Ionizable cationic lipids for RNA delivery

Abstract

The present disclosure describes compounds of formula (I) below, and pharma- ceutically acceptable salts thereof: [Formula 1] JPEG2024546431000410.jpg65169

Description

[Technical Field]

[0001] FIELD OF THE INVENTION Embodiments herein relate generally to lipids. Specifically, embodiments herein relate to novel lipids and lipid compositions that facilitate intracellular delivery of bioactive and therapeutic molecules. [Background technology]

[0002] The variety of nucleic acid-based therapeutics for targeted delivery creates challenges for lipid-based delivery vehicles. For example, nucleic acids are structurally diverse in size and type. Examples include DNA used in gene therapy, plasmids, small interfering nucleic acids (siNA), and microRNAs (miRNAs) for use in RNA interference (RNAi), antisense molecules, ribozymes, antagomirs, and aptamers.

[0003] The design and use of cationic lipids and ionizable cationic lipids for inclusion in such lipid-based delivery vehicles has shown great advantages. However, the use of these lipids can contribute to significant side effects when administered in vivo. One problem observed includes low biodegradability and clearance from target tissues, thus resulting in lipid accumulation in vivo. Another problem is that large amounts of lipids can cause adverse immunogenic effects, which can lead to discomfort in subjects and reduced therapeutic efficacy of active ingredients. A third problem associated with many cationic lipids is the low rate of effective delivery to the target, thus resulting in relatively low therapeutic efficacy or low efficacy. Finally, it is important that the cationic lipid in the delivery vehicle has a specially adjusted pKa, which can be formulated with nucleic acid-based therapeutic agents to protect the nucleic acid-based therapeutic agent from degradation during administration, and also be able to release the therapeutic agent when the vehicle reaches its target. Therefore, there is a need in the art for the development of new lipids that can meet the special needs of lipid-nucleic acid delivery systems.

[0004] Each of the following references is incorporated by reference herein in its entirety: International Patent Application PCT / US2014 / 066242, published as WO2015074085A1, International Patent Application PCT / US2015 / 030218, published as WO2016081029A1, U.S. Patent US10227302, U.S. Patent US10383952, and U.S. Patent US10526284, each of which discloses ionized cationic lipids for RNA delivery; WO20171 International Patent Application PCT / US2016 / 069493, published as PCT International Patent Application No. 17530A1, which discloses ionizable cationic lipids; International Patent Application PCT / US2019 / 025246, published as WO2019191780A1, which discloses lipid particles for nucleic acid delivery; and U.S. Patent Application No. 16 / 823212, published as US2020 / 0297634, which discloses methods for making lipid-encapsulated RNA nanoparticles. Summary of the Invention

[0005] The present disclosure provides lipids of formula (I) described herein that are useful for lipid-based delivery of nucleic acids and other therapeutic agents for treating diseases.These and other uses will be apparent to those skilled in the art.Additional features and advantages of the subject technology will be set forth in the following description, and in part will be apparent from the description, or may be learned by practice of the subject technology.The advantages of the subject technology will be realized and attained by the structures particularly pointed out in the written description and embodiments herein, as well as in the accompanying drawings.

[0006] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the subject technology.

[0007] In some embodiments, the disclosure provides a compound of formula I, or a pharmaceutically acceptable salt thereof: [ka] (I) During the ceremony, R 1 and R 2 are each independently H or C 1-6 is alkyl; or R 1 and R 2 are joined to form a saturated heterocyclic ring, R 1 is a linear C 1-4 alkylene; and R 2 is -(CH2) m (X) n - in which X is O, S or NR 9 where R 9 is H or C 1-6 is alkyl; m is 1, 2, 3, or 4; and n is 0 or 1; L1 is a linear C optionally substituted with 1 to 3 methyl groups. 1-6 is alkylene; Y is selected from the group consisting of: [ka] During the ceremony, Each asterisk (*) indicates an atom bonded to L2 and L3; and R 10 is H or C 1-6 is alkyl; L2 and L3 are each independently a linear C 1-8 is alkylene; L4, L5, L6, L7, L8, and L9 are each independently absent or -CH2-, with the proviso that At least two of L4, L6 and L8 are -CH2-; and at least two of L5, L7 and L9 are -CH2-; R 3 and R 4 are each independently H, methyl, or ethyl; and R 5, R 6 , R 7 , and R 8 are each independently selected from the group consisting of: Linear C 1-20 Alkyl, wherein each linear C 1-20 The alkyl is optionally Substituted with one or more substituents selected from the group consisting of: C 1-6 Alkyl, C 1-6 Alkoxy, and -F, wherein the C 1-6 Each of the alkyl substituents is C 1-3 optionally substituted with one or more groups selected from the group consisting of alkoxy and -F; C 3-8 monocycloalkyl, wherein the C 3-8 Each monocycloalkyl is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C 7-12 bicycloalkyl, wherein the C 7-12 Each of the bicycloalkyls is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; and C 6-10 aryl, wherein the C 6-10 Each of the aryls is C 1-6 Alkyl, C 1-6 a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C 3-8 monocycloalkyl, wherein the C 3-8 Each monocycloalkyl is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C 7-12 bicycloalkyl, wherein the C 7-12 Each of the bicycloalkyls is C1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; and C 6-10 aryl, wherein the C 6-10 Each of the aryls is C 1-6 Alkyl, C 1-6 is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F.

[0008] In some embodiments, the present disclosure provides lipid nanoparticles comprising a plurality of ligands, each ligand independently being a compound described herein, wherein the plurality of ligands self-assemble to form a lipid nanoparticle comprising an interior and an exterior.

[0009] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a compound described herein or a lipid nanoparticle described herein, and a pharmaceutically acceptable excipient.

[0010] In some embodiments, the disclosure provides a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound described herein, a lipid nanoparticle described herein, or a pharmaceutical composition described herein.

[0011] In some embodiments, the present disclosure provides a method of delivering a nucleic acid to a subject in need thereof, the method comprising encapsulating a therapeutically effective amount of a nucleic acid in a lipid nanoparticle described herein and administering the lipid nanoparticle to the subject.

[0012] I. Overview Various configurations of the subject technology will be readily apparent to those skilled in the art from this disclosure, and it will be understood that various configurations of the subject technology have been shown and described by way of example. It will be understood that the subject technology is capable of other and different configurations, and its several details can all be modified in various other respects without departing from the scope of the subject technology. Accordingly, the Summary and Detailed Description should be regarded as illustrative in nature and not restrictive.

[0013] The detailed description of the present invention is intended as a description of various configurations of the subject technology and is not intended to represent the only configurations in which the subject technology may be practiced. The detailed description includes specific details for the purpose of providing a thorough understanding of the subject technology. However, it will be apparent to those skilled in the art that the subject technology may be practiced without these specific details.

[0014] II. Definition At various places in the present specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include any and all individual subcombinations of the members of such groups and ranges. For example, "C 1-6 The term "alkyl" is specifically intended to individually disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.

[0015] The term "approximately" or "about" as applied to one or more values ​​of interest refers to a value similar to a stated reference value. In certain embodiments, unless otherwise stated or otherwise clear from the context, the term "approximately" or "about" refers to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction of (greater than or less than) the stated reference value (except where such number would exceed 100% of the possible values).

[0016] In the claims, articles such as "a," "an," and "the" may mean one or more unless indicated to the contrary or clear from the context. A claim or description containing "or" between one or more group members is deemed to be satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless indicated to the contrary or otherwise clear from the context. The present disclosure includes embodiments in which exactly one member of a group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which two or more, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0017] As used herein, the term "alkoxy," alone or in combination, refers to an alkyl ether radical, where alkyl is as defined below. An alkoxy group can have the general formula: alkyl-O-. With respect to alkyl groups, alkoxy groups include, for example, C 1-6 Alkoxy groups can have any suitable number of carbon atoms, such as, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 2-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, and the like. Alkoxy groups can be further optionally substituted as defined herein.

[0018] As used herein, "alkyl" refers to a straight- or branched-chain hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds). An alkyl group may have 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as "1 to 20" refers to each integer within the given range; for example, "1 to 20 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, but this definition also encompasses occurrences of the term "alkyl" where no numerical range is specified). An alkyl group may have 1 carbon, 2 carbons, 3 carbons, 4 carbons, 5 carbons, 6 carbons, 7 carbons, 8 carbons, 9 carbons, 10 carbons, 11 carbons, 12 carbons, 13 carbons, 14 carbons, 15 carbons, 16 carbons, 17 carbons, 18 carbons, 19 carbons, or 20 carbons. An alkyl group may be straight-chained or branched. Alkyl is, for example, C 1-2 , C 1-3 , C 1-4 , C 1-5 , C 1-6 , C 1-7 , C 1-8 , C 1-9 , C 1-10 , C 2-3 , C 2-4 , C 2-5 , C 2-6 , C 3-4 , C 3-5 , C 3-6 , C 4-5 , C 4-6 and C 5-6 The alkyl group may contain any number of carbons, such as 1 to 9 carbon atoms. The alkyl group may also be a medium-sized alkyl having 1 to 9 carbon atoms. The alkyl group may also be a lower alkyl having 1 to 6 carbon atoms. The alkyl group may also be a "C 1-4 By way of example only, "C alkyl" may be designated as such, or a similar designation. 1-4"Alkyl" indicates that there are 1 to 4 carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are not limited in any way to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like.

[0019] "Alkylene" refers to a straight-chain or branched saturated aliphatic radical, i.e., a divalent hydrocarbon radical, having the number of carbon atoms indicated and linking at least two other groups. The two moieties linked to the alkylene can be attached to the same atom or different atoms of the alkylene group. For example, a straight-chain alkylene is -(CH2) n- where "n" is 1, 2, 3, 4, 5, or 6. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene, and hexylene. Alkylene groups can be substituted or unsubstituted.

[0020] The term "lower alkyl" means a group having 1 to 6 carbons in the chain which may be straight or branched. Non-limiting examples of suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, and hexyl.

[0021] As used herein, the term “amino” refers to —N(R N1 )2, wherein each R N1 are independently H, OH, NO2, N(R N2 )2, SO2OR N2 , SO2R N2 , SOR N2, an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, cycloalkyl, alkylcycloalkyl, carboxyalkyl (e.g., optionally substituted with an O-protecting group, e.g., an optionally substituted arylalkoxycarbonyl group or any described herein), sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein), alkoxycarbonylalkyl (e.g., optionally substituted with an O-protecting group, e.g., an optionally substituted arylalkoxycarbonyl group or any described herein), heterocyclyl (e.g., heteroaryl), or alkylheterocyclyl (e.g., alkylheteroaryl), and these enumerated R N1 Each of the groups may be optionally substituted as defined herein for each group, or two R N1 can be combined to form a heterocyclyl or N-protecting group, and each R N2 is independently H, alkyl, or aryl. The amino group of the present disclosure can be unsubstituted amino (i.e., -NH) or substituted amino (i.e., -N(R')). In a preferred embodiment, amino is -NH or -NHR. N1 where R N1 are independently OH, NO2, NH2, N(R N2 )2, SO2OR N2 , SO2R N2 , SOR N2 , alkyl, carboxyalkyl, sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein), alkoxycarbonylalkyl (e.g., t-butoxycarbonylalkyl), or aryl, and each R N2 is H, C1- 20 Alkyl (e.g., C 1-6 alkyl), or C 1-10 It may be aryl.

[0022] The term "anionic lipid" refers to a lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

[0023] As used herein, the term "aryl," alone or in combination, refers to a carbocyclic aromatic system containing one, two, or three rings, wherein the rings may be linked together in a pendant manner or may be fused. The term "aryl" encompasses aromatic radicals such as, for example, benzyl, phenyl, naphthyl, anthracenyl, phenanthryl, indanyl, indenyl, annulenyl, azulenyl, tetrahydronaphthyl, and biphenyl. The C of this disclosure 6-10 The aryl is a C6 aryl, a C7 aryl, a C8 aryl, a C9 aryl, or a C 10 In some embodiments, C 6-10 The aryl is monocyclic, such as, for example, a phenyl group. 6-10 Aryl is bicyclic, such as, for example, biphenyl, naphthyl, or indanyl.

[0024] The phrase "at least one" preceding a list of items with the term "and" or "or" separating any of the items modifies the list as a whole, rather than each member (i.e., each item) of the list. The phrase "at least one" does not require the selection of at least one of each listed item; rather, the phrase allows for the inclusion of any one of the items, and / or at least one of any combination of the items, and / or at least one of each of the items. As an example, the phrase "at least one of A, B, and C" or "at least one of A, B, or C" refers to A only, B only, or C only, any combination of A, B, and C, and / or at least one of each of A, B, and C, respectively.

[0025] When the terms "include," "have," or similar terms are used in the description or claims, such terms are intended to be inclusive in the same manner as the term "comprise" when used as a transitional term in a claim.

[0026] The term "cationic lipid" refers to amphipathic lipids and their salts, each of which has a positive hydrophilic head group, one, two, three or more hydrophobic fatty acid or fatty alkyl chains, and a connector between these two domains. Ionizable cationic lipids or protonatable cationic lipids are typically protonated (i.e., positively charged) at pH values ​​below their pKa and substantially neutral at pH values ​​above their pKa. Preferred ionizable cationic lipids are lipids with a pKa lower than physiological pH, typically about 7.4. The cationic lipids of the present disclosure may also be referred to as titratable cationic lipids. Cationic lipids can be "amino lipids" with a protonatable tertiary amine (e.g., pH-titratable) head group. Some exemplary amino lipids may contain a C18 alkyl chain and an ether, ester, or ketal bond between the head group and the alkyl chain. Such cationic lipids include, but are not limited to, DSDMA, DODMA, DLinDMA, DLenDMA, γ-DLenDMA, DLin-K-DMA, DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2, and C2K), DLin-K-C3-DMA A, DLin-K-C4-DMA, DLen-C2K-DMA, y-DLen-C2K-DMA, DLin-M-C2-DMA (also known as MC2), DLin-M-C3-DMA (also known as MC3), and (DLin-MP-DMA) (also known as 1-Bl 1).

[0027] The term "comprising" is intended to be open, allowing for, but not requiring, the inclusion of additional elements or steps. When the term "comprising" is used herein, the terms "consisting of" and "consisting essentially of" are also corollarily included and invoked.

[0028] The term "commercially available chemicals" and chemicals used in the examples described herein may be obtained from standard commercial sources, such as Acros Organics (Pittsburgh, Pa.), Sigma-Adrich Chemical (Milwaukee, Wis.), Avocado Research (Lancashire, UK), Bionet (Cornwall, UK), Boron Molecular (Research Triangle Park, NC), Combi-Blocks (San Diego, Calif.), Eastman Organic Chemicals, Eastman Kodak Company (Rochester, NY), Fisher Scientific Co. (Pittsburgh, Pa.), Frontier Scientific (Logan, Utah), ICN Biomedicals, Inc. (Costa Mesa, Calif.), Lancaster Synthesis (Windham, NH), Maybridge Chemical Co. (Cornwall, UK), Pierce Chemical Co. (Rockford, Ill.), Riedel de Haen (Hannover, Germany), Spectrum Quality Products, Inc. (New Brunswick, NJ), TCI America (Portland, OR), and Wako Chemicals USA, Inc. (Richmond, VA).

[0029] The phrase "compounds described in the chemical literature" can be identified through reference books and databases covering chemical compounds and chemical reactions, as known to those skilled in the art. Suitable reference books and articles detailing the synthesis of reactants useful in preparing the compounds disclosed herein or providing references to articles describing the preparation of the compounds disclosed herein include, for example, "Synthetic Organic Chemistry", John Wiley and Sons, Inc. New York; S.R. Sandler et al., "Organic Functional Group Preparations," 2nd Ed., Academic Press, New York, 1983; H.O. House, "Modern Synthetic Reactions," 2nd Ed., W.A. Benjamin, Inc. Menlo Park, Calif., 1972; T.L. Glichrist, "Heterocyclic Chemistry," 2nd Ed., John Wiley and Sons, New York, 1992; J. March, "Advanced Organic Chemistry: Reactions, Mechanisms and Structure," 5th Ed., Wiley Interscience, New York. York, 2001; specific and similar reactants can also be identified through the index of known chemicals produced by the Chemical Abstract Service of the American Chemical Society, which is available in most public book and university libraries as well as online databases (for more information, contact the American Chemical Society, Washington, DC). Chemicals that are known but not commercially available in catalogs may be prepared by custom chemical synthesis companies, where many of the standard chemical supply companies (such as those listed above) offer custom synthesis services.

[0030] As used herein, the term "effective amount" of an agent is an amount sufficient to bring about a beneficial or desired result, e.g., a clinical result, and thus "effective amount" will depend on the context in which it is applied. For example, in the context of administering an agent to treat cancer, an effective amount of the agent is an amount sufficient to achieve treatment of cancer as defined herein, e.g., compared to the response obtained without administration of the agent.

[0031] The term " completely encapsulated " means that the nucleic acid (for example, mRNA) in nucleic acid-lipid particles is not significantly degraded after being exposed to serum or nuclease assay, which would significantly degrade free RNA.When completely encapsulated, in a treatment that would normally degrade 100% of free nucleic acid, preferably less than 25% of the nucleic acid in the particles is degraded, more preferably less than 10%, and most preferably less than 5% of the nucleic acid in the particles is degraded." Completely encapsulated " also means that the nucleic acid-lipid particles are not rapidly degraded into their component parts when administered in vivo.

[0032] The term "compound" is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted.

[0033] As used herein, the term "cycloalkyl," or "carbocycle," alone or in combination, refers to a saturated or partially saturated, monocyclic or bicyclic alkyl radical, wherein each cyclic moiety contains 3 to 12 carbon atom ring members, which may optionally be a benzo-fused ring system, which may be optionally substituted as defined herein. In some embodiments, a cycloalkyl may contain 3 to 8 carbon atoms, or 7 to 12 carbon atoms. Examples of such cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, octahydronaphthyl, 2,3-dihydro-1H-indenyl, adamantyl, and the like. As used herein, "bicyclic" and "tricyclic" are intended to include both fused ring systems, such as, for example, decahydronaphthalene, octahydronaphthalene, and polycyclic (multi-center) saturated or partially unsaturated types. The latter isomers are commonly exemplified by bicyclo[1.1.1]pentane, camphor, adamantane, and bicyclo[3.2.1]octane. In embodiments, the cycloalkyl ring is a monocyclic ring containing 3 to 8 carbon atoms. In embodiments, the monocyclic ring is 3, 4, 5, 6, 7, or 8 carbon atoms. In embodiments, the cycloalkyl ring is a bicyclic ring containing 7 to 12 carbon atoms. In embodiments, the bicyclic ring is 7, 8, 9, 10, 11, or 12 carbon atoms.

[0034] The term "delivery" refers to the act or method of delivering a compound, substance, entity, moiety, cargo, or payload.

[0035] As used herein, the term "fragment" refers to a portion. For example, a fragment of a protein may include a polypeptide obtained by digesting a full-length protein isolated from a cultured cell.

