Carbamoyl lipid having cyclic group at side chain, lipid nanoparticle thereof, and pharmaceutical composition thereof

Abstract

The present inventors have found a carbamoyl lipid having a cyclic group in a side chain, which can form lipid nanoparticles, and have revealed that the lipid nanoparticles containing, as a constituent, the carbamoyl lipid having a cyclic group in a side chain according to the present invention can express a protein in astrocytes or hepatocytes. The lipid nanoparticles containing, as a constituent, the carbamoyl lipid having a cyclic group in a side chain according to the present invention contain a nucleic acid inside thereof and are expected as a component of a pharmaceutical composition useful for prevention and / or treatment of an astrocyte-related disease.

Description

Carbamoyl lipid having a cyclic group in the side chain, lipid nanoparticles thereof, and pharmaceutical compositions thereof

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

[0002] Because nucleic acids such as DNA and mRNA are easily degraded in the body, lipid nanoparticles are used to transport nucleic acids such as DNA and mRNA 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 that have been launched or are in clinical development. Various lipids are used as the cationic lipid, one of the components, in each nucleic acid-lipid nanoparticle.

[0003] Cationic lipids (also called ionizable lipids) are thought to form lipid nanoparticles and surround anionic DNA, mRNA, etc. Optimal cationic lipids can encapsulate nucleic acids such as DNA and mRNA. Furthermore, cationic lipids are thought to be able to deliver nucleic acids such as DNA and mRNA to cells and tissues, thereby producing desired proteins and exerting their functions (Patent Document 1).

[0004] A lipid represented by the following general formula is described as a cationic lipid used in lipid nanoparticles (Patent Document 1).

[0005]

[0006] (For the symbols in the formula, see Patent Document 1.)

[0007] A lipid represented by the following general formula is described as a cationic lipid used in lipid nanoparticles (Patent Document 2).

[0008]

[0009] (For the symbols in the formula, see Patent Document 2.)

[0010] There are technologies that can induce differentiation of somatic cells into target cells without using pluripotent stem cells such as iPS cells. If specific cells, tissues, or organs can be made to produce proteins involved in diseases, it is expected that these will become effective therapeutic drugs.

[0011] Astrocytes (astroglia) are one of the most abundant cells in the central nervous system, and under normal circumstances, they play an important role in maintaining the homeostasis of the blood-brain barrier and in the formation of neuronal synapses, thereby supporting normal brain function (Frontiers in Cellular Neuroscience, 2022, vol. 16, p. 850866; Toxicologic Pathology, 2011, vol. 39(1), pp. 115-123).

[0012] However, after brain injury, astrocytes become activated, accumulate at the site of injury, and form gliosis. Gliosis is thought to inhibit neuronal regeneration and has been reported in a wide range of central nervous system disorders, including Alzheimer's disease and other neurodegenerative disorders (Neuron, 2014, vol. 81, pp. 229-248). Therefore, the development of effective drugs that are taken up by astrocytes and act on them could potentially provide new treatments for a wide range of central nervous system disorders.

[0013] International Publication No. 2013 / 185116 International Publication No. 2023 / 091490

[0014] The main object of the present invention is to provide a new cationic lipid that can be a component of lipid nanoparticles, and to create lipid nanoparticles for uptake of nucleic acids into cells and protein expression. Another object of the present 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. Another object of the present invention is to provide pharmaceutical compositions containing lipid nanoparticles encapsulating nucleic acids (e.g., mRNA) useful for the prevention and / or treatment of astrocyte-related diseases. A further object of the present invention is to provide lipid nanoparticles encapsulating nucleic acids (e.g., mRNA) that can be delivered to astrocytes and / or liver cells, and pharmaceutical compositions containing the same.

[0015] As a result of extensive research, the present inventors have discovered the compound of formula (I) of the present invention or a salt thereof, and have found that novel and useful lipid nanoparticles can be prepared by using this compound as a cationic lipid. Furthermore, by examining the ratios of the constituent components of lipid nanoparticles (e.g., cationic lipid, neutral lipid, and PEGylated lipid), they have found an appropriate composition ratio. The present invention can provide such cationic lipids, lipid nanoparticles, nucleic acid-lipid nanoparticles, and pharmaceutical compositions containing them. The present invention can provide lipid nanoparticles encapsulating nucleic acids (e.g., mRNA) that can be delivered to astrocytes and / or liver cells, and pharmaceutical compositions containing the same.

[0016] That is, the present invention relates to the following (1) to (16): (The present invention) (1) A compound of formula (I) or a salt thereof:

[0017]

[0018] (In the formula, L 1 and L 2 are the same or different and are -CH2-, -CH2CH2-, or a bond; L 3 is a bond or C 1-10 alkylene, M is -CH2- or absent, n is 1 or 2, where n is 1 when M is -CH2-, E 1 and E 2are the same or different and represent -C(=O)O-*, -OC(=O)-*, -OC(=O)O-*, -C(=O)-*, or a bond, * represents R at this position 1 or R 2 where E 1 and E 2 is -C(=O)O-* or -OC(=O)-*, and R 1 and R 2 are the same or different and are -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 , where R 1 and R 2 Either one of -N(-R x )R y In the case of -E 1 -R 1 and -E 2 -R 2 Either one of -OC(=O)-N(-R x )R y or -C(=O)-N(-R x )R y and R 1 and R 2 Either one of them is -NR y (-C(=O)R x ) or -NR y C(=O)CH(-R x )R Z In this case, the R 1 E binds to1 or R 2 E binds to 2 is a bond; and E 1 and E 2 If both of the above are -OC(=O)-*, then R 1 and R 2 are the same or different and are -CH2CH2CH(-OR x ) OR y or -CH2-(C 5-20 alkenyl); R x , R y , and R Z are the same or different, C 5-15 alkyl, R 3 is represented by the formulas (a) to (i):

[0019]

[0020] a group selected from the group consisting of R a is C 1-6 alkyl, R b is -CH2-C 1-6 Alkyl or -C(=O)CH2N(CH3)2, R c and R d are the same or different and are -CH3, -CH2CH3, or -CH2CH2OH, or R c If is H, then R d is -CH2C(=O)NH2, and L cd is -CH2-, -CH2CH2-, -CH2CH2CH2-, or -CH2CH(CH3)-, R e is H or OH, R f is H, R g is C 1-6 alkyl, where R f and R g may form a pyrrolidine ring together with the carbon atom and nitrogen atom to which they are attached, R h is C 1-6 alkyl, R i is C 1-6 Alkyl or -CH2CH2OH, R j and R k are the same or different, C 1-6 alkyl, Rl and R m are the same or different, C 1-6 alkyl, s and t are the same or different and are 1 or 2.

[0021] (1a-1-1) R 3 (1a-1-2) The compound or salt thereof according to (1), wherein R is any one of formulas (a) to (d) and (f) to (i). 3 The compound or salt thereof according to (1), wherein:

[0022] (1a-2) -E 1 -R 1 is -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 is -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), -OC(=O)-CHCH(-R x )R y , -OC(=O)-CH2CH2CH(-R x )R y , -OC(=O)-CH2CH2CH(-ORx ) OR y , -OC(=O)-C3H6-(C 1-6 alkylene)-CH=CH-CH=CH-(C 1-6 alkyl), or -OC(=O)O-CHCH(-R x )R y The compound or salt thereof according to any one of (1) to (1a-1-2), wherein

[0023] (1a-2-1) R 3 (1a-2-2) The compound or salt thereof according to (1a-2), wherein R is any one of formulas (a) to (d) and (f) to (i). 3 The compound or salt thereof according to (1a-2), wherein: is formula (e):

[0024] (1a-3) E 1 and E 2 are the same or different and are -C(=O)O-*, -OC(=O)-*, or -OC(=O)O-*, * represents R at this position 1 or R 2 where E 1 and E 2 is -C(=O)O-*, and R 1 and R 2 are the same or different and -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 However, the same or different, C 5-15 alkyl, R 3 is a group selected from the group consisting of formulas (a) to (h),

[0025]

[0026] R c and R dare both -CH3, -CH2CH3, or -CH2CH2OH, or R c If is H, then R d is -CH2C(=O)NH2, and L cd is -CH2- or -CH2CH2-, where R c , R d When all of are -CH3, -CH2CH3, or -CH2CH2OH, L cd The compound of formula (I) or a salt thereof according to any one of (1) to (1a-2-2), wherein each is -CH2-, -CH2CH2-, or -CH2CH2-,

[0027] (1a-3-1) R 3 (1a-3-2) The compound or salt thereof according to (1a-3), wherein R is any one of formulas (a) to (d) or (f) to (h). 3 The compound or salt thereof according to (1a-3), wherein: is formula (e):

[0028] (1a-4) -E 1 -R 1 is -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-CHCH(-R x )R y , -E 2 -R 2 is -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 The compound or salt thereof according to any one of (1a-3) to (1a-3-2), wherein

[0029] (1a-4-1) R 3(1a-4-2) The compound or salt thereof according to (1a-4), wherein R is any one of formulas (a) to (d) or (f) to (h). 3 The compound or salt thereof according to (1a-4), wherein: is formula (e):

[0030] (1a-5) L 1 But, the bond, L 2 is -CH2- or a bond, L 3 But 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 However, the same or different, C 5-15 alkyl, R 3 is formula (d), (e), (g), or (h):

[0031]

[0032] a group selected from the group consisting of R c and R d are the same or different and are -CH3, -CH2CH3, or -CH2CH2OH, L cd is -CH2-, -CH2CH2-, or -CH2CH2CH2-, R e But, H, R f But, H, Rg But C 1-6 alkyl, R i But C 1-6 alkyl, R j and R k However, the same or different, C 1-6 (1a-5-1) R is an alkyl group, and t is 1, or a salt thereof. 3 (1a-5-2) The compound or salt thereof according to (1a-5), wherein R is formula (d), (h), or (g). 3 The compound or salt thereof according to (1a-5), wherein: is formula (e):

[0033] (1a-6) L 3 C6 alkylene, -E 1 -R 1 is -C(=O)O-CH2CH(-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 , R c and R d are the same or different and are -CH3, -CH2CH3, or -CH2CH2OH, and L cd is -CH2-, -CH2CH2-, or -CH2CH2CH2-, where R c , R d When all of are -CH3, -CH2CH3, or -CH2CH2OH, L cd The compound or salt thereof according to (1a-5) to (1a-5-2), wherein: are -CH2-, -CH2CH2-, or -CH2CH2-, respectively.

[0034] (1b-1) -E 1 -R 1 or -E 2 -R 2 is one of -C(=O)O-CH2CH(-R x )R y and 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 (1b-2) R is a compound or a salt thereof according to (1), wherein R is an alkyl group. 3 where formulas (d), (e), (g), and (h):

[0035]

[0036] (1b-3) R is a group selected from the group consisting of the compound or salt thereof according to (1). c and R d are both -CH2CH3, and L cd is -CH2CH2- and R e is H and R f is H and R g C 1-6 alkyl, and R i is -CH3, t is 1, and R j and R k (1b-4) The compound or salt thereof according to (1b-2), wherein R is —CH2CH3. 3 The compound or salt thereof according to (1b-3), wherein R is formula (e): 3 (1b-5) The compound or salt thereof according to (1b-3), wherein R is formula (d) or (e): g (1b-6) The compound or salt thereof according to (1b-4), wherein R is —CH3. 3 is formula (h), or a salt thereof according to (1b-3).

[0037] (2) The compound is 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate, 2-nonylundecyl 8-[(1-methyl-L-prolyl)(3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}bicyclo[1.1.1]pentan-1-yl)amino]octanoate, 2-nonylundecyl 8-{(N,N-diethyl-β-alanyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate, 2-nonylundecyl 8-{(1,4-diethyl-1,4-diazepane-6-carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate, 2-nonylundecyl 8-{[(4-methylpiperazin-1-yl)acetyl][(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate, and 2-heptylnonyl (1S,4r)-4-[{8-[(2-heptylnonyl)oxy]-8-oxooctyl}(1-methyl-L-prolyl)amino]cyclohexane-1-carboxylate, or a salt thereof, according to (1).

[0038] (2-1) The compound is 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate, 2-nonylundecyl 8-[(1-methyl-L-prolyl)(3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}bicyclo[1.1.1]pentan-1-yl)amino]octanoate, 2-nonylundecyl 8-{(1-ethyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate, 2-nonylundecyl 8-{(N,N-diethyl-β-alanyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate, 2-nonylundecyl 8-{(1,4-diethyl-1,4-diazepane-6-carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate, 2-nonylundecyl 8-{[(4-methylpiperazin-1-yl)acetyl][(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate, 2-Nonylundecyl 8-[(1-methyl-L-prolyl){(1r,3S)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]octanoate, 2-Heptylnonyl (1S,4r)-4-[{8-[(2-heptylnonyl)oxy]-8-oxooctyl}(1-methyl-L-prolyl)amino]cyclohexane-1-carboxylate, 2-Nonylundecyl 8-{(1-ethyl-D-prolyl)[(1r,3R)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate 3-Decyltridecyl 8-{[(1r,3S)-3-{2-[(3-decyltridecyl)oxy]-2-oxoethyl}cyclobutyl](1-methyl-L-prolyl)amino}octanoate 2-{(1-ethyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}ethyl; or 4-octyldodecanoic acid 4-[(1-ethyl-L-prolyl){(1r,3S)-3-[({[(2-nonylundecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]butyl; or a salt thereof. (2-2) The compound is 2-nonylundecyl 8-{(1-ethyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate, or 2-nonylundecyl 8-{(N,N-diethyl-β-alanyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate, or a salt thereof.

[0039] (3) Lipid nanoparticles comprising the compound according to (1) or a salt thereof. (4) Lipid nanoparticles comprising the compound according to (1) or a salt thereof, a neutral lipid, and a PEGylated lipid. (5) Lipid nanoparticles according to (4), encapsulating a nucleic acid. (6) Lipid nanoparticles according to (5), wherein the nucleic acid is mRNA. (7) Lipid nanoparticles according to any one of (4) to (6), wherein the neutral lipid is a phospholipid and a sterol, the phospholipid is DPPC, DSPC, SOPC, DoPhPE, DOPS, or DHSM, the sterol is cholesterol, 7α-hydroxycholesterol, or β-sitosterol, and the PEGylated lipid is DMG-PEG2000, PEG monostearate, or C8 PEG2000 ceramide. (8) Lipid nanoparticles according to any one of (5) to (7), wherein the lipid nanoparticles are capable of expressing a protein in astrocytes. (9) The lipid nanoparticles according to any one of (5) to (8), wherein the nucleic acid is mRNA useful for the prevention and / or treatment of an astrocyte-related disease. (10) The lipid nanoparticles according to any one of (5) to (9), wherein the nucleic acid is mRNA encoding NeuroD1 protein, the compound of formula (I) or a salt thereof is 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate or a salt thereof, the neutral lipid is DSPC and cholesterol, and the PEGylated lipid is DMG-PEG2000. (11) The lipid nanoparticles according to any one of (5) to (10), wherein the nucleic acid is mRNA encoding NeuroD1 protein, comprising a base sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2. (12) The lipid nanoparticles according to any one of (3) to (11), comprising, based on the total amount of the lipid nanoparticles, the compound according to (1) or a salt thereof at a composition ratio of 20.0 to 80.0 mol%, a neutral lipid at 18.5 to 78.5 mol%, and a PEGylated lipid at 0.5 to 2.5 mol%. (13) The lipid nanoparticles according to any one of (3) to (11), comprising, based on the total amount of the lipid nanoparticles, the compound according to (1) or a salt thereof at a composition ratio of 30.0 to 60.0 mol%, a neutral lipid at 38.5 to 68.5 mol%, and a PEGylated lipid at 0.5 to 2.0 mol%.

[0040] (14) A pharmaceutical composition comprising the lipid nanoparticles according to any one of (5) to (13). (15) A pharmaceutical composition comprising the lipid nanoparticles according to any one of (5) to (13) and one or more pharmaceutically acceptable pharmaceutical additives. (16) The pharmaceutical composition according to (15), which is a pharmaceutical composition for preventing and / or treating an astrocyte-related disease. The present invention also relates to a pharmaceutical composition for preventing and / or treating an astrocyte-related disease, which contains the lipid nanoparticles according to any one of (5) to (13) containing the compound of formula (I) or a salt thereof. The pharmaceutical composition also encompasses an agent for preventing and / or treating an astrocyte-related disease, which contains the lipid nanoparticles according to any one of (5) to (13) containing the compound of formula (I) or a salt thereof. The present invention also relates to: use of lipid nanoparticles according to any one of (5) to (13) comprising the compound of formula (I) or a salt thereof for the manufacture of a pharmaceutical composition for the prevention and / or treatment of an astrocyte-related disease; lipid nanoparticles according to any one of (5) to (13) comprising the compound of formula (I) or a salt thereof for use in the prevention and / or treatment of an astrocyte-related disease; use of lipid nanoparticles according to any one of (5) to (13) comprising the compound of formula (I) or a salt thereof for the prevention and / or treatment of an astrocyte-related disease; and a method for the prevention and / or treatment of an astrocyte-related disease, comprising administering to a subject an effective amount of lipid nanoparticles according to any one of (5) to (13) comprising the compound of formula (I) or a salt thereof.

[0041] The present invention also relates to a method for delivering nucleic acids (e.g., nucleic acids useful for preventing and / or treating astrocyte-related diseases) to cells in a living body, particularly astrocytes, by using nucleic acid-lipid nanoparticles containing a compound of formula (I) or a salt thereof.The present invention relates to lipid nanoparticles encapsulating nucleic acids (e.g., mRNA) that can be delivered to astrocytes or liver cells, and pharmaceutical compositions containing the same.

[0042] "Astrocyte-associated diseases" are diseases in which astrocytes are involved, including cerebral infarction, cerebral hemorrhage, traumatic brain injury, neurodegenerative diseases, amyotrophic lateral sclerosis, Duchenne muscular dystrophy, Alexander disease, multiple sclerosis, spinal cord injury, and neuromyelitis optica. In some embodiments, these diseases include cerebral hemorrhage, traumatic brain injury, neurodegenerative diseases, amyotrophic lateral sclerosis, Duchenne muscular dystrophy, Alexander disease, multiple sclerosis, and neuromyelitis optica. Here, cerebral infarction includes, in some embodiments, perforating branch infarction, cerebral infarction with brain damage in the perforating branch-served area, subacute cerebral infarction, subacute to chronic cerebral infarction, chronic cerebral infarction, cerebral infarction with severe motor dysfunction, cerebral infarction with a modified Rankin Scale (mRS) of 2 or higher, cerebral infarction with an mRS of 4 or higher, and combinations thereof. Cerebral infarction includes perforating branch infarction, cerebral infarction with brain damage in the perforating branch-served area, subacute to chronic cerebral infarction, and cerebral infarction with severe motor dysfunction. Cerebral infarction is classified into the following stages: The "acute phase" refers to the period up to 72 hours after 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 72 hours or more after the onset of cerebral infarction. The "chronic phase" refers to the period one month or more after the onset of cerebral infarction. Here, "spinal cord injury" refers to, in one embodiment, spinal cord injury in the subacute to chronic phase, in another embodiment, spinal cord injury in the chronic phase, in one embodiment, spinal cord injury accompanied by quadriplegia, in one embodiment, spinal cord injury accompanied by motor dysfunction of the upper arm, in one embodiment, spinal cord injury including cervical spinal cord injury, and in one embodiment, spinal cord injury due to crush injury. Spinal cord injury is classified into the following phases: The "acute phase" refers to the period from immediately after the onset of spinal cord injury to less than two weeks. The "subacute phase" (also called the recovery phase) refers to the period from two weeks to less than four weeks after the onset of spinal cord injury. The "chronic phase" refers to four weeks or more after the onset of spinal cord injury.The "subacute to chronic phase" may, in one embodiment, be the period from two weeks after the onset of spinal cord injury, in one embodiment, be the period from four weeks after the onset of spinal cord injury, in one embodiment, be the period from three months after the onset of spinal cord injury, or in one embodiment, be the period from six months after the onset of spinal cord injury.

[0043] This specification includes the disclosures of Japanese Patent Application Nos. 2023-221885, filed December 27, 2023, and 2023-220683, filed December 27, 2023, which are priority applications of this application.

[0044] Furthermore, the present invention is not limited to the above-described embodiments, but also includes embodiments that appropriately combine the contents described in the detailed invention of the specification.

[0045] Lipids that are compounds of formula (I) or salts thereof can be used as one of the components of lipid nanoparticles to prepare lipid nanoparticles. The resulting lipid nanoparticles can incorporate nucleic acids into target cells, such as astrocytes, and express proteins. The lipid nanoparticles can incorporate nucleic acids useful for the prevention and / or treatment of astrocyte-related diseases into target cells and express proteins. In other words, pharmaceutical compositions useful for the prevention and / or treatment of astrocyte-related diseases can be provided. Pharmaceutical compositions containing the nucleic acid-lipid nanoparticles of the present invention can be used as agents for the prevention and / or treatment of astrocyte-related diseases.

[0046] Figure 1 shows the results of the modified Rankin Scale (mRS) evaluation of the daily changes in motor dysfunction after endothelin-1 treatment (induced cerebral ischemia) in the NeuroD1 (ND1) nucleic acid lipid nanoparticle group (ND1 mRNA) and the control group (Control mRNA) described in Test Example 4. The horizontal axis represents the number of days after endothelin-1 injection, and the vertical axis represents the mRS value (maximum 6). Open circles represent the mean mRS score at each evaluation time point in the ND1 nucleic acid lipid nanoparticle group. Note that mRS was evaluated every two weeks from day 28 after endothelin-1 injection, and the results on day 28 represent the mean scores for individual subjects evaluated on days 28 and 29. Error bars represent the standard error of the mean (SEM). Closed circles represent the mean mRS score for the control group at each evaluation time point. Note that the results on day 28 represent the mean scores for individual subjects evaluated on days 28 and 29. Nucleic acid lipid nanoparticles were administered on day 21 after endothelin-1 treatment.

[0047] The present invention will be described in detail below. In this specification, the following terms have the following meanings unless otherwise specified. The following definitions are intended to clarify the defined terms but are not intended to limit them. If a term used herein is not specifically defined, the term is used in the sense generally accepted by those skilled in the art. Unless otherwise specified, when a symbol in a chemical formula in this specification is used in other chemical formulas, the same symbol has the same meaning.

