Novel lipids for nucleic acid segment delivery

Abstract

Disclosed herein is a compound of formula (I) or a pharma- ceutically acceptable salt thereof, wherein X 1 , Y 1 , X 2 , Y 2 , a, b, c, d, e, and f are as defined herein. Also disclosed are lipid nanoparticles comprising a compound of formula (I) or a pharma- ceutically acceptable salt thereof; a pharmaceutical composition comprising a plurality of lipid nanoparticles comprising a compound of formula (I) or a pharma- ceutically acceptable salt thereof and a nucleic acid segment; and a method of delivering a nucleic acid segment, comprising administering a plurality of lipid nanoparticles comprising a compound of formula (I) or a pharma- ceutically acceptable salt thereof and a nucleic acid segment. [Formula 1] JPEG2024539964000071.jpg70160

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 271,960, filed on 26 October 2021. This application is incorporated herein by reference in its entirety with respect to all its purposes. [Background technology]

[0002] Nucleic acid segments such as oligonucleotides (e.g., RNA, e.g., messenger RNA [mRNA] and small interfering RNA [siRNA], antisense oligonucleotides [ASO], and DNA) have broad potential as novel therapeutic agents for various diseases and disorders. However, challenges remain in the administration of oligonucleotide therapeutics. Typical formulations involve encapsulating oligonucleotides in lipid nanoparticles (LNPs). LNP formulations typically include (a) an ionizable or cationic lipid or polymer material having a tertiary or quaternary amine to encapsulate polyanionic mRNA; (b) a zwitterionic lipid similar to lipids in cell membranes; (c) cholesterol to stabilize the lipid bilayer of the LNP; and (d) polyethylene glycol (PEG)-lipids that provide a hydration layer to the nanoparticles, improving colloidal stability and reducing protein absorption (see Non-Patent Literature 1).

[0003] In 2018, the FDA approved patisiran, the first RNA interference therapy, for the treatment of polyneuritis in individuals with hereditary trans-tyretin-mediated amyloidosis. It is delivered intravenously using an LNP incorporating an ionizable lipid (DLin-MC3-DMA, [MC3]). However, depending on the target organ, intended delivery route, and required therapeutic window, MC3 may not be the most suitable delivery system. Dose-limiting toxicity has been reported in studies in two toxicology-related test species, rats and monkeys. This is related to the MC3-based LNP formulation, not the delivered cargo (see Non-Patent Literature 2). More recently, lipid nanoparticle technology has been successfully applied to produce the first approved mRNA product for prophylactic vaccination against the SARS-CoV-2 virus (see, e.g., Non-Patent Literature 3). However, there remains a need to develop new ionizable lipids for use in lipid nanoparticle formulations for the delivery of oligonucleotide therapeutics. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Kowalski et al.,Molecular Therapy,27(4),(2019),710-728 [Non-Patent Document 2] Sedic et al.,Vet. Pathol.55(2),(2018),341-354 [Non-Patent Document 3] L. Schoenmaker et al., International Journal of Pharmaceutics, 601, (2021), May 120586 [Overview of the project] [Means for solving the problem]

[0005] In some embodiments, formula (I) is disclosed: [Chemical formula] is a compound or a pharmaceutically acceptable salt thereof, wherein a and b are each independently 3, 4, or 5; c and d are each independently 1, 2, or 3; e and f are each independently 0, 1, or 2; X is methylene or [Chemical formula] is, X 2 is methylene or [Chemical formula] is, g and h are each independently 1, 2, or 3; i and j are each independently 0, 1, or 2; Y 1 and Y 2 are each independently linear C 7~10 alkyl, linear C 7~10 alkenyl, or [Chemical formula] is, Z 1 and Z 2 are each independently linear C 7~10 alkyl, linear C 7~10 alkenyl, or [Chemical formula] is, R 1 and R 2 are each independently linear C 7~10 alkyl; R 3 and R 4 are each independently linear C 7~10 alkyl.

[0006] In some embodiments, the disclosed lipid nanoparticles include a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0007] In some embodiments, a pharmaceutical composition is disclosed comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a plurality of lipid nanoparticles comprising nucleic acid segments.

[0008] In some embodiments, a method is disclosed for treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.

[0009] In some embodiments, the disclosed pharmaceutically acceptable compositions described herein are used for the treatment of a disease or disorder. [Brief explanation of the drawing]

[0010] [Figure 1] This shows the expression of eGFP in rat lungs 24 hours after intratracheal administration of an LNP formulation containing MC3, MOD5, compound 4, and compound 2. [Figure 2] This shows the BALF neutrophil concentrations in rat BALF 24 hours after intratracheal administration of an LNP preparation containing MC3, MOD5, compound 4, and compound 2. [Figure 3] This shows the expression of eGFP in rat lungs 24 hours after intratracheal administration of an LNP preparation containing MC3 and compound 1. [Figure 4] This shows the BALF neutrophil concentration in rat BALF 24 hours after intratracheal administration of an LNP preparation containing MC3 and compound 1. [Figure 5A-B] This shows the expression of eGFP, as indicated by IHC, in both macrophages and type 1 epithelial cells after intratracheal administration of an LNP preparation containing compound 1. [Figure 6] The levels of eGFP in rat lungs 5 ​​and 24 hours after inhalation administration of an LNP formulation containing compound 1 are shown. [Figure 7]This shows the BALF neutrophil concentration in rat BALF 24 hours after inhalation administration of an LNP preparation containing compound 1. [Figure 8] This shows the expression of eGFP in mouse liver 24 hours after intravenous administration of an LNP formulation containing MC3, compound 2, and compound 3. [Figure 9] This shows the expression of eGFP in mouse spleens 24 hours after intravenous administration of an LNP preparation containing MC3, compound 2, and compound 3. [Figure 10] This shows the expression of eGFP in mouse lungs 24 hours after intravenous administration of an LNP formulation containing MC3, compound 2, and compound 3. [Figure 11] This shows the expression of eGFP in mouse kidneys 24 hours after intravenous administration of an LNP preparation containing MC3, compound 2, and compound 3. [Figure 12] This shows the expression of eGFP in mouse hearts 24 hours after intravenous administration of an LNP formulation containing MC3, compound 2, and compound 3. [Figure 13] This shows the expression of luciferase protein in mouse liver 24 hours after intravenous administration of an LNP preparation containing MC3, compound 2, and compound 3. [Figure 14] These images show IVIS images of the mouse heart, lungs, spleen, and liver 24 hours after intravenous administration of an LNP preparation containing MC3, compound 2, and compound 3. [Figure 15] This shows the hEPO protein concentration in mouse plasma 6 hours after intravenous administration of an LNP preparation containing MC3, compound 2, and compound 3. [Figure 16] This shows average cortical and striatal Luc expression in LoxP Luc reporter mice after intrastriatal administration of an LNP preparation containing MOD5 and compound 5. [Modes for carrying out the invention]

[0011] In some embodiments, formula (I) is disclosed: [ka] A compound of or a pharmaceutically acceptable salt thereof, wherein the formula is a and b are, independently, 3, 4, or 5; c and d are independently 1, 2, or 3; e and f are independently 0, 1, or 2; X 1 is methylene or [ka] And, X 2 is methylene or [ka] And, g and h are independently 1, 2, or 3; i and j are independently 0, 1, or 2; Y 1 and Y 2 Each of them is independently a linear C 7~10 Alkyl, linear C 7~10 Alkenil, or [ka] And, Z 1 and Z 2 Each of them is independently a linear C 7~10 Alkyl, linear C 7~10 Alkenil, or [ka] And, R 1 and R 2 Each of them is independently a linear C 7~10 It is alkyl; R 3 and R 4 Each of them is independently a linear C 7~10 It is alkyl.

[0012] In some embodiments of formula (I), X 1 and X 2Both are methylene compounds.

[0013] In some embodiments of formula (I), a and b are both 4; and c and d are both 2.

[0014] In some embodiments of equation (I), both e and f are 0.

[0015] In some embodiments of formulas I and II: Y 1 is a linear C 7~10 Alkyl or linear C 7~10 It is an alkenil; Y 2 teeth [ka] and; R 1 and R 2 Each of them is independently a linear C 7~10 It is alkyl.

[0016] In some embodiments of formula (I), Y 1 and Y 2 Each of them operates independently. [ka] and; R 1 and R 2 Each of them is independently a linear C 7~10 It is alkyl.

[0017] In some embodiments of formula (I), e and f are each independently 1 or 2. In some further embodiments of formula (I), Y 1 and Y 2 Each of them operates independently. [ka] And, R 1 and R 2 Each of them is independently a linear C 7~10It is alkyl.

[0018] In some embodiments, the compound of formula (I) is of formula (II): [ka] It is a compound of the formula, in which, k is 0, 1, or 2; Y 1 is a linear C 7~10 Alkyl, linear C 7~10 Alkenil, or [ka] And, Y 2 teeth [ka] And, R 1 and R 2 Each of them is independently a linear C 7~10 It is alkyl.

[0019] In some embodiments of formula (I), X 1 teeth [ka] and; X 2 teeth [ka] That is the case.

[0020] In some embodiments of formula (I), a and b are both 4; and c and d are both 2.

[0021] In some embodiments of formula (I), e and f are independently 1 or 2.

[0022] In some embodiments of formula (I), both g and h are 2.

[0023] In some embodiments of formula (I), both i and j are 1.

[0024] In some embodiments of formula (I), Y 1 and Y 2 Each of them operates independently. [ka] and; R 1 and R 2 Each of them is independently a linear C 7~10 It is alkyl.

[0025] In some embodiments, the compound of formula (I) is heptadecan-9-yl8-(7-(8-(nonyloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonan-3-yl)octanoate or a pharmaceutically acceptable salt thereof.

[0026] In some embodiments, the compound of formula (I) is heptadecan-9-yl(Z)-8-(7-(8-(nona-2-en-1-yloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonan-3-yl)octanoate or a pharmaceutically acceptable salt thereof.

[0027] In some embodiments, the compound of formula (I) is 3-heptyldodecyl 8-(7-(8-((3-octylundecyl)oxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonan-3-yl)octanoate or a pharmaceutically acceptable salt thereof.

[0028] In some embodiments, the compound of formula (I) is di(heptadecan-9-yl)8,8'-(9-oxa-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)dioctanoate or a pharmaceutically acceptable salt thereof.

[0029] In some embodiments, the compound of formula (I) is tetrakis(2-octyldecyl)3,3',3'',3'''-(((9-oxa-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrapropionate or a pharmaceutically acceptable salt thereof.

[0030] In some embodiments, the compound of formula (I) is heptadecan-9-yl8-(7-(8-(nonyloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-yl)octanoate.

[0031] In some embodiments, the compound of formula (I) is heptadecan-9-yl(Z)-8-(7-(8-(nona-2-en-1-yloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonan-3-yl)octanoate.

[0032] In some embodiments, the compound of formula (I) is 3-heptyldodecyl 8-(7-(8-((3-octylundecyl)oxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-yl)octanoate.

[0033] In some embodiments, the compound of formula (I) is di(heptadecan-9-yl)8,8'-(9-oxa-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)dioctanoate.

[0034] In some embodiments, the compound of formula (I) is tetrakis(2-octyldecyl)3,3',3'',3'''-(((9-oxa-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrapropionate.

[0035] As used herein, the term "Ci~j" indicates a range of carbon atoms, where i and j are integers, and the range of carbon atoms includes the endpoints (i.e., i and j) and each integer point between them, where j is greater than i. For example, C 7~10 This refers to a range of 7 to 10 carbon atoms, including 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, and 10 carbon atoms.

[0036] As used herein, the term "alkyl," whether used as part of another term or independently, refers to a saturated hydrocarbon chain. In one embodiment, the saturated hydrocarbon chain referred to is a straight-chain alkyl in which carbon atoms are linked in a single, unbranched chain. The term "Ci~j alkyl" refers to an alkyl having i to j carbon atoms. For example, "C 7~10 "Alkyl" refers to an alkyl group that has 7 to 10 carbon atoms.

[0037] As used herein, the term “alkenyl,” whether used as part of another term or independently, refers to an unsaturated hydrocarbon chain containing at least one double bond. In one embodiment, the unsaturated hydrocarbon chain referred to is a straight-chain alkenyl in which carbon atoms are linked in a single, unbranched chain. The term “Ci~j alkenyl” refers to an alkenyl having i~j carbon atoms. For example, C 7~10 An alkenyl refers to an alkenyl having 7 to 10 carbon atoms. In one embodiment, C 7~10 The alkenyl group contains one double bond.

