Compound or salt thereof, lipid nanoparticles, and pharmaceutical composition

Abstract

Provided is a compound represented by formula (1) or a salt thereof. In formula (1), RB1 to RB4 are each independently a C1-3 alkylene group. RB5 is a C1-3 alkylene group that may have a heteroatom. RL1 to RL3 are each independently a linear or branched saturated aliphatic hydrocarbon group that may have a substituent and that has a carbon number of not less than 8, or a linear or branched unsaturated aliphatic hydrocarbon group that may have a substituent and that has a carbon number of not less than 8. YS1 is a divalent linking group that may have an oxygen atom.

Description

Compounds or salts thereof, lipid nanoparticles, and pharmaceutical compositions

[0001] The present invention relates to compounds or salts thereof, lipid nanoparticles, and pharmaceutical compositions. This application claims priority under Japanese Patent Application No. 2025-007203, filed in Japan on January 17, 2025, the contents of which are incorporated herein by reference.

[0002] In recent years, RNA has attracted attention as a modality in gene therapy. RNA has a low risk of integration into the genome and is safer than DNA. Furthermore, once the RNA sequence is determined, it is easy to design, and it can target any gene or express any protein in the body. For these reasons, RNA has the potential to lead to safe and versatile drug discovery.

[0003] RNAs such as mRNA and siRNA (small interfering RNA) are immunogenic, easily degraded by enzymes, and not taken up by cells; therefore, simply administering them into the body does not produce the desired therapeutic effect. As a means of efficiently delivering RNA into target tissues or target cells, the use of lipid nanoparticles (LNPs) as carriers has been investigated. LNPs used in gene therapy and other applications are complexes of nucleic acids, which are the active ingredient, and lipids that protect these nucleic acids. Examples of nucleic acids that constitute LNPs include mRNA, siRNA, antisense oligonucleotides (ASOs), and DNA. As a result of the delivery of these nucleic acids into target tissues or target cells, the desired biological activity is exhibited.

[0004] Ionizable lipids are a major component of LNPs (Long Nucleoplasmic Nets). These ionizable lipids are a type of lipid molecule that exhibits charge neutrality at physiological pH and protonates in acidic regions. During LNP formation, ionizable lipids interact with nucleic acids, contributing to the efficient encapsulation of nucleic acids within the LNP. Furthermore, when LNPs are taken up by cells, ionizable lipids protonate in the acidic environment of the cell, causing membrane fusion between the lipid membrane of the LNP and the endosomal membrane, thus contributing to their escape from endosomes. Ionizable lipids are classified into unsaturated, multi-tail, and biodegradable types based on their structural characteristics. These structural characteristics contribute to the efficiency of the release of nucleic acids (the payload) within the cell and the reduction of LNP cytotoxicity.

[0005] For example, Patent Document 1 discloses a novel ionized lipid containing a biodegradable ester bond. The ionized lipid disclosed in Patent Document 1 exhibits excellent efficacy in nucleic acid delivery.

[0006] International Publication No. 2023 / 136689

[0007] The present invention aims to provide novel compounds or salts thereof, lipid nanoparticles, and pharmaceutical compositions that can be used as ionized lipids during the formation of LNPs, which can effectively exert the function of the encapsulated nucleic acids within cells.

[0008] In other words, the present invention encompasses the following embodiments: [1] A compound or a salt thereof represented by the following formula (1). [In formula (1), R Ｂ１ ~R Ｂ４ These are, independently, alkylene groups having 1 to 3 carbon atoms. Ｂ５ R is an alkylene group having 1 to 3 carbon atoms, which may have heteroatoms. L１ ~R L３ Each of these is independently a linear or branched saturated aliphatic hydrocarbon group having 8 or more carbon atoms, which may have substituents, or a linear or branched unsaturated aliphatic hydrocarbon group having 8 or more carbon atoms, which may have substituents. Ｓ１is a divalent linking group which may have an oxygen atom. [2] A compound represented by the following formula (2) or a salt thereof. [In formula (2), R Ｂ１ ~R Ｂ４ are each independently an alkylene group having 1 to 3 carbon atoms. R Ｂ５ is an alkylene group having 1 to 3 carbon atoms which may have a hetero atom. R L１１ ~R L１４ are each independently a linear or branched saturated aliphatic hydrocarbon group having 8 or more carbon atoms which may have a substituent, or a linear or branched unsaturated aliphatic hydrocarbon group having 8 or more carbon atoms which may have a substituent. Y Ｓ１１ , Y Ｓ１２ are each independently a divalent linking group which may have an oxygen atom. [3] The compound or a salt thereof according to [1] or [2], wherein R Ｂ５ is an alkylene group having 1 to 3 carbon atoms. [4] The compound or a salt thereof according to [1], wherein R L１ ~R L３ are each independently a group represented by the following formula (R-1) or the following formula (R-2). [In formula (R-1) and (R-2), R LX is a -OH group, -SH group, -OC(=O)R group (R is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms), or an amino group (-NH ２ ), R LY is a linear or branched saturated or unsaturated aliphatic hydrocarbon group having 4 or more carbon atoms, n1 is an integer of 1 to 3, n2 is an integer of 1 to 12, and * indicates the bonding position to the nitrogen atom. [5] The compound or a salt thereof according to [2], wherein R L１１ ~R L１４ are each independently a group represented by the following formula (R-1) or the following formula (R-2). [In formula (R-1) and (R-!2), R LX is a -OH group, -SH group, -OC(=O)R group (R is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms), or an amino group (-NH ２ ), R LY[6] The compound or salt thereof described in [2], wherein formula (2) is the following formula (2-1), formula (2-2), formula (2-3), or formula (2-4). [In formulas (2-1), (2-2), (2-3), (2-4), R Ｌ１ , R Ｌ２ At least one of them is a linear saturated aliphatic hydrocarbon group or an unsaturated aliphatic hydrocarbon group having 4 or more carbon atoms, R ＬＹ′ [7] The compound or a salt thereof according to [6], wherein p is a linear saturated aliphatic hydrocarbon group or an unsaturated aliphatic hydrocarbon group having 4 or more carbon atoms, p is an integer from 1 to 5, q is an integer from 1 to 11, r is an integer from 1 to 11, s is an integer from 1 to 3, and t is an integer from 1 to 11. [8] The compound or a salt thereof according to [6], wherein q is an integer from 2 to 7. [9] The compound or a salt thereof according to [6], wherein formula (2-1) is compound 7, compound 15, compound 18, compound 30, or compound 31 shown below.

[10] The compound or salt thereof described in [6], wherein formula (2-2) is compound 20, compound 21, compound 32, or compound 33 shown below.

[11] The above formula (2-3) is the compound 26 shown below, the compound described in [6] or a salt thereof.

[12] Lipid nanoparticles comprising nucleic acid and ionized lipid, wherein the ionized lipid is a compound or salt thereof as described in any one of [1] to

[11] .

[13] Lipid nanoparticles comprising nucleic acid and ionized lipid, wherein the ionized lipid is compound 4, compound 7, compound 11, compound 15, compound 18, compound 20, compound 21, compound 26, compound 30, compound 31, compound 32, or compound 33 as shown below.

[14] Lipid nanoparticles according to

[12] or

[13] , further comprising sterols.

[15] Lipid nanoparticles according to any one of

[12] to

[14] , wherein the nucleic acid is mRNA or siRNA.

[16] A pharmaceutical composition comprising lipid nanoparticles according to any one of

[12] to

[15] .

[0009] According to the present invention, it is possible to provide novel compounds or salts thereof, lipid nanoparticles, and pharmaceutical compositions that can be used as ionized lipids during the formation of LNPs that can effectively exert the function of the encapsulated nucleic acids within cells.

[0010] This figure shows the results of the mouse bioluminescence imaging evaluation test for Comparative Example 4. This figure shows the results of the mouse bioluminescence imaging evaluation test for Example 10. This figure shows the results of the mouse bioluminescence imaging evaluation test for Example 11. This figure shows the results of the mouse bioluminescence imaging evaluation test for Example 12. This figure shows the results of the mouse bioluminescence imaging evaluation test for Example 13. This figure shows the results of the mouse bioluminescence imaging evaluation test for Example 14. This figure shows the results of the mouse bioluminescence imaging evaluation test for Comparative Example 5. This figure shows the results of the mouse bioluminescence imaging evaluation test for Example 15.

[0011] <Compound or Salt Thereof> One aspect of the present invention is a compound or a salt thereof represented by the following formula (1).

[0012]

[0013] In formula (1), R Ｂ１ ~R Ｂ４ These are, independently, alkylene groups having 1 to 3 carbon atoms. Ｂ５ R is an alkylene group having 1 to 3 carbon atoms, which may have heteroatoms. Ｌ１ ~R Ｌ３ Each of these is independently a linear or branched saturated aliphatic hydrocarbon group having 8 or more carbon atoms, which may have substituents, or a linear or branched unsaturated aliphatic hydrocarbon group having 8 or more carbon atoms, which may have substituents. Ｓ１ This is a divalent linking group which may have an oxygen atom.

[0014] The compound represented by formula (1) comprises a head portion having a bicyclic structure containing a tertiary amine and a leg portion having eight or more carbon atoms. The compound represented by formula (1) functions as an ionized lipid and, together with other constituent lipids, forms LNPs that can encapsulate target substances such as mRNA. Specifically, in an acidic environment, the nitrogen atom of the head portion is protonated and becomes positively charged, interacting mainly with negatively charged target substances and forming LNPs together with other constituent lipids such as phospholipids, cholesterol, and PEG lipids, allowing the target substances to be encapsulated within the LNPs.

[0015] In formula (1), R Ｂ１ ~R Ｂ４ Each of these is independently an alkylene group having 1 to 3 carbon atoms, preferably an alkylene group having 1 to 2 carbon atoms, and more preferably a methylene group. In formula (1), R Ｂ５ R is an alkylene group having 1 to 3 carbon atoms, which may have heteroatoms. Ｂ５ Examples of heteroatoms that may be present include oxygen atoms, sulfur atoms, and nitrogen atoms, with oxygen atoms being preferred. In one embodiment of the present invention, in formula (1), R Ｂ５ The alkylene group having 1 to 3 carbon atoms is preferred, the alkylene group having 1 to 2 carbon atoms is more preferred, and the methylene group is even more preferred. That is, in the compound represented by formula (1), R Ｂ５ It is preferable that it does not have heteroatoms, R Ｂ５ A methylene group is more preferable. In formula (1), R Ｂ５ When the compound lacks heteroatoms, an imbalance between electron-donating and electron-withdrawing properties is more likely to occur within the bicyclo ring that constitutes the main skeleton, resulting in increased polarization within the ring. This increased polarization is thought to promote the localization of lone pairs of electrons in the tertiary amine, strengthening its interaction with proton-donating species in the external environment and improving its responsiveness to protonation behavior.