[0036] The term "hydrophobic lipid" refers to a compound having a non-polar group, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups optionally substituted with one or more aromatic, alicyclic, or heterocyclic groups. Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0037] The term "lipid" refers to organic compounds that contain esters of fatty acids and are characterized by being insoluble in water but soluble in many organic solvents. Lipids are generally divided into at least three classes: (1) "simple lipids," which include fats and oils and waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0038] The term "lipid delivery vehicle" refers to a lipid formulation that can be used to deliver a therapeutic nucleic acid (e.g., mRNA) to a target site of interest (e.g., a cell, tissue, organ, etc.). The lipid delivery vehicle can be a nucleic acid-lipid particle that can be formed from a cationic lipid, a non-cationic lipid (e.g., a phospholipid), a conjugated lipid that prevents particle aggregation (e.g., a PEG lipid), and optionally cholesterol. Typically, the therapeutic nucleic acid (e.g., mRNA) is encapsulated in the lipid portion of the particle, which can protect it from enzymatic degradation.

[0039] The term "lipid encapsulation" refers to lipid particles that provide a therapeutic nucleic acid, such as mRNA, that is fully encapsulated, partially encapsulated, or both. In preferred embodiments, the nucleic acid (e.g., mRNA) is fully encapsulated within the lipid particle.

[0040] The term "amphipathic lipid" or "amphiphilic lipid" refers to a material in which the hydrophobic portion of the lipid material orients toward the hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase. The hydrophilic character comes from the presence of polar or charged groups such as carbohydrate, phosphate, carboxylic acid, sulfato, amino, sulfhydryl, nitro, hydroxyl, and other similar groups. Hydrophobicity can be imparted by the inclusion of nonpolar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0041] The term "heteroalkyl," as used herein, alone or in combination, refers to a stable straight- or branched-chain, or cyclic hydrocarbon radical, or combinations thereof, fully saturated or containing one to three degrees of unsaturation, and consisting of a specified number of carbon atoms and one to three heteroatoms selected from the group consisting of O, N, and S, where the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized (i.e., bonded to four groups). The O, N, and S heteroatoms may be located at any interior position of the heteroalkyl group. Up to two heteroatoms may be consecutive, such as, for example, --CHNHOCH.

[0042] The term "linker" or "linking moiety" refers to a group of atoms, e.g., 10 to 100 atoms, which can be composed of atoms or groups such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker may be of sufficient length so as not to interfere with incorporation into the amino acid sequence. Examples of chemical groups that can be incorporated into the linker include, but are not limited to, alkyl, alkenyl, alkynyl, amido, amino, ether, thioether, ester, alkyl, heteroalkyl, aryl, or heterocyclyl, each of which can be optionally substituted as described herein. Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., monomeric units of ethylene or propylene glycol, e.g., diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran polymers. Other examples include, but are not limited to, cleavable moieties within the linker, such as, for example, a disulfide bond (-SS-) or an azo bond (-NN-), which can be cleaved using a reducing agent or photolysis. Non-limiting examples of selectively cleavable bonds include, for example, an amide bond, which can be cleaved by the use of tris(2-carboxyethyl)phosphine (TCEP) or other reducing agents, and / or photolysis, as well as ester bonds, which can be cleaved by, for example, acidic or basic hydrolysis.

[0043] The term mammal means a human or other mammal, or a human.

[0044] The term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a protein or polypeptide of interest and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded protein or polypeptide of interest.

[0045] The term "modified" refers to a change in the state or structure of a molecule of the present disclosure. Molecules may be modified in many ways, including chemically, structurally, and functionally. In one embodiment, a nucleic acid active ingredient is modified by the introduction of non-natural nucleosides and / or nucleotides, for example, as they relate to the natural ribonucleotides A, U, G, and C. Non-standard nucleotides, such as cap structures, may differ in chemical structure from the A, C, G, and U ribonucleotides, but are not considered "modified."

[0046] The term "naturally occurring" means existing in nature without artificial assistance.

[0047] The term "patient" refers to a subject seeking or needing treatment, a subject in need of treatment, a subject receiving treatment, or a subject to be treated, or a subject receiving care from a trained professional for a particular disease or condition.

[0048] The phrase "optionally substituted X" (e.g., optionally substituted alkyl) is intended to be equivalent to "X, where X is optionally substituted" (e.g., "alkyl, where the alkyl is optionally substituted"). It is not intended to imply that the character of "X" (e.g., alkyl) itself is optional.

[0049] As used herein, the phrase "pharmaceutically acceptable" is used to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0050] As used herein, the phrase "pharmaceutically acceptable excipient" refers to any component other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving an active compound), which has substantially non-toxic and non-inflammatory properties in patients. Excipients may include, for example, anti-adhesives, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), softeners, emulsifiers, fillers (diluents), film formers or coatings, flavors, flavorings, glidants (glidants), lubricants, preservatives, printing inks, absorbents, suspending or dispersing agents, sweeteners, and hydration water. Exemplary excipients include, but are not limited to, butylhydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0051] The phrase "pharmaceutically acceptable salt" refers to derivatives of the disclosed compounds, where the parent compound is modified by converting an existing acid or base moiety into its salt form (e.g., by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, cinnamate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, and the like. Salts include, for example, hydroxybenzoates, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, toluenesulfonates, undecanoates, and valerates. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Pharmaceutically acceptable salts of the present disclosure include conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.Generally, these salts can be prepared by reacting the free acid form or free base form of these compounds with a stoichiometric amount of a suitable base or acid in water or an organic solvent, or in a mixture of the two, and generally non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p.1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.

[0052] The term "pharmacokinetics" refers to any one or more characteristics of a molecule or compound as they relate to determining the fate of a substance administered to a living organism. Pharmacokinetics is divided into several areas, including the extent and rate of absorption, distribution, metabolism, and excretion. This is commonly referred to as ADME: (A) absorption is the process of a substance entering the blood circulation; (D) distribution is the dispersion or dissemination of a substance throughout the body's fluids and tissues; (M) metabolism (or biotransformation) is the irreversible conversion of a parent compound to daughter metabolites; and (E) excretion (or elimination) refers to the removal of a substance from the body. In rare cases, some drugs irreversibly accumulate in body tissues.

[0053] As used herein, the term "pharmaceutically acceptable solvate" refers to a compound of the present disclosure in which molecules of a suitable solvent are incorporated into the crystal lattice. A suitable solvent is physiologically tolerable at the administered dosage. For example, a solvate may be prepared by crystallization, recrystallization, or precipitation from a solution containing an organic solvent, water, or a mixture thereof. Examples of suitable solvents include ethanol, water (e.g., monohydrate, dihydrate, and trihydrate), N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N,N'-dimethylformamide (DMF), N,N'-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water is the solvent, the solvate is referred to as a "hydrate."

[0054] The term "phosphate" is used in its ordinary sense as understood by those skilled in the art, including its protonated form, e.g., [ka] Includes.

[0055] As used herein, the terms "monophosphate," "diphosphate," and "triphosphate" are used in their ordinary sense as understood by those skilled in the art and include the protonated forms.

[0056] The term "prevention" refers to partially or completely delaying the onset of an infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, features, or clinical signs of a particular infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, features, or signs of a particular infection, disease, disorder, and / or condition; partially or completely delaying the progression from an infection, a particular disease, disorder, and / or condition, and / or reducing the risk of developing pathology associated with an infection, disease, disorder, and / or condition.

[0057] The term "RNA" refers to a molecule containing at least one ribonucleotide residue. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribo-furanose moiety. This term includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications can include the addition of non-nucleotide material to the end of an interfering RNA or internally, for example, at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the present disclosure can also include non-naturally occurring nucleotides or non-standard nucleotides such as chemically synthesized nucleotides or deoxynucleotides. These modified RNAs can be referred to as analogs or analogs of naturally occurring RNA. As used herein, the terms "ribonucleic acid" and "RNA" refer to molecules containing at least one ribonucleotide residue, including siRNA, antisense RNA, single-stranded RNA, microRNA, mRNA, non-coding RNA, and polyvalent RNA.

[0058] The term "sample" or "biological sample" refers to a subset of its tissues, cells, or component parts (e.g., bodily fluids, including, but not limited to, blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic blood, urine, vaginal fluid, and semen). A sample may further include homogenates, lysates, or extracts prepared from a whole organism, or a subset of its tissues, cells, or component parts, or a fraction or portion thereof, including, but not limited to, plasma, serum, spinal fluid, lymph, external sections of skin, respiratory, intestinal, and reproductive tracts, tears, saliva, milk, blood cells, tumors, organs. A sample also refers to a culture medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecules.

[0059] The terms "significant" or "significantly" are used synonymously with the term "substantially."

[0060] The phrase "single unit dose" is a dose of any therapeutic agent administered in one dose / single / single route / single point of contact, i.e., a single administration event.

[0061] The term "siRNA" or small interfering RNA, sometimes known as short interfering RNA or silencing RNA, typically refers to a class of double-stranded non-coding RNA molecules, 18-27 base pairs in length, similar to miRNAs, that operate within the RNA interference (RNAi) pathway, which interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, thereby preventing translation.

[0062] The term solvate refers to a physical association of a compound of the present disclosure with one or more solvent molecules. This physical association involves varying degrees of ionic bonding, including hydrogen bonding. In certain cases, a solvate has the ability to isolate, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "Solvate" encompasses both solution-phase and isolable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates, and the like.

[0063] The term "stable" refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture and, preferably, is capable of being formulated into an effective therapeutic agent.

[0064] The terms "stabilize," "stabilized," and "stabilized region" mean to make stable or to become stable.

[0065] The term "substituted" means substitution with a specified group other than hydrogen, or, for example, with one or more independently selected groups, moieties, or radicals, each of which may be the same or different.

[0066] The term "substantially" refers to a qualitative condition indicating the extent or degree of a total or near-total characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of perfection inherent in many biological and chemical phenomena.

[0067] The phrase "substantially equal" refers to the time difference between doses, the term meaning ±2%.

[0068] The phrase "substantially simultaneously" refers to multiple doses and the term means within 2 seconds.

[0069] The phrase "suffering from" relates to an individual "suffering from" a disease, disorder, and / or condition having been diagnosed with or exhibiting one or more symptoms of the disease, disorder, and / or condition.

[0070] The phrase "susceptibility" refers to an individual who is "susceptible" to a disease and / or condition who has not been diagnosed with the disease, disorder, and / or condition and / or does not exhibit symptoms of the disease, disorder, and / or condition, but who has a tendency to develop the disease or its symptoms. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., cancer) may be characterized by one or more of the following: (1) a genetic mutation associated with the development of the disease, disorder, and / or condition, (2) a genetic polymorphism associated with the development of the disease, disorder, and / or condition, (3) an increase and / or decrease in the expression and / or activity of proteins and / or nucleic acids associated with the disease, disorder, and / or condition, (4) habits and / or lifestyle associated with the development of the disease, disorder, and / or condition, (5) a family history of the disease, disorder, and / or condition, and (6) exposure to and / or infection with a microorganism associated with the development of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition does not develop the disease, disorder, and / or condition.

[0071] The term "synthetic" means produced, prepared, and / or manufactured by the hand of man. Synthesis of polynucleotides or polypeptides or other molecules of the disclosure may be chemical synthesis or enzymatic synthesis.

[0072] The term "therapeutic agent" refers to any agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject.

[0073] The term "therapeutically effective amount" means the amount of agent (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) delivered that, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, is sufficient to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0074] The term "therapeutically effective outcome" means an outcome that is sufficient to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of an infection, disease, disorder, and / or condition in a subject suffering from or susceptible to the infection, disease, disorder, and / or condition.

[0075] The term "total daily dose" is the amount given or prescribed for a 24-hour period. It may be administered as a single unit dose.

[0076] The term "treatment" refers to the partial or complete alleviation, palliation, improvement, amelioration, delay in onset, inhibition of progression, reduction in severity, and / or reduction in incidence of one or more symptoms or characteristics of a particular infection, disease, disorder, and / or condition. For example, "treatment" of cancer may refer to inhibiting tumor survival, growth, and / or spread. Treatment may be administered to subjects who do not show signs of the disease, disorder, and / or condition and / or who show only early signs of the disease, disorder, and / or condition, with the intent of reducing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0077] The term "unmodified" refers to any substance, compound, or molecule before it has been altered in any way. Unmodified may, but does not necessarily, refer to a wild-type or naturally occurring biomolecule. A molecule may undergo a series of modifications, whereby each modified molecule may serve as an "unmodified" starting molecule for subsequent modifications.

[0078] The compounds described herein may be asymmetric (e.g., having one or more stereocenters). Unless otherwise specified, all stereoisomers, such as enantiomers and diastereomers, are intended. Compounds of the present disclosure containing asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods for preparing optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by enantioselective and / or stereoselective synthesis. Many geometric isomers of olefins, C-N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present disclosure. Cis and trans geometric isomers of the compounds of the present disclosure are described and may be isolated as a mixture of isomers or as separated isomeric forms.

[0079] The compounds of the present disclosure also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond and the simultaneous migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonation states with the same empirical formula and total charge. Examples of prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and cyclic forms in which protons can occupy two or more positions in heterocyclic ring systems, such as 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms may be in equilibrium or sterically locked into one form by appropriate substitution.

[0080] The compounds of the present disclosure also include all isotopes of atoms occurring in intermediate or final compounds. "Isotopes" refer to atoms with the same atomic number but different mass numbers resulting from different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium.

[0081] The compounds and salts of the disclosure can be prepared in combination with solvents or water molecules to form solvates and hydrates by routine methods.

[0082] The term "half-life" is the time required for a quantity, such as the concentration or activity of a nucleic acid or protein, to decrease to half of its value as measured at the beginning of a period of time.

[0083] The term "in vitro" refers to events that take place not in a living organism (e.g., an animal, plant, or microorganism) but in an artificial environment, e.g., in a test tube or reaction vessel, in a cell culture, in a petri dish, etc.

[0084] The term "in vivo" refers to events that take place within a living organism (e.g., an animal, plant, or microorganism, or cells or tissues thereof).

[0085] The term "monomer" refers to a single unit, e.g., a single nucleic acid, that can combine with another molecule of the same or different type to form an oligomer. In some embodiments, the monomer may be a non-locked nucleic acid, i.e., a UNA monomer.

[0086] The term "neutral lipid" refers to lipid species that exist in either an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0087] The term "non-cationic lipid" means an amphipathic lipid or a neutral lipid or an anionic lipid, as described herein.

[0088] The term "subject" or "patient" refers to any living organism to which a composition according to the present disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants.

[0089] The term "translatable" may be used interchangeably with the term "expressible" and refers to the ability of a polynucleotide or a portion thereof to be converted into a polypeptide by a host cell. As understood in the art, translation is the process by which ribosomes in the cytoplasm of a cell produce a polypeptide. In translation, messenger RNA (mRNA) is decoded by tRNA in the ribosomal complex to produce a specific amino acid chain or polypeptide. Furthermore, when used herein with respect to an oligomer, the term "translatable" means that at least a portion of the oligomer, for example, the coding region (also known as the coding sequence or CDS) of the oligomer sequence, has the ability to be converted into a protein or a fragment thereof.

[0090] While the present disclosure has been described in connection with certain specific embodiments and numerous details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the present disclosure includes additional embodiments and that some of the details described herein may be varied considerably without departing from the present disclosure. The present disclosure includes such additional embodiments, modifications, and equivalents. In particular, the present disclosure includes any combination of features, terms, or elements of the various exemplary components and examples.

[0091] III.Compound In some embodiments, the disclosure provides a compound of formula I, or a pharmaceutically acceptable salt thereof: [ka] (I) During the ceremony, R 1 and R 2 are each independently H or C 1-6 is alkyl; or R 1 and R 2 are joined to form a saturated heterocyclic ring, R 1 is a linear C 1-4 alkylene; and R 2 is -(CH2) m (X) n - in which X is O, S or NR 9 where R 9 is H or C 1-6 is alkyl; m is 1, 2, 3, or 4; and n is 0 or 1; L1 is a linear C optionally substituted with 1 to 3 methyl groups. 1-6 is alkylene; Y is selected from the group consisting of: [ka] During the ceremony, Each asterisk (*) indicates an atom bonded to L2 and L3; and R 10 is H or C 1-6 is alkyl; L2 and L3 are each independently a linear C 1-8 is alkylene; L4, L5, L6, L7, L8, and L9 are each independently absent or -CH2-, with the proviso that At least two of L4, L6 and L8 are -CH2-; and at least two of L5, L7 and L9 are -CH2-; R 3 and R 4are each independently H, methyl, or ethyl; and R 5 , R 6 , R 7 , and R 8 are each independently selected from the group consisting of: Linear C 1-20 Alkyl, wherein each linear C 1-20 The alkyl is optionally Substituted with one or more substituents selected from the group consisting of: C 1-6 Alkyl, C 1-6 Alkoxy, and -F, wherein the C 1-6 Each of the alkyl substituents is C 1-3 optionally substituted with one or more groups selected from the group consisting of alkoxy and -F; C 3-8 monocycloalkyl, wherein the C 3-8 Each monocycloalkyl is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C 7-12 bicycloalkyl, wherein the C 7-12 Each of the bicycloalkyls is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; and C 6-10 aryl, wherein the C 6-10 Each of the aryls is C 1-6 Alkyl, C 1-6 a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C 3-8 monocycloalkyl, wherein the C 3-8 Each monocycloalkyl is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C7-12 bicycloalkyl, wherein the C 7-12 Each of the bicycloalkyls is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; and C 6-10 aryl, wherein the C 6-10 Each of the aryls is C 1-6 Alkyl, C 1-6 is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F.

[0092] In some embodiments, the disclosure provides a compound of formula I, or a pharmaceutically acceptable salt thereof: [ka] (I) During the ceremony, R 1 and R 2 are each independently H or C 1-6 is alkyl; or R 1 and R 2 are joined to form a saturated heterocyclic ring, R 1 is a linear C 1-4 alkylene; and R 2 is -(CH2) m (X) n - in which X is O, S or NR 9 where R 9 is H or C 1-6 is alkyl; m is 1, 2, 3, or 4; and n is 0 or 1; L1 is a linear C optionally substituted with 1 to 3 methyl groups. 1-6 is alkylene; Y is selected from the group consisting of: [ka] During the ceremony, Each asterisk (*) indicates an atom bonded to L2 and L3; and R 10 is H or C 1-6 is alkyl; L2 and L3 are each independently a linear C 1-8 is alkylene; L4, L5, L6, L7, L8, and L9 are each independently absent or -CH2-, with the proviso that At least two of L4, L6 and L8 are -CH2-; and at least two of L5, L7 and L9 are -CH2-; R 3 and R 4 are each independently H, methyl, or ethyl; and R 5 , R 6 , R 7 , and R 8 are each independently selected from the group consisting of: Linear C 1-20 Alkyl, wherein each linear C 1-20 The alkyl is optionally Substituted with one or more substituents selected from the group consisting of: C 1-6 Alkyl, C 1-6 Alkoxy, and -F, wherein the C 1-6 Each of the alkyl substituents is C 1-3 optionally substituted with one or more groups selected from the group consisting of alkoxy and -F; C 3-8 monocycloalkyl, wherein the C 3-8 Each monocycloalkyl is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C 7-12 bicycloalkyl, wherein the C 7-12Each of the bicycloalkyls is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; and C 6-10 aryl, wherein the C 6-10 Each of the aryls is C 1-6 Alkyl, C 1-6 a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C 3-8 monocycloalkyl, wherein the C 3-8 Each monocycloalkyl is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C 7-12 bicycloalkyl, wherein the C 7-12 Each of the bicycloalkyls is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; and C 6-10 aryl, wherein the C 6-10 Each of the aryls is C 1-6 Alkyl, C 1-6 is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F.

