Urea or carbamate lipid having cyclic group in side chain, lipid nanoparticles thereof, and pharmaceutical composition thereof

Abstract

The present invention provides: a urea or carbamate lipid having a cyclic group in a side chain; lipid nanoparticles containing the lipid as a constituent component; and a pharmaceutical composition thereof. The present inventors have found a urea or carbamate lipid having a cyclic group in a side chain, and urea or carbamate lipid nanoparticles containing the lipid as a constituent component. It has been found that the lipid nanoparticles can express a protein in cells.

Description

Urea or carbamate lipid having a cyclic group in the side chain, lipid nanoparticles thereof, and pharmaceutical composition 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 thereof, lipid nanoparticles encapsulating nucleic acids, and compositions thereof.

[0002] Cationic lipids are thought to surround anionic molecules such as DNA and mRNA and form lipid nanoparticles. Lipid nanoparticles are used as carriers in drug delivery systems (DDS). Optimal lipid nanoparticles can encapsulate nucleic acids such as DNA and mRNA. Furthermore, cationic lipids can deliver nucleic acids such as DNA and mRNA to cells and tissues, allowing them to produce desired proteins and exert their functions. Examples of applications of lipid nanoparticles include COVID-19 vaccines and cancer vaccines, which have been launched or are in clinical development (Patent Document 1).

[0003] 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). If we can develop effective drugs that act by being taken up by astrocytes, it may be possible to provide new treatments for central nervous system diseases.

[0004] International Publication No. 2017 / 049245 International Publication No. 2024 / 143444

[0005] The present invention provides novel lipids useful as components of lipid nanoparticles, and lipid nanoparticles containing the lipids that can encapsulate nucleic acids and be taken up by target cells (e.g., astrocytes).

[0006] 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 also discovered novel lipid nanoparticles comprising the compound of formula (I) of the present invention or a salt thereof. The present invention can provide a pharmaceutical composition comprising lipid nanoparticles encapsulating a nucleic acid.

[0007] That is, the present invention relates to the following: (The present invention) (1) A compound of formula (I) or a salt thereof:

[0008] (In the formula, L 1 is -(CH2) q -(Ring D)-(CH2) p -*, * is R 1 one of p and q is 0 and the other is 0, 1 or 2; ring D is a group represented by the formula (Aa), (Ab), (Ac), (Ad) and (Ae):

[0009]

[0010] ** is a group selected from the group consisting of (CH2) p indicates that R 1 and R 2 are independently represented by the formulae (Ba), (Bb), (Bc), (Bd), (Be), (Bf), (Bg), (Bh), (Bi) and (Bj):

[0011]

[0012] a group selected from the group consisting of: 1 and R 2 are both (Bf) or (Bg), then ring D is formula (Ab) or (Ad); q is 1; p is 2; or ring A is formula (b), (c) or (d), and R 11a , R 11b independently, C 5-15 alkyl, R 12 is C 5-20 Alkenyl, L 4 is C 1-10 alkylene, the combination of G and Y (G, Y) is (O, CH), (bond, N) or (NCH3, CH), X is CH or N, ring A, X, Y and G together represent the formulas (a) to (f):

[0013]

[0014] a group selected from the group consisting of: R Z This bond is R Z indicates that it binds to R z is C 1-6 Alkyl, -C(O)-(C 1-6 alkylene)-N(C 1-6 alkyl)2 or -(C 1-6 (Alkylene)-NR 00 R 01 , R 00 and R 01 independently, C 1-6 Alkyl.) (1-1) R 1 and R 2 are independently a group selected from the group consisting of formulae (Ba), (Bb), (Bc), (Bd), (Be), (Bf), (Bg), (Bh), (Bi) and (Bj), and R 1 and R 2 (1-2) The compound of formula (I) or a salt thereof according to (1), wherein any one of R is a group selected from the group consisting of formulas (Ba), (Bb), (Bc), (Bd), (Bh), (Bi), and (Bj). 1 and R 2 are independently a group selected from the group consisting of formulae (Ba), (Bb), (Bc), (Bd), (Be), (Bf), (Bh), (Bi) and (Bj), and R 1 and R 2 (1-3) The compound of formula (I) or a salt thereof according to any one of (1) to (1-1), wherein either one of R is a group selected from the group consisting of formulas (Ba), (Bb), (Bc), (Bd), (Bh), (Bi), and (Bj). 1 and R 2 is independently a group selected from the group consisting of formulas (Ba), (Bd), (Bf), and (Bh), and R 1 and R 2is a group selected from the group consisting of formula (Ba), (Bd), and (Bh). (1-4) Compounds of formula (I) or salts thereof according to (1) to (1-3), wherein the combination of G and Y (G, Y) is (O, CH) or (NCH3, CH), X is N, and ring A, X, Y, and G together represent formula (b), (e), or (f). (1-5) Compounds of formula (I) or salts thereof according to (1) to (1-3), wherein the combination of G and Y (G, Y) is (bond, N), X is N, and ring A, X, Y, and G together represent formula (a). (1-6) Compounds of formula (I) or salts thereof according to (1) to (1-5), wherein ring D is formula (Aa) or (Ab). (1-7) R 1 and R 2 are all (Bf) or (Bg), and ring D is formula (Ab) or (Ad); q is 1; p is 2; or ring A is formula (b), (c), or (d). (2) A lipid nanoparticle comprising a compound of formula (I) according to (1) or a salt thereof. (3) A lipid nanoparticle comprising a compound of formula (I) according to (1) or a salt thereof, a neutral lipid, and a PEGylated lipid. (4) A lipid nanoparticle according to (2) or (3), which encapsulates a nucleic acid. (5) A lipid nanoparticle according to (4), in which the nucleic acid is mRNA. (6) A lipid nanoparticle according to (3) to (5), in which the neutral lipid is a phospholipid and a sterol, the phospholipid is DSPC, the sterol is cholesterol, and the PEGylated lipid is DMG-PEG2000. (7) The lipid nanoparticles according to any one of (2) to (6), comprising, based on the total amount of the lipid nanoparticles, the compound of formula (I) or a salt thereof at a composition ratio of 50 mol %, DSPC at 10 mol %, cholesterol at 38.5 mol %, and DMG-PEG2000 at 1.5 mol %. (8) The lipid nanoparticles according to any one of (2) to (7), which are capable of expressing a protein in astrocytes.

[0015] The present invention also relates to: a pharmaceutical composition comprising the lipid nanoparticles described in (2) to (8); a pharmaceutical composition comprising the lipid nanoparticles described in (2) to (8) and one or more pharmaceutically acceptable pharmaceutical additives; and a pharmaceutical composition for preventing and / or treating astrocyte-related diseases, which contains the lipid nanoparticles described in (2) to (8). The pharmaceutical composition also includes a preventive and / or therapeutic agent for astrocyte-related diseases, which contains the lipid nanoparticles described in (2) to (8). The present invention also relates to: use of the lipid nanoparticles described in (2) to (8) for producing a pharmaceutical composition for preventing and / or treating astrocyte-related diseases; lipid nanoparticles described in (2) to (8) for use in the prevention and / or treatment of astrocyte-related diseases; use of the lipid nanoparticles described in (2) to (8) for the prevention and / or treatment of astrocyte-related diseases; and a method for preventing and / or treating astrocyte-related diseases, which comprises administering an effective amount of the lipid nanoparticles described in (2) to (8) to a subject.

[0016] The present invention also relates to a method for delivering a nucleic acid to cells, particularly astrocytes, in a living body by using nucleic acid-lipid nanoparticles containing the compound of formula (I) or a salt thereof.

[0017] "Astrocyte-related diseases" include cerebral infarction, cerebral hemorrhage, traumatic brain injury, spinal cord injury, neurodegenerative diseases, amyotrophic lateral sclerosis, Duchenne muscular dystrophy, Alexander disease, multiple sclerosis, and neuromyelitis optica. In some embodiments, cerebral hemorrhage, traumatic brain injury, spinal cord injury, neurodegenerative diseases, amyotrophic lateral sclerosis, Duchenne muscular dystrophy, Alexander disease, multiple sclerosis, and neuromyelitis optica are included.

[0018] 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.

[0019] The lipid compound of formula (I) or a salt thereof can be used to prepare lipid nanoparticles, which can incorporate nucleic acids into target cells, such as astrocytes, and express proteins.

[0020] 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.

[0021] 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.

[0022] "Alkyl" refers to straight-chain or branched alkyl. In one embodiment, it is straight-chain 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 one embodiment, C 5-20 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 6-12 In some embodiments, alkyl is C 7-10 In some embodiments, alkyl is C 7-9 In some embodiments, alkyl is C 7-8 In some embodiments, alkyl is C 8-10 In some embodiments, alkyl is C 8-9 In some embodiments, the alkyl is a C alkyl. In some embodiments, the alkyl is a C 6-9In some embodiments, the alkyl is a C2 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 methyl or ethyl. In some embodiments, the alkyl is ethyl. In some embodiments, the alkyl is n-heptyl. 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. In some embodiments, the alkyl is n-decyl.

[0023] "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 5-8 In one embodiment, alkylene is C 5-7 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 C2 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 ethylene, n-hexanediyl, or n-heptanediyl. In one embodiment, the alkylene is ethylene. In one embodiment, the alkylene is n-hexanediyl. In one embodiment, the alkylene is n-heptanediyl.

[0024] "Alkenyl" means straight or branched alkenyl, C 5-20The 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 5-10 In some embodiments, alkenyl is -C 5-9 Alkylene -CH=CH-CH=CH-C 5-6 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 one embodiment, alkenyl is -C8 alkylene-(CH=CH-CH2)2-C4 alkyl (both double bonds are Z), -C8 alkylene-(CH=CH-CH2)2-C6 alkyl (both double bonds are Z), -C7 alkylene-CH=CH-C8 alkyl (both double bonds are Z), C7 alkylene-(CH=CH-CH2)2-C4 alkyl (both double bonds are Z), C7 alkylene-(CH=CH-CH2)3-C4 alkyl (both double bonds are Z), C3 alkylene-(CH=CH-CH2)4-C4 alkyl (both double bonds are Z), C3 alkylene-(CH=CH-CH2)5-C1 alkyl (both double bonds are Z), or C2 alkylene-(CH=CH-CH2)6-C1 alkyl (both double bonds are Z). In one embodiment, the alkenyl is -C7 alkylene-(CH=CH-CH2)2-C4 alkyl (both double bonds are Z). In one embodiment, the alkenyl is -C6 alkylene-(CH=CH-CH2)2-C4 alkyl (both double bonds are Z). In one embodiment, the alkenyl is -C8 alkylene-(CH=CH-CH2)2-C4 alkyl (both double bonds are Z).

[0025] "Halogen" means F, Cl, Br, or I.

[0026] "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, and in one embodiment, they are lipid nanoparticles that encapsulate mRNA.

[0027] "Particle size" refers to the particle size of lipid nanoparticles. For a static dispersion, the hydrodynamic diameter (Dh) is measured using a particle size analyzer (Zetasizer Nano ZSP, manufactured by Malvern Panalytical) using the Dynamic Light Scattering (DLS) technique, in accordance with ISO 22412. The "particle size" of lipid nanoparticles is calculated as the Z-average particle size. In one embodiment, the particle size of lipid nanoparticles is 10 nm to 1000 nm, in another embodiment, 30 nm to 500 nm, or in another embodiment, 30 nm to 250 nm. In one embodiment, it is 60 nm to 180 nm. In one embodiment, it is 80 nm to 150 nm. In one embodiment, it is 70 nm to 110 nm.