[0016] In formula (1), Y Ｓ１This is a divalent linking group which may have an oxygen atom. The divalent linking group is, for example, an alkylene group having 1 to 10 carbon atoms, preferably an alkylene group having 1 to 8 carbon atoms, and more preferably an alkylene group having 1 to 5 carbon atoms. Also, Y Ｓ１ The alkylene group may have a branched chain. "May have an oxygen atom" means that it is not particularly limited to any functional group containing an oxygen atom. Ｓ１ If is a divalent linking group containing an oxygen atom, Y Ｓ１ Examples include divalent linking groups containing a carbonyl group, an ether bond, a hydroxyl group, and an ester bond. The compound represented by formula (1) is preferably a divalent linking group containing a carbonyl group. In formula (1), Y Ｓ１ If is a divalent linking group having an oxygen atom, Y Ｓ１ Due to the electronic effects caused by the oxygen atom inside, the bicyclo ring and Y Ｓ１ The binding with is stabilized. As a result, degradation and structural changes during blood retention are suppressed, and overall stability in the blood is expected to be enhanced.

[0017] In formula (1), R Ｌ１ ~R Ｌ３ Each of these is independently a linear or branched saturated aliphatic hydrocarbon group having 8 or more carbon atoms, which may have substituents, or a linear or branched unsaturated aliphatic hydrocarbon group having 8 or more carbon atoms, which may have substituents.

[0018] In this specification, "may have substituents" means that a hydrogen atom (H) is substituted with a monovalent group, and a methylene group (-CH) is substituted with a monovalent group. ２ This includes both cases where the negative (-) is substituted with a divalent group.

[0019] R L１ ~R L３ The monovalent substituents that may be present are an -OH group, an -SH group, an -OC(=O)R group (where R is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms), or an amino group (-NH ２ ) are some examples.

[0020] R Ｌ１ ~R Ｌ３Divalent substituents that may be present include ether bonds (-O-), amide bonds ([-C(=O)-NH-], [-NH-C(=O)-]), ester bonds ([-C(=O)-O-], [-O-C(=O)-]), thioester bonds ([-C(=O)-S-], [-S-C(=O)-]), phosphate ester bonds (-P(=O)(OR')O-, where R' is a C1-C10 alkyl group which may have a hydrogen atom or an oxygen atom), carbonyl groups (-C(=O)-), Thiocarbonyl group (-C(=S)-), carbonate bond (-O-C(=O)-O-), thiocarbonate bond (-O-C(=O)-S-), dithiocarbonate bond (-S-C(=O)-S-), urethane bond ([-O-C(=O)-N-], [-N-C(=O)-O-]), urea bond (-N-C(=O)-N-), thiourethane bond ([-S-C(=O)-N-], [-N-C(=O)-S-]), thiourea bond (-N-C(=S)-N-), sulfonyl group (-S(=O)) ２ -), sulfonyl ester (-O-S (=O) ２ -), sulfamide ([-N-S (=O) ２ -], [-S (=O) ２ Examples include -N-), disulfide (-S-S-), or thioether bond (-S-).

[0021] R Ｌ１ ~R Ｌ３ The number of carbon atoms is 8 or more, preferably 10 or more, and more preferably 12 or more. Ｌ１ ~R Ｌ３ The number of carbon atoms is, for example, 20 or less, 18 or less, or 16 or less. Ｌ１ ~R Ｌ３ The above upper and lower limits for the number of carbon atoms can be combined in any way. Examples of combinations include 8 to 20, 10 to 18, and 12 to 16.

[0022] R Ｌ１ ~R Ｌ３ If R is an unsaturated aliphatic hydrocarbon group, Ｌ１ ~R Ｌ３ It is preferable that the group is an unsaturated aliphatic hydrocarbon group having a double bond, and more preferably an unsaturated aliphatic hydrocarbon group containing 1 to 3 double bonds.

[0023] In equation (1), R L１ ~R L３ Each of these is preferably a group represented by the following formula (R-1) or the following formula (R-2).

[0024]

[0025] In formulas (R-1) and (R-2), R LX This includes -OH group, -SH group, -OC(=O)R group (where R is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms), or amino group (-NH ２ ) and R LY n1 is a linear or branched saturated or unsaturated aliphatic hydrocarbon group having four or more carbon atoms, n1 is an integer from 1 to 3, n2 is an integer from 1 to 12, and * indicates the bond site with the nitrogen atom. Preferably, n2 is an integer from 2 to 7.

[0026] Formula (R-2) is preferably the formula (R-2-1) shown below.

[0027]

[0028] In formula (R-2-1), R Ｌ１ , R Ｌ２ At least one of them is a linear saturated aliphatic hydrocarbon group or an unsaturated aliphatic hydrocarbon group having 4 or more carbon atoms. That is, in one embodiment of the present invention, R in formula (R-2-1) Ｌ１ , R Ｌ２ Both may be linear saturated aliphatic hydrocarbon groups or unsaturated aliphatic hydrocarbon groups having 4 or more carbon atoms. In one embodiment of the present invention, R in formula (R-2-1) Ｌ１ , R Ｌ２ Either one of them may be a linear saturated aliphatic hydrocarbon group or an unsaturated aliphatic hydrocarbon group having 4 or more carbon atoms, and the other may be a linear saturated aliphatic hydrocarbon group or an unsaturated aliphatic hydrocarbon group having 1 to 3 carbon atoms.

[0029] In equation (1), R L１ ~R L３However, if the group is represented by the above formula (R-1) or (R-2), it is presumed that when lipid nanoparticles are formed, their stability will increase, and the function of the encapsulated nucleic acid can be effectively exerted within the cell.

[0030] The following are specific examples of compounds represented by formula (1).

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039]

[0040] [Method for producing the compound represented by formula (1)] The compound represented by formula (1) can be produced by introducing the leg portion to the head portion of the compound via a spacer. The spacer has a structure that bridges the head portion and the leg portion.

[0041] Suitable compounds for the head portion include 3,7-diazabicyclo[3.3.1]nonane (bispirin; CAS number: 280-74-0), 9-oxa-3,7-diazabicyclo[3.3.1]nonane (CAS number: 329-96-4), 3,7-diazabicyclo[3.3.2]decane (CAS number: 347861-81-8), and 3,7-diazabicyclo[3.3.3]undecane (CAS number: 477881-56-4). In one embodiment of the present invention, suitable compounds for the head portion include 3,7-diazabicyclo[3.3.1]nonane (bispirin; CAS number: 280-74-0), 3,7-diazabicyclo[3.3.2]decane (CAS number: 347861-81-8), and 3,7-diazabicyclo[3.3.3]undecane (CAS number: 477881-56-4).

[0042] Examples of compounds that can act as spacers include those represented by formula (S)-1 or formula (S)-2 shown below.

[0043]

[0044] In the above formula (S)-1, X is a halogen atom, and is F, Cl, Br or I. In the above formulas (S)-1 and (S)-2, Y ｓ Y in formula (1) above is a divalent linking group which may have an oxygen atom. Ｓ１ The explanation is the same as for the above formula (S)-1. In formula (S)-1, Ptg is an amine protecting group. Examples of Ptg include ter-butoxycarbonyl group (Boc group), 9-fluorenylmethyloxycarbonyl group (Fmoc group), benzyloxycarbonyl group (Cbz group), benzyl group (Bn group), allyloxycarbonyl group (Alloc group), and 2-nitrobenzenesulfonyl group (Ns group). Ptg is preferably Boc group, Fmoc group, Cbz group, Bn group, or Ns group, with Boc group and Ns group being particularly preferred.

[0045] As the compound represented by formula (S)-1 or formula (S)-2, for example, the following compounds can be used. However, the compound represented by formula (S)-1 or formula (S)-2 is not limited to the following examples. In the following examples, Ptg is a protecting group for an amine.

[0046]

[0047] As the compound that becomes the leg part, compounds represented by any of the following formulas (L)-1 to (L)-8 can be mentioned.

[0048]

[0049] In formula (L)-1, X is a halogen atom, which is F, Cl, Br or I. In formulas (L)-1 to (L)-8, R ＬＹ is a linear or branched saturated aliphatic hydrocarbon group or unsaturated aliphatic hydrocarbon group having 4 or more carbon atoms.

[0050] The compound represented by formula (L)-1 may be a compound represented by the following formula (L)-1-1, the compound represented by formula (L)-2 may be a compound represented by the following formula (L)-2-1, the compound represented by formula (L)-4 may be a compound represented by the following formula (L)-4-1, the compound represented by formula (L)-5 may be a compound represented by the following formula (L)-5-1, the compound represented by formula (L)-6 may be a compound represented by the following formula (L)-6-1, the compound represented by formula (L)-7 may be a compound represented by the following formula (L)-7-1, and the compound represented by formula (L)-8 may be a compound represented by the following formula (L)-8-1.

[0051]

[0052] In formulas (L)-1-1, (L)-2-1, (L)-4-1, (L)-5-1, (L)-6-1, (L)-7-1, (L)-8-1, at least one of R Ｌ１ and R Ｌ２ is a linear saturated aliphatic hydrocarbon group or unsaturated aliphatic hydrocarbon group having 4 or more carbon atoms. That is, in one aspect of the present invention, R Ｌ１ and R Ｌ２Both may be linear saturated aliphatic hydrocarbon groups or unsaturated aliphatic hydrocarbon groups having 4 or more carbon atoms. In one embodiment of the present invention, R Ｌ１ , R Ｌ２ Either one of them may be a linear saturated or unsaturated aliphatic hydrocarbon group having 4 or more carbon atoms, and the other may be a linear saturated or unsaturated aliphatic hydrocarbon group having 1 to 3 carbon atoms. In formulas (L)-1-1 and (L)-2-1, X is a halogen atom, and is F, Cl, Br, or I.

[0053] Examples of compounds that can be represented by any of formulas (L)-1 to (L)-8 include the following compounds. However, compounds represented by any of formulas (L)-1 to (L)-8 are not limited to the following examples.

[0054]

[0055]

[0056]

[0057] [Method 1 for producing the compound represented by formula (1)] The compound represented by formula (1) can be produced by the following production method 1.

[0058]

[0059] In the above manufacturing method 1, R Ｂ１ ~R Ｂ５ The explanation regarding Ptg is the same as above. In the above manufacturing method 1, Y ｓ R is a divalent linking group which may have an oxygen atom. In the above manufacturing method 1, ＬＹ This is a linear or branched saturated aliphatic hydrocarbon group having 8 or more carbon atoms, which may have substituents, or a linear or branched unsaturated aliphatic hydrocarbon group having 8 or more carbon atoms, which may have substituents.

[0060] [Method 2 for producing the compound represented by formula (1)] The compound represented by formula (1) can be produced by the following production method 2.