[0093] In some embodiments, R 1 is H or C 1-6 alkyl, and R 2 is C 1-6 alkyl or R 1 and R 2 are bonded to form the saturated heterocycle.

[0094] In some embodiments, Y is selected from the group consisting of: [ka]

[0095] In some embodiments, Y is: [ka]

[0096] In some embodiments, Y is: [ka]

[0097] In some embodiments, Y is: [ka]

[0098] In some embodiments, Y is [ka] and R 1 and R 2 When at least one of R is H, L is -CH- or -CHCH-. In some embodiments, regardless of the identity of Y, R 1 and R 2 At least one of is H, and L1 is -CH2- or -CH2CH2-.

[0099] In some embodiments, Y is: [ka]

[0100] In some embodiments, Y is: [ka]

[0101] In some embodiments, Y is: [ka]

[0102] In some embodiments, Y is: [ka]

[0103] In some embodiments, R 1 and R 2 are each independently, C 1-6 In some embodiments, R 1 and R 2 are each independently, C 1-3 In some embodiments, R 1 and R 2 are each methyl.

[0104] In some embodiments, R 1 and R 2 are linked to form a heterocycle. In some embodiments, the heterocycle is selected from the group consisting of: [ka] In the formula, each asterisk (*) indicates an atom bonded to L1.

[0105] In some embodiments, R 1 and R 2 are joined to form a heterocycle selected from the group consisting of: [ka]

[0106] In some embodiments, R 1 and R 2 are joined to form a heterocycle selected from the group consisting of: [ka]

[0107] In some embodiments, R 5 , R 6 , R 7 and R 8 are each independently selected from the group consisting of: Linear C 1-8 alkyl, wherein the linear C 1-8 Alkyl is optionally Substituted with one or more substituents selected from the group consisting of: C 1-3 Alkyl, C 1-3 Alkoxy, and -F, wherein the C 1-3 Each of the alkyl substituents is C 1-3 optionally substituted with one or more groups selected from the group consisting of alkoxy and -F; saturated C 3-6 monocycloalkyl, wherein the saturated C 3-6 Each monocycloalkyl is C 1-6 Alkyl, C 1-3 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; saturated C 7-12 Bicycloalkyl, wherein the saturated C 7-12 Each of the bicycloalkyls is C 1-6 Alkyl, C 1-3 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; and C 6-10 aryl, wherein the C 6-10 Each of the aryls is C 1-6 Alkyl, C 1-3 a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; saturated C 3-6 monocycloalkyl, wherein the C 3-6 Each monocycloalkyl is C 1-6 Alkyl, C 1-3optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; saturated C 7-12 Bicycloalkyl, wherein the saturated C 7-12 Each of the bicycloalkyls is C 1-6 Alkyl, C 1-3 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; and C 6-10 aryl, wherein the C 6-10 Each of the aryls is C 1-6 Alkyl, C 1-3 is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F.

[0108] In some embodiments, R 5 , R 6 , R 7 and R 8 are each independently a linear C 1-8 alkyl, in which case the linear C 1-8 The alkyl is optionally C 1-3 Alkyl, C 1-3 substituted with one or more substituents selected from the group consisting of alkoxy and -F, 1-3 Each of the alkyl substituents is optionally selected from C 1-3 In some embodiments, R is substituted with one or more groups selected from the group consisting of alkoxy and -F. 5 , R 6 , R 7 and R 8 are each independently one or more C 1-3 Linear C optionally substituted with alkyl 1-8 In some embodiments, R 5 , R 6 , R 7 , and R 8 each independently represents a linear C optionally substituted with 1 to 3 methyl groups 6-8 In some embodiments, R 5 , R 6 , R7 , and R 8 are each independently n-heptyl or n-octyl.

[0109] In some embodiments, R 5 , R 6 , R 7 , and R 8 are each independently saturated C 3-6 Linear C optionally substituted with monocycloalkyl 1-8 alkyl, in which case the saturated C 3-6 Monocycloalkyl is optionally C 1-6 Alkyl, C 1-3 In some embodiments, the saturated C 3-6 Each monocycloalkyl optionally contains one or more C 1-3 In some embodiments, the saturated C 3-6 Each monocycloalkyl is optionally substituted with 1 to 3 methyl groups.

[0110] In some embodiments, R 5 , R 6 , R 7 , and R 8 are each independently saturated C 7-12 Linear C optionally substituted with bicycloalkyl 1-8 alkyl, and the saturated C 7-12 Each bicycloalkyl is C 1-6 Alkyl, C 1-3 In some embodiments, the saturated C 7-12 Each bicycloalkyl optionally contains one or more C 1-3 In some embodiments, the saturated C 7-12 Each bicycloalkyl is optionally substituted with 1 to 3 methyl groups.

[0111] In some embodiments, R 5 , R 6 , R7 , and R 8 are each independently, C 6-10 Linear C optionally substituted with aryl 1-8 alkyl, and the C 6-10 Each aryl is C 1-6 Alkyl, C 1-3 In some embodiments, the C is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy and -F. 6-10 Each aryl may contain one or more C 1-3 In some embodiments, the C is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with alkyl. 6-10 Each aryl is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one to three methyl groups.

[0112] In some embodiments, R 5 , R 6 , R 7 , and R 8 are each independently saturated C 3-6 monocycloalkyl, and the C 3-6 Each monocycloalkyl is optionally selected from C 1-6 Alkyl, C 1-3 In some embodiments, the C is optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F. 3-6 Each monocycloalkyl optionally contains one or more C 1-3 In some embodiments, the C 3-6 Each monocycloalkyl is optionally substituted with 1 to 3 methyl groups. In some embodiments, optionally substituted C 3-6 Monocycloalkyl is cyclopentyl. In some embodiments, optionally substituted C 3-6 Monocycloalkyl is cyclohexyl.

[0113] In some embodiments, R 5 , R 6 , R 7 , and R8 are each independently saturated C 7-12 Bicycloalkyl, the saturated C 7-12 Each bicycloalkyl is optionally selected from C 1-6 Alkyl, C 1-3 In some embodiments, the C is substituted with one or more substituents selected from the group consisting of alkoxy and -F. 7-12 Each bicycloalkyl optionally contains one or more C 1-3 In some embodiments, the C 7-12 Each bicycloalkyl is optionally substituted with 1 to 3 methyl groups.

[0114] In some embodiments, R 5 , R 6 , R 7 , and R 8 are each independently, C 6-10 aryl, and the C 6-10 Each aryl is C 1-6 Alkyl, C 1-3 In some embodiments, the C is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy and -F. 6-10 Each aryl may contain one or more C 1-3 In some embodiments, the C is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with alkyl. 6-10 Each aryl is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with 1 to 3 methyl groups. In some embodiments, the optionally substituted C 6-10 Each aryl is an optionally substituted monocyclic aromatic hydrocarbon. In some embodiments, the optionally substituted monocyclic aromatic hydrocarbon is phenyl. In some embodiments, the optionally substituted C 6-10 Each aryl is an optionally substituted bicyclic aromatic hydrocarbon. In some embodiments, the optionally substituted bicyclic aromatic hydrocarbon is naphthyl.

[0115] In some embodiments, R 5 , R 6 , R 7 and R 8 are each independently a monocycloalkyl selected from the group consisting of: [ka] During the ceremony, Each asterisk (*) indicates an atom bonded to a carbonyl carbon; Each R 11 independently, C 1-6 is alkyl; Each R 12 independently, C 1-3 is alkoxy; Each R 13 is -F; each p is independently 0 to 11; each q is independently 0 to 11; and each r is independently 0 to 11; In the formula, the sum of p, q, and r is 11 or less.

[0116] In some embodiments, R 5 , R 6 , R 7 and R 8 are each independently a bicycloalkyl selected from the group consisting of: [ka] During the ceremony, Each asterisk (*) indicates an atom attached to a carbonyl carbon; and Each bicycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: (R 11 ) p , where each R 11 independently, C 1-6 alkyl, and each p is independently 0 to 13; (R 12 ) q , where each R 12 independently, C 1-3alkoxy, where each q is independently 0 to 13; and (R 13 ) r , where each R 13 is -F, and each r is independently 0 to 13; In the formula, the sum of p, q, and r is 13 or less.

[0117] In some embodiments, R 5 , R 6 , R 7 and R 8 are each independently a bicycloalkyl selected from the group consisting of: [ka] During the ceremony, Each asterisk (*) indicates an atom attached to a carbonyl carbon; and Each bicycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: (R 11 ) p , where each R 11 independently, C 1-6 alkyl, and each p is independently 0 to 15; (R 12 ) q , where each R 12 independently, C 1-3 alkoxy, where each q is independently 0 to 15; and (R 13 ) r , where each R 13 is -F, and each r is independently 0 to 15; In the formula, the sum of p, q, and r is 15 or less.

[0118] In some embodiments, R 5 , R 6 , R 7 and R 8 are each independently a bicycloalkyl selected from the group consisting of: [ka] During the ceremony, Each asterisk (*) indicates an atom attached to a carbonyl carbon; and Each bicycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: (R 11 ) p , where each R 11 independently, C 1-6 alkyl, and each p is independently 0 to 17; (R 12 ) q , where each R 12 independently, C 1-3 alkoxy, where each q is independently 0 to 17; and (R 13 ) r , where each R 13 is -F, and each r is independently 0 to 17; In the formula, the sum of p, q, and r is 17 or less.

[0119] In some embodiments, the sum of p, q, and r is 0.

[0120] In some embodiments, the sum of p, q, and r is one.

[0121] In some embodiments, R 5 , R 6 , R 7 and R 8 are each independently selected from the group consisting of: [ka] During the ceremony, Each asterisk (*) indicates an atom attached to a carbonyl carbon; Each R 14 are independently H or C 1-6 is alkyl; and Each R 15 are independently H or C 1-6 It is alkyl.

[0122] In some embodiments, R 14 are each independently H or methyl, and R 15 is H.

[0123] In some embodiments, R 5 , R 6 , R 7 and R 8 are each independently selected from the group consisting of: [ka] During the ceremony, Each asterisk (*) indicates an atom bonded to a carbonyl carbon; Each R 11 independently, C 1-6 is alkyl; Each R 12 independently, C 1-3 is alkoxy; Each R 13 is -F; each p is independently 0 to 5; each q is independently 0 to 5; and each r is independently 0 to 5; In the formula, the sum of p, q and r is 5 or less.

[0124] In some embodiments, R 5 , R 6 , R 7 and R 8 are each independently selected from the group consisting of: [ka] During the ceremony, Each asterisk (*) indicates an atom attached to a carbonyl carbon; and Each bicyclic aromatic hydrocarbon is optionally substituted with one or more substituents selected from the group consisting of: (R 11 ) p , where each R 11 independently, C 1-6 alkyl, and each p is independently 0 to 7; (R 12 ) q , where each R 12 independently, C 1-3 alkoxy, where each q is independently 0 to 7; and (R 13 ) r , where each R 13 is -F, and each r is independently 0 to 7; In the formula, the sum of p, q and r is 7 or less.

[0125] In some embodiments, the sum of p, q, and r is zero.

[0126] In some embodiments, the sum of p, q, and r is one.

[0127] In some embodiments, R 5 and R 6 are the same. In some embodiments, R 7 and R 8 are the same. In some embodiments, R 5 , R 6 , R 7 , and R 8 are the same.

[0128] In some embodiments, L is a straight chain unsubstituted alkylene. In some embodiments, L is propylene.

[0129] In some embodiments, L2 and L3 are each independently a linear C 1-5 In some embodiments, L2 and L3 are the same.

[0130] In some embodiments, L4 and L5 are the same. In some embodiments, L6 and L7 are the same. In some embodiments, L8 and L9 are the same. In some embodiments, L4, L5, L6, L7, L8, and L9 are each -CH2-. In some embodiments, L6, L7, L8, and L9 are each -CH2-, and L4 and L5 are absent. In some embodiments, L4, L5, L8, and L9 are each -CH2-, and L6 and L7 are absent. In some embodiments, L4, L5, L6, and L7 are each -CH2-, and L8 and L9 are absent.

[0131] In some embodiments, L4 and L5 are the same. In some embodiments, L6 and L7 are the same. In some embodiments, L8 and L9 are the same. In some embodiments, L4, L5, L6, L7, L8, and L9 are each -CH2-. In some embodiments, L6, L7, L8, and L9 are each -CH2-, and L4 and L5 are absent. In some embodiments, L4, L5, L8, and L9 are each -CH2-, and L6 and L7 are absent. In some embodiments, L4, L5, L6, and L7 are each -CH2-, and L8 and L9 are absent.

[0132] In some embodiments, R 3 and R 4 are each independently H or methyl. In some embodiments, R 3 and R 4 are each H. In some embodiments, R 3 and R 4 are each methyl.

[0133] In some embodiments, the compound is [ka] [ka] [ka] [ka] [ka] [ka] [ka] or a pharmaceutically acceptable salt thereof.

[0134] In some embodiments, the compound is lipid 1: [ka] or a pharmaceutically acceptable salt thereof.

[0135] In some embodiments, the compound is lipid 2: [ka] or a pharmaceutically acceptable salt thereof.

[0136] In some embodiments, the compound is lipid 3: [ka] or a pharmaceutically acceptable salt thereof.

[0137] In some embodiments, the compound is lipid 4: [ka] or a pharmaceutically acceptable salt thereof.

[0138] In some embodiments, the compound is lipid 5: [ka] or a pharmaceutically acceptable salt thereof.

[0139] In some embodiments, the compound is lipid 6: [ka] or a pharmaceutically acceptable salt thereof.

[0140] In some embodiments, the compound is lipid 6a: [ka] or a pharmaceutically acceptable salt thereof.

[0141] In some embodiments, the compound is lipid 7: [ka] or a pharmaceutically acceptable salt thereof.

[0142] In some embodiments, the compound is lipid 8: [ka] or a pharmaceutically acceptable salt thereof.

[0143] In some embodiments, the compound is lipid 9: [ka] or a pharmaceutically acceptable salt thereof.

[0144] In some embodiments, the compound is lipid 10: [ka] or a pharmaceutically acceptable salt thereof.

[0145] In some embodiments, the compound is lipid 11: [ka]

[0146] or a pharmaceutically acceptable salt thereof.

[0147] In some embodiments, the compound is lipid 12: [ka] or a pharmaceutically acceptable salt thereof.

[0148] In some embodiments, the compound is lipid 13: [ka] or a pharmaceutically acceptable salt thereof.

[0149] In some embodiments, the compound is lipid 14: [ka] or a pharmaceutically acceptable salt thereof.

[0150] In some embodiments, the compound is lipid 15: [ka] or a pharmaceutically acceptable salt thereof.

[0151] In some embodiments, the compound is lipid 16: [ka] or a pharmaceutically acceptable salt thereof.

[0152] In some embodiments, the compound is lipid 17: [ka] or a pharmaceutically acceptable salt thereof.

[0153] In some embodiments, the compound is lipid 18: [ka] or a pharmaceutically acceptable salt thereof.

[0154] In some embodiments, the compound is lipid 19: [ka] or a pharmaceutically acceptable salt thereof.

[0155] In some embodiments, the compound comprises lipid 20: [ka] or a pharmaceutically acceptable salt thereof.

[0156] In some embodiments, the compound is lipid 21: [ka] or a pharmaceutically acceptable salt thereof.

[0157] In some embodiments, the compound is lipid 22: [ka] or a pharmaceutically acceptable salt thereof.

[0158] In some embodiments, the compound is lipid 23: [ka] or a pharmaceutically acceptable salt thereof.

[0159] In some embodiments, the compound is lipid 24: [ka] or a pharmaceutically acceptable salt thereof.

[0160] In some embodiments, the compound is lipid 25: [ka] or a pharmaceutically acceptable salt thereof.

[0161] In some embodiments, the compound is lipid 26: [ka] or a pharmaceutically acceptable salt thereof.

[0162] In some embodiments, the compound is lipid 27: [ka] or a pharmaceutically acceptable salt thereof.

[0163] In some embodiments, the compound is lipid 28: [ka] or a pharmaceutically acceptable salt thereof.

[0164] In some embodiments, the compound is lipid 29: [ka] or a pharmaceutically acceptable salt thereof.

[0165] In some embodiments, the compound is lipid 30: [ka] or a pharmaceutically acceptable salt thereof.

[0166] In some embodiments, the compound is lipid 31: [ka] or a pharmaceutically acceptable salt thereof.

[0167] In some embodiments, the compound is lipid 32: [ka] or a pharmaceutically acceptable salt thereof.

[0168] In some embodiments, the compound is lipid 33: [ka] or a pharmaceutically acceptable salt thereof.

[0169] In some embodiments, the compound is lipid 34: [ka] or a pharmaceutically acceptable salt thereof.

[0170] In some embodiments, the compound is lipid 35: [ka] or a pharmaceutically acceptable salt thereof.

[0171] In some embodiments, the compound is lipid 36: [ka] or a pharmaceutically acceptable salt thereof.

[0172] In some embodiments, the compound is lipid 37: [ka] or a pharmaceutically acceptable salt thereof.

[0173] In some embodiments, the compound is lipid 38: [ka] or a pharmaceutically acceptable salt thereof.

[0174] In some embodiments, the compound is lipid 39: [ka] or a pharmaceutically acceptable salt thereof.

[0175] In some embodiments, the compound is lipid 40: [ka] or a pharmaceutically acceptable salt thereof.

[0176] In some embodiments, the compound is lipid 41: [ka] or a pharmaceutically acceptable salt thereof.

[0177] In some embodiments, the present invention provides a lipid composition comprising a nucleic acid and a compound of the present disclosure. In some embodiments, the nucleic acid is selected from siRNA, mRNA, self-replicating RNA, DNA plasmid, and antisense oligonucleotide. In some embodiments, the nucleic acid is mRNA or self-replicating RNA comprising a coding region encoding a therapeutic protein of interest. In some embodiments, the therapeutic protein of interest is an enzyme, antibody, antigen, receptor, or transporter. In some embodiments, the therapeutic protein of interest is a gene-editing enzyme. In some embodiments, the gene-editing enzyme is selected from TALEN, CRISPR, meganuclease, or zinc finger nuclease. In some embodiments, the lipid composition comprises a liposome, lipoplex, or lipid nanoparticle.