[0038] In some embodiments, the disclosed compound is of formula (I). In some embodiments, the disclosed compound is a pharmaceutically acceptable salt of the compound of formula (I). The term “pharmaceutically acceptable salt” includes acid addition salts or base salts that retain the biological efficacy and properties of the compound of formula (I) and are not typically biologically or otherwise undesirable. Pharmaceutically acceptable acid addition salts may be formed from inorganic and organic acids, for example, acetate, aspartate, benzoate, besylate, bromide / hydrobromide, bicarbonate / carbonate, bisulfate / sulfate, camphor sulfonate, chloride / hydrochloride, chlortheophyllonate, citrate, ethanedisulfonate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hydroiodide / iodide, isethionate, lactate, etc. These include ctobionates, lauryl sulfates, malates, maleates, malons, mandelates, mesilates, methylsulfates, naphthoates, napsylates, nicotinates, nitrates, octadecanoates, oleates, oxalates, palmitates, palmoates, phosphates / hydrogen phosphates / dihydrogen phosphates, polygalacturonates, propions, stearates, succinates, subsalicylates, sulfates / hydrogen sulfates, tartrates, tosylates, and trifluoroacetates. Examples of inorganic acids from which salts can be derived include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Examples of organic acids from which salts can be derived include acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, trifluoroacetic acid, and sulfosalicylic acid.

[0039] In some embodiments, lipid nanoparticles (LNPs) comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof are disclosed. In some embodiments, lipid nanoparticles (LNPs) comprising the compound of formula (I) are disclosed. The term “lipid nanoparticle” includes a high electron-density nanostructure core produced by microfluidic mixing of a lipid-containing solution in ethanol with an aqueous solution. The lipid nanoparticles disclosed herein can be constructed from any material used in conventional nanoparticle technology, such as ionizable lipids, neutral lipids, sterols, and polymer-conjugated lipids, when the net charge of the nanoparticles is about 0.

[0040] In some embodiments, the compound of formula (I) is an ionizable lipid suitable for lipid nanoparticles. Other non-limiting examples of ionizable lipids that can be combined with the compound of formula (I) in lipid nanoparticles include, for example, lipids containing a positive charge on an acidic scale within the physiological pH range, e.g., 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA (see, e.g., U.S. Patent No. 8,158,601)), 2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), Merck-32 (see, e.g., International Publication No. 2012 / 018754), Acuitas-5 (see, e.g., International Publication No. 2015 / 199952), KL-10 (see, e.g., U.S. Patent Application Publication No. 2012 / 0295832), C12-200 (see, e.g., Love, KT et al.) Examples include al., PNAS, 107:1864 (2009). Ionizable lipids may be present in the lipid nanoparticles in a molar percentage range of approximately 5% to 90%, for example, approximately 10% to 80%, for example, approximately 25% to 75%, for example, approximately 40% to 60%, or approximately 40% to 50%, for example, approximately 45% or 50%, relative to the total lipids present.

[0041] The term "neutral lipid" refers to lipids with a net charge of zero at physiological pH, such as lipids that exist in an uncharged form at physiological pH or in a neutral zwitterionic form, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), and combinations thereof. Neutral lipids may be present in lipid nanoparticles in a molar percentage of approximately 1% to 50%, for example, approximately 5% to 20%, for example, 7.5% to 12.5%, or for example, approximately 10% relative to the total lipids present. In some embodiments, the neutral lipid is DSPC. In some embodiments, the neutral lipid is DOPE. In some embodiments, the neutral lipid is DPPC. In some embodiments, the neutral lipid is DMPC.

[0042] The term "sterol" includes cholesterol, etc. Sterols can be present in lipid nanoparticles in amounts ranging from approximately 10% to approximately 90%, for example, approximately 20% to approximately 50%, for example, approximately 35% to 45%, or for example, approximately 38.5% of the total lipids. In some embodiments, the sterol is cholesterol.

[0043] The term "polymer-conjugated lipid" includes lipids containing both a lipid portion and a polymer portion, such as PEGylated lipids containing both a lipid portion and a polyethylene glycol portion. Non-limiting examples include dimyristoylphosphatidylethanolamine-poly(ethylene glycol) 2000 (DMPE-PEG2000), DPPE-PEG2000, DMG-PEG2000, DPG-PEG2000, PEG2000-c-DOMG, PEG2000-c-DOPG, etc. The molecular weight of the usable poly(ethylene glycol) can range from about 500 to about 10,000 Da, or about 1,000 to about 5,000 Da. In some embodiments, the polymer-conjugated lipid is DMPE-PEG2000. In some embodiments, the polymer-conjugated lipid is DPPE-PEG2000. In some embodiments, the polymer-conjugated lipid is DMG-PEG2000. In some embodiments, the polymer-conjugated lipid is DPG-PEG2000. In some embodiments, the polymer conjugate lipid is PEG2000-c-DOMG. In some embodiments, the polymer conjugate lipid is PEG2000-c-DOPG. The polymer conjugate lipid may be present in a molar percentage of about 1.5%, for example, ranging from about 0% to about 20%, for example, about 0.5% to about 5%, for example, about 1% to about 2%, relative to the total lipids present in the lipid nanoparticles.

[0044] In at least one embodiment of the present disclosure, lipid nanoparticles may be prepared by combining multiple lipid components. For example, lipid nanoparticles may be prepared by combining a compound of formula (I) or a pharmaceutically acceptable salt thereof, sterols, neutral lipids, and polymer conjugate lipids in a molar ratio of 50:40-x:10:x relative to the total lipids present. For example, lipid nanoparticles may be prepared by combining a compound of formula (I) or a pharmaceutically acceptable salt thereof, sterols, neutral lipids, and polymer conjugate lipids in a molar ratio of 50:37:10:3 (moles / moles), or for example 50:38.5:10:1.5 (moles / moles), or for example 50:39.5:10:0.5 (moles / moles), or 50:39.75:10:0.25 (moles / moles).

[0045] In another embodiment, lipid nanoparticles may be prepared using the compound of formula (I) or a pharmaceutically acceptable salt thereof, sterols (such as cholesterol), neutral lipids (such as DSPC), and polymer conjugate lipids (such as DMPE-PEG2000) in a molar ratio of approximately 50:38.5:10:1.5 (moles / mol) relative to the total lipids present. Yet another non-limiting example is lipid nanoparticles containing the compound of formula (I) or a pharmaceutically acceptable salt thereof, sterols (such as cholesterol), neutral lipids (such as DSPC), and polymer conjugate lipids (such as DMPE-PEG2000) in a molar ratio of approximately 47.7:36.8:12.5:3 (moles / mol) relative to the total lipids present. Another non-limiting example is a lipid nanoparticle containing the compound of formula (I) or a pharmaceutically acceptable salt thereof, sterols (such as cholesterol), neutral lipids (such as DSPC), and polymer conjugate lipids (such as DMPE-PEG2000) in a molar ratio of approximately 52.4:40.4:6.4:0.8 (moles / mol) relative to the total lipids present. In another embodiment, a non-limiting example is a lipid nanoparticle containing the compound of formula (I) or a pharmaceutically acceptable salt thereof, sterols (such as cholesterol), neutral lipids (such as DSPC), and polymer conjugate lipids (such as DMPE-PEG2000) in a molar ratio of approximately 53.5:41.2:4.6:0.7 (moles / mol) relative to the total lipids present. Another non-limiting example is lipid nanoparticles containing the compound of formula (I) or its pharmaceutically acceptable salt, sterols (such as cholesterol), neutral lipids (such as DSPC), and polymer conjugate lipids (such as DMPE-PEG2000) in a molar ratio of approximately 30:50:19:1 (mol / mol) relative to the total lipids present.

[0046] The selection of neutral lipids, sterols, and / or polymer-conjugated lipids constituting the lipid nanoparticles, and the relative molar ratios of such lipids to each other, may be determined by the characteristics of the selected lipids, the properties of the intended target cells, and the characteristics of the nucleic acid segments to be delivered. For example, in certain embodiments, the molar percentage of the compound of formula (I) or its pharmaceutically acceptable salt in the lipid nanoparticles may be greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, or greater than about 70% relative to the total lipids present. The molar percentage of neutral lipids in the lipid nanoparticles may be greater than about 5%, greater than about 10%, greater than about 20%, greater than about 30%, or greater than about 40% relative to the total lipids present. The molar percentage of sterols in the lipid nanoparticles may be greater than about 10%, greater than about 20%, greater than about 30%, or greater than about 40% relative to the total lipids present. The molar percentage of polymer-conjugated lipids in lipid nanoparticles may be more than approximately 0.25% of the total lipids present, for example, more than approximately 1%, more than approximately 1.5%, more than approximately 2%, more than approximately 5%, or more than approximately 10%.

[0047] According to this disclosure, lipid nanoparticles may comprise each of the compound of formula (I) or a pharmaceutically acceptable salt thereof, neutral lipids, sterols, and / or polymer-conjugated lipids in any desired useful orientation. For example, the core of a nanoparticle may comprise the compound of formula (I) or a pharmaceutically acceptable salt thereof alone or in combination with another ionizable lipid, sterol, and then one or more layers comprising neutral lipids and / or polymer-conjugated lipids may surround the core. For example, according to one embodiment, the core of a lipid nanoparticle may comprise a core comprising the compound of formula (I) or a pharmaceutically acceptable salt thereof and sterols (e.g., cholesterol) in any particular ratio, surrounded by a neutral lipid monolayer (e.g., DSPC) of any particular thickness, and further surrounded by an outer polymer-conjugated lipid monolayer of any particular thickness. In such an example, a nucleic acid segment may be incorporated into either the core or one of the subsequent layers, depending on the properties of the intended target cell and the characteristics of the nucleic acid segment to be delivered. The core and outer layer may further contain other components typically incorporated into lipid nanoparticles known in the art. Furthermore, it will be understood by those skilled in the art that liposomes are delivery vehicles having a vesicular structure different from the lipid nanoparticles disclosed herein. Liposome vesicles consist of lipid bilayers formed in the shape of hollow spheres containing an aqueous phase. For example, liposomes contain a lamellar phase, while lipid nanoparticles have a non-lamellar structure.

[0048] In addition, the molar percentage of the components of the lipid nanoparticles (e.g., the compound of formula (I) or its pharmaceutically acceptable salt, neutral lipid, sterol, and / or polymer conjugate lipid) constituting the lipid nanoparticles may be selected to provide specific physical parameters of the overall lipid nanoparticles, such as the surface area of ​​one or more lipids. For example, the molar percentage of the compound of formula (I) or its pharmaceutically acceptable salt, neutral lipid, sterol, and / or polymer conjugate lipid constituting the lipid nanoparticles may be selected to obtain a surface area per neutral lipid, such as DSPC. As a non-limiting example, the molar percentage of the compound of formula (I) or its pharmaceutically acceptable salt, neutral lipid, sterol, and / or polymer conjugate lipid may be about 1.0 nm. 2 ~about 2.0nm 2 For example, about 1.2 nm 2 The surface area per DSPC may be determined accordingly.

[0049] According to this disclosure, the lipid nanoparticles may further include nucleic acid segments that can be associated on the surface of the lipid nanoparticles and / or encapsulated within the same lipid nanoparticles.

[0050] The term “nucleic acid segment” is understood to mean any one or more nucleic acid segments selected from antisense oligonucleotides, DNA, mRNA, siRNA, Cas9 guide RNA complexes, or combinations thereof. Nucleic acid segments as used herein may be wild-type or modified. In at least one embodiment, a sex lipid nanoparticle may comprise several different nucleic acid segments. In yet another embodiment, the wild-type or modified nucleic acid segment encodes the polypeptide of interest. A modified nucleic acid segment comprises a nucleic acid segment having chemical modifications to any part of its structure so that the nucleic acid segment does not exist in nature. In some embodiments, the nucleic acid segment is RNA. In some embodiments, the nucleic acid segment is mRNA. In some embodiments, the nucleic acid segment is modified mRNA.

[0051] As used herein, the term “therapeutic dose” refers to the amount of nucleic acid segment sufficient to modulate protein expression in a target tissue and / or cell type. In some embodiments, the therapeutic dose of a nucleic acid segment is sufficient to treat a disease or disorder associated with the protein expressed by the nucleic acid segment.