[0061]

[0062] In the above manufacturing method 2, R Ｂ１ ~R Ｂ５ 、The explanations regarding Ptg and n2 are the same as above. In the above manufacturing method 2, Y ｓ is a divalent linking group that may have an oxygen atom. In the above manufacturing method 2, R ＬＹ is a linear or branched saturated aliphatic hydrocarbon group having 4 or more carbon atoms that may have a substituent, or a linear or branched unsaturated aliphatic hydrocarbon group having 4 or more carbon atoms that may have a substituent.

[0063] One aspect of the present invention is a compound or a salt thereof represented by the following formula (2).

[0064]

[0065] In formula (2), R Ｂ１ ~R Ｂ４ are each independently an alkylene group having 1 to 3 carbon atoms. R Ｂ５ is an alkylene group having 1 to 3 carbon atoms that may have a hetero atom. R L１１ ~R L１４ are each independently a linear or branched saturated aliphatic hydrocarbon group having 8 or more carbon atoms that may have a substituent, or a linear or branched unsaturated aliphatic hydrocarbon group having 8 or more carbon atoms that may have a substituent. Y Ｓ１１ 、Y Ｓ１２ are each independently a divalent linking group that may have an oxygen atom.

[0066] The compound represented by formula (2) comprises a head portion having a biring structure, a leg portion having eight or more carbon atoms, and a spacer connecting the head portion and the leg portion. In one embodiment of the present invention, the "head portion having a biring structure" in formula (2) means that the head portion is composed of two ring structures and does not contain any structural elements other than these two ring structures. The compound represented by formula (2) functions as an ionized lipid and can form LNPs together with other constituent lipids, thereby encapsulating target substances such as mRNA. Specifically, in an acidic environment, the nitrogen atom in the compound represented by formula (2) is protonated and becomes positively charged, interacting mainly with negatively charged target substances, and forming LNPs together with other constituent lipids such as phospholipids, cholesterol, and PEG lipids, thereby encapsulating the target substances within the LNPs.

[0067] In equation (2), R Ｂ１ ~R Ｂ４ The explanation for this is R in equation (1) above. Ｂ１ ~R Ｂ４ The explanation is similar to that for the following. In particular, R in equation (2) Ｂ１ ~R Ｂ４ It is preferable that it is a methylene group.

[0068] In equation (2), R Ｂ５ The explanation for this is R in equation (1) above. Ｂ５ This is similar to the explanation regarding R. Ｂ５ It is preferable that it does not have heteroatoms, R Ｂ５ It is more preferable that R is a methylene group. In formula (2), Ｂ５ When the compound lacks heteroatoms, an electron density imbalance is more likely to occur within the bicyclo ring that constitutes the main skeleton, leading to increased polarization within the ring. This polarization extends to the entire structure, including the spacer, and the electron density of the lone pairs of electrons in the tertiary amine becomes relatively higher. As a result, the interaction with proton-donating species in the external environment is strengthened, and the responsiveness of the tertiary amine to protonation behavior is thought to improve.

[0069] In equation (2), Y Ｓ１１ , Y Ｓ１２The explanation for this is Y in equation (1) above. Ｓ１ The explanation is similar to that for the following. In particular, Y in equation (2) Ｓ１１ , Y Ｓ１２ It is preferable that is a divalent linking group containing a carbonyl group. In formula (2), Y Ｓ１１ , Y Ｓ１２ If is a divalent linking group having an oxygen atom, then the bicyclo ring and Y Ｓ１１ , Y Ｓ１２ It is presumed that this stabilizes the binding and improves stability during bloodstream retention.

[0070] In equation (2), R L１１ ~R L１４ The explanation for this is R in equation (1) above. L１ ~R L３ The explanation is similar to that for . In equation (2), R L１１ ~R L１４ Preferably, each of these is independently a group represented by the above formula (R-1) or the above formula (R-2).

[0071] Formula (2) is preferably one of the following formulas (2-1) to (2-6).

[0072]

[0073] In equation (2-1), p is an integer from 1 to 5, preferably from 1 to 3. q is an integer from 1 to 11, preferably from 2 to 7. r is an integer from 1 to 11, preferably from 5 to 9.

[0074]

[0075] In equation (2-2), p is an integer from 1 to 5, preferably from 1 to 3. q is an integer from 1 to 11, preferably from 2 to 7. s is an integer from 1 to 3, preferably from 1. t is an integer from 1 to 11, preferably from 1 to 6.

[0076]

[0077] In equation (2-3), R Ｌ１ , R Ｌ２ At least one of them is a linear saturated aliphatic hydrocarbon group or an unsaturated aliphatic hydrocarbon group having four or more carbon atoms.ＬＹ′ R is a linear saturated aliphatic hydrocarbon group or an unsaturated aliphatic hydrocarbon group having 4 or more carbon atoms. p is an integer from 1 to 5, preferably from 1 to 3. q is an integer from 1 to 11, preferably from 2 to 7. In formula (2-3), R Ｌ１ , R Ｌ２ At least one of them preferably has 6 or more carbon atoms, and more preferably 7 or more. In formula (2-3), R Ｌ１ , R Ｌ２ The number of carbon atoms in at least one of the atoms is, for example, 20 or less, 18 or less, or 16 or less. Ｌ１ , R Ｌ２ Regarding the number of carbon atoms, the above upper and lower limits can be combined arbitrarily. Examples of combinations include 4 to 20, 6 to 18, and 7 to 16. In equation (2-3), R ＬＹ′ The carbon atoms preferably have 6 or more carbon atoms, and more preferably 7 or more carbon atoms. In formula (2-3), R ＬＹ′ The number of carbon atoms is, for example, 20 or less, 18 or less, or 16 or less. ＬＹ′ The above upper and lower limits for the number of carbon atoms can be combined in any way. Examples of combinations include 4 to 20, 6 to 18, and 7 to 16.

[0078]

[0079] In equation (2-4), p is an integer from 1 to 5, preferably from 1 to 3. q is an integer from 1 to 11, preferably from 2 to 7. r is an integer from 1 to 11, preferably from 5 to 9. s is an integer from 1 to 3, preferably from 1. t is an integer from 1 to 11, preferably from 1 to 6.

[0080]

[0081] In equation (2-5), R Ｌ１ , R Ｌ２ At least one of them is a linear saturated aliphatic hydrocarbon group or an unsaturated aliphatic hydrocarbon group having 4 or more carbon atoms. p is an integer from 1 to 5, preferably an integer from 1 to 3. q is an integer from 1 to 11, preferably an integer from 2 to 7. In formula (2-5), R Ｌ１ , RＬ２ At least one of them preferably has 6 or more carbon atoms, and more preferably 7 or more. In formula (2-5), R Ｌ１ , R Ｌ２ The number of carbon atoms in at least one of the atoms is, for example, 20 or less, 18 or less, or 16 or less. Ｌ１ , R Ｌ２ The above upper and lower limits for the number of carbon atoms can be combined in any way. Examples of combinations include 4 to 20, 6 to 18, and 7 to 16.

[0082]

[0083] In equation (2-6), R ＬＹ′ R is a linear saturated or unsaturated aliphatic hydrocarbon group having four or more carbon atoms. p is an integer from 1 to 5, preferably from 1 to 3. In formula (2-6), R ＬＹ′ The carbon atoms preferably have 6 or more carbon atoms, and more preferably 7 or more carbon atoms. In formula (2-6), R ＬＹ′ The number of carbon atoms is, for example, 20 or less, 18 or less, or 16 or less. ＬＹ′ The above upper and lower limits for the number of carbon atoms can be combined in any way. Examples of combinations include 4 to 20, 6 to 18, and 7 to 16.

[0084] Specific examples of compounds represented by formula (2) are shown below.

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] [Method for producing the compound represented by formula (2)] The compound represented by formula (2) can be produced by introducing the leg portion to the head portion of the compound via a spacer. The spacer has a structure that bridges the head portion and the leg portion.

[0108] Suitable compounds for the head portion include 3,7-diazabicyclo[3.3.1]nonane (bispirin; CAS number: 280-74-0), 9-oxa-3,7-diazabicyclo[3.3.1]nonane (CAS number: 329-96-4), 3,7-diazabicyclo[3.3.2]decane (CAS number: 347861-81-8), and 3,7-diazabicyclo[3.3.3]undecane (CAS number: 477881-56-4). In one embodiment of the present invention, suitable compounds for the head portion include 3,7-diazabicyclo[3.3.1]nonane (bispirin; CAS number: 280-74-0), 3,7-diazabicyclo[3.3.2]decane (CAS number: 347861-81-8), and 3,7-diazabicyclo[3.3.3]undecane (CAS number: 477881-56-4).

[0109] Examples of compounds that can act as spacers include compounds represented by formula (S)-1 or formula (S)-2 above.

[0110] [Method 11 for producing the compound represented by formula (2)] The compound represented by formula (2) can be produced by the following production method 11.

[0111]

[0112] In the above manufacturing method 11, R Ｂ１ ~R Ｂ５ The explanation regarding Ptg is the same as above. In the above manufacturing method 11, Y ｓ R is a divalent linking group which may have an oxygen atom. In the above manufacturing method 11, ＬＹ This is a linear or branched saturated aliphatic hydrocarbon group having 8 or more carbon atoms, which may have substituents, or a linear or branched unsaturated aliphatic hydrocarbon group having 8 or more carbon atoms, which may have substituents.

[0113] [Method 12 for producing the compound represented by formula (2)] The compound represented by formula (2) can be produced by the following production method 12.

[0114]

[0115] In the above manufacturing method 12, R Ｂ１ ~R Ｂ５ The explanation regarding Ptg and n2 is the same as above. In the above manufacturing method 12, Y ｓ R is a divalent linking group which may have an oxygen atom. In the above manufacturing method 12, ＬＹ This is a linear or branched saturated aliphatic hydrocarbon group having 4 or more carbon atoms, which may have substituents, or a linear or branched unsaturated aliphatic hydrocarbon group having 4 or more carbon atoms, which may have substituents.

[0116] [Method 13 for producing the compound represented by formula (2)] The compound represented by formula (2) can be produced by the following production method 13.

[0117]

[0118] In the above manufacturing method 13, R Ｂ１ ~R Ｂ５ The explanation for Ptg and n2 is the same as above. In the above manufacturing method 13, p is an integer from 1 to 5. In the above manufacturing method 13, R ＬＹ This is a linear or branched saturated aliphatic hydrocarbon group having 4 or more carbon atoms, which may have substituents, or a linear or branched unsaturated aliphatic hydrocarbon group having 4 or more carbon atoms, which may have substituents.

[0119] [Method 14 for producing the compound represented by formula (2)] The compound represented by formula (2) can be produced by the following production method 14.