[0178] IV. Lipid Formulations and Nanoparticles Lipid-based formulations Therapies based on the intracellular delivery of nucleic acids to target cells face both extracellular and intracellular barriers. In fact, naked nucleic acid materials cannot be easily administered systemically due to their toxicity, low stability in serum, rapid renal clearance, reduced uptake by target cells, phagocytic uptake, and their ability to activate immune responses—all characteristics that hinder their clinical development. When exogenous nucleic acid materials (e.g., mRNA) enter the human biological system, they are recognized as foreign pathogens by the reticuloendothelial system (RES) and removed from the blood circulation before having a chance to encounter target cells within or outside the vascular system. The half-life of naked nucleic acids in the bloodstream has been reported to be approximately several minutes (Kawabata K, Takakura Y, Hashida M Pharm Res. 1995 Jun;12(6):825-30). Chemical modifications and appropriate delivery methods can reduce uptake by the RES and protect nucleic acids from degradation by ubiquitous nucleases, which increases the stability and efficacy of nucleic acid-based therapies. In addition, RNA or DNA are anionic hydrophilic polymers that are also anionic on the surface, which makes them unfavorable for cellular uptake. Therefore, the success of nucleic acid-based therapy depends primarily on the development of vehicles or vectors that can efficiently and effectively deliver genetic material to target cells and obtain sufficient levels of expression in vivo with minimal toxicity.

[0179] Furthermore, upon internalization into target cells, nucleic acid delivery vectors face challenges due to intracellular barriers, including endosomal uptake, lysosomal degradation, unpackaging of the nucleic acid from the vector, translocation across the nuclear membrane (for DNA), and release in the cytoplasm (for RNA). Thus, successful nucleic acid-based therapy relies on the ability of the vector to deliver the nucleic acid to a target site inside the cell to achieve sufficient levels of the desired activity, such as gene expression.

[0180] While some gene therapies have successfully utilized viral delivery vectors (e.g., AAV), lipid-based formulations are increasingly recognized as one of the most promising delivery systems for RNA and other nucleic acid compounds due to their biocompatibility and ease of large-scale production. One of the most significant advances in lipid-based nucleic acid therapy occurred in August 2018, when patisiran (ALN-TTR02) was approved by the U.S. Food and Drug Administration (FDA) and the European Commission (EC) as the first siRNA therapeutic. ALN-TTR02 is an siRNA formulation based on so-called stable nucleic acid lipid particle (SNALP) transfection technology. Despite the success of patisiran, the delivery of nucleic acid therapeutics, including mRNA, via lipid formulations is still under development. The use of mRNA in lipid delivery vehicles has rapidly gained attention as a result of the COVID-19 pandemic, and several vaccines delivering mRNA encoding the COVID-19 spike protein have demonstrated strong protective capabilities. Such lipid-based mRNA vaccines include Pfizer and BioNtech's BNT162b2 and Moderna's mRNA-1273, which have received emergency use authorization worldwide.

[0181] Some art-recognized lipid formulation delivery vehicles for nucleic acid therapeutics include, in various embodiments, polymeric carriers (such as polyethyleneimine (PEI), lipid nanoparticles, and liposomes), nanoliposomes, ceramide-containing nanoliposomes, multivesicular liposomes, proteoliposomes, exosomes of both natural and synthetic origin, natural, synthetic, and semi-synthetic lamellar bodies, nanoparticles, micelles, and emulsions. Because these lipid formulations can vary in structure and composition and are expected to be a rapidly developing field, the art uses several different terms to describe a single type of delivery vehicle. At the same time, lipid formulation terminology varies throughout the scientific literature with respect to its intended meaning, and this inconsistent use has led to confusion regarding the exact meaning of some lipid formulation terms. Among several potential lipid formulations, liposomes, cationic liposomes, and lipid nanoparticles are specifically described in detail and defined herein for purposes of this disclosure.

[0182] Liposomes Conventional liposomes are vesicles consisting of at least one bilayer and an internal aqueous compartment. The bilayer membrane of liposomes is typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin, containing spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16:307-321, 1998). The bilayer membrane of liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). They generally exist as spherical vesicles and can range in size from 20 nm to several microns. Liposome formulations can be prepared as colloidal dispersions or lyophilized to reduce stability risks and improve the shelf life of liposome-based drugs. Methods for preparing liposome compositions are known in the art and are within the skill of those of ordinary skill in the art.

[0183] Liposomes with only one bilayer are called unilamellar, while liposomes with two or more bilayers are called multilamellar. The most common types of liposomes are small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), and multilamellar vesicles (MLVs). In contrast to liposomes, lysosomes, micelles, and reverse micelles are composed of a single lipid layer. While liposomes are generally considered to have a single internal compartment, some formulations can be multivesicular liposomes (MVLs), which consist of multiple discontinuous internal aqueous compartments separated by several non-concentric lipid bilayers.

[0184] Liposomes have long been recognized as drug delivery vehicles due to their excellent biocompatibility, given that liposomes are essentially analogs of biological membranes and can be prepared from both natural and synthetic phospholipids ( Int. J. Nanomedicine. 2014;9:1833-1843 ). In their use as drug delivery vehicles, liposomes have an aqueous core surrounded by a hydrophobic membrane, so hydrophilic solutes dissolved in the core cannot easily pass through the bilayer, and hydrophobic compounds associate with the bilayer. Therefore, liposomes can be loaded with hydrophobic and / or hydrophilic molecules. When liposomes are used to carry nucleic acids, such as RNA, the nucleic acid is contained within the liposomal compartment in the aqueous phase.

[0185] Cationic Liposomes Liposomes can be composed of cationic lipids, anionic lipids, and / or neutral lipids. As an important subclass of liposomes, cationic liposomes are liposomes made entirely or partially from positively charged lipids, more specifically, lipids containing both cationic groups and lipophilic moieties. In addition to the general characteristics of liposomes described above, the positively charged portion of the cationic lipids used in cationic liposomes offers several advantages and some unique structural features. For example, the lipophilic portion of cationic lipids is hydrophobic, and therefore orients itself away from the aqueous interior of the liposome and associates with other non-polar and hydrophobic species. Conversely, the cationic portion associates with polar molecules and species that can complex with the aqueous medium and, more importantly, the aqueous interior of the cationic liposome. For these reasons, cationic liposomes are increasingly being investigated for use in gene therapy due to their preference for negatively charged nucleic acids via electrostatic interactions, resulting in complexes that offer biocompatibility, low toxicity, and the potential for large-scale production required for in vivo clinical use. Cationic lipids suitable for use in cationic liposomes are listed herein below.

[0186] lipid nanoparticles In contrast to liposomes and cationic liposomes, lipid nanoparticles (LNPs) have a structure containing a single monolayer or bilayer of lipids that encapsulates a compound in the solid phase. Thus, unlike liposomes, lipid nanoparticles do not have an aqueous or other liquid phase within their interior. Rather, lipids from the bilayer or monolayer shell directly complex with the internal compound, thereby encapsulating it within the solid core. Lipid nanoparticles are typically spherical vesicles with a relatively uniform distribution of shapes and sizes. While sources differ regarding the size that qualifies a lipid particle as a nanoparticle, there is some overlapping agreement that lipid nanoparticles can have diameters ranging from 10 nm to 1000 nm. However, more commonly, they are considered to be smaller than 120 nm or even smaller than 100 nm.

[0187] For lipid nanoparticle nucleic acid delivery systems, the lipid shell can be formulated to contain ionic cationic lipids that can complex and associate with the negatively charged backbone of the nucleic acid core. Ionic cationic lipids with an apparent pKa value of less than about 7 have the advantage of complexing with the negatively charged backbone of the nucleic acid, providing the cationic lipid for loading into lipid nanoparticles at pH values ​​below the pKa of the positively charged ionic lipid. Then, at physiological pH values, the lipid nanoparticles can adopt a relatively neutral exterior, allowing for a significant increase in the circulatory half-life of the particles after intravenous administration. In the context of nucleic acid delivery, lipid nanoparticles offer many advantages over other lipid-based nucleic acid delivery systems, including high nucleic acid encapsulation efficiency, potent transfection, improved tissue penetration for delivering therapeutic agents, and low levels of cytotoxicity and immunogenicity.

[0188] Prior to the development of lipid nanoparticle delivery systems for nucleic acids, cationic lipids had been widely investigated as synthetic materials for the delivery of nucleic acid drugs. In these early efforts, nucleic acids were mixed together at physiological pH and then condensed with cationic lipids to form lipid-nucleic acid complexes known as lipoplexes. However, lipoplexes proved unstable and were characterized by a wide size distribution ranging from submicron scale to several microns. Lipoplexes such as Lipofectamine® reagent have found considerable utility for in vitro transfection. However, these first-generation lipoplexes have not proven useful in vivo. The large particle size and positive charge (imparted by cationic lipids) result in rapid plasma clearance, hemolysis, and other toxicities, as well as immune system activation.

[0189] In some embodiments, the lipid nanoparticles comprise a lipid of Formula I: [ka] (I) During the ceremony, R 1 and R 2 are each independently H or C 1-6 is alkyl; or R 1 and R 2 are joined to form a saturated heterocyclic ring, R 1 is a linear C 1-4 alkylene; and R 2 is -(CH2) m (X) n - in which X is O, S or NR 9 where R 9 is H or C 1-6 is alkyl; m is 1, 2, 3, or 4; and n is 0 or 1; L1 is a linear C optionally substituted with 1 to 3 methyl groups. 1-6 is alkylene; Y is selected from the group consisting of: [ka] During the ceremony, Each asterisk (*) indicates an atom bonded to L2 and L3; and R 10 is H or C 1-6 is alkyl; L2 and L3 are each independently a linear C 1-8 is alkylene; L4, L5, L6, L7, L8, and L9 are each independently absent or -CH2-, with the proviso that At least two of L4, L6 and L8 are -CH2-; and at least two of L5, L7 and L9 are -CH2-; R 3 and R 4 are each independently H, methyl, or ethyl; and R 5, R 6 , R 7 , and R 8 are each independently selected from the group consisting of: Linear C 1-20 Alkyl, wherein each linear C 1-20 The alkyl is optionally substituted with one or more substituents selected from the group consisting of: C 1-6 Alkyl, C 1-6 Alkoxy, and -F, wherein the C 1-6 Each of the alkyl substituents is C 1-3 optionally substituted with one or more groups selected from the group consisting of alkoxy and -F; C 3-8 monocycloalkyl, wherein the C 3-8 Each monocycloalkyl is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C 7-12 bicycloalkyl, wherein the C 7-12 Each of the bicycloalkyls is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; and C 6-10 aryl, wherein the C 6-10 Each of the aryls is C 1-6 Alkyl, C 1-6 a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C 3-8 monocycloalkyl, wherein the C 3-8 Each monocycloalkyl is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C 7-12 bicycloalkyl, wherein the C 7-12 Each of the bicycloalkyls is C 1-6Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; and C 6-10 aryl, wherein the C 6-10 Each of the aryls is C 1-6 Alkyl, C 1-6 is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F.

[0190] In some embodiments, the lipid nanoparticles comprise a lipid of Formula I: [ka] (I) During the ceremony, R 1 and R 2 are each independently H or C 1-6 is alkyl; or R 1 and R 2 are joined to form a saturated heterocyclic ring, R 1 is a linear C 1-4 alkylene; and R 2 is -(CH2) m (X) n - in which X is O, S or NR 9 where R 9 is H or C 1-6 is alkyl; m is 1, 2, 3, or 4; and n is 0 or 1; L1 is a linear C optionally substituted with 1 to 3 methyl groups. 1-6 is alkylene; Y is selected from the group consisting of: [ka] During the ceremony, Each asterisk (*) indicates an atom bonded to L2 and L3; and R 10 is H or C 1-6 is alkyl; L2 and L3 are each independently a linear C 1-8 is alkylene; L4, L5, L6, L7, L8, and L9 are each independently absent or -CH2-, with the proviso that At least two of L4, L6 and L8 are -CH2-; and at least two of L5, L7 and L9 are -CH2-; R 3 and R 4 are each independently H, methyl, or ethyl; and R 5 , R 6 , R 7 , and R 8 are each independently selected from the group consisting of: Linear C 1-20 Alkyl, wherein each linear C 1-20 The alkyl is optionally substituted with one or more substituents selected from the group consisting of: C 1-6 Alkyl, C 1-6 Alkoxy, and -F, wherein the C 1-6 Each of the alkyl substituents is C 1-3 optionally substituted with one or more groups selected from the group consisting of alkoxy and -F; C 3-8 monocycloalkyl, wherein the C 3-8 Each monocycloalkyl is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C 7-12 bicycloalkyl, wherein the C 7-12 Each of the bicycloalkyls is C 1-6 Alkyl, C 1-6optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; and C 6-10 aryl, wherein the C 6-10 Each of the aryls is C 1-6 Alkyl, C 1-6 a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C 3-8 monocycloalkyl, wherein the C 3-8 Each monocycloalkyl is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C 7-12 bicycloalkyl, wherein the C 7-12 Each of the bicycloalkyls is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; and C 6-10 aryl, wherein the C 6-10 Each of the aryls is C 1-6 Alkyl, C 1-6 is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F.

[0191] In some embodiments, any one or more lipids listed herein may be explicitly excluded.

[0192] In some embodiments, the present disclosure provides lipid nanoparticles comprising a plurality of ligands, each ligand independently being a compound described herein, wherein the plurality of ligands self-assemble to form a lipid nanoparticle comprising an interior and an exterior.

[0193] In some embodiments, the average size of the lipid nanoparticles is about 100 nm. In some embodiments, the average size of the lipid nanoparticles is less than about 100 nm. In some embodiments, the average particle size of the lipid nanoparticles is between about 40 nm and about 100 nm. In some embodiments, the average particle size of the lipid nanoparticles is between about 50 nm and about 90 nm. In some embodiments, the average particle size of the lipid nanoparticles is between about 55 nm and about 85 nm.

[0194] In some embodiments, the lipid nanoparticle further comprises a nucleic acid therein. In some embodiments, the nucleic acid is selected from siRNA, mRNA, self-replicating RNA, DNA plasmid, and antisense oligonucleotide. In some embodiments, the nucleic acid is mRNA or self-replicating RNA comprising a coding region encoding a therapeutic protein of interest. In some embodiments, the therapeutic protein of interest is an enzyme, antibody, antigen, receptor, or transporter. In some embodiments, the therapeutic protein of interest is a gene-editing enzyme. In some embodiments, the gene-editing enzyme is selected from TALEN, CRISPR, meganuclease, or zinc finger nuclease.

[0195] In some embodiments, the lipid nanoparticles further comprise siRNA or mRNA therein. In some embodiments, the lipid nanoparticles further comprise mRNA therein.

[0196] In some embodiments, the lipid nanoparticles further comprise a helper lipid, as described below. In some embodiments, the lipid nanoparticles further comprise a PEG-lipid conjugate, as described herein.

[0197] In some embodiments, the lipid nanoparticles comprise about 45 mol% to about 65 mol% of a compound of the present disclosure, about 2 mol% to about 15 mol% of a helper lipid, about 20 mol% to about 42 mol% of cholesterol, and about 0.5 mol% to about 3 mol% of a PEG-lipid conjugate. In some embodiments, the lipid nanoparticles comprise about 50 mol% to about 61 mol% of a compound of the present disclosure, about 5 mol% to about 9 mol% of a helper lipid, about 29 mol% to about 38 mol% of cholesterol, and about 1 mol% to about 2 mol% of a PEG-lipid conjugate. In some embodiments, the lipid nanoparticles comprise about 56 mol% to about 58 mol% of a compound of the present disclosure, about 6 mol% to about 8 mol% of DSPC, about 31 mol% to about 34 mol% of cholesterol, and about 1.25 mol% to about 1.75 mol% of a PEG-lipid conjugate.

[0198] In some embodiments, the lipid nanoparticles comprise about 50 mol% to about 61 mol% of a compound of the present disclosure, about 2 mol% to about 12 mol% of DSPC, about 25 mol% to about 42 mol% of cholesterol, and about 0.5 mol% to about 3 mol% of PEG2000-DMG. In some embodiments, the lipid nanoparticles comprise about 50 mol% to about 61 mol% of a compound of the present disclosure, about 5 mol% to about 9 mol% of DSPC, about 29 mol% to about 38 mol% of cholesterol, and about 1 mol% to about 2 mol% of PEG2000-DMG. In some embodiments, the lipid nanoparticles comprise about 56 mol% to about 58 mol% of a compound of the present disclosure, about 6 mol% to about 8 mol% of DSPC, about 31 mol% to about 34 mol% of cholesterol, and about 1.25 mol% to about 1.75 mol% of PEG2000-DMG.

[0199] In some embodiments, the lipid nanoparticles have a total lipid:nucleic acid weight ratio of about 50:1 to about 10:1. In some embodiments, the lipid nanoparticles have a total lipid:nucleic acid weight ratio of about 40:1 to about 20:1. In some embodiments, the lipid nanoparticles have a total lipid:nucleic acid weight ratio of about 35:1 to about 25:1. In some embodiments, the lipid nanoparticles have a total lipid:nucleic acid weight ratio of about 32:1 to about 28:1. In some embodiments, the lipid nanoparticles have a total lipid:nucleic acid weight ratio of about 31:1 to about 29:1.

[0200] In some embodiments, the lipid nanoparticles have a total lipid:mRNA weight ratio of about 50:1 to about 10:1. In some embodiments, the lipid nanoparticles have a total lipid:mRNA weight ratio of about 40:1 to about 20:1. In some embodiments, the lipid nanoparticles have a total lipid:mRNA weight ratio of about 35:1 to about 25:1. In some embodiments, the lipid nanoparticles have a total lipid:mRNA weight ratio of about 32:1 to about 28:1. In some embodiments, the lipid nanoparticles have a total lipid:mRNA weight ratio of about 31:1 to about 29:1.

[0201] In some embodiments, the lipid nanoparticles comprise a HEPES buffer at a pH of about 7.4. In some embodiments, the HEPES buffer has a concentration of about 7 mg / mL to about 15 mg / mL. In some embodiments, the lipid nanoparticles further comprise NaCl at about 2.0 mg / mL to about 4.0 mg / mL.

[0202] In some embodiments, the lipid nanoparticles further comprise one or more cryoprotectants. In some embodiments, the one or more cryoprotectants are selected from sucrose, glycerol, or a combination of sucrose and glycerol. In some embodiments, the lipid nanoparticles comprise a combination of sucrose at a concentration of about 70 mg / mL to about 110 mg / mL and glycerol at a concentration of about 50 mg / mL to about 70 mg / mL.

[0203] Lipid-nucleic acid formulations The nucleic acid or a pharmaceutically acceptable salt thereof can be incorporated into a lipid formulation (ie, a lipid-based delivery vehicle).

[0204] In the context of the present disclosure, lipid-based delivery vehicles typically function to transport a desired nucleic acid (such as siRNA, plasmid DNA, mRNA, or self-replicating RNA) to a target cell or tissue. The lipid-based delivery vehicle can be any suitable lipid-based delivery vehicle known in the art. In some embodiments, the lipid-based delivery vehicle is a liposome, a cationic liposome, or a lipid nanoparticle containing a nucleic acid. In some embodiments, the lipid-based delivery vehicle comprises a nanoparticle or bilayer of lipid molecules and a nucleic acid. In some embodiments, the lipid bilayer preferably further comprises a neutral lipid or polymer. In some embodiments, the lipid formulation preferably comprises a liquid medium. In some embodiments, the formulation preferably further encapsulates a nucleic acid. In some embodiments, the lipid formulation preferably further comprises a nucleic acid and a neutral lipid or polymer. In some embodiments, the lipid formulation preferably encapsulates a nucleic acid.