[0028] The term "cationic lipid" refers to a linear or branched fatty acid chain or fatty acid ester compound or salt thereof that can take on a cation (positive charge) in the molecule in response to pH, or a fatty acid chain or fatty acid ester compound or salt thereof that already takes on a cation (positive charge) in the molecule. In one embodiment, the lipid is a compound of formula (I) or a salt thereof.

[0029] "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.

[0030] 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, DoPhPE, DOPS, or DHSM. In one embodiment, the phospholipid is DSPC.

[0031] A "sterol" is a steroid alcohol, a compound having a hydroxy group on the A ring of the steroid skeleton. In some embodiments, the sterol is cholesterol or a corticosteroid. In some embodiments, 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 some embodiments, the sterol is cholesterol, 7α-hydroxycholesterol, or β-sitosterol. In some embodiments, the sterol is cholesterol.

[0032] "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 one embodiment, the PEGylated lipid is 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine-PEG (DLPE-PEG), 1,2-dimyristoyl-rac-glycero-3-methoxyPEG (DMG-PEG2000, also known as DMG-PEG), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-PEG (DMPE-PEG), 1,2-dipalmitoyl-rac-glycero-3-methoxyPEG (DPG -PEG), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine-PEG (DPPC-PEG), 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 some embodiments, the PEGylated lipid is DMG-PEG2000, PEG monostearate, or N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol)2000]} (C8 PEG2000 ceramide). In some embodiments, the PEGylated lipid is DMG-PEG2000.

[0033] "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.

[0034] 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 in 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, 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.

[0035] "Nucleic acid" refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In lipid nanoparticles encapsulating nucleic acids, the nucleic acid content is, in one embodiment, 0.001 to 60% by weight, in another embodiment, 0.1 to 40% by weight, in another embodiment, 1.0 to 25% by weight, and in another embodiment, 3.0 to 10% by weight, based on the total weight of the lipid nanoparticles. The 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).

[0036] "mRNA" refers to messenger ribonucleic acid, and may be any that can be translated into a desired protein. mRNA 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. An example of mRNA is NeuroD1 mRNA.

[0037] "MRNA encoding xxx protein" refers to RNA that includes a polynucleotide containing a nucleotide sequence encoding the xxx protein and can be translated into the xxx protein. Herein, mRNA encoding the xxx protein is also referred to as xxx mRNA. That is, mRNA encoding the xxx protein includes a polynucleotide that encodes the xxx protein in an expressible state. In one embodiment, the mRNA encoding the xxx protein of the present invention is a polynucleotide encoding the xxx protein that includes functional sequences for expressing the xxx protein (for example, including but not limited to, a 5' cap, CDS, 5' UTR, 3' UTR, and poly A tail).

[0038] 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 7It 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.

[0039] "UTR" refers to untranslated regions, and includes 5'UTR and 3'UTR. The 5'UTR and 3'UTR may be synthetic UTRs, derived from the gene to be expressed, or derived from a heterologous gene. The UTR may be naturally occurring, or may be a modified form in which 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. A UTR may comprise multiple UTRs connected directly or via a spacer sequence. The start codon and 5'UTR may contain a portion of the Kozak sequence (e.g., the sequence 5' from the start codon in the Kozak sequence).

[0040] "CDS" refers to a coding sequence, a region of DNA sequence that is translated into a protein, and may be codon-optimized.

[0041] A "poly A tail" is a polyadenylic acid tail that functions in mRNA stabilization, nuclear export, translation, etc. It is also called a poly A tail. 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, 100 to 140 bases, 70 to 120 bases, or 120 bases. In another embodiment, the poly A tail length is 120 bases. In another embodiment, the poly A tail length is 30 to 500 bases, or in another embodiment, 74 to 84, 75 to 85, 75 to 83, or 76 to 82 bases.

[0042] 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 sequence shown in SEQ ID NO: 1), as well as the nucleosides and nucleotides containing them, may be natural or modified, i.e., modified nucleotides, and / or may have other modifications (such as methylation).

[0043] 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%.

[0044] "Expression" 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, in the context of nucleic acids, i.e., nucleic acid sequences. As used herein, expression and production are used interchangeably, such as protein expression.

[0045] "NeuroD1" is a neuronal differentiation protein (Neuronal differentiation 1) and a basic helix-loop-helix (bHLH) transcription factor belonging to the NeuroD family. NeuroD1 plays a central role in inducing differentiation of neural stem cells into neurons. 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, with insertion, deletion, substitution, and / or addition of 1 to 50 amino acids (e.g., 1 to 2, 1 to 3, 1 to 5, 1 to 7, 1 to 10, or 1 to 30) and having transcription factor activity. In one embodiment, NeuroD1 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 2. The nucleotide sequence shown in SEQ ID NO: 1 is the CDS of the NeuroD1 gene.

[0046] "Sequence identity" refers to the percentage (%) of residues that match between a reference base or amino acid sequence and a target biological sequence when the sequence is aligned (usually the percentage of residues that match over the entire length of the target sequence). For example, it can be calculated using EMBOSS Needle (Nucleic Acids Res., 2015; Vol. 43: pW580-W584). Examples of parameters are (Gap Open Penalty = 10, Gap Extend Penalty = 0.5, Matrix = EBLOSUM62, End Gap Penalty = false).

[0047] 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.

[0048] 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. Embodiments of the compound are described below, and the present invention encompasses the compounds or salts thereof of these embodiments. (Embodiments of Cationic Lipids of the Present Invention) (1) A compound of formula (I) in which q is 0 and p is 0 or 1. A compound of formula (I) in which q is 0 and p is 0. A compound of formula (I) in which q is 0 and p is 1. (2) A compound of formula (I) in which ring D is formula (Aa), (Ab) or (Ae). A compound of formula (I) in which ring D is formula (Aa) or (Ab). A compound of formula (I) in which ring D is formula (Ae). A compound of formula (Aa):

[0049]

[0050] (** indicates (CH2) p indicates that R binds to R. 1 and R 2 are independently a group selected from the group consisting of formulae (Ba), (Bb), (Bc), (Bd), (Be), (Bf), (Bg), and (Bh), and one is a group selected from the group consisting of formulae (Ba), (Bb), (Bc), (Bd), and (Bh). 1 and R 2 are independently a group selected from the group consisting of formulae (Ba), (Bb), (Bc), (Bd), (Be), (Bf), (Bh), (Bi), and (Bj), and one is a group selected from the group consisting of formulae (Ba), (Bb), (Bc), (Bd), (Bh), (Bi), and (Bj). 1 and R 2are independently a group selected from the group consisting of formulae (Ba), (Bb), (Bc), (Bd), (Be), (Bf), (Bh), (Bi), and (Bj), and one is a group selected from the group consisting of formulae (Ba), (Bb), (Bc), (Bd), (Be), (Bh), (Bi), and (Bj). 1 and R 2 are independently a group selected from the group consisting of formulae (Ba), (Bd), (Bf), and (Bh), and one is a group selected from the group consisting of formulae (Ba), (Bd), and (Bh). 1 and R 2 are independently 1 is a group selected from (Ba), (Bf), and (Bh), and R 2 Compounds of formula (I) in which R is a group selected from (Bd) and (Bf). 1 and R 2 is a group selected from the group consisting of combinations of (Ba and Bf), (Ba and Bd), (Bf and Bd), and (Bh and Bf).

[0051]

[0052] R 1 and R 2 are both (Bf) or (Bg), and ring D is formula (Ab) or (Ad); q is 1; p is 2; or ring A is formula (b), (c) or (d). (4) R 11a , R 11b But independently, C 5-10 Compounds of formula (I) where R is alkyl. 11a , R 11b But independently, C 7-10 Compounds of formula (I) where R is alkyl. 11a , R 11b But independently, C 8-10 (5) R 12 But C 15-17 Compounds of formula (I) where R is alkenyl. 12 But -C 5-10 Alkylene -CH=CH-CH2-CH=CH-C5-10 A compound of formula (I) wherein -C is alkyl. 5-9 Alkylene -CH=CH-CH2-CH=CH-C 5-6 (6) L 4 is C2 alkylene, or C 5-7 A compound of formula (I) which is alkylene. 4 But C 2-3 Alkylene, or C 5-7 A compound of formula (I) which is alkylene. 4 is a C2 alkylene compound of formula (I). 4 is C 5-7 (7) Compounds of formula (I) in which the combination of G and Y (G, Y) is (O, CH) or (NCH3, CH), X is N, and ring A, X, Y and G together are represented by formula (b), (e) or (f). Compounds of formula (I) in which the combination of G and Y (G, Y) is (bond, N), X is N, and ring A, X, Y and G together are represented by formula (a):

[0053]

[0054] Compounds of formula (I) in the group Z This bond is R Z (8) Compounds of formula (I) in which the combination of G and Y (G, Y) is (bond, N), X is CH or N, and ring A, X, Y and G together are represented by formula (a), (c) or (d). z But C 1-6 Compounds of formula (I) where R is alkyl. z is a C2 alkyl group. (9) A compound of formula (I) that is a consistent combination of the groups described in (1) to (8) above.

[0055] Specific examples of the combination of the above aspects include the following compounds or salts thereof: 1 But -(CH2) q -(Ring D)-(CH2) p -*, q is 0, p is 0 or 1, ring D is formula (Aa), and R 1 and R 2are independently a group selected from the group consisting of formulae (Ba), (Bd), (Bf) and (Bh), and one is a group selected from the group consisting of formulae (Ba), (Bd) and (Bh), and R 11a , R 11b But independently C 5-10 is alkyl, and L 4 C 2- Alkylene or C 5-7 alkylene, the combination of G and Y (G, Y) is (bond, N), X is N, ring A, X, Y and G together represent the formula (a), and R z C 1-6 The compound of formula (I) wherein:

[0056] An embodiment of the present invention includes Example compounds, wherein the compound is any of the Example compounds or a salt thereof, i.e., a compound selected from Ex1, Ex2, Ex3, Ex4, Ex5, Ex6, Ex7, Ex8, Ex9, Ex10, Ex11, Ex12, Ex13, Ex14, Ex15, Ex16, Ex17, Ex18, Ex19, Ex20, Ex21, Ex22, Ex23, Ex24, Ex25, Ex26, Ex27, Ex28, Ex29, Ex30, Ex31, Ex32, Ex33, Ex34, Ex35, Ex36, Ex37, Ex38, Ex39, Ex40, Ex41, Ex42, Ex43, Ex44, Ex45, Ex46, Ex47, Ex48, Ex49, Ex50, Ex51, Ex52, Ex53, and Ex54, or a salt thereof.

[0057] In one embodiment, the specific compound of the present invention includes the following compound or a salt thereof:

[0058]

[0059] Or its salt.

[0060] 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.