[0052] In at least one embodiment, the weight ratio of the total lipid phase to the nucleic acid segment ranges from about 40:1 to about 1:1, for example, about 10:1. This corresponds to an approximate molar ratio of about 3:1 of the compound of formula (I) or its pharmaceutically acceptable salt to the nucleic acid monomer. In yet another example, the weight ratio of the total lipid phase to the nucleic acid segment ranges from about 30:1 to about 1:1, for example, about 20:1, which corresponds to an approximate molar ratio of about 6:1 of the compound of formula (I) or its pharmaceutically acceptable salt to the nucleic acid monomer. However, the relative molar ratio of the lipid phase and / or lipid phase components to the nucleic acid monomer may be determined by the properties of the intended target cell and the characteristics of the nucleic acid segment, and is not limited to the embodiments specified above. In some embodiments, the molar ratio of the compound of formula (I) or its pharmaceutically acceptable salt to the nucleic acid monomer is about 2.75:1 to 6:1. In some embodiments, the molar ratio of the compound of formula (I) or its pharmaceutically acceptable salt to the nucleic acid monomer is about 2.75:1. In some embodiments, the approximate molar ratio of the compound of formula (I) or its pharmaceutically acceptable salt to the nucleic acid monomer is about 3:1. In some embodiments, the molar ratio of the compound of formula (I) or its pharmaceutically acceptable salt to the nucleic acid monomer is about 5.5:1. In some embodiments, the approximate molar ratio of the compound of formula (I) or its pharmaceutically acceptable salt to the nucleic acid monomer is about 6:1.

[0053] In some embodiments, lipid nanoparticles have a z-average particle size ( <d> Z The z-average particle size is approximately 200 nm or less, for example, approximately 100 nm or less, or for example, approximately 75 nm or less. In at least one embodiment of the present disclosure, the lipid nanoparticles have a z-average particle size ranging from approximately 50 nm to approximately 100 nm, for example, approximately 60 nm to approximately 90 nm, or approximately 70 nm to approximately 80 nm, for example, approximately 75 nm.

[0054] In certain embodiments, the lipid nanoparticles have a nucleic acid segment encapsulation efficiency (%EE) of about 80% or more, for example, over 90%, and for example, ranging from about 95% to 100%. As used herein, the term "encapsulation efficiency" refers to the ratio of encapsulated nucleic acid segments in the lipid nanoparticles to the total nucleic acid segment content in the lipid nanoparticle composition, as measured by dissolving the lipid nanoparticles using a detergent, for example, Triton X-100.

[0055] The pharmaceutical compositions of this disclosure may further comprise at least one pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes compounds, materials, compositions, and / or dosage forms suitable for use in contact with human and animal tissues within the bounds of sound medical judgment, without excessive toxicity, irritation, allergic reactions, or other issues or complications, commensurate with a reasonable risk-benefit ratio.

[0056] The pharmaceutical composition may be in a form suitable for parenteral administration. Suitable parenteral administration methods include, but are not limited to, subcutaneous, intramuscular, and intravenous administration. The pharmaceutical composition may be in a form suitable for intratracheal infusion, bronchial infusion, and / or inhalation. The pharmaceutical liquid composition may be atomized using an inert gas for inhalation. The atomized suspension may be inhaled directly from an atomizer, or the atomizer may be attached to a face tent mask or an intermittent positive pressure breathing apparatus.

[0057] The amount of nucleic acid segment combined with one or more pharmaceutically acceptable carriers to produce a single dosage form will inevitably vary depending on the target being treated and the specific route of administration. For further information on routes of administration and administration plans, readers should refer to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

[0058] In one embodiment, the disclosure provides a method for administering a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a plurality of lipid nanoparticles containing a therapeutically effective amount of nucleic acid segments, to a subject requiring such administration.

[0059] The term "subject" includes warm-blooded mammals, such as primates, cattle, pigs, sheep, dogs, cats, rabbits, rats, and mice. In some embodiments, the subject is a primate, such as a human. In some embodiments, the subject requires treatment (for example, the subject would benefit biologically or medically from the treatment).

[0060] The lipid nanoparticles disclosed herein may further serve as drug delivery vehicles for the selective delivery of nucleic acid segments, such as antisense oligonucleotides, DNA, mRNA, siRNA, and Cas9-guide RNA complexes, to target cells and tissues. Thus, in one embodiment, a method for delivering nucleic acid segments to cells comprises contacting the cells in vitro or in vivo with a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a plurality of lipid nanoparticles comprising a therapeutically effective amount of nucleic acid segments. In some embodiments, the nucleic acid segments regulate polypeptide expression, for example, by increasing or decreasing expression, or by upregulating or downregulating expression.

[0061] Another embodiment provides a method for delivering a therapeutically effective amount of nucleic acid segment to a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a plurality of lipid nanoparticles containing a therapeutically effective amount of nucleic acid segment.

[0062] A pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a plurality of lipid nanoparticles containing nucleic acid segments, as disclosed herein, may be used to treat a wide variety of disorders and diseases characterized by underexpression of polypeptides, overexpression of polypeptides, and / or absence / presence of polypeptides in a subject. Disclosed is a method for treating a subject suffering from a disease or disorder, comprising administering to the subject a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a plurality of lipid nanoparticles containing a therapeutically effective amount of nucleic acid segments.

[0063] Furthermore, disclosed is the use of a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a plurality of lipid nanoparticles containing a therapeutically effective amount of nucleic acid segments, for treating a disease or disorder.

[0064] Furthermore, disclosed are pharmaceutical compositions used for the treatment of a disease or disorder, the pharmaceutical compositions comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a plurality of lipid nanoparticles comprising a therapeutically effective amount of nucleic acid segments.

[0065] Furthermore, disclosed is a method for regulating protein expression in cells, comprising administering a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and a plurality of lipid nanoparticles comprising nucleic acid segments, to a subject requiring such regulation. In at least one embodiment, protein expression can be increased by a factor of about 2 up to 24 hours. In another embodiment, protein expression can be increased by a factor of about 3 up to 72 hours. [Examples]

[0066] General method 1 ¹H NMR: 300MHz; Probe: ATM+Z PABBO BB-1H / D 5mm broadband liquid probe BBFO; Magnet: ULTRASHIELD™ 300; Crate: AVANCE III 300; Autosampler: SampleXpress™ 60; Software: Topspin 3. 400MHz; Probe: ATM+Z PABBO BB-1H / D 5mm broadband liquid probe BBFO; Magnet: ASCEND™ 400; Crate: AVANCE III 300; Autosampler: SampleXpress™ 60; Software: Topspin 3. All spectra were calibrated using TMS as an internal reference.

[0067] 1 ¹H NMR: 500MHz; Probe: 5mm Bruker Smart probe with ATM+Z PABBO 500S1-BBF-HD; Magnet: ASCEND® 500; Console: AVANCE Neo 500; Autosampler: SampleXpress® 60; Software: Topspin 4. Proton chemical shifts are expressed in parts per million (ppm, δ scale) and referenced to residual protium in NMR solvents (chloroform-d: δ7.26, methanol-d4: δ3.31, DMSO-d6: δ2.50). The data is expressed as follows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, dd=doublet of doublets, dt=doublet of triplets, m=multiplet, br=broad, app=apparent), integral, and coupling constant (J) (Hertz (Hz)).

[0068] LCMS: Shimadzu LCMS-2020 instrument connected to DAD detector, ELSD detector, and 2020EV MS; Column: Shim-pack XR-ODS C18 (50 × 3.0 mm, 2.2 μm); Eluent A: Water (0.05% TFA), Eluent B: MeCN (0.05% TFA); Gradient: 5-95% B 2.00 min, hold 0.70 min (Method A) or 60-95% B 1.00 min, hold 1.70 min (Method B); Flow rate: 1.20 mL / min; PDA detection (SPD-M20A) 190-400 nm. ESI mode mass spectrometer.

[0069] UPLC-MS was performed using a Waters Acquity UPLC and Waters SQD mass spectrometer (column temperature 30°C, UV detection = 210-400 nm, mass spectrometry = ESI with positive / negative switching) at a flow rate of 1 mL / min over 1.5 minutes (total runtime 2 minutes until equilibrium returns to starting conditions). Here, A = 0.1% formic acid in water, B = 0.1% formic acid in acetonitrile (for acid work), or A = 0.1% ammonium hydroxide in water, B = acetonitrile (for base work). The column used for acid analysis was Waters Acquity HSS T3 (1.8 mm, 2.1 × 30 mm), and the column used for base analysis was Waters Acquity BEH C18 (1.7 mm, 2.1 × 30 mm).

[0070] HPLC: DAD detector, Shimadzu LCMS-2020 instrument connected to CAD detector; Column Ascentis Express C18 (100 × 4.6 mm), 2.7 μm; Mobile phase A: Water (0.05% TFA), Mobile phase B: MeCN; Gradient 10-95% B 4.00 min, retention 8 min, or as indicated, flow rate 1.50 mL / min; Purity % as area.

[0071] Preparative HPLC equipment: Waters 2545 Binary Gradient Module, Waters 2767 Sample Manager, Waters 2489 UV / Visible Detector, Waters SQ Detector 2. Method A: Column XSelect CSH Prep C18 OBD Column, 19×250mm, 5μm; Mobile phase A: Water (0.05% TFA), Mobile phase B: MeCN; Flow rate: 25mL / min, gradient shown. Method B: Column SunFire C18 OBD, 19×250mm, 5μm; Mobile phase A: Water (0.05% TFA), Mobile phase B: MeCN; Flow rate: 60mL / min, gradient shown.

[0072] Abbreviation 1,2-DCE 1,2-dichloroethane ACN Acetonitrile CPME Cyclopentyl Methyl Ether DCM Dichloromethane DMSO (Dimethyl Sulfoxide) DIEA N,N-diisopropylethylamine DIPEA N,N-diisopropylethylamine DMAP N,N-dimethylaminopyridine <d> N Number average particle size <d> Z z-average particle size EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide EDCI 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EE% encapsulation effectiveness HCl ethyl acetate HPLC (High-Performance Liquid Chromatography) KI Potassium Iodide MC3(6Z,9Z,28Z,31Z)-heptatriaconto-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate NMP N-methyl-2-pyrrolidone PDI polydispersity index PE Petroleum Ether (30-50) PBS (phosphate-buffered saline) rt room temperature TEA (Triethylamine) THF (Tetrahydrofuran) UPLC Ultra-High-Speed ​​Liquid Chromatography Z-pot (zeta potential).

[0073] Example 1. Synthesis of Compound 1 [ka] Reagents: a) EDC, DIPEA, DMAP, DCM; b) K2CO3, KI, ACN; c) 4M HCl, Dioxane; d) EDC, DIPEA, DMAP, DCM; e) DIPEA, KI, ACN

[0074] Intermediate 1: Heptadecane-9-yl-8-bromooctanoate [ka] Step a: EDC (0.785 g, 4.09 mmol) was added in one go under argon to a DCM-stirred mixture (15 mL) of 8-bromooctanoic acid (0.522 g, 2.34 mmol), heptadecane-9-ol (0.5 g, 1.95 mmol), DMAP (0.048 g, 0.39 mmol), and DIPEA (1.396 ml, 7.99 mmol). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in siRNA (50 mL), and then washed sequentially with saturated aqueous NaHCO3 (50 mL) and saturated aqueous NaCl (50 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure to obtain the crude product. The resulting residue was purified by flash silica chromatography with an elution gradient of 0 and 20% siRNA (in hexane). The product fraction was concentrated to dryness under reduced pressure to obtain heptadecan-9-yl-8-bromooctanoate (0.714 g, 79%) as a colorless oil. ¹H NMR (500 MHz, chloroform-d, 27°C): 0.89 (6H,t), 1.27-1.70 (36H,m), 1.77-1.94 (2H,m), 2.21-2.37 (2H,t), 3.41 (2H,t), 4.78-4.95 (1H,m).