[0205] Provided herein are lipid formulations comprising one or more therapeutic nucleic acid molecules encapsulated within the lipid formulation. In some embodiments, the lipid formulation comprises a liposome. In some embodiments, the lipid formulation comprises a cationic liposome. In some embodiments, the lipid formulation comprises a lipid nanoparticle.

[0206] In some embodiments, the nucleic acid is fully encapsulated within the lipid portion of the lipid formulation, thereby making the nucleic acid in the lipid formulation resistant to nuclease degradation in aqueous solution. In other embodiments, the lipid formulations described herein are substantially non-toxic to mammals, such as humans.

[0207] The lipid formulations of the present disclosure also typically have a total lipid:nucleic acid ratio (mass / mass) of about 1:1 to about 100:1, about 1:1 to about 50:1, about 2:1 to about 45:1, about 3:1 to about 40:1, about 5:1 to about 38:1, about 6:1 to about 40:1, about 7:1 to about 35:1, about 8:1 to about 30:1, about 10:1 to about 25:1, about 8:1 to about 12:1, about 13:1 to about 17:1, about 18:1 to about 24:1, or about 20:1 to about 30:1. In some preferred embodiments, the total lipid:nucleic acid ratio (mass / mass) is about 10:1 to about 25:1. The ratio may be any value or subvalue within the recited range, including the endpoints.

[0208] The lipid formulations of the present disclosure typically have a diameter of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 95 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 20 nm, about 210 nm, about 220 nm, about 230 nm, about 240 nm, about 250 nm, about 260 nm, about 270 nm, about 280 nm, about 290 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 56 nm, about 57 nm, about 58 nm, about 59 nm, about 60 nm, about 61 nm, about 62 nm, about 63 nm, about 64 nm, about 65 nm, about 66 nm, about 67 nm, about 68 nm, about 69 nm, about 70 nm, about 7 The lipid nanoparticles of the present disclosure have an average diameter of about 0 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, or about 150 nm, and are substantially non-toxic. The diameter may be any value or subvalue within the recited range, including the end points. In addition, when nucleic acid is present in the lipid nanoparticles of the present disclosure, it is resistant to degradation by nuclease in aqueous solution.

[0209] In a preferred embodiment, the lipid formulation comprises a nucleic acid, a cationic lipid (e.g., one or more cationic lipids or salts thereof described herein), a phospholipid, and a conjugated lipid that inhibits particle aggregation (e.g., one or more PEG-lipid conjugates and / or other lipid conjugates of the present disclosure). The lipid formulation may also comprise cholesterol.

[0210] In some embodiments, the lipid nanoparticle further comprises a PEG-lipid conjugate. In some embodiments, the PEG-lipid conjugate is PEG-DMG. In some embodiments, the PEG-DMG is PEG2000-DMG.

[0211] In nucleic acid-lipid formulations, nucleic acid can be completely encapsulated in the lipid portion of formulation, thereby protecting nucleic acid from nuclease degradation.In a preferred embodiment, the lipid formulation comprising nucleic acid is completely encapsulated in the lipid portion of lipid formulation, thereby protecting nucleic acid from nuclease degradation.In certain cases, the nucleic acid in lipid formulation is not substantially decomposed after particles are exposed to nuclease at 37 ℃ for at least 20, 30, 45 or 60 minutes.In certain other cases, the nucleic acid in lipid formulation is not substantially decomposed after the formulation is incubated in serum at 37 ℃ for at least 30, 45 or 60 minutes, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 hours.In other embodiments, nucleic acid is complexed with the lipid portion of formulation.

[0212] In the context of nucleic acids, complete encapsulation can be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye whose fluorescence is enhanced when associated with nucleic acids. Encapsulation is determined by adding the dye to the lipid formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon the addition of a small amount of nonionic detergent. Detergent-mediated disruption of the lipid layer releases the encapsulated nucleic acid, allowing it to interact with the membrane-impermeable dye. Nucleic acid encapsulation may be calculated as E = (I0 - I) / I0, where I and I0 refer to the fluorescence intensity before and after the addition of detergent.

[0213] In other embodiments, the present disclosure provides nucleic acid-lipid compositions comprising a plurality of nucleic acid-liposomes, nucleic acid-cationic liposomes, or nucleic acid-lipid nanoparticles. In some embodiments, the nucleic acid-lipid composition comprises a plurality of nucleic acid-liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of nucleic acid-cationic liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of nucleic acid-lipid nanoparticles.

[0214] In some embodiments, the lipid formulation comprises nucleic acid that is fully encapsulated within the lipid portion of the formulation, thereby providing about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 30% to about 95%, about 40% to about 95%, about 50% to about 95%, about 60% to about 95%, about 70% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, or at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% (or any fraction or range therein) of the nucleic acid encapsulated therein. The amount may be any value or subvalue within the recited range, inclusive of the endpoints.

[0215] Depending on the intended use of the lipid formulation, the ratio of the components can be varied, and assays known in the art can be used to measure the delivery efficiency of a particular formulation.

[0216] According to some embodiments, the expressible polynucleotide, nucleic acid active agent, and mRNA construct can be lipid-formulated. The lipid formulation is preferably selected from, but not limited to, liposomes, cationic liposomes, and lipid nanoparticles. In a preferred embodiment, the lipid formulation is a cationic liposome or lipid nanoparticle (LNP), (a) nucleic acid (mRNA, siRNA, etc.); (b) a lipid of the present disclosure, which may be cationic; and (c) optionally, a non-cationic lipid (such as a neutral lipid); and (d) optionally, a sterol.

[0217] cationic lipids The lipid formulation preferably contains a cationic lipid suitable for forming cationic liposomes or lipid nanoparticles. Cationic lipids have been widely studied for nucleic acid delivery because they can bind to negatively charged membranes and induce uptake. Generally, cationic lipids are amphiphilic substances containing a positive hydrophilic head group, two (or more) lipophilic tails or steroid moieties, and a connector between these two domains. Preferably, the cationic lipid carries a net positive charge at approximately physiological pH. Cationic liposomes have traditionally been the most commonly used non-viral delivery system for oligonucleotides, including plasmid DNA, antisense oligos, and siRNA / small hairpin RNA-shRNA. Cationic lipids such as DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and DOTMA (N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-methylammonium sulfate) can form complexes or lipoplexes with negatively charged nucleic acids through electrostatic interactions, providing high in vitro transfection efficiency.

[0218] In the lipid formulations of the present disclosure, cationic lipids include, for example, N,N-dioleyl-N,N-di-9-cis-octadecenyl ammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammonium propane chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-dioleyloxy-3-trimethylaminopropane chloride salt), N-(1-(2,3- Dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-y-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP) ), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl) ), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its analogues, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (C12-200 ), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine (MC3 ether), 4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-1-amine (MC4 ether), or any combination thereof. Other cationic lipids include N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 3P-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamidoglycylcarboxyspermine (DOGS), 1,2-dioleyl-sn-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), and 2,2-dilinoleyl-4-dimethylaminoethyl-[1,Examples of suitable cationic lipids include, but are not limited to, 3]-dioxolane (XTC). In addition, commercially available preparations of cationic lipids, such as Lipofectin (including DOTMA and DOPE, available from GIBCO / BRL) and Lipofectamine (including DOSPA and DOPE, available from GIBCO / BRL), can be used.

[0219] Other suitable cationic lipids are described in WO 09 / 086558, WO 09 / 127060, WO 10 / 048536, WO 10 / 054406, WO 10 / 088537, WO 10 / 129709, and WO 2011 / 153493, U.S. Patent Publication Nos. 2011 / 0256175, 2012 / 0128760, and 2012 / 0027803, U.S. Patent No. 8,158,601, and Love et al., PNAS, 107(5), 1864-69, 2010, the contents of which are incorporated herein by reference.

[0220] Other suitable cationic lipids include those with alternative fatty acid groups and other dialkylamino groups, including those with different alkyl substituents (e.g., N-ethyl-N-methylamino- and N-propyl-N-ethylamino-).These lipids are part of a subcategory of cationic lipids called amino lipids.In some embodiments of the lipid formulations described herein, the cationic lipid is an amino lipid.In general, amino lipids with less saturated alkyl chains are easier to size for filter sterilization purposes, especially when the complex must be sized to less than about 0.3 microns. 14 ~C 22 Amino lipids containing unsaturated fatty acids with carbon chain lengths in the range of 0 to 1000 may be used. Other scaffolds may be used to separate the amino group from the fatty acid or fatty alkyl portion of the amino lipid.

[0221] In some embodiments, the cationic lipids of the present disclosure are ionizable and have at least one protonatable or deprotonatable group such that the lipid is positively charged at a pH below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably above physiological pH. It should be understood that the addition or removal of protons as a function of pH is an equilibrium process, and reference to a charged or neutral lipid refers to the nature of the predominant species; not all of the lipids need be present in the charged or neutral form. Lipids with more than one protonatable or deprotonatable group or that are zwitterionic are not excluded from use in the present disclosure. In certain embodiments, the protonatable lipid has a pKa of the protonatable group ranging from about 4 to about 11. In some embodiments, the ionizable cationic lipid has a pKa of about 5 to about 7. In some embodiments, the pKa of the ionizable cationic lipid is about 6 to about 7.

[0222] In some embodiments, the lipid formulation comprises a lipid of Formula I: [ka] (I) During the ceremony, R 1 and R 2 are each independently H or C 1-6 is alkyl; or R 1 and R 2 are joined to form a saturated heterocyclic ring, R 1 is a linear C 1-4 alkylene; and R 2 is -(CH2) m (X) n - in which X is O, S or NR 9 where R 9 is H or C 1-6 is alkyl; m is 1, 2, 3, or 4; and n is 0 or 1; L1 is a linear C optionally substituted with 1 to 3 methyl groups. 1-6 is alkylene; Y is selected from the group consisting of: [ka] During the ceremony, Each asterisk (*) indicates an atom bonded to L2 and L3; and R 10 is H or C 1-6 is alkyl; L2 and L3 are each independently a linear C 1-8 is alkylene; L4, L5, L6, L7, L8, and L9 are each independently absent or -CH2-, with the proviso that At least two of L4, L6 and L8 are -CH2-; and at least two of L5, L7 and L9 are -CH2-; R 3 and R 4 are each independently H, methyl, or ethyl; and R 5 , R 6 , R 7 , and R 8 are each independently selected from the group consisting of: Linear C 1-20 Alkyl, wherein each linear C 1-20 The alkyl is optionally Substituted with one or more substituents selected from the group consisting of: C 1-6 Alkyl, C 1-6 Alkoxy, and -F, wherein the C 1-6 Each of the alkyl substituents is C 1-3 optionally substituted with one or more groups selected from the group consisting of alkoxy and -F; C 3-8 monocycloalkyl, wherein the C 3-8 Each monocycloalkyl is C 1-6 Alkyl, C1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C 7-12 bicycloalkyl, wherein the C 7-12 Each of the bicycloalkyls is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; and C 6-10 aryl, wherein the C 6-10 Each of the aryls is C 1-6 Alkyl, C 1-6 a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C 3-8 monocycloalkyl, wherein the C 3-8 Each monocycloalkyl is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C 7-12 bicycloalkyl, wherein the C 7-12 Each of the bicycloalkyls is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; and C 6-10 aryl, wherein the C 6-10 Each of the aryls is C 1-6 Alkyl, C 1-6 is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F.

[0223] In some embodiments, the lipid formulation comprises a lipid of Formula I: [ka] (I) During the ceremony, R 1and R 2 are each independently H or C 1-6 is alkyl; or R 1 and R 2 are joined to form a saturated heterocyclic ring, R 1 is a linear C 1-4 alkylene; and R 2 is -(CH2) m (X) n - in which X is O, S or NR 9 where R 9 is H or C 1-6 is alkyl; m is 1, 2, 3, or 4; and n is 0 or 1; L1 is a linear C optionally substituted with 1 to 3 methyl groups. 1-6 is alkylene; Y is selected from the group consisting of: [ka] During the ceremony, Each asterisk (*) indicates an atom bonded to L2 and L3; and R 10 is H or C 1-6 is alkyl; L2 and L3 are each independently a linear C 1-8 is alkylene; L4, L5, L6, L7, L8, and L9 are each independently absent or -CH2-, with the proviso that At least two of L4, L6 and L8 are -CH2-; and at least two of L5, L7 and L9 are -CH2-; R 3 and R 4 are each independently H, methyl, or ethyl; and R 5 , R 6 , R 7, and R 8 are each independently selected from the group consisting of: Linear C 1-20 Alkyl, wherein each linear C 1-20 The alkyl is optionally substituted with one or more substituents selected from the group consisting of: C 1-6 Alkyl, C 1-6 Alkoxy, and -F, wherein the C 1-6 Each of the alkyl substituents is C 1-3 optionally substituted with one or more groups selected from the group consisting of alkoxy and -F; C 3-8 monocycloalkyl, wherein the C 3-8 Each monocycloalkyl is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C 7-12 bicycloalkyl, wherein the C 7-12 Each of the bicycloalkyls is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; and C 6-10 aryl, wherein the C 6-10 Each of the aryls is C 1-6 Alkyl, C 1-6 a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C 3-8 monocycloalkyl, wherein the C 3-8 Each monocycloalkyl is C 1-6 Alkyl, C 1-6 optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C 7-12 bicycloalkyl, wherein the C 7-12 Each of the bicycloalkyls is C 1-6 Alkyl, C 1-6optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; and C 6-10 aryl, wherein the C 6-10 Each of the aryls is C 1-6 Alkyl, C 1-6 is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F.

[0224] In some embodiments, any one or more lipids listed herein may be explicitly excluded.

[0225] Helper lipids and sterols The mRNA-lipid formulations of the present disclosure can include helper lipids, which can be referred to as neutral lipids, neutral helper lipids, non-cationic lipids, non-cationic helper lipids, anionic lipids, anionic helper lipids, or zwitterionic lipids. Lipid formulations, particularly cationic liposomes and lipid nanoparticles, have been found to have increased cellular uptake when helper lipids are present in the formulation. (Curr. Drug Metab. 2014; 15(9): 882-92). For example, some studies have shown that neutral and zwitterionic lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), dioleoyl-phosphatidyl-ethanoalamine (DOPE), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), are more fusogenic (i.e., facilitate fusion) than cationic lipids, potentially affecting the polymorphic characteristics of lipid-nucleic acid complexes, promoting the transition from lamellar to hexagonal phases, and thus triggering cell membrane fusion and disruption (Nanomedicine (Lond). 2014 Jan;9(1):105-20). Additionally, the use of helper lipids may help reduce any potential adverse effects of using many common cationic lipids, such as toxicity and immunogenicity.

[0226] Non-limiting examples of non-cationic lipids suitable for the lipid formulations of the present disclosure include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylcholine (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phosphatidylethanolamine (DOPE ... Examples of suitable phospholipids include dioleoylphosphatidylethanolamine (POPE), palmitoyloleylphosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably derived from fatty acids having C10-C24 carbon chains, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0227] In some embodiments, the helper lipid is selected from dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC), and phosphatidylcholine (PC). In some embodiments, the helper lipid is distearoylphosphatidylcholine (DSPC).

[0228] Additional examples of non-cationic lipids include sterols such as cholesterol and their derivatives. One study concluded that, as a helper lipid, cholesterol increases the charge spacing of the lipid layer that aligns with nucleic acids, making the charge distribution more closely match that of nucleic acids. (JRSoc.Interface.2012 Mar 7;9(68):548-561). Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholestanol, 5α-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs such as 5α-cholestan, cholestenone, 5α-cholestanone, and cholesteryl decanoate, and mixtures thereof. In a preferred embodiment, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether.

[0229] In some embodiments, the helper lipid present in the lipid formulation comprises or consists of a mixture of one or more phospholipids and cholesterol or its derivatives. In other embodiments, the helper lipid present in the lipid formulation comprises or consists of one or more phospholipids, for example, a cholesterol-free lipid formulation. In yet other embodiments, the helper lipid present in the lipid formulation comprises or consists of cholesterol or its derivatives, for example, a phospholipid-free lipid formulation. In some embodiments, the lipid nanoparticle further comprises cholesterol.

[0230] Other examples of helper lipids include non-phosphorus containing lipids such as, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, and sphingomyelin.

[0231] In some embodiments, the helper lipid comprises about 1 mol% to about 50 mol%, about 5 mol% to about 48 mol%, about 5 mol% to about 46 mol%, about 25 mol% to about 44 mol%, about 26 mol% to about 42 mol%, about 27 mol% to about 41 mol%, about 28 mol% to about 40 mol%, or about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, or about 39 mol% (or any fraction or range therein) of the total lipid present in the lipid formulation. In some embodiments, the helper lipid comprises about 1 mol% to about 20 mol%, about 2 mol% to about 12 mol%, about 5 mol% to about 9 mol%, or about 6 mol% to about 8 mol%.

[0232] In some embodiments, the total amount of helper lipids in the formulation includes two or more helper lipids, and the total amount of helper lipids comprises about 20 mol% to about 50 mol%, about 22 mol% to about 48 mol%, about 24 mol% to about 46 mol%, about 25 mol% to about 44 mol%, about 26 mol% to about 42 mol%, about 27 mol% to about 41 mol%, about 28 mol% to about 40 mol%, or about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, or about 39 mol% (or any fraction or range therein) of the total lipids present in the lipid formulation. In some embodiments, the helper lipid is a combination of DSPC and DOTAP. In some embodiments, the helper lipid is a combination of DSPC and DOTMA.

[0233] The cholesterol or cholesterol derivative in the lipid formulation may comprise up to about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, or about 60 mol% of the total lipid present in the lipid formulation. In some embodiments, the cholesterol or cholesterol derivative comprises about 15 mol% to about 45 mol%, about 20 mol% to about 40 mol%, about 30 mol% to about 40 mol%, or about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, or about 40 mol% of the total lipid present in the lipid formulation.

[0234] The percentage of helper lipid present in the lipid formulation is a target amount, and the actual amount of helper lipid present in the formulation may vary, for example, by ±5 mole %.

[0235] Mechanisms of action for cellular uptake of lipid formulations Lipid formulations for intracellular delivery of nucleic acids, particularly liposomes, cationic liposomes, and lipid nanoparticles, are designed for cellular uptake by penetrating target cells through the endocytic mechanism of the target cells, delivering the contents of the lipid delivery vehicle into the cytosol of the target cells (Nucleic Acid Therapeutics, 28(3):146-157, 2018). Specifically, in the case of the nucleic acid-lipid formulations described herein, the lipid formulation enters cells via receptor-mediated endocytosis. Prior to endocytosis, functionalized ligands, such as the lipid conjugates of the present disclosure, on the surface of the lipid delivery vehicle can be shed from the surface, which induces internalization into the target cell. During endocytosis, a portion of the cell's plasma membrane surrounds the vector and engulfs it into a vesicle, which then pinches the vesicle out of the cell membrane, enters the cytosol, and ultimately passes through the endolysosomal pathway. For delivery vehicles containing ionic cationic lipids, the increased acidity of endosomes as they age results in vehicles with a strong positive charge on the surface.The interaction between the delivery vehicle and the endosomal membrane then leads to a membrane fusion event, leading to the cytoplasmic delivery of the payload.For mRNA or self-replicating RNA payloads, the cell's own internal translation process then translates the RNA into the encoded protein.The encoded protein can then undergo further post-translational processing, including transport to the target organelle or intracellular location.