[0061] Furthermore, the compound of formula (I) or a salt thereof may have an asymmetric center or axial asymmetry, and therefore may exist as an enantiomer (optical isomer). The compound of formula (I) or a salt thereof encompasses both isolated individual enantiomers, such as the (R) form and the (S) form, and mixtures thereof (including racemic and non-racemic mixtures). In one embodiment, the enantiomer is "stereochemically pure." "Stereochemically pure" refers to a purity that can be recognized by those skilled in the art as being substantially stereochemically pure. In one embodiment, the 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.

[0062] 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.

[0063] Furthermore, the present invention also encompasses various hydrates and solvates, and crystalline polymorphic substances of the compound of formula (I) or a salt thereof.

[0064] 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. 2 Substitution 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.

[0065] 2. Lipid Nanoparticles Some embodiments of lipid nanoparticles comprising the compound of formula (I) of the present invention or a salt thereof are shown below. Note that in the following embodiments, "compound of formula (I) or a salt thereof" includes the embodiments described above in (Embodiments of cationic lipids of the present invention) in "compound of formula (I) or a salt thereof." (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. (3a) Lipid nanoparticles according to (3), wherein the nucleic acid is NeuroD1 mRNA.

[0066] (4) The lipid nanoparticles according to (2) to (3), wherein the neutral lipid is a phospholipid and a sterol. (4a) The lipid nanoparticles according to (4), wherein the neutral lipid is DSPC and cholesterol. (4b) The lipid nanoparticles according to (4), wherein the phospholipid is DSPC. (4c) The lipid nanoparticles according to (4), wherein the sterol is cholesterol. (5) The lipid nanoparticles according to (2) to (4), wherein the PEGylated lipid is DMG-PEG2000.

[0067] (6) Lipid nanoparticles that are a consistent combination of two or more of (1) to (5) described in (Embodiments of the lipid nanoparticles of the present invention). Lipid nanoparticles that are a consistent combination of the embodiment described in (Embodiments of the cationic lipids of the present invention) and the embodiment described in (Embodiments of the lipid nanoparticles of the present invention).

[0068] Specific examples of the combination of (6) above include the following: (7) Lipid nanoparticles containing the compound of formula (I) or a salt thereof at a composition ratio of 50 mol %, DSPC at 10 mol %, cholesterol at 38.5 mol %, and DMG-PEG2000 at 1.5 mol %, based on the total amount of the lipid nanoparticles.

[0069] 3. Pharmaceutical Compositions Some embodiments of pharmaceutical compositions comprising lipid nanoparticles containing the compound of formula (I) or a salt thereof, which is the compound of the present invention, and one or more pharmaceutically acceptable pharmaceutical additives are shown below. (Embodiments of the Pharmaceutical Compositions of the Present Invention) (1) A pharmaceutical composition comprising the lipid nanoparticles described in (Embodiment of the Lipid Nanoparticles of the Present Invention). (2) A pharmaceutical composition comprising the lipid nanoparticles described in (Embodiment of the Lipid Nanoparticles of the Present Invention) and one or more pharmaceutically acceptable pharmaceutical additives. (3) A pharmaceutical composition for the prevention and / or treatment of astrocyte-related diseases, which comprises the lipid nanoparticles described in (Embodiment of the Lipid Nanoparticles of the Present Invention).

[0070] 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.

[0071] 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.

[0072] 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.

[0073] Typically, when administered intravenously, 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.

[0074] 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.

[0075] 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 thought to be effective. The combination may be administered simultaneously, or separately, consecutively, or at a desired time interval. The simultaneous administration preparations may be formulated as a combined preparation or separately.

[0076] 4. Method for Producing Cationic Lipids 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. 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 produced by introducing the protecting group and then removing it as necessary.

[0077] The compound of formula (I) of the present invention or a salt thereof can be produced by the following production method. Representative production methods for the compound of formula (I) or a salt thereof are explained below. Each production method can also be performed with reference to the reference documents described. Hereinafter, the compound or a salt thereof will be described as a compound. Note that the production method of the present invention is not limited to the examples shown below.

[0078] (First manufacturing method)

[0079]

[0080] (Wherein, the combination of G and Y (G', Y) is (bond, N) or (NCH3, CH), and the combination of G and Y (G 0, Y) is (O, CH). Lv represents a leaving group.) Compound (I) of the present invention can be prepared by reacting compound (1-1) and compound (2) with an equivalent or excess amount of a carbonylation reagent in a reaction-inert solvent in the presence of a base, under cooling to heating, in one embodiment at temperatures from -20°C to 100°C, typically for 0.1 hour to 1 day. Examples of the solvent include halogenated hydrocarbons such as DCM and 1,2-dichloroethane, aromatic hydrocarbons such as toluene, ethers such as THF, diethyl ether, 1,4-dioxane, and CPME, DMF, DMSO, MeCN, ethyl acetate, and mixtures thereof. Examples of the carbonylation reagent include triphosgene, diphosgene, 4-nitrophenyl chloroformate, phenyl chloroformate, bis(pentafluorophenyl) carbonate, and CDI. Examples of the base include K2CO3, KOtBu, and DIPEA. Furthermore, compound (I) of the present invention can be produced by reacting compound (1-1) and compound (2) under the same conditions as above, followed by the use of an acid such as TFA at room temperature or under heating, in one embodiment at 40°C to 145°C. Compound (I) can also be produced by isolating the activated derivatives of the starting materials, compound (1-1) or compound (2). Examples of production methods via these activated derivatives are shown below. These methods are referred to as the route via compound (3a) and the route via compound (3c), respectively.

[0081] (Production Method 1 - Route via Compound (3a)) When compound (3a), an active derivative of compound (2), is used as the route, a two-step process is carried out: first, compound (3a) is produced from compound (2), and then compound (I) is produced from compound (3a). The process for producing compound (3a) from compound (2) is described below. Compound (3a) can be produced by converting compound (2) into a carbamoyl imidazole compound and then methylating it. In the first step, CDI is added to compound (2) and the reaction is carried out in the presence or absence of a base. EtN or pyridine can be used as the base, and THF, DCM, CPME, DMF, or the like can be used as the solvent, and the temperature can be from 0°C to reflux. In the next methylation step, the reaction can be carried out with a methylating agent such as CHI in a solvent such as MeCN, chloroform, or DCM at room temperature to 90°C for 12 to 36 hours. The process for producing compound (I) of the present invention from compound (3a) is described below. A mixture of compound (1-1) and compound (3a) in equal amounts or an excess of either is used, and the mixture is stirred in a reaction-inert solvent in the presence of a base under cooling to heating, typically at room temperature, or in one embodiment, at 70° C. to 160° C., for typically 0.1 hours to 5 days. NaH or DIPEA can be used as the base, and DMF, THF, CPME, DCM, or the like can be used as the solvent.

[0082] (Production Method 1 - Route via Compound (3c)) When compound (3c), an active derivative of compound (1-1), is used as the intermediate, a two-step process is carried out: first, compound (3c) is produced from compound (1-1), and then compound (I) is produced from compound (3c). The process for producing compound (3c) from compound (1-1) is described below. Compound (1-1) and compound (3b) are used in equal amounts, or either one in excess, and a mixture thereof is stirred in a reaction-inert solvent such as THF under cooling to heating, in one embodiment at 30°C to 100°C, usually for 0.1 hour to 5 days. The process for producing compound (I) of the present invention from compound (3c) is described below. Compound (3c) and compound (2) are used in equal amounts, or either one in excess, and a mixture thereof is stirred in the presence of a base in a reaction-inert solvent under cooling to heating, in one embodiment at 70°C to 160°C, usually for 0.1 hour to 5 days. Examples of the solvent include halogenated hydrocarbons such as chloroform, ethers such as THF, DMF, DMSO, ethyl acetate, acetonitrile, water, and mixtures thereof. Examples of the base include organic bases such as triethylamine, DIPEA, and N-methylmorpholine. In the first production method, the R Z is a protecting group, and then the protecting group is removed to form R Z (I) can also be prepared by adding the following by an appropriate reaction (the same applies to the following processes). Examples of the protecting group in Process 1 include a tert-butoxycarbonyl group and a benzyloxycarbonyl group. [Reference] Tetrahedron, 2005, Vol. 61, pp. 7153-7175

[0083] (Second manufacturing method)

[0084]

[0085] (Wherein, COOR 2 ' or OCOR 2 ' is R 2 Among these, (Bb), (Bc), (Bd), (Bf), (Bg), (Bi) and (Bj) groups are shown.)

[0086] The compound (I-2) of the present invention can be produced by the esterification reaction of compound (4-1) with compound (5-1). This reaction is carried out in a solvent by combining compound (4-1) with compound (5-1) 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., KCO, NaCO, KOH, etc.) may accelerate the reaction. Alternatively, the compound (I-2) can be produced by converting carboxylic acid (4-1) to a reactive derivative and then reacting it with alcohol (5-1). Examples of reactive derivatives of carboxylic acid include acid halides obtained by reaction with halogenating agents such as phosphorus oxychloride and 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 (4-1) can be carried out in a solvent at a temperature of, in one embodiment, -20°C to 60°C. The solvent is a halogenated hydrocarbon such as DCM. Similarly, compound (I-3) of the present invention can be produced by the esterification reaction of compound (4-2) with compound (5-2).

[0087] (Third production method)

[0088]

[0089] (Wherein, COOR 1 ' or OCOR 1 ' is R 1 Among these, (Bb), (Bc), (Bd), (Bf), (Bg), (Bi) and (Bj) groups are shown.)

[0090] The compound (I-3) of the present invention can be produced by the esterification reaction of the compound (6-1) and the compound (7-1). The compound (I-4) of the present invention can be produced by the esterification reaction of the compound (6-2) and the compound (7-2). The reaction can be carried out in the same manner as the esterification in the second production method.

[0091] (Raw material manufacturing method 1)

[0092]

[0093] (In the formula, Hal represents a halogen, and Prt represents a protecting group.) Compound (2) can be produced by reacting compound (8-1) with compound (9-1) and then deprotecting the resulting compound. Here, a leaving group such as a methanesulfonyloxy group can be used instead of a halogen. In this reaction, equal amounts of compound (8-1) and compound (9-1) or an excess of one are used, and the mixture is stirred in a reaction-inert solvent or without a solvent, under cooling to reflux, preferably at 0°C to 80°C, for typically 0.1 hours to 5 days. Examples of solvents include aromatic hydrocarbons such as toluene and xylene; ethers such as CPME, diethyl ether, THF, and 1,4-dioxane; halogenated hydrocarbons such as DCM and 1,2-dichloroethane; DMAc, NMP, DMF, DMSO, MeCN, ethyl acetate, and mixtures thereof. The reaction may be accelerated by the use of organic bases such as TEA, DIPEA, or N-methylmorpholine, or inorganic bases such as KI, NaI, TBAI, K2CO3, Na2CO3, or KOH. Compound (2) can also be produced under similar conditions using a compound lacking the protective group Prt of compound (8-1). Compound (2) can also be produced by reacting compound (8-2) with compound (9-2). [References] SR Sandler and W. Karo, "Organic Functional Group Preparations," 2nd ed., Vol. 1, Academic Press Inc., 1991. "Experimental Chemistry Lectures (5th ed.)," edited by the Chemical Society of Japan, Vol. 14, 2005 (Maruzen).