[0048] "Alkyl" refers to straight or branched alkyl. 1-6 Alkyl is an alkyl group containing 1 to 6 carbon atoms. 5-10 Alkyl is an alkyl having 5 to 10 carbon atoms. In some embodiments, alkyl is C 1-6 In some embodiments, alkyl is C 5-10 In some embodiments, alkyl is C 5-15 In some embodiments, alkyl is C 6-10 In some embodiments, alkyl is C 7-9 In some embodiments, alkyl is C 7-8 In some embodiments, alkyl is C8-9 In some embodiments, the alkyl is a C alkyl. In some embodiments, the alkyl is a C 6-9 In some embodiments, the alkyl is an alkyl. In some embodiments, the alkyl is a C6 alkyl. In some embodiments, the alkyl is a C8 alkyl. In some embodiments, the alkyl is a C9 alkyl. In some embodiments, the alkyl is n-hexyl. In some embodiments, the alkyl is n-heptyl. In some embodiments, the alkyl is n-octyl. In some embodiments, the alkyl is n-nonyl.

[0049] "Alkylene" refers to straight or branched alkylene. 5-10 Alkylene is an alkylene having 5 to 10 carbon atoms. In one embodiment, alkylene is C 1-10 In one embodiment, alkylene is C 1-6 In one embodiment, alkylene is C 5-8 In one embodiment, alkylene is C 7-9 In one embodiment, alkylene is C 6-8 It is alkylene. In one embodiment, the alkylene is C6 alkylene. In one embodiment, the alkylene is hexanediyl, heptanediyl, octanediyl, or nonanediyl. In one embodiment, the alkylene is heptanediyl, octanediyl, or nonanediyl. In one embodiment, the alkylene is heptane-1,7-diyl, octanediyl, or nonane-1,9-diyl. In one embodiment, the alkylene is n-hexane-1,6-diyl.

[0050] "Alkenyl" means straight or branched alkenyl, C 5-20 The alkenyl in the formula (I) is a straight-chain or branched alkenyl having 5 to 20 carbon atoms, such as vinyl, propenyl, butenyl, pentenyl, 1-methylvinyl, 1-methyl-2-propenyl, 1,3-butadienyl, 1,3-pentadienyl, etc. In one embodiment, the alkenyl is -C 5-10 Alkylene -CH=CH-CH=CH-C 5-10It is alkyl. In one embodiment, it is a straight-chain alkenyl. In one embodiment, the alkenyl 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 some embodiments, alkenyl is -C8 alkylene-(CH=CH-CH2)2-C4 alkyl (wherein both double bonds are in the Z configuration), -C8 alkylene-(CH=CH-CH2)2-C6 alkyl (wherein both double bonds are in the Z configuration), -C7 alkylene-CH=CH-C8 alkyl (wherein both double bonds are in the Z configuration), -C7 alkylene-(CH=CH-CH2)2-C4 alkyl (wherein both double bonds are in the Z configuration), -C7 alkylene-(CH=CH-CH2)3-C4 alkyl (wherein both double bonds are in the Z configuration), -C3 alkylene-(CH=CH-CH2)4-C4 alkyl (wherein both double bonds are in the Z configuration), -C3 alkylene-(CH=CH-CH2)5-C1 alkyl (wherein both double bonds are in the Z configuration), or -C2 alkylene-(CH=CH-CH2)6-C1 alkyl. (wherein both double bonds are Z-configuration). In one embodiment, the alkenyl is -C7 alkylene-(CH=CH-CH2)2-C4 alkyl (wherein both double bonds are Z-configuration). In one embodiment, the alkenyl is -C6 alkylene-(CH=CH-CH2)2-C4 alkyl (wherein both double bonds are Z-configuration). In one embodiment, the alkenyl is -C8 alkylene-(CH=CH-CH2)2-C4 alkyl (wherein both double bonds are Z-configuration). In one embodiment, "-CH2-(C 5-20 alkenyl)" is -C3H6-(C 1-6 alkylene)-CH=CH-CH2-CH=CH-(C 1-6 alkyl).

[0051] "Halogen" means F, Cl, Br, or I.

[0052] "Lipid nanoparticles" are nanoparticles whose main component is lipid. Typically, lipid nanoparticles contain cationic lipids, neutral lipids, and PEGylated lipids. In one embodiment, they are nucleic acid-lipid nanoparticles that further encapsulate nucleic acids. Furthermore, "nucleic acid-lipid nanoparticles" are lipid nanoparticles that encapsulate nucleic acids useful for the prevention and / or treatment of diseases, in one embodiment, lipid nanoparticles that encapsulate nucleic acids useful for the prevention and / or treatment of astrocyte-related diseases, and in one embodiment, lipid nanoparticles that encapsulate mRNA.

[0053] "Particle size" refers to the particle size of the lipid nanoparticles. For a dispersion in a stationary state, the hydrodynamic diameter (Dh) is measured using a particle size measurement device (Zetasizer (registered trademark) Nano ZSP or Ultra, manufactured by Malvern Panalytical) using the Dynamic Light Scattering (DLS) technique. This is in accordance with ISO 22412. The "particle size" of the lipid nanoparticles is calculated as the Z-average particle size. In one embodiment, the particle size of the lipid nanoparticles is 10 nm to 1,000 nm, in one embodiment, 30 nm to 500 nm, or in one embodiment, 30 nm to 250 nm. In one embodiment, it is 60 nm to 180 nm. In one embodiment, it is 70 nm to 110 nm. In one embodiment, it is 70 nm to 100 nm. In one embodiment, it is 80 nm to 100 nm. In one embodiment, it is 90 nm to 100 nm.

[0054] A "cationic lipid" is a compound or a salt thereof that can take on a cation (positive charge) in the molecule in response to pH and has a fatty acid chain. In one embodiment, the compound is represented by formula (I) or a salt thereof.

[0055] "Neutral lipids" are "phospholipids" and "sterols." In some embodiments, the neutral lipids are phospholipids and sterols, in some embodiments, the neutral lipids are phospholipids, and in some embodiments, the neutral lipids are sterols.

[0056] A "phospholipid" is a lipid having a phosphate ester group. Phospholipids include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), or sphingomyelin (SM), and combinations thereof. Phosphatidylcholine (PC) includes 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC). Phosphatidylethanolamine (PE) includes 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-di-O-phytanyl-sn-glycero-3-phosphoethanolamine (DoPhPE; CAS 150135-14-1), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE).Phosphatidylglycerol (PG) is 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DMPG), 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DPPG), or 1,2-distearoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DSPG), etc. Phosphatidylserine (PS) is phosphatidylserine (PS) or 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), etc. Sphingomyelin (SM) includes sphingomyelin (SM) or dihydrosphingomyelin (DHSM). In one embodiment, the phospholipid is DPPC, DSPC, SOPC, DOPE, DoPhPE, DOPS, or DHSM. In one embodiment, the phospholipid is DSPC. In one embodiment, no phospholipid is used, and the phospholipid is DOTAP or DOTMA.

[0057] A "sterol" is a steroid alcohol, a compound having a hydroxy group on the A ring of the steroid skeleton. In one embodiment, the sterol is cholesterol or a corticosteroid. In one embodiment, the sterol is cholesterol, 7α-hydroxycholesterol, brassicasterol, ergosterol, fecosterol, campesterol, sitosterol, β-sitosterol, stigmasterol, tomatidine, tomatine, ursolic acid, or a mixture thereof. It is also a combination of two or more types. In one embodiment, the sterol is cholesterol, 7α-hydroxycholesterol, campesterol, or β-sitosterol. In one embodiment, the sterol is cholesterol.

[0058] "PEGylated lipid" refers to a lipid having 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. In some embodiments, the PEGylated lipid is 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-PEG (DLPE-PEG), 1,2-dimyristoyl-rac-glycero-3-methoxy-PEG (DMG-PEG2000, also known as DMG-PEG), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-PEG (DMPE-PEG), 1,2-dipalmitoyl-rac-glycero-3-methoxy-PEG (DPG-PEG), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine-PEG (DPPC-PEG), or 1,2-distearoyl-rac-glycero-3-PEG. (DSG-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG (DSPE-PEG), PEG monostearate, or N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]}(C8 PEG2000 ceramide). In one embodiment, the PEGylated lipid is DMG-PEG2000, PEG monostearate, or N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]}(C8 PEG2000 ceramide). In another embodiment, the PEGylated lipid is DMG-PEG2000.

[0059] "Encapsulation rate" refers to the amount of nucleic acid incorporated into 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 amount of 100 mg of nucleic acid is incorporated into lipid nanoparticles, the encapsulation rate can be expressed as 98%. As used herein, "encapsulation" refers to being completely or substantially contained within or encompassed. In nucleic acid-lipid nanoparticles such as mRNA, the encapsulation rate of nucleic acid is measured by the method described in detail below. In some embodiments, the encapsulation rate of nucleic acid is 70% or more, in some embodiments 80% or more, in some embodiments 90% or more, in some embodiments 93% or more, and in some embodiments 95% or more.

[0060] The "N / P ratio" is the value obtained by dividing the number of moles (N) of amino groups of the cationic lipid in the nucleic acid-lipid nanoparticle by the number of moles (P) of phosphate of the RNA. In this specification, the N / P ratio was calculated assuming that the amino group is a nitrogen atom with an ACD / pKa GALAS calculated value greater than 6 using ACD / Percepta (ACD / Labs Release 2019.2.2 or 2023.1.1, registered trademark, Advanced Chemistry Development, Inc.). In one embodiment, the N / P ratio is 1 to 12, in another embodiment, the N / P ratio is 6 to 12, in another embodiment, the N / P ratio is 6 or 12, and in another embodiment, the N / P ratio is 6.

[0061] "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. In one embodiment, the nucleic acid includes mRNA. In one embodiment, the nucleic acid includes siRNA. In one embodiment, the nucleic acid includes Ascl1 mRNA (mRNA encoding Ascl1 protein is also referred to as Ascl mRNA, and hereinafter, mRNA encoding xxx protein is also referred to as xxx mRNA), Dlx2 mRNA, NeuroD1 mRNA (mRNA encoding NeuroD1 protein), Ngn2 mRNA, BDNF mRNA, NGF mRNA, or NT-3 mRNA. In one embodiment, the nucleic acid is NeuroD1 mRNA. In one embodiment, the nucleic acid is Ascl1 mRNA. In one embodiment, the nucleic acid is NeuroD1 mRNA containing a base sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO:2. In one embodiment, the nucleic acid is NeuroD1 mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 1. In one embodiment, the nucleic acid is NeuroD1 mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 3. In one embodiment, the nucleic acid is NeuroD1 mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 4. In one embodiment, the nucleic acid is NeuroD1 mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 5. In one embodiment, the nucleic acid is mRNA encoding NeuroD1 protein comprising a sequence for capping. In one embodiment, the nucleic acid is mRNA encoding NeuroD1 protein having a 5'-terminal sequence suitable for capping using CleanCap (registered trademark) or Reagent AG (TriLink BioTechnologies). In one embodiment, the nucleic acid is mRNA encoding NeuroD1 protein having a 5'-terminal sequence of AGG. In one embodiment, the nucleic acid is mRNA encoding NeuroD1 protein having a 5'-terminal sequence (positions 1 to 3 of SEQ ID NO: 4). In one embodiment, the mRNA is BDNF mRNA.In some embodiments, lipid nanoparticles contain two or more types of nucleic acids, and in some embodiments, lipid nanoparticles contain only one type of nucleic acid. In some embodiments, lipid nanoparticles encapsulating nucleic acids contain 0.001 to 60% by weight of nucleic acid, based on the total weight of the lipid nanoparticle, in some embodiments, 0.1 to 40% by weight, in some embodiments, 1.0 to 25% by weight, and in some embodiments, 3.0 to 10% by weight of nucleic acid. mRNA may 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). As used herein, "mRNA encoding a xxx protein" refers to RNA that contains a polynucleotide containing a base sequence encoding the xxx protein and can be translated into the xxx protein. As used herein, mRNA encoding a xxx protein is also referred to as xxx mRNA. In other words, it contains a polynucleotide that encodes the xxx protein in an expressible state. In one embodiment, in the present invention, the mRNA encoding the xxx protein is a polynucleotide encoding the xxx protein that includes functional sequences for expressing the xxx protein (e.g., including, but not limited to, a CDS, a 5'UTR, a 3'UTR, a 5' cap structure, and a polyA sequence).

[0062] The polynucleotides herein may be described as a base sequence containing "T" as a representative DNA sequence, but for example, in an RNA (e.g., mRNA) containing a base sequence specified by a particular SEQ ID NO, when the base sequence specified by the particular SEQ ID NO represents a DNA sequence, the base sequence is understood to be an RNA sequence in which each "T" in the DNA sequence is replaced 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 SEQ ID NOs: 1 and 3), as well as the nucleosides and nucleotides containing them, may be natural or modified, and / or may have other modifications (such as methylation), for each individual base, nucleoside, and nucleotide.

[0063] "mRNA" refers to messenger ribonucleic acid, and may be any that can be translated into a desired protein. Its structure includes a 5' cap, a 5' untranslated region (hereinafter, the untranslated region is also referred to as a UTR), a coding region (CDS), a 3' UTR, and a poly(A) tail.

[0064] The "5' cap" is a modified structure found at the 5' end of a protein that is involved in the stability of mature mRNA and translation initiation. 7 The structure in which the ribose 2' position of the first nucleoside of the mRNA is methylated in addition to the Cap-0 structure is called Cap-1, and the structure in which the ribose 2' positions of the first and second nucleosides are methylated is called Cap-2. Cap-0, Cap-1, and Cap-2 are each m 7 GpppNp, m 7 GpppN1mp,m 7 It is also referred to as GpppN1mpN2mp (N1 and N2 each represent a nucleoside, where m represents a 2'-O methyl group) (Nature Reviews Molecular Cell Biology 2014, vol. 15(5), pp. 313-326). In one embodiment, it is Cap-0, Cap-1, or Cap-2. In one embodiment, it is Cap-0. In one embodiment, it is Cap-1. In one embodiment, it is Cap-2.

[0065] "UTR" refers to untranslated regions, and includes 5'UTR and 3'UTR. The 5'UTR and 3'UTR may be derived from the gene to be expressed or from a heterologous gene. The UTR may be naturally occurring, or may be modified in that the sequence has been altered by inserting, deleting, substituting, and / or adding one or several (e.g., 2, 3, 4, 5, or 6) nucleotides relative to the naturally occurring UTR. As used herein, "UTR derived from a gene" includes not only naturally occurring UTRs but also such modified UTRs. A UTR may comprise multiple UTRs connected directly or via a spacer sequence. The start codon and 5'UTR may include a portion of a Kozak sequence (e.g., the sequence 5' from the start codon in the Kozak sequence). In one embodiment, the UTR is derived from a globin gene. In one embodiment, the UTR is derived from an α-globin gene. In one embodiment, the UTR is derived from a human α-globin gene. In one embodiment, the UTR is derived from a β-globin gene. In one embodiment, the 5'UTR and 3'UTR are UTRs from the human β-globin gene. In one embodiment, the 5'UTR and 3'UTR are UTRs from the human α-globin gene.

[0066] "CDS" refers to a coding sequence, a DNA sequence region that is translated into protein, and may be codon-optimized. The stop codon at the 3' end of the CDS may be any of TAA, TGA, or TAG, or multiple stop codons may be used consecutively (e.g., TAATGATAG).

[0067] A "modified nucleotide" is a nucleotide that has been modified. For example, it is a nucleotide that has been modified by methylation, atom exchange, double bond saturation, deamination, or substitution of an oxygen atom or the like with a sulfur atom. In one embodiment, it is a nucleotide with a modified nucleobase. In one embodiment, it is a nucleotide with a modified ribose. In one embodiment, it is a nucleotide with a modified phosphate group. In one embodiment, it is a nucleotide containing 1-methyladenosine, pseudouridine, N1-methylpseudouridine (also called 1-methylpseudouridine), dihydrouridine, 5-methoxycytidine, 5-methylcytidine, 7-methylguanosine, N6-methyladenosine, inosine, or thiouridine. In one embodiment, it is a nucleotide containing 1-methyladenosine. In one embodiment, it is a nucleotide containing a modified uridine. In one embodiment, it is a nucleotide containing pseudouridine or N1-methylpseudouridine. In one embodiment, it is a nucleotide containing pseudouridine. In one embodiment, it is a nucleotide containing N1-methylpseudouridine. In one embodiment, the modified nucleotide is a nucleotide containing dihydrouridine. In one embodiment, the modified nucleotide is a nucleotide containing inosine. In one embodiment, the modified nucleotide is a nucleotide containing 4-thiouridine. In this specification, the term "some or all" for modified nucleotides may include some of the specified modified nucleotides in the entire modified nucleotide sequence that are not substituted. The percentage of residues in the entire sequence in which a specific nucleotide is substituted with a modified nucleotide may be, but is not limited to, 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%, or 10-25%. In one aspect, "a portion" as a percentage of the number of residues substituted with modified nucleotides relative to the total number of residues may be, for example, but is not limited to, 60 to 99%, 70 to 99%, 80 to 99%, 90 to 99%, 95 to 99%, 60 to 99.9%, 70 to 99.9%, 80 to 99.9%, 90 to 99.9%, or 95 to 99.9%.

[0068] A "poly A tail" is a polyadenylic acid tail that functions in mRNA stabilization, nuclear export, translation, etc., and is a region of mRNA that contains multiple (but not limited to, typically 10 or more) consecutive adenosine monophosphates and is located downstream of the 3' UTR, for example, directly downstream (i.e., on the 3' side). It is also referred to as a poly A sequence. In one embodiment, the poly A tail length is 20 to 1000 bases. In another embodiment, the poly A tail length is 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. In one embodiment, the poly A tail length is 120 bases. In another embodiment, the poly A tail length is 79 bases. In one embodiment, the poly-A chain length is 30 to 500 bases (in one embodiment, 40 to 200, 50 to 200, 50 to 150, 50 to 100, 50 to 90, 60 to 150, 60 to 100, 60 to 90, 70 to 130, 70 to 120, 70 to 100, 70 to 90, 70 to 85, 70 to 80, 75 to 130, 75 to 120, 75 to 100, 75 to 90; in one embodiment, 74 to 84, 75 to 85, 75 to 83, or 76 to 82 bases). In one embodiment, the poly-A chain length is 120 bases. In one embodiment, the poly-A chain length is 79 bases. In one embodiment, the poly-A chain length is 74 to 84 bases. In one embodiment, the poly-A chain length is 75 to 85 bases.

[0069] As used herein, "expression" of a nucleic acid, i.e., a nucleic acid sequence, refers to the translation of mRNA into a polypeptide, the assembly of a protein from a polypeptide, or the post-translational modification of a polypeptide or protein. Also, as used herein, the terms expression and production are used interchangeably, such as protein expression.

[0070] "NeuroD1" is a neuronal differentiation protein (Neuronal differentiation 1) and 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 the differentiation of neural stem cells into neurons. Glial cells are classified into four types of cells: astrocytes, oligodendrocytes, ependymal cells, and microglia. It is known that ectopic expression of NeuroD1 in glial cells such as NG2 cells or astrocytes can convert glial cells into neurons (WO 2014 / 015261). In one embodiment, NeuroD1 is a protein consisting of an amino acid sequence that shares 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) sequence identity with the amino acid sequence set forth in SEQ ID NO: 2 and has transcription factor activity. In one embodiment, NeuroD1 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, which has 1 to 50 (e.g., 1 to 2, 1 to 3, 1 to 5, 1 to 7, 1 to 10, or 1 to 30) amino acid insertions, deletions, substitutions, and / or additions, and which has transcription factor activity. In one embodiment, NeuroD1 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 2.

[0071] "Transcription factor activity" refers to the ability of a protein to activate the transcription of its associated gene (i.e., transcription activation ability). Specifically, whether a protein has transcription factor activity can be determined by, for example, whether the expression level of the associated gene is increased or decreased in a recombinant vector compared to cells not transfected with the vector. Recombinant vectors can be prepared by incorporating a polynucleotide encoding a protein into a vector having any promoter, and the prepared recombinant vector can be introduced into cells to express the protein. For example, if the expression of the associated gene is increased in cells transfected with a vector containing a polynucleotide encoding NeuroD1, the NeuroD1 protein can be determined to have transcription factor activity.

[0072] "Sequence identity" refers to the percentage (%) of residues that match between sequences when a reference biological sequence (such as a nucleotide sequence or an amino acid sequence) and a target biological sequence are aligned (usually the percentage of matching residues relative to the entire length of the target sequence). "Sequence identity" can be calculated, for example, using EMBOSS Needle (Nucleic Acids Res., 2015; Vol. 43: pW580-W584) as the identity value obtained using the default parameters. The parameters are as follows (Gap Open Penalty = 10, Gap Extend Penalty = 0.5, Matrix = EBLOSUM62, End Gap Penalty = false).

[0073] "Perforator infarction" refers to a disease in which infarction occurs in the thalamus, caudate nucleus, putamen, globus pallidus, and / or internal capsule, which are innervated by perforator branches, resulting in functional impairment of the infarcted area. "Perforator branches" are small arteries that branch off from the middle cerebral artery, which constitutes the main cerebral artery, and innervate the basal ganglia, thalamus, and internal capsule. When a brain region is "innervated" by a blood vessel, it means that the blood supply to that brain region is provided by that blood vessel, and that the brain region is within the vascular territory of that vessel.

[0074] The "area innervated by the perforator branches" refers to the area innervated by the perforator branches, including the basal ganglia, thalamus, and internal capsule.

[0075] "Cerebral infarction with brain damage in the perforator branch-served area" refers to cerebral infarction in which brain damage caused by infarction occurs primarily in the perforator branch-served area. Cerebral infarction with brain damage in the perforator branch-served area includes not only cerebral infarction in which brain damage occurs in the perforator branch-served area due to infarction in the perforator branch-served area, but also cerebral infarction in which brain damage occurs secondarily in the perforator branch-served area, such as when cell necrosis caused by infarction occurring around the perforator branch-served area spreads to the perforator branch-served area, damaging the function of that area. As used herein, brain damage refers to functional damage of the brain caused by cell necrosis caused by infarction. Brain damage usually involves cell necrosis in the area with the brain damage. "Cerebral infarction with brain damage in the perforator branch-served area" may be cerebral infarction with cell necrosis in the perforator branch-served area.

