Novel lipids for delivery of nucleic acid segments

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 TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 271,960, filed October 26, 2021, which is incorporated by reference in its entirety for all 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 new therapeutic treatments for various diseases and disorders. However, challenges remain in the administration of oligonucleotide therapeutics. A typical formulation involves encapsulating the oligonucleotide in a lipid nanoparticle (LNP). LNP formulations typically contain (a) an ionizable or cationic lipid or polymeric material with tertiary or quaternary amines that encapsulates the polyanionic mRNA; (b) a zwitterionic lipid that resembles the lipids in cell membranes; (c) cholesterol that stabilizes the lipid bilayer of the LNP; and (d) polyethylene glycol (PEG)-lipids that provide a hydration layer to the nanoparticles, improve colloidal stability, and reduce protein absorption (see Non-Patent Document 1).

[0003] In 2018, the FDA approved the first RNA interference therapy, patisiran, for the treatment of polyneuropathy in people with hereditary transthyretin-mediated amyloidosis. It is delivered intravenously using LNPs 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 for all delivery systems. Dose-limiting toxicity was reported from studies in two toxicology-related test species, rats and monkeys. This was related to the MC3-based LNP formulation, not the delivered cargo (see Non-Patent Document 2). Also, recently, lipid nanoparticle technology was successfully applied to generate the first approved mRNA product for prophylactic vaccination against the SARS-COV-2 virus (see, for example, Non-Patent Document 3). However, there is still 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 Summary of the Invention [Means for solving the problem]

[0005] In some embodiments, disclosed is a compound of formula (I): [ka] or a pharma- ceutically 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 1 is methylene or [ka] and X 2 is methylene or [ka] and 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 a linear C 7~10 Alkyl, linear C 7~10 alkenyl, or [ka] and Z 1 and Z 2 are each independently a linear C 7~10 Alkyl, linear C 7~10 alkenyl, or [ka] and R 1 and R 2 are each independently a linear C 7~10 is alkyl; R 3 and R 4 are each independently a linear C 7~10 It is an alkyl.

[0006] In some embodiments, disclosed are lipid nanoparticles comprising a compound of formula (I) or a pharma- ceutically acceptable salt thereof.

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

[0008] In some embodiments, disclosed is a method of 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, disclosed is a pharmaceutical composition described herein for use in treating a disease or disorder. [Brief description of the drawings]

[0010] [Figure 1] 1 shows eGFP expression in rat lungs 24 hours after intratracheal administration of LNP formulations containing MC3, MOD5, Compound 4, and Compound 2. [Diagram 2] 1 shows BALF neutrophil concentrations in rat BALF 24 hours after intratracheal administration of LNP formulations containing MC3, MOD5, Compound 4, and Compound 2. [Diagram 3] 1 shows eGFP expression in rat lungs 24 hours after intratracheal administration of LNP formulations containing MC3 and Compound 1. [Figure 4] 1 shows BALF neutrophil concentrations in rat BALF 24 hours after intratracheal administration of an LNP formulation containing MC3 and Compound 1. [Figure 5A-B] 1 shows eGFP expression as demonstrated by IHC in both macrophages and type 1 epithelial cells following intratracheal administration of an LNP formulation containing Compound 1. [Figure 6] 1 shows the levels of eGFP in rat lungs at 5 and 24 hours after inhalation administration of an LNP formulation containing Compound 1. [Figure 7]1 shows the BALF neutrophil concentration in rat BALF 24 hours after inhalation administration of an LNP formulation containing Compound 1. [Figure 8] 1 shows eGFP expression in mouse liver 24 hours after intravenous administration of LNP formulations containing MC3, Compound 2, and Compound 3. [Figure 9] 1 shows eGFP expression in mouse spleens 24 hours after intravenous administration of LNP formulations containing MC3, Compound 2, and Compound 3. [Figure 10] 1 shows eGFP expression in mouse lungs 24 hours after intravenous administration of LNP formulations containing MC3, Compound 2, and Compound 3. [Figure 11] 1 shows eGFP expression in mouse kidneys 24 hours after intravenous administration of LNP formulations containing MC3, Compound 2, and Compound 3. [Figure 12] 1 shows eGFP expression in mouse hearts 24 hours after intravenous administration of LNP formulations containing MC3, Compound 2, and Compound 3. [Figure 13] 1 shows luciferase protein expression in mouse liver 24 hours after intravenous administration of LNP formulations containing MC3, Compound 2, and Compound 3. [Figure 14] IVIS images of mouse heart, lung, spleen, and liver 24 hours after intravenous administration of LNP formulations containing MC3, Compound 2, and Compound 3 are shown. [Figure 15] 1 shows hEPO protein concentrations in mouse plasma 6 hours after intravenous administration of LNP formulations containing MC3, Compound 2, and Compound 3. [Figure 16] 1 shows average cortical and striatal Luc expression in LoxP Luc reporter mice following intrastriatal administration of LNP formulations containing MOD5 and Compound 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] In some embodiments, disclosed is a compound of formula (I): [ka] or a pharma- ceutically 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 1 is methylene or [ka] and X 2 is methylene or [ka] and 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 a linear C 7~10 Alkyl, linear C 7~10 alkenyl, or [ka] and Z 1 and Z 2 are each independently a linear C 7~10 Alkyl, linear C 7~10 alkenyl, or [ka] and R 1 and R 2 are each independently a linear C 7~10 is alkyl; R 3 and R 4 are each independently a linear C 7~10 It is an alkyl.

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

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

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

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

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

[0018] In some embodiments, the compound of formula (I) has the formula (II): [ka] is a compound of the formula k is 0, 1, or 2; Y 1 is a linear C 7~10 Alkyl, linear C 7~10 alkenyl, or [ka] and Y 2 teeth [ka] and R 1 and R 2 are each independently a linear C 7~10 It is an alkyl.

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

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

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

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

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

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

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

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

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

[0031] In some embodiments, the compound of formula (I) is heptadecan-9-yl (Z)-8-(7-(8-(non-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]nonan-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" refers to 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 every integer point therebetween, where j is greater than i. For example, C 7~10 indicates a range of 7 to 10 carbon atoms, including 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, and 10 carbon atoms.

[0036] The term "alkyl" as used herein, whether used as part of another term or used independently, refers to a saturated hydrocarbon chain. In one embodiment, the saturated hydrocarbon chain referred to above is a straight chain alkyl in which the carbon atoms are linked in a single continuous chain without branching. 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 having 7 to 10 carbon atoms.