[0236] By controlling the composition and concentration of lipid conjugate, it is possible to control the rate at which lipid conjugate is exchanged from lipid preparation, and then the rate at which lipid preparation becomes fusogenic.In addition, other variables, including, for example, pH, temperature or ionic strength, can be used to change and / or control the rate at which lipid preparation becomes fusogenic.Other methods that can be used to control the rate at which lipid preparation becomes fusogenic will be clear to those skilled in the art after reading this disclosure.In addition, by controlling the composition and concentration of lipid conjugate, it is also possible to control the size of liposome or lipid particle.

[0237] Lipid formulation manufacturing There are many different methods for preparing lipid formulations containing nucleic acids. (Curr. Drug Metabol. 2014, 15, 882-892; Chem. Phys. Lipids 2014, 177, 8-18; Int. J. Pharm. Stud. Res. 2012, 3, 14-20) The following techniques are briefly described here: thin film hydration, double emulsion, reverse phase evaporation, microfluidic preparation, double asymmetric centrifugation, ethanol injection, detergent dialysis, spontaneous vesicle formation by ethanol dilution, and encapsulation in preformed liposomes.

[0238] Thin Film Hydration In the thin film hydration (TFH) or Bangham method, lipids are dissolved in an organic solvent and then evaporated using a rotary evaporator, resulting in the formation of a thin lipid layer. After layer hydration with an aqueous buffer solution containing the compound to be loaded, multilamellar vesicles (MLVs) are formed, which can be reduced in size to generate small unilamellar vesicles (LUVs) or large unilamellar vesicles (SUVs) by extrusion through a membrane or by sonication of the starting MLVs.

[0239] Double emulsion Lipid formulations can also be prepared through a double emulsion technique, which involves dissolving lipids in a water / organic solvent mixture. An organic solution containing aqueous droplets is mixed with excess aqueous medium, resulting in the formation of a water-in-oil-in-water (W / O / W) double emulsion. After vigorous mechanical shaking, some of the aqueous droplets collapse, resulting in large unilamellar vesicles (LUVs).

[0240] Reverse Phase Evaporation Reverse-phase evaporation (REV) can also be used to achieve nucleic acid-loaded LUVs. In this technique, a two-phase system is formed by dissolving phospholipids in an organic solvent and an aqueous buffer. The resulting suspension is then sonicated briefly until the mixture becomes a clear, one-phase dispersion. Lipid formulation is achieved after evaporation of the organic solvent under reduced pressure. This technique can be used to encapsulate a variety of large and small hydrophilic molecules, including nucleic acids.

[0241] Microfluidic preparation Unlike other bulk techniques, microfluidic methods offer the possibility of controlling the lipid hydration process. Methods can be classified into continuous-flow microfluidics and droplet-based microfluidics according to the way the flow is manipulated. In the microhydrodynamic focusing (MHF) method, which operates in continuous-flow mode, lipids are dissolved in isopropyl alcohol, which is then hydrodynamically focused in a microchannel cross-junction between two aqueous buffer streams. Vesicle size can be controlled by adjusting the flow rate, thus controlling the lipid solution / buffer dilution process. The method can be used to generate oligonucleotide (ON) lipid formulations by using a microfluidic device consisting of three inlet ports and one outlet port.

[0242] Double asymmetric centrifugation Double asymmetric centrifugation (DAC) differs from more conventional centrifugation by using an additional rotation around its own perpendicular axis. Efficient homogenization is achieved by creating two overlaying movements: the sample is pushed outward as in a conventional centrifuge, and then pushed toward the center of the vial by an additional rotation. A viscous vesicular phospholipid gel (VPC) is achieved by mixing the lipid and NaCl solution, which is then diluted to obtain a lipid formulation dispersion. Lipid formulation size can be adjusted by optimizing the DAC speed, lipid concentration, and homogenization time.

[0243] Ethanol injection The ethanol injection (EI) method can be used for nucleic acid encapsulation. This method involves the rapid injection of a lipid-dissolved ethanol solution into an aqueous medium containing the nucleic acid to be encapsulated through the use of a needle. As the phospholipids are dispersed throughout the medium, vesicles spontaneously form.

[0244] Detergent dialysis Nucleic acid can be encapsulated using detergent dialysis method.In brief, lipid and plasmid are solubilized in detergent solution of appropriate ionic strength, and after removing detergent by dialysis, stabilized lipid formulation is formed.Then, unencapsulated nucleic acid is removed by ion exchange chromatography, and vesicles are emptied by sucrose density gradient centrifugation.This technique is very sensitive to cationic lipid content and salt concentration of dialysis buffer, and this method is also difficult to scale up.

[0245] Spontaneous vesicle formation upon ethanol dilution Stable lipid formulations can also be generated via the spontaneous vesicle formation method by ethanol dilution, in which stepwise or dropwise ethanol dilution provides for the spontaneous formation of nucleic acid-loaded vesicles by the controlled addition of lipids dissolved in ethanol to a rapidly mixing aqueous buffer containing nucleic acid.

[0246] V. Pharmaceutical Compositions and Delivery Methods To promote in vivo nucleic acid activity (e.g., mRNA expression, or knockdown by ASO or siRNA), the lipid formulation delivery vehicle described herein can be combined with one or more additional nucleic acids, carriers, targeting ligands, or stabilizing reagents, or can be mixed with a suitable excipient into a pharmacological composition. Techniques for drug formulation and administration can be found in "Remington's Pharmaceutical Sciences" (Mack Publishing Co., Easton, Pa., latest edition).

[0247] The lipid formulations and pharmaceutical compositions of the present disclosure may be administered and dosed according to current medical practice, taking into account the clinical condition of the subject, administration site and administration method, administration schedule, subject's age, sex, weight, and other factors relevant to those skilled in the art.The "effective amount" for the purpose of this specification can be determined by relevant considerations such as experimental clinical research, pharmacology, clinical practice, and medical technology, as known to those skilled in the art.In some embodiments, the amount administered is effective to achieve at least some stabilization, improvement, or elimination of symptoms and other indicators that are selected by those skilled in the art as an appropriate measure of disease progression, regression, or improvement.For example, an appropriate amount and administration regimen is one that causes at least transient protein (e.g., enzyme) production.

[0248] The pharmaceutical compositions disclosed herein can be formulated using one or more excipients to (1) increase stability, (2) increase cell transfection, (3) allow for sustained or delayed release (e.g., from a depot formulation of the nucleic acid), (4) alter biodistribution (e.g., targeting the nucleic acid to a particular tissue or cell type), (5) increase the activity of the nucleic acid or protein expressed therefrom in vivo, and / or (6) alter the release profile of the nucleic acid or encoded protein in vivo.

[0249] Preferably, lipid formulations can be administered in a localized manner rather than systemically.Local delivery can affect different methods depending on the tissue to be targeted.For example, the aerosol containing the composition of the present disclosure can be inhaled (for delivery to nose, trachea or bronchus).

[0250] Pharmaceutical compositions can be administered to any desired tissue.In some embodiments, the nucleic acid delivered by lipid formulations or compositions of the present disclosure is active in the tissue to which lipid formulations and / or compositions are administered.In some embodiments, nucleic acid is active in tissues different from the tissue to which lipid formulations and / or compositions are administered.The example of tissue that nucleic acid can be delivered to includes but is not limited to lung, trachea and / or nasal cavity, muscle, liver, eye or central nervous system.

[0251] The pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such preparative methods include the step of bringing into association the active ingredient (i.e., nucleic acid) with an excipient and / or one or more other accessory ingredients. Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses.

[0252] Pharmaceutical compositions may additionally contain pharmaceutically acceptable excipients, which as used herein include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, and the like, appropriate for the particular dosage form desired.

[0253] In addition to conventional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, etc., excipients of the present disclosure can include, but are not limited to, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with primary DNA constructs or mRNA (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics, and combinations thereof.

[0254] Thus, the formulations described herein can include one or more excipients in an amount that together increase the stability of the nucleic acid in the lipid formulation, increase cell transfection with the nucleic acid (e.g., mRNA or siRNA), increase expression of the encoded protein, and / or alter the release profile of the encoded protein, or increase knockdown of the target naturally occurring nucleic acid. Additionally, nucleic acids can be formulated using self-assembling nucleic acid nanoparticles.

[0255] Various excipients for formulating pharmaceutical compositions and techniques for preparing compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006, the entirety of which is incorporated herein by reference).The use of conventional excipient vehicles can be contemplated within the scope of the embodiments of the present disclosure, except that any conventional excipient vehicle may be incompatible with the substance or its derivatives, such as by causing any undesirable biological effect or otherwise interacting in a harmful manner with any other component(s) of the pharmaceutical composition.

[0256] The dosage form of the composition of the present disclosure can be a solid, which can be reconstituted in liquid before administration.Solid can be administered as powder.In some embodiments, the pharmaceutical composition comprises a lyophilized nucleic acid-lipid formulation.

[0257] In preferred embodiments, the pharmaceutical compositions described herein may be formulated as a liquid suspension of the nucleic acid-lipid nanoparticles described herein. In some embodiments, the liquid suspension is in a buffer solution. In some embodiments, the buffer solution comprises a buffer selected from the group consisting of HEPES, MOPS, TES, and TRIS. In some embodiments, the buffer has a pH of about 7.4. In some preferred embodiments, the buffer is HEPES. In some further embodiments, the buffer solution further comprises a cryoprotectant. In some embodiments, the cryoprotectant is selected from a combination of a sugar and glycerol, or a sugar and glycerol. In some embodiments, the sugar is a dimeric sugar. In some embodiments, the sugar is sucrose. In some preferred embodiments, the buffer comprises HEPES, sucrose, and glycerol at a pH of 7.4. In some embodiments, the suspension is frozen during storage and thawed prior to administration. In some embodiments, the suspension is frozen at a temperature below about -70°C. In some embodiments, the suspension is diluted with sterile water prior to inhalable administration. In some embodiments, inhalable administration comprises diluting the suspension with about 1 volume to about 4 volumes of sterile water. In some embodiments, the lyophilized nucleic acid-lipid nanoparticle formulation can be resuspended in a buffer solution described herein.

[0258] The dosage form of the composition of the present disclosure can be a solid that can be reconstituted in a liquid before administration. The solid can be administered as a powder. The solid can be in the form of a capsule, tablet, or gel.

[0259] To formulate a composition for pulmonary delivery within the present disclosure, the nucleic acid-lipid formulation can be combined with various pharmaceutically acceptable additives, as well as bases or carriers for dispersing the nucleic acid-lipid formulation.Examples of additives include pH adjusters such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and mixtures thereof.Other additives include local anesthetics (e.g., benzyl alcohol), isotonicity agents (e.g., sodium chloride, mannitol, sorbitol), adsorption inhibitors (e.g., Tween 80), solubility enhancers (e.g., cyclodextrin and its derivatives), stabilizers (e.g., serum albumin), and reducing agents (e.g., glutathione).When the composition for mucosal delivery is liquid, the tonicity of the formulation, measured with reference to the tonicity of 0.9% (w / v) saline solution taken as 1, is typically adjusted to a value that does not induce substantially irreversible tissue damage to the mucosa at the administration site. Generally, the tonicity of the solution is adjusted to a value of 1 / 3 to 3, more typically 1 / 2 to 2, and most often 3 / 4 to 1.7.

[0260] The nucleic acid-lipid formulation may be dispersed in a base or vehicle, which may contain a hydrophilic compound capable of dispersing the nucleic acid-lipid formulation and any desired additives.The base may be selected from a wide range of suitable carriers, including but not limited to copolymers of polycarboxylic acids or their salts, carboxylic acid anhydrides (e.g., maleic anhydride) with other monomers (e.g., methyl (meth)acrylate, acrylic acid, etc.), hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives such as hydroxymethylcellulose, hydroxypropylcellulose, and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and their non-toxic metal salts.Biodegradable polymers are often selected as bases or carriers, such as polylactic acid, poly(lactic acid-glycolic acid) copolymers, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid) copolymers, and mixtures thereof.Alternatively or additionally, synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters, etc. can be used as carriers. Hydrophilic polymers and other carriers can be used alone or in combination, and the improved structural integrity can be imparted to the carrier by partial crystallization, ionic bonding, cross-linking, etc. The carrier can be provided in various forms, including fluid or viscous solution, gel, paste, powder, microsphere and film for direct application to the nasal mucosa.The use of selected carriers in this context can lead to the enhanced absorption of nucleic acid-lipid formulations.

[0261] Alternatively, the compositions of the present disclosure may contain pharmaceutically acceptable carrier materials required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, and wetting agents, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and mixtures thereof. For solid compositions, conventional non-toxic pharmaceutically acceptable carriers can be used, including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc.

[0262] According to the present disclosure, a therapeutically effective dose of a provided composition, when administered regularly, results in an increase in nucleic acid activity levels in a subject compared to baseline activity levels before treatment. Typically, activity levels are measured in a biological sample obtained from the subject, such as blood, plasma or serum, urine, or solid tissue extract. The baseline level can be measured immediately before treatment. In some embodiments, administration of a pharmaceutical composition described herein results in an increase in nucleic acid activity levels in a biological sample (e.g., plasma / serum or lung epithelial swab) of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to baseline levels before treatment. In some embodiments, administration of provided compositions results in an increase in nucleic acid activity levels in a biological sample (e.g., plasma / serum or lung epithelial swab) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to pre-treatment baseline levels for at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, or at least about 15 days.

[0263] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a compound described herein or a lipid nanoparticle described herein and a pharmaceutically acceptable excipient.

[0264] In some embodiments, the present disclosure provides a method of delivering a nucleic acid to a subject in need thereof, the method comprising encapsulating a therapeutically effective amount of a nucleic acid in a lipid nanoparticle described herein and administering the lipid nanoparticle to the subject.

[0265] In some embodiments, the present disclosure provides a method of delivering mRNA to a subject in need thereof, the method comprising encapsulating a therapeutically effective amount of mRNA in a lipid nanoparticle described herein and administering the lipid nanoparticle to the subject.

[0266] VI. Treatment method In some embodiments, the disclosure provides a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound described herein, a lipid nanoparticle described herein, or a pharmaceutical composition described herein. In some embodiments, the compound or lipid nanoparticle is administered intravenously or intramuscularly. In some embodiments, the compound or lipid nanoparticle is administered intravenously. In some embodiments, the compound or lipid nanoparticle is administered intramuscularly.

[0267] In some embodiments, methods of treating a disease in a subject in need thereof are provided, the methods comprising administering to the subject a lipid composition described herein. In some embodiments, the lipid composition is administered intravenously or intramuscularly. In some embodiments, the lipid composition is administered intravenously. In some embodiments, the lipid composition is administered intramuscularly.

[0268] In some embodiments, a method for treating a disease or disorder in a mammalian subject is provided.A therapeutically effective amount of a composition comprising a lipid, particularly a cationic lipid, a nucleic acid, an amphiphile, a phospholipid, cholesterol, and a PEG-conjugated cholesterol, as disclosed herein, can be administered to a subject with a disease or disorder associated with the expression or overexpression of a gene that can be reduced, decreased, downregulated, or silenced by the composition.The compositions described herein can be used in a method for treating cancer or inflammatory disease.The disease can be selected from the group consisting of central nervous system disorders, peripheral nervous system disorders, muscle atrophy, muscular dystrophy, immune disorders, cancer, kidney diseases, fibrotic diseases, genetic abnormalities, inflammation, and cardiovascular disorders.

[0269] In some embodiments, the present disclosure provides a method of expressing a protein or polypeptide in a target cell, the method comprising contacting the target cell with a lipid nanoparticle described herein, or a pharmaceutical composition described herein. In some embodiments, the protein or polypeptide is an antigen, and expression of the antigen provides an in vivo immunogenic response.

[0270] VII. Working Examples Example 1. Synthesis of lipid 1: ((4,4'-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetranonanoate [ka] Overall Scheme: [ka] [ka]

[0271] Synthesis of 1-1: 2-oxopropane-1,3-diyl dinonanoate [ka]

[0272] A solution of 1,3-dihydroxyacetone (6.8 g, 1 equiv.) in CHCl was added to a 500 mL three-neck round-bottom flask reactor under a N atmosphere. Stirring was initiated, and the temperature was adjusted to room temperature. Pyridine (17.9 g, 3 equiv.) was added to the reactor while maintaining the temperature at 25±5°C. 4-Dimethylaminopyridine (DMAP) (0.276 g, 0.03 equiv.) was added to the reactor while maintaining the temperature at 25±5°C. Nonanoyl chloride (20 g, 1.5 equiv.) was added dropwise to the reactor at 0-5°C. After the addition, the temperature was maintained at room temperature and the mixture was stirred for 6 hours. An additional 6.66 g of nonanoyl chloride (0.5 equiv.) was added dropwise to the reactor at 0-5°C. The reaction temperature was allowed to rise to room temperature and stirred overnight under nitrogen. The formed pyridine hydrochloride was removed by filtration and washed with CHCl. The combined filtrate and washes were then washed with 200 mL each of 5% aqueous NaHCO3, 0.1 N HCl, and brine. The solution was then dried over Na2SO4 and concentrated under vacuum. The residue was then crystallized from methanol (50 mL) to give a white solid. This afforded 16 g (59.7%) of white product. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): RT 2.26 min, m / z (calculated) 370.27, (found) 371.00 (M+H + ).

[0273] 1-2: Synthesis of 2-hydroxypropane-1,3-dinonanoate [ka]

[0274] A 250 mL three-necked round-bottom flask was charged with a solution of lipid-1-1 (9.6 g, 1.0 equiv.) in 100 mL of THF. To the solution, AcOH (2.02 g, 1.3 equiv.) was added at 0 °C. Then, NaBH3CN (1.96 g, 1.2 equiv.) was added to the mixture at 0 °C. The mixture was stirred at room temperature for 16 h under a nitrogen atmosphere. The reaction mixture was quenched with 100 mL of water. The mixture was extracted three times with 100 mL of EtOAc. The organic layers were combined, washed with brine (300 mL), and concentrated in vacuo to give 9.5 g (crude) of 1-2, which was used in the next step without further purification.