[0076] Certain embodiments of the compound of formula (I) or a salt thereof, which is the compound of the present invention, lipid nanoparticles containing the same, and pharmaceutical compositions are shown below. Note that all embodiments can be freely combined in any combination of two or more that is not contradictory. Even if a combination is not specifically described, one or more embodiments can be combined in a certain embodiment.

[0077] 1. Cationic Lipids Some embodiments of the compound of formula (I) or a salt thereof, which is the compound of the present invention, are shown below. The embodiments of the compound are described below. "Compound or a salt thereof" is described as "compound". (Embodiments of the cationic lipid of the present invention) (1) L 1 Compounds of formula (I) wherein L is -CH2-. 1 (2) A compound of formula (I) wherein L is a bond. 2 Compounds of formula (I) wherein L is -CH2-. 2 A compound of formula (I) wherein L is a bond. 2 (3) A compound of formula (I) wherein L is -CH2- or a bond. 3 C 6-9 A compound of formula (I) which is alkylene. 3is C6 alkylene. (4) A compound of formula (I) wherein M is absent and n is 1 or 2. A compound of formula (I) wherein M is absent and n is 1. A compound of formula (I) wherein M is absent and n is 2. A compound of formula (I) wherein M is -CH2- and n is 1. A compound of formula (I) wherein M is -CH2- or absent and n is 1. (5) E 1 is -C(=O)O-* or -OC(=O)-*, and * is R 1 The compound of formula (I) is shown to bind to E 1 is -OC(=O)-* or -OC(=O)O-*, and * is R 1 The compound of formula (I) is shown to bind to E 1 is -OC(=O)O-* or -C(=O)O-*, and * is R 1 The compound of formula (I) is shown to bind to E 1 is -C(=O)O-*, and * is R at this position. 1 The compound of formula (I) is shown to bind to E 1 is -OC(=O)- *, and * is R at this position. 1 The compound of formula (I) is shown to bind to E 1 is -OC(=O)O-*, and * is R at this position. 1 The compound of formula (I) is shown to bind to E 1 and E 2 are the same or different and are -C(=O)O-*, -OC(=O)-*, or -OC(=O)O-*, and * is R 1 or R 2 where E 1 and E 2 (6) A compound of formula (I), wherein either one of E is —C(═O)O—*. 2 is -C(=O)O-* or -OC(=O)-*, and * is R 2 The compound of formula (I) is shown to bind to E 2 is -OC(=O)-* or -OC(=O)O-*, and * is R 2 The compound of formula (I) is shown to bind to E 2is -OC(=O)O-* or -C(=O)O-*, and * is R 2 The compound of formula (I) is shown to bind to E 2 is -C(=O)O-*, and * is R at this position. 2 The compound of formula (I) is shown to bind to E 2 is -OC(=O)-*, and * is R at this position. 2 The compound of formula (I) is shown to bind to E 2 is -OC(=O)O-*, and * is R at this position. 2 (7) R 1 is -CH2CH(-R x )R y or -CH2-(C 5-15 alkenyl). 1 is -CH2CH(-R x )R y A compound of formula (I) wherein R 1 -CH2CH(-OR x ) OR y A compound of formula (I) wherein R 1 -CH2-(C 5-15 R 1 -CH2-(C 5-15 alkenyl). 1 is -CH2CH(-R x )R y A compound of formula (I) wherein R 1 and R 2 are the same or different and -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 (8) Compounds of formula (I) wherein R is aryl; 2 is -CH2CH(-R x )R y or -CH2-(C 5-15 alkenyl).2 is -CH2CH(-R x )R y , -CH2CH(-OR x ) OR y , -CH2-(C 5-15 alkyl), or -CH2-(C 5-15 alkenyl). 2 is -CH2CH(-R x )R y A compound of formula (I) wherein R 2 -CH2CH(-OR x ) OR y A compound of formula (I) wherein R 2 -CH2-(C 5-15 R 2 -CH2-(C 5-15 (9) -E 1 -R 1 or -E 2 -R 2 However, one of them is -C(=O)O-CH2CH(-R x )R y A compound of formula (I) wherein E 1 -R 1 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)-(C 5-15 alkenyl). 1 -R 1 -C(=O)O-CH2CH(-R x )R y A compound of formula (I) wherein E 1 -R 1 is -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-(C1-6 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 A compound of formula (I) wherein E 1 -R 1 is -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-CHCH(-R x )R y -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 1 -R 1 is -C(=O)O-CH2CH(-R x )R y or -OC(=O)O-CH2CH(-R x )R y A compound of formula (I) wherein: (10) -E 1 -R 1 or -E 2 -R 2 The other 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 alkenyl). 2 -R 2 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 alkenyl). 2 -R 2 is -C(=O)O-CH2CH(-R x )R y A compound of formula (I) wherein E 2 -R 2 is -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), -OC(=O)-CHCH(-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-CHCH(-R x )R y -E 2 -R 2 is -C(=O)O-CH2CH(-R x )R y , -C(=O)O-C3H6-(C 1-6 alkylene)-CH=CH-CH=CH-(C1-6 alkyl), or -OC(=O)-C3H6-(C 1-6 alkylene)-CH=CH-CH=CH-(C 1-6 -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 -E 2 -R 2 is -C(=O)O-CH2CH(-R x )R y (11) A compound of formula (I) wherein R x C 5-10 Compounds of formula (I) where R is alkyl. x C 7-9 Compounds of formula (I) where R is alkyl. x Compounds of formula (I) where R is C7 alkyl. x Compounds of formula (I) where R is C8 alkyl. x Compounds of formula (I) where R is C9 alkyl. x Compounds of formula (I) where R is C8 alkyl or C9 alkyl. x and R y However, the same or different, C 5-15 (12) R y C 5-10 Compounds of formula (I) where R is alkyl. y C 7-9 Compounds of formula (I) where R is alkyl. y Compounds of formula (I) where R is C7 alkyl. y Compounds of formula (I) where R is C8 alkyl. y (13) Compounds of formula (I) where R is C9 alkyl. 3 is the expression:

[0078]

[0079] Compounds of formula (I), wherein R is a group selected from the group consisting of (d), (e), (g), and (h). 3 A compound of formula (I) wherein R is a group selected from the group consisting of formulas (e), (g), and (h). 3 A compound of formula (I) wherein R is formula (d). 3 A compound of formula (I) wherein R is formula (e). 3 A compound of formula (I) wherein R is formula (g). 3 A compound of formula (I) wherein R is formula (h). 3 where formulas (d), (e), (g), and (h):

[0080]

[0081] Compounds of formula (I) wherein R is a group selected from the group consisting of 3 is a group selected from the group consisting of formulas (e), (g), and (h). 3 (14) R a (15) Compounds of formula (I) wherein R is —CH3. b -CH2-C 1-6 Compounds of formula (I) where R is alkyl. b (16) R c and R d are both -CH3, and L cd Compounds of formula (I) wherein R is -CH2-. c and R d are both -CH2CH3, and L cd Compounds of formula (I) wherein R is -CH2CH2-. c and R d are both -CH2CH2OH, and L cd Compounds of formula (I) wherein R is -CH2CH2-. c is H and R d Compounds of formula (I) where R is —CH2C(═O)NH2. c and R d are all -CH3, -CH2CH3, or -CH2CH2OH, or R c If is H, then Rd is -CH2C(=O)NH2, and L cd is -CH2- or -CH2CH2-, where R c , R d When all of are -CH3, -CH2CH3, or -CH2CH2OH, L cd (17) A compound of formula (I) wherein R is -CH2-, -CH2CH2-, or -CH2CH2-. e Compounds of formula (I) wherein R is H. e (18) Compounds of formula (I) wherein R is OH. f Compounds of formula (I) wherein R is H. (19) g Compounds of formula (I) wherein R is -CH3. f and R g together with the carbon and nitrogen atoms to which they are attached form a pyrrolidine ring. g C 1-6 (20) R h (21) Compounds of formula (I) wherein R is -CH2CH3. i Compounds of formula (I) wherein R is -CH3. i C 1-6 (22) R j , R k A compound of formula (I) where R j and R k are the same or different, C 1-6 (21) A compound of formula (I) wherein s is 1. (22) A compound of formula (I) wherein s is 1. (23) A compound of formula (I) wherein s is 1. (24) A compound of formula (I) wherein t is 1. (25) A compound of formula (I) which is a consistent combination of the groups described in (1) to (24) above.

[0082] Specific examples of the combination of the above aspects include the following compounds or salts thereof: 1 is a bond and L 2 is -CH2- and L 3 is C6 alkylene, M is absent, n is 1, and E 1 is -C(=O)O-* and E 2is -C(=O)O-* and R 1 is -CH2CH(-R x )R y and R 2 is -CH2CH(-R x )R y and R x and R y is C8 alkyl or C9 alkyl, and R 3 is the formula (e), and R e is H and R f is H and R g Compounds of formula (I) wherein L is CH3. 1 is a bond and L 2 is -CH2- and L 3 is a C6 alkylene, M is absent, n is 1, and -E 1 -R 1 -C(=O)O-CH2CH(-R x )R y where R x and R y is a C8 alkyl, and -E 2 -R 2 is -C(=O)O-CH2CH(-R x )R y where R x and R y is a C9 alkyl, and R 3 is the formula (e), and R e is H and R f is H and R g A compound of formula (I) wherein is CH3.

[0083] Specific embodiments of the compound of the present invention include the following compounds or salts thereof: 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate, 8-[(1-methyl-L-prolyl)(3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}bicyclo[1.1.2-nonylundecyl 8-{(1-ethyl-L-prolyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate, 2-nonylundecyl 8-{(N,N-diethyl-β-alanyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate, 2-nonylundecyl 8-{(1,4-diethyl-1,4-diazepan-6-carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate, 2-nonylundecyl 8-{[(4-methylpiperazin-1-yl)acetyl][(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate, 2-nonylundecyl 8-[(1-methyl-L-prolyl){(1r,3S)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]octanoate, 2-heptylnonyl (1S,4r)-4-[{8-[(2-heptylnonyl)oxy]-8-oxooctyl}(1-methyl-L-prolyl)amino]cyclohexane-1-carboxylate, 2-nonylundecyl 8-{(1-ethyl-D-prolyl)[(1r,3R)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate, 3-decyltridecyl 8-{[(1r,3S)-3-{2-[(3-decyltridecyl)oxy]-2-oxoethyl}cyclobutyl](1-methyl-L-prolyl)amino}octanoate, 2-{(1-ethyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}ethyl 4,4-bis(octyloxy)butanoate, or A compound which is 4-[(1-ethyl-L-prolyl){(1r,3S)-3-[({[(2-nonylundecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]butyl 4-octyldodecanoate, or a salt thereof.

[0084] Specific embodiments of the compound of the present invention include the following compounds or salts thereof: 2-nonylundecyl 8-{(1-ethyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate, or 2-nonylundecyl 8-{(N,N-diethyl-β-alanyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate, or a salt thereof.

[0085] In this specification, the compound of formula (I) or a salt thereof may be described in only one isomeric form, but the present invention also includes other isomers, and also includes isolated isomers and mixtures thereof.

[0086] Compounds of formula (I) may exist as tautomers or geometric isomers depending on the type of substituents. Although compounds of formula (I) may be described herein in only one isomeric form, the present invention encompasses other isomers, including isolated isomers and mixtures thereof. In this specification, the term "all double bonds are Z-configuration" refers to the (Z) configuration, i.e., the cis configuration. Furthermore, the term "all double bonds are E-configuration" refers to the (E) configuration, i.e., the trans configuration. Furthermore, compounds of formula (I) or salts thereof may have asymmetric centers or axial asymmetry, which may result in the existence of enantiomers (optical isomers). Compounds of formula (I) or salts thereof encompass both isolated individual enantiomers, such as the (R) configuration and the (S) configuration, and mixtures thereof (including racemic and non-racemic mixtures). In some embodiments, enantiomers are "stereochemically pure." "Stereochemically pure" refers to a purity that can be recognized by those skilled in the art as being substantially stereochemically pure. In some embodiments, an enantiomer is a compound having a stereochemical purity of, for example, 90% ee (enantiomeric excess) or more, 95% ee or more, 98% ee or more, or 99% ee or more.

[0087] The salt of the compound of formula (I) is a pharmaceutically acceptable salt, and may form an acid addition salt depending on the type of substituent. Specific examples include acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid, and 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, dibenzoyltartaric acid, ditoluoyltartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, aspartic acid, and glutamic acid.

[0088] Furthermore, the present invention also encompasses various hydrates and solvates, and crystalline polymorphic substances of the compound of formula (I) or a salt thereof.

[0089] The present invention also encompasses compounds of formula (I) or salts thereof that are pharmaceutically acceptable and labeled with one or more radioactive or non-radioactive isotopes. Suitable examples of isotopes used to isotopic label the compounds of the present 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) are included. Isotopically labeled compounds of the present invention, drugs, and / or substrates may be used in tissue distribution studies and other studies. For example, tritium ( 3 H), carbon-14 ( 14 Radioactive isotopes such as C may be used for this purpose due to their ease of labeling and detection. 2Substitution with positron-emitting isotopes (H) may be therapeutically advantageous due to increased metabolic stability (e.g., increased in vivo half-life, reduced dosage requirements, and fewer drug interactions). 11 C, 18 F, 15 O and 13 Substitution with an isotopically labeled N or the like can be used in positron emission tomography (PET) studies to examine substrate receptor occupancy. Isotopically labeled compounds of the present invention can generally be prepared by conventional methods known to those skilled in the art, or by methods similar to those described in the Examples or Preparations, using appropriate isotopically labeled reagents in place of unlabeled reagents.

[0090] 2. Lipid Nanoparticles Some embodiments of lipid nanoparticles containing the compound of formula (I) of the present invention or a salt thereof are shown below. Note that in the following embodiments, "a compound of formula (I) or a salt thereof" includes the embodiment of "a compound of formula (I) or a salt thereof" described above in (Embodiments of the cationic lipid of the present invention).

[0091] (Embodiments of lipid nanoparticles of the present invention) (1) Lipid nanoparticles comprising a compound of formula (I) or a salt thereof. (2) Lipid nanoparticles comprising a compound of formula (I) or a salt thereof, a neutral lipid, and a PEGylated lipid. (3) Lipid nanoparticles according to (2), which encapsulate a nucleic acid.

[0092] (4) The lipid nanoparticles according to (2) to (3), wherein the neutral lipid is a phospholipid and a sterol. The lipid nanoparticles according to (2) to (3), wherein the neutral lipid is DSPC and cholesterol. (4a) The lipid nanoparticles according to (4), wherein the phospholipid is DSPC. (4b) The lipid nanoparticles according to (4), wherein the sterol is cholesterol. (5) The lipid nanoparticles according to (3), wherein the PEGylated lipid is DMG-PEG2000.

[0093] (6) Lipid nanoparticles in which the composition ratio of each component is the molar percentage shown in (6a) to (6e) based on the total amount of the lipid nanoparticles. (6a) Lipid nanoparticles according to (3), in which the composition ratio of the compound of formula (I) or a salt thereof is 20.0 to 80.0 molar percentage based on the total amount of the lipid nanoparticles. Lipid nanoparticles containing the compound of formula (I) or a salt thereof, in which the composition ratio of the compound of formula (I) or a salt thereof is 30.0 to 60.0 molar percentage based on the total amount of the lipid nanoparticles. (6b) Lipid nanoparticles according to (3), in which the composition ratio of neutral lipids is 18.5 to 78.5 molar percentage based on the total amount of the lipid nanoparticles. Lipid nanoparticles according to (3), in which the composition ratio of neutral lipids is 38.5 to 68.5 molar percentage based on the total amount of the lipid nanoparticles. (6c) Lipid nanoparticles according to (6b), in which the composition ratio of phospholipids is 4.0 to 18.1 molar percentage based on the total amount of the lipid nanoparticles. (6b) The lipid nanoparticles according to (6b), wherein the composition ratio of phospholipids is 5.0 to 18.1 mol% based on the total amount of the lipid nanoparticles. (6d) The lipid nanoparticles according to (6b), wherein the composition ratio of sterols is 14.5 to 62.3 mol% based on the total amount of the lipid nanoparticles. (6b) The lipid nanoparticles according to (6b), wherein the composition ratio of sterols is 25.5 to 54.4 mol% based on the total amount of the lipid nanoparticles. (6e) The lipid nanoparticles according to (3), wherein the composition ratio of PEGylated lipids is 0.5 to 2.5 mol% based on the total amount of the lipid nanoparticles. The lipid nanoparticles according to (3), wherein the composition ratio of PEGylated lipids is 0.5 to 2.0 mol% based on the total amount of the lipid nanoparticles.

[0094] (7) The lipid nanoparticles according to (3), wherein the nucleic acid is mRNA. The lipid nanoparticles according to (3), wherein the nucleic acid is a nucleic acid useful for the prevention and / or treatment of an astrocyte-related disease. The lipid nanoparticles according to (3), wherein the nucleic acid is mRNA useful for the prevention and / or treatment of an astrocyte-related disease. The lipid nanoparticles according to (3), wherein the nucleic acid is Ascl1 mRNA, Dlx2 mRNA, NeuroD1 mRNA, Ngn2 mRNA, BDNF mRNA, NGF mRNA, or NT-3 mRNA. The lipid nanoparticles according to (3), wherein the nucleic acid is BDNF mRNA or NeuroD1 mRNA. The lipid nanoparticles according to (3), wherein the nucleic acid is NeuroD1 mRNA. (8) The lipid nanoparticles according to (7), wherein the nucleic acid is NeuroD1 mRNA comprising a nucleotide sequence encoding a protein having 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) sequence identity to a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2. The lipid nanoparticle according to (7), wherein the nucleic acid is NeuroD1 mRNA comprising a base sequence having 70% or more (e.g., 80% or more, 90% or more, 95% or more) sequence identity to the base sequence shown in SEQ ID NO: 1 or 3, and further encodes the amino acid sequence shown in SEQ ID NO: 2. The lipid nanoparticle according to (7), wherein the nucleic acid is NeuroD1 mRNA comprising a base sequence having 70% or more (e.g., 80% or more, 90% or more, 95% or more) sequence identity to the base sequence shown in SEQ ID NO: 1, and further encodes the amino acid sequence shown in SEQ ID NO: 2. The lipid nanoparticle according to (7), wherein the nucleic acid is NeuroD1 mRNA comprising a base sequence having 70% or more (e.g., 80% or more, 90% or more, 95% or more) sequence identity to the base sequence shown in SEQ ID NO: 3, and further encodes the amino acid sequence shown in SEQ ID NO: 2. (8a) The lipid nanoparticle according to (7), wherein the nucleic acid is NeuroD1 mRNA comprising a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 2. The lipid nanoparticle according to (7), wherein the nucleic acid is NeuroD1 mRNA comprising the base sequence shown in SEQ ID NO: 1. The lipid nanoparticle according to (7), wherein the nucleic acid is NeuroD1 mRNA comprising the base sequence shown in SEQ ID NO: 3.The lipid nanoparticle according to (7), wherein the nucleic acid is NeuroD1 mRNA comprising the nucleotide sequence shown in SEQ ID NO: 4. The lipid nanoparticle according to (7), wherein the nucleic acid is NeuroD1 mRNA comprising the nucleotide sequence shown in SEQ ID NO: 5. The lipid nanoparticle according to (7), wherein the nucleic acid is NeuroD1 mRNA comprising a nucleotide sequence having 70% or more (e.g., 80% or more, 90% or more, 95% or more) sequence identity to the nucleotide sequence shown at positions 44 to 1114 of SEQ ID NO: 4, and further encoding the amino acid sequence shown in SEQ ID NO: 2. The lipid nanoparticle according to (7), wherein the nucleic acid is NeuroD1 mRNA comprising a nucleotide sequence having 70% or more (e.g., 80% or more, 90% or more, 95% or more) sequence identity to the nucleotide sequence shown at positions 44 to 1114 of SEQ ID NO: 5, and further encoding the amino acid sequence shown in SEQ ID NO: 2.

[0095] (9) The lipid nanoparticle according to (7), wherein the nucleic acid is NeuroD1 mRNA. (9a) The lipid nanoparticle according to (9), wherein the 5' cap is Cap-0, Cap-1, or Cap-2. The lipid nanoparticle according to (9), wherein the 5' cap is Cap-1. (9b) The lipid nanoparticle according to (9), wherein the 5' UTR comprises a Kozak sequence. The lipid nanoparticle according to (9), wherein the 5' UTR or 3' UTR of the nucleic acid is derived from an alpha globin gene. The lipid nanoparticle according to (9), wherein the 5' UTR or 3' UTR of the nucleic acid is derived from a human alpha globin gene. The lipid nanoparticle according to (9), wherein the 5' UTR or 3' UTR of the nucleic acid is derived from a beta globin gene. The lipid nanoparticle according to (9), wherein the 5' UTR or 3' UTR is derived from a human beta globin gene. The lipid nanoparticle according to (9), wherein the 5' UTR comprises a Kozak sequence. The lipid nanoparticle according to (9), wherein the 5' UTR is derived from an alpha globin gene. The lipid nanoparticle according to (9), wherein the 5' UTR is derived from a human α globin gene. The lipid nanoparticle according to (9), wherein the 5' UTR is derived from a β globin gene. The lipid nanoparticle according to (9), wherein the 5' UTR is derived from a human β globin gene. The lipid nanoparticle according to (9), wherein the 3' UTR is derived from a human α globin gene. The lipid nanoparticle according to (9), wherein the 3' UTR is derived from a human β globin gene. (9c) The lipid nanoparticle according to (9), wherein the poly-A tail is NeuroD1 mRNA having 20 to 1000 bases. The lipid nanoparticle according to (9), wherein the poly-A tail is NeuroD1 mRNA having 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 nanoparticle according to (9), wherein the poly-A tail is NeuroD1 mRNA having 120 bases. The lipid nanoparticle according to (9), wherein the poly A tail is NeuroD1 mRNA having 79 bases. (9d) The lipid nanoparticle according to (9), comprising a modified nucleotide. The lipid nanoparticle according to (9), comprising N1-methylpseudouridine. The lipid nanoparticle according to (9), wherein some or all of the uridines are N1-methylpseudouridine. The lipid nanoparticle according to (9), wherein some of the uridines are substituted with N1-methylpseudouridine.(9) Lipid nanoparticles in which all uridines are substituted with N1-methylpseudouridine. (10) Lipid nanoparticles which are a consistent combination of two or more of (1) to (9) described above (Embodiments of lipid nanoparticles of the present invention). Lipid nanoparticles which are a consistent combination of an embodiment described in (Embodiments of cationic lipids of the present invention) and an embodiment described in (Embodiments of lipid nanoparticles of the present invention).