[0075] Intermediate 2: tert-butyl7-(8-(heptadecane-9-yloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-carboxylate [ka] Step b: Heptadecan-9-yl-8-bromooctanoate (0.808 g, 1.75 mmol) (intermediate 1) was added dropwise to a stirred acetonitrile mixture (10 mL) of tert-butyl-9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-carboxylate hydrochloride (0.386 g, 1.46 mmol), potassium carbonate (0.423 g, 3.06 mmol), and potassium iodide (0.048 g, 0.29 mmol) under argon. The resulting mixture was stirred at 80°C for 16 hours. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in siRNA (100 mL), and washed sequentially with saturated aqueous Na₂CO₃ (100 mL) and saturated aqueous NaCl (100 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated to dryness under reduced pressure to obtain the crude product. The resulting residue was purified by flash silica chromatography with an elution gradient of 0-100% in DCM (20% MeOH and 1% NH4OH in DCM). The product fraction was concentrated to dryness under reduced pressure to obtain tert-butyl 7-(8-(heptadecane-9-yloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-carboxylate (0.747 g, 84%) as a pale yellow oil. 1H NMR(500MHz,chloroform-d,22°C)0.88(6H,t),1.20-1.67(47H,m),2.02-2.19(2H,m),2.22-2.31(2H,t),2.3 5-2.49(2H,m),2.77-2.96(2H,m),3.18-3.38(2H,m),3.68-3.83(2H,m),3.92-4.17(2H,m),4.87(1H,m).

[0076] Intermediate 3: Heptadecan-9-yl8-(9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-yl)octanoate dihydrochloride [ka] Process c: HCl (2.250 ml, 9.00 mmol) in dioxane was added dropwise to a stirred dioxane mixture (10 mL) of tert-butyl 7-(8-(heptadecan-9-yloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-carboxylate (0.548 g, 0.90 mmol) (intermediate 2) under argon at 0°C. The resulting mixture was warmed and stirred at room temperature for 4 hours. The reaction mixture was concentrated to dryness under reduced pressure and washed with dioxane (3 × 50 mL) to obtain heptadecan-9-yl 8-(9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-yl)octanoate dihydrochloride (0.472 g, 96%) as a pale yellow oil. ¹H NMR (500 MHz, methanol-d4, 27°C): 0.90 (6H, t), 1.29 (38H, m), 2.24-2.39 (2H, t), 3.49-3.59 (3H, t), 4.85-4.91 (1H, m).

[0077] Intermediate 4: Nonyl-8-bromooctanoate [ka] Process d: EDC (2.79 g, 14.56 mmol) was added in one go to a stirred DCM solution (15 mL) containing 8-bromooctanoic acid (2.320 g, 10.40 mmol), nonane-1-ol (1.205 ml, 6.93 mmol), DMAP (0.169 g, 1.39 mmol), and DIPEA (2.54 ml, 14.56 mmol). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in siRNA (100 mL), and washed sequentially with saturated aqueous NaHCO3 (100 mL) and saturated aqueous NaCl (100 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure to obtain the crude product. The resulting residue was purified by flash silica chromatography with an eluent gradient of 0-20% siRNA (in hexane). The product fraction was concentrated to dryness under reduced pressure to obtain nonyl 8-bromooctanoate (1.580 g, 65.2%) as a colorless oil. ¹H NMR (500 MHz, 0001 chloroform-d, 27°C): 0.82-0.95 (3H,t), 1.23-1.51 (18H,m), 1.56-1.69 (4H,m), 1.80-1.92 (2H,m), 2.30 (2H,t), 3.33-3.48 (2H,t), 4.07 (2H,t).

[0078] Compound 1: Heptadecan-9-yl 8-(7-(8-(nonyloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-yl)octanoate [ka] Process e: Nonyl 8-bromooctanoate (0.144 g, 0.41 mmol) (intermediate 4) was added dropwise to a stirred acetonitrile mixture (5 mL) of heptadecan-9-yl 8-(9-oxa-3,7-diazabicyclo[3.3.1]nonan-3-yl)octanoate dihydrochloride (0.2 g, 0.34 mmol) (intermediate 3) and DIPEA (0.486 ml, 2.78 mmol) under argon. The resulting mixture was stirred at 80°C for 16 hours. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in siRNA (50 mL), and washed sequentially with saturated aqueous NaHCO3 (50 mL) and saturated aqueous NaCl (50 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure to obtain the crude product. The resulting residue was purified twice by flash silica chromatography with an elution gradient of 0-100% in DCM (20% MeOH and 1% NH4OH in DCM). The product fraction was concentrated to dryness under reduced pressure to obtain heptadecan-9-yl8-(7-(8-(nonyloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonan-3-yl)octanoate (0.119 g, 44.5%) as a pale yellow oil. 1H NMR (500MHz, methanol-d4, 27°C)0.90(9H,t),1.23-1.43(48H,m),1.48-1.68(14H,m),2.24-2.34(8H,m),2.41-2.52(4H,br d),2.87(4H,br d),3.83-3.92(2H,m),4.07(2H,s),4.88(1H,m).UPLC,ms detection(ES+)([M+H]+)=777.8 Da;RT ELSD=1.91 min.

[0079] Example 2. Synthesis of Compound 2 [ka] Reagents: a) 4M HCl, dioxane; b) EDC, DIPEA, DMAP, DCM; c) DIPEA, KI, ACN

[0080] Intermediate 1: 9-Oxa-3,7-diazabicyclo[3.3.1]nonanedihydrochloride [ka] Step a: HCl (2 ml, 65.83 mmol) was added dropwise to a stirred 1,4-dioxane mixture (8 mL) containing tert-butyl 9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-carboxylate hydrochloride (0.3 g, 1.13 mmol) under argon. The reaction mixture was stirred overnight at room temperature until a precipitate formed. The reaction mixture was concentrated to dryness under reduced pressure to obtain 9-oxa-3,7-diazabicyclo[3.3.1]nonane (0.220 g, 97%) as a white powder. ¹H NMR (500 MHz, methanol-d4, 27°C) 3.51 (8H, m), 4.41 (2H, br t).

[0081] Intermediate 2: Heptadecane-9-yl-8-bromooctanoate [ka] Step b: EDC (0.785 g, 4.09 mmol) was added in one go under argon to a stirred DCM mixture (15 mL) containing 8-bromooctanoic acid (0.522 g, 2.34 mmol), heptadecane-9-ol (0.5 g, 1.95 mmol), DMAP (0.048 g, 0.39 mmol), and DIPEA (1.396 ml, 7.99 mmol). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in siRNA (50 mL), and washed sequentially with saturated aqueous NaHCO3 (50 mL) and saturated aqueous NaCl (50 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure to obtain the crude product. The resulting residue was purified by flash silica chromatography with an elution gradient of 0-20% siRNA (in hexane). The product fraction was concentrated to dryness under reduced pressure to obtain heptadecan-9-yl-8-bromooctanoate (0.714 g, 79%) as a colorless oil. ¹H NMR (500 MHz, chloroform-d, 27°C): 0.89 (6H,t), 1.27-1.70 (36H,m), 1.77-1.94 (2H,m), 2.21-2.37 (2H,t), 3.41 (2H,t), 4.78-4.95 (1H,m).

[0082] Compound 2: Di(heptadecan-9-yl)8,8'-(9-oxa-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)dioctanoate [ka] Process c: Heptadecan-9-yl-8-bromooctanoate (0.285 g, 0.62 mmol) (intermediate 2) was added dropwise to a stirred acetonitrile mixture (5 mL) of 9-oxa-3,7-diazabicyclo[3.3.1]nonanedihydrochloride (0.04 g, 0.20 mmol) (intermediate 1), DIPEA (0.142 ml, 0.82 mmol), and potassium iodide (6.60 mg, 0.04 mmol) under argon at 25°C. The resulting mixture was stirred at 80°C for 16 hours. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in siRNA (50 mL), and washed sequentially with saturated aqueous Na₂CO₃ (50 mL) and saturated aqueous NaCl (50 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated to dryness under reduced pressure to obtain the crude product. The resulting residue was purified by flash silica chromatography, with an elution gradient of 0 to 100% in DCM (20% MeOH and 1% NH4OH in DCM). The product fraction was concentrated to dryness under reduced pressure to obtain di(heptadecan-9-yl)8,8'-(9-oxa-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)dioctanoate (0.062 g, 34.9%) as a pale yellow oil. 1H NMR(500MHz,chloroform-d,27°C)0.89(12H,t),1.15-1.72(76H,m),2.28(8H,m),2.41-2.50(4H,m),2.76-2.84(4H,m),3.88(2H,br s),4.87(2H,m).UPLC,ms detection(ES+)([M+H]+)=890.2;RT ELSD=2.27min.

[0083] Example 3. Synthesis of Compound 3 [ka] Reagents: a) NaH, DMF; b) LiCl, H2O, DMSO; c) LiAlH4, THF; d) Acryloyl chloride, TEA, DCM; e) DIPEA, KI, ACN; f) HCl, Dioxane; g) TEA, IPA.

[0084] Intermediate 1: Dimethyl 2,2-dioctylmalonate [ka] Step a: Sodium hydride (3.48 g, 87.05 mmol) was slowly suspended in anhydrous DMF under nitrogen, and the mixture was cooled to 0°C. Dimethyl malonate (4.33 ml, 37.85 mmol) and 1-bromooctane (19.61 ml, 113.54 mmol) (25 mL each in DMF) were successively added, and the mixture was stirred at room temperature for 3 hours. After the reaction, water (250 mL) was added, and the aqueous layer was extracted with (Et2O) (3 × 100 mL). The combined organic layers were successively washed with water (100 mL) and saturated aqueous NaCl (100 mL). The organic layers were dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure to obtain the crude product. The resulting residue was purified by flash silica chromatography with an elution gradient of 0 and 50% siRNA (in hexane). The product fraction was concentrated to dryness under reduced pressure to obtain dimethyl 2,2-dioctylmalonate (8.89 g, 65.9%) as a pale yellow oil. ¹H NMR (500 MHz, chloroform-d, 27°C) showed 0.85-0.92 (6H,3), 1.27 (24H,m), 1.77-1.93 (4H,m), 3.62-3.77 (6H,s).

[0085] Intermediate 2: Methyl 2-octyldecanoate [ka] Step b: A DMSO mixture (80 mL) containing dimethyl 2,2-dioctylmalonate (9.2 g, 25.80 mmol) (intermediate 1), lithium chloride (1.422 g, 33.54 mmol), and water (0.604 g, 33.54 mmol) was stirred under reflux for 24 hours. After cooling to room temperature, water (150 mL) was added to quench the reaction. The reaction mixture was extracted with Et2O (3 × 50 mL). The combined organic layers were sequentially washed with water (3 × 50 mL). The organic layers were dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure to obtain the crude product. The resulting residue was purified by flash silica chromatography with an elution gradient of 0 and 50% siRNA (in hexane). The product fraction was concentrated to dryness under reduced pressure to obtain methyl 2-octyldecanoate (7.30 g, 95%) as a yellow liquid. ¹H NMR (500 MHz, chloroform-d, 27°C): 0.89 (6H, t), 1.18-1.71 (28H, m), 2.28-2.39 (1H, m), 3.68 (3H, s).

[0086] Intermediate 3: 2-octyldecane-1-ol [ka] Process c: To a THF solution (100 mL) of methyl 2-octyldecanoate (7.3 g, 24.45 mmol) (intermediate 2), lithium aluminum hydride (14.67 ml, 29.35 mmol) was added dropwise at 0°C. Subsequently, the reaction mixture was warmed and stirred at room temperature for 24 hours. After completion, 3 M HCl (50 mL) was added to quench the reaction. The reaction mixture was diluted with water (100 mL) and DCM (100 mL). The layers were separated, and the aqueous layer was extracted with DCM (3 × 50 mL). The combined organic layers were washed with saturated aqueous NaCl (100 mL). The organic layers were dried over Na₂SO₄, filtered, and concentrated to dryness under reduced pressure to obtain the crude product. The resulting residue was purified by flash silica chromatography with an elution gradient of 0 and 40% siRNA (in hexane). The product fraction was concentrated to dryness under reduced pressure to obtain 2-octyldecane-1-ol (4.56 g, 68.9%) as a yellow liquid. ¹H NMR (500 MHz, chloroform-d, 27°C): 0.89 (6H, t), 1.16-1.39 (28H, m), 1.42-1.50 (1H, m), 3.55 (2H, br s).