[0275] 1-3: Synthesis of dimethyl 4,4'-(benzylazanediyl)dibutyrate [ka]

[0276] A 1 L three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere was charged with acetonitrile (208 mL), K2CO3 (31 g, 2.3 equiv.), benzylamine (10.3 g, 1.0 equiv.), and methyl 4-bromobutyrate (40 g, 2.3 equiv.) at 25 °C. The mixture was heated to 80 °C and stirred for 15 h. The reaction was cooled to 25 °C. The flask was charged with water (200 mL, 20 V) and extracted with EtOAc (2 × 200 mL). The organic phase was dried (Na2SO4) and concentrated under reduced pressure at 35 °C. This afforded 1-3 (23 g, crude) as a crude product, which was used subsequently without further purification. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature 2.26 min, m / z (calculated) 370.27, (observed) 371.00 (M+H).

[0277] 1-4: Synthesis of dimethyl 4,4'-((tert-butoxycarbonyl)azanediyl)dibutyrate [ka]

[0278] EtOH (230 mL, 10 V), 1-3 (23.0 g, 1.0 equiv.), (Boc)O (18.0 g, 1.1 equiv.), and Pd / C (2.3 g, 10% w / w) were placed in a 1 L hydrogenation autoclave at ambient temperature. The mixture was stirred at room temperature under a hydrogen atmosphere of 5 atm for 16 h. TLC showed that 1-3 was completely consumed. The reaction mixture was filtered and concentrated under vacuum at 40 °C to give 22 g of crude 1-4.

[0279] 1-5: Synthesis of 4,4'-((tert-butoxycarbonyl)azanediyl)dibutyric acid [ka]

[0280] A 500 mL three-neck round-bottom flask purged and maintained with an inert nitrogen atmosphere was charged with a solution of 1-4 (22 g, 1.0 equiv.) in ethanol (110 mL). Aqueous NaOH (6 M, 110 mL, 5 V) was added at room temperature. The reaction mixture was stirred at 60 °C for 2 h. The reaction was then diluted with brine (220 mL, 10 V) and extracted with t-BuOH / n-heptane (2:1, 220 mL, 2×) to remove organic impurities. The aqueous phase was acidified to approximately pH 3 by adding 3 M aqueous HCl and then extracted with t-BuOH:n-heptane (2:1) (220 mL). The organic phase was concentrated under reduced pressure. The residue was slurried with diethyl ether (44 mL, 2 V) and filtered. The filter cake was collected to give 1-5 (14 g, 43% yield over three steps) as a white solid.

[0281] 1-6: Synthesis of ((4,4'-((tert-butoxycarbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetranonanoate [ka]

[0282] A 250 mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere was charged with a solution of 1-5 (3.7 g, 1.00 equiv.) in 100 mL of CHCl. ​​To this solution was added 1-2 (9.5 g, 2.00 equiv.), DMAP (4.69 g, 3 equiv.), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (7.35 g, 3 equiv.) at 0 °C. The reaction mixture was stirred overnight at 25 °C. The reaction was then quenched with 200 mL of 10% aqueous citric acid. The organic phase was washed with 200 mL of brine. The mixture was dried over anhydrous sodium sulfate and concentrated in vacuo to give 12 g of 1-6, which was used in the next step without further purification. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature 2:46 min, m / z (calculated) 997.71, (found) 1020.6 (M+Na).

[0283] 1-7: Synthesis of bis(4-((1,3-bis(nonanoyloxy)propan-2-yl)oxy)-4-oxobutyl)ammonium chloride [ka]

[0284] A solution of 1-6 (12 g, 1.00 equiv.) in EtOAc (68 mL) was placed in a 500 mL round-bottom flask purged and maintained under an inert nitrogen atmosphere. To this solution, EtOAc / HCl (15 mL, 5.00 equiv., 4 M) was added dropwise at 0–10 °C. The resulting solution was stirred at room temperature for 5 h. The resulting mixture was concentrated in vacuo. The residue was diluted with CHCl (100 mL), and 25 g of silica gel (type: ZCX-2, 100–200 mesh) was added. The mixture was concentrated in vacuo and then applied to an atmospheric silica gel column using a CHCl / MeOH gradient from 1:0 to 15:1. The product eluent was collected from 20:1 to 15:1 and concentrated in vacuo. This afforded 4.2 g of 1-7 as a light yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature 1.50 min, m / z (calculated) 897.65, (found) 898.6 (M+H).

[0285] Lipid 1: Synthesis of ((4,4'-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetranonanoate [ka]

[0286] A solution of 1-7 (4.2 g, 1 equiv.) in CHCl (150 mL) was placed in a 250-mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere. To this mixture was added triphosgene (1.33 g, 1 equiv.) at room temperature. Subsequently, pyridine (1.78 g, 5.00 equiv.) was added dropwise with stirring at 0 °C. The mixture was stirred at room temperature for 4 hours and then concentrated in vacuo. The residue was dissolved in pyridine (600 mL). To this solution was added 3-(dimethylamino)propane-1-thiol (0.92 g, 1.20 equiv.) dropwise over 10 minutes with stirring at 0 °C. The resulting solution was stirred at room temperature overnight. The resulting mixture was concentrated in vacuo. The residue was diluted with 100 mL of EtOAc. The mixture was washed with 2 × 100 mL of 10% citric acid and 2 × 100 mL of NaHCO. The mixture was washed with 100 mL of brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was dissolved in 100 mL of CHCl, and 12 g of silica gel (type: ZCX-2, 100-200 mesh) was added. The mixture was concentrated under vacuum and then applied to an atmospheric silica gel column using a CHCl / MeOH gradient from 1:0 to 15:1. The product eluent was collected from 20:1 to 15:1 and concentrated under vacuum. The product was dissolved in 36 mL of n-heptane (20 V) and 0.09 g of activated carbon powder was added. The mixture was stirred at room temperature for 4 hours and then filtered. 0.09 g of activated carbon powder was added to the filtrate, and the mixture was stirred at room temperature for an additional 4 hours. The mixture was filtered. 0.045 g of activated carbon powder was added to the filtrate, and the mixture was stirred at room temperature overnight. The mixture was filtered. A mixture of methanol (60 mL) and water (20 mL) was added to the filtrate. The mixture was stirred at room temperature for 30 minutes, the phases were separated, and the upper phase was collected. A mixture of methanol (60 mL) and water (20 mL) was added to the n-heptane phase. The mixture was stirred at room temperature for 30 minutes, the phases were separated, and the upper phase was collected and concentrated to give 1.5 g (31.9%) of lipid 1 (HPLC purity: 96.7%, 205 nm). ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B, 8 min, 0.7 min hold): room temperature 7.07 min, m / z (calculated) 1042.71, (observed) 1043.8 (M+H). 1H NMR (300MHz, CDCl3): δ5.27(m, 2H), 4.33(dd, J=11.9, 4.4Hz, 4H), 4.17(dd, J=11.9, 5.7Hz, 4H), 3.41(brm, 4H), 2.94(t, J=7.3Hz, 2H), 2.10-2.35(20H), 1.92(s, 6H), 1.65(m, 8H), 1.10-1.57(40H), 0.96-0.85(12H).

[0287] Example 2. Synthesis of lipid 2: ((4,4'-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetraoctanoate [ka] Overall Scheme: [ka]

[0288] 2-1: Synthesis of 2-oxopropane-1,3-dioctanoate [ka]

[0289] A 2 L three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere was charged with a solution of 1,3-dihydroxyacetone (50 g, 1.0 equiv.) and octanoyl chloride (225.8 g, 2.5 equiv.) in CHCl3 (900 mL). Pyridine (175.8 g, 4.0 equiv.) was added to the reactor while maintaining the temperature at 0 °C for 40 min. The mixture was stirred overnight at room temperature under nitrogen. The formed pyridine hydrochloride was removed by filtration and washed with CHCl2. The combined filtrate and washings were then washed with 200 mL of 5% aqueous NaHCO3 and brine (200 mL). The solution was then dried over NaSO4 and concentrated under vacuum. The crude product was slurried in n-heptane (125 mL) for 30 min and filtered. This afforded 98 g (52% yield) of 2-1 as a colorless oil. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature 2.09 min, m / z (calculated) 342.24, (observed) 343.45 (M+H + ).

[0290] 2-2: Synthesis of 2-hydroxypropane-1,3-diyl dioctanoate [ka]

[0291] A 500 mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere was charged with a solution of 2-1 (20 g, 1.0 equiv.) in 200 mL of THF. To this solution, AcOH (4.56 g, 1.3 equiv.) was added at 0 °C, and then NaBHCN (4.3 g, 1.2 equiv.) was added to the mixture at 0 °C. The mixture was stirred at room temperature for 4 h. The reaction mixture was quenched with 200 mL of water. The mixture was extracted with CHCl (3 × 200 mL), and the combined organic phases were washed with an aqueous solution of 5% NaHCO (500 mL), brine (500 mL), and then dried over NaSO. Filtration and concentration under vacuum gave crude 2-2 (19.5 g), which was used in the next step without purification.

[0292] 2-3: Synthesis of ((4,4'-((tert-butoxycarbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetraoctanoate [ka]

[0293] A solution of 1-5 (8.4 g, 1.0 equiv.) followed by 2-2 in CHCl (175 mL) was added to a 500 mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere, and the solution was cooled in an ice-water bath. To this solution, DMAP (3.55 g, 1.0 equiv.) and EDCI (22.3 g, 4.0 equiv.) were added at 0° C. The reaction mixture was stirred at 25° C. overnight. The reaction was then quenched with 200 mL of 10% aqueous citric acid. The organic layer was separated, washed with brine (200 mL), and dried over anhydrous sodium sulfate. Filtration and concentration under vacuum gave crude 2-3, which was dissolved in CHCl, and the solution was adsorbed onto 54 g of silica gel (type: ZCX-2, 100–200 mesh, 6.43 w / w) and purified on a silica gel column (270 g of silica gel, type: ZCX-2, 100–200 mesh, 32.14 w / w) using a 100:0 to 90:10 petroleum ether / ethyl acetate gradient. Product-containing fractions were pooled, combined, and concentrated under reduced pressure to give 15 g (55% over two steps) of 2-3 as a light yellow oil.

[0294] 2-4: Synthesis of bis(4-((1,3-bis(octanoyloxy)propan-2-yl)oxy)-4-oxobutyl)ammonium chloride [ka]

[0295] A solution of 2-3 (15 g, 1.0 equiv.) in EtOAc (85.5 mL) was placed in a 500 mL round-bottom flask purged and maintained with an inert nitrogen atmosphere, and the mixture was cooled in an ice-water bath. To the solution, HCl in EtOAc (80 mL, 10.0 equiv., 2 mol / L) was added dropwise at 0-10°C. The resulting solution was stirred at room temperature overnight. The mixture was concentrated in vacuo. This afforded 13 g (93% yield) of 2-4 as a light yellow oil, which was used in the next reaction without further purification. ELSD A: water / 0.05% TFA; B: CH3CN / 0.05% TFA 95:5-5:95 A / B, 3 min, 0.7 min hold): RT 0.746 min, m / z (calculated) 842.60, (found) 842.71 (M+H + ).

[0296] Lipid 2: Synthesis of ((4,4'-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetraoctanoate [ka]

[0297] A 500-mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere was charged with 2-4 (8 g, 1 equiv.) dissolved in CHCl (280 mL), and the solution was cooled in an ice-water bath. To this mixture was added triphosgene (2.82 g, 1 equiv.), followed by dropwise addition of pyridine (3.76 g, 5.00 equiv.) with stirring at 0 °C. The mixture was stirred at room temperature for 4 hours and then concentrated under vacuum. The residue was dissolved in pyridine (160 mL), and the solution was cooled in an ice-water bath under nitrogen. To this solution was added dropwise 3-(dimethylamino)propane-1-thiol (1.356 g, 1.20 equiv.) with stirring at 0 °C for 10 minutes. The resulting solution was stirred at room temperature overnight. The mixture was concentrated under vacuum, and the residue was dissolved in CHCl (200 mL). The mixture was washed with 10% aqueous citric acid (2 × 100 mL), 5% NaHCO (2 × 100 mL), and brine (100 mL). The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. The crude product was dissolved in CHCl (25 mL), adsorbed onto 12 g of silica gel (type: ZCX-2, 100–200 mesh, 6.43 w / w), and purified on a silica gel column (80 g of silica gel, type: ZCX-2, 100–200 mesh, 32.14 w / w) using a gradient of CHCl / MeOH from 100:0 to 97:3. Fractions containing the pure product were analyzed, pooled, and concentrated under reduced pressure. The product thus obtained was dissolved in 36 mL of n-heptane, and 0.22 g of activated carbon powder was added. The mixture was stirred at room temperature for 4 h and then filtered. The filtrate was concentrated under vacuum. This gave 3.8 g (42%) of 2 as a viscous pale yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 2 min, 0.7 min hold): room temperature 1.48 min, m / z (calculated) 986.65, (found) 987.4 (M+H). 1H NMR (300MHz, CDCl3) δ5.26(m, 2H), 4.32(dd, J=11.9, 4.4Hz, 4H), 4.16(dd, J=11.9, 5.7Hz, 4H), 3.40(br m, 4H), 2.93(t, J=7.3Hz, 2H), 2.39-2.25(18H), 1.91-1.59(16H), 1.06-1.45(32H), 0.913-0.869(12H).

[0298] Example 3. Synthesis of Lipid 3: Bis(1,3-bis(nonanoyloxy)propan-2-yl)5-((4-(dimethylamino)butanoyl)oxy)nonanedioic acid salt HCl salt [ka] Overall Scheme: [ka]

[0299] 3-2: Synthesis of bis(1,3-bis(nonanoyloxy)propan-2-yl)5-oxonanedioate [ka]

[0300] A solution of 3-1 (7.04 g, 1.0 equiv.) in CHCl (100 mL) was placed in a 250 mL three-neck round-bottom flask purged and maintained with an inert nitrogen atmosphere and cooled in an ice-water bath under nitrogen. Subsequently, 1-2 (25.95 g, 2.0 equiv.), DMAP (4.26 g, 1.0 equiv.), and EDCI (20.09 g, 3.0 equiv.) were added at 0 °C. The resulting solution was stirred at room temperature for 16 h. The reaction mixture was adsorbed onto 90 g of silica gel (type: ZCX-2, 100-200 mesh, 6.43 w / w) and purified on a silica gel column (900 g, type: ZCX-2, 100-200 mesh) using a 100:0 to 90:10 PE / EA gradient. Fractions containing pure product were pooled, concentrated in vacuo, and dried over PO to give 19.7 g (62.2%) of 3-2 as a yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature 0.87 min, m / z (calculated) 910.63, (found) 933.35 (M+Na).

[0301] 3-3: Synthesis of bis(1,3-bis(nonanoyloxy)propan-2-yl) 5-hydroxynonanedioate [ka]

[0302] A 250 mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere was charged with a solution of 3-2 (10.7 g, 1.0 equiv.) in THF (100 mL, 10V) and cooled in an ice-water bath. Subsequently, HOAc (7.96 g, 11.3 equiv.) and NaBHCN (8.88 g, 12.0 equiv.) were added at 0 °C. The resulting solution was stirred at room temperature for 16 h. The reaction was then quenched by the addition of water (100 mL, 10V). The resulting solution was extracted with ethyl acetate (3 × 100 mL), and the organic layers were combined. The resulting mixture was washed with brine (2 × 100 mL). The mixture was dried over anhydrous sodium sulfate, and the organic phase was concentrated in vacuo. The reaction mixture was adsorbed onto 40 g of silica gel (type: ZCX-2, 100-200 mesh, 6.43 w / w) and purified on a silica gel column (400 g, type: ZCX-2, 100-200 mesh) using a 100:0 to 80:20 PE / EA gradient. Fractions containing pure product were pooled, concentrated under vacuum, and dried over PO to give 7.42 g (69.2%) of 3-3 as a yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature, 0.87 min, m / z (calculated) 913.27, (found) 935.35 (M+Na).

[0303] Lipid 3: Synthesis of bis(1,3-bis(nonanoyloxy)propan-2-yl)5-((4-(dimethylamino)butanoyl)oxy)nonanedioic acid salt HCl salt [ka]

[0304] A solution of 3-3 (7.42 g, 1.0 equiv.) in CHCl (110 mL) was placed in a 250 mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere and cooled in an ice-water bath. 4-(Dimethylamino)butanoic acid-HCl salt (1.63 g, 1.2 equiv.), DMAP (0.4 g, 0.4 equiv.), and EDCI (3.74 g, 2.4 equiv.) were added in portions at 0 °C. The resulting solution was stirred at room temperature for 16 h. Silica gel (40 g, Type: ZCX-2, 100-200 mesh, 6.43 w / w) was added to the reaction mixture, and the mixture was adsorbed onto the silica gel. The mixture was purified on a silica gel column (300 g, Type: ZCX-2, 100-200 mesh) using a gradient of 100:0 to 75:25 EtOAc / THF. Fractions containing pure product were pooled and concentrated in vacuo to give 1.9 g of 3 (26.3%) as a yellow oil. ELSD A:water / 0.05% TFA:B:CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature 1.89 min, m / z (calculated) 1025.74, (found) 1026.55 (M+H). 1 H NMR (300MHz, CDCl3): δ5.21 (m, 2H), 4.89 (brs, 1H), 4.34 (m, 4H), 4.13 (m, 4H), 2.27-2.37 (16H), 2.22(s, 6H), 2.04(brm, 2H), 1.80(m, 2H), 1.59-1.74(14H), 1.15-1.28(40H), 0.85-0.96(12H).

[0305] Example 4. Synthesis of lipid 4: bis(1,3-bis(octanoyloxy)propan-2-yl)5-((4-(dimethylamino)butanoyl)thio)nonanedioate [ka] Overall Scheme: [ka] [ka]

[0306] 4-1: Synthesis of bis(1,3-bis(octanoyloxy)propan-2-yl)5-oxonanedioate [ka]

[0307] A 1 L three-necked round-bottom flask was charged with a solution of 3-1 (20.0 g, 98.909 mmol, 1.00 equiv), 2-2 (68.2 g, 197.818 mmol, 2 equiv), and DMAP (36.3 g, 296.727 mmol, 3 equiv) in CHCl (600 mL) and cooled in an ice-water bath under nitrogen. EDCI (56.9 g, 296.727 mmol, 3 equiv) was then added in several batches at 0 °C. The resulting solution was stirred at room temperature for 16 h. The reaction was then quenched by the addition of aqueous HCl (1 mol / L, 75 mL). The resulting solution was extracted with CHCl (200 mL). The combined organic phase was washed with brine (2 × 200 mL). The mixture was dried over anhydrous sodium sulfate and concentrated. The crude material was dissolved in CHCl (200 mL), adsorbed onto silica gel (108 g, type: ZCX-2, 100-200 mesh), and purified on a silica gel column (720 g, type: ZCX-2, 100-200 mesh) using a 100:0 to 90:10 PE / EA gradient. Fractions containing pure product were pooled and concentrated in vacuo to give 50 g (53.2%) of 4-1 as a yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature 1.74 min, m / z (calculated) 854.58, (found) 877.75 (M+Na).