[0094] (Raw material manufacturing method 2)

[0095]

[0096] Compound (4-1) can be produced by deprotecting compound (11-1). Compound (11-1) can be produced from compound (1) and compound (10-1). It can be produced in the same manner as in Production Method 1. Compound (4-2) can be produced by deprotecting compound (11-2). Compound (11-2) can be produced from compound (1) and compound (10-2). It can be produced in the same manner as in Production Method 1.

[0097] (Raw material manufacturing method 3)

[0098]

[0099] (wherein R represents a protecting group for a carboxylic acid. For example, C 1-6 The compound (6-1) and the compound (6-2) can be prepared in the same manner as in the raw material preparation method 2.

[0100] (Raw material manufacturing method 4)

[0101]

[0102] (In the formula, -O(C=O)OCH2R 1 ' is R 1 The term "Ba" refers to a group represented by (Ba) or (Be). Among compounds (8-1), compound (8-1-1) can be produced by reacting compound (15), obtained from compound (3b) and compound (14), with compound (16). Both are carbonation reactions, and the same conditions as those used to produce compound (3c) from compound (1-1) and compound (3b) in (Production Method 1) can be used. In the reaction to obtain compound (15) from compound (3b) and compound (14), equal amounts of compound (3b) and compound (14) or an excess of either are used, and the mixture is stirred in a reaction-inert solvent in the presence of a base under cooling to reflux, preferably at 0°C to 80°C, for typically 0.1 hours to 5 days. Examples of solvents that can be used include DCM, CPME, and mixtures thereof. Examples of bases that can be used include DMAP and pyridine. The same conditions can also be used for the reaction to obtain compound (8-1-1) from compound (15) and compound (16).

[0103] 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.

[0104] 5. Method for Producing Lipid Nanoparticles 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 mixture 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.

[0105] 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 include 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.

[0106] 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 another embodiment, about −80° C. to about −20° C., in another embodiment, about −40° C. to about −20° C., and in another embodiment, 4° C. or lower. In one embodiment, the dispersion medium is PBS.

[0107] 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.

[0108] 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.

[0109] 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, 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 BioTechnologies) capping technology.

[0110] 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).

[0111] (Capping) Capping can be performed using vaccinia capping enzyme, 2'O-methyltransferase, CleanCap, etc. If the sequence transcribed by IVT does not contain a poly(A) tail, a poly(A) addition reaction can also be performed.

[0112] (Plasmid Preparation) Linearized plasmids can be prepared using the following steps. A plasmid can be prepared by inserting any UTR and any base sequence into a plasmid (e.g., a high-copy plasmid for E. coli (pUC18 or pCU18, etc.)) and amplifying it using E. coli or other bacteria. After purification, the plasmid can be linearized 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). Transcribed RNA can be purified using a silica gel column or the like. RNA containing poly(A) tails can be purified using an Oligo dT column. Linearized plasmids can be purified using PureLink® (Thermo Fisher Scientific) or the like.

[0113] Test Example 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. In the test examples and the like below in this specification, the following abbreviations may be used. (Abbreviations) B-27: B-27 serum-free supplement, DMEM: Dulbecco's modified Eagle's medium, DMEM / F-12: Dulbecco's modified Eagle's medium / nutrient mixture F-12, DMG-PEG2000: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000, 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, FLuc: luciferase (firefly), LNP: lipid nanoparticles, mRNA: messenger ribonucleic acid, PBS: phosphate-buffered saline, RLU: relative light unit, Sham group: sham control group, TBS: Tris-buffered saline

[0114] Test Example 1: In vitro luciferase assay (evaluation of lipid nanoparticle delivery using mouse primary astrocytes) The compound of formula (I) or a salt thereof constituting the lipid nanoparticles was evaluated for its nucleic acid delivery 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 delivery efficiency of the test drug (nucleic acid-lipid nanoparticles) was calculated by evaluating the expression level of luciferase encoded by the mRNA. Two to three wells were used for each group. The method and results are shown below.

[0115] (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 (Jackson Laboratory Japan) mice, P1-P10, were used.

[0116] (Addition of FLuc nucleic acid-lipid nanoparticles to mouse primary astrocytes) Mouse primary astrocytes obtained as described above were seeded at 2000 cells / well on poly-D-lysine-coated 96-well plates (IWAKI, Greiner Bio-One, and CORNING) 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 (Thermo Fisher Scientific) was used at 100 μL / well. The next day, 100 μL of the test drug (FLuc nucleic acid-lipid nanoparticles) diluted with PBS and medium was added to a final concentration of 0.8 μg / mL, and the plates were cultured overnight at 200 μL / well. The next day, all medium was removed, and 100 μL of the ONE-Glo Luciferase assay system (Promega) was diluted 2-fold with PBS and added to each well. After mixing for 1 minute, 80 μL of each solution was transferred to a 96-well white plate (Thermo Fisher Scientific), and luminescence was detected using EnVision (Revvity). RLU (Relative Light Unit) was used as an activity indicator.

[0117] (Expression Evaluation) As a result of the above test, luciferase activity was observed in FLuc nucleic acid-lipid nanoparticles containing a representative example compound of the present invention as a constituent. This indicated that the FLuc nucleic acid-lipid nanoparticles were taken up into cells, translated, and expressed luciferase protein. The results for nucleic acid-lipid nanoparticles containing example compounds as constituents are shown in the table below. Here, NUM indicates each lipid nanoparticle containing an example compound of the compound of formula (I) or a salt thereof as a constituent. ExA-L0 (A is a number) indicates lipid nanoparticles containing the example compound ExA of the compound of formula (I) or a salt thereof and having the composition L0. L0 indicates lipid nanoparticles containing ExA, DSPC, cholesterol, and DMG-PEG2000 (molar ratio 50 / 10 / 38.5 / 1.5) as constituents. In the NUM, for example, "NUM: Ex1-L0 to Ex54-L0" refers to lipid nanoparticles containing, as a constituent component, example compounds Ex1 to Ex54 of the compound of formula (I) or a salt thereof, and having a lipid composition of L0. The average RLU values ​​for each group of FLuc nucleic acid-lipid nanoparticles were calculated and are shown in the table below. The rows in the table below indicate FLuc nucleic acid-lipid nanoparticles and their RLU. For example, "NUM: Ex1-L0" indicates FLuc nucleic acid-lipid nanoparticles that encapsulate FLuc mRNA, contain example compound Ex1 of the compound of formula (I) or a salt thereof as a constituent component, and have a lipid composition of L0. This indicates that the RLU was 16,560,300.

[0118]

[0119] Test Example 2: In vivo luciferase assay (Test Method) The delivery efficiency of nucleic acid-lipid nanoparticles in the hippocampus was evaluated using IVIS spectrum (Revvity). FLuc nucleic acid-lipid nanoparticles were administered intracerebrally to 6-12 week-old BALB / c mice (Jackson Laboratory Japan, n = 2-3) under isoflurane anesthesia (isoflurane inhalation anesthetic solution "VTRS"; Viatris, hereinafter). For intrahippocampal administration, the administration coordinates were determined based on the Mouse Brain Atlas (Academic Press), 2.0 mm posterior and 1.5 mm right of the Bregma, at a depth of 2.0 mm. Under isoflurane anesthesia, the hair was shaved with clippers, and 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, 200 μL / mouse of 15 mg / mL luciferin (Promega) was administered intraperitoneally. 20 minutes later, luminescence was monitored using IVIS spectrum. The acquired luminescence values ​​were converted to photons / sec using Living Image software and used for analysis.

[0120] (Expression Evaluation) As a result of the above test, luciferase activity was observed in the nucleic acid-lipid nanoparticles of a representative example of the present invention. This indicated that the FLuc nucleic acid-lipid nanoparticles were taken up into cells in the hippocampus, translated, and expressed luciferase protein. The table below shows the luciferase luminescence value data for the FLuc nucleic acid-lipid nanoparticles (the data is the average luminescence value of 2 to 3 FLuc nucleic acid-lipid nanoparticles evaluated). In the table below, each row represents the results for each lipid nanoparticle. For example, "NUM: Ex1-L0" refers to lipid nanoparticles containing FLuc mRNA and the example compound Ex1, which has the same lipid composition as Ex1-L0. The luminescence value of this lipid nanoparticle is 55,170,000.

[0121]

[0122] 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.

[0123] 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, DMAc: N,N-dimethylacetamide, 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, IPA: 2-propanol, KOtBu: potassium tert-butoxide, MeCN: acetonitrile, NMP: N-methyl-2-pyrrolidone, TBAI: tetrabutylammonium iodide, TEA: triethylamine, TFA: trifluoroacetic acid, THF: tetrahydrofuran, NUM: Example number or Production Example number (for example, / 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. The compound or salt thereof of the Example number was produced by sequentially synthesizing the compound or salt thereof of the Production Example number. For example, the compound of Example 1 was produced from the compound or salt thereof of Production Example 1-1, sequentially via the compound or salt thereof of Production Example 1-2, the compound or salt thereof of Production Example 1-3, the compound or salt thereof of Production Example 1-4, and the compound or salt thereof of Production Example 1-5. The compound or salt thereof of Production Example 14-1 was produced from the compound or salt thereof of Production Example 55-1, via the compound or salt thereof of Production Example 55-2, and the compound or salt thereof of Production Example 55-3. Production Example 55-1 was produced from the compound or salt thereof of Production Example 47-1. STR: Chemical structure; DAT: Physicochemical data; NMR: 500 MHz １The chemical shift δ value in ppm for H-NMR is shown (the NMR brackets indicate the measurement solvent; for example, (CDCl3) is a measurement in deuterated chloroform. The NMR signal indicates a representative signal). s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, ESI+: m / z value in ESI-MS+. 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 in mol / L is represented as M. For example, 1M aqueous sodium hydroxide solution means a 1 mol / L aqueous sodium hydroxide solution.

[0124] Preparation Example 1-1: DCM (100 mL) was added to 2-nonylundecan-1-ol (6 g). To this mixture, 8-bromooctanoic acid (4.94 g), HATU (9.18 g), DMAP (245 mg), and DIPEA (8.64 mL) were added at room temperature. The reaction mixture was stirred at room temperature for 16 hours. Saturated aqueous sodium bicarbonate was added to the reaction mixture at room temperature. The separated aqueous layer was extracted with chloroform, and the combined organic layer was purified on neutral silica gel (hexane / ethyl acetate) and then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to obtain 2-nonylundecyl 8-bromooctanoate (9.35 g) as an oil.

[0125] Preparation Example 1-2: To a mixture of 2-octyldecan-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 the reaction mixture was stirred at room temperature for 68 hours. The reaction mixture was concentrated under reduced pressure, and hexane was added to the resulting residue. 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.

[0126] Preparation Example 1-3: 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 sequentially, and the reaction mixture was stirred at room temperature for 2 days. The reaction mixture was concentrated under reduced pressure, diluted with an ethyl acetate-hexane mixed solvent, 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 {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}methyl 2-octyldecyl carbonate (1.18 g) as an oil.

[0127] Preparation Example 1-4: 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, and the reaction mixture was stirred 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 (882.8 mg) as an oil.

[0128] Preparation Example 1-5: 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 potassium iodide (20 mg) were added under an argon atmosphere. The reaction mixture was stirred in an oil bath at 80°C for 2.5 days. After cooling, the reaction mixture was 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.