[0120]

[0121] In the above manufacturing method 14, R Ｂ１ ~R Ｂ５ The explanation regarding Ptg and n2 is the same as above. In the above manufacturing method 14, Y ｓ R is a divalent linking group which may have an oxygen atom. In the above manufacturing method 14, ＬＹ１ , R ＬＹ２These are not identical, and each may have substituents, and are a linear or branched saturated aliphatic hydrocarbon group having 4 or more carbon atoms, or a linear or branched unsaturated aliphatic hydrocarbon group having 4 or more carbon atoms, and may have substituents.

[0122]

[0123] In the above manufacturing method 15, R Ｂ１ ~R Ｂ５ The explanations regarding Ptg and n2 are the same as above. In the above manufacturing method 15, p is an integer from 1 to 5. In the above manufacturing method 15, R ＬＹ１ , R ＬＹ２ These are not identical, and each may have substituents, and are a linear or branched saturated aliphatic hydrocarbon group having 4 or more carbon atoms, or a linear or branched unsaturated aliphatic hydrocarbon group having 4 or more carbon atoms, and may have substituents.

[0124] <Lipid Nanoparticles> One aspect of the present invention is lipid nanoparticles (LNPs) comprising nucleic acids and ionized lipids. The ionized lipids contained in the lipid nanoparticles according to one aspect of the present invention are the compound represented by formula (1) above or a salt thereof, or the compound represented by formula (2) above or a salt thereof. The lipid nanoparticles of the present invention (also referred to as LNPs of the present invention) should be interpreted as including all forms comprising nucleic acids and ionized lipids. For example, liposomes, lipid complexes, Liporex, etc., are within the scope of the interpretation of lipid nanoparticles.

[0125] The lipid component of the LNP of the present invention may include phospholipids and other lipids. Phospholipids that may be included in the LNP of the present invention include, for example, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine, CAS number: 816-94-4) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, CAS number: 4004-05- 1) 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC: 1,2-dilinooleoyl-sn-glycero-3-phosphocholine), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC: 1,2-dimyristoyl-sn-glycero-phosphocholine), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC: 1,2-dioleoyl-sn-glycero-3-p phosphocholine (CAS number: 4235-95-4), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, CAS number: 63-89-8), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC: 1,2-diundecanoyl-sn-glycero-phosphocholine), 1-palmitoyl-2-oleoyl -sn-glycero-3-phosphocholine (POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, CAS number: 26853-31-6), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChems PC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-Didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, 1,2-SOPC(1-Stearoyl-2-Oleoyl-sn-glycero-3-P Examples include, but are not limited to, C, CAS number: 56421-10-4), 1,2-SOPE (1-Stearoyl-2-Oleoyl-sn-glycero-3-PE, CAS number: 6418-95-7), DEPC (1,2-Dierucoyl-sn-glycero-3-PC, CAS number: 51779-95-4), 1,2-OPPC (1-Oleoyl-2-Palmitoyl-sn-glycero-3-PC, CAS number: 59491-62-2), and 1,2-SMPC (1-Stearoyl-2-Myristoyl-sn-glycero-3-PC, CAS number: 20664-02-2). In a preferred embodiment, the phospholipid may be DSPC, DOPE, DOPC, 1,2-DPPC, 1,2-SOPC, 1,2-SOPE, DEPC, 1,2-OPPC, 1,2-POPC, and 1,2-SMPC.

[0126] Furthermore, in one embodiment, the LNP of the present invention may contain lipids other than ionized lipids and phospholipids as constituent lipids of the LNP. Examples of such other lipids include PEG lipids and sterols.

[0127] In one embodiment, the LNP of the present invention may further contain PEG lipids (also referred to as "PEG-modified lipids"). In this specification, "PEG lipid" means any lipid modified with a PEG (polyethylene glycol) group. Examples of PEG lipids that may be included in the LNP of the present invention include 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-5000 (DMG-PEG5000), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG2000), and Cremophore (CREMOPHOR Examples include, but are not limited to, polyoxyethylene sorbitan monooleate (EL), etc. In a preferred embodiment, the PEG lipid may be DMG-PEG5000, DMG-PEG2000, and ALC-0159. PEG lipids as constituent lipids of LNPs play a role in preventing aggregation between particles and improving blood stability. In the present invention, other PEG substitute lipids that perform the same role and are modified with groups such as polysarcosine, polyoxazoline, and polyvinylpyrrolidone may be used.

[0128] In one embodiment, the LNP of the present invention may contain sterols (also referred to as "steroid alcohols"). In this specification, "sterol" means a subgroup of steroids that can be produced by plants, animals, or fungi. Examples of sterols that may be included in the LNP of the present invention include, but are not limited to, cholesterol, ergosterol, campesterol, oxysterol, antrosterol, desmosterol, nicasterol, sitosterol, and stigmasterol. In one preferred embodiment, the sterol may be cholesterol.

[0129] When the LNP of the present invention contains ionized lipids, phospholipids, sterols, and PEG lipids, the content ratio of each lipid component to the total amount of constituent lipids in the LNP may be as follows: The lower limit of the content ratio of ionized lipids may be 5 mol% or more, 6 mol% or more, 7 mol% or more, 8 mol% or more, 9 mol% or more, 10 mol% or more, 15 mol% or more, 20 mol% or more, 25 mol% or more, 30 mol% or more, 35 mol% or more, 40 mol% or more, or 50 mol% or more. The upper limit of ionized lipids may be 75 mol% or less, 70 mol% or less, 65 mol% or less, 60 mol% or less, 55 mol% or less, 50 mol% or less, 45 mol% or less, 40 mol% or less, 35 mol% or less, 30 mol% or less, 25 mol% or less, 20 mol% or less, 15 mol% or less, or 10 mol% or less. The ionized lipid content is preferably 5 to 75 mol%, more preferably 10 to 65 mol%, and even more preferably 10 to 60 mol%. The lower limit of the phospholipid content may be 5 mol% or more, 6 mol% or more, 7 mol% or more, 8 mol% or more, 9 mol% or more, 10 mol% or more, 15 mol% or more, 20 mol% or more, 25 mol% or more, 30 mol% or more, or 35 mol% or more. The upper limit of the phospholipid content may be 40 mol% or less, 35 mol% or less, 30 mol% or less, 25 mol% or less, 20 mol% or less, 15 mol% or less, or 10 mol% or less. The phospholipid content is preferably 5 to 40 mol%, more preferably 5 to 30 mol%. The lower limit of the sterol content can be 15 mol% or more, 20 mol% or more, 25 mol% or more, 30 mol% or more, 35 mol% or more, 40 mol% or more, 45 mol% or more, 50 mol% or more, 55 mol% or more, 60 mol% or more, 65 mol% or more, 70 mol% or more, or 75 mol% or more. The upper limit of the sterol content can be 90 mol% or less, 85 mol% or less, 80 mol% or less, 75 mol% or less, 70 mol% or less, 65 mol% or less, 60 mol% or less, 55 mol% or less, 50 mol% or less, 45 mol% or less, 40 mol% or less, 35 mol% or less, 30 mol% or less, 25 mol% or less, or 20 mol% or less. Preferably, the sterol content is 15 to 90 mol%, more preferably 20 to 90 mol%.The lower limit of the PEG lipid content ratio may be 0.5 mol% or more, 1.0 mol% or more, 1.5 mol% or more, 2.0 mol% or more, 2.5 mol% or more, 3.0 mol% or more, 3.5 mol% or more, or 4.0 mol% or more. The upper limit of the PEG lipid content ratio may be 5.0 mol% or less, 4.5 mol% or less, 4.0 mol% or less, 3.5 mol% or less, 3.0 mol% or less, 2.5 mol% or less, 2.0 mol% or less, 1.5 mol% or less, or 1.0 mol% or less. Preferably, the PEG lipid content ratio is 0.5 to 5 mol%, more preferably 0.5 to 4 mol%.

[0130] Furthermore, in one embodiment, the LNP of the present invention may contain components other than nucleic acids and lipids. Examples of such components include, but are not limited to, surfactants, hyaluronic acid, or derivatives thereof.

[0131] In one embodiment, the LNP of the present invention may contain a surfactant. Examples of surfactants that may be included in the LNP of the present invention include, but are not limited to, polyoxyethylene sorbitan monooleate (e.g., polysorbate 80), polyoxyethylene polyoxypropylene glycol (e.g., Pluronic® F68), sorbitan fatty acid esters (e.g., sorbitan monolaurate, sorbitan monooleate), polyoxyethylene derivatives (e.g., polyoxyethylene hydrogenated castor oil 60, polyoxyethylene lauryl alcohol), glycerin fatty acid esters, or polyethylene glycol alkyl ethers. In one preferred embodiment, the surfactant included in the LNP of the present invention is polyoxyethylene polyoxypropylene glycol, glycerin fatty acid ester, or polyethylene glycol alkyl ether.

[0132] In one embodiment, the LNP of the present invention may further contain hyaluronic acid or a derivative thereof in addition to nucleic acids and lipids. Examples of hyaluronic acid derivatives include compounds obtained by dehydrating and condensing hyaluronic acid on the hydroxyl group of fatty acid glyceryl.

[0133] In one embodiment, the average particle diameter of the LNP of the present invention may have average particle diameters of about 30 nm to about 250 nm, about 30 nm to about 200 nm, about 30 nm to about 170 nm, about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, and about 70 nm to about 80 nm. In a preferred embodiment, the average particle diameter of the LNP of the present invention may be about 30 nm to about 200 nm.

[0134] Where used herein, the term "approximately" refers to a value similar to the reference value when applied to the value of interest. The term "approximately" means a range of values ​​that fall within ±10% of the reference value.

[0135] In one embodiment, the lower limit of the polydispersity index (PDI) of the LNP of the present invention may be 0.001 or higher, but is not limited thereto. The upper limit of the PDI of the LNP of the present invention is preferably 0.35 or lower, more preferably 0.30 or lower, even more preferably 0.20 or lower, even more preferably 0.18 or lower, and also preferably 0.12 or lower.

[0136] In the present invention and this specification, the average particle size of LNP refers to the volume-based D50 particle size (nm) measured by dynamic light scattering. The PDI of LNP is calculated from the particle size distribution measured by dynamic light scattering.

[0137] <Method for Producing Lipid Nanoparticles> The method for producing lipid nanoparticles according to this embodiment (hereinafter sometimes referred to as "the method for producing the present invention") is a method for producing LNPs of the present invention that include at least ionized lipids, phospholipids, sterols, and PEG lipids as constituent lipids, and comprises the following steps: a step of preparing a lipid solution containing ionized lipids, phospholipids, sterols, and PEG lipids; a step of preparing a nucleic acid solution containing nucleic acids; and a step of mixing the lipid solution and the nucleic acid solution.

[0138] The ionized lipids, phospholipids, sterols, and PEG lipids used are those incorporated into the LNP of the present invention as described above.

