence shown in Serial No. 2. The nucleic acid is the lipid nanoparticle described in (7) containing a NeuroD1 mRNA that has a sequence identity of 70% or more (e.g., 80% or more, 90% or more, 95% or more) with respect to the base sequence shown in Serial No. 1 and encodes the amino acid sequence shown in Serial No. 2. The nucleic acid is a NeuroD1 mRNA containing a base sequence that has more than 70% (e.g., more than 80%, more than 90%, more than 95%) sequence identity with respect to the base sequence shown in sequence number 3 and encoding the amino acid sequence shown in sequence number 2, as described in (7) of the lipid nanoparticles.

[0187] (8a) The nucleic acid is a lipid nanoparticle of (7) containing a base sequence encoding a protein composed of the amino acid sequence shown in Serial No. 2. The nucleic acid is a lipid nanoparticle of (7) containing a NeuroD1 mRNA containing the base sequence shown in Serial No. 1. The nucleic acid is a lipid nanoparticle of (7) containing a NeuroD1 mRNA containing the base sequence shown in Serial No. 3. The nucleic acid is a lipid nanoparticle of (7) containing a NeuroD1 mRNA containing the base sequence shown in Serial No. 4. The nucleic acid is a lipid nanoparticle of (7) containing a NeuroD1 mRNA containing the base sequence shown in Serial No. 5. The nucleic acid is a NeuroD1 mRNA containing a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, 95% or more) relative to the base sequence shown in positions 44 to 1114 of Serial No. 4 and encoding the amino acid sequence shown in Serial No. 2. The nucleic acid is a NeuroD1 mRNA containing a base sequence that has more than 70% (e.g., more than 80%, more than 90%, more than 95%) sequence identity with respect to the base sequence shown at positions 44 to 1114 of sequence number 5 and encodes the lipid nanoparticles described in (7) of sequence number 2.

[0188] (9) The nucleic acid is NeuroD1 mRNA and the lipid nanoparticles described in (7) are lipid nanoparticles.

[0189] (9a) The lipid nanoparticles of (9) with a 5' cap of Cap-0, Cap-1, or Cap-2. The lipid nanoparticles of (9) with a 5' cap of Cap-1.

[0190] (9b) Lipid nanoparticles of (9) with a 5'UTR containing a Kozak sequence. The 5'UTR or 3'UTR of the nucleic acid is derived from the α-globin gene of the lipid nanoparticles of (9). The 5'UTR or 3'UTR of the nucleic acid is derived from the human α-globin gene of the lipid nanoparticles of (9). The 5'UTR or 3'UTR of the nucleic acid is derived from the β-globin gene of the lipid nanoparticles of (9). The 5'UTR or 3'UTR of the 5'UTR is derived from the human β-globin gene of the lipid nanoparticles of (9). The 5'UTR contains a Kozak sequence of the lipid nanoparticles of (9). The 5'UTR is derived from the α-globin gene of the lipid nanoparticles of (9). The 5'UTR is derived from the human α-globin gene of the lipid nanoparticles of (9). The 5'UTR is derived from the β-globin gene of the lipid nanoparticles of (9). The 5'UTR is derived from the human β-globin gene of the lipid nanoparticles of (9). The 3'UTR is derived from the human α-globin gene of the lipid nanoparticles of (9). The 3'UTR is derived from the lipid nanoparticles described in (9) of the human β-globin gene.

[0191] (9c) The lipid nanoparticles described in (9) are NeuroD1 mRNA with a polyA chain of 20 to 1000 bases. The lipid nanoparticles described in (9) are NeuroD1 mRNA with a polyA chain of 30 to 500 bases, 50 to 200 bases, 60 to 150 bases, 70 to 130 bases, 70 to 120 bases, 70 to 80 bases, 120 bases, or 79 bases. The lipid nanoparticles described in (9) are NeuroD1 mRNA with a polyA chain of 120 bases. The lipid nanoparticles described in (9) are NeuroD1 mRNA with a polyA chain of 79 bases.

[0192] (9d) Lipid nanoparticles of (9) containing modified nucleotides. Lipid nanoparticles of (9) containing N1-methylpseuuridine. Lipid nanoparticles of (9) in which some or all of the uridine is N1-methylpseuuridine. Lipid nanoparticles of (9) in which some of the uridine is replaced with N1-methylpseuuridine. Lipid nanoparticles of (9) in which all of the uridine is replaced with N1-methylpseuuridine.

[0193] (10) Lipid nanoparticles that are two or more non-contradictory combinations of (1) to (9) described in the above (methods of lipid nanoparticles of the present invention). Lipid nanoparticles that are non-contradictory combinations of the methods described in (methods of cationic lipids of the present invention) and the methods described in (methods of lipid nanoparticles of the present invention).

[0194] As a combination of the above (10), specifically, the following methods can be listed as examples.

[0195] (10a) The nucleic acid is mRNA encoding NeuroD1 protein, the lipid nanoparticles are a compound of formula (I) or a salt thereof of 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonyl undecyl ester or a salt thereof, the neutral lipid is DSPC and cholesterol, the PEGylated lipid is DMG-PEG2000, and the lipid nanoparticles containing a compound of formula (I) or a salt thereof, neutral lipid and PEGylated lipid encapsulated nucleic acid are included.

[0196] (10b) The nucleic acid is the lipid nanoparticle described in (10a) which contains a base sequence encoding a protein consisting of the amino acid sequence shown in sequence number 2 and an mRNA encoding the NeuroD1 protein.

[0197] (10c) The nucleic acid is the lipid nanoparticle described in (8a) which contains the mRNA encoding the NeuroD1 protein as shown in (10a) containing the base sequence shown in sequence number 1.

[0198] (10d) The nucleic acid is the lipid nanoparticle described in (8a) which contains the mRNA encoding the NeuroD1 protein as shown in (10a) containing the base sequence shown in sequence number 3.

[0199] (10e) The nucleic acid is the lipid nanoparticle described in (8a) which contains the mRNA encoding the NeuroD1 protein as shown in (10a) containing the base sequence shown in sequence number 4.

[0200] (10f) The nucleic acid is the lipid nanoparticle described in (8a) which contains the mRNA encoding the NeuroD1 protein as shown in (10a) containing the base sequence shown in sequence number 5.

[0201] In (10g)(10a)~(10f), lipid nanoparticles containing 20.0~70.0 mol% of 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonyl undecyl ester or its salt, neutral lipids (i.e. DSPC and cholesterol) in a composition ratio of 27.0~79.5 mol%, and DMG-PEG2000 in a composition ratio of 0.5~3.0 mol%.

[0202] In (10h)(10a) to (10f), lipid nanoparticles containing 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonyl undecyl ester or its salt, at a composition ratio of 35.0 to 50.0 mol% based on the total amount of lipid nanoparticles, neutral lipids (i.e., DSPC and cholesterol) at a composition ratio of 47.5 to 64.0 mol%, and DMG-PEG2000 at a composition ratio of 1.0 to 2.5 mol%.

[0203] (10i) Lipid nanoparticles containing nucleic acids and neutral lipids, PEGylated lipids and compounds of formula (I) or their salts, wherein the nucleic acid is a nucleotide sequence in which the 1st to 3rd positions are 5' end sequences suitable for the capping method of CleanCap (registered trademark) Reagent AG (TriLink BioTechnologies), the 4th to 43rd positions are 5' UTRs, the 44th to 1114th positions are the CDS (Sequence No. 3) of the human NeuroD1 gene, the 1115th to 1120th positions are two consecutive stop codons, the 1121st to 1231st positions are 3' UTRs, and the 1232nd to 1310th positions are mRNA encoding the NeuroD1 protein corresponding to the poly A chain.

[0204] As a specific example of the lipid nanoparticles included in this invention, the following methods can be listed.

[0205] (11a) Lipid nanoparticles encapsulating nucleic acids and containing neutral lipids, PEGylated lipids, and compounds of formula (I) or their salts, wherein, The nucleic acid is the mRNA encoding human ND1 (here, the 5' cap structure is Cap-1, the CDS is the CDS of the human ND1 gene (sequence number 1), and the poly-A chain is 120 bases long). In the lipid nanoparticles, the compound of formula (I) or its salt is 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonyl undecyl ester or its salt, the neutral lipid is DSPC and cholesterol, and the PEGylated lipid is DMG-PEG2000.

[0206] (11b) Lipid nanoparticles encapsulating nucleic acids and containing neutral lipids, PEGylated lipids, and compounds of formula (I) or their salts, wherein, The nucleic acid is mRNA encoding human ND1. (Here, the 5' cap structure is Cap-1, the 5' UTR originates from the human α-globin gene, the CDS is the CDS of the human ND1 gene (Sequence No. 3), the 3' UTR originates from the human α-globin gene, and the polyA chain consists of a 79-nucleotide polynucleotide sequence (Sequence No. 4) in which all uridines are N1-methylpseudouridine (Sequence No. 5). Here, in the base sequence shown in Sequence No. 4 or Sequence No. 5, positions 1-3 correspond to AGG, positions 4-43 correspond to the 5' UTR, positions 44-1114 correspond to the CDS of the human NeuroD1 gene (Sequence No. 3), positions 1115-1120 correspond to two consecutive stop codons, positions 1121-1231 correspond to the 3' UTR, and positions 1232-1310 correspond to the polyA sequence.) In the lipid nanoparticles, the compound of formula (I) or its salt is 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonyl undecyl ester or its salt, the neutral lipid is DSPC and cholesterol, and the PEGylated lipid is DMG-PEG2000.

[0207] (11c) Lipid nanoparticles encapsulating nucleic acids and containing neutral lipids, PEGylated lipids, and compounds of formula (I) or their salts, wherein, The nucleic acid is mRNA encoding human ND1 (here, the 5' cap structure is Cap-1, consisting of the base sequence shown in positions 1-1231 of sequence number 4 or the base sequence shown in positions 1-1231 of sequence number 5, and a poly-A chain). In the lipid nanoparticles, the compound of formula (I) or its salt is 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonyl undecyl ester or its salt, the neutral lipid is DSPC and cholesterol, and the PEGylated lipid is DMG-PEG2000.

[0208] (11d) Lipid nanoparticles described in (11a) to (11c) containing nucleic acids and neutral lipids, PEGylated lipids and compounds of formula (I) or salts thereof, wherein, based on the total amount of lipid nanoparticles, they contain 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonyl undecyl ester or a salt thereof at a composition ratio of 50 mol%, DSPC and cholesterol at a composition ratio of 48.5 mol%, and DMG-PEG2000 at a composition ratio of 1.5 mol%.

[0209] 3. Pharmaceutical Composition

[0210] The following illustrates one manner in which a pharmaceutical composition comprises lipid nanoparticles containing a compound of formula (I) as a compound of the present invention or a salt thereof, and one or more pharmaceutically acceptable pharmaceutical additives.

[0211] (The method of preparation of the pharmaceutical composition of the present invention)

[0212] (1) A pharmaceutical composition containing lipid nanoparticles as described in any one of (3) to (11) of the present invention.

[0213] (2) A pharmaceutical composition containing the lipid nanoparticles of the present invention as described in any one of (3) to (11) and one or more pharmaceutically acceptable pharmaceutical additives.

[0214] (3) The pharmaceutical composition described in (1) or (2) for the prevention and / or treatment of astrocyte-related diseases.

[0215] As one example of a specific pharmaceutical composition included in this invention, the following methods can be listed.

[0216] (A) A pharmaceutical composition comprising encapsulated nucleic acids and lipid nanoparticles containing neutral lipids, PEGylated lipids, and a compound of formula (I) or a salt thereof, wherein, The nucleic acid is mRNA encoding the NeuroD1 protein. In the lipid nanoparticles, the compound of formula (I) or its salt is 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonyl undecyl ester or its salt, the neutral lipid is DSPC and cholesterol, and the PEGylated lipid is DMG-PEG2000.

[0217] (B) A pharmaceutical composition comprising lipid nanoparticles containing an encapsulated nucleic acid and a compound of formula (I) or a salt thereof, a neutral lipid, and a PEGylated lipid, wherein, Nucleic acid is mRNA containing a base sequence that encodes the NeuroD1 protein, consisting of the amino acid sequence shown in SEQ ID NO: 2. In the lipid nanoparticles, the compound of formula (I) or its salt is 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonyl undecyl ester or its salt, the neutral lipid is DSPC and cholesterol, and the PEGylated lipid is DMG-PEG2000.

[0218] (C) A pharmaceutical composition comprising lipid nanoparticles containing encapsulated nucleic acids and containing a compound of formula (I) or a salt thereof, neutral lipids, and PEGylated lipids, wherein, The nucleic acid is the mRNA encoding human ND1 (here, the 5' cap structure is Cap-1, the CDS is the CDS of the human ND1 gene (sequence number 1), and the poly-A chain is 120 bases long). In the lipid nanoparticles, the compound of formula (I) or its salt is 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonyl undecyl ester or its salt, the neutral lipid is DSPC and cholesterol, and the PEGylated lipid is DMG-PEG2000.

[0219] (D) A pharmaceutical composition comprising lipid nanoparticles containing an encapsulated nucleic acid and a compound of formula (I) or a salt thereof, a neutral lipid, and a PEGylated lipid, wherein, The nucleic acid is mRNA encoding human ND1. (Here, the 5' cap structure is Cap-1, the 5' UTR originates from the human α-globin gene, the CDS is the CDS of the human ND1 gene (Sequence No. 3), the 3' UTR originates from the human α-globin gene, and the polyA chain consists of a 79-nucleotide polynucleotide sequence (Sequence No. 4) in which all uridines are N1-methylpseudouridine (Sequence No. 5). Here, in the base sequence shown in Sequence No. 4 or Sequence No. 5, positions 1-3 correspond to AGG, positions 4-43 correspond to the 5' UTR, positions 44-1114 correspond to the CDS of the human NeuroD1 gene (Sequence No. 3), positions 1115-1120 correspond to two consecutive stop codons, positions 1121-1231 correspond to the 3' UTR, and positions 1232-1310 correspond to the polyA sequence.) In the lipid nanoparticles, the compound of formula (I) or its salt is 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonyl undecyl ester or its salt, the neutral lipid is DSPC and cholesterol, and the PEGylated lipid is DMG-PEG2000.

[0220] (E) A pharmaceutical composition comprising lipid nanoparticles containing an encapsulated nucleic acid and a compound of formula (I) or a salt thereof, a neutral lipid, and a PEGylated lipid, wherein, The nucleic acid is mRNA encoding human ND1 (here, the 5' cap structure is Cap-1, consisting of the base sequence shown in positions 1-1231 of sequence number 4 or the base sequence shown in positions 1-1231 of sequence number 5, and a poly-A chain). In the lipid nanoparticles, the compound of formula (I) or its salt is 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonyl undecyl ester or its salt, the neutral lipid is DSPC and cholesterol, and the PEGylated lipid is DMG-PEG2000.

[0221] The route of administration of the pharmaceutical composition is not limited, and examples include non-oral administration such as intracerebral, intra-articular, intravenous, intramuscular, subcutaneous, via injection, mucosal solution, or inhalation. Generally, when administered intracerebrally, it is delivered to the central nervous system. One route of administration is intraparenchymal administration. Another route is intraventricular administration. Another route is intraspinal administration. Another route is intramedullary administration. Another route is intracanal administration. Another route is intraspinal administration.

[0222] Typically, when administering the drug to the brain, one method is to administer approximately 0.001 to 10 mg of the lipid nanoparticle-containing pharmaceutical composition of the present invention per 1 kg of brain tissue; another method is to administer approximately 0.001 to 50 mg of the lipid nanoparticle-containing pharmaceutical composition of the present invention per 1 kg of brain tissue; and yet another method is to administer approximately 0.001 to 100 mg of the lipid nanoparticle-containing pharmaceutical composition of the present invention per 1 kg of brain tissue, administered once daily to in multiple divided doses. The dosage can be appropriately determined based on various factors such as disease, symptoms, age, and gender.

[0223] Typically, when administering the pharmaceutical composition of the present invention intramuscularly, a daily dose of about 1 to 10 mg / kg of body weight is suitable, and it is administered once daily to in multiple divided doses. The dosage can be appropriately determined based on various circumstances, taking into account the disease, symptoms, age, gender, etc.

[0224] Although the dosage forms, administration sites, excipients, and additives vary, the pharmaceutical composition of the present invention contains, as one method, 5 to 50% by weight of lipid nanoparticles based on the total weight of the pharmaceutical composition. Regarding the content of lipid nanoparticles, etc., one method is 3 to 70% by weight, and another method is 10 to 50% by weight.