[0037] The term "alkenyl" as used herein, whether used as part of another term or used independently, refers to an unsaturated hydrocarbon chain containing at least one double bond. In one embodiment, the unsaturated hydrocarbon chain referred to above is a straight chain alkenyl in which the carbon atoms are linked in a single continuous chain without branching. The term "Ci-j alkenyl" refers to an alkenyl having i to j carbon atoms. For example, C 7~10 Alkenyl refers to an alkenyl having 7 to 10 carbon atoms. 7~10 The alkenyl group contains one double bond.

[0038] In some embodiments, disclosed are compounds of formula (I). In some embodiments, disclosed are pharma- ceutically acceptable salts of compounds of formula (I). The term "pharma-ceutically acceptable salts" includes acid addition or base salts that retain the biological effectiveness and properties of compounds of formula (I) and are typically not biologically or otherwise undesirable. Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids, such as acetate, aspartate, benzoate, besylate, bromide / hydrobromide, bicarbonate / carbonate, hydrogen sulfate / sulfate, camphorsulfonate, chloride / hydrochloride, chlortheophyllonate, citrate, ethanedisulfonate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hydroiodide / iodide, isethionate, lactate, laurate, arginate, arginine ... Examples of suitable salts include but are not limited to, carbohydrates, oleates, oxalates, palmitates, palmoates, phosphates / hydrogenphosphates / dihydrogenphosphates, polygalacturonates, propionates, stearates, succinates, subsalicylates, sulfates / hydrogensulfates, tartrates, tosylates, and trifluoroacetates. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, 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, sulfosalicylic acid, and the like.

[0039] In some embodiments, disclosed are lipid nanoparticles (LNPs) comprising a compound of formula (I) or a pharma- ceutically acceptable salt thereof. In some embodiments, disclosed are lipid nanoparticles (LNPs) comprising a compound of formula (I). The term "lipid nanoparticles" includes an electron-dense 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, provided that the net charge of the nanoparticle 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 may be combined with compounds of formula (I) in lipid nanoparticles include lipids that contain a positive charge on the acidic scale, e.g., in the physiological pH range, such as 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA (see, e.g., U.S. Pat. No. 8,158,601), 2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), Merck-32 (see, e.g., WO 2012 / 018754), Acuitas-5 (see, e.g., WO 2015 / 199952), KL-10 (see, e.g., U.S. Pat. App. Pub. No. 2012 / 0295832), C12-200 (see, e.g., Love, KT et al., J. Am. Soc. Soc., 19 ... al., PNAS, 107:1864 (2009)). The ionizable lipid may be present in a molar percentage amount ranging from about 5% to about 90%, for example, from about 10% to about 80%, for example, from about 25% to about 75%, for example, from about 40% to about 60%, from about 40% to about 50%, for example, about 45% or about 50%, relative to the total lipid present in the lipid nanoparticle.

[0041] The term "neutral lipid" includes lipids with zero net charge at physiological pH, e.g., lipids that exist in uncharged form or neutral zwitterionic form at physiological pH, e.g., distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), etc., and combinations thereof. The neutral lipid may be present in a molar percentage amount ranging from about 1% to about 50%, e.g., about 5% to about 20%, e.g., 7.5% to about 12.5%, e.g., about 10%, relative to the total lipid present in the lipid nanoparticle. 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 and the like. The sterol may be present in a molar percentage amount ranging from about 10% to about 90%, such as about 20% to about 50%, such as about 35% to about 45%, for example about 38.5%, relative to the total lipid present in the lipid nanoparticle. In some embodiments, the sterol is cholesterol.

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

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

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

[0046] The selection of neutral lipids, sterols, and / or polymer-conjugated lipids that compose the lipid nanoparticles, and the relative molar ratios of the lipids to each other can be determined by the characteristics of the selected lipids, the nature of the intended target cells, and the characteristics of the nucleic acid segment to be delivered.For example, in certain embodiments, the molar percentage of the compound of formula (I) or its pharma- ceutically acceptable salt in the lipid nanoparticles can 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 can 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 can 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 lipid in the lipid nanoparticle can be greater than about 0.25%, e.g., greater than about 1%, greater than about 1.5%, greater than about 2%, greater than about 5%, or greater than about 10%, relative to the total lipid present.

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

[0048] In addition, the mole percentages of the lipid nanoparticle components (e.g., the compound of formula (I) or its pharma- ceutically acceptable salt, neutral lipid, sterol, and / or polymer-conjugated lipid) that make up the lipid nanoparticle may be selected to provide a particular physical parameter of the lipid nanoparticle as a whole, such as the surface area of ​​one or more lipids. For example, the mole percentages of the compound of formula (I) or its pharma-ceutically acceptable salt, neutral lipid, sterol, and / or polymer-conjugated lipid that make up the lipid nanoparticle may be selected to provide a surface area per neutral lipid, such as DSPC. As a non-limiting example, the mole percentages of the compound of formula (I) or its pharma-ceutically acceptable salt, neutral lipid, sterol, and / or polymer-conjugated lipid may be selected to provide a surface area per neutral lipid, such as DSPC, of ​​about 1.0 nm 2 ~about 2.0nm 2 , for example, about 1.2 nm 2 The surface area per DSPC may be determined to give

[0049] In accordance with the present disclosure, the lipid nanoparticles may further comprise a nucleic acid segment, which may be associated on the surface of the lipid nanoparticle and / or encapsulated within the same lipid nanoparticle.

[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. The nucleic acid segments herein may be wild-type or modified. In at least one embodiment, the lipid nanoparticles may contain multiple different nucleic acid segments. In yet another embodiment, the wild-type or modified nucleic acid segments encode a polypeptide of interest. The modified nucleic acid segment includes a nucleic acid segment having chemical modifications at any part of its structure such that the nucleic acid segment does not occur 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] The term "therapeutically effective amount" as used herein refers to an amount of a nucleic acid segment sufficient to modulate protein expression in a target tissue and / or cell type. In some embodiments, a therapeutically effective amount of a nucleic acid segment is an amount 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 the compound of formula (I) or a pharma- ceutically acceptable salt thereof to the nucleic acid monomer of about 3:1. 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 the compound of formula (I) or a pharma- ceutically acceptable salt thereof to the nucleic acid monomer of about 6:1. However, the relative molar ratio of the lipid phase and / or lipid phase components to the nucleic acid monomer is not limited within the scope of the above-specified embodiments, since it can be determined by the nature of the intended target cell and the characteristics of the nucleic acid segment. In some embodiments, the molar ratio of the compound of formula (I) or a pharma- ceutically acceptable salt thereof 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 a pharma- ceutically acceptable salt thereof to the nucleic acid monomer is about 2.75:1. In some embodiments, the approximate molar ratio of the compound of formula (I) or a pharma- ceutically acceptable salt thereof to the nucleic acid monomer is about 3:1. In some embodiments, the approximate molar ratio of the compound of formula (I) or a pharma- ceutically acceptable salt thereof to the nucleic acid monomer is about 5.5:1. In some embodiments, the approximate molar ratio of the compound of formula (I) or a pharma- ceutically acceptable salt thereof to the nucleic acid monomer is about 6:1.