[0096] Specific examples of the combination of (10) above include the following: (10a) Lipid nanoparticles encapsulating a nucleic acid, the lipid nanoparticles comprising a compound of formula (I) or a salt thereof, a neutral lipid, and a PEGylated lipid, wherein the nucleic acid is an mRNA encoding NeuroD1 protein, and the compound of formula (I) or a salt thereof is 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate or a salt thereof, the neutral lipid is DSPC and cholesterol, and the PEGylated lipid is DMG-PEG2000. (10b) Lipid nanoparticles according to (10a), the nucleic acid is an mRNA encoding NeuroD1 protein, the base sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2. (10c) The lipid nanoparticle according to (8a), wherein the nucleic acid is mRNA encoding the NeuroD1 protein according to (10a), comprising the nucleotide sequence shown in SEQ ID NO: 1. (10d) The lipid nanoparticle according to (8a), wherein the nucleic acid is mRNA encoding the NeuroD1 protein according to (10a), comprising the nucleotide sequence shown in SEQ ID NO: 3. (10e) The lipid nanoparticle according to (8a), wherein the nucleic acid is mRNA encoding the NeuroD1 protein according to (10a), comprising the nucleotide sequence shown in SEQ ID NO: 4. (10f) The lipid nanoparticle according to (8a), wherein the nucleic acid is mRNA encoding the NeuroD1 protein according to (10a), comprising the nucleotide sequence shown in SEQ ID NO: 5. (10g) Lipid nanoparticles according to (10a) to (10f), comprising, based on the total amount of the lipid nanoparticles, 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate or a salt thereof at a composition ratio of 20.0 to 70.0 mol %, neutral lipid (i.e., DSPC and cholesterol) at 27.0 to 79.5 mol %, and DMG-PEG2000 at 0.5 to 3.0 mol %.(10h) Lipid nanoparticles according to (10a) to (10f), comprising 35.0 to 50.0 mol% of 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate or a salt thereof, 47.5 to 64.0 mol% of a neutral lipid (i.e., DSPC and cholesterol), and 1.0 to 2.5 mol% of DMG-PEG2000, based on the total amount of the lipid nanoparticles. (10i) Lipid nanoparticles encapsulating a nucleic acid, the nucleic acid comprising, in its base sequence, nucleotides 1 to 3 of which are a 5'-end sequence suitable for capping using CleanCap (registered trademark) Reagent AG (TriLink BioTechnologies), nucleotides 4 to 43 of which are 5'-UTR, nucleotides 44 to 1114 of which are the CDS of the human NeuroD1 gene (SEQ ID NO: 3), nucleotides 1115 to 1120 of which are two consecutive stop codons, nucleotides 1121 to 1231 of which are 3'-UTR, and nucleotides 1232 to 1310 of which are mRNA encoding the NeuroD1 protein corresponding to a poly(A) tail, the lipid nanoparticles comprising a neutral lipid, a PEGylated lipid, and the compound of formula (I) or a salt thereof.

[0097] Specific embodiments of lipid nanoparticles encompassed by the present invention include the following: (11a) Lipid nanoparticles encapsulating a nucleic acid and comprising a neutral lipid, a PEGylated lipid, and a compound of formula (I) or a salt thereof, wherein the nucleic acid is mRNA encoding human ND1 (wherein the 5' cap structure is Cap-1, the CDS is the CDS of the human ND1 gene (SEQ ID NO: 1), and the polyA tail is 120 bases), and the lipid nanoparticles comprise 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate or a salt thereof, the neutral lipid is DSPC and cholesterol, and the PEGylated lipid is DMG-PEG2000. (11b) The nucleic acid is an mRNA encoding human ND1 (wherein the 5' cap structure is Cap-1, the 5' UTR is derived from the human α-globin gene, the CDS is the CDS of the human ND1 gene (SEQ ID NO: 3), the 3' UTR is derived from the human α-globin gene, and the poly(A) tail consists of a nucleotide sequence (SEQ ID NO: 4) containing 79 polynucleotides in which all uridines are N1-methylpseudouridines (SEQ ID NO: 5), wherein, in the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5, nucleotides 1 to 3 correspond to AGG, positions 4 to 43 correspond to the 5' UTR, positions 44 to 1114 correspond to the CDS of the human NeuroD1 gene (SEQ ID NO: 3), positions 1115 to 1120 correspond to two consecutive stop codons, positions 1121 to 1231 correspond to the 3' UTR, and positions 1232 to 1310 correspond to the poly(A) sequence); Lipid nanoparticles encapsulating a nucleic acid and comprising a neutral lipid, a PEGylated lipid, and a compound of formula (I) or a salt thereof, wherein the lipid nanoparticles are 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate or a salt thereof, the neutral lipid is DSPC and cholesterol, and the PEGylated lipid is DMG-PEG2000.(11c) Lipid nanoparticles encapsulating a nucleic acid and comprising a neutral lipid, a PEGylated lipid, and a compound of formula (I) or a salt thereof, wherein the nucleic acid is mRNA encoding human ND1 (wherein the 5' cap structure is Cap-1 and consists of the base sequence shown at positions 1 to 1231 of SEQ ID NO: 4 or the base sequence shown at positions 1 to 1231 of SEQ ID NO: 5, and a poly A chain), and the lipid nanoparticles are 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate or a salt thereof, the neutral lipid is DSPC and cholesterol, and the PEGylated lipid is DMG-PEG2000. (11d) Lipid nanoparticles according to any one of (11a) to (11c), which contain a nucleic acid encapsulated therein and which comprise a neutral lipid, a PEGylated lipid, and the compound of formula (I) or a salt thereof, and which contain 50 mol % of 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate or a salt thereof, 48.5 mol % of DSPC and cholesterol, and 1.5 mol % of DMG-PEG2000, based on the total amount of the lipid nanoparticles.

[0098] 3. Pharmaceutical Compositions Some embodiments of pharmaceutical compositions containing lipid nanoparticles containing the compound of formula (I) of the present invention or a salt thereof, and one or more pharmaceutically acceptable excipients are shown below.

[0099] (Embodiments of the pharmaceutical composition of the present invention) (1) (Embodiments of the lipid nanoparticles of the present invention) A pharmaceutical composition comprising the lipid nanoparticles according to any one of (3) to (11). (2) (Embodiments of the lipid nanoparticles of the present invention) A pharmaceutical composition comprising the lipid nanoparticles according to any one of (3) to (11) and one or more pharmaceutically acceptable pharmaceutical additives. (3) The pharmaceutical composition according to (1) or (2) for the prevention and / or treatment of an astrocyte-related disease.

[0100] Specific embodiments of pharmaceutical compositions encompassed by the present invention include the following: (A) A pharmaceutical composition comprising lipid nanoparticles encapsulating a nucleic acid and comprising a neutral lipid, a PEGylated lipid, and a compound of formula (I) or a salt thereof, wherein the nucleic acid is mRNA encoding NeuroD1 protein, and the lipid nanoparticles contain the compound of formula (I) or a salt thereof, 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate or a salt thereof, the neutral lipid is DSPC and cholesterol, and the PEGylated lipid is DMG-PEG2000. (B) A pharmaceutical composition comprising lipid nanoparticles encapsulating a nucleic acid and comprising a compound of formula (I) or a salt thereof, a neutral lipid, and a PEGylated lipid, wherein the nucleic acid is mRNA encoding NeuroD1 protein, the base sequence of which encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, and in the lipid nanoparticles, the compound of formula (I) or a salt thereof is 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate or a salt thereof, the neutral lipid is DSPC and cholesterol, and the PEGylated lipid is DMG-PEG2000. (C) A pharmaceutical composition comprising lipid nanoparticles encapsulating a nucleic acid and comprising 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 (wherein the 5' cap structure is Cap-1, the CDS is the CDS of the human ND1 gene (SEQ ID NO: 1), and the polyA tail is 120 bases), and in the lipid nanoparticles, the compound of formula (I) or a salt thereof is 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate or a salt thereof, the neutral lipid is DSPC and cholesterol, and the PEGylated lipid is DMG-PEG2000.(D) the nucleic acid is an mRNA encoding human ND1 (wherein the 5' cap structure is Cap-1, the 5' UTR is derived from the human α globin gene, the CDS is the CDS of the human ND1 gene (SEQ ID NO: 3), the 3' UTR is derived from the human α globin gene, and the poly(A) tail consists of a nucleotide sequence (SEQ ID NO: 4) containing 79 polynucleotides in which all uridines are N1-methylpseudouridines (SEQ ID NO: 5), wherein, in the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5, nucleotides 1 to 3 correspond to AGG, positions 4 to 43 correspond to the 5' UTR, positions 44 to 1114 correspond to the CDS of the human NeuroD1 gene (SEQ ID NO: 3), positions 1115 to 1120 correspond to two consecutive stop codons, positions 1121 to 1231 correspond to the 3' UTR, and positions 1232 to 1310 correspond to the poly(A) sequence); A pharmaceutical composition comprising lipid nanoparticles encapsulating a nucleic acid and comprising a compound of formula (I) or a salt thereof, a neutral lipid, and a PEGylated lipid, wherein the lipid nanoparticles are 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate or a salt thereof, the neutral lipid is DSPC and cholesterol, and the PEGylated lipid is DMG-PEG2000. (E) A pharmaceutical composition comprising lipid nanoparticles encapsulating a nucleic acid and comprising 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 (wherein the 5' cap structure is Cap-1 and consists of the base sequence shown at positions 1 to 1231 of SEQ ID NO: 4 or the base sequence shown at positions 1 to 1231 of SEQ ID NO: 5, and a poly A chain), and in the lipid nanoparticles, the compound of formula (I) or a salt thereof is 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate or a salt thereof, the neutral lipid is DSPC and cholesterol, and the PEGylated lipid is DMG-PEG2000.

[0101] The administration form of the pharmaceutical composition is not limited, and examples include parenteral administration such as intracerebral, intraarticular, intravenous, intramuscular, or subcutaneous injections, transmucosal liquids, and inhalants. Typically, administration into the brain results in delivery to the central nervous system. In some embodiments, administration is intracerebral parenchymal administration. In some embodiments, administration is intraventricular administration. In some embodiments, administration is intraspinal administration. In some embodiments, administration is intrathecal administration. In some embodiments, administration is intraspinal parenchymal administration.

[0102] Typically, when administered intracerebrally, the pharmaceutical composition containing the lipid nanoparticles of the present invention is administered in one embodiment at a dose of about 0.001 to 10 mg per kg of brain, in another embodiment at a dose of about 0.001 to 50 mg per kg of brain, or in another embodiment at a dose of about 0.001 to 100 mg per kg of brain, administered once or multiple times daily. The dose is determined appropriately for each individual case, taking into consideration the disease, symptoms, age, sex, etc.

[0103] Typically, when administered intramuscularly, the daily dose of the pharmaceutical composition of the present invention is approximately 1 to 10 mg / kg of body weight, administered once or in divided doses per day. The dose is determined appropriately for each individual case, taking into account the disease, symptoms, age, sex, etc.

[0104] Although it varies depending on the route of administration, dosage form, administration site, and types of excipients and additives, the pharmaceutical composition of the present invention contains lipid nanoparticles in an amount of 5 to 50% by weight based on the total weight of the pharmaceutical composition, and the content of lipid nanoparticles, etc. is in an amount of 3 to 70% by weight, and in an amount of 10 to 50% by weight, in another embodiment.

[0105] The pharmaceutical composition of the present invention can be used in combination with various therapeutic or preventive agents for diseases for which the pharmaceutical composition is believed to be effective. The combination may be administered simultaneously, separately, consecutively, or at a desired time interval. The simultaneous administration preparations may be formulated as a combined preparation or separately.

[0106] 4. Method for Producing the Cationic Lipid of the Present Invention The compound of formula (I) or a salt thereof, which is the compound of the present invention, can be produced by various known synthetic methods, taking advantage of characteristics based on its basic structure or the type of substituent. In this case, depending on the type of functional group, it may be effective from a manufacturing technology perspective to replace the functional group with an appropriate protecting group (a group that can be easily converted to the functional group) at the stage from the raw material to the intermediate. Examples of such protecting groups include those described in "Greene's Protective Groups in Organic Synthesis" (5th ed., 2014) by PGM Wuts and TW Greene, and can be appropriately selected and used depending on the reaction conditions. In such a method, the desired compound can be obtained by introducing the protecting group, carrying out the reaction, and then removing the protecting group as necessary.

[0107] Representative methods for producing the compound of formula (I) or a salt thereof are described below. Each method can be performed by referring to the references attached to the description. However, the production methods of the present invention are not limited to the examples shown below.

[0108] The compound of the present invention or a salt thereof can be produced by the following production method. (Production Method 1)

[0109]

[0110] Compound (I) of the present invention can be produced by the amidation reaction of compound (1) with compound (2). This reaction is carried out in a solvent containing a mixture of compound (1) and compound (2) and a condensing agent. Examples of the solvent include aromatic hydrocarbons such as benzene, toluene, or xylene, halogenated hydrocarbons such as DCM, DMF, DMSO, and mixtures thereof. Examples of the condensing agent include HATU, EDCI·HCl, and the like. Additives (e.g., HOBt, DMAP) may promote the reaction. Organic bases (e.g., TEA, DIPEA, etc.) and inorganic bases (e.g., KCO, NaCO, or KOH) may further promote the reaction. Alternatively, the compound can be produced by converting carboxylic acid (1) to a reactive derivative and then reacting it with amine (2). Examples of reactive derivatives of carboxylic acid include acid halides obtained by reaction with a halogenating agent such as phosphorus oxychloride or thionyl chloride, mixed acid anhydrides obtained by reaction with isobutyl chloroformate, and activated 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 of, in one embodiment, -20°C to 60°C. The solvent can be a halogenated hydrocarbon such as DCM, an aromatic hydrocarbon, an ether, or the like. In this reaction, instead of compound (1), R protected with a protecting group can be used. 3 After using a group that can be converted to 3 Deprotection or the desired reaction is carried out from the group that can be converted to R 3 In this reaction, compound (I) can be produced by derivatizing compound (I) into 3 After carrying out this reaction using a group that can be converted to R 3 Compound (I) can also be produced by derivatizing the above-mentioned aryl group to the above-mentioned aryl group. [References] SR Sandler and W. Karo, "Organic Functional Group Preparations," Academic Press Inc., 1991, 2nd edition, Vol. 1. "Experimental Chemistry Lectures (5th edition)," edited by the Chemical Society of Japan, Vol. 16, 2005, Maruzen.

[0111] (Second manufacturing method)

[0112]

[0113] The compound (I-1) of the present invention can be produced by the esterification reaction of compound (3-1) with compound (4-2). This reaction is carried out in a solvent containing a mixture of compound (3-1) and compound (4-2) and a condensing agent. Examples of the solvent include halogenated hydrocarbons such as DCM, DMF, DMSO, and mixtures thereof. Examples of the condensing agent include HATU, EDCI·HCl, CDI, DPPA, and phosphorus oxychloride. Additives (e.g., HOBt, DMAP) may promote the reaction. Organic bases (e.g., TEA, DIPEA, etc.) and inorganic bases (e.g., K2CO3, Na2CO3, or KOH) may further promote the reaction. Alternatively, the compound can be produced by converting carboxylic acid (4-2) to a reactive derivative and then reacting it with alcohol (3-1). Examples of reactive derivatives of carboxylic acids include acid halides obtained by reaction with a halogenating agent such as phosphorus oxychloride or thionyl chloride, mixed acid anhydrides obtained by reaction with isobutyl chloroformate, and activated 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 of, in some embodiments, -20°C to 60°C. The solvent is a halogenated hydrocarbon such as DCM. Compound (I-2) of the present invention can be produced by esterification of compound (3-2) with compound (4-1). This reaction can be carried out under the same conditions as in Production Method 2.

[0114] (Raw material manufacturing method 1)

[0115]

[0116] (where Lv represents a leaving group.) Compound (2) can be produced by the reaction of compound (5) with compound (6). Examples of the leaving group Lv include halogen, methanesulfonyloxy, and the like. This reaction involves reacting a mixture of equal amounts of compound (5) and compound (6) or an excess of one in a solvent. In one embodiment, the reaction temperature is 0°C to 100°C. In another embodiment, the temperature is 50°C to 90°C. Examples of solvents include halogenated hydrocarbons such as DCM and 1,2-dichloroethane, DMF, DMSO, MeCN, CPME, and mixtures thereof. An organic base (such as TEA or DIPEA), an inorganic base (such as KCO, NaCO, or KOH), or an additive (such as KI or NaI) can further promote the reaction. The starting compound (6) can be produced by constructing E2 by esterification or carbonation of the corresponding starting material. The esterification can be carried out under the conditions described above. [References] SR Sandler and W. Karo, "Organic Functional Group Preparations", 1991, 2nd edition, Vol. 1, Academic Press Inc.; "Experimental Chemistry Lectures (5th edition)", edited by the Chemical Society of Japan, Vol. 14, 2005, Maruzen.

[0117] (Raw material manufacturing method 2)

[0118]

[0119]

[0120] Compound (3-1) can be produced by alkylating compound (5) with compound (7-1), amidating the resulting compound (8-1) with compound (1), and deprotecting the resulting compound (9-1). The alkylation reaction can be carried out in the same manner as in Method 1. The amidation can be carried out in the same manner as in Method 1. Compound (3-2) can be produced in the same manner as in the production method for compound (3-1). Deprotection can be carried out by standard deprotection. See Greene and Wuts, "Protective Groups in Organic Synthesis," 3rd Edition, John Wiley & Sons Inc., 1999.

[0121] (Raw material manufacturing method 3)

[0122]

[0123]

[0124] (In the formula, Pr represents a protecting group.)

[0125] Compound (5-4) can be produced from compound (5-1), which is one embodiment of compound (5) protected with a protecting group, by esterification, carbonate, or carbamate formation. Compound (5-3) can also be produced from compound (10-1) and compound (11-1'). Compound (10-1') can be produced from compound (10-1). Compound (5-3) can be produced from compound (10-1') and compound (11-1). Compound (11-1') can be produced from compound (11-1). Compound (5-4) can be produced from compound (10-1') and compound (12). In the above production method, compound (10-1') and compound (11-1') are isolated and then subjected to the next reaction, but depending on the compound, the next reaction can be carried out without isolation. (Starting Material Production Method 4)

[0126]

[0127] (In the formula, Pr represents a protecting group.)

[0128] Compound (5-7) can be produced from compound (5-5), which is one embodiment of compound (5) protected with a protecting group, by amidation.

[0129] The compound of formula (I) or a salt thereof is isolated and purified as a free compound, a salt thereof, a hydrate, a solvate, or a crystalline polymorph. The compound of formula (I) or a salt thereof can also be produced by a conventional salt formation reaction. Isolation and purification are carried out using conventional procedures such as extraction, fractional crystallization, and various fractional chromatography (e.g., silica gel chromatography). Various isomers can be produced by selecting appropriate starting compounds, or can be separated by taking advantage of differences in physicochemical properties between isomers. For example, optical isomers can be obtained by common optical resolution methods of racemates (e.g., fractional crystallization leading to diastereomeric salts with optically active bases or acids, chromatography using chiral columns, etc.), or can also be produced from appropriate optically active starting compounds.

[0130] 5. Method for Producing Lipid Nanoparticles of the Present Invention Lipid nanoparticles can be produced by adding components such as nucleic acid to the compound of formula (I) or its salt, neutral lipid, and PEGylated lipid components, and dispersing them in a medium. For example, the compound of formula (I) or its salt, DSPC, sterol, and PEGylated lipid are dissolved in a solvent to form an oil phase, which is then mixed or suspended in an aqueous phase such as a buffer solution containing nucleic acid. The solvent in the mixed solution is then removed by dialysis, ultrafiltration, or other techniques to obtain lipid nanoparticles. Pharmaceutical compositions can also be produced by known methods using additives such as the excipients described above in addition to nucleic acid-lipid nanoparticles.

[0131] 6. Method for Manufacturing Pharmaceutical Compositions Pharmaceutical compositions can be prepared by commonly used methods using pharmaceutical additives (excipients, etc.) commonly used in the art, i.e., pharmaceutical excipients, etc. Pharmaceutical additives are not limited to the components contained in pharmaceutical compositions, and may contain preservatives, stabilizers, antioxidants, antiseptics, and other additives in addition to excipients. Other additives may also be added to the pharmaceutical composition. Pharmaceutical compositions may also contain pharmaceutically acceptable vehicles.

[0132] The pharmaceutical composition is refrigerated or frozen for storage and / or transportation. In one embodiment, the temperature is about −150° C. to about 0° C., in one embodiment, about −80° C. to about −20° C., in one embodiment, about −40° C. to about −20° C., and in one embodiment, 4° C. or below. In one embodiment, the solution is PBS.

[0133] Injectable preparations preferably contain 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, for example, alcohols such as ethanol. Such compositions may further contain an isotonic agent, an antiseptic, a wetting agent, an emulsifier, a dispersant, a stabilizer, or a solubilizing agent. These are sterilized, for example, by filtration through a bacteria-retaining filter, addition of a sterilizing agent, or irradiation. Alternatively, these can be prepared as sterile solid compositions, which can be dissolved or suspended in sterile water or a sterile injectable solvent before use.

[0134] Transmucosal preparations such as inhalants and nasal preparations are liquid and can be prepared according to conventional methods. For example, known excipients, as well as pH adjusters, preservatives, surfactants, lubricants, stabilizers, thickeners, etc., may be added as appropriate. Administration can be performed using a suitable inhalation or insufflation device. For example, known devices such as metered dose inhalers or nebulizers can be used to administer the compound alone or as a powder of a formulated mixture, or as a solution or suspension in combination with pharmaceutically acceptable excipients. Dry powder inhalers and the like may be for single or multiple administrations. They may also be in the form of a pressurized aerosol spray using a suitable propellant, for example, a suitable gas such as a chlorofluoroalkane or carbon dioxide.