[0225] The pharmaceutical compositions of the present invention can be used in combination with various therapeutic or preventative agents for diseases in which the pharmaceutical compositions are believed to be effective. This combination can be administered simultaneously, or separately and continuously, or at desired time intervals. Simultaneously administered formulations can be combination agents or separately formulated.

[0226] 4. Method for manufacturing cationic lipids of the present invention

[0227] Compounds of formula (I) or salts thereof, which are compounds of the present invention, can be manufactured using various known synthetic methods based on the characteristics of their basic structure or the types of substituents. In this case, it is sometimes technically effective to replace the functional group with a suitable protecting group (a group that can be easily converted into the functional group) in advance from the starting material to the intermediate stage, depending on the type of functional group.

[0228] Examples of such protecting groups include those described in "Greene's Protective Groups in Organic Synthesis" (5th edition, 2014) by PGM Wuts and TW Greene. The appropriate protecting group can be selected based on the reaction conditions. After introducing the protecting group and reacting, the protecting group can be removed as needed to obtain the desired compound.

[0229] The following describes representative methods for manufacturing compounds of formula (I) or their salts. Each manufacturing method can also be performed with reference to the references appended to this description. It should be noted that the manufacturing methods of the present invention are not limited to the examples shown below.

[0230] The compounds of the present invention or their salts can be manufactured by the following methods.

[0231] (Manufacturing Method 1)

[0232] The compound (I) of the present invention can be produced by an amidation reaction of compound (1) and compound (2).

[0233] The reaction is carried out in a solvent with a mixture of compounds (1) and (2) and a condensing agent. Examples of solvents include aromatic hydrocarbons such as benzene, toluene, or xylene; halogenated hydrocarbons such as DCM; DMF; DMSO; and mixtures thereof. Examples of condensing agents include HATU and EDCI·HCl. Additives (e.g., HOBt, DMAP) sometimes facilitate the reaction. Organic bases (such as TEA or DIPEA) and inorganic bases (such as K2CO3, Na2CO3, or KOH) sometimes further advance the reaction.

[0234] Alternatively, it can be produced by converting carboxylic acid (1) into a reactive derivative and then reacting it with amine (2).

[0235] Examples of reactive derivatives of carboxylic acids include: acyl halides obtained by reacting with halogenating agents such as phosphorus oxychloride or thionyl chloride; mixed acid anhydrides obtained by reacting with isobutyl chloroformate; and reactive esters obtained by condensation with HOBt. The reaction of these reactive derivatives with compound (2) can be carried out in a solvent at a temperature ranging from -20°C to 60°C. Solvents include halogenated hydrocarbons such as DCM, aromatic hydrocarbons, and ethers.

[0236] It should be noted that in this reaction, a compound protected by a protected group that can be converted to R can also be used instead of compound (1). 3 Following the protected group, the group protected by the protected group can be converted into R. 3 The group undergoes deprotection and a desired reaction to derive R. 3 To produce compound (I). In this reaction, it is also possible to use a compound that can be converted to R instead of compound (1). 3 After the group undergoes this reaction, it is derivatized into R. 3 To manufacture compound (I).

[0237] [References] SR Sandler and W. Karo, *Organic Functional Group Preparations*, Academic Press Inc., 2nd edition, Volume 1, 1991. *Lectures on Experimental Chemistry* (5th edition), edited by the Chemical Society of Japan, 2005, Volume 16, Maruzen.

[0238] (Second Manufacturing Method)

[0239] The compound (I-1) of the present invention can be produced by the esterification reaction of compound (3-1) and compound (4-2).

[0240] The reaction is carried out in a solvent with a mixture of compounds (3-1) and (4-2) and a condensing agent. Solvents include halogenated hydrocarbons such as DCM, DMF, DMSO, and mixtures thereof. Condensing agents include HATU, EDCI·HCl, CDI, DPPA, and phosphorus oxychloride. Additives (e.g., HOBt, DMAP) sometimes facilitate the reaction. Organic bases (such as TEA or DIPEA) and inorganic bases (K₂CO₃, Na₂CO₃, or KOH) sometimes further promote the reaction.

[0241] Alternatively, it can be produced by reacting a carboxylic acid (4-2) with an alcohol (3-1) after converting it into a reactive derivative. Examples of reactive derivatives of carboxylic acids include: acyl halides obtained by reacting with halogenating agents such as phosphorus oxychloride and thionyl chloride; mixed acid anhydrides obtained by reacting with isobutyl chloroformate; and reactive esters obtained by condensation with HOBt. The reaction of these reactive derivatives with compound (3-1) can be carried out in a solvent at a temperature ranging from -20°C to 60°C. The solvent can be a halogenated hydrocarbon such as DCM.

[0242] The compound (I-2) of this invention can be prepared by esterification of compound (3-2) with compound (4-1). This reaction can be carried out under the same conditions as the second preparation method.

[0243] (Raw material manufacturing method 1)

[0244] (In the formula, Lv represents the leaving group.)

[0245] Compound (2) can be produced by reacting compound (5) with compound (6). Examples of leaving groups Lv include halogens, methanesulfonyloxy groups, etc.

[0246] In this reaction, equal or one-excess amounts of compounds (5) and (6) are used to react a mixture of them in a solvent. The reaction temperature is, in one case, 0°C to 100°C. Alternatively, it is 50°C to 90°C. Solvents include halogenated hydrocarbons such as DCM, 1,2-dichloroethane, DMF, DMSO, MeCN, CPME, and mixtures thereof. Organic bases (such as TEA or DIPEA) and inorganic bases (K2CO3, Na2CO3, or KOH), or additives (KI or NaI), sometimes further promote the reaction.

[0247] It should be noted that the starting material compound (6) can be manufactured by esterification or carbonate reaction of the corresponding starting material to form E2. Esterification can be performed under the conditions described above.

[0248] [References] SR Sandler and W. Karo, "Organic Functional Group Preparations", 2nd edition, Volume 1, 1991, Academic Press Inc.; Japan Chemical Society (ed.), "Lectures on Experimental Chemistry (5th edition)", Volume 14, 2005, Maruzen.

[0249] (Raw material manufacturing method 2)

[0250] Compound (3-1) can be prepared by alkylation of compound (5) with compound (7-1), amidation of the resulting compound (8-1) with compound (1), and deprotection of the resulting compound (9-1). The alkylation reaction can be carried out in the same manner as in method 1 of the preparation of the starting material. The amidation reaction can be carried out in the same manner as described in method 1. Compound (3-2) can be prepared in the same manner as in the preparation of compound (3-1). Deprotection can be carried out by conventional deprotection. See Greene and Wuts, “Protective Groups in Organic Synthesis,” 3rd ed., John Wiley & Sons Inc., 1999.

[0251] (Raw material manufacturing method 3)

[0252] (In the formula, Pr represents a protecting group.)

[0253] Compounds (5-1) to (5-4), which are protected by a protecting group as a form of compound (5), can be manufactured by esterification, carbonate esterification, or carbamate esterification. Compound (5-3) can also be manufactured by compound (10-1) and compound (11-1'). Compound (10-1') can be manufactured by compound (10-1). Compound (5-3) can be manufactured by compound (10-1') and compound (11-1). Compound (11-1') can be manufactured by compound (11-1). Compound (5-4) can be manufactured by compound (10-1') and compound (12). It should be noted that the above manufacturing method shows an example of temporarily isolating compound (10-1') and compound (11-1') before proceeding with subsequent reactions; however, depending on the compound, isolation may not be performed before proceeding with subsequent reactions.

[0254] (Raw material manufacturing method 4)

[0255] (In the formula, Pr represents a protecting group.)

[0256] Compounds (5-5) to (5-7), which are protected by a protecting group as a form of compound (5), can be manufactured by amidation.

[0257] Compounds of formula (I) or their salts can be isolated or purified to free compounds, their salts, hydrates, solvates, or polymorphs. Compounds of formula (I) or their salts can also be prepared by salt-forming reactions using conventional methods. Isolation and purification can be performed using conventional procedures such as extraction, fractional crystallization, and various fractional chromatography methods (silica gel chromatography, etc.).

[0258] Various isomers can be manufactured by selecting suitable starting material compounds, or they can be separated by utilizing the differences in physicochemical properties between isomers. For example, optical isomers can be obtained by conventional optical resolution methods for racemates (e.g., stepwise crystallization of diastereomer salts of optically active bases or acids, chromatography using chiral columns, etc.), or they can be manufactured from suitable optically active starting material compounds.

[0259] 5. Method for manufacturing lipid nanoparticles of the present invention

[0260] Lipid nanoparticles can be manufactured by adding components such as nucleic acids to the compounds of formula (I) or their salts, neutral lipids and PEGylated lipids and dispersing them in a medium.

[0261] For example, a compound of formula (I) or its salt, DSPC, sterol, and PEGylated lipids are dissolved in a solvent to form an oil phase, which is then mixed with or suspended in an aqueous phase such as a buffer solution containing nucleic acids. The solvent in the mixed solution is then removed by methods such as dialysis or ultrafiltration, thereby obtaining lipid nanoparticles.

[0262] In addition, regarding the pharmaceutical composition, the nucleic acid lipid nanoparticles can be manufactured using the aforementioned excipients and other additives by known methods.

[0263] 6. Method for manufacturing pharmaceutical compositions

[0264] Pharmaceutical compositions can be prepared using pharmaceutical additives (excipients, etc.), i.e., pharmaceutical excipients, by conventionally used methods. Pharmaceutical additives are not limited to the components contained in the pharmaceutical composition; in addition to excipients, they may also contain preservatives, stabilizers, antioxidants, preservatives, etc. Furthermore, other additives may be added to the pharmaceutical composition. Additionally, pharmaceutically acceptable media may be added to the pharmaceutical composition.

[0265] The pharmaceutical composition may be refrigerated or frozen for storage and / or transport. Regarding temperature, one option is about -150°C to about 0°C, another option is about -80°C to about -20°C, a third option is about -40°C to about -20°C, and a fourth option is below 4°C. As a possible solution, PBS is used.

[0266] Injectable preparations preferably comprise sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Aqueous solvents include, for example, distilled water for injection or physiological saline. Non-aqueous solvents include alcohols such as ethanol. Such compositions may also contain isotonic agents, preservatives, wetting agents, emulsifiers, dispersants, stabilizers, or co-solvents. These can be sterilized, for example, by filtration through a bacterial trap, by the application of a bactericide, or by irradiation. Alternatively, these can be formulated as sterile solid compositions and dissolved or suspended in sterile water or sterile solvents for injection before use.

[0267] Inhalers, nasal inhalers, and other mucosal preparations can be manufactured using liquid formulations according to existing known methods. For example, known excipients, pH adjusters, preservatives, surfactants, lubricants, stabilizers, thickeners, etc., can be appropriately added. Administration can be performed using suitable inhalation or blowing devices. For example, known devices such as metered-dose inhalers or nebulizers can be used to administer the compound alone or as a formulated mixture in powder form, or it can be combined with pharmaceutically acceptable drug additives and administered in solution or suspension form. Dry powder inhalers can be single- or multiple-dose dry powder inhalers. Alternatively, pressurized aerosol sprays using suitable propellants, such as chlorofluorocarbons or carbon dioxide, can also be used.

[0268] 7. Methods for manufacturing nucleic acids

[0269] Nucleic acids, such as mRNA, can be manufactured using techniques known in the field. Examples are listed below, but are not limited to. mRNA can be manufactured using a linear plasmid as a DNA template via in vitro transcription (IVT). Known manufacturing methods include (i) post-transcriptional capping (the steps of which are performed in the order of plasmid preparation, in vitro transcription, and 5' capping) and (ii) co-transcriptional capping (the steps of which are performed in the order of plasmid preparation, in vitro transcription, and simultaneous 5' capping). In co-transcriptional capping, Cap-0 can be added to the 5' end of the RNA when using the anti-reverse cap analog (ARCA) technique, and Cap-1 can be added to the 5' end of the RNA when using the CleanCap (registered trademark, TriLink) capping technique.

[0270] (In vitro transcription)

[0271] In IVT, the reaction typically uses transcription buffer, nucleoside triphosphates (NTPs), RNase inhibitors, and polymerases (e.g., T7 RNA polymerase). NTPs can be either natural or non-natural (modified).

[0272] (Add a hat)

[0273] Capping can be performed using methods such as vaccinia capping enzyme, 2'O-methyltransferase, or CleanCap. Even when the sequence obtained from IVT transcription does not contain a polyA sequence, polyA addition can still be performed. It should be noted that, regarding the sequence, when using the capping method with CleanCap (registered trademark) Reagent AG (TriLink BioTechnologies), the suitable 5' end sequence for this method is, for example, AGG (positions 1-3 of sequence number 4).

[0274] (Plasmid preparation)

[0275] Linear plasmids can be prepared using the following steps. Plasmids with arbitrary UTRs and arbitrary base sequences (e.g., the base sequence encoding the NeuroD1 protein) inserted into them (e.g., high-copy plasmids for E. coli such as pUC18 or pCU18) can be prepared and amplified using E. coli, etc. After purification, the plasmids can be used to prepare linear plasmids using restriction enzymes and buffers. After purification, the linear plasmids can be used directly as DNA templates. Alternatively, they can be used as DNA templates after polymerase chain reaction (PCR).

[0276] (purification)

[0277] mRNA and its intermediates in the manufacturing process can be purified using methods known in the field. Examples are listed below, but are not limited to. DNA templates can be removed using deoxyribonuclease I (DNase I). Transcribed RNA can be purified using silica gel columns, etc. RNA containing polyA sequences can be purified using Oligo dT columns. Linear plasmids can be purified using PureLink (registered trademark) (Thermo Fisher Scientific), etc.

[0278] Test case

[0279] The pharmacological activity of nucleic acid lipid nanoparticles composed of compounds of formula (I) or their salts was confirmed by the following tests. In the test examples and other materials described below, the following abbreviations are sometimes used.

[0280] (abbreviation)

[0281] B-27: B-27 serum-free supplement; BDNF: Brain-derived neurotrophic factor; DMEM: Durbeco-modified Igor medium; DMG-PEG2000: 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol 2000; DOTAP: 1,2-dioleoyloxy-3-trimethylammonium propane; DOTMA: 1,2-di-O-octadecenyl-3-trimethylammonium propane; DSPC: 1,2-distearate-sn-glycerol-3-phosphocholine; eGFP: enhanced green fluorescent protein; FBS: fetal bovine serum; FLuc nucleic acid lipid nanoparticles: lipid nanoparticles encapsulating FLuc mRNA; ND1 nucleic acid lipid nanoparticles: encapsulating NeuroD1. Lipid nanoparticles of mRNA, FLuc: luciferase (firefly), LNP: lipid nanoparticles, mRNA: messenger ribonucleic acid, ND1: NeuroD1, PBS: phosphate buffer, RLA: relative luciferase activity, RLU: relative luminescence, tris buffer: tris(hydroxymethyl)aminomethane buffer, FLuc eGFP mixed mRNA: mRNA composed of FLuc mRNA and eGFP mRNA (also known as FLuc eGFP mixed nucleic acid), FLuc eGFP mixed nucleic acid lipid nanoparticles: lipid nanoparticles containing mRNA composed of FLuc mRNA and eGFP mRNA.

[0282] Experimental Example 1: In vitro luciferase assay

[0283] Experiment 1-1 (Evaluation of the introduction of lipid nanoparticles using primary mouse astrocytes)

[0284] For compounds of formula (I) or their salts constituting lipid nanoparticles, the efficiency of nucleic acid delivery into mouse primary astrocytes was evaluated. Nucleic acid lipid nanoparticles were fabricated using FLuc mRNA (TriLink BioTechnologies) and NanoAssemblr (a registered trademark, manufactured by Precision NanoSystems). The delivery efficiency of the test drug (nucleic acid lipid nanoparticles) was calculated by evaluating the expression level of the luciferase encoded by the mRNA. It should be noted that each group was tested in 3 wells. The methods and results are shown.

[0285] (Obtaining primary mouse astrocytes)

[0286] Primary mouse astrocytes were obtained according to Lynette C. Foo., Purification of Rat and Mouse Astrocytes by Immunopanning. Cold Spring Harbor Protocols, 2013, Vol. 5, pp. 421-432. Cell resuscitation was performed at 5% CO2. Newborn mice P1-P10 from C57BL / 6J (Jackson Laboratories, Japan) were used.