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

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

[0055] The pharmaceutical composition of the present disclosure may further comprise at least one pharma- ceutically acceptable carrier.As used herein, the term "pharma-ceutically acceptable carrier" includes compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues, within the scope of sound medical judgment, without excessive toxicity, irritation, allergic reaction, or other problems or complications, commensurate with a reasonable risk / benefit ratio.

[0056] The pharmaceutical composition may be in a form suitable for parenteral administration. For example, suitable parenteral administration includes, but is not limited to, subcutaneous administration, intramuscular administration, and intravenous administration. The pharmaceutical composition may be in a form suitable for intratracheal instillation, bronchial instillation, and / or inhalation. The pharmaceutical liquid composition may be nebulized by using an inert gas for inhalation. The nebulized suspension may be directly inhaled from the nebulizer, or the nebulizer may be attached to a face mask or an intermittent positive pressure breathing device.

[0057] The amount of nucleic acid segment combined with one or more pharma- ceutically acceptable carriers to produce a single dosage form will necessarily vary depending on the subject being treated and the particular route of administration. For further information regarding routes of administration and dosing regimens, the reader is referred 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 of administering to a subject in need thereof a pharmaceutical composition comprising a compound of formula (I) or a pharma- ceutically acceptable salt thereof and a therapeutically effective amount of a plurality of lipid nanoparticles comprising a nucleic acid segment.

[0059] The term "subject" includes warm-blooded mammals, such as primates, cows, 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 is in need of treatment (e.g., the subject will benefit biologically or medically from the treatment).

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

[0061] Another embodiment provides a method of delivering a therapeutically effective amount of a 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 pharma- ceutically acceptable salt thereof and a plurality of lipid nanoparticles comprising a therapeutically effective amount of the nucleic acid segment.

[0062] A pharmaceutical composition comprising a compound of formula (I) or a pharma- ceutically acceptable salt thereof and a plurality of lipid nanoparticles comprising a nucleic acid segment as disclosed herein can be used to treat a wide variety of disorders and diseases characterized by underexpression of a polypeptide in a subject, overexpression of a polypeptide in a subject, and / or absence / presence of a polypeptide in a subject. Accordingly, disclosed is a method of 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 pharma- ceutically acceptable salt thereof and a therapeutically effective amount of a plurality of lipid nanoparticles comprising a nucleic acid segment.

[0063] Further disclosed is the use of a pharmaceutical composition comprising a compound of formula (I) or a pharma- ceutically acceptable salt thereof and a therapeutically effective amount of a plurality of lipid nanoparticles comprising a nucleic acid segment for treating a disease or disorder.

[0064] Further disclosed is a pharmaceutical composition for use in treating a disease or disorder, the pharmaceutical composition comprising a compound of formula (I) or a pharma- ceutical acceptable salt thereof, and a therapeutically effective amount of a plurality of lipid nanoparticles comprising a nucleic acid segment.

[0065] Further disclosed is a method of regulating protein expression in a cell, comprising administering to a subject in need thereof a pharmaceutical composition comprising a compound of formula (I) or a pharma- ceutically acceptable salt thereof and a plurality of lipid nanoparticles comprising a nucleic acid segment. In at least one embodiment, protein expression can be increased by about 2-fold for up to 24 hours. In another embodiment, protein expression can be increased by about 3-fold for up to 72 hours. EXAMPLES

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

[0067] 1 H NMR: 500 MHz; Probe: 5 mm 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 are referenced to residual protium in the NMR solvent (chloroform-d: δ 7.26, methanol-d4: δ 3.31, DMSO-d6: δ 2.50). Data are 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) in Hertz (Hz).

[0068] LCMS: Instrument Shimadzu LCMS-2020 coupled with 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. Mass spectrometer in ESI mode.

[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) with a solvent gradient of 2 to 98% B over 1.5 min (total run time 2 min with equilibrium returning to starting conditions) at a flow rate of 1 mL / min, where A=0.1% formic acid in water, B=0.1% formic acid in acetonitrile (for acid workup), or A=0.1% ammonium hydroxide in water, B=acetonitrile (for base workup). The column used for the analysis of acids was a Waters Acquity HSS T3 (1.8 mm, 2.1×30 mm) and the column used for the analysis of bases was a Waters Acquity BEH C18 (1.7 mm, 2.1×30 mm).

[0070] HPLC: Instrument Shimadzu LCMS-2020 coupled with DAD detector, 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 to 95% B 4.00 min, hold 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, 19×250 mm, 5 μm; Mobile Phase A Water (0.05% TFA), Mobile Phase B MeCN; Flow Rate 25 mL / min, gradient as indicated. Method B Column SunFire C18 OBD, 19×250 mm, 5 μm; Mobile Phase A Water (0.05% TFA), Mobile Phase B MeCN; Flow Rate 60 mL / min, gradient as indicated.