[0139] In the production method of the present invention, first, a lipid solution containing ionized lipids, phospholipids, sterols, and PEG lipids is prepared. Specifically, ionized lipids, phospholipids, sterols, and PEG lipids are added to an organic solvent in a desired molar ratio. The organic solvent is not particularly limited as long as it is an organic solvent capable of dissolving all of the ionized lipids, phospholipids, sterols, and PEG lipids. Examples of such organic solvents include alcoholic solvents such as methanol, ethanol, propanol, isopropanol, and butanol; ester-based solvents such as ethyl acetate and butyl acetate; ketone-based solvents such as acetone, methyl ethyl ketone, and methyl isobutyl ketone; and chlorine-based solvents such as chloroform. Alternatively, a mixed solvent of two or more of these organic solvents, or a mixed solvent of one or more of these organic solvents with water, may also be used. In the production method of the present invention, from the standpoint of relatively high safety for living organisms, an alcoholic solvent or a mixed solvent of water and an alcoholic solvent is preferred, ethanol or a mixed solvent of ethanol and water is more preferred, and ethanol is particularly preferred.

[0140] Furthermore, the nucleic acid solution in the production method of the present invention can be prepared by mixing a nucleic acid such as DNA or RNA with an aqueous solvent capable of dissolving the nucleic acid. The aqueous solvent is not particularly limited as long as it can dissolve the nucleic acid, and any solvent may be used. Examples of aqueous solvents include water and buffer solutions (e.g., citrate buffer solution, acetate buffer solution, etc.), but are not limited to these. In one preferred embodiment, the aqueous solvent is an acetate buffer solution.

[0141] In one embodiment, the pH of the aqueous solvent used in the manufacturing method of the present invention is usually 3.0 to 10.0, preferably 3.0 to 8.0, more preferably 3.0 to 7.0, and even more preferably 3.5 to 5.5.

[0142] In the manufacturing method of the present invention, the mixing of the lipid solution and the nucleic acid solution may be carried out by any means as long as the lipid solution and the nucleic acid solution are uniformly mixed. Examples of means for mixing the lipid solution and the nucleic acid solution include, but are not limited to, pipetting and microfluidic mixing devices. In a preferred embodiment, a microfluidic mixing device is used in the manufacturing method of the present invention.

[0143] In one embodiment, the lipid solution and the nucleic acid solution are mixed using a microfluidic mixing device. Using a microfluidic mixing device allows for rapid and thorough mixing of the lipid solution and the nucleic acid solution. The microfluidic mixing device used in the manufacturing method of the present invention may be any device capable of mixing the lipid solution and the nucleic acid solution, thereby producing the desired LNPs. One example is, but is not limited to, the NanoAssemblr (manufactured by Precision Nanosystems).

[0144] In microfluidic mixing, rapid and thorough mixing is typically achieved by bringing multiple sample solutions into contact at different flow rates, thereby enhancing the diffusion effect between the flows of the sample solutions. The flow rate ratio between the two solutions can affect the size of the LNPs prepared. In one embodiment, in the mixing of a lipid solution and a nucleic acid solution, the flow rate ratio of the lipid solution to the nucleic acid solution ([flow rate of lipid solution (mL / min)] : [flow rate of nucleic acid solution (mL / min)]) is typically 1:10 to 10:1, preferably 1:2 to 3:1, but is not limited to these values.

[0145] In one embodiment, the mixture of lipid solution and nucleic acid solution mixed by a microfluidic mixing device may be subjected to downsizing or purification as needed. Such downsizing or purification can be carried out using known methods such as ultrafiltration or filtration using filters.

[0146] <Pharmaceutical composition containing lipid nanoparticles> One aspect of the present invention provides a pharmaceutical composition containing the LNP of the present invention (hereinafter sometimes referred to as "the pharmaceutical composition of the present invention").

[0147] The amount of LNP of the present invention included in the pharmaceutical composition of the present invention is not particularly limited. The lower limit of the amount of LNP of the present invention included in the pharmaceutical composition of the present invention is usually 0.01% by mass or more, preferably 0.1% by mass or more, 0.5% by mass or more, 1.0% by mass or more, 1.5% by mass or more, 2.0% by mass or more, 2.5% by mass or more, 3.0% by mass or more, 3.5% by mass or more, 4.0% by mass or more, 4.5% by mass or more, 5.0% by mass or more, 5.5% by mass or more, 6.0% by mass or more, 6.5% by mass or more, 7.0% by mass or more, 7.5% by mass or more, 8.0% by mass or more, 8.5% by mass or more, 9.0% by mass or more, or 9.5% by mass or more, but is not limited to these. Furthermore, the upper limit of the blending amount is usually 100% by mass or less, and preferably may be 99.9% by mass or less, 99.0% by mass or less, 95.0% by mass or less, 90.0% by mass or less, 85.0% by mass or less, 80.0% by mass or less, 75.0% by mass or less, 70.0% by mass or less, 65.0% by mass or less, 60.0% by mass or less, 55.0% by mass or less, 50.0% by mass or less, 45.0% by mass or less, 40.0% by mass or less, 35.0% by mass or less, 30.0% by mass or less, 25.0% by mass or less, 20.0% by mass or less, 15.0% by mass or less, or 10.0% by mass or less, but is not limited to these.

[0148] The pharmaceutical composition of the present invention may contain components other than the LNP of the present invention. Examples of such components include pharmaceutically acceptable carriers. A pharmaceutically acceptable carrier generally means an inert and non-toxic solid or liquid filler, diluent, or encapsulating material that does not react with the active ingredient. Examples of pharmaceutically acceptable carriers used in the pharmaceutical composition of the present invention include, but are not limited to, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and mixtures thereof. Furthermore, in order to bring the physical properties of the pharmaceutical composition of the present invention closer to physiological conditions, the pharmaceutical composition of the present invention may contain pharmaceutically acceptable auxiliary substances. Examples of such auxiliary substances include, but are not limited to, pH adjusters, buffers, tension adjusters, wetting agents, etc.

[0149] The pharmaceutical composition of the present invention may be administered orally or parenterally. Parenteral administration may include, but is not limited to, transdermal, subcutaneous, intravenous, intra-arterial, intramuscular, intraperitoneal, vaginal, and intranasal administration. The number of administrations may be a single dose or multiple doses.

[0150] The target population to which the pharmaceutical composition of the present invention can be applied is not particularly limited. Examples of target populations to which the pharmaceutical composition of the present invention can be applied include, but are not limited to, mammals (humans, chimpanzees, dogs, cats, horses, cattle, sheep, goats, rats, mice, rabbits, pigs, etc.). In a preferred embodiment, the target population may be humans.

[0151] The present invention will be described in more detail in the following examples, but the present invention is not limited in any way by these examples.

[0152] Unless otherwise specified, silica gel 60N (63-210 μm) manufactured by Kanto Kagaku Co., Ltd. was used as the support for silica gel column chromatography. NMR spectra were measured using tetramethylsilane (0 ppm) as an internal reference, with the total δ values ​​expressed in ppm, using an AVANCE NEO400 (Bruker). MS spectra were measured using a JMS-S3000 (JEOL).

[0153] <Synthesis Example 1> First, compound 3 (1-1.2,2'-(3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)bis(ethan-1-amine)) was synthesized. Compound 1 (3,7-diazabicyclo[3.3.1]nonane) was synthesized using the method described in Tetrahedron Letters 53 (2012) 623-626.

[0154] 0.5 g (4 mmol) of compound 1 and 1.8 g (8 mmol) of 2-(tert-Butoxycarbonylamino)ethyl Bromide were dissolved in 10 mL of acetonitrile, and 1.1 g (10 mmol) of sodium carbonate was added. The mixture was reacted at room temperature for 16 hours. The remaining sodium carbonate was removed by filtration, and the crude reaction solution was concentrated under vacuum. After purification by column chromatography (chloroform / methanol = 85 / 15), 1 g (2.5 mmol, 62%) of compound 2 was obtained. Subsequently, 1 g (2.5 mmol) of compound 2 was dissolved in 10 mL of dichloromethane, and 1.9 mL (25 mmol) of TFA was added. The mixture was reacted at room temperature for 16 hours. After concentrating the reaction solution, it was neutralized with saturated saline solution and 5 N sodium hydroxide aqueous solution, and then extracted with chloroform. The organic layer was separated, dehydrated with sodium sulfate, and concentrated to obtain 0.5 g (2.5 mmol) of compound 3. This reaction is shown in (RF)-1 below.

[0155]

[0156] 1-2. Nonyl 5-bromopentanoate was synthesized according to the synthesis method for nonyl 8-bromooctanoate described in the Supplemental Information of Molecular Therapy, 2018, 26(6), 509. This reaction is shown in (RF)-2 below.

[0157]

[0158] 212 mg (1 mmol) of compound 3 and 1.8 g (5.8 mmol) of nonyl 5-bromopentanoate were dissolved in 10 ml of acetonitrile, and 1.4 g (10 mmol) of sodium carbonate and a small amount of potassium iodide were added. The mixture was heated under reflux for 19 hours. After cooling to room temperature, the sodium carbonate was filtered off, and the reaction mixture was concentrated. The resulting crude solution was purified by column chromatography (chloroform / methanol = 9 / 1) to obtain 72.4 mg (0.65 mmol, 6%) of compound 4 (tetranonyl 5,5',5'',5'''-(((3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)bis(ethane-2,1-diyl))bis(azanetriyl))tetrapentanoate). This reaction is shown in (RF)-3 below. The NMR results of compound 4 are as follows. 1 H NMR (400 MHz, CDCl3) δppm 4.04 (m, 8H), 3.50-2.25 (br, 34H), 1.90 (br, 2H), 1.75-1.25(br, 72H), 0.89(m, 12H) MALDI-TOF MS [M+Na]: m / z calcd for C67H128N4O8Na 1139.9624,found 1140.0144

[0159]

[0160] <Synthesis Example 2> First, compound 6 (2-1. 1,1'-(3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)bis(2-aminoethan-1-one)) was synthesized. 0.5 g (4 mmol) of compound 1, 1.8 g (10 mmol) of N-(tert-Butoxycarbonyl)glycine, 1.9 g (10 mmol) of 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride (EDC・HCl), 5.2 g (40 mmol) of N,N-Diisopropylethylamine (DIPEA), and 0.24 g (2 mmol) of 4-Dimethyl aminopyridine (DMAP) were dissolved in 20 mL of dichloromethane and reacted at room temperature for 20 hours. After neutralizing the reaction mixture with saturated saline and 2N hydrochloric acid, the organic layer was separated. After concentrating the organic layer, it was purified by column chromatography (chloroform / methanol = 9 / 1) to obtain 1.1 g (2.6 mmol, 65%) of compound 5. Subsequently, 1.1 g (2.6 mmol) of compound 5 was dissolved in 10 mL of dichloromethane, 2 mL (26 mmol) of TFA was added, and the mixture was reacted at room temperature for 4 hours. After concentrating the reaction mixture, saturated saline solution and 5 N sodium hydroxide aqueous solution were added to neutralize the reaction mixture, and then chloroform was added for extraction. The organic layer was separated, dehydrated with sodium sulfate, and concentrated to obtain 106 mg (0.4 mmol, 17%) of compound 6. This reaction is shown in (RF)-4 below.