[0287] (Experimental Methods)

[0288] Primary mouse astrocytes obtained by the above method were seeded at 2000 cells / well in a poly-D-lysine-coated 96-well plate (IWAKI, Greiner) and cultured overnight at 37°C with 5% CO2.

[0289] As the culture medium, DMEM / F-12 medium (Thermo Fisher Scientific) containing 2% B-27 (Thermo Fisher Scientific), 10% FBS (Cytiva), and 1% penicillin-streptomycin (PS) (Thermo Fisher Scientific) was used at 100 μL / well. The next day, 100 μL of the test drug (FLuc nucleic acid lipid nanoparticles) diluted with PBS and the medium was added to achieve a final concentration of 0.8 μg / mL, and the mixture was incubated overnight at 200 μL / well. The next day, after removing all the medium, 100 μL of the ONE-Glo luciferase assay system (Promega) was added to each well after a 2-fold dilution with PBS. After mixing for 1 minute, 80 μL was transferred to each well of a 96-well white plate (Thermo Fisher Scientific), and luminescence was detected using an Envision (Revvity) instrument. RLU (Relative Light Unit) was used as an activity indicator.

[0290] (Expressing an evaluation)

[0291] The results of the above experiments showed that luciferase activity was observed in nucleic acid lipid nanoparticles containing compounds from the representative embodiments of the present invention as constituent components. This indicates that lipid nanoparticles encapsulating FLuc mRNA were taken into cells and translated to express the luciferase protein. The results for nucleic acid lipid nanoparticles (nucleic acid: Fluc mRNA) containing compounds from the embodiments are shown below. Here, NUM represents each lipid nanoparticle containing compounds from the embodiments of formula (I) or their salts as constituent components. ExA-LB (A and B are numbers) represent lipid nanoparticles containing ExA from the embodiments of formula (I) or their salts, and LB. L1 represents lipid nanoparticles containing ExA, DSPC, cholesterol, and DMG-PEG2000 (molar ratio 50 / 10 / 38.5 / 1.5) as constituent components. For example, Ex1-L1 to Ex5-L1 represent lipid nanoparticles with a lipid composition of L1, which are composed of the compound of formula (I) or its salt.

[0292] [Table 1]

[0293] [Table 2]

[0294] The following table shows the results for nucleic acid lipid nanoparticles (nucleic acid: Fluc mRNA) composed of the compounds of the examples. In the NUM section of the table below, Ex1-L1 to Ex1-L54 represent Ex1-L1-FLuc mRNA to Ex1-L54-FLuc mRNA, respectively. That is, Ex1-L1 to Ex1-L54 are lipid nanoparticles containing compound Ex1 of the examples, which encapsulate FLuc mRNA and have the lipid compositions described below. The average RLU of each group of nucleic acid lipid nanoparticle expression was calculated, and the relative value (RLA (Relative Luciferase Activity)) is shown when Ex1-L1 is set to 1. It should be noted that Ex1-L1a, Ex1-L1b, and Ex1-L1c in the table below all represent Ex1-L1 from different batches, used as RLA. Ex1-L2, Ex1-L3, Ex1-L5, Ex1-L8, Ex1-L12, Ex1-L16, Ex1-L21, Ex1-L24, Ex1-L26, Ex1-L29, Ex1-L35, Ex1-L39, Ex1-L45, Ex1-L47, and Ex1-L48 in Ex1-L2 to Ex1-L48 show the relative values ​​when Ex1-L1a is set to 1. Ex1-L4, Ex1-L6, Ex1-L7, Ex1-L9 to Ex1- L11, Ex1-L13~Ex1-L15, Ex1-L17~Ex1-L20, Ex1-L22, Ex1-L23, Ex1-L25, Ex1-L27, Ex1-L28, Ex1-L30~Ex1-L34, Ex1-L36~Ex1-L38, Ex1-L40~Ex1-L44, and Ex1-L46 show the relative values ​​when Ex1-L1b is set to 1, while Ex1-L51~Ex1-L54 show the relative values ​​when Ex1-L1c is set to 1. It should be noted that RLA was not calculated for Ex1-L49 and Ex1-L50, but the results of the above experiments confirm the expression of a certain amount of luciferase protein.

[0295] [Table 3]

[0296] Experimental Examples 1-2 (Evaluation of Lipid Nanoparticle Delivery Using Hepa1-6 Cells)

[0297] For compounds of formula (I) or their salts constituting lipid nanoparticles, the nucleic acid delivery efficiency of the lipid nanoparticles into the Hepa1-6 cell line (ATCC, mouse hepatocellular carcinoma cell line) was evaluated. As mRNA, nucleic acid lipid nanoparticles were prepared using NanoAssemblr (a registered trademark, Precision NanoSystems) with a mixture of FLuc mRNA and eGFP mRNA (molar ratio 1:1) (TriLink BioTechnologies). The delivery efficiency of the test drug (nucleic acid lipid nanoparticles) was calculated by evaluating the expression level of the luciferase encoded by the mRNA. It should be noted that each group was tested in 3 wells. The methods and results are shown below.

[0298] (Experimental Methods)

[0299] Hepa1-6 cells were seeded at 2000 cells / well in 96-well plates (Corning) and cultured overnight at 37°C with 5% CO2. As the culture medium, 100 μL / well was used in DMEM (Sigma) containing 10% FBS (Cytiva) and 1% penicillin-streptomycin (PS) (Thermo Fisher Scientific). The next day, 100 μL of the test drug (FLuc_eGFP nucleic acid lipid nanoparticles) diluted with PBS and culture medium was added to achieve a final concentration of 0.8 μg / mL, and the cells were cultured at 200 μL / well for two nights. After 2 days, all culture medium was removed, and 100 μL of the ONE-Glo luciferase assay system (Promega) diluted 2-fold with PBS was added to each well. After mixing for 3 minutes, 80 μL was transferred to each well of a 96-well white plate (Nunc), and luminescence was detected using an Envision (Revvity) assay. RLU (Relative Light Unit) was used as an activity indicator.

[0300] (Expressing an evaluation)

[0301] The results of the above experiments showed that luciferase activity was observed in nucleic acid lipid nanoparticles containing compounds from the representative embodiments of the present invention as constituent components. This indicates that lipid nanoparticles containing a mixture of FLuc mRNA and eGFP mRNA were taken into cells and translated to express the luciferase protein. The results of nucleic acid lipid nanoparticles (nucleic acid: FLuc eGFP mixed mRNA) containing the compounds from the embodiments are shown below. Ex6-L1 in the NUM of the table below represents Ex6-L1-FLuc eGFP mixed mRNA. Ex6-L1 is a lipid nanoparticle containing compound Ex6 of the embodiments, with the same lipid composition as Ex1-L1, containing a mixture of FLuc eGFP mixed mRNA. Similarly, Ex7-L1 in the NUM of the table below is a lipid nanoparticle containing compound Ex7 of the embodiments, containing Ex7-L1-FLuc eGFP mixed mRNA. In addition, the average RLU of each group of nucleic acid lipid nanoparticle expression is shown below.

[0302] [Table 4]

[0303] Experimental Example 2: In vivo luciferase assay

[0304] (Experimental Methods)

[0305] The delivery efficiency of nucleic acid lipid nanoparticles into the brain was evaluated using an IVIS spectrometer (Revvity). In 6-12 week old BALB / c mice (Jackson Laboratories, Japan; CLEA, Japan n=3), nucleic acid lipid nanoparticles were administered into the brain parenchyma under isoflurane anesthesia (isoflurane inhalation anesthetic "VTRS"; Mylan Pharmaceutical Co., Ltd., hereinafter the same). During intraparenchymal administration, the drug delivery coordinates were determined based on mouse brain maps (Academic Press) at a depth of 2.5 mm, 0.0 mm anterior-posterior to the anterior fontanelle and 2.0 mm to the left. Under isoflurane anesthesia, after shaving with clippers, the skull was fixed using a brain positioning and fixation device (KOPF), and a hole with a diameter of approximately 1 mm was drilled using a drill bit (Foredom). 0.3 mg / mL of the test drug (FLuc nucleic acid lipid nanoparticles) was loaded into an Ito syringe (Ito Seisakusho), and 1 μL was administered to the designated coordinates using an infusion pump (Narishige) at a flow rate of 0.2 μL / min. After administration, the animal was left to stand for 1 minute, then the needle was slowly withdrawn, and the scalp was sutured. One day later, under isoflurane anesthesia, 250 μL / animal was administered intraperitoneally with 15 mg / mL fluorescein (Promega). Luminescence was confirmed 20 minutes later using an IVIS spectrometer. The obtained luminescence values ​​were quantified in photons per second using Living Image software for analysis.

[0306] (Expressing an evaluation)

[0307] The results of the above experiments showed that luciferase activity was observed in the lipids of the representative embodiments of the present invention. This indicates that the lipid nanoparticles containing FLuc mRNA were taken up into cells in the brain and translated to express the luciferase protein. The expression data of the nucleic acid lipid nanoparticles are shown in the table below (the average of three individuals of the evaluated FLuc nucleic acid lipid nanoparticles). For example, Ex1-L1 listed in NUM of the table below is Ex1-L1-FLuc mRNA. That is, Ex1-L1 is a lipid nanoparticle containing compound Ex1 of the examples, which has the same lipid composition as Ex1-L1 and contains FLuc mRNA.

[0308] [Table 5]

[0309] Experimental Example 3: In vitro transformation of primary rat astrocytes into nerve cells

[0310] (Addition of nucleic acid lipid nanoparticles to primary rat astrocytes)

[0311] Primary rat astrocytes were obtained and used according to Lynette C. Foo. Purification of Rat and Mouse Astrocytes by Immunopanning. Cold Spring Harbor Protocols, 2013, Vol. 5, pp. 421-432. Cell resuscitation was performed at 5% CO2. Newborn Wistar rat pups (CLEA, Japan, P1-P10) were used. Primary rat astrocytes were seeded at 40,000 cells / well in poly-D-lysine-coated 96-well plates (Corning, 354640) and cultured overnight at 37°C with 5% CO2. During culture, 100 μL / well was prepared using DMEM / F-12 medium (Thermo Fisher Scientific, 11320033) containing 2% B-27 (registered trademark) supplement (Thermo Fisher Scientific, A1895601), 10% fetal bovine serum (FBS, Cytiva, SH30070.03), and 1% penicillin-streptomycin (PS) (Thermo Fisher Scientific, 15070063).

[0312] The following day, all culture medium was removed, and ND1 nucleic acid lipid nanoparticles (Ex1-L1-ND1 mRNA) diluted with differentiation conversion medium were added to achieve a final concentration of 0.5 μg / mL, followed by incubation. In the control, PBS was diluted with differentiation conversion medium before addition. The differentiation conversion medium was Neurobasal Medium (Thermo Fisher Scientific, 21103049) containing 2% B-27 (trademark) supplement, 1% Glutamax (trademark) supplement (Thermo Fisher Scientific, 35050061), 2% fetal bovine serum, 1% MEM non-essential amino acids (Fujifilm, 139-15651), 1% PS, and 0.02% BDNF solution. It should be noted that the BDNF solution was prepared by dissolving BDNF powder (PeproTech, 450-02) in pure water to achieve a concentration of 100 μg / mL.

[0313] On the day following the addition of the ND1 nucleic acid lipid nanoparticles (Day 1) and on Day 3, all culture media were replaced using differentiation conversion medium.

[0314] (Confirmation of NeuroD1 and TUJ1 protein expression using Western blotting)

[0315] Eight hours after the addition of the ND1 nucleic acid lipid nanoparticles, and on day 7, the differentiation conversion medium was removed, cells were washed with PBS, and lysed using RIPA buffer (Thermo Fisher Scientific, 89901). Protein quantification was performed using the Lowry method, and 10 μg of each protein was subjected to electrophoresis. NeuroD1 protein expression was detected by Western blotting using an anti-NeuroD1 antibody (Abcam, ab60704). Additionally, TUJ1 protein expression was detected by Western blotting using an anti-TUJ1 antibody (Abcam, ab78078).

[0316] The results showed that ND1 protein was expressed within 8 hours of the addition of ND1 nucleic acid lipid nanoparticles. This indicates that the ND1 nucleic acid lipid nanoparticles were ingested and translated into ND1 protein, which was then expressed in rat primary astrocytes. Furthermore, it was shown that TUJ1 protein expression increased from 8 hours after ND1 nucleic acid lipid nanoparticle addition compared to the PBS-added sample, and this increase continued until day 7 after ND1 nucleic acid lipid nanoparticle addition. These results demonstrate that using lipid nanoparticles containing Ex1-L1 can deliver ND1 mRNA to rat primary astrocytes, resulting in ND1 protein expression from the ND1 mRNA within the cells. Due to the functionalization of this protein, the rat primary astrocytes were transformed into neural cells.

[0317] Experimental Example 4: Effect of ND1 Nucleic Acid Lipid Nanoparticles on a Cynomolgus Monkey Cerebral Infarction Model

[0318] (The creation of a model of cerebral infarction in cynomolgus monkeys)

[0319] Six male cynomolgus monkeys (aged 3 years and older, New Japan Science) were anesthetized via intramuscular injection (IM) at a dose of approximately 10 mg / kg body weight of ketamine hydrochloride (ketalar 500 mg for intramuscular injection; Daiichi Sankyo). The anesthetized monkeys were intubated and anesthesia was maintained by inhalation of isoflurane (Isoflurane inhalation anesthetic solution "VTRS"; Mylan Pharmaceutical Co., Ltd.) through the trachea under artificial or spontaneous respiration. The hair on the anesthetized monkeys' heads was shaved with clippers, and the monkeys were then secured to a brain fixation device (Narishige Co., Ltd.). Subsequently, while considering bleeding, the scalp of the monkeys was incised, and the anterior fontanelle, the intersection of the sagittal and coronal sutures on the anterior surface of the skull, was identified. The injection site was indicated as follows, using the anterior fontanelle (B: 0 mm, ML coordinate: 0 mm (center)) as the reference point.

[0320] • B: The distance along the front-to-back axis with the former halogen point as the reference point (positive values ​​indicate the distance to the front).

[0321] • L: Distance to the left from the base point of the ML coordinate system

[0322] • D: The depth of the brain in a vertically downward direction from the dura mater.

[0323] For six male cynomolgus monkeys, endothelin-1 (Peptide Research Institute, No. 4198-v) solution was injected. Specifically, for the cynomolgus monkey skull, the anterior fontanelle was used as the base point for injection, and holes with a diameter of about 1 mm (total of 4) were drilled at positions (i) B: 2.0 mm, L: 8.0 mm, (ii) B: 5.0 mm, L: 8.0 mm, (iii) B: 9.0 mm, L: 5.0 mm, and (iv) B: 9.0 mm, L: 12.0 mm. A microsyringe (HAMILTON) connected to a microsyringe (Narishige) was inserted through each hole, and the needle tip was left in place at a depth (D) of 19.0 mm (as described in (i) and (ii) above), 13.0 mm (as described in (iii) above), or 15.0 mm (as described in (iv) above) from the dura mater. Endothelin-1 was injected into a total of four sites corresponding to the locations described above for each individual using a microsyringe. Endothelin-1 was injected at a concentration of 2.0 μg / μL (prepared by dissolving 1% acetic acid (Wako, 017-00256) in Milli-Q water) at an injection rate of 1.5 μL / min, with a volume of 30 μL injected at each site. After injection, the syringe was allowed to stand for approximately 20 minutes to prevent leakage. The injection sites (i) to (iv) targeted (i) the ventrolateral nucleus and ventroposterior nucleus of the thalamus, (ii) the globus pallidus, (iii) the caudate nucleus, and (iv) the putamen, respectively. By injecting endothelin into these sites as described above, obstruction of the internal capsule was achieved in addition to the target sites. This created a cerebral infarction model with obstruction of the basal ganglia, thalamus, and internal capsule. This model can evaluate perforator infarction, cerebral infarction with brain dysfunction in the perforator innervation area, subacute to chronic cerebral infarction, and cerebral infarction with severe motor dysfunction.

[0324] (Administration of ND1 nucleic acid lipid nanoparticles to the model)

[0325] Six individuals with a cerebral infarction model established using the above method were divided into two groups (control group n=3 and ND1 nucleic acid lipid nanoparticle group n=3) on day 21 after endothelin-1 injection (chronic phase) in a manner that did not produce inter-group differences in mRS efficacy (see table below). It should be noted that the control group (3 individuals) received Ex1-L1-eGFP mRNA, while the ND1 nucleic acid lipid nanoparticle group (3 individuals) received Ex1-L1-ND1 mRNA.