[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 EtOAc Ethyl acetate HPLC High Performance Liquid Chromatography KI Potassium iodide MC3 (6Z,9Z,28Z,31Z)-Heptatriacont-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 Performance 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 portion to a stirred mixture of 8-bromooctanoic acid (0.522 g, 2.34 mmol), heptadecan-9-ol (0.5 g, 1.95 mmol), DMAP (0.048 g, 0.39 mmol), and DIPEA (1.396 ml, 7.99 mmol) in DCM (15 mL) under argon. The resulting mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in EtOAc (50 mL), and then washed successively 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 give the crude product. The resulting residue was purified by flash silica chromatography, elution gradient 0 to 20% EtOAc in hexanes. The product fractions were concentrated to dryness under reduced pressure to give 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-Butyl 7-(8-(heptadecan-9-yloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-carboxylate [ka] Step b: Heptadecane-9-yl 8-bromooctanoate (0.808 g, 1.75 mmol) (Intermediate 1) was added dropwise to a stirred mixture 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) in acetonitrile (10 mL) under argon. The resulting mixture was stirred at 80° C. for 16 h. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in EtOAc (100 mL), and washed successively 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 give the crude product. The resulting residue was purified by flash silica chromatography, elution gradient 0-100% in DCM (20% MeOH and 1% NH4OH in DCM). The product fractions were concentrated to dryness under reduced pressure to give tert-butyl 7-(8-(heptadecan-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-yl 8-(9-oxa-3,7-diazabicyclo[3.3.1]nonan-3-yl)octanoate dihydrochloride [ka] Step c: HCl in dioxane (2.250 ml, 9.00 mmol) was added dropwise to a stirred mixture 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) in dioxane (10 mL) under argon at 0° C. The resulting mixture was warmed and stirred at room temperature for 4 h. The reaction was concentrated to dryness under reduced pressure and washed with dioxane (3×50 mL) to give 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 (500MHz, 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 portion to a stirred solution of 8-bromooctanoic acid (2.320 g, 10.40 mmol), nonan-1-ol (1.205 ml, 6.93 mmol), DMAP (0.169 g, 1.39 mmol), and DIPEA (2.54 ml, 14.56 mmol) in DCM (15 mL). The resulting mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in EtOAc (100 mL), and washed successively 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 give the crude product. The resulting residue was purified by flash silica chromatography, eluent gradient 0-20% EtOAc in hexanes. The product fractions were concentrated to dryness under reduced pressure to give 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]nonan-3-yl)octanoate [ka] Process e: Nonyl 8-bromooctanoate (0.144 g, 0.41 mmol) (Intermediate 4) was added dropwise to a stirred mixture of heptadecane-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) in acetonitrile (5 mL) under argon. The resulting mixture was stirred at 80° C. for 16 h. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in EtOAc (50 mL), and washed successively 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 give the crude product. The resulting residue was purified twice by flash silica chromatography, elution gradient 0-100% in DCM (20% MeOH and 1% NH4OH in DCM). The product fractions were concentrated to dryness under reduced pressure to give heptadecan-9-yl 8-(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]nonane dihydrochloride [ka] Step a: HCl (2 ml, 65.83 mmol) was added dropwise to a stirred mixture of tert-butyl 9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-carboxylate hydrochloride (0.3 g, 1.13 mmol) in 1,4-dioxane (8 mL) under argon. The reaction was stirred overnight at room temperature until a precipitate formed. The reaction mixture was concentrated to dryness under reduced pressure to give 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: Heptadecan-9-yl 8-bromooctanoate [ka] Step b: EDC (0.785 g, 4.09 mmol) was added in one portion to a stirred DCM mixture (15 mL) of 8-bromooctanoic acid (0.522 g, 2.34 mmol), heptadecan-9-ol (0.5 g, 1.95 mmol), DMAP (0.048 g, 0.39 mmol), and DIPEA (1.396 mL, 7.99 mmol) under argon. The resulting mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in EtOAc (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 give the crude product. The resulting residue was purified by flash silica chromatography, elution gradient 0-20% EtOAc in hexanes. The product fractions were concentrated to dryness under reduced pressure to give 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] Step c: Heptadecane-9-yl 8-bromooctanoate (0.285 g, 0.62 mmol) (Intermediate 2) was added dropwise to a stirred mixture of 9-oxa-3,7-diazabicyclo[3.3.1]nonane dihydrochloride (0.04 g, 0.20 mmol) (Intermediate 1), DIPEA (0.142 ml, 0.82 mmol), and potassium iodide (6.60 mg, 0.04 mmol) in acetonitrile (5 mL) at 25° C. under argon. The resulting mixture was stirred at 80° C. for 16 h. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in EtOAc (50 mL), and washed successively 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 give the crude product. The resulting residue was purified by flash silica chromatography, elution gradient 0 to 100% in DCM (20% MeOH and 1% NH4OH in DCM). The product fractions were concentrated to dryness under reduced pressure to give 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) in DMF (25 mL each) were added successively and the mixture was stirred at room temperature for 3 h. After the reaction, water (250 mL) was added and the aqueous layer was extracted with (Et2O) (3×100 mL). The combined organic layers were washed successively with water (100 mL) and saturated aqueous NaCl (100 mL). The organic layer was dried over Na2SO4, filtered and concentrated to dryness under reduced pressure to give the crude product. The resulting residue was purified by flash silica chromatography, elution gradient 0 to 50% EtOAc in hexanes. The product fractions were concentrated to dryness under reduced pressure to give dimethyl 2,2-dioctylmalonate (8.89 g, 65.9%) as a pale yellow oil. 1H NMR (500 MHz, chloroform-d, 27 °C) 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 mixture of 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) in DMSO (80 mL) was stirred at reflux for 24 h. 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 washed in turn with water (3×50 mL). The organic layers were dried over Na2SO4, filtered, and concentrated to dryness under reduced pressure to give the crude product. The resulting residue was purified by flash silica chromatography, elution gradient 0 to 50% EtOAc in hexanes. The product fractions were concentrated to dryness under reduced pressure to give methyl 2-octyldecanoate (7.30 g, 95%) as a yellow liquid. 1H NMR (500MHz, 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-Octyldecan-1-ol [ka] Step c: To a solution of methyl 2-octyldecanoate (7.3 g, 24.45 mmol) (Intermediate 2) in THF (100 mL) was added lithium aluminum hydride (14.67 ml, 29.35 mmol) dropwise at 0° C. The reaction was then warmed and stirred at room temperature for 24 h. Upon completion, 3M 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 layer was dried over Na2SO4, filtered and concentrated to dryness under reduced pressure to give the crude product. The resulting residue was purified by flash silica chromatography, elution gradient 0 to 40% EtOAc in hexanes. The product fractions were concentrated to dryness under reduced pressure to give 2-octyldecan-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 mixture of 2-octyldecan-1-ol (1 g, 3.70 mmol) (Intermediate 3) and TEA (2.113 ml, 15.16 mmol) in DCM (20 mL) at 0° C. under nitrogen. The resulting mixture was stirred at room temperature for 16 h. 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 give the crude product. The resulting residue was purified by flash silica chromatography, elution gradient 0 to 100% EtOAc in hexanes. The product fractions were concentrated to dryness under reduced pressure to give 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: tert-Butyl (4-bromobutyl)carbamate (0.502 g, 1.99 mmol) was added in one portion to a stirred mixture of 9-oxa-3,7-diazabicyclo[3.3.1]nonane dihydrochloride (0.1 g, 0.50 mmol), DIPEA (0.521 ml, 2.98 mmol), and potassium iodide (0.017 g, 0.10 mmol) in acetonitrile (5 mL) under nitrogen. The resulting mixture was stirred at 80° C. for 16 h. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in EtOAc (50 mL), and washed successively 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 give the crude product. The resulting residue was purified by flash silica chromatography, elution gradient 0 to 100% in DCM (20% MeOH and 1% NH4OH in DCM). The product fractions were concentrated to dryness under reduced pressure to give 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(butan-1-amine) tetrahydrochloride [ka] Process f: A solution of HCl in dioxane (4 M, 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 inert atmosphere and stirred at room temperature for 16 h. 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 give 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 in one portion to a stirred mixture of 4,4'-(9-oxa-3,7-diazabicyclo[3.3.1]nonane-3,7-diyl)bis(butan-1-amine) tetrahydrochloride (0.05 g, 0.12 mmol) (Intermediate 5) and TEA (0.134 ml, 0.96 mmol) in iPrOH (4 mL). The resulting mixture was stirred at 80° C. for 3 days. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in EtOAc (50 mL), and washed successively 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 give the crude product. The resulting residue was purified by flash silica chromatography, elution gradient 0 to 100% in DCM (20% MeOH and NH4OH in DCM). The product fractions were concentrated to dryness under reduced pressure to give 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 portion to a stirred DCM mixture (15 mL) of 8-bromooctanoic acid (0.522 g, 2.34 mmol), heptadecan-9-ol (0.5 g, 1.95 mmol), DMAP (0.048 g, 0.39 mmol), and DIPEA (1.396 ml, 7.99 mmol) under argon. The resulting mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in EtOAc (50 mL), and washed successively 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 give the crude product. The resulting residue was purified by flash silica chromatography, elution gradient 0 to 20% EtOAc in hexanes. The product fractions were concentrated to dryness under reduced pressure to give 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-Butyl 7-(8-(heptadecan-9-yloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonane-3-carboxylate [ka] Step b: Heptadecane-9-yl 8-bromooctanoate (0.808 g, 1.75 mmol) (Intermediate 1) was added dropwise to a stirred mixture 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) in acetonitrile (10 mL) under argon. The resulting mixture was stirred at 80° C. for 16 h. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in EtOAc (100 mL), and washed successively 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 give the crude product. The resulting residue was purified by flash silica chromatography, elution gradient 0 to 100% in DCM (20% MeOH and 1% NH4OH in DCM). The product fractions were concentrated to dryness under reduced pressure to give tert-butyl 7-(8-(heptadecan-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-yl 8-(9-oxa-3,7-diazabicyclo[3.3.1]nonan-3-yl)octanoate dihydrochloride [ka] Step c: HCl in dioxane (2.250 ml, 9.00 mmol) was added dropwise to a stirred mixture 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) in dioxane (10 mL) under argon at 0° C. The resulting mixture was warmed and stirred at room temperature for 4 h. The reaction was concentrated to dryness under reduced pressure and washed with dioxane (3×50 mL) to give 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 (500MHz, 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-yl 8-bromooctanoate [ka] Process d: EDC (2.83 g, 14.76 mmol) was added in one portion to a stirred DCM mixture (15 mL) of 8-bromooctanoic acid (2.353 g, 10.55 mmol), DIPEA (3.68 ml, 21.09 mmol), (Z)-non-2-en-1-ol (1 g, 7.03 mmol), and DMAP (0.172 g, 1.41 mmol) under argon. The resulting mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in EtOAc (100 mL), and washed successively 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 give the crude product. The resulting residue was purified by flash silica chromatography, elution gradient 0 to 100% EtOAc in hexanes. The product fractions were concentrated to dryness under reduced pressure to give (Z)-non-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-(non-2-en-1-yloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonan-3-yl)octanoate [ka] Process e: Heptadecane-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 at 20° C. under nitrogen. The resulting mixture was stirred at 20° C. for 30 min. (Z)-Nona-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 h. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in EtOAc (50 mL) and washed successively 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 give the crude product. The resulting residue was purified by flash silica chromatography, elution gradient 0 to 100% in DCM (20% MeOH and 1% NH4OH in DCM). The product fractions were concentrated under reduced pressure to give heptadecan-9-yl (Z)-8-(7-(8-(non-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 solution of sodium hydride (2.51 g, 62.88 mmol) in THF (50 mL) at 0° C. under nitrogen. The resulting mixture was stirred at 0° C. for 30 min. Heptadecan-9-one (2 g, 7.86 mmol) was added to the mixture and warmed to 30° C. under nitrogen. The resulting mixture was stirred under reflux for 18 h. After cooling to room temperature, the reaction mixture was quenched with water (100 mL), extracted with EtOAc (3×50 mL), and the combined organic layers were dried over MgSO4, filtered, and concentrated to dryness under reduced pressure to give a yellow oil. The resulting residue was purified by flash silica chromatography, elution gradient 0 to 40% EtOAc in hexanes. 1H NMR was used to determine the purity of each fraction. The combined product fractions were concentrated to dryness under reduced pressure to give ethyl 3-octylundec-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-octylundecanoate [ka] Step b: Ethyl 3-octylundec-2-enoate (2.0 g, 6.16 mmol) (Intermediate 1) and platinum(IV) oxide (0.028 g, 0.12 mmol) in CHCl3 / MeOH (5:1) (30 mL) were stirred under a balloon of hydrogen at atmospheric pressure for 16 h. The reaction mixture was filtered through Celite. The resulting residue was purified by flash silica chromatography, elution gradient 0 to 40% EtOAc in hexanes. Separation was difficult due to overlap with the heptadecan-9-one starting material. 1H NMR was used to determine the purity of each fraction. The combined product fractions were concentrated to dryness under reduced pressure to give ethyl 3-octylundecanoate (1.540 g, 77%) as a colorless oil. 1H NMR (500MHz, 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-Octylundecan-1-ol [ka] Step c: Lithium aluminum hydride (7.75 ml, 7.75 mmol) was added slowly to a stirred solution of ethyl 3-octylundecanoate (2.3 g, 7.04 mmol) (Intermediate 2) in THF (20 mL) at 0° C. under nitrogen. The resulting mixture was stirred at room temperature for 18 h. After completion, the reaction was cooled to 0° C., quenched with 3M HCl (100 mL), extracted with EtOAc (3×50 mL), and the combined organic layers were dried over MgSO4, filtered, and concentrated to dryness under reduced pressure to give a yellow oil. The resulting residue was purified by flash silica chromatography, elution gradient 0 to 40% EtOAc in hexanes. The product fractions were concentrated to dryness under reduced pressure to give 3-octylundecan-1-ol (1.354 g, 67.6%) as a colorless oil. 1H NMR (500MHz, 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-Octylundecyl 8-bromooctanoate [ka] Process d: EDC (0.707 g, 3.69 mmol) was added in one portion to a stirred DCM mixture (10 mL) of 8-bromooctanoic acid (0.470 g, 2.11 mmol), DIPEA (0.645 ml, 3.69 mmol), 3-octylundecan-1-ol (0.5 g, 1.76 mmol) (Intermediate 3), and DMAP (0.043 g, 0.35 mmol) under argon. The resulting mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in EtOAc (50 mL), and washed successively 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 give the crude product. The resulting residue was purified by flash silica chromatography, elution gradient 0 to 100% EtOAc in hexanes. The product fractions were concentrated to dryness under reduced pressure to give 3-octylundecyl 8-bromooctanoate (0.545 g, 63.3%) as a pale yellow oil. 1H NMR (500 MHz, chloroform-d, 27 °C) 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-Octylundecyl 8-bromooctanoate (0.302 g, 0.62 mmol) (Intermediate 4) was added dropwise to a stirred mixture of 9-oxa-3,7-diazabicyclo[3.3.1]nonane dihydrochloride (0.04 g, 0.20 mmol), DIPEA (0.142 ml, 0.82 mmol), and potassium iodide (6.60 mg, 0.04 mmol) in acetonitrile (5 mL) at 25° C. under argon. The resulting mixture was stirred at 80° C. for 16 h. The reaction mixture was concentrated to dryness under reduced pressure, redissolved in EtOAc (50 mL), and washed successively 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 give the crude product. The resulting residue was purified by flash silica chromatography, elution gradient 0 to 100% in DCM (20% MeOH and 1% NH4OH in DCM). The product fractions were concentrated to dryness under reduced pressure to give 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) formulations containing eGFP mRNA LNPs were prepared using a microfluidic setup, NanoAssemblr (Precision NanoSystems Inc.). Briefly, lipid stocks were dissolved in ethanol and mixed in appropriate molar ratios to obtain a lipid concentration of 12.5 mM. The size of LNPs was determined by DLS measurements 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 LNPs is shown in Table 1. Lipid solutions (99.5%) in ethanol were prepared with four different lipid components: ionizable lipids, i.e., MC3, MOD5, compound 1, compound 2, or compound 4; cholesterol (Sigma-Aldrich); DSPC (distearoylphosphatidylcholine, Avanti Polar Lipids Inc); and polymer-conjugated lipids. The lipid ratio in all experiments was ionizable lipid / cholesterol / DSPC / polymer-conjugated 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, and a more detailed description 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 solution of 50 mM citrate. The mRNA and lipid solutions were mixed by a microfluidic mixing system of NanoAssemblr (Precision Nanosystems, Vancouver, BC, Canada) at a mixing ratio of aqueous solution:EtOH=3:1 and a constant flow rate of 12 mL / min. The 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 at the time of mixing (see Table 1). The LNPs were dialyzed overnight against 500× sample volume using a Slide-A-Lyzer G2 dialysis cassette from Thermo Scientific with a molecular weight cutoff of 10K.