[0129] Example 1 To a mixture of 2-nonylundecyl 8-({(1r,3r)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino)octanoate (162 mg) and CPME (4 mL), bis(pentafluorophenyl)carbonate (234 mg) was added, and the reaction mixture was stirred at room temperature for 2 hours. To this mixture was added 1-ethyl-1,4-diazepane (0.3 mL) at room temperature. The mixture was stirred in an oil bath at 80°C for 16 hours. After cooling to room temperature, the reaction mixture was diluted with a hexane-ethyl acetate (1:1) mixed solvent and washed with saturated aqueous sodium hydrogen carbonate, 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 then by amino silica gel column chromatography (hexane / ethyl acetate) to give 2-nonylundecyl 8-[(4-ethyl-1,4-diazepane-1-carbonyl){(1r,3r)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]octanoate (46 mg) as an oil.

[0130] Preparation Example 2-1: CPME (4 mL) was added to 6-bromohexyl 4,4-bis(octyloxy)butanoate (250 mg). To this mixture, [(1r,3r)-3-aminocyclobutyl]methyl 2-octyldecyl carbonate (392 mg) in MeCN (4 mL), DIPEA (0.21 mL), and potassium iodide (8 mg) were added at room temperature. The mixture was stirred in an oil bath at 80°C for 2 days. After cooling to room temperature, saturated aqueous sodium bicarbonate and hexane 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 obtained residue was purified by amino silica gel column chromatography (hexane / ethyl acetate) to give 6-({(1r,3r)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino)hexyl 4,4-bis(octyloxy)butanoate (281 mg) as an oil.

[0131] Preparation Example 2-2 CPME (4 mL) was added to 6-({(1r,3r)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino)hexyl 4,4-bis(octyloxy)butanoate (281 mg). CDI (166 mg) was added to this mixture at room temperature. This mixture was stirred in an oil bath at 50°C for 20 hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give 6-[(1H-imidazole-1-carbonyl){(1r,3r)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]hexyl 4,4-bis(octyloxy)butanoate (286 mg) as an oil.

[0132] Preparation Example 2-3 CPME (5 mL) was added to 6-[(1H-imidazole-1-carbonyl){(1r,3r)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]hexyl 4,4-bis(octyloxy)butanoate (286 mg). Methyl iodide (0.2 mL) was added to this mixture at room temperature. This mixture was stirred in an oil bath at 50° C. for 18 hours. MeCN (2 mL) and methyl iodide (0.39 mL) were added to this mixture. This mixture was stirred in an oil bath at 50° C. for 18 hours. The reaction mixture was concentrated under reduced pressure to give 1-[(6-{[4,4-bis(octyloxy)butanoyl]oxy}hexyl){(1r,3r)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}carbamoyl]-3-methyl-1H-imidazol-3-ium iodide (305 mg) as an oil.

[0133] Example 2 DCM (3 mL) was added to 1-[(6-{[4,4-bis(octyloxy)butanoyl]oxy}hexyl){(1r,3r)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}carbamoyl]-3-methyl-1H-imidazol-3-ium iodide (155 mg). To this mixture were added 1-ethyl-1,4-diazepane (0.025 mL) and DIPEA (0.063 mL) at room temperature. The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was purified by silica gel column chromatography (chloroform / methanol) and then by amino silica gel column chromatography (hexane / ethyl acetate / methanol) to give 6-[(4-ethyl-1,4-diazepane-1-carbonyl){(1r,3r)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]hexyl 4,4-bis(octyloxy)butanoate (62 mg) as an oil.

[0134] Preparation Example 3-1: DCM (1000 mL) was added to 2-octyldecan-1-ol (50 g) at room temperature. To this mixture, {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}acetic acid (50.8 g), HATU (71 g), DMAP (2.25 g), and DIPEA (78.3 mL) were added at room temperature, and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure. Heptane and 90% aqueous methanol were added to the concentrated residue, and the separated organic layer was washed with 90% aqueous methanol and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (heptane / ethyl acetate) to give 2-octyldecyl {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}acetate (79.2 g) as an oil.

[0135] Production Example 3-2: Toluene (200 mL) was added to 2-octyldecyl {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}acetate (40 g), and the mixture was stirred at room temperature. TFA (30 mL) was added to the mixture, and the mixture was stirred at an external temperature of 45°C for 18 hours. After cooling to room temperature, the reaction mixture was diluted with toluene (200 mL), and this diluted solution was added dropwise to a 20% aqueous potassium carbonate solution and stirred. The organic layer was isolated and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (heptane / ethyl acetate) to obtain 2-octyldecyl [(1r,3r)-3-aminocyclobutyl]acetate (14.5 g) as an oil.

[0136] Preparation Example 3-3: Benzyl 2-bromoethyl ether (3 g) was added with 2-octyldecyl [(1r,3r)-3-aminocyclobutyl]acetate (10.6 g) and MeCN (90 mL) at room temperature. DIPEA (6 mL) was added to this mixture at room temperature, and the mixture was stirred at an external temperature of 75°C for 67 hours. After completion of the reaction, the reaction mixture was cooled to room temperature, and heptane and water were added. The separated organic layer was collected and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (heptane / ethyl acetate) to give 2-octyldecyl [(1r,3r)-3-{[2-(benzyloxy)ethyl]amino}cyclobutyl]acetate (1.3 g) as an oil.

[0137] Preparation Example 3-4: To a mixture of 2-octyldecyl [(1r,3r)-3-{[2-(benzyloxy)ethyl]amino}cyclobutyl]acetate (0.88 g) and CPME (10 mL) was added bis(pentafluorophenyl)carbonate (2.02 g) at room temperature, and the reaction mixture was stirred at room temperature for 4 hours. To the reaction mixture was added bis(pentafluorophenyl)carbonate (0.34 g), and the reaction mixture was stirred at room temperature for 1.5 hours. To the reaction mixture was added 1-ethyl-1,4-diazepane (5.51 g) and CPME (3 mL), and the reaction mixture was stirred at an external temperature of 90°C for 17.5 hours. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate and washed twice with 5% aqueous potassium carbonate. The organic layer was concentrated under reduced pressure. The resulting residue was purified by amino silica gel column chromatography (heptane / ethyl acetate) to give 2-octyldecyl [(1r,3r)-3-{[2-(benzyloxy)ethyl](4-ethyl-1,4-diazepane-1-carbonyl)amino}cyclobutyl]acetate (870 mg) as an oil.

[0138] Preparation Example 3-5: To 2-octyldecyl [(1r,3r)-3-{[2-(benzyloxy)ethyl](4-ethyl-1,4-diazepane-1-carbonyl)amino}cyclobutyl]acetate (850 mg), acetic acid (4 mL), methanol (4 mL), and 20% palladium hydroxide on carbon (approximately 50% wet with water) (56 mg) were added at room temperature, and the mixture was stirred under a hydrogen atmosphere at the same temperature for 15.5 hours. To the reaction mixture, acetic acid (2 mL) and 20% palladium hydroxide on carbon (approximately 50% wet with water) (18 mg) were added, and the mixture was stirred under a hydrogen atmosphere at room temperature for 1.5 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. To the resulting residue, acetic acid (4 mL) and 20% palladium hydroxide on carbon (approximately 50% wet with water) (55 mg) were added, and the mixture was stirred under a hydrogen atmosphere at room temperature for 1.5 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by aminosilica gel column chromatography (chloroform / methanol) to give 2-octyldecyl {(1r,3r)-3-[(4-ethyl-1,4-diazepane-1-carbonyl)(2-hydroxyethyl)amino]cyclobutyl}acetate (525 mg) as an oil.

[0139] Example 3: 2-Octyldecyl {(1r,3r)-3-[(4-ethyl-1,4-diazepane-1-carbonyl)(2-hydroxyethyl)amino]cyclobutyl}acetate (268 mg) was mixed with 4,4-bis(decyloxy)butanoic acid (260 mg), HATU (247 mg), DMAP (6 mg), DIPEA (0.162 mL), and DCM (5 mL), and the reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with chloroform and washed with saturated aqueous sodium bicarbonate and water. The organic layer was concentrated under reduced pressure, and the residue was purified by aminosilica gel column chromatography (heptane / ethyl acetate). After concentration under reduced pressure, the resulting oil was diluted with heptane and washed with methanol. The heptane layer was concentrated under reduced pressure to give 2-{(4-ethyl-1,4-diazepane-1-carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}ethyl 4,4-bis(decyloxy)butanoate (125 mg) as an oil.

[0140] Preparation Example 4-1: CPME (15 mL) was added to 4-nitrophenyl 2-octyldecyl carbonate (500 mg). To this mixture, pyridine (1.9 mL), tert-butyl [(1r,3r)-3-hydroxycyclobutyl]carbamate (645 mg), and DMAP (28 mg) were added at room temperature. The reaction mixture was stirred in a 50°C oil bath for 18 hours. To the reaction mixture, tert-butyl [(1r,3r)-3-hydroxycyclobutyl]carbamate (430 mg) was added at room temperature. The reaction mixture was stirred in a 50°C oil bath for 20 hours. After cooling to room temperature, hexane 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 (hexane / ethyl acetate) to give (1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl 2-octyldecyl carbonate (487 mg) as an oil.

[0141] Preparation Example 4-2: DCM (3.2 mL) was added to (1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl 2-octyldecyl carbonate (487 mg). TFA (1.6 mL) was added to this mixture at room temperature. The reaction mixture was stirred at room temperature for 18 hours. Chloroform and saturated aqueous sodium bicarbonate were 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 to give (1r,3r)-3-aminocyclobutyl 2-octyldecyl carbonate (381 mg) as an oil.

[0142] Preparation Example 4-3: To a mixture of (1r,3r)-3-aminocyclobutyl 2-octyldecyl carbonate (378 mg) and 6-bromohexyl 4,4-bis(octyloxy)butanoate (250 mg) in CPME (4 mL) was added MeCN (4 mL), DIPEA (0.21 mL), and potassium iodide (8 mg) at room temperature. The reaction mixture was stirred in an 80°C oil bath for 2 days. After cooling to room temperature, saturated aqueous sodium bicarbonate and hexane were added. The separated organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by aminosilica gel column chromatography (hexane / ethyl acetate) to give 6-{[(1r,3r)-3-({[(2-octyldecyl)oxy]carbonyl}oxy)cyclobutyl]amino}hexyl 4,4-bis(octyloxy)butanoate (301 mg) as an oil.

[0143] Preparation Example 4-4: To a mixture of 6-{[(1r,3r)-3-({[(2-octyldecyl)oxy]carbonyl}oxy)cyclobutyl]amino}hexyl 4,4-bis(octyloxy)butanoate (301 mg) and DCM (6 mL) was added CDI (181 mg) at room temperature. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give 6-{(1H-imidazole-1-carbonyl)[(1r,3r)-3-({[(2-octyldecyl)oxy]carbonyl}oxy)cyclobutyl]amino}hexyl 4,4-bis(octyloxy)butanoate (330 mg) as an oil.