[0161]

[0162] Using 106 mg (0.44 mmol) of compound 6, 290 mg (0.25 mmol, 58%) of compound 7 (tetranonyl 5,5',5'',5''-(((-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)bis(2-oxoethane-2,1-diyl))bis(azanetriyl))tetrapentanoate) was synthesized using the same procedure as in (RF)-3 above. This reaction is shown in (RF)-5 below. The NMR measurement results for compound 7 are as follows. 1H NMR (400 MHz, CDCl3) δppm 4.78, 4.16 (dd, 4H), 4.05 (m, 8H), 3.57, 3.33, 2.97, 2.81 (dd, 8H), 2.55-1.80 (br, 20H), 1.70-1.20 (br, 72H), 0.89(m, 12H)MALDI-TOF MS [M+Na]: m / z calcd for C67H124N4O10Na 1167.9210, found 1167.9371

[0163]

[0164] <Synthesis Example 3> First, compound 10 (3-1. 2-(3,7-diazabicyclo[3.3.1]nonan-3-yl)ethan-1-amine) was synthesized. Compound 8 (tert-Butyl 3,7-diazabicyclo[3.3.1]nonane-3-carboxylate, CAS NO. 227940-72-9) was purchased from ChemScene LLC as a reagent from Sigmaaldrich and used. 4.5 g (20 mmol) of compound 8 and 4.5 g (20 mmol) of 2-(tert-Butoxycarbonylamino)ethyl Bromide were dissolved in 30 ml of acetonitrile, and 3.2 g (30 mmol) of sodium carbonate was added, and the mixture was heated and stirred at 40°C for 16 hours. The reaction mixture was cooled to room temperature, the remaining sodium carbonate was removed by filtration, and the crude reaction solution was concentrated under vacuum. It was then purified by column chromatography (chloroform / methanol = 95 / 5) to obtain 6.7 g (18 mmol, 90%) of compound 9. Subsequently, 185 mg (0.5 mmol) of compound 9 was dissolved in 5 mL of dichloromethane, 0.38 mL (5 mmol) of TFA was added, and the mixture was reacted at room temperature for 16 hours. After concentrating the reaction mixture, it was neutralized with saturated saline solution and 5N sodium hydroxide aqueous solution, and then extracted with chloroform. The organic layer was separated, dehydrated with sodium sulfate, and concentrated to obtain 80 mg (0.49 mmol) of compound 10. This reaction is shown in (RF)-6 below.

[0165]

[0166] 80 mg (0.49 mmol) of compound 10 and 368 mg (2 mmol) of 1,2-Epoxydodecane were dissolved in 10 ml of ethanol, and 202 mg (2 mmol) of triethylamine was added. The mixture was heated at 80°C for 16 hours with stirring. After cooling to room temperature, the reaction mixture was concentrated. The resulting crude solution was purified by column chromatography (chloroform / methanol = 85 / 15) to obtain 169 mg (0.23 mmol, 47%) of compound 11 (1,1'-((2-(7-(2-hydroxydodecyl)-3,7-diazabicyclo[3.3.1]nonan-3-yl)ethyl)azanediyl)bis(dodecan-2-ol)). This reaction is shown in (RF)-7 below. The NMR results of compound 11 are as follows. 1H NMR (400 MHz, CDCl3) δppm 4.00-2.00 (br, 23H), 1.75 (br, 2H), 1.50-1.20 (br, 54H), 0.88(m, 9H) MALDI-TOF MS [M+H]: m / z calcd for C45H92N3O3 721.7133,found 721.6721

[0167]

[0168] <Synthesis Example 4> First, heptadecan-9-yl 5-bromopentanoate was synthesized according to the synthesis method for heptadecan-9-yl 8-bromooctanoate described in the Supplemental Information of Molecular Therapy, 2018, 26(6), 509. This reaction is shown in (RF)-8 below.

[0169]

[0170] 85 mg (0.5 mmol) of compound 10 and 622 mg (1.5 mmol) of heptadecan-9-yl 5-bromopentanoate were dissolved in 5 ml of acetonitrile and 5 ml of tetrahydrofuran, and 318 mg (3 mmol) of sodium carbonate was added. The mixture was heated under reflux for 16 hours. After cooling to room temperature, the sodium carbonate was filtered off, and the reaction mixture was concentrated. The resulting crude solution was purified by column chromatography (chloroform / methanol = 95 / 5) to obtain 243 mg (0.2 mmol, 41%) of compound 12 (di(heptadecan-9-yl) 5,5'-((2-(7-(5-(heptadecan-9-yloxy)-5-oxopentyl)-3,7-diazabicyclo[3.3.1]nonan-3-yl)ethyl)azanediyl)dipentanoate). This reaction is shown in (RF)-9 below. The NMR measurement results for compound 12 are as follows: ¹H NMR (400 MHz, CDCl3) δppm 4.84 (m, 3H), 4.70-2.75 (br, 12H), 2.50-1.80 (br, 12H), 1.75-1.10 (br, 100H), 0.88 (m, 18H) MALDI-TOF MS [M+H]: m / z calcd for C75H146N3O6 1185.1206, found 1185.0507

[0171]

[0172] <Synthesis Example 5> First, compound 14 (1,1'-(3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)bis(3-aminopropan-1-one)) was synthesized. Using 0.5 g (4 mmol) of compound 1, 1.60 g (3.4 mmol, 85%) of compound 13 was obtained using the same procedure as for compound 5, except that N-(tert-Butoxycarbonyl)-β-alanine was used instead of N-(tert-Butoxycarbonyl)glycine. Next, using 468 mg (1.0 mmol) of compound 13, 268 mg (1.0 mmol, 100%) of compound 14 was obtained using the same procedure as for compound 6. This reaction is shown in (RF)-10 below.

[0173]

[0174] Using 268 mg (1.0 mmol) of compound 14, 591 mg (0.50 mmol, 50%) of compound 15 was synthesized using the same procedure as in (RF)-5 above. This reaction is shown in (RF)-11 below. The NMR measurement results for compound 15 are as follows. 1 H NMR (400 MHz, CDCl3) δppm 4.80,(d, 2H), 4.05 (t, 8H),4.00(d,2H), 3.29(d, 2H),2.79(d,4H) 2.72-2.32 (br, 14H),2.31(t,8H) ,1.92(d,4H),1.70-1.20 (br, 72H), 0.89(t, 12H) MALDI-TOF MS [M+Na]: m / z calcd for C69H128N4O10Na 1195.9523, found 1195.8949

[0175]

[0176] <Synthesis Example 6> First, compound 17 (1,1'-(3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)bis(4-aminobutane-1-one)) was synthesized. Using 0.5 g (4 mmol) of compound 1, 1.50 g (3.0 mmol, 75%) of compound 16 was obtained using the same procedure as for compound 5, except that N-(tert-Butoxycarbonyl)-4-aminobutyric acid was used instead of N-(tert-Butoxycarbonyl)glycine. Next, using 497 mg (1.0 mmol) of compound 16, 296 mg (1.0 mmol, 100%) of compound 17 was obtained using the same procedure as for compound 6. This reaction is shown in (RF)-12 below.

[0177]

[0178] Using 296 mg (1.0 mmol) of compound 17, 629 mg (0.50 mmol, 50%) of compound 18 was synthesized using the same procedure as in (RF)-5 above. This reaction is shown in (RF)-13 below. The NMR measurement results for compound 18 are as follows. 1 H NMR (400 MHz, CDCl3) δppm 4.79(d,2H), 4.05(t,8H),3.99 (d, 2H),3.29(d, 2H),2.81(d,4H) 2.72-2.32 (br, 22H),1.93(d,4H),1.70-1.20 (br, 76H), 0.89(m, 12H) MALDI-TOF MS [M+K]: m / z calcd for C71H132N4O10K 1239.9575, found 1239.9988

[0179]

[0180] <Synthesis Example 7> Using 240 mg (1.0 mmol) of compound 6, compound 19 was synthesized in 241 mg (0.15 mmol, 6.7%) using the same procedure as in (RF)-5, except that heptadecan-9-yl 5-bromopentanoate was used instead of nonyl 5-bromopentanoate. This reaction is shown in (RF)-14 below. The NMR measurement results for compound 19 are as follows. 1 H NMR (400 MHz, CDCl3) δppm 4.85 (t, 4H), 4.78(d,2H), 4.17(d,2H) 3.58(d,2H),3.33(d,2H),2.96(d,2H),2.80(d,2 H),2.47(m,4H),2.35(m,4H),2.26(t,8H),1.85-1.95(br,4H),1.68-1.18 (br, 128H), 0.88(t, 24H) MALDI-TOF MS [M+H]: m / z calcd for C99H189N4O10 1595.4432, found 1594.3438

[0181]

[0182] <Synthesis Example 8> cis-2-Nonenyl 5-bromopentanoate was synthesized using the same procedure as in (RF)-8, except that cis-2-nonen-1-ol was used instead of 9-nonanol. This reaction is shown in (RF)-15 below.

[0183]

[0184] Compound 20 was synthesized in 552 mg (0.49 mmol, 49%) using 240 mg (1.0 mmol) of compound 6, following the same procedure as in (RF)-5, except that cis-2-Nonenyl 5-bromopentanoate was used instead of nonyl 5-bromopentanoate. This reaction is shown in (RF)-16 below. The NMR results of compound 20 are as follows. 1 H NMR (400 MHz, CDCl3) δppm 5.63 (m, 4H), 5.51(m,4H),4.77(d,2H)4.61(d,8H),4.14(d,2H),3.56,2.96(dd,4H),3.32,2.79(dd, 4H),2.47(m,4H),2.35(m,4H),2.29(t,8H),2.09(q,8H),1.85-1.95(br,4H),1.68-1.18 (br, 48H), 0.88(t, 12H) MALDI-TOF MS [M+H]: m / z calcd for C67H117N4O10 1137.8764, found 1137.7794

[0185]

[0186] Compound 21 was synthesized in 327 mg (0.28 mmol, 28%) using 268 mg (1.0 mmol) of compound 14, following the same procedure as in (RF)-11, except that cis-2-Nonenyl 5-bromopentanoate was used instead of nonyl 5-bromopentanoate. This reaction is shown in (RF)-17 below. The NMR results of compound 20 are as follows. 1H NMR (400 MHz, CDCl3) δppm 5.62 (m, 4H), 5.51 (m, 4H), 4.80 (d, 2H) 4.61 (d, 8H), 3.99 (d, 2H), 3.28 (d, 2H), 2.79 (d, 2H), 2.7 4(m,2H),2.60(m,2H),2.35(m,20H),2.00(q,8H),1.85-1.95(br,4H),1.68-1.18 (br, 48H), 0.88(t, 12H) MALDI-TOF MS [M+H]: m / z calcd for C69H121N4O10 1165.9077, found 1165.8164

[0187]

[0188] <Synthesis Example 9> First, compound 22 (N-[(2-Nitrophenyl)sulfonyl]-β-alanine) was synthesized. 4.45 g (50 mmol) of β-alanine and 59.5 g (460 mmol) of N,N-Diisopropylethylamine (DIPEA) were dissolved in 250 mL of a mixed solvent of THF and 250 mL of water, and the mixture was stirred at 0°C for 5 minutes. A solution of 12.9 g (55 mmol) of 2-Nitrobenzenesulfonyl Chloride dissolved in 50 mL of THF was added dropwise to the mixed solution, and the mixture was reacted at 0°C for 1 hour and then at 25°C for 1 hour. The resulting reaction mixture was concentrated using a rotary evaporator, and then separated by adding 300 mL of chloroform and 100 mL of 1N hydrochloric acid. The organic layer was dried over sodium sulfate, concentrated using a rotary evaporator, and purified by column chromatography with chloroform:methanol = 9:1 (Vol) as the developing solvent to obtain a white powder. The obtained white powder was further purified by chloroform recrystallization to obtain 5.91 g (21.5 mmol, 43%) of compound 22. This reaction is shown in (RF)-18 below.