[0326] In the diagnosis and treatment of stroke in humans, the mRS (human clinical version) listed in the table below is widely used as an indicator of physical impairment. The mRS (monkey version) is an indicator formed by modifying the mRS (human clinical version) for monkey use. As an indicator used in monkey motor function assessment, the National Institutes of Health Stroke Scale (NIHSS), which is used to assess the neurological severity of stroke in humans, is known to be suitable for the Non-Human Primate Stroke Scale (NHPSS) in non-human primate models. The mRS (monkey version) can also assess the persistence of motor function impairment, just like the NHPSS. In the assessment of the persistence of motor function impairment in this model, the mRS can be used.

[0327] [Table 6]

[0328] 1 Mild paralysis: No obvious paralysis is observed, but slight paralysis symptoms are visible.

[0329] 2 Paralysis: Obvious paralysis can be observed.

[0330] 3 Mild reduction in voluntary movement: No obvious reduction in voluntary movement is observed, but symptoms of reduced voluntary movement are slightly visible.

[0331] 4 Reduced voluntary movement: A clear reduction in voluntary movement is observed.

[0332] 5 Level of consciousness (monkey): judged based on the level of responsiveness to humans.

[0333] 6 Sitting position / Lady-side position: at least one of sitting position or lateral position.

[0334] 7 Mild decrease in level of consciousness: No obvious decrease in level of consciousness is observed, but symptoms of a slight decrease in level of consciousness are visible.

[0335] 8 Decreased level of consciousness: A clear decrease in the level of consciousness can be observed.

[0336] For each group of individuals with cerebral infarction, anesthesia was administered via intramuscular injection of ketamine hydrochloride at a dose of approximately 10 mg / kg body weight, and maintained by inhalation of isoflurane. While under anesthesia, the hair on the head was shaved with clippers, and the individual was then secured to a brain fixation device. The surgical area was then disinfected, and the scalp was incised to expose the anterior fontanelle, taking into account bleeding.

[0337] In each group of individuals with cerebral infarction models, endothelin-1 was injected into four wells at a total of four sites during the creation of the cerebral infarction model. The microsyringe was left in place so that the needle tip reached the same depth (D) as when injecting endothelin-1. Ex1-L1-ND1 mRNA was administered to three individuals, and Ex1-L1-eGFP mRNA was administered to three individuals. Each nucleic acid-lipid nanoparticle was prepared at a concentration of 0.3 mg / mL using a solvent (PBS or Tris buffer) and administered at a rate of 30 μL / site at a rate of 1.5 μL / min. After administration, the syringe was allowed to stand for approximately 20 minutes.

[0338] (Observation of motor function)

[0339] For each individual in the ND1 nucleic acid lipid nanoparticle group and control group in the above-mentioned (administration of ND1 nucleic acid lipid nanoparticles to the model), mRS-based observations were performed before endothelin-1 injection treatment (day 0) and on days 1, 7, 14, 21, 22, 28, 42, 56, 70, and 84 after endothelin-1 injection treatment. It should be noted that performance evaluations were performed every two weeks after day 28 after endothelin-1 injection treatment, serving as the performance evaluation on day 28 after endothelin-1 injection (day 7 after nucleic acid lipid nanoparticle administration), and on days 28 or 29 after endothelin-1 injection (day 7 or 8 after nucleic acid lipid nanoparticle administration). These results are considered as the results on day 28 after endothelin-1 injection (day 7 after nucleic acid lipid nanoparticle administration) in this example.

[0340] The results showed that on day 1 after administration, there was no difference in motor function between the ND1 nucleic acid lipid nanoparticle group and the control group, and the same motor dysfunction as before administration of the nucleic acid lipid nanoparticles was observed in both groups. However, the control group also showed no improvement in motor function on day 84 after endothelin-1 injection (day 63 after administration of the nucleic acid lipid nanoparticles). In contrast, the ND1 nucleic acid lipid nanoparticle group began to show recovery of motor function after day 70 after endothelin-1 injection (day 49 after administration of the nucleic acid lipid nanoparticles), and significant recovery of motor function was confirmed on day 84 after endothelin-1 injection (day 63 after administration of the nucleic acid lipid nanoparticles). Figure 1 This indicates that administering ND1 nucleic acid lipid nanoparticles can restore motor dysfunction associated with brain disorders.

[0341] Based on the above results, the administration of ND1 nucleic acid lipid nanoparticles can treat cerebral infarction (especially perforator infarction, cerebral infarction with brain dysfunction in the perforator innervation area, subacute to chronic cerebral infarction, and cerebral infarction with severe motor dysfunction).

[0342] Experimental Example 5: Effect of ND1 Nucleic Acid Lipid Nanoparticles on a Spinal Cord Injury Model

[0343] (Evaluation Method)

[0344] Regarding the effects on spinal cord injury, a spinal cord injury model can be used for evaluation. The state of motor dysfunction in the spinal cord injury model can be evaluated using the Field Rating Scale. The Field Rating Scale can be used after modification of the Original Open Field Rating Scale (see PLoS ONE, 2011, Vol. 6, 11, e27706). When an individual cannot maintain a sitting posture and the total score on the Field Rating Scale is less than 10, it can be judged that the animal has limb paralysis.

[0345] [Table 7]

[0346] [Table 8]

[0347] [Table 9] 1. Forelimbs protruding from the cage bottom (forelimbs unable to move): When in a prone position in a cage with a grid-like bottom, the forelimbs dangle from the gaps in the grid on the bottom of the cage and cannot be raised. 2. Elbow extended above the cage bottom: When in a prone position in the cage described above, the elbow can be raised above the bottom surface of the cage through the gaps in the grid. 3. Extending from the wrist to the bottom of the cage: When in a prone position in the cage described above, the wrist can be raised from the gaps in the grid at the bottom of the cage to above the bottom surface. 4. Hands not extending out of the cage bottom: When in a prone position in the cage described above, the forelimbs are able to support the body without extending out of the gaps in the cage bottom grid. 5. Hind limbs protruding from the cage bottom (hind limbs unable to move): When in a prone position in the cage described above, the hind limbs dangle from the gaps in the cage bottom grille and cannot be raised. 6. Knee-length extension above the cage bottom: When in a prone position in the cage described above, the knees can be raised above the bottom of the cage through the gaps in the grid. 7. Extending up to the ankles above the bottom of the cage: When in a prone position in the cage described above, the ankles can be raised above the bottom of the cage through the gaps in the grid. 8. Feet not protruding from the cage bottom: When in a prone position in the cage described above, the hind limbs are able to support the body without dangling from the gaps in the cage bottom grille. (Creation of a spinal cord injury model) For common marmosets, ketamine hydrochloride and xylazine were used for endotracheal anesthesia. An Atom feeding tube (6Fr) was inserted into the anesthetized marmoset as an endotracheal tube, and anesthesia was maintained by inhalation of isoflurane. The fur on the back of the anesthetized marmoset was shaved with clippers, and the marmoset was placed prone on a 37°C warming pad and kept immobilized. The skin of the neck was incised, connective tissue was removed to expose the vertebrae, and the vertebral arch containing the spinous process of cervical vertebra C5 was removed to expose the spinal cord (cervical spinal cord).

[0348] Using a 2.5mm circular contact head installed on the impactor, the spinal cord is compressed from both sides to the dorsal surface of the middle and cervical spinal cord exposed by removing the vertebral arch of the C5 cervical vertebra, on the left and right sides of the midline of the spinal cord, under a pressure of 280 kdyn for several seconds each.

[0349] Following the compression, to inhibit spasms and contractures, as part of rehabilitation training, several flexion-extension exercises were performed on the finger joints, wrist (ankle), elbow (knee), and shoulder (hip) joints of the left and right forelimbs of the common marmoset. Starting from the 5th to 7th day after the compression, rehabilitation training was implemented 1 to 2 times a day during the observation period.

[0350] (Administering nucleic acid lipid nanoparticles to the damaged area)

[0351] For common marmosets after crushing, motor dysfunction was evaluated using the Field Rating Scale. Individuals who were unable to maintain a sitting position several weeks after crushing and whose total Field Rating Scale score was less than 10 were divided into a control group and a drug administration group in a way that made the scores between groups equal.

[0352] After compression, under anesthesia administered with ketamine hydrochloride and xylazine, and maintained with isoflurane inhalation, the compressed area was exposed again. Nucleic acid lipid nanoparticles were administered using a syringe attached to a microinjector to a depth of approximately 1 mm on the dorsal surface of the cervical spinal cord, approximately 1 mm from the center of the compression site on the left side of the spinal cord. Nucleic acid lipid nanoparticles were then administered to the center of the compression site on the right side of the spinal cord in the same manner. After administration, the syringe was left to stand for several minutes, and then the muscle and skin were sutured. Several individuals in the drug administration group were administered the nucleic acid lipid nanoparticle solution, while several individuals in the control group were administered eGFP mRNA.

[0353] While assessing motor dysfunction using the Field Rating Scale, grip strength of both forelimbs was measured. Grip strength was measured using a device consisting of a basket easily grasped by the marmoset's forelimbs attached to a digital dynamometer (hereinafter referred to as grip strength test). Specifically, the marmoset grasped the basket, and the test administrator pulled on the marmoset until it released the basket. The force with which the marmoset gripped the basket was measured using the digital dynamometer, and this measurement was taken as the marmoset's grip strength. Motor dysfunction scoring and grip strength testing were performed at the same time point, and the marmoset was observed and evaluated for several weeks after drug administration.

[0354] Example

[0355] The following examples further illustrate in detail the method for manufacturing compounds of formula (I) or their salts.

[0356] It should be noted that the present invention is not limited to the compounds described in the examples. Furthermore, the methods for manufacturing the raw material compounds are shown in the manufacturing examples. Additionally, the methods for manufacturing compounds of formula (I) or their salts are not limited to the methods described in the specific examples; compounds of formula (I) or their salts can also be manufactured by combinations of these manufacturing methods or by methods obvious to those skilled in the art.

[0357] In addition, the following abbreviations are sometimes used in this specification, examples, manufacturing examples and tables.

[0358] CDI: 1,1'-carbonyldiimidazole, CPME: cyclopentylmethyl ether, DCM: dichloromethane, DIPEA: N,N-diisopropylethylamine, DMAP: 4-(dimethylamino)pyridine, DMF: N,N-dimethylformamide, DMSO: dimethyl sulfoxide, DPPA: diphenyl azidophosphate, EDCI·HCl: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EtOH: ethanol, HATU: 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine 3-Oxide hexafluorophosphate, HOBt: 1-hydroxybenzotriazole, MeCN: acetonitrile, TEA: triethylamine, TFA: trifluoroacetic acid, NUM: Example number or manufacturing example number ( / HCl indicates that the example or manufacturing example is a hydrochloride salt), REF: Manufacturing example number or example number with reference to the manufacturing method, PEx: Manufacturing example number, Ex: Example number, STR: Chemical structural formula, DAT: Physicochemical data, NMR: Indicates 500MHz 1The chemical shift δ value of H-NMR (ppm) (the value in parentheses in NMR indicates the solvent used in the determination, e.g., (CDCl3) is the value measured in deuterated chloroform. The NMR signal represents a representative signal). s: singlet, t: triplet, m: multiplet, br: broad peak, ESI+: m / z value in ESI-MS+, CI+: m / z value in CI-MS+, HPLC Rt: retention time (minutes) in high-performance liquid chromatography (measured using a CHIRAL ART Amylose-SA 250mm × 4.6mm ID, S-3μm, with 10mM ammonium acetate in 2-propanol as the mobile phase, at a column temperature of 12℃ and a flow rate of 0.25mL / min using a Corona (registered trademark) electro-cavitation detector (CAD). It should be noted that in this specification, the names of compounds sometimes use naming software such as ACD / Name (registered trademark, Advanced Chemistry Development, Inc.). Additionally, for convenience, concentrations in mol / L are expressed as M. For example, 1M sodium hydroxide aqueous solution refers to a 1 mol / L sodium hydroxide aqueous solution.

[0359] Manufacturing Example 1-1

[0360] 8-Bromooctanoic acid (2.97 g), DIPEA (4.7 mL), HATU (5.4 g), and DMAP (135 mg) were added to a mixture of 2-nonylundecane-1-ol (3.25 g) and DCM (40 mL) in a water bath, and the mixture was stirred at room temperature for 6.5 hours. After adding chloroform and water to the reaction mixture, the organic layer was separated, and the aqueous layer was extracted with chloroform. The combined organic layers were washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give 2-nonylundecane 8-bromooctanoic acid (5.27 g) as an oil.

[0361] Manufacturing Examples 1-2

[0362] To a mixture of 2-octyldecane-1-ol (500 mg) and DCM (5 mL), {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}acetic acid (511 mg), DMAP (24 mg), DIPEA (798 μL), and HATU (918 mg) were added, and the mixture was stirred at room temperature for 6 hours. Chloroform and water were added to the reaction mixture, the organic layer was separated, and the aqueous layer was extracted with chloroform. The combined organic layers were washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give 2-octyldecyl {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}acetic acid 879 mg as an oil.

[0363] Manufacturing Examples 1-3

[0364] TFA (1.5 mL) was added to an 8 mL mixture of DCM containing {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}acetate 2-octyldecyl ester (877 mg), and the mixture was stirred for 6 hours at room temperature. The reaction mixture was then added to a mixture of chloroform and a saturated aqueous solution of sodium bicarbonate, and the organic layer was separated. The aqueous layer was extracted with chloroform. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 2-octyldecyl [(1r,3r)-3-aminocyclobutyl]acetate 2-octyldecyl ester (661 mg) as an oil.

[0365] Manufacturing Examples 1-4

[0366] A mixture of 2-[(1r,3r)-3-aminocyclobutyl]acetic acid 2-octyldecyl ester (660 mg) and a mixture of 2-nonyl undecyl 8-bromooctanoic acid ester (430 mg) and CPME (4 mL), DIPEA (731 μL), and KI (30 mg) was added to a MeCN (4 mL) mixture at room temperature, and the mixture was stirred in an oil bath at 80 °C for 48 hours. The reaction mixture was then added to a mixture of chloroform and a saturated aqueous sodium bicarbonate solution, and the organic layer was separated. The aqueous layer was extracted with chloroform. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by amino silica gel column chromatography (hexane / ethyl acetate), and the crude purified product was further purified by silica gel column chromatography (chloroform / methanol) to give 2-nonyl undecyl 8-{[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid ester (452 ​​mg) as an oil.

[0367] Example 1

[0368] At room temperature, a mixture of 2-nonyl undecyl ester of 8-{[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid (1200 mg) and DCM (24 mL) was added to 1-methyl-L-proline (11.8% water, 290 mg), HATU (910 mg), DMAP (27 mg), and DIPEA (510 μL), and stirred at room temperature for 4 hours. Water was added to the reaction mixture, the organic layer was separated, and the mixture was concentrated under reduced pressure. Heptane and a 90% aqueous methanol solution were added to the resulting residue, the heptane layer was separated, and the mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (heptane / ethyl acetate), and the crude purified product was purified by amino silica gel column chromatography (heptane / ethyl acetate) to obtain 1.2 g of 2-nonyl undecyl octanoic acid ester of 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid in the form of an oil.

[0369] Example 2

[0370] A mixture of 2-nonyl undecyl octanoate (120 mg) and DCM (3 mL) was added to a mixture of 1-methyl-D-proline monohydrate (26 mg), HATU (68 mg), and DIPEA (0.038 mL), and stirred at room temperature for 15 hours. The reaction mixture was purified by silica gel column chromatography (chloroform / methanol), and the crude purified product was purified by amino silica gel column chromatography (hexane / ethyl acetate) to give 2-nonyl undecyl octanoate (129 mg) as an oil.

[0371] Manufacturing Example 4-1

[0372] To a mixture of 2-octyldecane-1-ol (620 mg) and DCM (20 mL), {3-[(tert-butoxycarbonyl)amino]bicyclo[1.1.1]pentan-1-yl}acetic acid (500 mg), HATU (950 mg), DIPEA (0.54 mL), and DMAP (25 mg) were added, and the mixture was stirred at room temperature for 40 hours. The reaction mixture was diluted with ethyl acetate and washed with a saturated aqueous sodium bicarbonate solution. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give 2-octyldecyl {3-[(tert-butoxycarbonyl)amino]bicyclo[1.1.1]pentan-1-yl}acetic acid 900 mg as an oil.