[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 the mRNA lipid nanoparticles was determined by dynamic light scattering measurements using a Zetasizer Nano ZS from Malvern Instruments Ltd to obtain the z-average particle size directly. Number-based particle size distributions and means were calculated using a particle refractive index of 1.45.

[0106] The mRNA encapsulation and concentration were determined using the Ribo-Green assay. Encapsulation in all samples was typically 90-99%. The final mRNA concentration and encapsulation efficiency percentage (%EE) were measured by Quant-it Ribogreen Assay Kit (ThermoFischer Scientific Inc.) using Triton-X100 to destroy the LNPs. The 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 formulations to rats In vivo studies were performed in the AAALAC-accredited animal facility of AstraZeneca, Gothenburg, Sweden, under approval by the Animal Ethics Committee of Gothenburg (no. 82-2015). Male Wistar Han rats were purchased (Charles River Germany Limited) and caged in groups of four on shavings with food (R70 Rat and Mouse chow (Lantmaennen, Stockholm, Sweden) immediately upon arrival and provided with drinking water drawn from the water mains ad libitum. The animals were approximately 10 weeks old at the start of dosing. The environment was maintained with a target temperature of 19-24°C and a relative humidity of 40-70% on a 12-h light / dark cycle. The animals were acclimated to the housing conditions for at least 5 days before any experimental procedure. Animals receiving the inhalation dose were further habituated to the inhalation restraint procedure by gradually increasing the duration of exposure to the restraint procedure up to the maximum anticipated duration of each study, for a maximum of 5 days before the start of dosing.

[0112] Compound 1 LNP formulation of eGFP mRNA was administered by a single inhalation dose at a target lung dose of 0.02 mg / kg eGFP or by a single intratracheal administration. A group of the same size served as a placebo control in which animals were exposed to phosphate buffered saline by inhalation or intratracheal installation. The expression level of eGFP in rat lungs at 24 hours after intratracheal administration is shown in Figure 3, and the BALF neutrophil concentration in rat BALF at 24 hours after intratracheal administration is shown in Figure 4. The expression level of eGFP in rat lungs at 5 hours and 24 hours after inhalation administration is shown in Figure 6, and the BALF neutrophil concentration in rat BALF at 24 hours after inhalation administration is shown in Figure 7. Furthermore, immunohistochemistry (IHC) studies show the expression of eGFP not only in macrophages but also in type 1 epithelial cells (see Figures 5A and 5B).

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

[0114] Inhalation Aerosols were generated using an Aerogen Solo vibrating mesh nebulizer (Galway Ireland). The nebulizer was filled with 5.6 mL of vehicle or eGFP mRNA in LNP formulation and nebulized at approximately 60 μl / min for the duration of the dosing period. Rats were placed in a rodent restraint and then placed in an AstraZeneca designed inhalation dosing system. Animals were monitored throughout the experimental procedure for any signs of clinically unremarkable morbid effects after dosing or any abnormal changes in body weight noted. 24 hours after the final dose, rats were sacrificed by sedation with isoflurane anesthesia followed by cutting of the vena cava and removal of the heart.