[0135] 7. Methods for Producing Nucleic Acids Nucleic acids, such as mRNA, can be produced using techniques known in the art. Examples include, but are not limited to, the following. mRNA can be produced by in vitro transcription (IVT) using a linearized plasmid as a DNA template. Known production methods include (i) post-transcriptional capping (which involves plasmid preparation, in vitro transcription, and 5'-capping, in that order) and (ii) co-transcriptional capping (which involves plasmid preparation, in vitro transcription, and 5'-capping simultaneously, in that order). In the co-transcriptional capping method, Cap-0 is added to the 5' end of RNA using anti-reverse cap analog (ARCA) technology, and Cap-1 is added to the 5' end of RNA using CleanCap (registered trademark, TriLink) capping technology.

[0136] In vitro transcription (IVT) typically involves a transcription buffer, nucleoside triphosphates (NTPs), an RNase inhibitor, and a polymerase (e.g., T7 RNA polymerase). NTPs can be either natural or non-natural (modified).

[0137] (Capping) Capping can be performed using vaccinia capping enzyme, 2'O-methyltransferase, CleanCap, or other methods. If the sequence transcribed by IVT does not contain a polyA sequence, a polyA addition reaction can also be performed. Regarding the sequence, when using CleanCap (registered trademark) Reagent AG (TriLink BioTechnologies), a 5'-terminal sequence suitable for the capping method using CleanCap (registered trademark) Reagent AG (TriLink BioTechnologies) is, for example, AGG (positions 1 to 3 of SEQ ID NO: 4).

[0138] (Plasmid Preparation) Linearized plasmids can be prepared using the following steps. A plasmid can be prepared by inserting any UTR and any base sequence (e.g., a base sequence encoding NeuroD1 protein) into a plasmid (e.g., a high-copy plasmid for E. coli (pUC18 or pCU18, etc.)), and then amplifying the plasmid using E. coli or similar. After purification, the linearized plasmid can be prepared using restriction enzymes and buffer. After purification, the linearized plasmid can be used directly as a DNA template. Alternatively, the linearized plasmid can be used as a DNA template after polymerase chain reaction (PCR). (Purification) mRNA and intermediates in its production process can be purified using methods known in the art. Examples include, but are not limited to, the following: The DNA template can be removed using deoxyribonuclease I (DNase I). The transcribed RNA can be purified using a silica gel column, etc. RNA containing a poly(A) sequence can be purified using an Oligo dT column. Linearized plasmids can be purified using PureLink (registered trademark) (Thermo Fisher Scientific) or the like. Test Example

[0139] The pharmacological activity of nucleic acid-lipid nanoparticles composed of the compound of formula (I) or a salt thereof was confirmed by the following test. The following abbreviations may be used in the following test examples and the like in this specification. (Abbreviations) B-27: B-27 serum-free supplement, BDNF: brain-derived neurotrophic factor, DMEM: Dulbecco's modified Eagle's medium, DMG-PEG2000: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000, DOTAP: 1,2-dioleoyloxy-3-trimethylammonium propane, DOTMA: 1,2-di-O-octadecenyl-3-trimethylammonium propane, DSPC: 1,2-distearoyl-sn-glycero-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: NeuroD1 Lipid nanoparticles encapsulating mRNA, FLuc: luciferase (firefly), LNP: lipid nanoparticles, mRNA: messenger ribonucleic acid, ND1: NeuroD1, PBS: phosphate buffer solution, RLA: relative luciferase activity, RLU: relative light unit, Tris buffer solution: tris(hydroxymethyl)aminomethane buffer solution, FLuc eGFP mixed mRNA: mRNA mixed with FLuc mRNA and eGFP mRNA (also called FLuc eGFP mixed nucleic acid), FLuc eGFP mixed nucleic acid lipid nanoparticles: lipid nanoparticles encapsulating mRNA mixed with FLuc mRNA and eGFP mRNA.

[0140] Test Example 1 In vitro luciferase assay

[0141] Test Example 1-1 (Evaluation of lipid nanoparticle introduction using mouse primary astrocytes) The compound of formula (I) or a salt thereof constituting lipid nanoparticles was evaluated for its nucleic acid introduction efficiency into mouse primary astrocytes. FLuc mRNA (TriLink BioTechnologies) was used as the mRNA, and nucleic acid-lipid nanoparticles were prepared using NanoAssemblr (registered trademark, Precision NanoSystems). The introduction efficiency of the test drug (nucleic acid-lipid nanoparticles) was calculated by evaluating the expression level of luciferase encoded by the mRNA. Each group was tested in three wells. The method and results are shown below.

[0142] (Obtaining Mouse Primary Astrocytes) Mouse primary 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. However, cell recovery was performed under 5% CO2. Neonatal C57BL / 6J mice (Jackson Laboratory Japan) P1-P10 were used. (Test Method) Mouse primary astrocytes obtained using the above method were seeded at 2000 cells / well on a poly-D-lysine-coated 96-well plate (IWAKI, Greiner) and cultured overnight at 37°C under 5% CO2. 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 as the medium, at a concentration of 100 μL per well. The following day, 100 μL of the test drug (FLuc nucleic acid lipid nanoparticles) diluted with PBS and medium was added to the wells at a final concentration of 0.8 μg / mL, and the mixture was incubated overnight at 200 μL per well. After removing the medium, the ONE-Glo Luciferase assay system (Promega) was diluted 2-fold with PBS and added at 100 μL per well. After mixing for 1 minute, 80 μL of the mixture was transferred to a 96-well white plate (Thermo Fisher Scientific), and luminescence was detected using an Envision (Revvity) microscope. Relative Light Units (RLU) were used as an activity indicator.

[0143] (Expression Evaluation) As a result of the above test, luciferase activity was observed in nucleic acid-lipid nanoparticles containing representative example compounds of the present invention as constituents. This suggests that lipid nanoparticles encapsulating FLuc mRNA are taken up into cells, translated, and express luciferase protein. The table below shows the results for nucleic acid-lipid nanoparticles (nucleic acid: Fluc mRNA) containing example compounds as constituents. Here, NUM indicates each lipid nanoparticle containing an example compound of the compound of formula (I) or a salt thereof as a constituent. ExA-LB (A and B are numbers) indicates lipid nanoparticles containing the example compound ExA of the compound of formula (I) or a salt thereof and having the composition of LB. L1 indicates lipid nanoparticles containing ExA, DSPC, cholesterol, and DMG-PEG2000 (molar ratio 50 / 10 / 38.5 / 1.5) as constituents. For example, Ex1-L1 to Ex5-L1 refer to lipid nanoparticles containing example compounds Ex1, Ex2, Ex3, Ex4, and Ex5 of the compound of formula (I) or a salt thereof as constituent components, and having a lipid composition of L1.

[0144]

[0145]

[0146] The table below shows the results for nucleic acid-lipid nanoparticles (nucleic acid: Fluc mRNA) containing example compounds as components. Ex1-L1 to Ex1-L54 listed under NUM in the table below represent Ex1-L1-FLuc mRNA to Ex1-L54-FLuc mRNA, respectively. That is, Ex1-L1 to Ex1-L54 are lipid nanoparticles containing example compound Ex1, each of which encapsulates FLuc mRNA and has the lipid composition described below. The average RLU value for each group of nucleic acid-lipid nanoparticle expression was calculated, and the relative value (RLA (Relative Luciferase Activity)) when Ex1-L1 was set to 1 was shown. Note that Ex1-L1a in the table below , Ex1-L1b, and Ex1-L1c all indicate Ex1-L1 prepared in different lots. Of Ex1-L2 to Ex1-L48, 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 are the values ​​of Ex1-L4, Ex1-L6, and Ex1-L7 when Ex1-L1a is set as 1. The RLA values ​​for Ex1-L7, Ex1-L9 to Ex1-L11, Ex1-L13 to Ex1-L15, Ex1-L17 to Ex1-L20, Ex1-L22, Ex1-L23, Ex1-L25, Ex1-L27, Ex1-L28, Ex1-L30 to Ex1-L34, Ex1-L36 to Ex1-L38, Ex1-L40 to Ex1-L44, and Ex1-L46 are shown relative to the value for Ex1-L1b, which is set to 1. The RLA values ​​for Ex1-L51 to Ex1-L54 are shown relative to the value for Ex1-L1c, which is set to 1. Although the RLA values ​​for Ex1-L49 and Ex1-L50 were not calculated, the above test confirmed that they express a certain amount of luciferase protein.

[0147]

[0148] Test Example 1-2 (Evaluation of lipid nanoparticle delivery using Hepa1-6 cells)

[0149] The nucleic acid delivery efficiency of lipid nanoparticles containing the compound of formula (I) or its salt was evaluated for Hepa1-6 cell line (ATCC, mouse hepatoma cell line). A mixture of FLuc mRNA and eGFP mRNA (molar ratio 1:1) (TriLink BioTechnologies) was used as the mRNA, and nucleic acid-lipid nanoparticles were prepared using NanoAssemblr (registered trademark, Precision NanoSystems). The delivery efficiency of the test drug (nucleic acid-lipid nanoparticles) was calculated by evaluating the expression level of luciferase encoded by the mRNA. Each group was tested in three wells. The method and results are shown below. (Test Method) Hepa1-6 cells were seeded at 2000 cells / well in a 96-well plate (Corning) and cultured overnight at 37°C under 5% CO2. DMEM medium (Sigma) containing 10% FBS (Cytiva) and 1% penicillin-streptomycin (PS) (Thermo Fisher Scientific) was used as the culture medium, at 100 μL per well. The next day, 100 μL of the test drug (FLuc_eGFP nucleic acid lipid nanoparticles) diluted in PBS and medium was added to the wells at a final concentration of 0.8 μg / mL, and the cells were incubated at 200 μL per well for two nights. After two days, the medium was removed, and 100 μL of the ONE-Glo Luciferase assay system (Promega) was diluted 2x with PBS and added to each well. After mixing for 3 minutes, 80 μL of the solution was transferred to a 96-well white plate (Nunc), and luminescence was detected using an Envision (Revvity) microscope. Relative Light Units (RLU) were used as an activity indicator.

[0150] (Expression Evaluation) As a result of the above test, luciferase activity was observed in nucleic acid-lipid nanoparticles containing a representative example compound of the present invention as a component. This suggests that lipid nanoparticles encapsulating a mixture of FLuc mRNA and eGFP mRNA are taken up into cells, translated, and express luciferase protein. The table below shows the results of nucleic acid-lipid nanoparticles (nucleic acid:FLuc eGFP mixed mRNA) containing example compounds as components. Ex6-L1 listed under NUM in the table below indicates the Ex6-L1-FLuc eGFP mixed mRNA. Ex6-L1 refers to lipid nanoparticles containing the example compound Ex6, which has the same lipid composition as Ex1-L1 and encapsulates a mixture of FLuc eGFP mixed mRNA. Similarly, Ex7-L1 listed under NUM in the table below refers to lipid nanoparticles containing the example compound Ex7, which encapsulates the Ex7-L1-FLuc eGFP mixed mRNA. The table below also shows the average RLU for each group of nucleic acid-lipid nanoparticles.

[0151]

[0152] Test Example 2: In vivo luciferase assay (Test Method) The delivery efficiency of nucleic acid-lipid nanoparticles into the brain was evaluated using IVIS spectrum (Revvity). Nucleic acid-lipid nanoparticles were administered intracerebrally to 6-12-week-old BALB / c mice (Jackson Laboratory Japan, Japan CLEA, n=3) under isoflurane anesthesia (isoflurane inhalation anesthetic solution "VTRS"; Mylan Pharmaceuticals Co., Ltd.; the same applies below). For intracerebral administration, the administration coordinates were determined based on the Mouse Brain Atlas (Academic Press) as follows: 0.0 mm anterior-posterior, 2.0 mm leftward, and 2.5 mm deep from Bregma. Under isoflurane anesthesia, the hair was shaved with clippers, and then the skull was fixed in place using a stereotaxic apparatus (KOPF) and a hole approximately 1 mm in diameter was drilled using a drill (Foredom). An Ito syringe (Ito Manufacturing) was filled with 0.3 mg / mL of the test drug (FLuc nucleic acid lipid nanoparticles), and 1 μL was administered at the designated coordinates using an injection pump (Narishige) at a flow rate of 0.2 μL / min. After 1 minute of administration, the needle was slowly withdrawn and the scalp sutured. One day later, under isoflurane anesthesia, 250 μL of 15 mg / mL luciferin (Promega) was administered intraperitoneally at 250 μL / mouse. Luminescence was monitored 20 minutes later using IVIS spectrum. Luminescence values ​​were converted to photons / sec using Living Image software and used for analysis.

[0153] (Expression Evaluation) As a result of the above test, luciferase activity was observed in the lipids of the representative examples of the present invention. This indicates that lipid nanoparticles encapsulating FLuc mRNA are taken up into cells in the brain, translated, and express luciferase protein. The table below shows expression data of nucleic acid-lipid nanoparticles (average values ​​of three evaluated FLuc nucleic acid-lipid nanoparticles). For example, Ex1-L1 listed as NUM in the table below is Ex1-L1-FLuc mRNA. In other words, Ex1-L1 is a lipid nanoparticle containing the example compound Ex1, which has the same lipid composition as Ex1-L1 and encapsulates FLuc mRNA.

[0154]

[0155] Test Example 3: In vitro Conversion of Rat Primary Astrocytes to Neurons (Addition of Nucleic Acid-Lipid Nanoparticles to Rat Primary Astrocytes) Rat primary 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. However, cell recovery was performed under 5% CO2. Neonatal Wistar rats (CLEA Japan, P1-P10) were used. The obtained rat primary astrocytes were seeded at 40,000 cells / well on a poly-D-lysine-coated 96-well plate (Corning, 354640) and cultured overnight at 37°C under 5% CO2. For the culture, 100 μL / well of DMEM / F-12 medium (Thermo Fisher Scientific, 11320033) containing 2% B-27® supplement (Thermo Fisher Scientific, A1895601), 10% fetal bovine serum (FBS, Cytiva, SH30070.03), and 1% penicillin-streptomycin (PS) (Thermo Fisher Scientific, 15070063) was used.

[0156] The next day, the entire culture medium was removed, and ND1 nucleic acid lipid nanoparticles (Ex1-L1-ND1 mRNA) diluted with transdifferentiation medium were added to a final concentration of 0.5 μg / mL. PBS was added as a control, diluted with transdifferentiation medium. The transdifferentiation medium was Neurobasal™ Medium (Thermo Fisher Scientific, 21103049) containing 2% B-27® supplement, 1% GlutaMAX™ 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. The BDNF solution was prepared by dissolving BDNF powder (PeproTech, 450-02) in purified water to a concentration of 100 μg / mL. On the day following the addition of the ND1 nucleic acid-lipid nanoparticles (day 1) and on day 3, the entire medium was replaced with transdifferentiation medium.

[0157] (Confirmation of NeuroD1 and TUJ1 protein expression by immunoblotting) Eight hours after the addition of the ND1 nucleic acid-lipid nanoparticles, on day 7, the transdifferentiation medium was removed, the cells were washed with PBS, and the cells were lysed using RIPA Buffer (Thermo Fisher Scientific, 89901). After protein quantification by the Lowry assay, 10 μg of each protein was electrophoresed, and NeuroD1 protein expression was detected by immunoblotting using an anti-NeuroD1 antibody (Abcam, ab60704). TUJ1 protein expression was also detected by immunoblotting using an anti-TUJ1 antibody (Abcam, ab78078).

[0158] As a result, ND1 protein expression was observed 8 hours after addition of ND1 nucleic acid-lipid nanoparticles. These results indicate that ND1 nucleic acid-lipid nanoparticles are taken up by rat primary astrocytes, translated, and ND1 protein is expressed in rat primary astrocytes. Furthermore, TUJ1 protein expression increased compared to PBS-treated samples 8 hours after addition of ND1 nucleic acid-lipid nanoparticles, and continued to increase even 7 days after addition of ND1 nucleic acid-lipid nanoparticles. These results indicate that lipid nanoparticles containing Ex1-L1 can deliver ND1 mRNA to rat primary astrocytes, that the ND1 mRNA converts into ND1 protein in rat primary astrocytes, and that the protein functions to convert rat primary astrocytes into neurons.

[0159] Test Example 4: Effect of ND1 Nucleic Acid Lipid Nanoparticles on a Cynomolgus Monkey Cerebral Infarction Model (Preparation of a Cynomolgus Monkey Cerebral Infarction Model) Six male cynomolgus monkeys (aged 3 years or older, Shin Nippon Kayaku) were anesthetized by intramuscular injection (i.m.) of ketamine hydrochloride (Ketalar intramuscular injection 500 mg; Daiichi Sankyo Propharma) at a dose of approximately 10 mg / kg body weight. The anesthetized cynomolgus monkeys were intubated, and maintenance anesthesia was performed by inhaling isoflurane (isoflurane inhalation anesthetic solution "VTRS"; Mylan Pharmaceuticals Co., Ltd.) through the trachea under artificial respiration or spontaneous breathing. The hair on the head of the anesthetized cynomolgus monkey was shaved with clippers, and the cynomolgus monkey was then fixed in a stereotaxic apparatus (Narishige Co., Ltd.). The skin on the head of the cynomolgus monkey was then incised, taking care to prevent bleeding, and the bregma, the intersection of the sagittal and coronal sutures at the anterior surface of the skull, was identified. The injection site is indicated as follows, with bregma (B: 0 mm, ML coordinate: 0 mm (midline)) as the base point. ・B: Distance in the anterior-posterior axis direction from bregma as the base point (positive numbers indicate anterior distance). ・L: Distance to the left from the base point of the ML coordinate. ・D: Depth within the brain in a vertical downward direction from the dura mater.

[0160] Endothelin-1 (Peptide Institute, code 4198-v) solution was injected into six male cynomolgus monkeys. Specifically, four holes approximately 1 mm in diameter were drilled into the skull of each cynomolgus monkey at the following positions, with bregma as the injection point: (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 total of four holes). A microsyringe (HAMILTON) connected to a microinjector (Narishige) was inserted through each hole and placed so that the needle tip was positioned at a depth (D) of 19.0 mm (see (i) and (ii) above), 13.0 mm (see (iii) above), or 15.0 mm (see (iv) above) below the dura mater. Endothelin-1 was injected into four sites per animal, corresponding to the above locations, using a microsyringe. Endothelin-1 was dissolved at a concentration of 2.0 μg / μL in 1% acetic acid (prepared by diluting acetic acid (Wako, 017-00256) with Milli-Q® water) at a volume of 30 μL per site at an injection rate of 1.5 μL / min. After the injection, the syringe was left stationary for approximately 20 minutes to prevent leakage. The injection sites (i) to (iv) above targeted the (i) ventral lateral thalamic nucleus and ventral posterior thalamic nucleus, (ii) globus pallidus, (iii) caudate nucleus, and (iv) putamen, respectively. Injecting endothelin into these injection sites as described above caused damage to the internal capsule in addition to the targeted sites. In this way, a cerebral infarction model with basal ganglia, thalamic, and internal capsule damage was created. This model can be used to evaluate perforator infarction, cerebral infarction with brain damage in the area supplied by the perforator, subacute to chronic cerebral infarction, and cerebral infarction with severe motor dysfunction.

[0161] (Administration of ND1 nucleic acid lipid nanoparticles to the model) Six individuals from the cerebral infarction model prepared by the above method were divided into two groups (control group n=3, ND1 nucleic acid lipid nanoparticle group n=3) on day 21 (chronic phase) after endothelin-1 injection treatment, so that there would be no difference between the groups in terms of mRS monkeys (see table below). The control group (3 individuals) was administered Ex1-L1-eGFP mRNA, and the ND1 nucleic acid lipid nanoparticle group (3 individuals) was administered Ex1-L1-ND1 mRNA.

[0162] In human stroke treatment, the mRS (for human clinical use) listed in the table below is widely used as an index of physical disability. The mRS (for monkeys) is an index modified for monkeys from the mRS (for human clinical use). A known index used to score motor dysfunction in monkeys is the Non-Human Primate Stroke Scale (NHPSS), an assessment scale that adapts the National Institutes of Health Stroke Scale (NIHSS), a scale used to assess the neurological severity of stroke in humans, to a non-human primate model. Like the NHPSS, the mRS (for monkeys) can assess the persistence of motor dysfunction, and the mRS can be used to assess the persistence of motor dysfunction in this model.

[0163]

[0164] 1 Mild paralysis: No obvious paralysis is observed, but slight symptoms of paralysis are observed. 2 Paralysis: When obvious paralysis is observed. 3 Mild hypoactivity: No obvious hypoactivity of locomotor activity is observed, but slight symptoms of hypoactivity are present. 4 Hypoactivity: When a clear decrease in locomotor activity is observed. 5 Level of consciousness (monkey): Determined based on level of responsiveness to humans. 6 Sitting / lying position: At least one of sitting and lying positions. 7 Mildly decreased level of consciousness: No obvious decrease in level of consciousness is observed, but there are slight symptoms of a decrease in level of consciousness. 8Decreased level of consciousness: When a clear decrease in level of consciousness is observed.

[0165] Each group of cerebral infarction model animals was anesthetized with ketamine hydrochloride (approximately 10 mg / kg body weight) via intravenous injection and maintained with isoflurane inhalation. Under continuous anesthesia, the hair on the head was shaved with clippers, and the model animals were fixed in a stereotaxic apparatus. Next, the surgical field was disinfected, and the scalp was incised to expose the bregma, taking care to prevent bleeding.

[0166] In each group of cerebral infarction model animals, a microsyringe was placed at each of the four holes where endothelin-1 was injected during the creation of the cerebral infarction model, with the needle tip positioned at the same depth (D) as when endothelin-1 was injected. Three animals were injected with Ex1-L1-ND1 mRNA, and three with Ex1-L1-eGFP mRNA. Each nucleic acid-lipid nanoparticle was adjusted to 0.3 mg / mL in solvent (PBS or Tris buffer solution) and administered at 30 μL per site at an injection rate of 1.5 μL / min. After administration, the syringe was left undisturbed for approximately 20 minutes.