[0373] Manufacturing Example 4-2

[0374] TFA (1.5 mL) was added to a mixture of {3-[(tert-butoxycarbonyl)amino]bicyclo[1.1.1]pentan-1-yl}acetate 2-octyldecyl ester (900 mg) and DCM (8 mL) under ice-cooling, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with chloroform and neutralized by adding a saturated aqueous solution of sodium bicarbonate under ice-cooling. The mixture was extracted with chloroform, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform / methanol) to give 2-octyldecyl (3-aminobicyclo[1.1.1]pentan-1-yl)acetate (685 mg) as an oil.

[0375] Manufacturing Example 4-3

[0376] Add 2-octyldecyl 8-bromooctanoate (450 mg), CPME (6 mL), DIPEA (0.4 mL), and KI (15 mg) to a mixture of (3-aminobicyclo[1.1.1]pentan-1-yl)acetic acid 2-octyldecyl ester (680 mg) and MeCN (6 mL), and stir in an oil bath at 80 °C for 3.5 days under an argon atmosphere. After the reaction mixture was allowed to cool naturally, it was diluted with ethyl acetate and washed with water. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give 2-nonyluncealkyl 8-[3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}bicyclo[1.1.1]pentan-1-yl)amino]octanoate 2-nonyluncealkyl ester (482 mg) as an oil.

[0377] Example 4

[0378] A mixture of 2-nonyl undecyl ester of 8-[(3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}bicyclo[1.1.1]pentan-1-yl)amino]octanoic acid (150 mg) and DCM (3 mL) was added with 1-methyl-L-proline (11.8% water, 33 mg), HATU (84 mg), and DIPEA (0.031 mL), and stirred at room temperature for 24 hours. The reaction mixture was purified by silica gel column chromatography (chloroform / methanol), and the crude purified product was purified by amino silica gel column chromatography (hexane / ethyl acetate) to give 2-nonyl undecyl ester of 8-[(1-methyl-L-prolyl)(3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}bicyclo[1.1.1]pentan-1-yl)amino]octanoic acid (128 mg) as an oil.

[0379] Manufacturing Example 5-1

[0380] DIPEA (1.7 mL) was added to a mixture of (9Z,12Z)-octadec-9,12-dien-1-ol (1.5 mL) and DCM (15 mL) at room temperature. Chloroacetyl chloride (0.58 mL) was added dropwise under ice cooling, and the mixture was stirred at room temperature for 1 hour. Water was added to the reaction mixture, and the separated aqueous layer was extracted with chloroform. The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give (9Z,12Z)-octadec-9,12-dien-1-yl chloroacetate (1.47 g) as an oil.

[0381] Example 7

[0382] To a mixture of 2-nonyl undecyl octanoate (120 mg) and DCM (3 mL), N,N-diethyl-β-alanine (26 mg), HATU (68 mg), and DIPEA (0.038 mL) were added, and the mixture was stirred at room temperature for 18 hours. The reaction mixture was purified by silica gel column chromatography (chloroform / methanol), and the crude purified product was purified by amino silica gel column chromatography (hexane / ethyl acetate / methanol) to give 2-nonyl undecyl octanoate (119 mg) as an oil.

[0383] Manufacturing Example 9-1

[0384] DIPEA (1.1 mL) was added to a mixture of tert-butyl aziridine-3-carboxylate hydrochloride (500 mg), N,N-dimethylglycine (320 mg), HATU (1.18 g), and DCM (10 mL), and the mixture was stirred at room temperature for 18 hours. A saturated aqueous sodium bicarbonate solution was added to the reaction mixture, and the separated organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol / 28% ammonia), and the crude purified product was further purified by amino silica gel column chromatography (hexane / ethyl acetate / methanol) to give 1-(N,N-dimethylglycyl)aziridine-3-carboxylate tert-butyl ester (350 mg) as an oil.

[0385] Manufacturing Example 9-2

[0386] TFA (2.2 mL) was added to a mixture of 1-(N,N-dimethylglycyl)azacyclobutane-3-carboxylate (350 mg) and DCM (2 mL) at room temperature, and the mixture was stirred for 18 hours at room temperature. TFA (1.1 mL) was then added to the reaction mixture, and the mixture was stirred for 2 hours at room temperature. The reaction mixture was then concentrated under reduced pressure. This process was repeated four times, and the resulting residue was then added with 4 M hydrogen chloride / 1,4-di(N,N-dimethylglycyl)azine-3-carboxylate. The process involved alkylating a solution (5 mL) and concentrating it under reduced pressure, thereby yielding 1-(N,N-dimethylglycyl)azacyclobutane-3-carboxylate (492 mg) as an oily crude product.

[0387] Manufacturing Example 12-1

[0388] DIPEA (1.82 mL) and iodoethane (0.51 mL) were added to a mixture of methyl 1,4-diazacycloheptan-6-carboxylate hydrochloride (491 mg) and MeCN (10 mL) at room temperature, and the mixture was stirred in an oil bath at 60 °C for 10 hours. After the reaction mixture was allowed to cool naturally to room temperature, it was concentrated under reduced pressure. Ethyl acetate was added to the residue, and the mixture was stirred at room temperature for 15 minutes. The insoluble matter was then filtered off. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by amino silica gel column chromatography (hexane / ethyl acetate / methanol). The crude purified product was then purified by silica gel column chromatography (chloroform / methanol) to give methyl 1,4-diethyl-1,4-diazacycloheptan-6-carboxylate (167 mg) as an oil.

[0389] Example 12

[0390] Add 0.25 mL of 1 M sodium hydroxide aqueous solution to a mixture of methyl 1,4-diethyl-1,4-diazacycloheptan-6-carboxylate (35 mg) and methanol (2.5 mL) at room temperature and stir for 10 hours at room temperature. Add 0.25 mL of 1 M hydrochloric acid to the reaction mixture and concentrate under reduced pressure. Add methanol / chloroform to the residue and concentrate under reduced pressure. Repeat this operation three times. Add 62 mg of HATU, a mixture of 110 mg of 2-nonyl undecyl octanoate and DCM (3 mL), and 0.035 mL of DIPEA to the residue and stir for 10 hours at room temperature. The reaction mixture was purified by silica gel column chromatography (chloroform / methanol), and the crude purified product was purified by amino silica gel column chromatography (hexane / ethyl acetate / methanol) to give 2-nonyl undecyl ester of 8-{(1,4-diethyl-1,4-diazacycloheptane-6-carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid (74 mg) as an oil.

[0391] Manufacturing Example 14-1

[0392] A mixture of (1r,3r)-3-(hydroxymethyl)cyclobutyl]carbamate tert-butyl ester (500 mg) and DCM (30 mL) was added with (9Z,12Z)-octadec-9,12-dienoic acid (0.81 mL), DIPEA (0.64 mL), EDCI·HCl (0.72 g), and DMAP (61 mg), and stirred at room temperature for 18 hours. The reaction mixture was diluted with DCM, washed with brine, and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give (9Z,12Z)-octadec-9,12-dienoic acid {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}methyl ester (1.06 g) as an oil.

[0393] Manufacturing Example 16-1

[0394] A mixture of [(1r,3r)-3-aminocyclobutyl]acetic acid 2-octyldecyl ester (1.06 g) and CPME (8 mL), along with DIPEA (0.6 mL), was added to a mixture of [(2-bromoethoxy)methyl]benzene (300 mg) and MeCN (8 mL) at room temperature. The mixture was stirred in an oil bath at 80 °C for 40 hours. After the reaction mixture was allowed to cool naturally to room temperature, ethyl acetate and a saturated aqueous sodium bicarbonate solution were added. The separated organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by amino silica gel column chromatography (ethyl acetate / hexane) to give [(1r,3r)-3-{[2-(benzyloxy)ethyl]amino}cyclobutyl]acetic acid 2-octyldecyl ester (474 ​​mg) as an oil.

[0395] Manufacturing Example 16-3

[0396] Palladium-activated carbon (Pd 10%) (26 mg) was added to a mixture of [(1S,3r)-3-{[2-(benzyloxy)ethyl](1-methyl-L-prolyl)amino}cyclobutyl]acetic acid 2-octyldecyl ester (263 mg) and 2-propanol (5 mL) under argon atmosphere and at room temperature, and stirred for one weekend under hydrogen atmosphere and at room temperature. Palladium-activated carbon (Pd 10%) (26 mg) was subsequently added to the reaction mixture under argon atmosphere and at room temperature, and stirred for 24 hours under hydrogen atmosphere and at room temperature at 3 atm. Palladium-activated carbon (Pd 10%) (26 mg) and acetic acid (0.072 mL) were subsequently added to the reaction mixture under argon atmosphere and at room temperature, and stirred for 24 hours under hydrogen atmosphere and at room temperature at 3 atm. Palladium-activated carbon (Pd 10%) (26 mg) was added to the reaction mixture under argon atmosphere and at room temperature, and the mixture was stirred for 24 hours under hydrogen atmosphere at 3 atm and at room temperature. The reaction mixture was filtered through diatomaceous earth (registered trademark), and the filtrate was concentrated under reduced pressure. Ethyl acetate and saturated sodium bicarbonate aqueous solution were added to the residue, the organic layer was separated, washed with saturated brine, dried with anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform / methanol) to give 140 mg of {(1S,3r)-3-[(2-hydroxyethyl)(1-methyl-L-prolyl)amino]cyclobutyl}acetic acid 2-octyldecyl ester.

[0397] Example 16

[0398] Add HATU (64 mg), DIPEA (0.045 mL), a mixture of {(1S,3r)-3-[(2-hydroxyethyl)(1-methyl-L-prolyl)amino]cyclobutyl}acetate 2-octyldecyl ester (70 mg) and DCM (1 mL), and DMAP (1.6 mg) to a mixture of (9Z,12Z)-octadec-9,12-dienoic acid (0.053 mL) and DCM (1 mL) at room temperature, and stir for 18 hours at room temperature. The reaction mixture was purified by silica gel column chromatography (chloroform / methanol), and the crude purified product was purified by amino silica gel column chromatography (hexane / ethyl acetate / methanol) to give (9Z,12Z)-octadec-9,12-dienoic acid 2-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}ethyl ester (78 mg) as an oil.

[0399] Example 21

[0400] At room temperature, a mixture of 2-nonyl undecyl ester of 8-{[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid (151 mg) and DCM (3 mL) was added to (4-methylpiperazin-1-yl)acetic acid (39.4 mg), DIPEA (0.096 mL), and HATU (92.7 mg), and stirred for 3 hours at room temperature. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, and the aqueous layer was extracted with chloroform. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified by amino silica gel column chromatography (hexane / ethyl acetate) to give 2-nonyl undecyl ester of 8-{[(4-methylpiperazin-1-yl)acetyl][(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid (132 mg) as an oil.

[0401] Manufacturing Example 22-2

[0402] TFA (3 mL) was added to a mixture of 4-({8-[(2-nonylundecyl)oxy]-8-oxooctyl}[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]carbamoyl)piperidine-1-carboxylic acid tert-butyl ester (614 mg) and DCM (6 mL) under ice-cooling conditions, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with chloroform, and after being made alkaline by adding saturated sodium bicarbonate solution under ice-cooling conditions, it was extracted with chloroform. The organic layer was dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and the residue was purified by amino silica gel column chromatography (hexane / ethyl acetate) to give 8-{[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl](piperidine-4-carbonyl)amino}octanoic acid 2-nonylundecyl ester (521 mg) as an oil.

[0403] Example 22

[0404] Sodium triacetoxyborohydride (131 mg) was added to a mixture of 2-nonyl undecyl octanoate (165 mg) and DCM (5 mL) at room temperature, and the mixture was stirred for 10 minutes at room temperature. Acetaldehyde (0.1 mL) was added to the reaction mixture under ice cooling, and the mixture was stirred for 17 hours at room temperature. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, and the mixture was extracted with chloroform. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform / methanol), and the crude purified product was purified by amino silica gel column chromatography (hexane / ethyl acetate) to obtain 2-nonyl undecyl ester of 8-{(1-ethylpiperidine-4-carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid (139 mg) in the form of an oil.

[0405] Manufacturing Example 24-1

[0406] Pyridine (6 mL), 4-nitrobenzene chloroformate (2.98 g), and DMAP (45 mg) were added to a mixture of 2-octyldecane-1-ol (2 g) and DCM (60 mL) under ice-cooling conditions, and the mixture was stirred at room temperature for 68 hours. The reaction mixture was concentrated under reduced pressure, and hexane was added to the resulting residue to remove insoluble matter. The filtrate was concentrated under reduced pressure to give 2.96 g of 4-nitrobenzene carbonate-2-octyldecyl carbonate as an oil.

[0407] Manufacturing Example 24-2

[0408] A mixture of 4-nitrophenyl carbonate = 2-octyldecyl carbonate (1.4 g) and DCM (40 mL) was added with pyridine (5 mL), [(1r,3r)-3-hydroxycyclobutyl]carbamate tert-butyl ester (1.81 g), and DMAP (80 mg), and stirred at room temperature for 2 days. The reaction mixture was concentrated under reduced pressure, diluted with ethyl acetate / hexane, and washed with saturated sodium bicarbonate aqueous solution, water, and saturated brine. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give 2-octyldecyl carbonate = (1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl ester (1.33 g) as an oil.

[0409] Manufacturing Example 25-1

[0410] Pyridine (5 mL), [(1r,3r)-3-(hydroxymethyl)cyclobutyl]carbamate (1.95 g), and DMAP (80 mg) were added to a mixture of 4-nitrobenzene carbonate-2-octyldecyl carbonate (1.4 g) and DCM (40 mL), and the mixture was stirred at room temperature for 2 days. The reaction mixture was concentrated under reduced pressure, diluted with ethyl acetate / hexane, and washed with saturated sodium bicarbonate solution, water, and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give methyl carbonate-2-octyldecyl carbonate (1.18 g) as an oil.

[0411] Manufacturing Example 25-2

[0412] TFA (5 mL) was added to a mixture of methyl carbonate ={(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}methyl ester = 2-octyldecyl ester (1.18 g) and DCM (10 mL) under ice-cooling conditions, and the mixture was stirred at room temperature for 2 hours. After neutralization with saturated aqueous sodium bicarbonate solution, the reaction mixture was extracted with chloroform. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by amino silica gel column chromatography (hexane / ethyl acetate) to give methyl carbonate =[(1r,3r)-3-aminocyclobutyl]methyl ester = 2-octyldecyl ester (883 mg) as an oil.

[0413] Manufacturing Example 25-3

[0414] Under an argon atmosphere, a mixture of methyl carbonate = [(1r,3r)-3-aminocyclobutyl]methyl ester = 2-octyldecyl carbonate (882 mg) and MeCN (8 mL) was added to 2-nonylundecyl 8-bromooctanoate (560 mg), CPME (8 mL), DIPEA (0.5 mL), and KI (20 mg), and stirred in an oil bath at 80 °C for 2.5 days. The reaction mixture was allowed to cool naturally, diluted with ethyl acetate, and washed with saturated aqueous sodium bicarbonate solution and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by amino silica gel column chromatography (hexane / ethyl acetate) to give 2-nonylundecyl 8-({(1r,3r)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino)octanoate (630 mg) as an oil.

[0415] Example 25

[0416] To a mixture of 2-nonyl undecyl ester of 8-({(1r,3r)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino)octanoic acid (160 mg) and DCM (3 mL), 1-methyl-L-proline monohydrate (35 mg), HATU (90 mg), and DIPEA (70 μ L) were added, and the mixture was stirred at room temperature for 14 hours. The reaction mixture was diluted with ethyl acetate / hexane (1 / 1) and washed with saturated aqueous sodium bicarbonate solution, water, and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform / methanol), and the crude purified product was purified by amino silica gel column chromatography (hexane / ethyl acetate) to obtain 2-nonyl undecyl ester of 8-[(1-methyl-L-prolyl){(1r,3S)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]octanoic acid (147 mg) as an oil.

[0417] Example 26

[0418] 2-Nonylundecyl octanoate (750 mg) was added to a mixture of 3-chloropropionyl chloride (360 mg), DIPEA (1 mL), and DCM (15 mL) at room temperature and stirred for 2 hours. Heptane and 90% aqueous methanol were added to the reaction mixture, the heptane layer was separated, washed with 90% aqueous methanol, and concentrated under reduced pressure to give 2-nonylundecyl octanoate (850 mg) as an oily crude product. 150 mg of 2-nonyl undecyl octanoate was added to a mixture of diethanolamine (0.61 g) and EtOH (3 mL) at room temperature and stirred for 20 hours. The reaction mixture was further stirred in an oil bath at 55 °C for 5 hours and then allowed to cool naturally to room temperature. Heptane and water were added to the reaction mixture, the organic layer was separated, washed with 90% methanol aqueous solution, and concentrated under reduced pressure. The residue was purified by amino silica gel column chromatography (heptane / ethyl acetate) to give 100 mg of 2-nonyl undecyl octanoate as an oil.