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

[0116] BAL Sampling Bronchial (Broncheo) alveolar lavage (BAL) was performed by manual irrigation of the entire lung. After exposing the trachea, a polyethylene tube (PE120) was inserted and ligated with a 1-0 silk suture. The tube was connected to a syringe prefilled with 4 ml of room temperature PBS, and PBS was slowly injected into the lung. 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 BAL was kept on ice until centrifugation (Hettich ROTANTA 46R, 1200 rpm, 10 min, 4° C.). After centrifugation, the supernatant was collected and aliquoted onto 96-well plates (0.15 mL / well, 5 plates) and kept on dry ice (0.1 ml / well). The plates were stored at a minimum of −75° C. for any further analysis. The cell pellet was resuspended in 0.5 ml PBS and kept on ice for cell counting. The total and differential cell numbers were counted using an automated SYSMEX XT-1800i Vet (Sysmex Corporation, Kobe, Japan).

[0118] Organ collection The right lobe was tied off, dissected free of non-lung tissue, freed from blood clots, and rinsed with saline. The right lobe was detached with a stitch, released, and rinsed with PBS to remove any blood contamination. The superior and middle lobes were weighed and collected in 7 mL precellys tubes for eGFP mRNA analysis. The inferior 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 saved 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 plastic lidded container.

[0119] Example 8. In vivo intravenous administration of LNP-eGFP mRNA formulations to mice In accordance with EU Directive 2010 / 63 / EU, all studies were conducted under the authority of the Home Office UK ethical and management standards and under the authority of the 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 similar procedures described in Example 6. Characterization is shown in Table 2. LNP formulations containing 0.4 mg / kg eGFP mRNA were administered via tail vein injection. Mice were euthanized 24 hours after administration and the following organs were extracted: liver, spleen, lungs, kidneys, and heart. Organs were collected in cryovials and flash 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, organs / tissues were thawed on ice and rinsed with 1x DPB to remove any blood. Organs / tissues were cut into slices of approximately 100-200 mg and transferred to 2 mL tubes containing 0.5-1 mL ice-cold 1x Cell Extraction Buffer PTR (from ELISA kit ab171581) with protease and phosphatase inhibitors (ThermoFisher 78445) and 5 mm stainless steel beads (Qiagen). Organ / tissue samples were homogenized for 3 min on a Tissue Lyser II (Qiagen) set at a frequency of 30 1 / s, then transferred to a clean tube and incubated on ice for 20 min. Samples were subsequently centrifuged at 18000xg for 20 min at 4°C and the clear homogenates were transferred to clean tubes, aliquoted and stored at -80°C until further use.

[0122] Quantification of eGFP in organs by ex vivo ELISA eGFP expression levels in liver, spleen, lung, kidney, 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: 1x Cell Extraction Buffer PTR with protease and phosphatase inhibitors (ThermoFisher 78445), 1x Wash Buffer PT, 1x Antibody Diluent, Antibody Cocktail, and EGFP standard curve. If further dilutions of tissue homogenates were required, this was performed with ice-cold 1x Cell Extraction Buffer PTR. 50 μl of diluted samples and eGFP standards were then added to each well, followed by 50 μl of antibody cocktail. The plate was then sealed and incubated for 1 hour at room temperature on a plate shaker set at 400 rpm. After incubation, each well was washed three times with 350 μl of 1x Wash Buffer PT. After the final washing step, excess liquid was removed by blotting the plate against a clean paper towel. Then, 100 μl of TMB substrate was added to each well, and the plate was covered with aluminum foil and incubated for 10 minutes on a plate shaker set at 400 rpm. Finally, 100 μl of stop solution was added to each well, the plate was shaken for 1 minute, and absorbance at 450 nm was recorded using an Envision microplate reader (Perkin Elmer). The eGFP standard curve was fitted to a sigmoidal 4PL curve using GraphPad Prism 9, and ng eGFP protein was estimated from the curve.

[0123] eGFP amounts were normalized by total tissue protein, as determined by 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 to each well, and the contents were quickly mixed for 30 seconds on a plate shaker. Plates were then covered and incubated at 37° C. for 30 minutes. After cooling the plates to room temperature, absorbance at 562 nm was recorded using an Envision microplate reader (Perkin Elmer). Total protein (mg) was estimated from a BSA standard curve. Data are reported as mean ng eGFP / mg of tissue protein ± SD.

[0124] Example 9. In vivo intravenous administration of LNP co-formulations 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 mixing mRNA dissolved in nuclease-free water, 100 mM citrate buffer (pH 3), and nuclease-free water to give a solution of 50 mM citrate. Lipid solutions (99.5%) in ethanol were prepared by a similar procedure described in Example 6 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 (dimyristoylphosphatidylethanolamine-poly(ethylene glycol) 2000, NOF Corporation). Characterization of LNPs is shown in Table 3. The total lipid concentration in all experiments was 12.5 mM. The mRNA and lipid solutions were mixed by 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 and 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 at 4 °C against PBS (pH 7.4). The volume of the PBS buffer was 500-1000 times the sample fraction volume. The next day, the sample was taken from the cassette by a syringe and needle. The needle was then replaced by a 0.2 μm syringe filter and the sample was filter-sterilized into a sterile tube.From this sample, 10 μL was diluted with 990 μl PBS buffer pH 7.4 and used to measure the intensity-average particle size and polydispersity index (PDI) by Malvern ZetaSizer (ZetaSizer Nano ZS, Malvern Instruments Inc., Westborough, MA, USA). The final mRNA concentration and encapsulation efficiency percentage (%EE) were measured by Quant-it Ribogreen Assay Kit (ThermoFischer Scientific Inc.) using Triton-X100 to destroy the LNPs.