[0167] (Observation of motor function) In the above (administration of ND1 nucleic acid lipid nanoparticles to the model), observations based on the mRS were performed on each individual in the ND1 nucleic acid lipid nanoparticle group and the control group 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. Note that behavioral evaluations were performed every two weeks from day 28 after endothelin-1 injection treatment onwards, but behavioral evaluations on day 28 after endothelin-1 injection (day 7 after administration of nucleic acid lipid nanoparticles) were evaluated on days 28 or 29 after endothelin-1 injection (day 7 or 8 after administration of nucleic acid lipid nanoparticles). In this example, these results were taken as the results on day 28 after endothelin-1 injection (day 7 after administration of nucleic acid lipid nanoparticles). As a result, in both the ND1 nucleic acid lipid nanoparticle group and the control group, there was no difference in motor function on day 1 after administration, and motor dysfunction similar to that before administration of nucleic acid lipid nanoparticles was observed. However, in the control group, motor function did not improve even on day 84 after endothelin-1 injection (day 63 after administration of nucleic acid lipid nanoparticles), whereas in the ND1 nucleic acid lipid nanoparticle group, motor function began to recover from day 70 after endothelin-1 injection (day 49 after administration of nucleic acid lipid nanoparticles), and a significant recovery of motor function was confirmed on day 84 after endothelin-1 injection (day 63 after administration of nucleic acid lipid nanoparticles) (Figure 1). These results demonstrate that administration of ND1 nucleic acid lipid nanoparticles can restore motor dysfunction associated with brain damage.

[0168] Based on the above results, administration of ND1 nucleic acid lipid nanoparticles can treat cerebral infarction (particularly perforator branch infarction, cerebral infarction with brain damage in the area supplied by the perforator branches, subacute to chronic cerebral infarction, and cerebral infarction presenting with severe motor dysfunction).

[0169] Test Example 5: Effect of ND1 nucleic acid lipid nanoparticles on a spinal cord injury model (Evaluation method) The effect on spinal cord injury can be evaluated using a spinal cord injury model. The state of motor dysfunction in a spinal cord injury model can be evaluated using the Field Rating Scale as an index. The Field Rating Scale can be used by modifying the Original Open Field Rating Scale (see PLoS ONE, 2011, Vol. 6, 11, e27706). If an animal cannot maintain a sitting position and the total score on the Field Rating Scale is less than 10 points, the animal can be determined to have quadriplegia.

[0170]

[0171]

[0172]

[0173] *1 Forelimbs sticking out from the cage floor (cannot move forelimbs): When placed in prone position in a cage with a lattice floor, the forelimbs fall through the gaps in the lattice on the cage floor and cannot be lifted up. *2 Elbows sticking out from the cage floor: When placed in prone position in the aforementioned cage, the elbows can be lifted up above the floor through the gaps in the lattice on the cage floor. *3 Wrists sticking out from the cage floor: When placed in prone position in the aforementioned cage, the wrists can be lifted up above the floor through the gaps in the lattice on the cage floor. *4 Hands not sticking out from the cage floor: When placed in prone position in the aforementioned cage, the forelimbs do not stick out through the gaps in the lattice on the cage floor and the body can be supported. *5 Hindlimbs sticking out from the cage floor (cannot move hindlimbs): When placed in prone position in the aforementioned cage, the hindlimbs fall through the gaps in the lattice on the cage floor and cannot be lifted up. *6 Knees protruding from the cage floor: When placed in prone position in the cage, the knees can be raised above the floor through the gaps in the grid on the cage floor. *7 Ankles protruding from the cage floor: When placed in prone position in the cage, the ankles can be raised above the floor through the gaps in the grid on the cage floor. *8 Feet not protruding from the cage floor: When placed in prone position in the cage, the hind legs do not protrude through the gaps in the grid on the cage floor, and the body can be supported.

[0174] (Creation of a spinal cord injury model) Common marmosets are anesthetized with ketamine hydrochloride and xylazine. An endotracheal intubation tube, an Atom feeding catheter (6Fr), is inserted into the trachea of ​​the anesthetized marmoset, and maintenance anesthesia is administered with inhalation of isoflurane. The back of the anesthetized marmoset is shaved and the marmoset is placed face down on a 37°C heating mat. The skin in the neck is incised, the connective tissue is removed to expose the vertebrae, and the vertebral arch, including the spinous process of cervical vertebra C5, is excised to expose the spinal cord (cervical cord).

[0175] A 2.5 mm round tip was attached to the impactor, and the spinal cord was crushed on both the left and right sides of the spinal cord midline on the dorsal surface of the cervical spinal cord in the area exposed by resection of the lamina of the C5 cervical vertebra, with a pressure of 280 kdyn applied for several seconds each.

[0176] After the crush injury, in order to prevent spasticity and contracture, rehabilitation is carried out by bending and straightening the joints of the fingers, wrists (ankles), elbows (knees), and shoulders (hips) of both the front and back limbs of the common marmoset several times. Rehabilitation begins 5 to 7 days after the crush injury and is carried out once or twice a day, every day during the observation period.

[0177] (Administration of nucleic acid-lipid nanoparticles to the damaged area) After the crush injury, common marmosets are evaluated for motor dysfunction using the Field Rating Scale, and those that are unable to maintain a sitting position several weeks after the crush injury and have a total Field Rating Scale score of less than 10 points are divided into a control group and a drug-administered group so that there is no difference in scores between the groups.

[0178] After the crush injury, the crushed area was re-exposed under induction anesthesia with ketamine hydrochloride and xylazine, followed by maintenance anesthesia with isoflurane inhalation. Using a syringe connected to a microinjector, nucleic acid-lipid nanoparticles were administered approximately 1 mm below the dorsal surface of the cervical spinal cord at the center of the compression site on the left side of the spinal cord. Next, similar administration was performed at the center of the compression site on the right side of the spinal cord. The syringe was left in place for several minutes after administration, after which the muscles and skin were sutured. Nucleic acid-lipid nanoparticle solution was administered to several animals in the drug-treated group, and eGFP mRNA was administered to several animals in the control group.

[0179] Motor dysfunction is assessed using the Field Rating Scale, and at the same time, the grip strength of both forelimbs is measured. Grip strength is measured using a device consisting of a digital force meter attached to a cage that is easy for the marmoset's forelimbs to grasp (hereinafter referred to as the grip test). Specifically, the marmoset grasps the cage, and the tester pulls the marmoset until the marmoset releases the cage. The force with which the marmoset grips the cage is measured using the digital force meter, and this measurement value is used as the marmoset's grip strength. Scoring of motor dysfunction and the grip test are performed at the same time, and observation and evaluation are carried out for several weeks after drug administration.

[0180] The production method of the compound of formula (I) or a salt thereof is explained in more detail below based on examples. Note that the present invention is not limited to the compounds described in the examples. In addition, the production methods of the starting compounds are shown in the respective production examples. In addition, the production method of the compound of formula (I) or a salt thereof is not limited to the production methods of the specific examples, and the compound of formula (I) or a salt thereof can also be produced by a combination of these production methods or by methods that are obvious to those skilled in the art.

[0181] The following abbreviations may be used in the present specification, examples, preparation examples, and tables: CDI: 1,1'-carbonyldiimidazole, CPME: cyclopentyl methyl ether, DCM: dichloromethane, DIPEA: N,N-diisopropylethylamine, DMAP: 4-(dimethylamino)pyridine, DMF: N,N-dimethylformamide, DMSO: dimethyl sulfoxide, DPPA: diphenylphosphoryl azide, EDCI.HCl: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, EtOH: ethanol, HATU: 1-[bis(dimethylamino)methylene]- 1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, HOBt: 1-hydroxybenzotriazole, MeCN: acetonitrile, TEA: triethylamine, TFA: trifluoroacetic acid, NUM: Example number or Production Example number ( / HCl indicates that the example or production example is a hydrochloride salt), REF: Production Example number or Example number based on the production method, PEx: Production Example number, Ex: Example number, STR: chemical structure, DAT: physicochemical data, NMR: 500 MHz １The H-NMR chemical shift δ value (ppm) is shown (the NMR brackets indicate the solvent used for measurement; for example, (CDCl3) is measured in deuterated chloroform. The NMR signal indicates a representative signal). s: singlet, t: triplet, m: multiplet, br: broad, ESI+: m / z value in ESI-MS+, CI+: m / z value in CI-MS+, HPLC Rt: high-performance liquid chromatography retention time (min) (measured using CHIRAL ART Amylose-SA 250 mm × 4.6 mm ID, S-3 μm, 10 mM ammonium acetate in 2-propanol as the mobile phase, column temperature 12 °C, flow rate 0.25 mL / min, Corona (registered trademark) charged aerosol detector (CAD)). Note that in this specification, naming software such as ACD / Name (registered trademark, Advanced Chemistry Development, Inc.) may be used to name compounds. For convenience, the concentration (mol / L) is expressed as M. For example, 1M sodium hydroxide aqueous solution means a 1 mol / L sodium hydroxide aqueous solution.

[0182] Preparation Example 1-1: 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-nonylundecan-1-ol (3.25 g) and DCM (40 mL) in a water bath, followed by stirring at room temperature for 6.5 hours. Chloroform and water were added to the reaction mixture, and the organic layer was separated. The aqueous layer was extracted with chloroform. The combined organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to yield 2-nonylundecyl 8-bromooctanoate (5.27 g) as an oil.

[0183] Preparation Example 1-2: To a mixture of 2-octyldecan-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 at room temperature and stirred 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 layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give 2-octyldecyl {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}acetate (879 mg) as an oil.

[0184] Preparation Example 1-3: To a mixture of 2-octyldecyl {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}acetate (877 mg) and DCM (8 mL), TFA (1.5 mL) was added at room temperature, followed by stirring at room temperature for 6 hours. The reaction mixture was added to a mixture of chloroform and saturated aqueous sodium bicarbonate, the organic layer was separated, and the aqueous layer was extracted with chloroform. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield 2-octyldecyl [(1r,3r)-3-aminocyclobutyl]acetate (661 mg) as an oil.

[0185] Preparation Example 1-4: To a mixture of 2-octyldecyl [(1r,3r)-3-aminocyclobutyl]acetate (660 mg) and MeCN (4 mL), a mixture of 2-nonylundecyl 8-bromooctanoate (430 mg) and CPME (4 mL), DIPEA (731 μL), and KI (30 mg) were added at room temperature, followed by stirring in an oil bath at 80°C for 48 hours. The reaction mixture was added to a mixture of chloroform and saturated aqueous sodium bicarbonate, after which the organic layer was separated and the aqueous layer was extracted with chloroform. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by amino silica gel column chromatography (hexane / ethyl acetate), and the crude product was then purified by silica gel column chromatography (chloroform / methanol) to give 2-nonylundecyl 8-{[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (452 ​​mg) as an oil.

[0186] Example 1: To a mixture of 2-nonylundecyl 8-{[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (1200 mg) and DCM (24 mL) was added 1-methyl-L-proline (11.8% water content, 290 mg), HATU (910 mg), DMAP (27 mg), and DIPEA (510 μL) at room temperature, followed by stirring at room temperature for 4 hours. Water was added to the reaction mixture, and the organic layer was separated and concentrated under reduced pressure. Heptane and 90% aqueous methanol were added to the resulting residue, and the heptane layer was separated and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (heptane / ethyl acetate), and then the crude product was purified by amino silica gel column chromatography (heptane / ethyl acetate) to give 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (1.2 g) as an oil.

[0187] Example 2: To a mixture of 2-nonylundecyl 8-{[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (120 mg) and DCM (3 mL), 1-methyl-D-proline monohydrate (26 mg), HATU (68 mg), and DIPEA (0.038 mL) were added and stirred at room temperature for 15 hours. The reaction mixture was purified by silica gel column chromatography (chloroform / methanol), and the crude product was purified by aminosilica gel column chromatography (hexane / ethyl acetate) to give 2-nonylundecyl 8-{(1-methyl-D-prolyl)[(1r,3R)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (129 mg) as an oil.

[0188] Preparation Example 4-1: To a mixture of 2-octyldecan-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 stirred at room temperature for 40 hours. The reaction mixture was diluted with ethyl acetate and washed with saturated aqueous sodium bicarbonate. 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 afford 2-octyldecyl {3-[(tert-butoxycarbonyl)amino]bicyclo[1.1.1]pentan-1-yl}acetate (900 mg) as an oil.

[0189] Preparation Example 4-2: To a mixture of 2-octyldecyl {3-[(tert-butoxycarbonyl)amino]bicyclo[1.1.1]pentan-1-yl}acetate (900 mg) and DCM (8 mL), TFA (1.5 mL) was added under ice-cooling, followed by stirring at room temperature for 2 hours. The reaction mixture was diluted with chloroform and neutralized with saturated aqueous 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 resulting 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.

[0190] Preparation Example 4-3: 2-Nonylundecyl 8-bromooctanoate (450 mg), CPME (6 mL), DIPEA (0.4 mL), and KI (15 mg) were added to a mixture of 2-octyldecyl (3-aminobicyclo[1.1.1]pentan-1-yl)acetate (680 mg) and MeCN (6 mL), and the mixture was stirred in an oil bath at 80°C under an argon atmosphere for 3.5 days. The reaction mixture was allowed to cool, then diluted with ethyl acetate and washed with water. 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-nonylundecyl 8-[(3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}bicyclo[1.1.1]pentan-1-yl)amino]octanoate (482 mg) as an oil.

[0191] Example 4 To a mixture of 2-nonylundecyl 8-[(3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}bicyclo[1.1.1]pentan-1-yl)amino]octanoate (150 mg) and DCM (3 mL), 1-methyl-L-proline (11.8% water content, 33 mg), HATU (84 mg), and DIPEA (0.031 mL) were added and stirred at room temperature for 24 hours. The reaction mixture was purified by silica gel column chromatography (chloroform / methanol), and then the crude product was purified by amino silica gel column chromatography (hexane / ethyl acetate) to give 2-nonylundecyl 8-[(1-methyl-L-prolyl)(3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}bicyclo[1.1.1]pentan-1-yl)amino]octanoate (128 mg) as an oil.

[0192] Preparation Example 5-1: To a mixture of (9Z,12Z)-octadeca-9,12-dien-1-ol (1.5 mL) and DCM (15 mL) was added DIPEA (1.7 mL) at room temperature, and chloroacetyl chloride (0.58 mL) was added dropwise under ice cooling. 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 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 (hexane / ethyl acetate) to afford (9Z,12Z)-octadeca-9,12-dien-1-yl chloroacetate (1.47 g) as an oil.

[0193] Example 7: To a mixture of 2-nonylundecyl 8-{[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}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 stirred at room temperature for 18 hours. The reaction mixture was purified by silica gel column chromatography (chloroform / methanol), and the crude product was purified by aminosilica gel column chromatography (hexane / ethyl acetate / methanol) to give 2-nonylundecyl 8-{(N,N-diethyl-β-alanyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (119 mg) as an oil.

[0194] Preparation Example 9-1: To a mixture of tert-butyl azetidine-3-carboxylate monohydrochloride (500 mg), N,N-dimethylglycine (320 mg), HATU (1.18 g), and DCM (10 mL) was added DIPEA (1.1 mL) and stirred at room temperature for 18 hours. Saturated aqueous sodium bicarbonate 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% aqueous ammonia), and the crude product was purified by aminosilica gel column chromatography (hexane / ethyl acetate / methanol) to give tert-butyl 1-(N,N-dimethylglycyl)azetidine-3-carboxylate (350 mg) as an oil.

[0195] Preparation Example 9-2: To a mixture of tert-butyl 1-(N,N-dimethylglycyl)azetidine-3-carboxylate (350 mg) and DCM (2 mL), TFA (2.2 mL) was added at room temperature and stirred for 18 hours. TFA (1.1 mL) was added to the reaction mixture, which was then stirred at room temperature for 2 hours. The reaction mixture was then concentrated under reduced pressure. The resulting residue was added to a 4M solution of hydrogen chloride in 1,4-dioxane (5 mL), and concentrated under reduced pressure. This procedure was repeated four times to yield 1-(N,N-dimethylglycyl)azetidine-3-carboxylic acid hydrochloride (492 mg) as an oily crude product.

[0196] Preparation Example 12-1: To a mixture of methyl 1,4-diazepane-6-carboxylate hydrochloride (491 mg) and MeCN (10 mL), DIPEA (1.82 mL) and ethyl iodide (0.51 mL) were added at room temperature, followed by stirring in an oil bath at 60 °C for 10 hours. The reaction mixture was allowed to cool to room temperature and then concentrated under reduced pressure. Ethyl acetate was added to the residue, and the mixture was stirred at room temperature for 15 minutes. Insoluble material was then removed by filtration. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by aminosilica gel column chromatography (hexane / ethyl acetate / methanol). The crude product was then purified by silica gel column chromatography (chloroform / methanol) to give methyl 1,4-diethyl-1,4-diazepane-6-carboxylate (167 mg) as an oil.

[0197] Example 12: To a mixture of methyl 1,4-diethyl-1,4-diazepane-6-carboxylate (35 mg) and methanol (2.5 mL), 1M aqueous sodium hydroxide (0.25 mL) was added at room temperature, followed by stirring at room temperature for 10 hours. 1M hydrochloric acid (0.25 mL) was added to the reaction mixture, which was then concentrated under reduced pressure. This procedure of adding methanol / chloroform to the residue and concentrating under reduced pressure was repeated three times. HATU (62 mg), a mixture of 2-nonylundecyl 8-{[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (110 mg) and DCM (3 mL), and DIPEA (0.035 mL) were added to the resulting residue, followed by stirring at room temperature for 10 hours. The reaction mixture was purified by silica gel column chromatography (chloroform / methanol), and then the crude product was purified by amino silica gel column chromatography (hexane / ethyl acetate / methanol) to give 2-nonylundecyl 8-{(1,4-diethyl-1,4-diazepane-6-carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (74 mg) as an oil.

[0198] Preparation Example 14-1: To a mixture of tert-butyl [(1r,3r)-3-(hydroxymethyl)cyclobutyl]carbamate (500 mg) and DCM (30 mL), (9Z,12Z)-octadeca-9,12-dienoic acid (0.81 mL), DIPEA (0.64 mL), EDCI·HCl (0.72 g), and DMAP (61 mg) were added and stirred at room temperature for 18 hours. The reaction mixture was diluted with DCM and washed with brine. 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 afford {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}methyl (9Z,12Z)-octadeca-9,12-dienoate (1.06 g) as an oil.

[0199] Preparation Example 16-1: To a mixture of [(2-bromoethoxy)methyl]benzene (300 mg) and MeCN (8 mL) was added a mixture of [(1r,3r)-3-aminocyclobutyl]2-octyldecyl acetate (1.06 g) and CPME (8 mL), and DIPEA (0.6 mL) at room temperature. The mixture was stirred in an oil bath at 80 °C for 40 hours. After cooling to room temperature, ethyl acetate and saturated aqueous sodium bicarbonate were added to the reaction mixture. 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 aminosilica gel column chromatography (ethyl acetate / hexane) to give 2-octyldecyl [(1r,3r)-3-{[2-(benzyloxy)ethyl]amino}cyclobutyl]acetate (474 ​​mg) as an oil.

[0200] Preparation Example 16-3: To a mixture of 2-octyldecyl [(1S,3r)-3-{[2-(benzyloxy)ethyl](1-methyl-L-prolyl)amino}cyclobutyl]acetate (263 mg) and 2-propanol (5 mL) was added palladium on activated carbon (Pd 10%) (26 mg) under an argon atmosphere at room temperature, and the mixture was stirred at room temperature under a hydrogen atmosphere over the weekend. To the reaction mixture was added palladium on activated carbon (Pd 10%) (26 mg) under an argon atmosphere at room temperature, and the mixture was stirred at room temperature under a hydrogen atmosphere at 3 atmospheres for 24 hours. To the reaction mixture was added palladium on activated carbon (Pd 10%) (26 mg) and acetic acid (0.072 mL) under an argon atmosphere at room temperature, and the mixture was stirred at room temperature under a hydrogen atmosphere at 3 atmospheres for 24 hours. To the reaction mixture was added palladium on activated carbon (Pd 10%) (26 mg) under an argon atmosphere at room temperature, and the mixture was stirred at room temperature under a hydrogen atmosphere at 3 atmospheres for 24 hours. The reaction mixture was filtered through Celite®, and the filtrate was concentrated under reduced pressure. Ethyl acetate and saturated aqueous sodium bicarbonate were added to the resulting residue, and the organic layer was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol) to give 2-octyldecyl {(1S,3r)-3-[(2-hydroxyethyl)(1-methyl-L-prolyl)amino]cyclobutyl}acetate (140 mg) as an oil.

[0201] Example 16: To a mixture of (9Z,12Z)-octadeca-9,12-dienoic acid (0.053 mL) and DCM (1 mL), HATU (64 mg), DIPEA (0.045 mL), a mixture of 2-octyldecyl {(1S,3r)-3-[(2-hydroxyethyl)(1-methyl-L-prolyl)amino]cyclobutyl}acetate (70 mg) and DCM (1 mL), and DMAP (1.6 mg) were added at room temperature, followed by stirring at room temperature for 18 hours. The reaction mixture was purified by silica gel column chromatography (chloroform / methanol), and the crude product was then purified by amino silica gel column chromatography (hexane / ethyl acetate / methanol) to give (9Z,12Z)-octadeca-9,12-dienoic acid 2-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}ethyl (9Z,12Z)-octadeca-9,12-dienoate (78 mg) as an oil.

[0202] Example 21: To a mixture of 2-nonylundecyl 8-{[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (151 mg) and DCM (3 mL), (4-methylpiperazin-1-yl)acetic acid (39.4 mg), DIPEA (0.096 mL), and HATU (92.7 mg) were added at room temperature, and the mixture was stirred at room temperature for 3 hours. 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 then concentrated under reduced pressure. The obtained residue was purified by amino silica gel column chromatography (hexane / ethyl acetate) to give 2-nonylundecyl 8-{[(4-methylpiperazin-1-yl)acetyl][(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (132 mg) as an oil.

[0203] Preparation Example 22-2 To a mixture of tert-butyl 4-({8-[(2-nonylundecyl)oxy]-8-oxooctyl}[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]carbamoyl)piperidine-1-carboxylate (614 mg) and DCM (6 mL), TFA (3 mL) was added under ice-cooling, followed by stirring at room temperature for 2 hours. The reaction mixture was diluted with chloroform, made basic with saturated aqueous sodium bicarbonate under ice-cooling, and then extracted with chloroform. The organic layer was dried over anhydrous magnesium sulfate and then 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-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl](piperidine-4-carbonyl)amino}octanoate (521 mg) as an oil.