[0419] Example 28

[0420] 8-{3-chloro-N-[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]propaneamide}2-nonylundecyl octanoate (120 mg), an intermediate of Example 26, was added to a mixture of glycine hydrochloride (520 mg), DIPEA (2 mL), and EtOH (2 mL) at room temperature, and the mixture was stirred for 2 hours at room temperature. The reaction mixture was further stirred in an oil bath at 55°C for 17 hours, then stirred in an oil bath at 75°C for 2 hours, and allowed to cool naturally to room temperature. Heptane and water were added to the reaction mixture, the organic layer was separated, washed with 90% methanol aqueous solution, and concentrated under reduced pressure. The residue was purified by amino silica gel column chromatography (heptane / ethyl acetate) to give 2-nonyl undecyl ester of 8-{[N-(2-amino-2-oxoethyl)-β-alanyl][(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid (45 mg) as an oil.

[0421] Manufacturing Example 30-1

[0422] 8-Bromooctanoic acid (554 mg), DIPEA (0.891 mL), HATU (1.01 g), and DMAP (27.2 mg) were added to a mixture of 2-heptylnonane-1-ol (501 mg) and DCM (10 mL) in a water bath, and the mixture was stirred at room temperature for 6.5 hours. After adding chloroform and water to the reaction mixture, the separated aqueous layer was extracted with chloroform. The combined organic layers were dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give 0.787 g of 2-heptylnonyl 8-bromooctanoic acid as an oil.

[0423] Manufacturing Example 30-2

[0424] (1r,4r)-4-[(tert-butoxycarbonyl)amino]cyclohexane-1-carboxylic acid (767 mg), DIPEA (0.898 mL), HATU (1.19 g), and DMAP (26.6 mg) were added to a mixture of 2-heptylnonane-1-ol (508 mg) and DCM (20 mL) in a water bath, and the mixture was stirred at room temperature for 24 hours. Chloroform and water were added to the reaction mixture, and the separated aqueous layer was extracted with chloroform. The combined organic layers were dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give 2-heptylnonyl (1r,4r)-4-[(tert-butoxycarbonyl)amino]cyclohexane-1-carboxylic acid as an oil (0.761 g).

[0425] Manufacturing Example 30-3

[0426] DCM (10 mL) and TFA (1.3 mL) were added to (1r,4r)-4-[(tert-butoxycarbonyl)amino]cyclohexane-1-carboxylic acid 2-heptylnonyl ester (0.76 g) at room temperature, and the reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with chloroform and neutralized with saturated sodium bicarbonate aqueous solution. The separated aqueous layer was extracted with chloroform. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by amino silica gel column chromatography (hexane / ethyl acetate) to give (1r,4r)-4-aminocyclohexane-1-carboxylic acid 2-heptylnonyl ester (570 mg) as an oil.

[0427] Manufacturing Example 30-4

[0428] A mixture of CPME (4 mL), MeCN (5 mL), DIPEA (0.26 mL), KI (24.5 mg), 2-heptylnonyl 8-bromooctanoate (331 mg), and CPME (1 mL) was added to (1r,4r)-4-aminocyclohexane-1-carboxylic acid 2-heptylnonyl ester (570 mg) at room temperature and stirred in an oil bath at 80 °C for 2 days. The reaction mixture was cooled to room temperature, and chloroform, water, and a saturated sodium bicarbonate aqueous solution were added. The separated aqueous layer was extracted with chloroform. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate), and the crude purified product was purified by amino silica gel column chromatography (hexane / ethyl acetate) to obtain (1r,4r)-4-({8-[(2-heptylnonyl)oxy]-8-oxooctyl}amino)cyclohexane-1-carboxylic acid 2-heptylnonyl ester (456 mg) as an oil.

[0429] Example 30

[0430] Add 1-methyl-L-proline monohydrate (36.4 mg), HATU (93.2 mg), and DIPEA (0.053 mL) to a mixture of (1r,4r)-4-({8-[(2-heptylnonyl)oxy]-8-oxooctyl}amino)cyclohexane-1-carboxylic acid 2-heptylnonyl ester (151 mg) and DCM (3 mL), and stir at room temperature for 24 hours. Add 1-methyl-L-proline monohydrate (18.1 mg), DIPEA (0.026 mL), and HATU (47.4 mg) to the reaction mixture, and stir at room temperature for 5 hours. Add chloroform, water, and a saturated sodium bicarbonate aqueous solution to the reaction mixture, and extract the separated aqueous layer with chloroform. Concentrate the combined organic layers under reduced pressure, add a mixture of ethyl acetate and water / saturated brine (1 / 1) to the residue, dry the separated organic layer with anhydrous sodium sulfate, and concentrate under reduced pressure. The residue was purified by silica gel column chromatography (chloroform / methanol), and the crude purified product was purified by amino silica gel column chromatography (hexane / ethyl acetate) to obtain (1S,4r)-4-[{8-[(2-heptylnonyl)oxy]-8-oxooctyl}(1-methyl-L-prolyl)amino]cyclohexane-1-carboxylic acid 2-heptylnonyl ester (123 mg) as an oil.

[0431] Example 31

[0432] Add 1-ethyl-D-proline (57 mg), HATU (151 mg), and DIPEA (0.085 mL) to a mixture of 8-{[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonyl undecyl ester (160 mg) and DCM (6 mL), and stir at room temperature for one weekend. Purify the reaction mixture by silica gel column chromatography (chloroform / methanol), and then purify the crude product by amino silica gel column chromatography (hexane / ethyl acetate / methanol) to obtain 8-{(1-ethyl-D-prolyl)[(1r,3R)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonyl undecyl ester (133 mg) as an oil.

[0433] Manufacturing Example 34-1

[0434] Palladium-activated carbon (Pd 10%) (40 mg) was added to a mixture of (1R,2R)-2-aminocyclopentane-1-carboxylic acid hydrochloride (200 mg), methanol (4 mL), and formaldehyde (37% aqueous solution, 0.33 mL) under an argon atmosphere at room temperature, and the mixture was stirred for 18 hours under a hydrogen atmosphere at room temperature. The reaction mixture was filtered through diatomaceous earth (registered trademark), and the filtrate was concentrated under reduced pressure. The reaction mixture was subjected to three cycles of addition of chloroform / methanol to the resulting residue and concentration under reduced pressure, followed by the addition of diisopropyl ether and stirring for 10 minutes at room temperature. The resulting solid was filtered off and dried under reduced pressure to give (1R,2R)-2-(dimethylamino)cyclopentane-1-carboxylic acid hydrochloride (217 mg) as a solid.

[0435] Example 39

[0436] A mixture of tert-butyl 4-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}butyrate (181 mg) and DCM (0.5 mL) was added to a solution of 4M hydrogen chloride / ethyl acetate (1.4 mL), and the mixture was stirred at room temperature for 18 hours. The reaction mixture was then concentrated under reduced pressure. A mixture of the resulting oil (175 mg) and DCM (5 mL) was then added to a solution of (9Z,12Z)-octadec-9,12-dien-1-ol (0.11 mL), HATU (130 mg), DMAP (3 mg), and DIPEA (0.15 mL), and the mixture was stirred at room temperature for 18 hours. The reaction mixture was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform / methanol), and the crude purified product was purified by amino silica gel column chromatography (hexane / ethyl acetate / methanol). The crude purified product was then purified by silica gel column chromatography (hexane / ethyl acetate / methanol) to obtain 4-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}butyric acid (9Z,12Z)-octadec-9,12-dien-1-yl ester (48 mg) as an oil.

[0437] Manufacturing Example 49-1

[0438] 8-Bromooctanoic acid (396 mg), DIPEA (0.633 mL), HATU (738 mg), and DMAP (19 mg) were added to a mixture of 3-decyltridecyl-1-ol (504 mg) and DCM (10 mL) in a water bath, and the mixture was stirred at room temperature for 7 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, and the mixture was extracted with chloroform. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give 3-decyltridecyl 8-bromooctanoic acid (783 mg) as an oil.

[0439] Manufacturing Example 49-2

[0440] To a mixture of 3-decyltridecane-1-ol (502 mg) and DCM (10 mL), {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}acetic acid (405 mg), DIPEA (0.63 mL), HATU (731 mg), and DMAP (18.2 mg) were added at room temperature, and the mixture was stirred for 24 hours at room temperature. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, and the mixture was extracted with chloroform. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give 3-decyltridecyl ester of {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}acetic acid (763 mg) as an oil.

[0441] Manufacturing Example 49-3

[0442] TFA (5 mL) was added to a mixture of {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}acetic acid 3-decyl tridecyl ester (763 mg) and DCM (15 mL) under ice-cooling, and the mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with chloroform and then made alkaline by adding saturated sodium bicarbonate solution under ice-cooling. The reaction mixture was extracted with chloroform, and the organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by amino silica gel column chromatography (hexane / ethyl acetate) to give [(1r,3r)-3-aminocyclobutyl]acetic acid 3-decyl tridecyl ester (585 mg) as an oil.

[0443] Manufacturing Example 49-4

[0444] DIPEA (0.277 mL) and KI (11 mg) were added to a mixture of [(1r,3r)-3-aminocyclobutyl]acetic acid 3-decyltridecyl ester (585 mg), 8-bromooctanoic acid 3-decyltridecyl ester (353 mg), MeCN (10 mL), and CPME (10 mL) at room temperature, and the mixture was stirred in an oil bath at 80 °C for 48 hours. After the reaction mixture was allowed to cool naturally at room temperature, a saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform / methanol) to give 8-{[(1r,3r)-3-{2-[(3-decyltridecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 3-decyltridecyl ester (393 mg) as an oil.

[0445] Example 49

[0446] At room temperature, a mixture of 3-decyltridecyl octanoate (129 mg) and DCM (5 mL) was added with 1-methyl-L-proline (11.8% water, 25.1 mg), HATU (64.9 mg), and DIPEA (0.036 mL), and stirred for 4 hours at room temperature. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, and the mixture was extracted with chloroform. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform / methanol), and the crude purified product was purified by amino silica gel column chromatography (hexane / ethyl acetate) to obtain 3-decyltridecyl octanoate (131 mg) as an oil.

[0447] Manufacturing Example 55-1

[0448] A mixture of [(1r,3r)-3-{[2-(benzyloxy)ethyl]amino}cyclobutyl]acetic acid 2-octyldecyl ester (540 mg), DCM (5.4 mL), 1-ethyl-L-proline (225 mg), HATU (999 mg), and DIPEA (550 μL) was stirred at room temperature for 5 hours. Heptane and 90% aqueous methanol were added to the reaction mixture, and the separated organic layer was washed with 90% aqueous methanol and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (heptane / ethyl acetate) to give [(1S,3r)-3-{[2-(benzyloxy)ethyl](1-ethyl-L-prolyl)amino}cyclobutyl]acetic acid 2-octyldecyl ester (550 mg) as an oil.

[0449] Manufacturing Example 55-2

[0450] At room temperature, 100 mg of 20% palladium hydroxide-activated carbon (containing 50% water) was added to a mixture of [(1S,3r)-3-{(1-ethyl-L-prolyl)amino}cyclobutyl]acetic acid 2-octyldecyl ester (500 mg) and acetic acid (4.2 mL). After purging the reaction system with nitrogen, the mixture was stirred for 3 hours at room temperature under a hydrogen atmosphere. The reaction mixture was filtered, and the filtrate was added to a mixture of ethyl acetate and water, with potassium carbonate added until the pH of the aqueous layer reached above 10. After obtaining the organic layer, the mixture was washed with water and concentrated under reduced pressure. The residue was purified by amino silica gel column chromatography (heptane / ethyl acetate) to give 320 mg of {(1S,3r)-3-[(1-ethyl-L-prolyl)(2-hydroxyethyl)amino]cyclobutyl]acetic acid 2-octyldecyl ester as an oil.

[0451] Example 55

[0452] At room temperature, 200 mg of 2-octyldecyl acetate and 4 mL of DCM were added to a mixture of 2-(1S,3r)-3-[(1-ethyl-L-prolyl)(2-hydroxyethyl)amino]cyclobutyl}acetate and 4,4-bis(octyloxy)butyric acid (250 mg), HATU (345 mg), DMAP (4.4 mg), and DIPEA (0.186 mL), and the mixture was stirred for 21.5 hours at room temperature. The reaction mixture was concentrated under reduced pressure, and heptane and 90% aqueous methanol were added to the residue. The separated organic layer was washed twice with 90% aqueous methanol. The 90% aqueous methanol layer was extracted again with heptane, and the combined organic layers were concentrated under reduced pressure. The residue was purified by amino silica gel column chromatography (heptane / ethyl acetate), and the crude purified product was further purified by silica gel column chromatography (heptane / ethyl acetate) to obtain 4,4-bis(octyloxy)butyric acid 2-{(1-ethyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}ethyl ester (151 mg) as an oil.

[0453] Manufacturing Example 56-1

[0454] Pyridine (5.4 mL), 4-nitrobenzene chloroformate (2.7 g), and DMAP (40 mg) were added to a mixture of 2-nonylundecane-1-ol (2 g) and DCM (60 mL) under ice-cooling conditions, and the mixture was stirred at room temperature for 2 days. The reaction mixture was concentrated under reduced pressure, and hexane was added to the resulting residue to remove insoluble matter. The filtrate was concentrated under reduced pressure to give 2.87 g of 4-nitrobenzene chloroformate-2-nonylundecyl ester as an oil.

[0455] Manufacturing Example 56-2

[0456] Pyridine (0.4 mL), DMAP (60 mg), and tert-butyl [(1r,3r)-3-(hydroxymethyl)cyclobutyl]carbamate (0.5 g) were added to a mixture of 4-nitrobenzene carbonate-2-nonylundecyl ester (1.4 g) and DCM (13 mL) at room temperature, and the mixture was stirred for 4 days at room temperature. Chloroform and a saturated aqueous solution of sodium bicarbonate were added to the reaction mixture, and the separated aqueous layer was extracted with chloroform. The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give methyl carbonate-2-nonylundecyl ester (1.29 g) as an oil.

[0457] Manufacturing Example 56-3

[0458] TFA (5 mL) was added to a mixture of methyl carbonate ={(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}methyl ester = 2-nonylundecyl ester (1.28 g) and DCM (10 mL) under ice-cooling, and the mixture was stirred at room temperature for 2.5 hours. The reaction mixture was then added to a mixture of chloroform and a saturated aqueous solution of sodium bicarbonate under water-cooling, and the organic layer was separated. The aqueous layer was extracted with chloroform. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by amino silica gel column chromatography (hexane / ethyl acetate), thereby giving methyl carbonate =[(1r,3r)-3-aminocyclobutyl]methyl ester = 2-nonylundecyl ester (916 mg) as an oil.

[0459] Manufacturing Example 58-1

[0460] 4-Octyldodecanoic acid (1 g), DIPEA (1.4 mL), HATU (1.58 g), and DMAP (42 mg) were added to a mixture of 0.7 mL of 4-bromobutane-1-ol and 10 mL of DCM under ice-cooling conditions, and the mixture was stirred at room temperature for 7 hours. Chloroform and water were added to the reaction mixture, the organic layer was separated, and the aqueous layer was extracted with chloroform. The combined organic layers were dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give 0.817 g of 4-octyldodecanoic acid 4-bromobutyl ester as an oil.

[0461] Manufacturing Example 58-2

[0462] Under an argon atmosphere, a mixture of methyl carbonate = [(1r,3r)-3-aminocyclobutyl]methyl ester = 2-nonylundecyl ester (471 mg) and CPME (4 mL) was added to 4-octyldodecanoic acid 4-bromobutyl ester (250 mg), MeCN (4 mL), DIPEA (0.25 mL), and KI (10 mg), and stirred in an oil bath at 80 °C for 2 days. The reaction mixture was allowed to cool naturally, diluted with ethyl acetate, and washed with saturated sodium bicarbonate aqueous solution and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by amino silica gel column chromatography (hexane / ethyl acetate) to give 4-octyldodecanoic acid 4-({(1r,3r)-3-[({[(2-nonylundecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino)butyl ester (333 mg) as an oil.