[0189]

[0190] Using 1.01 g (8 mmol) of compound 1, compound 23 was obtained in 3.55 g (5.6 mmol, 70%) using the same procedure as for compound 5, except that compound 22 was used instead of N-(tert-Butoxycarbonyl)glycine. Subsequently, compound 24 was synthesized in 876 mg (0.67 mmol, 67%) using 0.639 g (1 mmol) of compound 23, except that potassium carbonate was used instead of sodium carbonate, using the same procedure as for (RF)-14. This reaction is shown in (RF)-19 below.

[0191]

[0192] 789 mg (0.6 mmol) of compound 24, 267 mg (1.32 mmol) of 1-Dodecanethiol, and 1.17 g (3.6 mmol) of cesium carbonate were dissolved in 10 mL of acetonitrile and reacted at 50°C for 16 hours. The resulting reaction solution was concentrated using a rotary evaporator and then purified by column chromatography with chloroform:methanol = 90:10 (vol) as the developing solvent to obtain 321 mg (0.34 mmol, 57%) of compound 25. This reaction is shown in (RF)-20 below.

[0193]

[0194] Using 142 mg (0.15 mmol) of compound 25, 37.7 mg (0.027 mmol, 18%) of compound 26 was synthesized using the same procedure as in (RF)-13. This reaction is shown below in (RF)-21. The NMR measurement results for compound 26 are as follows. 1 H NMR (400 MHz, CDCl3) δppm 4.83 (m, 4H), 4.04(t,4H), 3.99(d,2H) 3.29(d,2H),2.82-2.20(br,26H),2.08-1.87(br,4H),1.74-1.20 (br, 100H), 0.88(t, 18H) MALDI-TOF MS [M+Na]: m / z calcd for C85H160N4O10Na 1420.2027, found 1420.0941

[0195]

[0196] <Synthesis Example 10> First, compound 28 (3-1. 2-(3,7-diazabicyclo[3.3.1]nonan-3-yl)propan-1-amine) was synthesized. Using 0.905 g (4 mmol) of compound 8, 0.816 g (2 mmol, 50%) of compound 27 was obtained using the same procedure as for compound 9, except that 5-(tert-Butoxycarbonylamino)propyl Bromide was used instead of 2-(tert-Butoxycarbonylamino)ethyl Bromide. Subsequently, using 0.411 g (1 mmol) of compound 27, 0.150 g (0.71 mmol, 71%) of compound 28 was obtained using the same procedure as for compound 10. This reaction is shown in (RF)-22 below.

[0197]

[0198] Using 106 mg (0.5 mmol) of compound 28, 46.5 mg (0.06 mmol, 12%) of compound 29 was synthesized using the same procedure as in (RF)-7. This reaction is shown in (RF)-23 below. The NMR measurement results for compound 29 are as follows. 1 H NMR (400 MHz, CDCl3) δppm 4.00-2.00 (br, 21H), 1.75-1.90(br,4H),1.70-1.15 (br, 60H), 0.88(t, 9H) MALDI-TOF MS [M+H]: m / z calcd for C48H98N3O3 764.7603,found 764.6900

[0199]

[0200] Nonyl 4-bromobutanoate, Nonyl 6-bromohexanoate, cis-2-Nonenyl 4-bromobutanoate, and cis-2-Nonenyl 6-bromohexanoate were synthesized using the same procedure as (RF)-2 and (RF)-15, except that 4-bromobutyric acid and 6-bromohexanoic acid were used as starting materials.

[0201] Compounds 30, 31, 32, and 33 were synthesized using the same procedure as in (RF)-17, except that Nonyl 4-bromobutanoate, Nonyl 6-bromohexanoate, cis-2-Nonenyl 4-bromobutanoate, and cis-2-Nonenyl 6-bromohexanoate were used as starting materials, with compound 14 as the starting material. This reaction is shown in (RF)-24 below.

[0202]

[0203] [Test Example 1] Synthetic compounds 4, 7, and 11, and commercially available lipid 244cis (4-[2-[bis[9-[(2Z)-2-nonen-1-yloxy]-9-oxononyl]amino]ethyl]-1-piperazinenonanoic acid, (2Z)-2-nonen-1-yl ester, CAS number: 2956402-64-3) were used as ionized lipids, DSPC (1,2-distearoyl-sn-glycero-3-phosphatidylcholine, CAS number: 816-94-4) was used as the phospholipid, cholesterol was used as the sterol, and DMG-PEG2000 was used as the PEG lipid to prepare LNPs containing mRNA, and their physical properties and cellular activity were investigated. The mRNA used was the mRNA encoding the luciferase protein FLuc (SEQ ID NO: 1).

[0204] [Test Example 2] As ionized lipids, synthesized compound 4, compound 7, compound 11, and commercially available lipid 244cis(4-[2-[bis[9-[(2Z)-2-nonen-1-yloxy]-9-oxononyl]amino]ethyl]-1-piperazinenonanoic acid, (2Z)-2-nonen-1-yl Using ester, CAS number: 2956402-64-3, the phospholipids used were DSPC (1,2-distearoyl-sn-glycero-3-phosphatidylcholine, CAS number: 816-94-4), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, CAS number: 4004-05-1), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine, CAS number: 4235-95-4), 1,2-DPPC (1,2-Dipalmitoyl-sn-glycero-3-PC, CAS number: 63-89-8), 1,2-SOPC (1-Stearoyl-2-Oleoyl-sn-glycero-3-PC, CAS number: 56421-10-4), and 1,2-SOPE (1-Ste aroyl-2-Oleoyl-sn-glycero-3-PE, CAS number: 6418-95-7), DEPC (1,2-Dierucoyl-sn-glycero-3-PC, CAS number: 517) 79-95-4), 1,2-OPPC (1-Oleoyl-2-Palmitoyl-sn-glycero-3-PC, CAS number: 59491-62-2) 1,2-POPC (1-Palmitoyl- LNPs encapsulating mRNA were prepared using 2-Oleoyl-sn-glycero-3-PC (CAS number: 26853-31-6) and 1,2-SMPC (1-Stearoyl-2-Myristoyl-sn-glycero-3-PC, CAS number: 20664-02-2), cholesterol as the sterol, and DMG-PEG2000 as the PEG lipid, and their physical properties and cellular activity were investigated. The mRNA used was the mRNA encoding the luciferase protein FLuc (SEQ ID NO: 1).

[0205] (Preparation of LNPs) A lipid mixture was prepared by mixing ionized lipids, phospholipids, cholesterol, and PEG lipids in a ratio of 50:10:38.5:1.5 mol%, respectively. This mixture was dissolved in 99.5% ethanol to obtain a 20 mmol / L lipid solution. In addition, mRNA encoding FLuc was dissolved in acetate buffer (25 mM sodium acetate, pH 4) to obtain a nucleic acid solution. The concentration of the nucleic acid solution was adjusted so that the NP ratio (ratio of amino groups in ionized lipids to phosphate groups in nucleic acids) was 6.

[0206] The obtained lipid solution and nucleic acid solution were mixed by pipetting at room temperature in a volume ratio of 1:2 to obtain a dispersion. The obtained dispersion was diluted 20-fold with D-PBS(-) buffer (Fujifilm Wako Pure Chemical Industries, Ltd.) and subjected to centrifugal ultrafiltration using a centrifugal filter unit (Amicon Ultra-4, Millipore). Subsequently, the solution after centrifugal ultrafiltration was further filtered using a 0.22 μm syringe filter (Millipore) to prepare LNPs. The obtained LNPs were stored at 4°C.

[0207] (Measurement of mRNA inclusion rate) A portion of the prepared LNP solution was taken, and the LNPs were dissolved by adding 0.5% Triton X-100. The nucleic acid concentration was measured using the nucleic acid quantification kit "Quant-iT RiboGreen RNA Assay Kit" (Thermo Fisher Scientific). In addition, a portion of the prepared LNP solution was taken, and the nucleic acid concentration was measured in the same manner without adding Triton X-100 to determine the concentration of mRNA that was not encapsulated in the LNPs. From these measurement results, the mRNA inclusion rate (%) in the LNPs was calculated.

[0208] (Measurement of average particle size of LNPs) The average particle size of LNPs was measured by dynamic light scattering. Specifically, diluted samples were added to a measurement plate, and laser light (633 nm) was irradiated using a dynamic light scattering device (DynaPro PlateReader III, Wyatt Technology) to measure the particle size (nm) and PDI.

[0209] (Cell assay) AAVpro 293T cells, pre-cultured using FBS-DMEM (DMEM containing 10% FBS and 1 mM SP), were seeded into 96-well plates at a density of 80,000 cells / cm² and cultured until 70–80% confluence (37°C, 5% CO2). 24 hours after seeding, the AAVpro 293T cells were transfected by adding the prepared LNP to each well and further cultured. 24 hours after transfection, Steady-Glo® Luciferase Assay System (Promega) was added, and luminescence intensity was analyzed using BioTek Synergy Neo2 (Agilent Technologies).

[0210] (Cytotoxicity) AAVpro 293T cells, pre-cultured using FBS-DMEM (DMEM containing 10% FBS and 1 mM SP), were seeded into 96-well plates at a density of 80,000 cells / cm² and cultured until 70–80% confluence (37°C, 5% CO2). Twenty-four hours after seeding, the prepared LNPs were added to each well to transfect the AAVpro 293T cells, and the cells were further cultured. Twenty-four hours after transfection, Cell Counting Kit-8 (Dojin Chemical Co., Ltd.) was added, and the cells were cultured for approximately 60 minutes before the absorbance was analyzed using BioTek Synergy Neo2 (Agilent Technologies). As a control, LNPs prepared in the same manner except without nucleic acid encapsulation were transfected and the absorbance was analyzed in the same manner. The relative absorbance (%) of each cell was defined as the cell viability (%), with the absorbance of the control cells set to 100%.