[0087] Intermediate 4: 2-octyldecyl acrylate [ka] Process d: Acryloyl chloride (0.358 ml, 4.44 mmol) was added dropwise to a stirred DCM mixture (20 mL) of 2-octyldecane-1-ol (1 g, 3.70 mmol) (intermediate 3) and TEA (2.113 ml, 15.16 mmol) under nitrogen at 0°C. The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure, diluted with DCM (100 mL), and washed with saturated aqueous NaCl (100 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure to obtain the crude product. The resulting residue was purified by flash silica chromatography with an elution gradient of 0 and 100% siRNA (in hexane). The product fraction was concentrated to dryness under reduced pressure to obtain 2-octyldecyl acrylate (0.850 g, 70.8%) as a pale yellow oil. ¹H NMR (500 MHz, chloroform-d, 27°C): 0.89 (6H, t), 1.23-1.35 (28H, m), 1.61-1.75 (1H, m), 4.07 (2H, d), 5.82 (1H, d), 6.13 (1H, dd), 6.40 (1H, d).

[0088] Intermediate 5: di-tert-butyl((9-oxa-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)bis(butane-4,1-diyl))dicarbamate [ka] Process e: 0.502 g, 1.99 mmol of tert-butyl(4-bromobutyl) carbamate was added in one go under nitrogen to a 5 mL stirred acetonitrile mixture of 9-oxa-3,7-diazabicyclo[3.3.1]nonanedihydrochloride (0.1 g, 0.50 mmol), DIPEA (0.521 mL, 2.98 mmol), and potassium iodide (0.017 g, 0.10 mmol). The resulting mixture was stirred at 80°C for 16 hours. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in siRNA (50 mL), and washed sequentially with saturated aqueous NaHCO3 (50 mL) and saturated aqueous NaCl (50 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure to obtain the crude product. The resulting residue was purified by flash silica chromatography, using a DCM elution gradient of 0 to 100% (20% MeOH and 1% NH4OH in DCM). The product fraction was concentrated to dryness under reduced pressure to obtain di-tert-butyl((9-oxa-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)bis(butane-4,1-diyl))dicarbamate (0.095 g, 40.6%) as a pale yellow oil. ¹H NMR (500 MHz, methanol-d4) δ ppm: 1.4 (18H, s), 1.5-1.6 (8H, m), 2.3 (4H, m), 2.4-2.6 (4H, m), 3.0-3.1 (8H, m), 3.8-3.9 (2H, m), 4.8 (2H, m).

[0089] Intermediate 6: 4,4'-(9-oxa-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)bis(butane-1-amine)tetrahydrochloride [ka] Process f: A HCl solution of dioxane (4M, 10.30 mmol, 2.58 mL) was added to di-tert-butyl((9-oxa-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)bis(butane-4,1-diyl))dicarbamate (0.097 g, 0.21 mmol) (intermediate 5) under an inert atmosphere, and the mixture was stirred at room temperature for 16 hours. After rotary evaporation, the reaction residue was diluted twice with dioxane (4 mL) and the solvent was removed under reduced pressure. The solid was concentrated to dryness under reduced pressure to obtain 4,4'-(9-oxa-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)bis(butane-1-amine)tetrahydrochloride as a white solid. 1H NMR (500MHz, methanol-d4) δ ppm 1.7(4H,m)1.7-1.8(4H,m)2.7-2.8(4H,m)2.9-3.0(8H,m)3.3-3.3(2H,m)3.5(4H,br d)4.1(2H,br s).

[0090] Compound 3: Tetrakis(2-octyldecyl)3,3',3'',3'''-(((9-oxa-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrapropionate [ka] Process g: 2-Octyldecyl acrylate (0.468 g, 1.44 mmol) (intermediate 4) was added all at once to a stirred iPrOH mixture (4 mL) of 4,4'-(9-oxa-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)bis(butane-1-amine)tetrahydrochloride (0.05 g, 0.12 mmol) (intermediate 5) and TEA (0.134 ml, 0.96 mmol). The resulting mixture was stirred at 80°C for 3 days. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in SiO (50 mL), and washed sequentially with saturated aqueous NaHCO3 (50 mL) and saturated aqueous NaCl (50 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure to obtain the crude product. The resulting residue was purified by flash silica chromatography, with an elution gradient of 0 to 100% in DCM (20% MeOH and NH4OH in DCM). The product fraction was concentrated to dryness under reduced pressure to obtain tetrakis(2-octyldecyl)3,3',3'',3'''-(((9-oxa-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrapropionate (0.027 g, 14.06%) as an orange oil. 1H NMR(500MHz, methanol-d4, 27°C)0.84-0.99(24H,t),1.21-1.41(116H,m),1.46-1.56(4H,m),1.66(8H,m), 2.43-2.52(10H,m),2.73-2.82(10H,m),2.98-3.08(4H,m),3.42-3.52(4H,m),4.00(8H,d),4.18(2H,br s).MS detection (ES+)([M+H]+)=1568.4.

[0091] Example 4. Synthesis of Compound 4 [ka] Reagents: a) EDC, DIPEA, DMAP, DCM; b) K2CO3, KI, ACN; c) 4M HCl, Dioxane; d) EDC, DIPEA, DMAP, DCM; e) DIPEA, KI, ACN

[0092] Intermediate 1: Heptadecane-9-yl-8-bromooctanoate [ka] Step a: EDC (0.785 g, 4.09 mmol) was added in one go under argon to a stirred DCM mixture (15 mL) containing 8-bromooctanoic acid (0.522 g, 2.34 mmol), heptadecane-9-ol (0.5 g, 1.95 mmol), DMAP (0.048 g, 0.39 mmol), and DIPEA (1.396 ml, 7.99 mmol). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in siRNA (50 mL), and washed sequentially with saturated aqueous NaHCO3 (50 mL) and saturated aqueous NaCl (50 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure to obtain the crude product. The resulting residue was purified by flash silica chromatography with an elution gradient of 0 and 20% siRNA (in hexane). The product fraction was concentrated to dryness under reduced pressure to obtain heptadecan-9-yl-8-bromooctanoate (0.714 g, 79%) as a colorless oil. ¹H NMR (500 MHz, chloroform-d, 27°C): 0.89 (6H,t), 1.27-1.70 (36H,m), 1.77-1.94 (2H,m), 2.21-2.37 (2H,t), 3.41 (2H,t), 4.78-4.95 (1H,m).

[0093] Intermediate 2: tert-butyl7-(8-(heptadecane-9-yloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-carboxylate [ka] Step b: Heptadecan-9-yl-8-bromooctanoate (0.808 g, 1.75 mmol) (intermediate 1) was added dropwise to a stirred acetonitrile mixture (10 mL) of tert-butyl-9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-carboxylate hydrochloride (0.386 g, 1.46 mmol), potassium carbonate (0.423 g, 3.06 mmol), and potassium iodide (0.048 g, 0.29 mmol) under argon. The resulting mixture was stirred at 80°C for 16 hours. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in siRNA (100 mL), and washed sequentially with saturated aqueous Na₂CO₃ (100 mL) and saturated aqueous NaCl (100 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated to dryness under reduced pressure to obtain the crude product. The resulting residue was purified by flash silica chromatography, with an elution gradient of 0 to 100% in DCM (20% MeOH and 1% NH4OH in DCM). The product fraction was concentrated to dryness under reduced pressure to obtain tert-butyl 7-(8-(heptadecane-9-yloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-carboxylate (0.747 g, 84%) as a pale yellow oil. 1H NMR(500MHz,chloroform-d,22°C)0.88(6H,t),1.20-1.67(47H,m),2.02-2.19(2H,m),2.22-2.31(2H,t),2.3 5-2.49(2H,m),2.77-2.96(2H,m),3.18-3.38(2H,m),3.68-3.83(2H,m),3.92-4.17(2H,m),4.87(1H,m).

[0094] Intermediate 3: Heptadecan-9-yl8-(9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-yl)octanoate dihydrochloride [ka] Process c: HCl (2.250 ml, 9.00 mmol) in dioxane was added dropwise to a stirred dioxane mixture (10 mL) of tert-butyl 7-(8-(heptadecan-9-yloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-carboxylate (0.548 g, 0.90 mmol) (intermediate 2) under argon at 0°C. The resulting mixture was warmed and stirred at room temperature for 4 hours. The reaction mixture was concentrated to dryness under reduced pressure and washed with dioxane (3 × 50 mL) to obtain heptadecan-9-yl 8-(9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-yl)octanoate dihydrochloride (0.472 g, 96%) as a pale yellow oil. ¹H NMR (500 MHz, methanol-d4, 27°C): 0.90 (6H, t), 1.29 (38H, m), 2.24-2.39 (2H, t), 3.49-3.59 (3H, t), 4.85-4.91 (1H, m).

[0095] Intermediate 4: (Z)-nona-2-en-1-il8-bromooctanoart [ka] Process d: EDC (2.83 g, 14.76 mmol) was added in one go under argon to a stirred DCM mixture (15 mL) containing 8-bromooctanoic acid (2.353 g, 10.55 mmol), DIPEA (3.68 ml, 21.09 mmol), (Z)-nona-2-en-1-ol (1 g, 7.03 mmol), and DMAP (0.172 g, 1.41 mmol). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in siRNA (100 mL), and washed sequentially with saturated aqueous NaHCO3 (100 mL) and saturated aqueous NaCl (100 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure to obtain the crude product. The resulting residue was purified by flash silica chromatography with an elution gradient of 0 and 100% siRNA (in hexane). The product fraction was concentrated to dryness under reduced pressure to obtain (Z)-nona-2-en-1-yl-8-bromooctanoate (1.930 g, 79%) as a pale yellow oil. 1H NMR (500 MHz, chloroform-d, 27 o C)0.79-0.95(3H,t),1.21-1.50(14H,m),1.58-1.69(2H,m),1.72-1.93(2H,m) ,2.11(2H,m),2.31(2H,t),3.31-3.65(2H,t),4.63(2H,d),5.48-5.73(2H,m).

[0096] Compound 4: Heptadecan-9-yl(Z)-8-(7-(8-(nona-2-en-1-yloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonan-3-yl)octanoate [ka] Process e: Heptadecan-9-yl-8-(9-oxa-3,7-diazabicyclo[3.3.1]nonan-3-yl)octanoate dihydrochloride (intermediate 3), dissolved in 1:1 acetonitrile / CPME (10 mL) and DIPEA (0.212 mL, 1.21 mmol), was added under nitrogen at 20°C. The resulting mixture was stirred at 20°C for 30 minutes. (Z)-nonan-2-en-1-yl-8-bromooctanoate (0.123 g, 0.35 mmol) (intermediate 4) was added dropwise to the stirred solution under nitrogen. The resulting mixture was heated to 80°C and stirred under nitrogen for 18 hours. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in siRNA (50 mL), and washed sequentially with saturated aqueous NaHCO3 (50 mL) and saturated aqueous sodium chloride (50 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure to obtain the crude product. The resulting residue was purified by flash silica chromatography, with an elution gradient of 0 to 100% in DCM (20% MeOH and 1% NH4OH in DCM). The product fraction was concentrated under reduced pressure to obtain heptadecan-9-yl(Z)-8-(7-(8-(nona-2-en-1-yloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonan-3-yl)octanoate (0.214 g, 93%) as a pale yellow oil. 1H NMR (500MHz, methanol-d4)0.86-1.00(9H,t),1.23-1.46(44H,m),1.56(8H,m),1.64(4H,m),2.15(2H,q),2.24-2.38(8H,m),2.51(4H,br d),2.92(4H,br d),3.91(2H,br s),4.64(2H,d),4.88-4.93(1H,m),5.49-5.71(2H,m).UPLC,ms detection(ES+)([M+H]+)=776.0Da;RT ELSD=1.88 min.

[0097] Example 5. Synthesis of Compound 5 [ka] Reagents a) Triethyl phosphonoacetate, NaH, THF; b) Pt(IV)O2, H2, CHCl3 / MeOH; c) LiAlH4, THF; d) EDC, DIPEA, DMAP, DCM; e) DIPEA, KI, ACN

[0098] Intermediate 1: Ethyl 3-octylundec-2-enoate [ka] Step a: Triethyl phosphonoacetate (12.59 ml, 62.88 mmol) was slowly added to a stirred THF solution (50 mL) of sodium hydride (2.51 g, 62.88 mmol) under nitrogen at 0°C. The resulting mixture was stirred at 0°C for 30 minutes. Heptadecane-9-one (2 g, 7.86 mmol) was added to the mixture and the mixture was heated to 30°C under nitrogen. The resulting mixture was stirred under reflux for 18 hours. After cooling to room temperature, the reaction mixture was quenched with water (100 mL), extracted with toluene (3 × 50 mL), and the combined organic layer was dried over MgSO4, filtered, and concentrated to dryness under reduced pressure to obtain a yellow oil. The resulting residue was purified by flash silica chromatography with an elution gradient of 0 and 40% toluene (in hexane). The purity of each fraction was determined by 1H NMR. The combined product fraction was concentrated to dryness under reduced pressure to obtain ethyl 3-octyl undeca-2-enoate (1.850 g, 72.5%) as a colorless oil. ¹H NMR (500 MHz, chloroform-d): 0.89 (6H, t), 1.21-1.53 ​​(27H, m), 2.13 (2H, t), 2.53-2.65 (2H, t), 4.15 (2H, q), 5.62 (1H, s).