[0204] Example 22: To a mixture of 2-nonylundecyl 8-{[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl](piperidine-4-carbonyl)amino}octanoate (165 mg) and DCM (5 mL), sodium triacetoxyborohydride (131 mg) was added at room temperature and stirred at room temperature for 10 minutes. Acetaldehyde (0.1 mL) was added to the reaction mixture under ice cooling and stirred at room temperature for 17 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, which was then extracted with chloroform. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (chloroform / methanol), and then the crude product was purified by amino silica gel column chromatography (hexane / ethyl acetate) to give 2-nonylundecyl 8-{(1-ethylpiperidine-4-carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (139 mg) as an oil.

[0205] Preparation Example 24-1: To a mixture of 2-octyldecane-1-ol (2 g) and DCM (60 mL), pyridine (6 mL), 4-nitrophenyl chloroformate (2.98 g), and DMAP (45 mg) were added under ice cooling and stirred at room temperature for 68 hours. The reaction mixture was concentrated under reduced pressure, and hexane was added to the resulting residue. Insoluble matter was filtered off. The filtrate was concentrated under reduced pressure to give 4-nitrophenyl 2-octyldecyl carbonate (2.96 g) as an oil.

[0206] Preparation Example 24-2: To a mixture of 2-octyldecyl 4-nitrophenyl carbonate (1.4 g) and DCM (40 mL), pyridine (5 mL), tert-butyl [(1r,3r)-3-hydroxycyclobutyl]carbamate (1.81 g), and DMAP (80 mg) were added 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 aqueous sodium bicarbonate, water, and saturated brine. 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 (1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl carbonate (1.33 g) as an oil.

[0207] Preparation Example 25-1: To a mixture of 4-nitrophenyl 2-octyldecyl carbonate (1.4 g) and DCM (40 mL), pyridine (5 mL), tert-butyl [(1r,3r)-3-(hydroxymethyl)cyclobutyl]carbamate (1.95 g), and DMAP (80 mg) were added 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 aqueous sodium bicarbonate, water, and saturated brine. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}methyl 2-octyldecyl carbonate (1.18 g) as an oil.

[0208] Preparation Example 25-2: To a mixture of {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}methyl 2-octyldecyl carbonate (1.18 g) and DCM (10 mL), TFA (5 mL) was added under ice cooling, followed by stirring at room temperature for 2 hours. The reaction mixture was neutralized with saturated aqueous sodium bicarbonate and then extracted with chloroform. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by aminosilica gel column chromatography (hexane / ethyl acetate) to give [(1r,3r)-3-aminocyclobutyl]methyl 2-octyldecyl carbonate (883 mg) as an oil.

[0209] Preparation Example 25-3: To a mixture of [(1r,3r)-3-aminocyclobutyl]methyl 2-octyldecyl carbonate (882 mg) and MeCN (8 mL), 2-nonylundecyl 8-bromooctanoate (560 mg), CPME (8 mL), DIPEA (0.5 mL), and KI (20 mg) were added under an argon atmosphere, and the mixture was stirred in an oil bath at 80°C for 2.5 days. The reaction mixture was allowed to cool, diluted with ethyl acetate, and washed with saturated aqueous sodium bicarbonate and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by aminosilica 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.

[0210] Example 25: To a mixture of 2-nonylundecyl 8-({(1r,3r)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino)octanoate (160 mg) and DCM (3 mL), 1-methyl-L-proline monohydrate (35 mg), HATU (90 mg), and DIPEA (70 μL) were added, followed by stirring at room temperature for 14 hours. The reaction mixture was diluted with ethyl acetate / hexane (1 / 1) and washed with saturated aqueous sodium bicarbonate, water, and saturated brine. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol), and the crude product was then purified by amino silica gel column chromatography (hexane / ethyl acetate) to give 2-nonylundecyl 8-[(1-methyl-L-prolyl){(1r,3S)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]octanoate (147 mg) as an oil.

[0211] Example 26: 2-nonylundecyl 8-{[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (750 mg) was added to a mixture of 3-chloropropionyl chloride (360 mg), DIPEA (1 mL), and DCM (15 mL) at room temperature, followed by stirring at room temperature for 2 hours. Heptane and 90% aqueous methanol were added to the reaction mixture, and the heptane layer was separated, washed with 90% aqueous methanol, and then concentrated under reduced pressure to give 2-nonylundecyl 8-{3-chloro-N-[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]propanamido}octanoate (850 mg) as an oily crude product. To a mixture of diethanolamine (0.61 g) and EtOH (3 mL) was added 2-nonylundecyl 8-{3-chloro-N-[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]propanamide}octanoate (150 mg) at room temperature, followed by stirring at room temperature for 20 hours. The reaction mixture was stirred for an additional 5 hours in a 55°C oil bath and then allowed to cool to room temperature. Heptane and water were added to the reaction mixture, and the organic layer was separated, washed with 90% aqueous methanol, and then concentrated under reduced pressure. The obtained residue was purified by amino silica gel column chromatography (heptane / ethyl acetate) to give 2-nonylundecyl 8-{[N,N-bis(2-hydroxyethyl)-β-alanyl][(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (100 mg) as an oil.

[0212] Example 28: To a mixture of glycinamide hydrochloride (520 mg), DIPEA (2 mL), and EtOH (2 mL) was added 2-nonylundecyl 8-{3-chloro-N-[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]propanamide}octanoate (120 mg), an intermediate from Example 26, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was stirred in a 55°C oil bath for an additional 17 hours, then in a 75°C oil bath for 2 hours, and then allowed to cool to room temperature. Heptane and water were added to the reaction mixture, and the organic layer was separated, washed with 90% aqueous methanol, and then concentrated under reduced pressure. The obtained residue was purified by amino silica gel column chromatography (heptane / ethyl acetate) to give 2-nonylundecyl 8-{[N-(2-amino-2-oxoethyl)-β-alanyl][(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (45 mg) as an oil.

[0213] Preparation Example 30-1: 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-heptylnonan-1-ol (501 mg) and DCM (10 mL) in a water bath and stirred at room temperature for 6.5 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 then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to yield 2-heptylnonyl 8-bromooctanoate (0.787 g) as an oil.

[0214] Preparation Example 30-2: To a mixture of 2-heptylnonan-1-ol (508 mg) and DCM (20 mL), (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 in a water bath and 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 resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to afford 2-heptylnonyl (1r,4r)-4-[(tert-butoxycarbonyl)amino]cyclohexane-1-carboxylate (0.761 g) as an oil.

[0215] Preparation Example 30-3: DCM (10 mL) and TFA (1.3 mL) were added to 2-heptylnonyl (1r,4r)-4-[(tert-butoxycarbonyl)amino]cyclohexane-1-carboxylate (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 aqueous sodium bicarbonate. The separated aqueous layer was extracted with chloroform. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by aminosilica gel column chromatography (hexane / ethyl acetate) to afford 2-heptylnonyl (1r,4r)-4-aminocyclohexane-1-carboxylate (570 mg) as an oil.

[0216] Preparation Example 30-4: 2-Heptylnonyl (1r,4r)-4-aminocyclohexane-1-carboxylate (570 mg) was added to CPME (4 mL), MeCN (5 mL), DIPEA (0.26 mL), KI (24.5 mg), and a mixture of 2-heptylnonyl 8-bromooctanoate (331 mg) and CPME (1 mL) at room temperature, followed by stirring in an oil bath at 80 °C for 2 days. The reaction mixture was cooled to room temperature, and chloroform, water, and saturated aqueous sodium bicarbonate were added to the reaction mixture. The separated aqueous layer was extracted with chloroform. The combined organic layers were dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate), and then the crude product was purified by amino silica gel column chromatography (hexane / ethyl acetate) to give 2-heptylnonyl (1r,4r)-4-({8-[(2-heptylnonyl)oxy]-8-oxooctyl}amino)cyclohexane-1-carboxylate (456 mg) as an oil.

[0217] Example 30: To a mixture of 2-heptylnonyl (1r,4r)-4-({8-[(2-heptylnonyl)oxy]-8-oxooctyl}amino)cyclohexane-1-carboxylate (151 mg) and DCM (3 mL), 1-methyl-L-proline monohydrate (36.4 mg), HATU (93.2 mg), and DIPEA (0.053 mL) were added and stirred at room temperature for 24 hours. 1-Methyl-L-proline monohydrate (18.1 mg), DIPEA (0.026 mL), and HATU (47.4 mg) were added to the reaction mixture, and the mixture was stirred at room temperature for 5 hours. Chloroform, water, and saturated aqueous sodium bicarbonate were added to the reaction mixture, and the separated aqueous layer was extracted with chloroform. The combined organic layer was concentrated under reduced pressure. To the resulting residue, a mixture of ethyl acetate and water / saturated brine (1 / 1) was added. The separated organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol), and the crude product was then purified by amino silica gel column chromatography (hexane / ethyl acetate) to give 2-heptylnonyl (1S,4r)-4-[{8-[(2-heptylnonyl)oxy]-8-oxooctyl}(1-methyl-L-prolyl)amino]cyclohexane-1-carboxylate (123 mg) as an oil.

[0218] Example 31: To a mixture of 2-nonylundecyl 8-{[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (160 mg) and DCM (6 mL), 1-ethyl-D-proline (57 mg), HATU (151 mg), and DIPEA (0.085 mL) were added and stirred at room temperature over the weekend. The reaction mixture was purified by silica gel column chromatography (chloroform / methanol), and the crude product was purified by aminosilica gel column chromatography (hexane / ethyl acetate / methanol) to give 2-nonylundecyl 8-{(1-ethyl-D-prolyl)[(1r,3R)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (133 mg) as an oil.

[0219] Preparation Example 34-1: Palladium-activated carbon (Pd 10%) (40 mg) was added to a mixture of (1R,2R)-2-aminocyclopentane-1-carboxylic acid monohydrochloride (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 Celite®, and the filtrate was concentrated under reduced pressure. The resulting residue was added with chloroform / methanol and concentrated under reduced pressure three times, followed by addition of diisopropyl ether and stirring at room temperature for 10 minutes. The resulting solid was collected by filtration and dried under reduced pressure to give (1R,2R)-2-(dimethylamino)cyclopentane-1-carboxylic acid hydrochloride (217 mg) as a solid.

[0220] Example 39: To a mixture of tert-butyl 4-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}butanoate (181 mg) and DCM (0.5 mL) was added 4 M hydrogen chloride / ethyl acetate solution (1.4 mL) at room temperature, followed by stirring at room temperature for 18 hours. The reaction mixture was concentrated under reduced pressure. To a mixture of the resulting oil (175 mg) and DCM (5 mL) was added (9Z,12Z)-octadeca-9,12-dien-1-ol (0.11 mL), HATU (130 mg), DMAP (3 mg), and DIPEA (0.15 mL) at room temperature, followed by stirring at room temperature for 18 hours. The reaction mixture was then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol), and then the crude product was purified by amino silica gel column chromatography (hexane / ethyl acetate / methanol). The crude product was then purified by silica gel column chromatography (hexane / ethyl acetate / methanol) to give (9Z,12Z)-octadeca-9,12-dien-1-yl 4-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}butanoate (48 mg) as an oil.

[0221] Preparation Example 49-1: 8-Bromooctanoic acid (396 mg), DIPEA (0.633 mL), HATU (738 mg), and DMAP (19 mg) were added to a mixture of 3-decyltridecan-1-ol (504 mg) and DCM (10 mL) in a water bath and stirred at room temperature for 7 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, which was then extracted with chloroform. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give 3-decyltridecyl 8-bromooctanoate (783 mg) as an oil.

[0222] Preparation Example 49-2: To a mixture of 3-decyltridecan-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 stirred for 24 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, which was then extracted with chloroform. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give 3-decyltridecyl {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}acetate (763 mg) as an oil.

[0223] Preparation Example 49-3: To a mixture of 3-decyltridecyl {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}acetate (763 mg) and DCM (15 mL), TFA (5 mL) was added under ice-cooling, followed by stirring at room temperature for 4 hours. The reaction mixture was diluted with chloroform and then made basic by adding saturated aqueous sodium bicarbonate 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 resulting residue was purified by aminosilica gel column chromatography (hexane / ethyl acetate) to give 3-decyltridecyl [(1r,3r)-3-aminocyclobutyl]acetate (585 mg) as an oil.

[0224] Preparation Example 49-4: To a mixture of 3-decyltridecyl [(1r,3r)-3-aminocyclobutyl]acetate (585 mg), 3-decyltridecyl 8-bromooctanoate (353 mg), MeCN (10 mL), and CPME (10 mL) was added DIPEA (0.277 mL) and KI (11 mg) at room temperature, followed by stirring in an 80°C oil bath for 48 hours. After cooling at room temperature, saturated aqueous 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 resulting residue was purified by silica gel column chromatography (chloroform / methanol) to afford 3-decyltridecyl 8-{[(1r,3r)-3-{2-[(3-decyltridecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (393 mg) as an oil.

[0225] Example 49: To a mixture of 3-decyltridecyl 8-{[(1r,3r)-3-{2-[(3-decyltridecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (129 mg) and DCM (5 mL), 1-methyl-L-proline (11.8% water content, 25.1 mg), HATU (64.9 mg), and DIPEA (0.036 mL) were added at room temperature, followed by stirring at room temperature for 4 hours. Saturated aqueous sodium bicarbonate was added to the reaction mixture, which was then extracted with chloroform. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol), and the crude product was then purified by amino silica gel column chromatography (hexane / ethyl acetate) to give 3-decyltridecyl 8-{[(1r,3S)-3-{2-[(3-decyltridecyl)oxy]-2-oxoethyl}cyclobutyl](1-methyl-L-prolyl)amino}octanoate (131 mg) as an oil.

[0226] Preparation Example 55-1: A mixture of 2-octyldecyl [(1r,3r)-3-{[2-(benzyloxy)ethyl]amino}cyclobutyl]acetate (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 afford 2-octyldecyl [(1S,3r)-3-{[2-(benzyloxy)ethyl](1-ethyl-L-prolyl)amino}cyclobutyl]acetate (550 mg) as an oil.

[0227] Preparation Example 55-2: To a mixture of 2-octyldecyl [(1S,3r)-3-{[2-(benzyloxy)ethyl](1-ethyl-L-prolyl)amino}cyclobutyl]acetate (500 mg) and acetic acid (4.2 mL) was added 20% palladium hydroxide on activated carbon (50% wet) (100 mg) at room temperature. The reaction mixture was purged with nitrogen and stirred under a hydrogen atmosphere at room temperature for 3 hours. The reaction mixture was filtered, and the filtrate was added to a mixture of ethyl acetate and water. Potassium carbonate was added until the pH of the aqueous layer reached 10 or higher. The organic layer was separated, washed with water, and concentrated under reduced pressure. The resulting residue was purified by aminosilica gel column chromatography (heptane / ethyl acetate) to give 2-octyldecyl {(1S,3r)-3-[(1-ethyl-L-prolyl)(2-hydroxyethyl)amino]cyclobutyl}acetate (320 mg) as an oil.

[0228] Example 55: To a mixture of 2-octyldecyl {(1S,3r)-3-[(1-ethyl-L-prolyl)(2-hydroxyethyl)amino]cyclobutyl}acetate (200 mg) and DCM (4 mL), 4,4-bis(octyloxy)butanoic acid (250 mg), HATU (345 mg), DMAP (4.4 mg), and DIPEA (0.186 mL) were added at room temperature, followed by stirring at room temperature for 21.5 hours. The reaction mixture was concentrated under reduced pressure, and then heptane and 90% aqueous methanol were added to the resulting 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 layer was concentrated under reduced pressure. The resulting residue was purified by amino silica gel column chromatography (heptane / ethyl acetate), and then the crude product was purified by silica gel column chromatography (heptane / ethyl acetate) to give 2-{(1-ethyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}ethyl 4,4-bis(octyloxy)butanoate (151 mg) as an oil.

[0229] Preparation Example 56-1: To a mixture of 2-nonylundecane-1-ol (2 g) and DCM (60 mL), pyridine (5.4 mL), 4-nitrophenyl chloroformate (2.7 g), and DMAP (40 mg) were added under ice cooling and stirred at room temperature for 2 days. The reaction mixture was concentrated under reduced pressure, and hexane was added to the resulting residue. Insoluble matter was filtered off. The filtrate was concentrated under reduced pressure to give 4-nitrophenyl 2-nonylundecyl carbonate (2.87 g) as an oil.

[0230] Preparation Example 56-2: To a mixture of 4-nitrophenyl 2-nonylundecyl carbonate (1.4 g) and DCM (13 mL), pyridine (0.4 mL), DMAP (60 mg), and tert-butyl [(1r,3r)-3-(hydroxymethyl)cyclobutyl]carbamate (0.5 g) were added at room temperature, and the mixture was stirred at room temperature for 4 days. Chloroform and saturated aqueous sodium bicarbonate were added to the reaction mixture, and the separated aqueous layer was extracted with chloroform. The combined organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}methyl 2-nonylundecyl carbonate (1.29 g) as an oil.

[0231] Preparation Example 56-3: To a mixture of {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}methyl 2-nonylundecyl carbonate (1.28 g) and DCM (10 mL), TFA (5 mL) was added under ice-cooling and stirred at room temperature for 2.5 hours. The reaction mixture was added to a mixture of chloroform and saturated aqueous sodium bicarbonate under water-cooling. The organic layer was separated, and the aqueous layer was extracted with chloroform. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by aminosilica gel column chromatography (hexane / ethyl acetate) to give [(1r,3r)-3-aminocyclobutyl]methyl 2-nonylundecyl carbonate (916 mg) as an oil.

[0232] Preparation Example 58-1: 4-Octyldodecanoic acid (1 g), DIPEA (1.4 mL), HATU (1.58 g), and DMAP (42 mg) were added to a mixture of 4-bromobutan-1-ol (0.7 mL) and DCM (10 mL) under ice-cooling, followed by stirring 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 then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to yield 4-bromobutyl 4-octyldodecanoate (0.817 g) as an oil.

[0233] Preparation Example 58-2: To a mixture of [(1r,3r)-3-aminocyclobutyl]methyl 2-nonylundecyl carbonate (471 mg) and CPME (4 mL), 4-bromobutyl 4-octyldodecanoate (250 mg), MeCN (4 mL), DIPEA (0.25 mL), and KI (10 mg) were added under an argon atmosphere, and the mixture was stirred in an oil bath at 80°C for 2 days. The reaction mixture was allowed to cool, then diluted with ethyl acetate and washed with saturated aqueous sodium bicarbonate and saturated brine. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by aminosilica gel column chromatography (hexane / ethyl acetate) to give 4-({(1r,3r)-3-[({[(2-nonylundecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino)butyl 4-octyldodecanoate (333 mg) as an oil.

[0234] Example 58: To a mixture of 4-({(1r,3r)-3-[({[(2-nonylundecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino)butyl 4-octyldodecanoate (100 mg) and DCM (2 mL), 1-ethyl-L-proline (22 mg), HATU (58 mg), and DIPEA (35 μL) were added at room temperature, followed by stirring at room temperature for 3 days. The reaction mixture was purified by silica gel column chromatography (chloroform / methanol), and the crude product was purified by amino silica gel column chromatography (hexane / ethyl acetate) to give 4-[(1-ethyl-L-prolyl){(1r,3S)-3-[({[(2-nonylundecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]butyl 4-octyldodecanoate (101 mg) as an oil.

[0235] Preparation Example 59-1: To a mixture of tert-butyl [(1r,3r)-3-(2-hydroxyethyl)cyclobutyl]carbamate (915 mg) and DCM (30 mL), pyridine (1.4 mL) and 4-nitrophenyl chloroformate (1.29 g) were added under ice-cooling, followed by stirring for 30 minutes under ice-cooling and then at room temperature for 18 hours. Silica gel was added to the reaction mixture, which was then 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 4-nitrophenyl carbonate (1.77 g) as a solid.

[0236] Preparation Example 59-2: To a mixture of 2-{(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}ethyl 4-nitrophenyl carbonate (1.77 g) and DCM (40 mL), pyridine (8 mL), 2-octyldecan-1-ol (3.78 g), and DMAP (114 mg) were added 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, saturated aqueous sodium bicarbonate, and saturated brine. The organic layer was dried over anhydrous sodium sulfate and then 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 2-octyldecyl carbonate (1.94 g) as an oil.

[0237] Preparation Example 63-1: To a mixture of tert-butyl [(1r,3r)-3-(hydroxymethyl)cyclobutyl]carbamate (300 mg) and DCM (9 mL), DMAP (2 mg), 4-nitrophenyl chloroformate (602 mg), and pyridine (0.6 mL) were added under ice-cooling and stirred at room temperature for 18 hours. N-Octyloctan-1-amine (2.3 mL) was added to this reaction mixture at room temperature and stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, and hexane / ethyl acetate and saturated aqueous sodium bicarbonate were added to the resulting residue. The separated organic layer was washed with saturated aqueous sodium bicarbonate, water, and saturated brine, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate / hexane) to give {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}methyl di(octyl)carbamate (612 mg) as an oil.

[0238] Preparation Example 66-1: To a mixture of 4-nitrophenyl 2-nonylundecyl carbonate (500 mg) and DCM (15 mL), DIPEA (1.8 mL), 6-bromohexan-1-ol (0.74 mL), and DMAP (13 mg) were added at room temperature and stirred at room temperature for 18 hours. 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 cooling to room temperature, DMAP (526 mg) was added and the mixture was stirred at room temperature over the weekend. Hexane and saturated aqueous sodium bicarbonate were added to the reaction mixture, and the separated organic layer was washed with saturated aqueous sodium bicarbonate, 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 afford 6-bromohexyl 2-nonylundecyl carbonate (344 mg) as an oil.

[0239] Preparation Example 72-1: To a mixture of 4-nitrophenyl 2-octyldecyl carbonate (500 mg) and CPME (15 mL), pyridine (1.9 mL), tert-butyl [(1r,4r)-4-hydroxycyclohexyl]carbamate (371 mg), and DMAP (561 mg) were added at room temperature and stirred in an oil bath at 50°C for 18 hours. After cooling to room temperature, hexane / ethyl acetate and saturated aqueous sodium bicarbonate were added to the reaction mixture. The separated organic layer was washed with 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 afford (1r,4r)-4-[(tert-butoxycarbonyl)amino]cyclohexyl 2-octyldecyl carbonate (495 mg) as an oil.