[0211] Table 1 shows the measurement results for the average particle size (nm), PDI, mRNA inclusion rate (%), luminescence intensity, and cell viability (%) of each LNP prepared in Test Example 1. Under the experimental conditions of this experiment, a luminescence intensity of 3000 or higher is preferable, and 16000 or higher is more preferable. In all examples, the LNPs showed no problems with mRNA inclusion rate, and both cell viability and luminescence intensity were good. In particular, cells administered with the LNP from Example 1 showed high luminescence intensity, indicating that this LNP had excellent cell viability.

[0212]

[0213] Table 2 shows the phospholipid that performed best among the LNPs prepared in Test Example 2 using different combinations of ionized lipids with phospholipids, along with the measurement results for the average particle size (nm), PDI, mRNA inclusion rate (%), luminescence intensity, and cell viability (%) of that LNP. Under the experimental conditions of this experiment, a luminescence intensity of 25,000 or higher is preferred, and 350,000 or higher is more preferred. In all of the examples, a significant increase in luminescence intensity was observed with the selection of phospholipids, and in particular, cells administered with the LNP of Example 1 showed very high luminescence intensity, indicating that this LNP had excellent cell viability.

[0214]

[0215] [Test Example 3] Synthetic compounds 7, 15, 18, 20, 21, and 26, as well as commercially available lipids 244cis and SM102 (8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester, CAS number: 2089251-47-6) were used as ionized lipids, DSPC was used as the phospholipid, cholesterol as the sterol, and DMG-PEG2000 was used as the PEG lipid to prepare LNPs containing mRNA, and their physical properties and cellular activity were investigated. The mRNA used was the mRNA encoding the luciferase protein FLuc (SEQ ID NO: 1).

[0216] (Preparation of LNPs) A lipid mixture was prepared by mixing ionized lipids, phospholipids, cholesterol, and PEG lipids in a ratio of 50:10:38.5:1.5 mol%, respectively. This mixture was dissolved in 99.5% ethanol to obtain a 20 mmol / L lipid solution. In addition, mRNA encoding FLuc was dissolved in acetate buffer (25 mM sodium acetate, pH 4) to obtain a nucleic acid solution. The concentration of the nucleic acid solution was adjusted so that the NP ratio (ratio of amino groups in ionized lipids to phosphate groups in nucleic acids) was 6.

[0217] The obtained lipid solution and nucleic acid solution were mixed at room temperature in a volume ratio of 1:3 using a microfluidic device (NanoAssemblr (registered trademark) Ignite NxGen (registered trademark) cartridges, manufactured by Neppagene Co., Ltd.) to obtain a dispersion. The obtained dispersion was diluted five-fold with D-PBS(-) buffer (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) or citrate buffer (20 mM citrate, pH 4), and centrifugal ultrafiltration was performed using a centrifugal filter unit (Amicon Ultra-4 or Amicon Ultra-15, manufactured by Millipore). Subsequently, the solution after centrifugal ultrafiltration was further filtered using a 0.22 μm syringe filter (manufactured by Millipore) to prepare LNPs. The obtained LNPs were stored at 4°C. The obtained LNPs were subjected to the same methods as in Test Examples 1 and 2 for measuring mRNA inclusion rate, measuring the average particle size of the LNPs, cell assays, and cytotoxicity.

[0218]

[0219] When compounds 7, 15, 18, 20, 21, and 26 were used as ionized lipids (Examples 4, 5, 6, 7, 8, and 9), the luminescence intensity was higher and cell activity was superior compared to Comparative Example 2. Furthermore, when compounds 15 and 21 were used (Examples 5 and 8), the luminescence intensity was more than twice as high not only compared to Comparative Example 2 but also compared to Comparative Example 3 using SM102.

[0220] (Mouse bioluminescence imaging evaluation test) Female BALB / cAJcl nu / nu 7-week-old mice were administered lipid nanoparticles encapsulating CleanCap FLuc mRNA (TriLink BioTechnologies) via tail vein (10 mL / kg i.v.). The dose was 0.01 mg / kg for commercially available lipids and 0.5 mg / kg for synthesized lipids. Four hours and fifty minutes after administering lipid particles, luciferin (15 mg / mL) was administered to mice (150 mg / kg i.p.), and the mice were placed in an IVIS (In Vivo Imaging System) device (IVIS® SpectrumCT, manufactured by Caliper Lifesciences) under isoflurane inhalation anesthesia. Ten minutes after luciferin administration, luminescence images were taken from the ventral (supine) side of the mice, and then each organ (liver, kidney, spleen, lung, brain, lymph nodes) was excised and imaged for in vitro imaging. The amount of luminescence was quantified using IVIS software, and evaluated using the Total Flux value. A higher amount of luminescence indicates stronger expression of the protein encoded by the RNA encapsulated in the lipid particles.

[0221] Figures 1-8 show the results of mouse bioluminescence imaging evaluation tests for each LNP. All tests were performed with n=3.

[0222] The captured images were analyzed using IVIS software to quantify the light emission, and the results were evaluated using the Total Flux value. The results are shown in Table 4.

[0223]

[0224] As shown in Table 4, compared to Comparative Example 4, Examples 10-14 demonstrated higher selectivity for delivery to the spleen, and Examples 10-12 and 14 also demonstrated higher selectivity for delivery to lymph nodes. Furthermore, Examples 11, 12, and 14 also demonstrated high selectivity for delivery to the lungs.

[0225] The mouse bioluminescence imaging evaluation test was conducted in the same manner as described above, except that the dose of compound 21 and the commercially available lipid was set to 0.1 mg / kg. The results are shown in Table 5.

[0226]

[0227] As shown in Table 5, it was confirmed that Example 15 showed higher luminescence in the spleen and lymph nodes compared to commercially available lipids.

[0228] When the compound or salt thereof of the present invention is used as an ionizing lipid for LNPs, it can more effectively exert the function of the encapsulated nucleic acid within the cell. For this reason, the compound or salt thereof of the present invention is particularly useful as a carrier for transporting functional nucleic acids such as mRNA and siRNA to target cells, and is especially useful in fields such as nucleic acid drugs.

Claims

1. A compound or salt thereof represented by the following formula (1). [In formula (1), R Ｂ１ ~R Ｂ４ These are, independently, alkylene groups having 1 to 3 carbon atoms. Ｂ５ R is an alkylene group having 1 to 3 carbon atoms, which may have heteroatoms. L１ ~R L３ Each of these is independently a linear or branched saturated aliphatic hydrocarbon group having 8 or more carbon atoms, which may have substituents, or a linear or branched unsaturated aliphatic hydrocarbon group having 8 or more carbon atoms, which may have substituents. Ｓ１ This is a divalent linking group that may contain an oxygen atom.

2. A compound or a salt thereof represented by the following formula (2). [In formula (2), R Ｂ１ to R Ｂ４ are each independently an alkylene group having 1 to 3 carbon atoms. R Ｂ５ is an alkylene group having 1 to 3 carbon atoms which may have a hetero atom. R L１１ to R L１４ are each independently a linear or branched saturated aliphatic hydrocarbon group having 8 or more carbon atoms which may have a substituent, or a linear or branched unsaturated aliphatic hydrocarbon group having 8 or more carbon atoms which may have a substituent. Y Ｓ１１ , Y Ｓ１２ are each independently a divalent linking group which may have an oxygen atom. ] 3. The aforementioned R Ｂ５ The compound or salt thereof according to claim 1 or 2, wherein is an alkylene group having 1 to 3 carbon atoms.

4. The aforementioned R L１ ~R L３ The compound or salt thereof according to claim 1, wherein each of these is independently a group represented by the following formula (R-1) or the following formula (R-2). [In formulas (R-1) and (R-2), R LX This includes -OH group, -SH group, -OC(=O)R group (where R is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms), or amino group (-NH ２ ) and R LY [where n1 is a linear or branched saturated or unsaturated aliphatic hydrocarbon group having 4 or more carbon atoms, n1 is an integer from 1 to 3, n2 is an integer from 1 to 12, and * indicates the bond site with the nitrogen atom.] 5. The aforementioned R L１１ ~R L１４ The compound or salt thereof according to claim 2, wherein each of these is independently a group represented by the following formula (R-1) or the following formula (R-2). [In formulas (R-1) and (R-2), R LX This includes -OH group, -SH group, -OC(=O)R group (where R is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms), or amino group (-NH ２ ) and R LY [where n1 is a linear or branched saturated or unsaturated aliphatic hydrocarbon group having 4 or more carbon atoms, n1 is an integer from 1 to 3, n2 is an integer from 1 to 12, and * indicates the bond site with the nitrogen atom.] 6. The compound or salt thereof according to claim 2, wherein formula (2) is formula (2-1), formula (2-2), formula (2-3), or formula (2-4). [In formulas (2-1), (2-2), (2-3), (2-4), R Ｌ１ , R Ｌ２ At least one of them is a linear saturated aliphatic hydrocarbon group or an unsaturated aliphatic hydrocarbon group having 4 or more carbon atoms, R ＬＹ‘ [where p is a linear saturated or unsaturated aliphatic hydrocarbon group having 4 or more carbon atoms, p is an integer from 1 to 5, q is an integer from 1 to 11, r is an integer from 1 to 11, s is an integer from 1 to 3, and t is an integer from 1 to 11.] 7. The compound or salt thereof according to claim 6, wherein p is an integer from 1 to 3.

8. The compound or salt thereof according to claim 6, wherein q is an integer from 2 to 7.

9. The compound or salt thereof according to claim 6, wherein formula (2-1) is compound 7, compound 15, compound 18, compound 30, or compound 31 as shown below.

10. The compound or salt thereof according to claim 6, wherein formula (2-2) is compound 20, compound 21, compound 32, or compound 33 as shown below.

11. The compound or salt thereof according to claim 6, wherein formula (2-3) is the compound 26 shown below.

12. Lipid nanoparticles comprising nucleic acid and ionized lipid, wherein the ionized lipid is the compound or salt thereof described in claim 1 or 2.

13. Lipid nanoparticles comprising nucleic acid and ionized lipid, wherein the ionized lipid is compound 4, compound 7, compound 11, compound 15, compound 18, compound 20, compound 21, compound 26, compound 30, compound 31, compound 32, or compound 33 as shown below.

14. Lipid nanoparticles according to claim 12, further comprising sterols.

15. The lipid nanoparticle according to claim 12, wherein the nucleic acid is mRNA or siRNA.

16. A pharmaceutical composition comprising lipid nanoparticles as described in claim 12.

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