[0099] Intermediate 2: Ethyl 3-octyl undecanoate [ka] Step b: Ethyl 3-octyl undecane-2 enoate (2.0 g, 6.16 mmol) (intermediate 1) and platinum(IV) oxide (0.028 g, 0.12 mmol) were stirred under a hydrogen balloon at atmospheric pressure for 16 hours. The reaction mixture was filtered through Celite. The resulting residue was purified by flash silica chromatography with an elution gradient of 0 and 40% siRNA (in hexane). Separation was difficult due to overlap with the heptadecane-9-one starting material. The purity of each fraction was determined by 1H NMR. The combined product fraction was concentrated to dryness under reduced pressure to obtain ethyl 3-octyl undecane (1.540 g, 77%) as a colorless oil. ¹H NMR (500 MHz, chloroform-d, 27°C): 0.83-0.95 (6H, t), 1.16-1.39 (31H, m), 1.79-1.92 (1H, br t), 2.22 (2H, d), 4.13 (2H, q).

[0100] Intermediate 3: 3-octylundecane-1-ol [ka] Process c: Lithium aluminum hydride (7.75 ml, 7.75 mmol) was slowly added under nitrogen at 0°C to a stirred THF solution (20 mL) of ethyl 3-octyl undecanoate (2.3 g, 7.04 mmol) (intermediate 2). The resulting mixture was stirred at room temperature for 18 hours. After completion, the reaction mixture was cooled to 0°C, quenched with 3 M HCl (100 mL), extracted with siRNA (3 × 50 mL), and the combined organic layer was dried over MgSO4, filtered, and concentrated to dryness under reduced pressure to obtain a yellow oil. The resulting residue was purified by flash silica chromatography with an elution gradient of 0 and 40% siRNA (in hexane). The product fraction was concentrated to dryness under reduced pressure to obtain 3-octyl undecane-1-ol (1.354 g, 67.6%) as a colorless oil. ¹H NMR (500 MHz, chloroform-d, 27°C): 0.89 (6H, t), 1.18-1.36 (28H, m), 1.38-1.46 (1H, br t), 1.53 (2H, q), 3.67 (2H, t).

[0101] Intermediate 4:3-octylundecyl8-bromooctanoate [ka] Process d: EDC (0.707 g, 3.69 mmol) was added in one go under argon to a stirred DCM mixture (10 mL) containing 8-bromooctanoic acid (0.470 g, 2.11 mmol), DIPEA (0.645 mL, 3.69 mmol), 3-octylundecane-1-ol (0.5 g, 1.76 mmol) (intermediate 3), and DMAP (0.043 g, 0.35 mmol). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in siRNA (50 mL), and washed sequentially with saturated aqueous NaHCO3 (100 mL) and saturated aqueous NaCl (50 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure to obtain the crude product. The resulting residue was purified by flash silica chromatography with an elution gradient of 0 and 100% RINKAN (in hexane). The product fraction was concentrated to dryness under reduced pressure to obtain 3-octylundecyl 8-bromooctanoate (0.545 g, 63.3%) as a pale yellow oil. ¹H NMR (500 MHz, chloroform-d, 27°C) showed 0.83-0.98 (6H,t), 1.20-1.49 (35H,m), 1.53-1.70 (4H,m), 1.79-1.92 (2H,m), 2.30 (2H,t), 3.41 (2H,t), 4.03-4.15 (2H,t).

[0102] Compound 5: Bis(3-octylundecyl)8,8'-(9-oxa-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)dioctanoate [ka] Process e: 3-Octylundecyl8-bromooctanoate (0.302 g, 0.62 mmol) (intermediate 4) was added dropwise to a stirred acetonitrile mixture (5 mL) of 9-oxa-3,7-diazabicyclo[3.3.1]nonanedihydrochloride (0.04 g, 0.20 mmol), DIPEA (0.142 ml, 0.82 mmol), and potassium iodide (6.60 mg, 0.04 mmol) under argon at 25°C. The resulting mixture was stirred at 80°C for 16 hours. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in siRNA (50 mL), and washed sequentially with saturated aqueous Na2CO3 (50 mL) and saturated aqueous NaCl (50 mL). The organic layer was dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure to obtain the crude product. The resulting residue was purified by flash silica chromatography, with an elution gradient of 0 to 100% in DCM (20% MeOH and 1% NH4OH in DCM). The product fraction was concentrated to dryness under reduced pressure to obtain bis(3-octylundecyl)8,8'-(9-oxa-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)dioctanoate (0.054 g, 28.6%) as a pale yellow oil. 1H NMR (500MHz, chloroform-d, 27°C) 0.89 (12H, t), 1.16-1.69 (80H, m), 1.73-1.85 (2H, m), 2.20-2.26 (4H, br t),2.26-2.31(4H,t),2.40-2.52(4H,m),2.82(4H,br d),3.82-3.94(2H,br t),4.09(4H,t).UPLC,ms detection(ES+)([M+H]+)=945.7;RT ELSD=2.43 min.

[0103] Example 6. Preparation of lipid nanoparticle (LNP) formulation containing eGFP mRNA LNPs were prepared using a microfluidic setup and NanoAssemblr (Precision NanoSystems Inc.). Briefly, lipid stocks were dissolved in ethanol and mixed in an appropriate molar ratio to obtain a lipid concentration of 12.5 mM. LNP size was determined by DLS measurement using a Zetasizer Nano ZS from Malvern Instruments Ltd. The number-weighted particle size distribution was calculated using a particle refractive index of 1.45. The characterization of the LNPs is shown in Table 1. Lipid solutions (99.5%) in ethanol were prepared using four different lipid components: ionizable lipids, namely MC3, MOD5, compound 1, compound 2, or compound 4; cholesterol (Sigma-Aldrich); DSPC (distearoylphosphatidylcholine, Avanti Polar Lipids Inc.); and polymer conjugate lipids. The lipid ratio in all experiments was ionizable lipid / cholesterol / DSPC / polymer conjugate lipid (50 / 38.5 / 10 / 1.5 mol%). The total lipid concentration in all experiments was 12.5 mM. MOD5 is an ionizable lipid with the structure shown below; a more detailed explanation can be found in Sabnis et al, Mol Therapy, Vol 26, 6, 2018, 1509-1519. [ka]

[0104] A citrate buffer solution of eGFP mRNA (purchased from TriLink Biotechnologies) was prepared by mixing mRNA dissolved in MilliQ water, 100 mM citrate buffer (pH 3), and MilliQ water to obtain a 50 mM citrate solution. The mRNA and lipid solutions were mixed using a NanoAssemblr (Precision Nanosystems, Vancouver, BC, Canada) microfluidic mixing system at a mixing ratio of aqueous solution:EtOH = 3:1 and a constant flow rate of 12 mL / min. At the time of mixing, mRNA in the citrate buffer solution was prepared so that the ratio of nitrogen atoms on the ionizable lipid to phosphorus atoms on the mRNA chain (N / P ratio) was 3:1 or 5:1 (see Table 1). The LNPs were dialyzed overnight using a Thermo Scientific Slide-A-Lyzer G2 dialysis cassette for a sample volume of 500 × with a molecular weight cutoff of 10 K.

[0105] The first 0.2–0.35 mL and the final 0.05–0.1 mL of the prepared LNP suspension were discarded, while the remaining volume was collected as the sample fraction. The size of mRNA lipid nanoparticles was determined by dynamic light scattering measurement using a Malvern Instruments Ltd Zetasizer Nano ZS to directly obtain the z-average particle size. The number-based particle size distribution and average were calculated using a particle refractive index of 1.45.

[0106] mRNA encapsulation and concentration were determined using the Ribo-Green assay. Encapsulation in all samples was typically 90–99%. Final mRNA concentration and encapsulation efficiency percentage (%EE) were measured using the Quant-it Ribogreen Assay Kit (ThermoFischer Scientific Inc.) with Triton-X100 for LNP disruption. mRNA encapsulation efficiency was calculated according to the following formula:

number

[0107] Table 1 summarizes the characterization of LNP formulations containing MC3, MOD5, compound 1, compound 2, or compound 4.

[0108] [Table 1]

[0109] DMPE-PEG2000 is dimyristoylphosphatidylethanolamine-poly(ethylene glycol)2000 (obtained from NOF Corporation).

[0110] DMG-PEG2000 is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

[0111] Example 7. In vivo intratracheal administration of LNP-eGFP mRNA preparation to rats. In vivo studies were conducted at AAALAC-accredited animal facilities in AstraZeneca, Gothenburg, and Sweden, under the approval of the Animal Ethics Committee of Gothenburg (no. 82-2015). Male Wistar Han rats were purchased (Charles River Germany Limited) and, immediately upon arrival, placed in cages of four on wood shavings with feed (R70 Rat and Mouse chow (Lantmaennen, Stockholm, Sweden)), with free access to drinking water drawn from the water main. The animals were approximately 10 weeks old at the start of administration. The environment was maintained at a target temperature of 19–24°C and a relative humidity of 40–70% with a 12-hour light / dark cycle. The animals were acclimated to the conditions of the containment area for at least 5 days before any experimental procedure. Animals that received the inhalation dose were further acclimated to the inhalation-restraint procedure by gradually increasing the period of exposure to the restraint procedure up to the maximum expected duration of each study, up to a maximum of 5 days before the start of administration.

[0112] The eGFP mRNA compound 1 LNP preparation was administered by a single inhalation dose of 0.02 mg / eGFPkg to the target lung, or by a single intratracheal administration. A group of the same size animals was used as a placebo control, exposed to phosphate-buffered saline by inhalation or intratracheal installation. Figure 3 shows the eGFP expression level in rat lungs 24 hours after intratracheal administration, and Figure 4 shows the BALF neutrophil concentration in rat BALF 24 hours after intratracheal administration. Figure 6 shows the eGFP expression level in rat lungs 5 ​​and 24 hours after inhalation administration, and Figure 7 shows the BALF neutrophil concentration in rat BALF 24 hours after inhalation administration. Furthermore, immunohistochemistry (IHC) tests showed eGFP expression not only in macrophages but also in type 1 epithelial cells (see Figures 5A and 5B).

[0113] The eGFP mRNA formulations MC3, MOD5, compound 2, and compound 4 LNP formulations described in Example 6 were each administered by a single intratracheal dose of 0.1 mg / eGFPkg to the target lung. A group of the same size animals was used as a placebo control, exposed to phosphate-buffered saline via intratracheal placement. Figure 1 shows the eGFP expression levels in rat lungs at 24 hours, and Figure 2 shows the BALF neutrophil concentration in rat BALF at 24 hours.

[0114] Inhalation administration Aerosols were generated using an Aerogen Solo vibrating mesh nebulizer (Galway Ireland). The nebulizer was filled with 5.6 mL of vehicle or LNP preparation containing eGFP mRNA and atomized at approximately 60 μl / min for the duration of administration. Rats were placed in rodent restraints and then in an inhalation administration system designed by AstraZeneca. Animals were monitored throughout the experimental procedure for any signs of post-administration pathological effects without clinical findings or any notable abnormal changes in body weight. 24 hours after the final dose, rats were euthanized by isoflurane sedation, followed by vena cava cutting and cardiac removal.

[0115] Intratracheal administration Rats were anesthetized with an isoflurane mixture (air / oxygen and 3.5% isoflurane), placed in a supine position at a 30-40° angle, and then the eGFP mRNA or vehicle from the LNP preparation was infused using a modified metal cannula with a bolus valve at the top. After administration, the rats were placed in a cage in a supine position with their heads up until consciousness was restored. The animals were monitored throughout the experimental procedure for any signs of post-administration pathological effects without clinical findings or any notable abnormal changes in body weight. Twenty-four hours after the final dose, the rats were euthanized by isoflurane sedation, followed by vena cava transection and cardiac removal.