[0144] Preparation Example 4-5: To a mixture of 6-{(1H-imidazole-1-carbonyl)[(1r,3r)-3-({[(2-octyldecyl)oxy]carbonyl}oxy)cyclobutyl]amino}hexyl 4,4-bis(octyloxy)butanoate (330 mg), chloroform (3 mL), and MeCN (3 mL) was added methyl iodide (0.23 mL) at room temperature. The reaction mixture was stirred in an oil bath at 50°C for 18 hours. The reaction mixture was concentrated under reduced pressure to give 1-{(6-{[4,4-bis(octyloxy)butanoyl]oxy}hexyl)[(1r,3r)-3-({[(2-octyldecyl)oxy]carbonyl}oxy)cyclobutyl]carbamoyl}-3-methyl-1H-imidazol-3-ium iodide (380 mg) as a solid.

[0145] Example 4 DCM (3 mL) was added to 1-{(6-{[4,4-bis(octyloxy)butanoyl]oxy}hexyl)[(1r,3r)-3-({[(2-octyldecyl)oxy]carbonyl}oxy)cyclobutyl]carbamoyl}-3-methyl-1H-imidazol-3-ium iodide (190 mg). To this mixture were added 1-ethyl-1,4-diazepane (0.031 mL) and DIPEA (0.078 mL) at room temperature. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was purified by silica gel column chromatography (chloroform / methanol) and then by amino silica gel column chromatography (hexane / ethyl acetate / methanol) to give 6-{(4-ethyl-1,4-diazepane-1-carbonyl)[(1r,3r)-3-({[(2-octyldecyl)oxy]carbonyl}oxy)cyclobutyl]amino}hexyl 4,4-bis(octyloxy)butanoate (141 mg) as an oil.

[0146] Preparation Example 5-1: DCM (9 mL) was added to tert-butyl [(1r,3r)-3-(hydroxymethyl)cyclobutyl]carbamate (300 mg). To this mixture, DMAP (2 mg), 4-nitrophenyl chloroformate (602 mg), and pyridine (0.6 mL) were added under ice cooling. The reaction mixture was stirred at room temperature for 18 hours. N-Octyloctan-1-amine (2.3 mL) was added to the reaction mixture at room temperature. The reaction mixture was 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 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 (hexane / ethyl acetate) to give {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}methyl di(octyl)carbamate (612 mg) as an oil.

[0147] Preparation Example 5-2 DCM (4 mL) was added to {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}methyl di(octyl)carbamate (612 mg). TFA (2 mL) was added to this mixture at room temperature. The reaction mixture was stirred at room temperature for 6 hours. A saturated aqueous solution of 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 to obtain [(1r,3r)-3-aminocyclobutyl]methyl di(octyl)carbamate (481 mg) as an oil.

[0148] Preparation Example 5-3: 2-nonylundecyl 8-bromooctanoate (325 mg) / CPME (4 mL) was added to [(1r,3r)-3-aminocyclobutyl]methyl di(octyl)carbamate (476 mg). To this mixture, MeCN (4 mL), DIPEA (0.28 mL), and potassium iodide (11 mg) were added at room temperature. The reaction mixture was stirred in an 80°C oil bath for 38 hours. After cooling to room temperature, saturated aqueous sodium bicarbonate and hexane 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 (hexane / ethyl acetate) to give 2-nonylundecyl 8-{[(1r,3r)-3-({[di(octyl)carbamoyl]oxy}methyl)cyclobutyl]amino}octanoate (388 mg) as an oil.

[0149] Preparation Example 5-4 DCM (4 mL) was added to 2-nonylundecyl 8-{[(1r,3r)-3-({[di(octyl)carbamoyl]oxy}methyl)cyclobutyl]amino}octanoate (205 mg). CDI (126 mg) was added to this mixture at room temperature. The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give 2-nonylundecyl 8-{[(1r,3r)-3-({[di(octyl)carbamoyl]oxy}methyl)cyclobutyl](1H-imidazole-1-carbonyl)amino}octanoate (224 mg) as an oil.

[0150] Preparation Example 5-5: Chloroform (2.5 mL) and MeCN (2.5 mL) were added to 2-nonylundecyl 8-{[(1r,3r)-3-({[di(octyl)carbamoyl]oxy}methyl)cyclobutyl](1H-imidazole-1-carbonyl)amino}octanoate (224 mg). Methyl iodide (0.16 mL) was added to this mixture at room temperature. The reaction mixture was stirred in an oil bath at 50°C for 18 hours. The reaction mixture was concentrated under reduced pressure to give 1-([(1r,3r)-3-({[di(octyl)carbamoyl]oxy}methyl)cyclobutyl]{8-[(2-nonylundecyl)oxy]-8-oxooctyl}carbamoyl)-3-methyl-1H-imidazol-3-ium iodide (260 mg) as an oil.

[0151] Example 5 1-([(1r,3r)-3-({[di(octyl)carbamoyl]oxy}methyl)cyclobutyl]{8-[(2-nonylundecyl)oxy]-8-oxooctyl}carbamoyl)-3-methyl-1H-imidazol-3-ium iodide (260 mg) was added with DCM (3 mL). To this mixture was added 1-ethyl-1,4-diazepane (0.043 mL) and DIPEA (0.11 mL) at room temperature. The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was purified by silica gel column chromatography (chloroform / methanol) and then by amino silica gel column chromatography (hexane / ethyl acetate / methanol) to give 2-nonylundecyl 8-{[(1r,3r)-3-({[di(octyl)carbamoyl]oxy}methyl)cyclobutyl](4-ethyl-1,4-diazepane-1-carbonyl)amino}octanoate (205 mg) as an oil.

[0152] Preparation Example 6-2: To a mixture of triphosgene (0.94 g) and DCM (10 mL), a mixture of methyl (1r,4r)-4-[(8-ethoxy-8-oxooctyl)amino]cyclohexane-1-carboxylate (927 mg), DIPEA (0.6 mL), and DCM (10 mL) was slowly added dropwise under water cooling. The reaction mixture was stirred at room temperature for 2.5 hours. To the reaction mixture, a mixture of 1-methyl-1,4-diazepane (2.48 mL), DIPEA (4.8 mL), and DCM (10 mL) was slowly added dropwise under water cooling, and the reaction mixture was stirred at room temperature for 2 days. The reaction mixture was added to a mixture of chloroform and saturated aqueous sodium bicarbonate at room temperature and then separated. The aqueous layer was extracted with chloroform, and the resulting organic layers were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by amino silica gel column chromatography (hexane / ethyl acetate) to give methyl (1r,4r)-4-[(8-ethoxy-8-oxooctyl)(4-methyl-1,4-diazepane-1-carbonyl)amino]cyclohexane-1-carboxylate (1.31 g) as an oil.

[0153] Preparation Example 6-3: To a mixture of methyl (1r,4r)-4-[(8-ethoxy-8-oxooctyl)(4-methyl-1,4-diazepane-1-carbonyl)amino]cyclohexane-1-carboxylate (1.31 g), THF (15 mL), and EtOH (15 mL) was added 1M aqueous sodium hydroxide (11 mL) at room temperature, and the reaction mixture was stirred at room temperature for 3 hours. 1M hydrochloric acid (11 mL) was added to the reaction mixture under ice cooling, and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was suspended in chloroform / methanol (4 / 1). The suspension was dried over anhydrous sodium sulfate and then concentrated under reduced pressure to give (1r,4r)-4-[(7-carboxyheptyl)(4-methyl-1,4-diazepane-1-carbonyl)amino]cyclohexane-1-carboxylic acid (1.22 g) as a solid.

[0154] Example 6: (1r,4r)-4-[(7-carboxyheptyl)(4-methyl-1,4-diazepane-1-carbonyl)amino]cyclohexane-1-carboxylic acid (150 mg) was added with HATU (410 mg), DMAP (9 mg), DMF (0.5 mL), DIPEA (0.37 mL), and 3-hexylnonan-1-ol (202 mg) in DCM (1.5 mL) at room temperature. The reaction mixture was stirred at room temperature for 4 days. The reaction mixture was added to a mixture of saturated aqueous sodium bicarbonate and ethyl acetate, and the separated aqueous layer was extracted with ethyl acetate. The combined organic layers were washed twice with saturated brine:water (1:1), then once with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by amino silica gel column chromatography (hexane / ethyl acetate) to obtain 3-hexylnonyl (1r,4r)-4-[{8-[(3-hexylnonyl)oxy]-8-oxooctyl}(4-methyl-1,4-diazepane-1-carbonyl)amino]cyclohexane-1-carboxylate (154.2 mg) as an oil.

[0155] Preparation Example 7-1: DCM (15 mL) and DIPEA (1.7 mL) were added to (9Z,12Z)-octadeca-9,12-dien-1-ol (1.5 mL) at room temperature. Chloroacetyl chloride (0.58 mL) was added dropwise to this mixture under ice cooling. The reaction mixture was stirred at room temperature for 1 hour. Water was added to the reaction mixture. The separated aqueous layer was extracted with chloroform, and 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 obtain (9Z,12Z)-octadeca-9,12-dien-1-yl chloroacetate (1.47 g) as an oil.

[0156] Preparation Example 13-2: To a mixture of tert-butyl [(1r,3r)-3-(hydroxymethyl)cyclobutyl]carbamate (500 mg) and DCM (30 mL), linoleic acid (0.81 mL), DIPEA (0.64 mL), EDCI·HCl (0.72 g), and DMAP (61 mg) were added sequentially, and the reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with DCM and washed with 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 {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}methyl (9Z,12Z)-octadeca-9,12-dienoate (1.06 g) as an oil.

[0157] Preparation Example 14-2: DCM (2 mL) was added to tert-butyl 4-[methyl({8-[(2-nonylundecyl)oxy]-8-oxooctyl}[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]carbamoyl)amino]azepane-1-carboxylate (189 mg), and then TFA (0.14 mL) was added to the mixture at room temperature. The reaction mixture was stirred at room temperature for 18 hours. Chloroform and saturated aqueous sodium bicarbonate were added to the reaction mixture at room temperature. The separated organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give 2-nonylundecyl 8-{[(azepan-4-yl)(methyl)carbamoyl][(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (169 mg) as an oil.

[0158] Example 14: To 2-nonylundecyl 8-{[(azepan-4-yl)(methyl)carbamoyl][(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (169 mg) was added IPA (2 mL) and MeCN (1 mL). To this mixture was added 10% palladium on activated carbon (17 mg) at room temperature under an argon atmosphere. This mixture was stirred at room temperature under a hydrogen atmosphere for 24 hours. The reaction mixture was filtered through Celite® and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol) and then by amino silica gel column chromatography (hexane / ethyl acetate / methanol) to give 2-nonylundecyl 8-{[(1-ethylazepan-4-yl)(methyl)carbamoyl][(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (141 mg) as an oil.

[0159] Preparation Example 15-1: DCM (6 mL) was added to diethylamine (0.42 mL). tert-Butyl 3-formylazetidine-1-carboxylate (250 mg) and acetic acid (0.077 mL) were added to this mixture at room temperature. This mixture was stirred at room temperature for 1 hour. Sodium triacetoxyborohydride (858 mg) was added to this mixture under ice-cooling. The reaction mixture was stirred at room temperature for 2.5 hours. Chloroform and saturated aqueous sodium bicarbonate were added to the reaction mixture under ice-cooling, and the mixture was stirred at room temperature for 15 minutes. The separated 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) and then by amino silica gel column chromatography (hexane / ethyl acetate) to give tert-butyl 3-[(diethylamino)methyl]azetidine-1-carboxylate (134 mg) as an oil.