[0125] [Table 3]

[0126] Animal testing: Experiments were performed in wild-type (WT) and LDLr knockout (KO) mice. All experiments were performed in accordance with the 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 bred in-house. 20 C57bl6 LDLR- / - and 40 WT mice were included in the study (N>5 per group). Animals were used at an average body weight of 25 g. 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 LNP as described above, or PBS as a control. Blood samples were collected in EDTA tubes via tail vein bleeding at t=6 h prior to sacrifice and via retro-orbital plexus bleeding at t=24 h. Mice were injected subcutaneously with 5 mg / kg luciferin substrate (RediJect D-Luciferin Bioluminescent Substrate from PerkinElmer) 20 min prior to termination. Heart, lungs, spleen, and liver were collected immediately after termination and imaged using IVIS Spectrum (PerkinElmer). Total luminescence from each organ was quantified using LivingImage (PerkinElmer). The resulting IVIS images are shown in FIG. 14. For hEPO analysis, blood samples were spun down and plasma was analyzed using a Human Erythropoietin Quantikine IVD ELISA Kit (R&D System). Data is reported as hEPO in ng / ml and samples were analyzed in triplicate. FIG. 13 shows luciferase protein expression in WT and KO mouse livers at 24 hours after intravenous administration of LNP formulations containing MC3, compound 2, and compound 3, while FIG. 15 shows hEPO protein concentrations in WT and KO mouse plasma at 6 hours after intravenous administration of LNP formulations containing MC3, compound 2, and compound 3.

[0127] Example 10. In vivo intrastriatal administration of LNP formulations of luciferase mRNA to mice MOD5 and compound 5 (AZ8608) LNP formulations containing luciferase mRNA were prepared by similar procedures described in Examples 6 and 9, where 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%. Characterization of the LNPs is shown in Table 4.

[0128] In-house generated reporter mice (LoxP Luc) containing a cyclic recombinase enzyme (Cre) inducible luciferase expression cassette were generated by standard random integration gene addition transgenesis. Prior to LNP administration, LoxP Luc reporter mice were anesthetized with isoflurane 4.0 / 1.5 O2 i. Mice were subcutaneously injected with 2 units (20 μl) of diluted Comforion Vet (10 mg / ml; 100 μg / mouse) under the neck skin with an insulin syringe (BD U-100). The fur was then shaved and the skull was cleaned with a Descutan swab (4% chlorhexidine) before the animal was placed on a stereotaxic board with a heating pad. The mouse was attached appropriately, the skull was adjusted to a horizontal position, and the skull was covered with a plastic film with an open hole. During surgery, a 6-8 mm incision was made in the midline of the skull, the area around the bregma was dried, and a drill was placed on the location after calculating the exact coordinates. One or two small holes were drilled into the skull on either side of the midline, and a Hamilton syringe for thigmotaxis was attached to the pump. The syringe was placed over the drill hole and slowly lowered deep to the right side to inject 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 per hemisphere) into the local brain 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 with tissue adhesive placed along the incision. Animals were observed and weighed daily. The recovery process and wound healing were closely followed for 3-5 days. After 7 days, ex vivo brain luciferase analysis of both hemispheres was performed. Figure 16 shows the cortical and striatal mean 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, whole brains were dissected, and striatum and cortex were isolated, weighed, and placed in separate tubes and frozen until further analysis. To measure LNP-mediated functional delivery levels, proteins were extracted from mouse brain tissues using a Qiagen TissueLyser as recommended by the manufacturer. Tissues were crushed, homogenized with a moving pestle, and centrifuged to remove non-soluble tissue debris from the suspension. Supernatants were transferred to separate tubes, and protein concentrations were determined by Bradford assay experiments. For 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. Samples were mixed, and the resulting luminescence signal was measured by a luminometer. Luminescence signals were normalized to tissue weight.< / d> < / d> < / d>

Claims

1. Formula (I): 【Chemistry 1】 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 １ is methylene or 【Chemistry 2】 and X ２ is methylene or 【Transformation 3】 and 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 a linear C ７～１０ Alkyl, linear C ７～１０ alkenyl, or 【Chemistry 4】 and Z １ and Z ２ are each independently a linear C ７～１０ Alkyl, linear C ７～１０ alkenyl, or 【Transformation 5】 and R １ and R ２ are each independently a linear C ７～１０ is alkyl; R ３ and R ４ are each independently a linear C ７～１０ is alkyl, The compound or a pharmaceutically acceptable salt thereof.

2. X １ and X ２ and R are both methylene, or a pharmaceutically acceptable salt thereof.

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

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

5. Y １ is a linear C ７～１０ Alkyl or linear C ７～１０ alkenyl; Y ２ teeth 【Transformation 6】 and R １ and R ２ are each independently a linear C ７～１０ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R is alkyl.

6. Y １ and Y ２ are each independently 【Transformation 7】 and R １ and R ２ are each independently a linear C ７～１０ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R is alkyl.

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

8. Y １ and Y ２ are each independently 【Transformation 8】 and R １ and R ２ are each independently a linear C ７～１０ 8. The compound of claim 7, or a pharmaceutically acceptable salt thereof, wherein: R is alkyl;

9. Formula (II): 【Chemistry 9】 is a compound of the formula k is 0, 1, or 2; Y １ is a linear C ７～１０ Alkyl, linear C ７～１０ alkenyl, or 【Chemistry 10】 and Y ２ teeth 【Chemistry 11】 and R １ and R ２ are each independently a linear C ７～１０ is alkyl, 2. The compound of claim 1 or a pharmaceutically acceptable salt thereof.

10. X １ teeth 【Chemistry 12】 and X ２ teeth 【Chemistry 13】 2. The compound of claim 1, wherein:

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

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

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

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

15. Y １ and Y ２ are each independently 【Chemistry 14】 and R １ and R ２ are each independently a linear C ７～１０ is alkyl, 11. The compound of 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]nonan-3-yl)octanoate; Heptadecan-9-yl (Z)-8-(7-(8-(non-2-en-1-yloxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonan-3-yl)octanoate; 3-heptyldodecyl 8-(7-(8-((3-octylundecyl)oxy)-8-oxooctyl)-9-oxa-3,7-diazabicyclo[3.3.1]nonan-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; 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from:

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

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

19. 19. The lipid nanoparticle of claim 18, wherein the neutral lipid is selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), or a combination thereof.

20. The lipid nanoparticle of claim 18, wherein the sterol is cholesterol.

21. The lipid nanoparticle of claim 18, wherein the polymer-conjugated 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 nanoparticle of claim 17, further comprising distearoylphosphatidylcholine (DSPC), cholesterol, and DMPE-PEG2000.

23. The lipid nanoparticle of claim 17, further comprising a therapeutic agent.

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

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

26. The lipid nanoparticle of claim 25, wherein the RNA is a modified mRNA.

27. 24. A pharmaceutical composition comprising a plurality of lipid nanoparticles according to claim 23.

28. 28. The pharmaceutical composition of claim 27 for use in the treatment of a disease or disorder.

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