[0116] BAL sampling Broncheo-alveolar lavage (BAL) was performed by manually perfusing the entire lung. After exposing the trachea, a polyethylene tube (PE120) was inserted and ligated with 1-0 silk sutures. The tube was connected to a syringe pre-filled with 4 ml of PBS at room temperature, and the PBS was slowly injected into the lung. The fluid was recollected by slow aspiration into the syringe. This procedure was performed twice. The final BAL fluid was transferred to a test tube (4 ml, polypropylene [PP]).

[0117] The tubes containing the BAL samples were weighed (assuming 1 gram equals 1 ml). The BALs were cooled on ice until centrifugation (Hettich Rotanta 46R, 1200 rpm, 10 minutes, 4°C). After centrifugation, the supernatant was collected and divided into 96-well plates (0.15 mL / well, 5 plates), and held on dry ice (0.1 ml / well). The plates were stored at a minimum of -75°C for any further analysis. The cell pellets were resuspended in 0.5 ml of PBS and held on ice for cell counting. Processing was performed immediately. The total number of cells and the difference in cell count were counted using the automated SYSMEX XT-1800i Vet (Sysmex Corporation, Kobe, Japan).

[0118] Organ collection The right lung lobe was ligated and separated, excised from non-lung tissue, blood clots were removed, and it was rinsed with saline. The right lobe was detached using sutures, released, rinsed with PBS to remove any blood contamination. The upper and middle lobes were weighed and collected in 7 mL precellys tubes for eGFP mRNA analysis. The lower vena cava lobe was weighed and collected in 7 mL precellys tubes for eGFP protein analysis. All right lobe samples were snap-frozen in liquid nitrogen. Samples were conserved and stored at a minimum of -80 °C until further processing for analysis. The left lobe was inflated with formalin and placed in excess formalin in a container with a plastic lid.

[0119] Example 8. In vivo intravenous administration of LNP-eGFP mRNA formulation to mice In accordance with EU Directive 2010 / 63 / EU, all studies were conducted in accordance with Home Office U.K. ethical and regulatory standards and under the authority of a UK Project license, reviewed and approved by the Animal Welfare and Ethical Review Body (AWERB). Wild-type female BALB / c mice (6 - 8 weeks old) were purchased from Charles River, UK and housed in the AstraZeneca animal facility. All mice were injected with 100 μl of MC3, and compound 2 and compound 3 LNP formulations were prepared by a similar procedure as described in Example 6. Characterization is shown in Table 2. The LNP formulation containing 0.4 mg / eGFP mRNA kg was administered via tail vein injection. Mice were euthanized 24 hours after administration, and the following organs: liver, spleen, lung, kidney, and heart were extracted. Organs were collected in cryovials and snap-frozen. The expression levels of eGFP in mouse organs at 24 hours are shown in Figure 8 (liver), Figure 9 (spleen), Figure 10 (lung), Figure 11 (kidney), and Figure 12 (heart).

[0120]

Table 2

[0121] Tissue homogenization Organ / tissue homogenization and cell lysis were performed prior to ELISA. Briefly, the organ / tissue was thawed on ice, rinsed with 1×DPB to remove any blood. The organ / tissue was cut into slices of approximately 100 - 200 mg and transferred to a 2 mL tube containing protease and phosphatase inhibitors (ThermoFisher 78445) and 0.5 - 1 mL of ice-cold 1×Cell Extraction Buffer PTR (from ELISA kit ab171581) with 5 mm stainless steel beads (Qiagen). The organ / tissue sample was homogenized on a Tissue Lyser II (Qiagen) set at a frequency of 30 1 / s for 3 minutes, then transferred to a clean tube and incubated on ice for 20 minutes. Subsequently, the sample was centrifuged at 18000×g for 20 minutes at 4°C, and the clear homogenate was transferred to a clean tube, aliquoted, and stored at -80°C until further use.

[0122] Quantification of eGFP in organs by ex vivo ELISA eGFP expression levels in the liver, spleen, lungs, kidneys, and heart were measured using the GFP SimpleStep ELISA® Kit (ab171581) according to the manufacturer's instructions. All reagents were equilibrated to room temperature before use. The following buffers were prepared: 1× cell extraction buffer PTR with protease and phosphatase inhibitors (ThermoFisher 78445), 1× wash buffer PT, 1× antibody diluent, antibody cocktail, and EGFP standard curve. If further dilution of tissue homogenates was required, this was done using ice-cold 1× cell extraction buffer PTR. Subsequently, 50 μl of diluted sample and eGFP standard were added to each well, followed by 50 μl of antibody cocktail. The plate was then sealed and incubated at room temperature for 1 hour on a plate shaker set to 400 rpm. After incubation, each well was washed three times with 350 μl of 1× wash buffer PT. After the final washing step, excess liquid was removed by blotting the plate onto a clean paper towel. Next, 100 μl of TMB substrate was added to each well, the plate was covered with aluminum foil, and incubated for 10 minutes on a plate shaker set to 400 rpm. Finally, 100 μl of stop solution was added to each well, the plate was shaken for 1 minute, and the absorbance at 450 nm was recorded using an Envision microplate reader (Perkin Elmer). The eGFP standard curve was fitted to a sigmoid 4PL curve using a GraphPad Prism 9, and the ng eGFP protein was estimated from the curve.

[0123] eGFP levels were normalized by total tissue protein using a BCA assay (Pierce 23225) according to the manufacturer's instructions. Briefly, 25 μl of diluted tissue homogenate or BSA standard was added to each well, followed by 200 μl of working reagent, and the contents were rapidly mixed on a plate shaker for 30 seconds. The plate was then covered and incubated at 37°C for 30 minutes. After the plate cooled to room temperature, absorbance at 562 nm was recorded using an Envision microplate reader (Perkin Elmer). Total protein (mg) was estimated from the BSA standard curve. Data are reported as mean ng eGFP / mg ± SD of tissue protein.

[0124] Example 9. In vivo intravenous administration of a co-formulation of hEPO and luciferase mRNA to mice. Preparation of lipid nanoparticle (LNP) formulations containing hEPO and luciferase mRNA: Solutions of hEPO (human EPO) mRNA and luciferase mRNA (both purchased from TriLink Biotechnologies) were mixed in a 1:1 ratio in citrate buffer by combining mRNA dissolved in nuclease-free water, 100 mM citrate buffer (pH 3), and nuclease-free water to obtain a 50 mM citrate solution. Lipid solutions (99.5%) in ethanol were prepared with four different lipid components: ionizable lipids, namely MC3 or compound 2 or compound 3; cholesterol (Sigma-Aldrich); DSPC (distearoylphosphatidylcholine, Avanti Polar Lipids Inc); and DMPE-PEG2000 (dimiristoylphosphatidylethanolamine-poly(ethylene glycol) 2000, NOF Corporation) using a similar procedure to that described in Example 6. The characterization of LNPs is shown in Table 3. The total lipid concentration in all experiments was 12.5 mM. mRNA and lipid solutions were mixed using a NanoAssemblr (Precision Nanosystems, Vancouver, BC, Canada) microfluidic mixing system at a mixing ratio of aqueous solution:EtOH = 3:1 and a constant flow rate of 12 mL / min. At the time of mixing, the molar ratio of ionizable lipids to phosphorus atoms on the mRNA chain was equal to 6. The first 0.2–0.35 mL and the final 0.05–0.1 mL of the prepared LNP suspension were discarded, while the remaining volume was collected as the sample fraction. The sample volume was immediately transferred to a Slide-a-lyzer G2 dialysis cassette (10000 MWCO, ThermoFischer Scientific Inc.) and dialyzed overnight in PBS (pH 7.4) at 4°C. The volume of PBS buffer was 500–1000 times the volume of the sample fraction. The following day, the sample was taken from the cassette using a syringe and needle. Next, the needle was replaced with a 0.2 μm syringe filter, and the sample was filter-sterilized in a sterile tube.From this sample, 10 μL was diluted with 990 μL of PBS buffer at pH 7.4 and used to measure the intensity-average particle size and polydispersity index (PDI) using a Malvern ZetaSizer (ZetaSizer Nano ZS, Malvern Instruments Inc., Westborough, MA, USA). The final mRNA concentration and encapsulation efficiency percentage (%EE) were measured using a Quant-it Ribogreen Assay Kit (ThermoFischer Scientific Inc.) with Triton-X100 to disrupt LNPs.

[0125] [Table 3]

[0126] Animal experiments: The experiments were performed in wild-type (WT) and LDLr knockout (KO) mice. All experiments were conducted in accordance with Swedish Animal Welfare and approved by the Ethical Committee for Laboratory Animals in Gothenburg, Sweden. Equal numbers of male and female C57bl6 LDLR- / - mice and their wild-type littermates were raised in-house. Twenty C57bl6 LDLR- / - and forty WT mice were included in the study (N≧5 per group). Animals were used at an average body weight of 25g. Animals were restrained and intravenously injected with 0.15 mg / hEPO mRNAkg + 0.15 mg / Luc mRNAkg (0.3 mg / total mRNAkg) co-formulated in the aforementioned LNP, or PBS as a control. Blood samples were collected in EDTA tubes via tail vein collection at t=6 and via orbital plexus collection at t=24. Mice were subcutaneously injected with 5 mg / kg of luciferin substrate (PerkinElmer's RediJect D-Luciferin Bioluminescent Substrate) 20 minutes before the end of the study. The heart, lungs, spleen, and liver were collected immediately after the end of the study and imaged using IVIS Spectrum (PerkinElmer). Total luminescence from each organ was quantified using LivingImage (PerkinElmer). The resulting IVIS images are shown in Figure 14. For hEPO analysis, blood samples were spun down and plasma was analyzed using the Human Erythropoietin Quantikine IVD ELISA Kit (R&D System). Data were reported as hEPO in ng / ml, and samples were analyzed in three replicates. Figure 13 shows the expression of luciferase protein in the livers of WT and KO mice 24 hours after intravenous administration of the LNP formulation containing MC3, compound 2, and compound 3, while Figure 15 shows the hEPO protein concentration in the plasma of WT and KO mice 6 hours after intravenous administration of the LNP formulation containing MC3, compound 2, and compound 3.

[0127] Example 10. In vivo intrastriatal administration of luciferase mRNA LNP preparation to mice. LNP formulations containing luciferase mRNA, MOD5 and compound 5 (AZ8608), were prepared by similar procedures described in Examples 6 and 9. Here, the lipid components were MOD5 / cholesterol / DSPC / DMPE-PEG = 50 / 38.5 / 10 / 1.5 mol%; compound 5 / cholesterol / DSPC / DMPE-PEG = 42.5 / 40 / 16 / 1.5 mol%. The characterization of the LNPs is shown in Table 4.

[0128] Our proprietary reporter mice (LoxP Luc) containing a cyclic recombinase enzyme (Cre)-inducible luciferase expression cassette were generated by standard randomized gene insertion transgenesis. Before LNP administration, the LoxP Luc reporter mice were anesthetized with isoflurane 4.0 / 1.5 O2 i. The mice were subcutaneously injected under the skin of the neck with 2 units (20 μl) of Comforion Vet (10 mg / ml; 100 μg / mouse) diluted with insulin syringe (BD U-100). Next, the fur was shaved, the skull was washed with a Decutan swab (4% chlorhexidine), and the animals were placed on a stereotactic board with a heating pad. The mice were properly attached, the skull was adjusted to a horizontal position, and the skull was covered with a perforated plastic film. During the surgical procedure, a 6-8 mm incision was made within the midline of the skull, the area around the anterior vertex was dried, and after calculating the precise coordinates, the drill was placed on that location. One or two small holes were drilled into the skull on both sides of the midline, and a Hamilton syringe for contact chemotaxis was attached to the pump. The syringe was placed over the drilled hole and slowly lowered deep to the right, injecting 5 μl of LNP solution at a flow injection pump rate of 0.5 μl / min per hole. Mice were administered 1.7 mg RNA (Cas9 mRNA / gRNA1 / gRNA2 50 / 25 / 25 w / w) by two injections of 5 mL per mouse (one injection per hemisphere) into the focal striatum. The injector was held in place for 3 minutes after each injection, then the syringe was slowly raised and both the pump and syringe were removed. To close the skin over the perforation, 2-3 stitches (Suture Polysorb 6-0) were used along with tissue adhesive placed along the incision. Animals were observed and weighed daily. The recovery process and wound healing were closely monitored for 3-5 days. After 7 days, ex vivo brain luciferase analysis was performed on both hemispheres. Figure 16 shows the mean cortical and striatal Luc expression levels in LoxP Luc reporter mice after intrastriatal administration of LNP formulations containing MOD5 and compound 5.