[0240] Preparation Example 73-1: N-decyldecan-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, followed by stirring at room temperature for 18 hours. The reaction mixture was concentrated under reduced pressure, and ethyl acetate / hexane and saturated aqueous sodium bicarbonate were added to the resulting residue. The separated organic layer was washed with water and saturated brine, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate / chloroform) to afford tert-butyl [(1r,3r)-3-{2-[di(decyl)amino]-2-oxoethyl}cyclobutyl]carbamate (755 mg) as a solid.

[0241] Preparation Example 74-1: To a mixture of tert-butyl [(1r,3r)-3-(hydroxymethyl)cyclobutyl]carbamate (500 mg) and DCM (10 mL), DIPEA (0.85 mL) and methanesulfonyl chloride (0.29 mL) were added 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 over anhydrous sodium sulfate and then concentrated under reduced pressure. To a mixture of the resulting solid (818 mg) and MeCN (10 mL), DIPEA (0.85 mL) and undecane-1-amine (2.66 mL) were added at room temperature. The mixture was stirred in an oil bath at 50°C for 2 hours and in an oil bath at 80°C for 16 hours. After cooling to room temperature, water and chloroform were added. The separated organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure. The obtained residue was purified by amino silica gel column chromatography (ethyl acetate / hexane), and then the crude product was purified by silica gel column chromatography (chloroform / methanol) to give tert-butyl {(1r,3r)-3-[(undecylamino)methyl]cyclobutyl}carbamate (644 mg) as a solid.

[0242] Preparation Example 74-2: To a mixture of tert-butyl {(1r,3r)-3-[(undecylamino)methyl]cyclobutyl}carbamate (644 mg) and DCM (10 mL), undecanoic acid (409 mg), HATU (830 mg), and DIPEA (0.47 mL) were added at room temperature, followed by stirring at room temperature for 16 hours. 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 tert-butyl {(1r,3r)-3-[(N-undecylundecanamido)methyl]cyclobutyl}carbamate (895 mg) as an oil.

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[0288] Example 76 Production of nucleic acid-lipid nanoparticles (Raw materials for nucleic acid-lipid nanoparticles) DOTAP is manufactured by NOF Corporation (product name: COATSOME (registered trademark) CL-8181TA), DOTMA is manufactured by NOF Corporation (product name: COATSOME (registered trademark) CL-E8181TA), DSPC is manufactured by NOF Corporation (product name: COATSOME (registered trademark) MC-8080), DPPC is manufactured by NOF Corporation (product name: COATSOME (registered trademark) MC-6060), DHSM is manufactured by Nippon Fine Chemicals Co., Ltd., SOPC is manufactured by Avanti Polar Lipids (catalog number: 850467P), DoPhPE is manufactured by Avanti Polar Lipids (catalog number: 999985P), DOPS (catalog number: 38-1810) from Larodan, DOPE (product name: COATSOME® ME-8181) from NOF Corporation, cholesterol (product name: Cholesterol HP) from Nippon Fine Chemicals, β-sitosterol (catalog number: S1270) from Sigma-Aldrich, 7α-hydroxycholesterol (catalog number: 700034P) from Avanti Polar Lipids, campesterol (catalog number: 306-01391) from Tama Biochemical Co., Ltd., DMG-PEG2000 (SUNBRIGHT® GM-020) from NOF Corporation, C8 PEG2000 ceramide (Avanti Polar) Lipids (catalog number: 880170P), PEG monostearate (polyethylene glycol monostearate) was from Fujifilm Wako Pure Chemical Industries, Ltd. (catalog number: 320-32585), and mRNA (FLuc mRNA, ND1 mRNA, eGFP mRNA) was from TriLink BioTechnologies.

[0289] (Preparation of Nucleic Acid-Lipid Nanoparticles) For Ex1-L1 to Ex1-L54, the cationic lipid compound of formula (I) or its salt, phospholipid, sterol, and PEGylated lipid were dissolved in ethanol at the lipid composition and N / P ratios shown in the table below to obtain an oil phase. Fluc mRNA was diluted with 10 mM citrate buffer (pH 4) to a concentration of 85 μg / mL to obtain an aqueous phase. The oil and aqueous phases were mixed in a microfluidic device (NanoAssemblr®, Precision NanoSystems) so that the volume ratio of oil phase to aqueous phase was 1:3. The mixture was then diluted 2-fold or 3-fold with phosphate-buffered saline (PBS) to obtain a nucleic acid-lipid nanoparticle dispersion. This dispersion was dialyzed against PBS to remove ethanol, and then concentrated by ultrafiltration to obtain the desired concentration to obtain nucleic acid-lipid nanoparticles of the example lipids. The table below shows the particle size and encapsulation rate of nucleic acid-lipid nanoparticles (nucleic acid:Fluc mRNA). Ex1-L1 to Ex1-L54 listed in NUM in the table below represent Ex1-L1-FLuc mRNA to Ex1-L54-FLuc mRNA, respectively.

[0290]

[0291]

[0292] Abbreviations in the table: CHO: Cholesterol, DMG: DMG-PEG2000, 7α-OH-CHO: 7α-hydroxycholesterol, PEG Ceramide: C8 PEG2000 ceramide, PEG Monostearate: Polyethylene Glycol Monostearate

[0293] Ex1-L1-FLuc mRNA was pharmacologically evaluated in Test Example 1, and the ratios of each component with a fluorescence intensity of 0.01 or greater and 0.1 or greater were calculated, assuming its fluorescence intensity as reference 1. The results showed that the composition ratio of Ex1 was 20.0-80.0 mol% and 30.0-60.0 mol%, respectively, based on the total amount of lipid nanoparticles. The composition ratios of neutral lipids were 18.5-78.5 mol% and 38.5-68.5 mol%, respectively, based on the total amount of lipid nanoparticles. The composition ratios of PEGylated lipids were 0.5-2.5 mol% and 0.5-2.0 mol%, respectively, based on the total amount of lipid nanoparticles.

[0294] (Raw material for ND1 nucleic acid lipid nanoparticles - ND1 nucleic acid) Two types of ND1 mRNA encoding human ND1 ((i) mRNA (hereinafter referred to as "ND1-1") consisting of a base sequence including 5'UTR and 3'UTR provided by TriLink BioTechnologies, CDS (SEQ ID NO: 1) of the human ND1 gene and a 120-base polyA sequence, and having a 5' cap structure of Cap-1; and (ii) mRNA (hereinafter referred to as "ND1-2") consisting of a base sequence including 5'UTR derived from the human α-globin gene, 3'UTR derived from the human α-globin gene, CDS (SEQ ID NO: 3) of the human ND1 gene and a polyA sequence of 79 polynucleotides (SEQ ID NO: 4) in which all uridines have been replaced with N1-methylpseudouridines, and having a 5' cap structure of Cap-1)) were prepared (commissioned to TriLink BioTechnologies). In the base sequence shown in SEQ ID NO: 4 or 5, nucleotides 1 to 3 correspond to AGG, nucleotides 4 to 43 correspond to the 5'UTR, nucleotides 44 to 1114 correspond to the CDS of the human NeuroD1 gene (SEQ ID NO: 3), nucleotides 1115 to 1120 correspond to two consecutive stop codons, nucleotides 1121 to 1231 correspond to the 3'UTR, and nucleotides 1232 to 1310 correspond to the poly(A) sequence. FLuc mRNA and eGFP mRNA were manufactured by TriLink BioTechnologies (product names: CleanCap® FLuc mRNA and CleanCap® EGFP mRNA).

[0295] (Preparation of ND1 nucleic acid lipid nanoparticles) ND1 nucleic acid lipid nanoparticles were specifically prepared by the following method. The cationic lipid Example Compound Ex1, DSPC, cholesterol, and DMG-PEG2000 were dissolved in ethanol at an N / P ratio of 6 to obtain an oil phase. 10 mM citrate buffer (pH 4) containing the above mRNA was used as the aqueous phase, and an oil phase was added so that the volume ratio of the oil phase to the aqueous phase was oil phase:aqueous phase = 1:3. The mixture was mixed using a microfluidic device (NanoAssemblr (registered trademark, Precision NanoSystems), and the mixture was diluted 2-fold with PBS to obtain a dispersion of ND1 nucleic acid lipid nanoparticles. Ethanol was removed by dialysis of this dispersion. Subsequently, the mixture was concentrated by ultrafiltration to obtain ND1 nucleic acid lipid nanoparticles adjusted to the desired concentration. Note that in Test Example 4, the ND1 nucleic acid lipid nanoparticles were specifically prepared by the following method. The cationic lipid Example Compound Ex1, DSPC (manufactured by Nippon Fine Chemicals Co., Ltd.), cholesterol (manufactured by Merck Ltd.), and DMG-PEG2000 (manufactured by Merck Ltd.) were dissolved in ethanol at an N / P ratio of 6 to obtain an oil phase. 10 mM citrate buffer (pH 4) containing the above mRNA was used as the aqueous phase, and the volume ratio of the oil phase to the aqueous phase was oil phase: aqueous phase = 1:3. The mixture was mixed using a microfluidic device (NanoAssemblr (registered trademark), Ignite+ (registered trademark) Precision NanoSystems). The mixture was diluted with Tris buffer solution to obtain a dispersion of ND1 nucleic acid lipid nanoparticles. The pH of this dispersion was adjusted to around 7 using 0.1 M Tris buffer solution, and then purified using tangential flow filtration (TFF) to obtain ND1 nucleic acid lipid nanoparticles prepared to the desired concentration. The nucleic acid-lipid nanoparticles were prepared using 10 mM citrate buffer (pH 4) and consisted of Ex1, DSPC, cholesterol, and DMG-PEG2000 (molar ratio 50 / 10 / 38.5 / 1.5) and were designated Ex1-L1-ND1 mRNA. Similarly, lipid nanoparticles encapsulating eGFP mRNA and with a lipid composition of Ex1-L1 were designated Ex1-L1-eGFP mRNA.

[0296] (Raw materials for Fluc-eGFP mixed nucleic acid-lipid nanoparticles) Fluc mRNA encoding firefly luciferase protein and eGFP mRNA (TriLink BioTechnologies) encoding green fluorescent protein were mixed at a molar ratio of 1:1 to obtain Fluc-eGFP mixed mRNA.

[0297] (Preparation of Fluc eGFP-mixed nucleic acid-lipid nanoparticles) Fluc eGFP-mixed nucleic acid-lipid nanoparticles were specifically prepared by the following method. The example compound (Ex6 or Ex7), DSPC, cholesterol, and DMG-PEG2000 were dissolved in ethanol at an N / P ratio of 6 to obtain an oil phase. The aqueous phase was prepared using 10 mM citrate buffer (pH 4) containing the above mRNA, and an oil phase was added so that the volume ratio of the aqueous phase to the oil phase was 1:3. The mixture was mixed using a microfluidic device (NanoAssemblr (registered trademark), Precision NanoSystems). The mixture was diluted 2-fold with PBS to obtain a dispersion of Fluc eGFP-mixed nucleic acid-lipid nanoparticles. The ethanol was removed by dialysis of this dispersion. The mixture was then concentrated by ultrafiltration to obtain Fluc eGFP-mixed nucleic acid-lipid nanoparticles adjusted to the desired concentration.

[0298] The nucleic acid-lipid nanoparticles were prepared using 10 mM citrate buffer (pH 4) and contained Fluc eGFP mixed nucleic acid-lipid nanoparticles composed of an example compound (Ex6 or Ex7), DSPC, cholesterol, and DMG-PEG2000 (molar ratio 50 / 10 / 38.5 / 1.5). These nanoparticles are referred to as Ex6-L1-Fluc eGFP mRNA and Ex7-L1-Fluc eGFP mRNA, respectively.

[0299] (Measurement of Particle Diameter) The particle diameter of the nucleic acid-lipid nanoparticles was measured for the nucleic acid-lipid nanoparticle dispersion using a particle diameter measuring device (Zetasizer (registered trademark) Nano ZSP or Ultra, manufactured by Malvern Panalytical).

[0300] (Evaluation of mRNA Encapsulation Rate) The mRNA encapsulation rate was measured in a nucleic acid-lipid nanoparticle dispersion diluted to an mRNA concentration of approximately 150-1000 ng / mL. 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) measured using Quant-iT RiboGreen RNA Reagent (Thermo Fisher Scientific) was used to determine the mRNA concentration present in the nucleic acid-lipid nanoparticle external solution. Furthermore, the mRNA concentration (B) measured after dilution with 2% Triton X-100 was used to determine the total mRNA concentration in the composition. The mRNA encapsulation rate was then calculated using the following formula (F1): Encapsulation rate (%) = 100 - (A / B) × 100 ... (F1). The table below shows the particle size and encapsulation rate of nucleic acid-lipid nanoparticles (nucleic acid: Fluc mRNA). Ex1-L1 to Ex75-L1 listed in NUM in the table below refer to Ex1-L1-FLuc mRNA to Ex75-L1-FLuc mRNA, respectively.

[0301]

[0302]

[0303] The table below shows the particle size and encapsulation rate of the nucleic acid-lipid nanoparticles (nucleic acid:Fluc eGFP mixed mRNA) used in Test Example 1-2.

[0304]

[0305] The table below shows the particle size and mRNA encapsulation rate of the nucleic acid-lipid nanoparticles (nucleic acid: Fluc mRNA) used in Test Example 2.

[0306]

[0307] The table below shows the particle size and mRNA encapsulation rate of the nucleic acid-lipid nanoparticles (nucleic acid: NeuroD1 mRNA) used in Test Example 4.

[0308]

[0309] The compound of formula (I) or its salt was able to form lipid nanoparticles. Lipid nanoparticles containing the compound of formula (I) or its salt were taken up by astrocytes or liver cells. Pharmaceutical compositions can also be produced using lipid nanoparticles of the compound of formula (I) or its salt 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, etc.

[0310] SEQ ID NO: 1: Human NeuroD1 gene (CDS) SEQ ID NO: 2: Human NeuroD1 protein SEQ ID NO: 3: Human NeuroD1 gene (CDS) SEQ ID NO: 4: Human NeuroD1 mRNA sequence (ND1-2) SEQ ID NO: 5: Human NeuroD1 mRNA sequence (ND1-2) containing modified nucleotides

Claims

1. A compound of formula (I) or a salt thereof: (wherein L 1 and L 2 are the same or different and are -CH2-, -CH2CH2-, or a bond, L 3 is a bond, or C 1-10 alkylene, M is -CH2- or does not exist, n is 1 or 2, provided that when M is -CH2-, n is 1, E 1 and E 2 are the same or different and are -C(=O)O-*, -OC(=O)-*, -OC(=O)O-*, -C(=O)-*, or a bond, * indicates bonding at this position to R 1 or R 2 and, wherein either one of E 1 and E 2 is -C(=O)O-* or -OC(=O)-*, R 1 and R 2 are the same or different and are -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 , provided that when either one of R 1 and R 2 is -N(-R x )R y , either one of E 1 -R 1 and E 2 -R 2 is -OC(=O)-N(-R x )R y or -C(=O)-N(-R x )R y ; wherein R 1 and R 2 are each independently selected from the group consisting of -NR y (-C(=O)R x ) or -NR y C(=O)CH(-R x )R Z ; when R 1 is bonded to E 1 or R 2 is bonded to E 2 is a bond; and when both E 1 and E 2 are -OC(=O)-*, R 1 and R 2 are the same or different and are -CH2CH2CH(-OR x )OR y or -CH2-(C 5-20 alkenyl); R x , R y , and R Z are the same or different and are C 5-15 alkyl, R 3 is a group selected from the group consisting of formulas (a) to (i): R a is C 1-6 alkyl, R b is -CH2-C 1-6 alkyl or -C(=O)CH2N(CH3)2, R c and R d are the same or different and are -CH3, -CH2CH3, or -CH2CH2OH, or when R c is H, R d is -CH2C(=O)NH2, L cd is -CH2-, -CH2CH2-, -CH2CH2CH2-, or -CH2CH(CH3)-, R e is H or OH, R f is H, R g is C 1-6 alkyl, where R f and R g may be integrated with the carbon and nitrogen atoms to which they are attached to form a pyrrolidine ring, R h is C 1-6 alkyl, R i is C 1-6 alkyl or -CH2CH2OH, R j and R k are the same or different and are C 1-6 alkyl, R l and R m are the same or different and are C 1-6 alkyl, s and t are the same or different and are 1 or 2.) 2. -E 1 -R 1 is -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 is -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), -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 The compound or a salt thereof according to claim 1, wherein it is as described above.

3. E 1 and E 2 is, independently or identically, -C(=O)O-*, -OC(=O)-*, or -OC(=O)O-*, where * is R 1 or R 2 at this position, and where E 1 and E 2 one of which is -C(=O)O-*, R 1 and R 2 is, independently or identically, -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 is, independently or identically, C 5-15 alkyl, R 3 is a group selected from the group consisting of formulas (a) to (h), R c and R d are both -CH3, -CH2CH3, or -CH2CH2OH, or, when R c is H, R d is -CH2C(=O)NH2, and L cd is -CH2- or -CH2CH2-, where, when R c , R d are both -CH3, -CH2CH3, or -CH2CH2OH, L cd is -CH2-, -CH2CH2-, or -CH2CH2-, respectively, for the compound of formula (I) according to claim 1 or a salt thereof.

4. -E 1 -R 1 is -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 is -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), which is the compound or a salt thereof according to claim 3.

5. L 1 is a bond, L 2 is -CH2- or a bond, L 3 is 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 are the same or different and are C 5-15 alkyl, R 3 is a group selected from the group consisting of formula (d), (e), (g), or (h): R c and R d are the same or different and are -CH3, -CH2CH3, or -CH2CH2OH, L cd is -CH2-, -CH2CH2-, or -CH2CH2CH2-, R e is H, R f is H, R g is C 1-6 alkyl, R i is C 1-6 alkyl, R j and R k are the same or different and are C 1-6 alkyl, t is 1, the compound according to claim 2 or a salt thereof.

6. L 3 is C6 alkylene, -E 1 -R 1 where -R x is -C(=O)O-CH2CH(-R y )R x )R y or -OC(=O)O-CH2CH(-R 2 )R 2 where -R x )R y R c and R d are the same or different and are -CH3, -CH2CH3, or -CH2CH2OH, and L cd is -CH2-, -CH2CH2-, or -CH2CH2CH2-, where R c , R d are both -CH3, -CH2CH3, or -CH2CH2OH, L cd is -CH2-, -CH2CH2-, or -CH2CH2- respectively, and is the compound or a salt thereof according to claim 5.

7. The compound is 8-{[(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl, 8-[(1-methyl-L-prolyl)(3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}bicyclo[1.1.1] (Pentan-1-yl)amino]octanoic acid 2-nonylundecyl, 8-{ (1-ethyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl, 8-{ (N,N-diethyl-β-alanyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl, 8-{ (1,4-diethyl-1,4-diazepane-6-carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl, 8-{ [(4-methylpiperazin-1-yl)acetyl][(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl, 8-[(1-methyl-L-prolyl){(1r,3S)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]octanoic acid 2-nonylundecyl, (1S,4r)-4-[{8-[(2-heptylnonyl)oxy]-8-oxooctyl}(1-methyl-L-prolyl)amino]cyclohexane-1-carboxylic acid 2-heptylnonyl, 8-{ (1-ethyl-D-prolyl)[(1r,3R)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoic acid 2-nonylundecyl, 8-{ [(1r,3S)-3-{2-[(3-decyltridecyl)oxy]-2-oxoethyl}cyclobutyl](1-methyl-L-prolyl)amino}octanoic acid 3-decyltridecyl, 4,4-bis(octyloxy)butanoic acid 2-{ (1-ethyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}ethyl, and 4-octyldodecanoic acid 4-[(1-ethyl-L-prolyl){(1r,3S)-3-[({[(2-nonylundecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]butyl, the compound according to claim 1 selected from the group consisting of or a salt thereof.

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

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

10. The lipid nanoparticle according to claim 9, encapsulating a nucleic acid.

11. The lipid nanoparticle according to claim 10, wherein the nucleic acid is mRNA.

12. The neutral lipid is a phospholipid and a sterol, the phospholipid is DPPC, DSPC, SOPC, DoPhPE, DOPS, or DHSM, the sterol is cholesterol, 7α-hydroxy cholesterol, or β-sitosterol, and the PEGylated lipid is DMG-PEG2000, PEG monostearate, or C8 PEG2000 ceramide. The lipid nanoparticle according to claim 11.

13. The lipid nanoparticle according to claim 12, capable of expressing a protein in astrocytes.

14. The lipid nanoparticle according to claim 13, wherein the nucleic acid is mRNA useful for the prevention and / or treatment of astrocyte-related diseases.

15. The nucleic acid is mRNA encoding NeuroD1 protein, the compound of formula (I) or a salt thereof is 8-{ (1-methyl-L-prolyl) [(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl} cyclobutyl]amino} octanoic acid 2-nonylundecyl or a salt thereof, the neutral lipid is DSPC and cholesterol, and the PEGylated lipid is DMG-PEG2000. The lipid nanoparticle according to claim 11.

16. The lipid nanoparticle according to claim 10, wherein the nucleic acid is mRNA encoding NeuroD1 protein comprising a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO:

2.

17. The lipid nanoparticle according to claim 15, wherein the nucleic acid is mRNA encoding NeuroD1 protein comprising a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO:

2.

18. The lipid nanoparticle according to claim 10, comprising the compound according to claim 1 or a salt thereof in a composition ratio of 20.0 to 80.0 mol%, a neutral lipid in a composition ratio of 18.5 to 78.5 mol%, and a PEGylated lipid in a composition ratio of 0.5 to 2.5 mol%, based on the total amount of the lipid nanoparticle.

19. The lipid nanoparticles according to claim 10, comprising the compound or a salt thereof in a molar ratio of 30.0 to 60.0 mol% based on the total amount of the lipid nanoparticles, neutral lipid in a molar ratio of 38.5 to 68.5 mol%, and PEGylated lipid in a molar 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. Use of the lipid nanoparticles according to any one of claims 10 to 19 for the manufacture of a pharmaceutical composition for the prevention and / or treatment of astrocyte-related diseases.

24. Use of the lipid nanoparticles according to any one of claims 10 to 19 for 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 administering an effective amount of the lipid nanoparticles according to any one of claims 10 to 19 to a subject.

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