[0160] Preparation Example 15-2 DCM (2 mL) was added to tert-butyl 3-[(diethylamino)methyl]azetidine-1-carboxylate (134 mg). TFA (0.42 mL) was added to this mixture at room temperature. The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was concentrated under reduced pressure to give N-[(azetidin-3-yl)methyl]-N-ethylethanamine trifluoroacetate (204 mg) as an oil.

[0161] Preparation Example 17-3: To tert-butyl 4-{(4-ethyl-1,4-diazepane-1-carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}butanoate (259 mg) was added 4 M hydrogen chloride / 1,4-dioxane solution (2.9 mL). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was concentrated under reduced pressure. The resulting residue was azeotroped once with 1,4-dioxane to give 4-{(4-ethyl-1,4-diazepane-1-carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}butanoic acid hydrochloride (247 mg) as an oil.

[0162] Example 17: 4-{(4-ethyl-1,4-diazepane-1-carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}butanoic acid hydrochloride (100 mg) was added to DCM (3 mL). To this mixture were added (2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-ol (0.076 mL), HATU (87 mg), DMAP (2 mg), and DIPEA (0.078 mL). The mixture was stirred at room temperature for 18 hours. The reaction mixture was purified by silica gel column chromatography (chloroform / methanol), then by amino silica gel column chromatography (hexane / ethyl acetate / methanol), and then by diol silica gel column chromatography (hexane / ethyl acetate / methanol) to give (2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl 4-{(4-ethyl-1,4-diazepane-1-carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}butanoate (82 mg) as an oil.

[0163] Preparation Example 20-1: DCM (9 mL) was added to 2-octyldecyl 4-nitrophenyl carbonate (300 mg). To this mixture, pyridine (1.1 mL), 4-(tert-butoxycarbonylamino)piperidine (413 mg), and DMAP (16 mg) were added at room temperature. The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was concentrated under reduced pressure. Hexane and ethyl acetate were added to the resulting residue, and the mixture was washed with saturated aqueous sodium bicarbonate, water, and saturated brine. It 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-octyldecyl 4-[(tert-butoxycarbonyl)amino]piperidine-1-carboxylate (309 mg) as an oil.

[0164] Preparation Example 23-4: 4-Octyldodecanoic acid (1 g), DIPEA (1.4 mL), HATU (1.58 g), and DMAP (42 mg) were added to a mixture of 4-bromo-1-butanol (0.7 mL) and DCM (10 mL) under ice cooling, in that order. The reaction mixture was then stirred at room temperature for 7 hours. Chloroform and water were added to the reaction mixture, and the aqueous layer was extracted with chloroform. The resulting organic layers were combined, dried over anhydrous magnesium sulfate, and 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.

[0165] Preparation Example 27-1: To a mixture of tert-butyl [(1r,3r)-3-(2-hydroxyethyl)cyclobutyl]carbamate (915 mg) and DCM (30 mL) was added pyridine (1.4 mL) and 4-nitrophenyl chloroformate (1.29 g) under ice-cooling. The reaction mixture was stirred for 30 minutes under ice-cooling and then at room temperature for 18 hours. Silica gel was added to the reaction mixture, and the mixture was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give 2-{(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}ethyl 4-nitrophenyl carbonate (1.77 g) as a solid.

[0166] Preparation Example 27-2: 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 sequentially, and the reaction mixture was stirred at room temperature for 5 days. The reaction mixture was concentrated, diluted with a mixed solvent of ethyl acetate and 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 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.

[0167] Example 27: To a mixture of 2-nonylundecyl 8-[(1H-imidazole-1-carbonyl){(1r,3r)-3-[2-({[(2-octyldecyl)oxy]carbonyl}oxy)ethyl]cyclobutyl}amino]octanoate (164 mg), MeCN (3 mL), and CPME (3 mL) was added methyl iodide (0.5 mL), and the reaction mixture was stirred in an oil bath at 50°C for 20 hours. The reaction mixture was allowed to cool, and the solvent was concentrated under reduced pressure. To the resulting residue was added a mixture of 1-ethyl-1,4-diazepane (132 μL), DCM (20 mL), and DIPEA (0.31 mL), and the reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (chloroform / methanol / 28% aqueous ammonia) and then by amino silica gel column chromatography (hexane / ethyl acetate) to give 2-nonylundecyl 8-[(4-ethyl-1,4-diazepane-1-carbonyl){(1r,3r)-3-[2-({[(2-octyldecyl)oxy]carbonyl}oxy)ethyl]cyclobutyl}amino]octanoate (139 mg) as an oil.

[0168] Preparation Example 33-2: To a mixture of 2-octyldecyl [(1r,3r)-3-{[4-(benzyloxy)butyl]amino}cyclobutyl]acetate (500 mg) and CPME (5 mL) was added bis(pentafluorophenyl)carbonate (1.1 g) at room temperature, and the reaction mixture was stirred at room temperature for 5.5 hours. To this mixture was added 1-ethyl-1,4-diazepane (2.5 g) and CPME (3 mL) at room temperature. The reaction mixture was stirred at an external temperature of 45°C for 13 hours, then at an external temperature of 85°C for 1 hour. At the same temperature, 1-ethyl-1,4-diazepane (1 g) was added and the mixture was stirred for 18 hours. After cooling the reaction mixture to room temperature, heptane and water were added to the mixture, and the mixture was separated to obtain the organic layer. The organic layer was washed with 90% aqueous methanol and then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (heptane / ethyl acetate) to give crude [(1r,3r)-3-{[4-(benzyloxy)butyl](4-ethyl-1,4-diazepane-1-carbonyl)amino}cyclobutyl]2-octyldecyl acetate (330 mg). To a mixture of [(1r,3r)-3-{[4-(benzyloxy)butyl](4-ethyl-1,4-diazepane-1-carbonyl)amino}cyclobutyl]2-octyldecyl acetate (330 mg) and acetic acid (2 mL) was added 20% palladium hydroxide on carbon (approximately 50% water-wet) (33 mg) at room temperature. The atmosphere in the reaction system was purged with nitrogen, then purged with hydrogen, and the mixture was stirred at an external temperature of 45 °C for 3 hours. The mixture was cooled to room temperature and purged with nitrogen. The filtrate from the filtered reaction mixture was added to a container containing ethyl acetate and water, and potassium carbonate was added to this mixture until the pH reached 10 or higher. The separated organic layer was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (heptane / ethyl acetate) to give 2-octyldecyl {(1r,3r)-3-[(4-ethyl-1,4-diazepane-1-carbonyl)(4-hydroxybutyl)amino]cyclobutyl}acetate (220 mg) as an oil.

[0169] Preparation Example 47-1: Under an argon atmosphere, bis(pentafluorophenyl)carbonate (640 mg) and potassium carbonate (860 mg) were added to a mixture of benzyl 3-hydroxyazetidine-1-carboxylate (440 mg) and CPME (15 mL) at room temperature. This mixture was stirred at room temperature for 2.5 hours. To this mixture was added a mixture of 2-nonylundecyl 8-{[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (1000 mg) and CPME (15 mL) at room temperature, and the reaction mixture was stirred at room temperature for 18 hours. To this mixture was added a mixture of bis(pentafluorophenyl)carbonate (640 mg), benzyl 3-hydroxyazetidine-1-carboxylate (440 mg), potassium carbonate (860 mg), and CPME (15 mL) that had been stirred at room temperature for 2 hours. The reaction mixture was stirred at room temperature for 20 hours. The reaction mixture was diluted with ethyl acetate, and anhydrous sodium sulfate was added to the mixture. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by aminosilica gel column chromatography (hexane / ethyl acetate) followed by silica gel column chromatography (hexane / ethyl acetate) to give benzyl 3-[({8-[(2-nonylundecyl)oxy]-8-oxooctyl}[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]carbamoyl)oxy]azetidine-1-carboxylate (1.31 g) as an oil.

[0170] Production Example 47-2 To a mixture of benzyl 3-[({8-[(2-nonylundecyl)oxy]-8-oxooctyl}[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]carbamoyl)oxy]azetidine-1-carboxylate (1.31 g) and IPA (30 mL) was added 10% palladium-activated carbon (approximately 50% water-wet) (268 mg) under an argon atmosphere, and the reaction mixture was stirred at room temperature for 5 hours under a hydrogen atmosphere. The insoluble material was removed by filtration through Celite (registered trademark), and the filtrate was concentrated under reduced pressure to give 2-nonylundecyl 8-({[(azetidin-3-yl)oxy]carbonyl}[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino)octanoate (1.03 g) as an oil.

[0171] Example 47: To a mixture of 2-nonylundecyl 8-({[(azetidin-3-yl)oxy]carbonyl}[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino)octanoate (175 mg) and DCM (3 mL), N,N-dimethylglycine (27 mg), HATU (105 mg), and DIPEA (50 μL) were added, and the reaction mixture was stirred at room temperature for 15 hours. The reaction mixture was purified by silica gel column chromatography (chloroform / methanol) and then by amino silica gel column chromatography (hexane / ethyl acetate) to give 2-nonylundecyl 8-{({[1-(N,N-dimethylglycyl)azetidin-3-yl]oxy}carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (125 mg) as an oil.

[0172] Example 49: To a mixture of 2-octyldecyl [(1r,3r)-3-{[2-(benzyloxy)ethyl]({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)amino}cyclobutyl]acetate (410 mg) and acetic acid (3 mL) was added 20% palladium hydroxide on carbon (approximately 50% wet with water) (158.2 mg) at room temperature, the atmosphere in the reaction system was replaced with nitrogen, and stirring was initiated at the same temperature. The atmosphere in the system was then replaced with hydrogen, and the reaction mixture was stirred at room temperature for 3 hours and 45 minutes. The reaction mixture was extracted with chloroform and a saturated aqueous solution of sodium bicarbonate, and the aqueous layer was further extracted with chloroform. The resulting chloroform layers were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol) to give 2-octyldecyl {(1r,3r)-3-[({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)(2-hydroxyethyl)amino]cyclobutyl}acetate (245.4 mg) as a crude product. To a mixture of 4,4-bis(octyloxy)butanoic acid (84.7 mg), HATU (101 mg), DMAP (2 mg), and DCM (2 mL) was added DIPEA (0.073 mL), followed by 2-octyldecyl {(1r,3r)-3-[({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)(2-hydroxyethyl)amino]cyclobutyl}acetate (130.4 mg) and DCM (2 mL) at room temperature, and the reaction mixture was stirred at room temperature for 22 hours and 20 minutes. To the reaction mixture was added DIPEA (0.052 mL), 4,4-bis(octyloxy)butanoic acid (69.8 mg), HATU (85.5 mg), and DCM (2 mL) at room temperature, and the mixture was stirred at room temperature for 4 hours. To this mixture were added heptane and a 90% aqueous solution of methanol, and the separated heptane layer was washed with a 90% aqueous solution of methanol, and the resulting heptane layer was concentrated under reduced pressure.The resulting residue was purified by amino silica gel chromatography (heptane / toluene / ethyl acetate) to give 2-{({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}ethyl 4,4-bis(octyloxy)butanoate (80.9 mg) as an oil.