[0463] Example 58

[0464] To a mixture of 4-octyldodecanoic acid 4-({(1r,3r)-3-[({[(2-nonylundecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino)butyl ester (100 mg) and DCM (2 mL), 1-ethyl-L-proline (22 mg), HATU (58 mg), and DIPEA (35 μ L) were added, and the mixture was stirred at room temperature for 3 days. The reaction mixture was purified by silica gel column chromatography (chloroform / methanol), and the crude purified product was purified by amino silica gel column chromatography (hexane / ethyl acetate) to give 4-octyldodecanoic acid 4-[(1-ethyl-L-prolyl){(1r,3S)-3-[({[(2-nonylundecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]butyl ester (101 mg) as an oil.

[0465] Manufacturing Example 59-1

[0466] Pyridine (1.4 mL) and 4-nitrobenzene chloroformate (1.29 g) were added to a mixture of [(1r,3r)-3-(2-hydroxyethyl)cyclobutyl]carbamate (915 mg) and DCM (30 mL) under ice-cooling conditions. The mixture was stirred for 30 min under ice-cooling conditions, followed by stirring at room temperature for 18 h. Silica gel was added to the reaction mixture, and the mixture was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give 2-{(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}ethyl carbonate-4-nitrobenzene ester (1.77 g) as a solid.

[0467] Manufacturing Example 59-2

[0468] A mixture of 2-{(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}ethyl ester=4-nitrobenzene ester (1.77 g) and DCM (40 mL) was added with pyridine (8 mL), 2-octyldecane-1-ol (3.78 g), and DMAP (114 mg), and stirred at room temperature for 5 days. The reaction mixture was concentrated under reduced pressure, diluted with ethyl acetate / hexane, and washed with saturated aqueous ammonium chloride solution, saturated aqueous sodium bicarbonate solution, and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give 2-{(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}ethyl ester=2-octyldecyl ester (1.94 g) as an oil.

[0469] Manufacturing Example 63-1

[0470] DMAP (2 mg), 4-nitrobenzene chloroformate (602 mg), and pyridine (0.6 mL) were added to a mixture of tert-butyl [(1r,3r)-3-(hydroxymethyl)cyclobutyl]carbamate (300 mg) and DCM (9 mL) under ice-cooling conditions, and the mixture was stirred at room temperature for 18 hours. N-octyloctane-1-amine (2.3 mL) was added to the reaction mixture at room temperature, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, and hexane / ethyl acetate and saturated sodium bicarbonate aqueous solution were added to the resulting residue. The separated organic layer was washed with saturated sodium bicarbonate aqueous solution, water, and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give di(octyl)carbamate {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}methyl ester (612 mg) as an oil.

[0471] Manufacturing Example 66-1

[0472] DIPEA (1.8 mL), 6-bromohexane-1-ol (0.74 mL), and DMAP (13 mg) were added to a mixture of 4-nitrobenzene carbonate-2-nonylundecyl ester (500 mg) and DCM (15 mL) at room temperature, and the mixture was stirred for 18 hours at room temperature. CPME (15 mL) was added to the reaction mixture, and the mixture was stirred in an oil bath at 80 °C for 7 hours. After the reaction mixture was allowed to cool naturally to room temperature, DMAP (526 mg) was added, and the mixture was stirred for one weekend at room temperature. Hexane and a saturated aqueous sodium bicarbonate solution were added to the reaction mixture, and the separated organic layer was washed with a saturated aqueous sodium bicarbonate solution, water, and saturated brine. After drying with anhydrous sodium sulfate, the layer was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give 6-bromohexane carbonate-2-nonylundecyl ester (344 mg) as an oil.

[0473] Manufacturing Example 72-1

[0474] At room temperature, pyridine (1.9 mL), [(1r,4r)-4-hydroxycyclohexyl]carbamate tert-butyl ester (371 mg), and DMAP (561 mg) were added to a mixture of 4-nitrobenzene carbonate = 2-octyldecyl ester (500 mg) and CPME (15 mL), and the mixture was stirred in an oil bath at 50 °C for 18 hours. After the reaction mixture was allowed to cool naturally to room temperature, hexane / ethyl acetate and a saturated aqueous solution of sodium bicarbonate were added to the reaction mixture. The separated organic layer was washed with water and saturated brine, dried with anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give 495 mg of 4-(1r,4r)-4-[(tert-butoxycarbonyl)amino]cyclohexyl ester = 2-octyldecyl ester as an oil.

[0475] Manufacturing Example 73-1

[0476] N-decyldecane-1-amine (545 mg), HATU (697 mg), and DIPEA (0.39 mL) were added to a mixture of {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}acetic acid (350 mg) and DCM (7 mL) at room temperature, and the mixture was stirred for 18 hours at room temperature. The reaction mixture was concentrated under reduced pressure, and ethyl acetate / hexane and saturated sodium bicarbonate aqueous solution were added to the residue. The separated organic layer was washed with water and saturated brine, dried with anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate / chloroform) to give tert-butyl [(1r,3r)-3-{2-[di(decyl)amino]-2-oxoethyl}cyclobutyl]carbamate (755 mg) as a solid.

[0477] Manufacturing Example 74-1

[0478] DIPEA (0.85 mL) and methanesulfonyl chloride (0.29 mL) were added to a mixture of [(1r,3r)-3-(hydroxymethyl)cyclobutyl]carbamate (500 mg) and DCM (10 mL) under ice-cooling. The reaction was initiated under ice-cooling and stirred for 1.5 hours. Water and chloroform were added to the reaction mixture under ice-cooling. The separated organic layer was dried with anhydrous sodium sulfate and concentrated under reduced pressure. DIPEA (0.85 mL) and undecane-1-amine (2.66 mL) were added to a mixture of the resulting solid (818 mg) and MeCN (10 mL) at room temperature. The mixture was stirred in an oil bath at 50 °C for 2 hours and then in an oil bath at 80 °C for 16 hours. After the reaction mixture was allowed to cool naturally to room temperature, water and chloroform were added. The separated organic layer was washed with saturated brine, dried with anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by amino silica gel column chromatography (ethyl acetate / hexane), and the crude purified product was purified by silica gel column chromatography (chloroform / methanol) to obtain tert-butyl {(1r,3r)-3-[(undecylamino)methyl]cyclobutyl}carbamate (644 mg) in solid form.

[0479] Manufacturing Example 74-2

[0480] Undecanoic acid (409 mg), HATU (830 mg), and DIPEA (0.47 mL) were added to a mixture of {(1r,3r)-3-[(N-undecylamino)methyl]cyclobutyl}carbamate (644 mg) and DCM (10 mL) at room temperature, and the mixture was stirred for 16 hours at room temperature. The reaction mixture was concentrated under reduced pressure, and the resulting residue was loaded onto amino silica gel and purified by silica gel column chromatography (ethyl acetate / hexane) to give {(1r,3r)-3-[(N-undecylundecylamide)methyl]cyclobutyl}carbamate (895 mg) as an oil.

[0481] [Table 10]

[0482] [Table 11]

[0483] [Table 12]

[0484] [Table 13]

[0485] [Table 14]

[0486] [Table 15]

[0487] [Table 16]

[0488] [Table 17]

[0489] [Table 18]

[0490] [Table 19]

[0491] [Table 20]

[0492] [Table 21]

[0493] [Table 22]

[0494] [Table 23]

[0495] [Table 24]

[0496] [Table 25]

[0497] [Table 26]

[0498] [Table 27]

[0499] [Table 28]

[0500] [Table 29]

[0501] [Table 30]

[0502] [Table 31]

[0503] [Table 32]

[0504] [Table 33]

[0505] [Table 34]

[0506] [Table 35]

[0507] [Table 36]

[0508] [Table 37]

[0509] [Table 38]

[0510] [Table 39]

[0511] [Table 40]

[0512] [Table 41]

[0513] [Table 42]

[0514] [Table 43]

[0515] [Table 44]

[0516] [Table 45]

[0517] [Table 46]

[0518] [Table 47]

[0519] [Table 48]

[0520] [Table 49]

[0521] [Table 50]

[0522] [Table 51]

[0523] [Table 52]

[0524] [Table 53]

[0525] [Table 54]

[0526] Example 76 Fabrication of Nucleic Acid Lipid Nanoparticles

[0527] (Raw material for nucleic acid lipid nanoparticles)

[0528] DOTAP uses products manufactured by Nippon Yu Co., Ltd. (product name: COATSOME (registered trademark) CL-8181TA), DOTMA uses products manufactured by Nippon Yu Co., Ltd. (product name: COATSOME (registered trademark) CL-E8181TA), DSPC uses products manufactured by Nippon Yu Co., Ltd. (product name: COATSOME (registered trademark) MC-8080), DPPC uses products manufactured by Nippon Yu Co., Ltd. (product name: COATSOME (registered trademark) MC-6060), DHSM uses products manufactured by Nippon Seika Co., Ltd., SOPC uses products manufactured by Avanti Polar Lipids Co., Ltd. (product catalog number: 850467P), and DoPhPE uses products manufactured by Avanti Polar Lipids Co., Ltd. Lipids (catalog number: 999985P), DOPS (Larodan, catalog number: 38-1810), DOPE (COATSOME ME-8181, registered trademark), cholesterol (CholesterolHP, Nippon Seika Co., Ltd.), β-sitosterol (Sigma-Aldrich, catalog number: S1270), 7α-hydroxycholesterol (Avanti Polar Lipids, catalog number: 700034P), campesterol (Tama Biochemical Co., Ltd., catalog number: 306-01391), DMG-PEG2000 (SUNBRIGHT GM-020, registered trademark), and C8 PEG2000 ceramide (Avanti Polar) are all manufactured by Lipids (catalog number: 999985P). Lipids (catalog number: 880170P), PEG monostearate (polyethylene glycol monostearate) is manufactured by Fujifilm and Koujun Pharmaceutical Co., Ltd. (catalog number: 320-32585), and mRNA (FLuc mRNA, ND1 mRNA, eGFP mRNA) is manufactured by TriLink BioTechnologies.

[0529] (Preparation of nucleic acid lipid nanoparticles)

[0530] In Ex1-L1 to Ex1-L54, compounds of formula (I) or their salts, phospholipids, sterols, and PEGylated lipids, which are cationic lipids, were dissolved in ethanol according to the lipid composition ratios and N / P ratios shown in the table below to obtain an oil phase. FLuc mRNA was diluted to 85 μg / mL with 10 mM citrate buffer (pH 4) to obtain an aqueous phase. The oil phase to aqueous phase volume ratio was adjusted to 1:3 using a microfluidic apparatus (NanoAssemblr, a registered trademark, manufactured by Precision NanoSystems). The mixture was diluted 2- or 3-fold with phosphate-buffered saline (PBS) to obtain a dispersion of nucleic acid lipid nanoparticles. Ethanol was removed by dialysis of the dispersion relative to PBS, and the concentration was achieved by ultrafiltration to obtain nucleic acid lipid nanoparticles of any concentration, thus yielding the lipids of the examples. The particle size and encapsulation efficiency of the nucleic acid lipid nanoparticles (nucleic acid: Fluc mRNA) are shown below. The NUM entries in the table below list Ex1-L1 to Ex1-L54 as Ex1-L1-FLuc mRNA to Ex1-L54-FLuc mRNA, respectively.

[0531] [Table 55]

[0532] [Table 56]

[0533] Abbreviations in the table: CHO: Cholesterol, DMG: DMG-PEG2000, 7α-OH-CHO: 7α-hydroxycholesterol, PEGCeramide: C8 PEG2000 ceramide, PEG Monostearate: Polyethylene glycol monostearate

[0534] For Ex1-L1-FLuc mRNA, pharmacological evaluation was performed using Example 1, and the ratios of components with fluorescence intensities above 0.01 and above 0.1 were calculated when its fluorescence intensity was set as baseline 1. The results showed that, based on the total amount of lipid nanoparticles, the composition ratio of Ex1 was 20.0–80.0 mol% and 30.0–60.0 mol%. Based on the total amount of lipid nanoparticles, the composition ratio of neutral lipids was 18.5–78.5 mol% and 38.5–68.5 mol%. Based on the total amount of lipid nanoparticles, the composition ratio of PEGylated lipids was 0.5–2.5 mol% and 0.5–2.0 mol%.

[0535] (ND1 nucleic acid is the raw material for ND1 nucleic acid lipid nanoparticles)

[0536] Two types of ND1 mRNA encoding human ND1 were produced: (i) an mRNA consisting of a 5' UTR and 3' UTR provided by TriLink BioTechnologies, a CDS of the human ND1 gene (Sequence No. 1), and a 120-base polyA sequence with a 5' cap structure of Cap-1 (hereinafter referred to as "ND1-1"), and (ii) an mRNA consisting of a 5' UTR derived from the human α-globin gene, a 3' UTR derived from the human α-globin gene, a CDS of the human ND1 gene (Sequence No. 3), and a 79-nucleotide polyA sequence (Sequence No. 4) in which all uridines were replaced with N1-methylpseuuridines (Sequence No. 5) (hereinafter referred to as "ND1-2") (commissioned by TriLink BioTechnologies). It should be noted that in the base sequence shown in sequence number 4 or sequence number 5, positions 1-3 of the nucleotides correspond to AGG, positions 4-43 correspond to the 5' UTR, positions 44-1114 correspond to the CDS of the human NeuroD1 gene (sequence number 3), positions 1115-1120 correspond to two consecutive stop codons, positions 1121-1231 correspond to the 3' UTR, and positions 1232-1310 correspond to the polyA sequence. The FLuc mRNA and eGFP mRNA were manufactured by TriLink BioTechnologies (product names: CleanCap (registered trademark) FLuc mRNA and CleanCap (registered trademark) EGFP mRNA).

[0537] (Preparation of ND1 nucleic acid lipid nanoparticles)

[0538] The preparation of ND1 nucleic acid lipid nanoparticles was specifically carried out by the following method. Compounds Ex1, DSPC, cholesterol, and DMG-PEG2000 (used as cationic lipids) were dissolved in ethanol at an N / P ratio of 6 to obtain an oil phase. A 10 mM citrate buffer (pH 4) containing the aforementioned mRNA was added as the aqueous phase to achieve a volume ratio of oil phase to aqueous phase of 1:3. The mixture was then mixed using a microfluidic device (NanoAssemblr, Precision NanoSystems). The mixture was diluted 2-fold with PBS to obtain a dispersion of ND1 nucleic acid lipid nanoparticles. Ethanol was removed by dialysis of the dispersion. The dispersion was then concentrated by ultrafiltration to obtain ND1 nucleic acid lipid nanoparticles of arbitrary concentration.

[0539] It should be noted that, in Experimental Example 4, the preparation of ND1 nucleic acid lipid nanoparticles was specifically carried out by the following method. Compound Ex1 (a cationic lipid), DSPC (manufactured by Nippon Seika Co., Ltd.), cholesterol (manufactured by Merck Co., Ltd.), and DMG-PEG2000 (manufactured by Merck Co., Ltd.) were dissolved in ethanol at an N / P ratio of 6 to obtain an oil phase. A 10 mM citrate buffer (pH 4) containing the above mRNA was used as the aqueous phase, and the oil phase to aqueous phase volume ratio was adjusted to 1:3 using a microfluidic apparatus (NanoAssemblr, Ignite+, PrecisionNanoSystems). The mixture was diluted with Tris buffer to obtain a dispersion of ND1 nucleic acid lipid nanoparticles. The pH of this dispersion was adjusted to approximately 7 with 0.1 M Tris buffer, and then purified using tangential flow filtration (TFF) to obtain ND1 nucleic acid lipid nanoparticles prepared at any concentration.

[0540] As nucleic acid lipid nanoparticles, lipid nanoparticles composed of Ex1, DSPC, cholesterol, and DMG-PEG2000 (molar ratio 50 / 10 / 38.5 / 1.5) prepared using 10 mM citrate buffer (pH 4) are referred to as Ex1-L1-ND1 mRNA. Similarly, lipid nanoparticles containing eGFP mRNA and with a lipid composition of Ex1-L1 are referred to as Ex1-L1-eGFP mRNA.

[0541] (Raw material for Fluc eGFP mixed nucleic acid lipid nanoparticles)

[0542] Flux mRNA encoding firefly luciferase protein and eGFP mRNA encoding green fluorescent protein (TriLink BioTechnologies) were mixed in a 1:1 molar ratio to obtain Flux eGFP mixed mRNA.