[0129] [Table 4]

[0130] Protein Extraction and Luciferase Assay One week after LNP administration, LoxP Luc reporter mice were sacrificed, and the whole brain was dissected. The striatum and cortex were isolated, weighed, placed in separate tubes, and frozen until further analysis. To measure the LNP-mediated functional delivery level, proteins were extracted from mouse brain tissues using a Qiagen TissueLyser according to the manufacturer's recommendations. The tissue was crushed, homogenized by moving a pestle, centrifuged, and the insoluble tissue residue was removed from the suspension. The supernatant was transferred to separate tubes, and the protein concentration was determined by Bradford assay experiment. For the luciferase assay, 20 μl of each supernatant was added to a microplate (white OptiPlate-96), and 100 μl of D-luciferin was added to each well. The samples were mixed, and the resulting luminescence signal was measured using a luminometer. The luminescence signal was normalized to the tissue weight. The present invention includes the following embodiments. [Section 1] Equation (I): [C1] TIFF0007909600000056.tif85161 A compound of or a pharmaceutically acceptable salt thereof, wherein the formula is a and b are, independently, 3, 4, or 5; c and d are independently 1, 2, or 3; e and f are independently 0, 1, or 2; X 1 is methylene or [C2] TIFF0007909600000057.tif41161 And, X 2 is methylene or [C3] TIFF0007909600000058.tif44161 And, g and h are independently 1, 2, or 3; i and j are independently 0, 1, or 2; Y 1 and Y 2 Each of them is independently a linear C 7~10 Alkyl, linear C 7~10 Alkenil, or [C4] TIFF0007909600000059.tif27161 And, Z 1 and Z 2 Each of them is independently a linear C 7~10 Alkyl, linear C 7~10 Alkenil, or [5] TIFF0007909600000060.tif27161 And, R 1 and R 2 Each of them is independently a linear C 7~10 It is alkyl; R 3 and R 4 Each of them is independently a linear C 7~10 It is alkyl. compound. [Section 2] X 1 and X 2 The compound described in item 1, in which both are methylene. [Section 3] The compound according to item 1 or 2, wherein a and b are both 4; and c and d are both 2. [Section 4] A compound described in any one of items 1 to 3, wherein both e and f are 0. [Section 5] Y 1 is a linear C 7~10 Alkyl or linear C 7~10 It is an alkenil; Y 2 teeth [6] TIFF0007909600000061.tif28161 and; R 1 and R 2 Each of them is independently a linear C 7~10 A compound that is alkyl, as described in any one of items 1 to 4. [Section 6] Y 1 and Y 2 Each of them operates independently. [7] TIFF0007909600000062.tif27161 and; R 1 and R 2 Each of them is independently a linear C 7~10 A compound that is alkyl, as described in any one of items 1 to 4. [Section 7] A compound according to any one of claims 1 to 3, wherein e and f are each independently 1 or 2. [Section 8] Y 1 and Y 2 Each of them operates independently. [8] TIFF0007909600000063.tif27161 and; R 1 and R 2 Each of them is independently a linear C 7~10 A compound that is alkyl, as described in item 7. [Section 9] Formula (II): [9] TIFF0007909600000064.tif85161 It is a compound of the formula, in which, k is 0, 1, or 2; Y 1 is a linear C 7~10 Alkyl, linear C 7~10 Alkenil, or [C10] TIFF0007909600000065.tif27161 And, Y 2 teeth [C11] TIFF0007909600000066.tif27161 And, R 1 and R 2 Each of them is independently a linear C 7~10 It is alkyl. A compound described in any one of items 1 to 6 or 8. [Section 10] X 1 teeth [C12] TIFF0007909600000067.tif42161 and; X 2 teeth [C13] TIFF0007909600000068.tif42161 The compound described in item 1. [Section 11] The compound described in item 10, wherein a and b are both 4; and c and d are both 2. [Section 12] The compound according to item 10 or 11, wherein e and f are each independently 1 or 2. [Section 13] The compound described in any one of items 10 to 12, wherein both g and h are 2. [Section 14] A compound described in any one of items 10 to 13, wherein both i and j are 1. [Section 15] Y 1 and Y 2 Each of them operates independently. [C14] TIFF0007909600000069.tif28161 And, R 1 and R 2 Each of them is independently a linear C 7~10 It is alkyl. A compound listed in any one of items 10 to 14. [Section 16] Heptadecan-9-yl8-(7-(8-(nonyloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-yl)octanoate; Heptadecan-9-yl(Z)-8-(7-(8-(nona-2-ene-1-yloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-yl)octanoate; 3-Heptyldodecyl 8-(7-(8-((3-octylundecyl)oxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-yl)octanoate; di(heptadecan-9-yl)8,8'-(9-oxa-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)dioctanoate; and Tetrakis(2-octyldecyl)3,3',3'',3'''-(((9-oxa-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrapropionate; A compound as described in item 1, selected from a pharmaceutically acceptable salt thereof. [Section 17] Lipid nanoparticles comprising a compound described in any one of items 1 to 16, or a pharmaceutically acceptable salt thereof. [Section 18] Lipid nanoparticles according to item 17, further comprising at least one neutral lipid, at least one sterol, and at least one polymer-conjugated lipid. [Section 19] The lipid nanoparticles according to item 18, wherein the neutral lipid is selected from distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylethanolamine (DOPE), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC), or a combination thereof. [Section 20] The lipid nanoparticles according to item 18 or 19, wherein the sterol is cholesterol. [Section 21] The polymer conjugate lipid is selected from DMPE-PEG2000, DPPE-PEG2000, DMG-PEG2000, DPG-PEG2000, PEG2000-c-DOMG, PEG-C-DOPG, or a combination thereof, as described in any one of claims 18, 19, or 20. [Section 22] The lipid nanoparticles further comprise distearoylphosphatidylcholine (DSPC), cholesterol, and DMPE-PEG2000, as described in any one of claims 17 to 21. [Section 23] Lipid nanoparticles according to any one of items 17 to 22, further comprising a therapeutic agent. [Section 24] The therapeutic agent is a nucleic acid segment, as described in item 23, lipid nanoparticles. [Section 25] The lipid nanoparticles described in item 24, wherein the nucleic acid segment is RNA. [Section 26] The lipid nanoparticles described in item 25, wherein the RNA is modified mRNA. [Section 27] A pharmaceutical composition comprising a plurality of lipid nanoparticles as described in any one of items 23 to 26. [Section 28] A method for treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described in item 27. [Section 29] Use of the pharmaceutical composition described in paragraph 28 for treating a disease or disorder. [Section 30] A pharmaceutical composition as described in item 27, used for the treatment of a disease or disorder. < / d> < / d> < / d>

Claims

1. Equation (I): 【Chemistry 1】 A compound of or a pharmaceutically acceptable salt thereof, wherein the formula is a and b are independently 3, 4, or 5; c and d are independently 1, 2, or 3; e and f are independently 0, 1, or 2; X １ is methylene or 【Chemistry 2】 And, X ２ is methylene or 【Transformation 3】 And, g and h are independently 1, 2, or 3; i and j are independently 0, 1, or 2; Y １ and Y ２ Each of them is independently a linear C ７～１０ Alkyl, linear C ７～１０ Alkenil, or 【Chemistry 4】 And, Z １ and Z ２ each independently is a straight-chain C ７～１０ alkyl, straight-chain C ７～１０ alkenyl, or 【Transformation 5】 And, R １ and R ２ Each of them is independently a linear C ７～１０ It is alkyl; R ３ and R ４ Each of them is independently a linear C ７～１０ It is alkyl. A compound or a pharmaceutically acceptable salt thereof.

2. X １ and X ２ The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein both are methylene.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein a and b are both 4; and c and d are both 2.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein both e and f are 0.

5. Y １ is a linear C ７～１０ Alkyl or linear C ７～１０ It is an alkenil; Y ２ teeth 【Transformation 6】 And; R １ and R ２ Each of them is independently a linear C ７～１０ The compound according to claim 1, or a pharmaceutically acceptable salt thereof, which is alkyl.

6. Y １ and Y ２ Each of them operates independently. 【Transformation 7】 And; R １ and R ２ Each of them is independently a linear C ７～１０ The compound according to claim 1, or a pharmaceutically acceptable salt thereof, which is alkyl.

7. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein e and f are each independently 1 or 2.

8. Y １ and Y ２ Each of them operates independently. 【Transformation 8】 And; R １ and R ２ Each of them is independently a linear C ７～１０ The compound according to claim 7, or a pharmaceutically acceptable salt thereof, which is alkyl.

9. Formula (II): 【Chemistry 9】 It is a compound of the formula, in which, k is 0, 1, or 2; Y １ is a linear C ７～１０ Alkyl, linear C ７～１０ Alkenil, or 【Chemistry 10】 And, Y ２ teeth 【Chemistry 11】 And, R １ and R ２ Each of them is independently a linear C ７～１０ It is alkyl. The compound according to claim 1 or a pharmaceutically acceptable salt thereof.

10. X １ teeth 【Chemistry 12】 And; X ２ teeth 【Chemistry 13】 The compound according to claim 1 or a pharmaceutically acceptable salt thereof.

11. The compound according to claim 10 or a pharmaceutically acceptable salt thereof, wherein a and b are both 4; and c and d are both 2.

12. The compound according to claim 10 or a pharmaceutically acceptable salt thereof, wherein e and f are each independently 1 or 2.

13. The compound according to claim 10 or a pharmaceutically acceptable salt thereof, wherein both g and h are 2.

14. The compound according to claim 10 or a pharmaceutically acceptable salt thereof, wherein both i and j are 1.

15. Y １ and Y ２ Each of them operates independently. 【Chemistry 14】 And, R １ and R ２ Each of them is independently a linear C ７～１０ It is alkyl. The compound according to claim 10 or a pharmaceutically acceptable salt thereof.

16. Heptadecan-9-yl 8-(7-(8-(nonyloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-yl)octanoate; Heptadecan-9-yl(Z)-8-(7-(8-(nona-2-en-1-yloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-yl)octanoate; 3-Heptyldodecyl 8-(7-(8-((3-octylundecyl)oxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-yl)octanoate; di(heptadecan-9-yl)8,8'-(9-oxa-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)dioctanoate; and Tetrakis(2-octyldecyl)3,3',3'',3'''-(((9-oxa-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrapropionate; A compound according to claim 1 or a pharmaceutically acceptable salt thereof, selected from the above.

17. Lipid nanoparticles comprising a compound according to any one of claims 1 to 16, or a pharmaceutically acceptable salt thereof.

18. The lipid nanoparticle according to claim 17, further comprising at least one neutral lipid, at least one sterol, and at least one polymer-conjugated lipid.

19. The lipid nanoparticles according to claim 18, wherein the neutral lipid is selected from distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylethanolamine (DOPE), dipalmitoyl phosphatidylcholine (DPPC), dimyristoyl phosphatidylcholine (DMPC), or a combination thereof.

20. The lipid nanoparticles according to claim 18, wherein the sterol is cholesterol.

21. The lipid nanoparticle according to claim 18, wherein the polymer conjugate lipid is selected from DMPE-PEG2000, DPPE-PEG2000, DMG-PEG2000, DPG-PEG2000, PEG2000-c-DOMG, PEG-C-DOPG, or a combination thereof.

22. The lipid nanoparticles according to claim 17, further comprising distearoylphosphatidylcholine (DSPC), cholesterol, and DMPE-PEG2000.

23. Lipid nanoparticles according to claim 17, further comprising a therapeutic agent.

24. The lipid nanoparticle according to claim 23, wherein the therapeutic agent is a nucleic acid segment.

25. The lipid nanoparticle according to claim 24, wherein the nucleic acid segment is RNA.

26. The lipid nanoparticle according to claim 25, wherein the RNA is modified mRNA.

27. A pharmaceutical composition comprising a plurality of lipid nanoparticles as described in claim 23.

28. A pharmaceutical composition according to claim 27, for use in the treatment of a disease or disorder.

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