[0173] Preparation Example 51-1: To a mixture of 4-nitrophenyl 2-nonylundecyl carbonate (500 mg) and DCM (15 mL) was added DIPEA (1.8 mL), 6-bromo-1-hexanol (0.74 mL), and DMAP (13 mg) at room temperature. The reaction mixture was stirred at room temperature for 18 hours. CPME (15 mL) was added to the 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 to the mixture, and the reaction 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 (hexane / ethyl acetate) to give 6-bromohexyl 2-nonylundecyl carbonate (344 mg) as an oil.

[0174] Preparation Example 55-1: To a mixture of 2-octyldecyl [(1r,3r)-3-aminocyclobutyl]acetate (660 mg) and MeCN (4 mL), 2-nonylundecyl 8-bromooctanoate (430 mg), CPME (4 mL), DIPEA (731 μL), and potassium iodide (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.

[0175] Preparation Example 55-2: To a mixture of 2-nonylundecyl 8-{[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (200 mg) and DCM (3 mL) was added CDI (61 mg) at room temperature, and the reaction mixture was stirred at room temperature overnight. CDI (63 mg) was added to the reaction mixture at room temperature, and the reaction mixture was stirred at room temperature for 2 hours. CDI (66 mg) was added to the reaction mixture at room temperature, and the reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was purified by silica gel chromatography (hexane / ethyl acetate) to obtain 2-nonylundecyl 8-{(1H-imidazole-1-carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (222.9 mg) as an oil.

[0176] Preparation Example 55-3: Chloroform (15 mL), MeCN (15 mL), and methyl iodide (2.28 mL) were added to 2-nonylundecyl 8-{(1H-imidazole-1-carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (3.21 g) and the mixture was stirred in an oil bath at 50° C. for 16 hours. The reaction mixture was concentrated under reduced pressure to give 3-methyl-1-({8-[(2-nonylundecyl)oxy]-8-oxooctyl}[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]carbamoyl)-1H-imidazol-3-ium iodide (3.71 g) as a viscous foam.

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[0219] Example 56 Production of FLuc nucleic acid-lipid nanoparticles (Raw materials for nucleic acid-lipid nanoparticles) DSPC was manufactured by NOF Corporation (product name: COATSOME (registered trademark) MC-8080), cholesterol was manufactured by Nippon Fine Chemical Co., Ltd. (product name: Cholesterol HP) or Merck (product name: Synthechol (registered trademark) Synthetic Cholesterol), DMG-PEG2000 was manufactured by NOF Corporation (SUNBRIGHT (registered trademark) GM-020), and mRNA was manufactured by TriLink BioTechnologies (product name: CleanCap (registered trademark) FLuc mRNA).

[0220] (Preparation of nucleic acid-lipid nanoparticles) Cationic lipid (ExA), phospholipid, sterol, and PEGylated lipid were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 and an N / P ratio of 6 to obtain an oil phase. FLuc mRNA was diluted with 10 mM citrate buffer (pH 4) to obtain an aqueous phase at 85 μg / mL. The oil and aqueous phases were mixed in a microfluidic device (NanoAssemblr (registered trademark), Precision NanoSystems) so that the volume ratio of the oil phase to the aqueous phase was oil phase: aqueous phase = 1:3, and the mixture was diluted 2-fold with phosphate-buffered saline (PBS) to obtain a dispersion of nucleic acid-lipid nanoparticles. This dispersion was dialyzed to remove ethanol, and then concentrated by ultrafiltration to obtain a desired concentration, thereby obtaining nucleic acid-lipid nanoparticles of the example lipid.

[0221] (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, manufactured by Malvern Panalytical).

[0222] (Evaluation of mRNA Encapsulation Rate) The mRNA encapsulation rate was measured in nucleic acid-lipid nanoparticle dispersions diluted to an mRNA concentration of approximately 150-1000 ng / mL. Specifically, the nucleic acid-lipid nanoparticle dispersion obtained as described above was diluted with TE buffer (10 mM Tris / 1 mM EDTA, pH 8.0, Thermo Fisher Scientific) and measured using Quant-iT RiboGreen RNA Reagent (Thermo Fisher Scientific). The mRNA concentration (A) measured in the nucleic acid-lipid nanoparticle external solution was used as the mRNA concentration. The nucleic acid-lipid nanoparticle dispersion was diluted with 2% Triton X-100 and the mRNA concentration (B) measured was used as 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 particle size and mRNA encapsulation rate of the nucleic acid-lipid nanoparticles are shown in the table below. In the table below, each row indicates the encapsulation rate and particle size of each nucleic acid-lipid nanoparticle. For example, the Ex1-L0 listed in NUM indicates that the nucleic acid-lipid nanoparticles encapsulating FLuc mRNA and having a lipid composition of L0, including Ex1, have an encapsulation rate of 95.9% and a particle size of 100.7 nm.

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[0225] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention. This application is based on a Japanese patent application (Patent Application No. 2024-123745) filed on July 30, 2024, the entirety of which is incorporated by reference.

[0226] The urea or carbamate lipid of the compound of formula (I) or its salt of the present invention can form lipid nanoparticles and encapsulate nucleic acids. The lipid nanoparticles obtained from the urea or carbamate lipid are taken up by astrocytes. Pharmaceutical compositions can also be provided using lipid nanoparticles containing the urea or carbamate lipid of the compound of formula (I) or its salt.

[0227] SEQ ID NO: 1: Human NeuroD1 gene (CDS) SEQ ID NO: 2: Human NeuroD1 protein

Claims

1. A compound of formula (I) or a salt thereof: (In the formula, L 1 is -(CH2) q -(Ring D)-(CH2) p -*, * is R 1 one of p and q is 0 and the other is 0, 1 or 2; ring D is a group represented by the formula (Aa), (Ab), (Ac), (Ad) and (Ae): ** is a group selected from the group consisting of (CH2) p indicates that R 1 and R 2 are independently represented by the formulae (Ba), (Bb), (Bc), (Bd), (Be), (Bf), (Bg), (Bh), (Bi) and (Bj): a group selected from the group consisting of: 1 and R 2 are both (Bf) or (Bg), then ring D is formula (Ab) or (Ad); q is 1; p is 2; or ring A is formula (b), (c) or (d), and R 11a , R 11b independently, C 5-15 alkyl, R 12 is C 5-20 Alkenyl, L 4 is C 1-10 alkylene, the combination of G and Y (G, Y) is (O, CH), (bond, N) or (NCH3, CH), X is CH or N, ring A, X, Y and G together represent the formulas (a) to (f): a group selected from the group consisting of: R Z This bond is R Z indicates that it binds to R z is C 1-6 Alkyl, -C(O)-(C 1-6 alkylene)-N(C 1-6 alkyl)2 or -(C 1-6 (Alkylene)-NR 00 R 01 , R 00 and R 01 independently, C 1-6 Alkyl.) 2. R 1 and R 2 are independently a group selected from the group consisting of formulae (Ba), (Bb), (Bc), (Bd), (Be), (Bf), (Bg), (Bh), (Bi) and (Bj), and R 1 and R 2 The compound of formula (I) or a salt thereof according to claim 1, wherein any one of the following is a group selected from the group consisting of formulae (Ba), (Bb), (Bc), (Bd), (Bh), (Bi) and (Bj):

3. R 1 and R 2 are independently a group selected from the group consisting of formulae (Ba), (Bb), (Bc), (Bd), (Be), (Bf), (Bh), (Bi) and (Bj), and R 1 and R 2 The compound of formula (I) or a salt thereof according to claim 2, wherein any one of the following is a group selected from the group consisting of formulae (Ba), (Bb), (Bc), (Bd), (Bh), (Bi) and (Bj):

4. R 1 and R 2 is independently a group selected from the group consisting of formulas (Ba), (Bd), (Bf), and (Bh), and R 1 and R 2 The compound of formula (I) or a salt thereof according to claim 2, wherein either one of the following is a group selected from the group consisting of formulae (Ba), (Bd), and (Bh):

5. A compound of formula (I) or a salt thereof according to claim 2, wherein the combination of G and Y (G, Y) is (O, CH) or (NCH3, CH), X is N, and ring A, X, Y and G taken together are represented by formula (b), (e) or (f).

6. A compound of formula (I) or a salt thereof according to claim 2, wherein the combination of G and Y (G, Y) is (bond, N), X is N, and ring A, X, Y and G together represent formula (a).

7. A compound of formula (I) or a salt thereof according to claim 5 or 6, wherein ring D is formula (Aa) or (Ab).

8. R 1 and R 2 are both (Bf) or (Bg), ring D is formula (Ab) or (Ad); q is 1; p is 2; or ring A is formula (b), (c) or (d), or a salt thereof.

9. L 1 But -(CH2) q -(Ring D)-(CH2) p -*, q is 0, p is 0 or 1, ring D is formula (Aa), and R 1 and R 2 are independently a group selected from the group consisting of formulae (Ba), (Bd), (Bf) and (Bh), and one is a group selected from the group consisting of formulae (Ba), (Bd) and (Bh), and R 11a , R 11b But independently C 5-10 is alkyl, and L 4 C 2- Alkylene or C 5-7 alkylene, the combination of G and Y (G, Y) is (bond, N), X is N, ring A, X, Y and G together represent the formula (a), and R z C 1-6 The compound of formula (I) or a salt thereof according to claim 7, wherein the aryl group is alkyl.

10. 2-nonylundecyl 8-[(4-ethyl-1,4-diazepane-1-carbonyl){(1r,3r)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]octanoate, 6-[(4-ethyl-1,4-diazepane-1-carbonyl){(1r,3r)-3-[({[(2-octyldecyl)oxy]carbonyl}oxy)methyl]cyclobutyl}amino]hexyl 4,4-bis(octyloxy)butanoate, 2-{(4-ethyl-1,4-diazepane-1-carbonyl)[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}ethyl 4,4-bis(decyloxy)butanoate, 2. The compound according to claim 1, which is 6-{(4-ethyl-1,4-diazepane-1-carbonyl)[(1r,3r)-3-({[(2-octyldecyl)oxy]carbonyl}oxy)cyclobutyl]amino}hexyl 4,4-bis(octyloxy)butanoate, or 2-nonylundecyl 8-{[(1r,3r)-3-({[di(octyl)carbamoyl]oxy}methyl)cyclobutyl](4-ethyl-1,4-diazepane-1-carbonyl)amino}octanoate, or a salt thereof.

11. Lipid nanoparticles comprising the compound of formula (I) or a salt thereof according to claim 1.

12. A lipid nanoparticle comprising the compound of formula (I) or a salt thereof according to claim 1, a neutral lipid and a PEGylated lipid.

13. The lipid nanoparticle of claim 12, which encapsulates a nucleic acid.

14. The lipid nanoparticle of claim 13, wherein the nucleic acid is mRNA.

15. The lipid nanoparticles according to claim 14, wherein the neutral lipid is a phospholipid and a sterol, the phospholipid is DSPC, the sterol is cholesterol, and the PEGylated lipid is DMG-PEG2000.

16. The lipid nanoparticle of claim 15, wherein the lipid nanoparticle is capable of expressing a protein in an astrocyte.

17. A pharmaceutical composition comprising lipid nanoparticles according to any one of claims 11 to 16.

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