[0543] (Preparation of Fluc eGFP mixed nucleic acid lipid nanoparticles)

[0544] The preparation of FluceGFP mixed nucleic acid lipid nanoparticles was specifically carried out by the following method. The compound of the examples (Ex6 or Ex7), DSPC, cholesterol, and DMG-PEG2000 were dissolved in ethanol at an N / P ratio of 6 to obtain an oil phase. A 10 mM citrate buffer (pH 4) containing the above mRNA was added as the aqueous phase to achieve a volume ratio of oil phase:water phase of 1:3. The mixture was then mixed using a microfluidic device (NanoAssemblr, a registered trademark, manufactured by Precision NanoSystems). The mixture was diluted 2-fold with PBS to obtain a dispersion of FluceGFP mixed nucleic acid lipid nanoparticles. Ethanol was removed by dialysis of this dispersion. Subsequently, the dispersion was concentrated by ultrafiltration to obtain FluceGFP mixed nucleic acid lipid nanoparticles adjusted to an arbitrary concentration.

[0545] As nucleic acid lipid nanoparticles, the Fluc eGFP mixed nucleic acid lipid nanoparticles prepared using 10mM citrate buffer (pH4) and composed of the compound of the example (Ex6 or Ex7), DSPC, cholesterol, and DMG-PEG2000 (molar ratio 50 / 10 / 38.5 / 1.5) are respectively named Ex6-L1-Fluc eGFP mRNA and Ex7-L1-Fluc eGFP mRNA.

[0546] (Particle size determination)

[0547] The particle size of the nucleic acid lipid nanoparticles was measured using a particle size analyzer (Zetasizer NanoZSP or Ultra, Malvern Panalytical).

[0548] (Evaluation of mRNA encapsulation rate)

[0549] The encapsulation rate of mRNA in the nucleic acid lipid nanoparticle dispersions obtained by diluting the nucleic acid lipid nanoparticles to achieve an mRNA concentration of approximately 150–1000 ng / mL was determined. Specifically, the nucleic acid lipid nanoparticles obtained as described above were diluted with TE buffer (10 mM Tris / 1 mM EDTA, pH 8.0), and the mRNA concentration (A) was measured using a Quant-iT RiboGreen RNA Reagent (Thermo Fisher Scientific), which was taken as the mRNA present in the extracellular fluid of the nucleic acid lipid nanoparticles. Additionally, the nucleic acid lipid nanoparticles were diluted with 2% Triton X-100, and the mRNA concentration (B) was measured, which was taken as the total mRNA concentration in the composition. The encapsulation rate of mRNA was then calculated using the following formula (F1).

[0550] Inclusion rate (%) = 100-(A / B)×100…(F1)

[0551] The table below shows the particle size and encapsulation efficiency of nucleic acid lipid nanoparticles (nucleic acid: Fluc mRNA). In the NUM column of the table below, Ex1-L1 to Ex75-L1 represent Ex1-L1-FLuc mRNA to Ex75-L1-FLuc mRNA, respectively.

[0552] [Table 57]

[0553] [Table 58]

[0554] The following table shows the particle size and encapsulation efficiency of the nucleic acid lipid nanoparticles (nucleic acid: Fluc eGFP mixed mRNA) used in Experiment Examples 1-2.

[0555] [Table 59]

[0556] The following table shows the particle size and mRNA encapsulation rate of the nucleic acid lipid nanoparticles (nucleic acid: Fluc mRNA) used in Experiment Example 2.

[0557] [Table 60]

[0558] The following table shows the particle size and mRNA encapsulation rate of the nucleic acid lipid nanoparticles (nucleic acid: NeuroD1 mRNA) used in Experiment Example 4.

[0559] [Table 61]

[0560] Industrial availability

[0561] Compounds of formula (I) or their salts can form lipid nanoparticles. Lipid nanoparticles containing compounds of formula (I) or their salts can be taken up into astrocytes or hepatocytes. Lipid nanoparticles containing compounds of formula (I) or their salts can also be used to manufacture pharmaceutical compositions by encapsulating nucleic acids. Lipid nanoparticles and pharmaceutical compositions encapsulating nucleic acids such as mRNA are expected to be useful for the prevention and / or treatment of astrocyte-related diseases.

[0562] Sequence List Free Text

[0563] Sequence number 1: Human NeuroD1 gene (CDS)

[0564] Serial number 2: Human NeuroD1 protein

[0565] Serial number 3: Human NeuroD1 gene (CDS)

[0566] Sequence number 4: Human NeuroD1 mRNA sequence (ND1-2)

[0567] Sequence number 5: Human NeuroD1 mRNA sequence containing modified nucleotides (ND1-2)

Claims

1. A compound of formula (I) or a salt thereof, In the formula, L 1 and L 2 Whether they are the same or different, they are -CH2-, -CH2CH2- or bonds. L 3 For key or C 1-10 Alkylene M is -CH2- or does not exist. n is 1 or 2, in, When M is -CH2-, n is 1. E 1 and E 2 Same or different, -C(=O)O- -OC(=O)- -OC(=O)O- -C(=O)- OR key, This indicates that at this location, R 1 Or R 2 bonding, Among them, E 1 and E 2 Either of them is -C(=O)O- or -OC(=O)- , R 1 and R 2 Same or different, for -CH(-R) x )R y -CH2CH(-R) x )R y -CH2CH2CH(-R) x )R y -CH2CH(-OR) x OR y -CH2CH2CH(-OR) x OR y -CH2-(C 5-15 alkyl), -CH2-(C 5-20 alkenyl), -N(-R) x )R y -NR y (-C(=O)R x ) or -NR y C(=O)CH(-R x )R Z , Among them, R 1 and R 2 Any one of them is -N(-R) x )R y At that time, E 1 -R 1 and E 2 -R 2 Either of them is -OC (=O) -N (-R) x )R y or -C(=O)-N(-R) x )R y , R 1 and R 2 Either of them is -NR y (-C(=O)R x ) or -NR y C(=O)CH(-R x )R Z At that time, with the R 1 bonded E 1 Or with R 2 bonded E 2 As key, and, E 1 and E 2 All are -OC (=O)- At that time, R 1 and R 2 Same or different, -CH2CH2CH(-OR) x OR y or -CH2-(C 5-20 alkenyl), R x R y and R Z Same or different, C 5-15 alkyl, R 3 To select groups from the group consisting of free formulas (a) to (i), R a C 1-6 alkyl, R b -CH2-C 1-6 Alkyl or -C(=O)CH2N(CH3)2, R c and R d The same or different, is -CH3, -CH2CH3 or -CH2CH2OH, or, R c When it is H, R d It is -CH2C(=O)NH2, L cd It can be -CH2-, -CH2CH2-, -CH2CH2CH2-, or -CH2CH(CH3)-. R e It is H or OH. R f For H, R g C 1-6 alkyl, Among them, R f and R g They can form pyrrolidine rings together with the carbon and nitrogen atoms they are bonded to. R h C 1-6 alkyl, R i C 1-6 Alkyl or -CH2CH2OH, R j and R k Same or different, C 1-6 alkyl, R l and R m Same or different, C 1-6 alkyl, s and t can be the same or different, and the result is 1 or 2.

2. The compound or a salt thereof according to claim 1, wherein, -E 1 -R 1 -C(=O)O-CH2CH(-R) x )R y -C(=O)O-CH2CH2CH(-R) x )R y -OC(=O)-C3H6-(C 1-6 (alkylene)-CH=CH-CH=CH-(C 1-6 Alkyl), -OC(=O)-N(-R) x )R y -OC(=O)O-CH(-R) x )R y -OC(=O)O-CH2CH(-R) x )R y -NR y (-C(=O)R x ) or -NR y C(=O)CH(-R x )R Z , -E 2 -R 2 -C(=O)O-CH2CH(-R) x )R y -C(=O)O-CH2CH2CH(-R) x )R y -C(=O)O-C3H6-(C 1-6 (alkylene)-CH=CH-CH=CH-(C 1-6 Alkyl group), -OC(=O)-CH2CH(-R) x )R y -OC(=O)-CH2CH2CH(-R) x )R y -OC(=O)-CH2CH2CH(-OR) x OR y -OC(=O)-C3H6-(C 1-6 (alkylene)-CH=CH-CH=CH-(C 1-6 Alkyl) or -OC(=O)O-CH2CH(-R x )R y .

3. The compound of formula (I) according to claim 1, or a salt thereof, wherein, E 1 and E 2 Same or different, -C(=O)O- -OC(=O)- or-OC(=O)O- , This indicates that at this location, R 1 Or R 2 bonding, Among them, E 1 and E 2 Either of them is -C(=O)O- , R 1 and R 2 Same or different, -CH2CH(-R) x )R y -CH2CH(-OR) x OR y -CH2CH2CH(-OR) x OR y -CH2-(C 5-15 alkyl) or -CH2-(C 5-20 alkenyl), R x and R y Same or different, C 5-15 alkyl, R 3 To select groups from the group consisting of free formulas (a) to (h), R c and R d All are -CH3, -CH2CH3, or -CH2CH2OH, or, R c When it is H, R d It is -CH2C(=O)NH2, L cd It is -CH2- or -CH2CH2-. Among them, R c R d When all are -CH3, -CH2CH3 or -CH2CH2OH, L cd They are -CH2-, -CH2CH2-, or -CH2CH2-, respectively.

4. The compound or a salt thereof according to claim 3, wherein, -E 1 -R 1 -C(=O)O-CH2CH(-R) x )R y -OC(=O)-C3H6-(C 1-6 (alkylene)-CH=CH-CH=CH-(C 1-6 Alkyl) or -OC(=O)O-CH2CH(-R x )R y , -E 2 -R 2 -C(=O)O-CH2CH(-R) x )R y -C(=O)O-C3H6-(C 1-6 (alkylene)-CH=CH-CH=CH-(C 1-6 Alkyl) or -OC(=O)-C3H6-(C 1-6 (alkylene)-CH=CH-CH=CH-(C 1-6 alkyl).

5. The compound or a salt thereof according to claim 2, wherein, L 1 As key, L 2 For -CH2- or bond, L 3 C 1-6 Alkylene -E 1 -R 1 is -C(=O)O-CH2CH(-R x )R y 、-C(=O)O-CH2CH2CH(-R x )R y 、-OC(=O)O-CH(-R x )R y or -OC(=O)O-CH2CH(-R x )R y , -E 2 -R 2 is -C(=O)O-CH2CH(-R x )R y , -C(=O)O-CH2CH2CH(-R x )R y , -OC(=O)-CH2CH2CH(-R x )R y or -OC(=O)-CH2CH2CH(-OR x )OR y , R x and R y Same or different, C 5-15 alkyl, R 3 The groups selected are those from the group consisting of free formulas (d), (e), (g), or (h). R c and R d Whether they are the same or different, they are -CH3, -CH2CH3, or -CH2CH2OH. L cd It can be -CH2-, -CH2CH2-, or -CH2CH2CH2-. R e For H, R f For H, R g C 1-6 alkyl, R i C 1-6 alkyl, R j and R k Same or different, C 1-6 alkyl, t is 1.

6. The compound or a salt thereof according to claim 5, wherein, L 3 It is a C6 alkylene group. -E 1 -R 1 -C(=O)O-CH2CH(-R) x )R y or -OC(=O)O-CH2CH(-R) x )R y , -E 2 -R 2 -C(=O)O-CH2CH(-R) x )R y , R c and R d Whether they are the same or different, they are -CH3, -CH2CH3, or -CH2CH2OH. L cd It can be -CH2-, -CH2CH2-, or -CH2CH2CH2-. Among them, R c R d When all are -CH3, -CH2CH3 or -CH2CH2OH, L cd They are -CH2-, -CH2CH2-, or -CH2CH2-, respectively.

7. The compound or a salt thereof according to claim 1, wherein, The compounds are selected from the group consisting of the following: 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester; 2-Nonylundecyl octanoate of 8-[(1-methyl-L-prolyl)(3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}bicyclo[1.1.1]pentan-1-yl)amino]octanoate; 8-{(1-ethyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester; 8-{(N,N-diethyl-β-alanyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester; 8-{(1,4-diethyl-1,4-diazacycloheptane-6-carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester; 8-{[(4-methylpiperazin-1-yl)acetyl][(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester; 8-[(1-methyl-L-prolyl){(1r,3S)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]octanoic acid 2-nonylundecyl ester; (1S,4r)-4-[{8-[(2-heptaylnonyl)oxy]-8-oxooctyl}(1-methyl-L-prolyl)amino]cyclohexane-1-carboxylic acid 2-heptaylnonyl ester; 8-{(1-ethyl-D-prolyl)[(1r,3R)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl ester; 8-{[(1r,3S)-3-{2-[(3-decyltridecyl)oxy]-2-oxoethyl}cyclobutyl](1-methyl-L-prolyl)amino}octanoic acid 3-decyltridecyl ester; 4,4-Bis(octyloxy)butyric acid 2-{(1-ethyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}ethyl ester; and 4-Octylododecanoic acid 4-[(1-ethyl-L-prolyl){(1r,3S)-3-[({[(2-nonylundecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]butyl ester.

8. A lipid nanoparticle comprising the compound of claim 1 or a salt thereof.

9. A lipid nanoparticle comprising the compound of claim 1 or a salt thereof, a neutral lipid, and a PEGylated lipid.

10. The lipid nanoparticles according to claim 9, wherein the nanoparticles contain nucleic acids.

11. The lipid nanoparticles according to claim 10, wherein, Nucleic acid is mRNA.

12. The lipid nanoparticles according to claim 11, wherein, Neutral lipids are phospholipids and sterols. Phospholipids include DPPC, DSPC, SOPC, DoPhPE, DOPS, or DHSM. Sterols include cholesterol, 7α-hydroxycholesterol, or β-sitosterol. The PEGylated lipids are DMG-PEG2000, PEG monostearate, or C8 PEG2000 ceramide.

13. The lipid nanoparticles according to claim 12, wherein, Lipid nanoparticles can express proteins within astrocytes.

14. The lipid nanoparticles according to claim 13, wherein, Nucleic acids are mRNAs that are useful for the prevention and / or treatment of astrocyte-related diseases.

15. The lipid nanoparticles according to claim 11, wherein, The nucleic acid is mRNA encoding the NeuroD1 protein. The compound of formula (I) or its salt is 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonyl undecyl ester or its salt, Neutral lipids are DSPC and cholesterol. The PEGylated lipid was DMG-PEG2000.

16. The lipid nanoparticles according to claim 10, wherein, Nucleic acid is mRNA containing a base sequence that encodes a protein consisting of the amino acid sequence shown in Serial Number 2, encoding the NeuroD1 protein.

17. The lipid nanoparticles according to claim 15, wherein, Nucleic acid is mRNA containing a base sequence that encodes a protein consisting of the amino acid sequence shown in Serial Number 2, encoding the NeuroD1 protein.

18. The lipid nanoparticles according to claim 10, wherein, Based on the total amount of lipid nanoparticles, the compound of claim 1 or its salt is contained in a composition ratio of 20.0 to 80.0 mol%, neutral lipids are contained in a composition ratio of 18.5 to 78.5 mol%, and PEGylated lipids are contained in a composition ratio of 0.5 to 2.5 mol%.

19. The lipid nanoparticles according to claim 10, wherein, Based on the total amount of lipid nanoparticles, the compound of claim 1 or its salt is contained in a composition ratio of 30.0 to 60.0 mol%, neutral lipids are contained in a composition ratio of 38.5 to 68.5 mol%, and PEGylated lipids are contained in a composition ratio of 0.5 to 2.0 mol%.

20. A pharmaceutical composition comprising the lipid nanoparticles according to any one of claims 10 to 19.

21. A pharmaceutical composition comprising the lipid nanoparticles according to any one of claims 10 to 19 and one or more pharmaceutically acceptable pharmaceutical additives.

22. The pharmaceutical composition according to claim 21, which is a pharmaceutical composition for the prevention and / or treatment of astrocyte-related diseases.

23. The use of the lipid nanoparticles according to any one of claims 10 to 19 in the manufacture of pharmaceutical compositions for the prevention and / or treatment of astrocyte-related diseases.

24. The use of the lipid nanoparticles according to any one of claims 10 to 19 in the prevention and / or treatment of astrocyte-related diseases.

25. The lipid nanoparticles according to any one of claims 10 to 19, for use in the prevention and / or treatment of astrocyte-related diseases.

26. A method for the prevention and / or treatment of astrocyte-related diseases, comprising the step of administering an effective amount of the lipid nanoparticles according to any one of claims 10 to 19 to a subject.

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