[0308] 4-2: Synthesis of bis(1,3-bis(octanoyloxy)propan-2-yl) 5-hydroxynonanedioate [ka]

[0309] A 1 L three-necked round-bottom flask was charged with a solution of 4-1 (50.0 g, 58.469 mmol, 1.00 equiv) in THF (500 mL). This solution was cooled in an ice-water bath under nitrogen, and HOAc (35.1 g, 584.686 mmol, 10 equiv) was added at 0 °C. To this was added NaBHCN (36.7 g, 584.686 mmol, 10 equiv) in several portions at 0 °C. The resulting solution was stirred at room temperature for 3 h. The reaction was then quenched by the addition of water (200 mL). The resulting solution was extracted with dichloromethane (1000 mL). The organic phase was dried over anhydrous NaSO and concentrated. The crude material was adsorbed onto silica gel (160 g, type: ZCX-2, 100-200 mesh) and purified on a silica gel column (400 g, type: ZCX-2, 100-200 mesh) using a 100:0 to 90:10 PE / EA gradient. Fractions containing pure product were pooled and concentrated in vacuo to give 20 g (40%) of 4-2 as a yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature, 1.00 min, m / z (calculated) 856.59, (found) 879.70 (M+Na).

[0310] 4-3: Synthesis of bis(1,3-bis(octanoyloxy)propan-2-yl)5-((methylsulfonyl)oxy)nonanedioate [ka]

[0311] A 500 mL three-necked round-bottom flask was charged with a solution of 4-2 (25.0 g, 29.166 mmol, 1.00 equiv) and EtN (5.9 g, 58.331 mmol, 2 equiv) in CHCl (250 mL) and cooled in an ice-water bath under nitrogen. Subsequently, MsCl (5.0 g, 43.748 mmol, 1.5 equiv) was added dropwise with stirring at 0 °C for 20 min. The resulting solution was stirred at room temperature for 3 h. The reaction was then quenched by the addition of water / ice (100 mL). The resulting solution was extracted with CHCl (2 × 100 mL). The combined organic phase was washed with brine (100 mL) and dried over NaSO. After concentration, 24 g (crude) of 4-3 was obtained as a light yellow oil, which was used directly in the next reaction. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 2 min, 0.7 min hold): room temperature 1.71 min, m / z (calculated) 934.57, (found) 957.65 (M+Na).

[0312] 4-4: Synthesis of bis(1,3-bis(octanoyloxy)propan-2-yl) 5-mercaptononanedioate [ka]

[0313] A 500 mL three-necked round-bottom flask was charged with a solution of 4-3 (30.0 g, 32.077 mmol, 1.00 equiv) in DMF (300, 10V), and the mixture was cooled in an ice-water bath under nitrogen. Subsequently, NaSH (9.0 g, 160.383 mmol, 5.00 equiv) was added in three portions over 1.5 h at 0 °C. The resulting solution was stirred at room temperature for 5 h. The reaction was then quenched by the addition of water / ice (100 mL). The mixture was extracted with EtOAc (3 × 100 mL). The combined organic phase was washed with brine (2 × 100 mL). The mixture was dried over anhydrous sodium sulfate and concentrated. This gave 10 g (crude) of 4-4 as a light yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 2 min, 0.7 min hold): room temperature 1.17 min, m / z (calculated) 872.57, (observed) 895.70 (M+Na).

[0314] Lipid 4: Synthesis of bis(1,3-bis(octanoyloxy)propan-2-yl)5-((4-(dimethylamino)butanoyl)thio)nonanedioate [ka]

[0315] A 250 mL three-necked round-bottom flask was charged with a solution of 4-4 (12.0 g, 13.742 mmol, 1.00 equiv), 4-(dimethylamino)butanoic acid (2.2 g, 16.490 mmol, 1.2 equiv), and DMAP (2.0 g, 16.490 mmol, 1.2 equiv) in DCM (120 mL). The solution was cooled in an ice-water bath under nitrogen. EDCI (3.16 g, 16.490 mmol, 1.2 equiv) was then added in several batches at 0 °C. The resulting solution was stirred at room temperature for 16 h. The reaction was then quenched by the addition of aqueous HCl (1 mol / L, 50 mL). The resulting solution was extracted with dichloromethane (2 × 100 mL). The combined organic phase was washed with brine (2 × 100 mL), dried over anhydrous sodium sulfate, and then concentrated. The crude material was adsorbed onto silica gel (25 g, type: ZCX-2, 100-200 mesh) and purified on a silica gel column (200 g, type: ZCX-2, 100-200 mesh) with a 100:0 to 25:1 CHCl / MeOH gradient. Fractions containing pure product were pooled and concentrated in vacuo to give 1.5 g (6.5% yield over three steps) of lipid 4 as a light yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 2 min, 0.7 min hold): room temperature 1.15 min, m / z (calculated) 985.66, (found) 986.55 (M+Na). 1 H-NMR (400MHz, CDCl3): δ5.26(m, 2H), 4.31(m, 4H), 4.15(m, 4H), 3.53(brs, 1H), 2.61(t, J=7.5Hz, 2H) , 2.39-2.28(14H), 2.24(s, 6H), 1.85(m, 2H), 1.787-1.512(16H), 1.34-1.29(32H), 0.98-0.80(12H).

[0316] Example 5. Synthesis of lipid 5: bis(1,3-bis(nonanoyloxy)propan-2-yl) 4-((4-(dimethylamino)butanoyl)oxy)heptanedioate [ka] Overall Scheme: [ka]

[0317] 5-1:4-oxoheptanedioic acid [ka]

[0318] To a three-necked round-bottom flask, EtOH (25 mL, 5 V) and diethyl-4-oxo-heptanethioate (5 g, 1 equiv.) were added at room temperature under nitrogen. The mixture was cooled in an ice-water bath, and then aqueous sodium hydroxide solution (6 N, 25 mL) was slowly added to the mixture at 0 °C. The resulting solution was then warmed and stirred at 60 °C for 2 h. After cooling to room temperature, brine (50 mL) and CHCl (50 mL) were added to the mixture and stirred for 10 min, after which the aqueous phase was separated. The pH of the aqueous phase was adjusted to 3-4 using 3 N HCl. The mixture was extracted with CHCl (100 mL). The organic phase was dried over anhydrous MgSO and then filtered. Concentration under vacuum gave 5-1 (3.2 g, 84.6% yield) as a light yellow solid. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 2 min, 0.7 min hold): room temperature 0.81 min, m / z (calculated) 174.05, (found) 197.06 (M+Na).

[0319] 5-2: Synthesis of 3,3'-(1,3-dithiolane-2,2-diyl)dipropionic acid [ka]

[0320] To a three-necked round-bottom flask, CHCl (32 mL), 5-1 (3.2 g, 1 equiv.), and ethane-1,2-dithiol (2.1 g, 1.2 equiv.) were added in one portion at room temperature. The mixture was cooled in an ice-water bath under nitrogen, and BF.EtO (6.48 g, 2.5 equiv.) was slowly added to the mixture at 0 °C. The resulting solution was stirred at 20 °C for 16 h. The solid was collected by filtration. The solid was dried under vacuum to give 5-2 (4 g, 88% yield) as a light yellow solid. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 2 min, 0.7 min hold): room temperature 0.20 min, m / z (calculated) 250.03, (found) 268.2 (M+Na).

[0321] 5-3: Synthesis of ((3,3'-(1,3-dithiolane-2,2-diyl)bis(propanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetranonanoate [ka]

[0322] To a three-necked round-bottom flask were added CHCl (100 mL), 5-2 (5 g, 1.0 equiv.), 1-2 (16.37 g, 2.2 equiv.), and DMAP (2.44 g, 1 equiv.) in succession. The mixture was cooled in an ice-water bath under nitrogen, and then EDCI (8.42 g, 2.2 equiv.) was added to the reaction mixture in several portions at 0 °C. The resulting solution was stirred at 20 °C for 16 h. The reaction was quenched with 10% aqueous citric acid (50 mL). The organic phase was separated, washed with 10% aqueous citric acid (50 mL), brine (50 mL), dried over anhydrous MgSO, and then filtered. Concentration in vacuo gave crude 5-3, which was dissolved in CHCl (50 mL). The solution was adsorbed onto silica gel (50 g, type: ZCX-2, 100-200 mesh) and purified on a silica gel column (200 g, type: ZCX-2, 100-200 mesh) using a 100:0 to 98:2 PE / EA gradient. Fractions containing pure product were pooled and concentrated in vacuo to give 5-3 (16.1 g, 84% yield) as a colorless oil. (Due to poor ionization, no mass was observed. Therefore, the molecule was used in the next step without further characterization.)

[0323] 5-4: Synthesis of bis(1,3-bis(nonanoyloxy)propan-2-yl) 4-oxoheptanedioate [ka]

[0324] Acetone (400 mL) and 5-3 (16 g, 1.0 equiv.) were added to a three-necked round-bottom flask, and the solution was cooled to −20° C. under nitrogen. A solution of NBS (11.87 g, 4 equiv.) in acetone (80 mL) was added dropwise to the reaction mixture at −20° C. over 15 min. The resulting solution was stirred at −20° C. for 1 h. The reaction was quenched with HO (320 mL) and allowed to warm to room temperature. The acetone was removed by concentration under vacuum, and the mixture was extracted with EtOAc (160 mL). The organic phase was dried over NaSO, filtered, and the solvent was removed under reduced pressure. The crude material was dissolved in CHCl (75 mL), adsorbed onto silica gel (30 g, type: ZCX-2, 100-200 mesh), and purified on a silica gel column (200 g, type: ZCX-2, 100-200 mesh) using a 100:0 to 97:3 PE / EA gradient. Fractions containing pure product were pooled and concentrated in vacuo to give 5-4 (10.3 g, 70% yield) as a colorless oil. 1 H NMR (300MHz, CDCl3) δ5.23(q, J=5.0Hz, 2H), 4.30(dd, J=11.9, 4.4Hz, 4H), 4.16(dd, J=12.0, 5.7Hz, 4H), 2.78(t, J =6.5Hz, 4H), 2.63(t, J=6.6Hz, 4H), 2.33(t, J=7.5Hz, 8H), 1.67-1.53(10H), 1.37-1.24(38H), 0.94-0.84(m, 12H).

[0325] 5-5: Synthesis of bis(1,3-bis(nonanoyloxy)propan-2-yl) 4-hydroxyheptanedioate [ka]

[0326] A 500 mL four-neck round-bottom flask purged and maintained with an inert nitrogen atmosphere was charged with 5-4 (10.3 g, 1 equiv.) and THF (100 mL). To this was added AcOH (7.0 g, 10 equiv.), and the solution was cooled in an ice-water bath. Subsequently, NaBHCN (7.32 g, 10 equiv.) was added in several batches at 0 °C. The resulting solution was stirred at 25 °C for 18 h. The reaction was quenched with H O (400 mL). The mixture was extracted with EtOAc (100 mL), and the organic phase was separated, dried over Na SO , filtered, and concentrated in vacuo. The crude product 5-5 was dissolved in CHCl and adsorbed onto silica gel (30 g, type: ZCX-2, 100-200 mesh) and purified on a silica gel column (150 g, type: ZCX-2, 100-200 mesh) using a 100:0 to 95:5 PE / EA gradient. Fractions containing the pure product were pooled and concentrated in vacuo to give 5-5 (6 g, 70% yield) as a colorless oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 5 min, 0.7 min hold): room temperature 3.48 min, m / z (calculated) 884.62, (found) 907.35 (M+Na).

[0327] Lipid 5: Synthesis of bis(1,3-bis(nonanoyloxy)propan-2-yl) 4-((4-(dimethylamino)butanoyl)oxy)heptanedioate [ka]

[0328] To a three-necked round-bottom flask were added 5-5 (4 g, 1 equiv.), 4-(dimethylamino)butanoic acid (0.99 g, 1.3 equiv.), DMAP (0.39 g, 0.7 equiv.), and CHCl (60 mL) in succession. The mixture was cooled in an ice-water bath under nitrogen, and then EDCI (1.21 g, 1.4 equiv.) was added to the reaction mixture in several portions at 0 °C. The resulting solution was stirred at 20 °C for 16 h. The reaction was quenched with 10% aqueous citric acid (40 mL). The organic phase was separated, washed with 10% aqueous citric acid (40 mL), brine (40 mL), dried over anhydrous MgSO, and then filtered. The solvent was removed in vacuo, the residue was dissolved in CHCl (25 mL), and the crude material was adsorbed onto silica gel (10 g, type: ZCX-2, 100-200 mesh) and purified on a silica gel column (50 g, type: ZCX-2, 100-200 mesh) using a 100:0 to 80:1 CHCl / MeOH gradient. Fractions containing pure product were pooled and concentrated in vacuo to give 5 (1.2 g, 27% yield) as a light yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 2 min, 0.7 min hold): room temperature 1.82 min, m / z (calculated) 997.71, (found) 998.56 (M+H). 1 H-NMR (300MHz, CDCl3): δ5.24(m, 2H), 4.96(dd, J=8.1, 4.2Hz, 1H), 4.30(m, 4H), 4.15(m, 4H) ), 2.41-2.25(20H), 1.80-1.92(6H), 1.67-1.54(10H), 1.02-1.49(40H), 0.94-0.84(12H).

[0329] Example 6. Synthesis of lipid 6a [ka] Overall Scheme: [ka]

[0330] 6-1: Synthesis of (S)-3-(benzyloxy)propane-1,2-diyl dinonanoate [ka]

[0331] To a three-neck flask, CHCl (60 mL), (R)-3-(benzyloxy)propane-1,2-diol (3 g, 1 equiv.), and nonanoyl chloride (6.4 g, 2.2 equiv.) were added in one portion at room temperature, and the mixture was cooled in an ice-water bath under nitrogen. Pyridine (3.90 g, 3 equiv.) was added to the reaction mixture over 10 minutes at 0°C. The resulting solution was stirred at 20°C for 16 hours. The reaction was then quenched by adding 30 mL of water and stirred for 10 minutes. The organic phase was separated. The aqueous layer was extracted with CHCl (75 mL). The combined organic layers were dried over anhydrous sodium sulfate and then filtered. Concentration in vacuo gave crude 6-1, which was dissolved in CHCl (50 mL), adsorbed onto 20 g of silica gel (type: ZCX-2, 100–200 mesh, 6.43 w / w), and purified on a silica gel column (100 g of silica gel, type: ZCX-2, 100–200 mesh, 32.14 w / w) using a gradient of 100:0 to 50:1 petroleum ether / EtOAc. Fractions containing pure product were analyzed, pooled, and concentrated under reduced pressure to give 6-1 (6.0 g, 80% yield) as a colorless oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 2 min, 0.7 min hold): RT 1.1 min, m / z (calculated) 462.33, (found) 485.2 (M+Na).

[0332] 6-2: Synthesis of (S)-3-hydroxypropane-1,2-diyl dinonanoate [ka]

[0333] A three-necked flask was charged with MeOH (60 mL), 6-1 (6 g, 1 equiv.), and Pd / C (0.6 g, 10 wt%) under a nitrogen atmosphere at room temperature. The flask was evacuated and flushed with hydrogen three times. The mixture was stirred under an atmosphere of hydrogen (balloon) at room temperature for 16 h. After filtration, the filtrate was concentrated to dryness in vacuo to give 6-2 (3.1 g, 64% yield) as a colorless oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 2 min, 0.7 min hold): room temperature 0.89 min, m / z (calculated) 3722.29, (found) 395.3 (M+Na).

[0334] 6-3: Synthesis of (2R,2'R)-((3,3'-((tert-butoxycarbonyl)azanediyl)bis(propanoyl))bis(oxy))bis(propane-3,1,2-triyl)tetranonanoate [ka]

[0335] To a three-necked round-bottom flask were added CHCl (20 mL), 3,3′-((tert-butoxycarbonyl)azanediyl)dipropionic acid (commercially available, 1 g, 1.0 equiv.), 6-2 (3.10 g, 2.2 equiv.), and DMAP (0.47 g, 1 equiv.) successively, and the mixture was cooled in an ice-water bath under nitrogen. EDCI (1.60 g, 2.2 equiv.) was added to the reaction mixture in several portions over 10 min at 0°C. The resulting solution was stirred at 20°C for 16 h. The reaction was quenched with 10% aqueous citric acid (10 mL). The organic phase was separated, washed with 10% aqueous citric acid (10 mL), brine (10 mL), dried over anhydrous MgSO, and then filtered. Concentration in vacuo gave crude 6-3, which was dissolved in CHCl (10 mL), adsorbed onto 5 g of silica gel (type: ZCX-2, 100–200 mesh, 6.43 w / w), and purified on a silica gel column (25 g of silica gel, type: ZCX-2, 100–200 mesh, 32.14 w / w) using a gradient of 100:0 to 50:1 petroleum ether / EtOAc. Fractions containing pure product were analyzed, pooled, and concentrated under reduced pressure to give 6-3 (3 g, 81% yield) as a yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature 2.35 min, m / z (calculated) 969.68, (found) 992.5 (M+Na).

[0336] 6-4: Synthesis of (2R,2'R)-((3,3'-azanediylbis(propanoyl))bis(oxy))bis(propane-3,1,2-triyl)tetranonanoate [ka]

[0337] To a three-neck flask, CHCl (60 mL) and 6-3 (3 g, 1 equiv.) were added in one portion at room temperature. The mixture was cooled in an ice-water bath under nitrogen, and then TFA (4.5 mL) was slowly added to the reaction mixture at 0–5°C. The reaction mixture was stirred at 25°C for 2 h. The reaction was then quenched by adding 5% aqueous sodium carbonate (10 wt%, 30 mL). The organic phase was separated. The organic phase was washed with brine (2 × 30 mL), dried over anhydrous MgSO, filtered, and concentrated to dryness to give 6-4 (2.5 g, 94% yield) as a yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature, 0.87 min, m / z (calculated) 869.62, (observed) 892.40 (M+Na).

[0338] Synthesis of lipid 6a [ka]

[0339] To a three-neck flask, CHCl (50 mL, 20 V) and 6-4 (2.50 g, 1 equiv.) were added in one portion at room temperature. The mixture was then cooled in an ice-water bath under nitrogen. Triphosgene (0.85 g, 1 equiv.) was then added in several portions to the reaction mixture at 0–5 °C. Pyridine (1.13 g, 5 equiv.) was slowly added to the reaction mixture over a period of 2 ± 0.5 h. After the addition, the reaction mixture was stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure, and the residue was dissolved in anhydrous pyridine (50 mL, 20 V) and cooled in an ice bath under nitrogen. To this, 3-(dimethylamino)-1-propanethiol hydrochloride (0.41 g, 1.2 equiv.) was added at 0 °C. After the addition, the above mixture was stirred at room temperature for 18 h. The solvent was removed by rotary evaporation under vacuum. The mixture was diluted with CHCl (50 mL). The organic phase was washed with 10% aqueous citric acid (3 × 25 mL). The organic phase was dried over anhydrous MgSO4 and then filtered. Concentration under vacuum gave crude lipid 6a, which was adsorbed onto 10 g of silica gel (type: ZCX-2, 100–200 mesh, 6.43 w / w) and purified on a silica gel column (50 g of silica gel, type: ZCX-2, 100–200 mesh, 32.14 w / w) using a 100:0 to 98:2 CHCl2 / MeOH gradient. Fractions containing pure product were analyzed, pooled, and concentrated under reduced pressure to give 6 (1.2 g, 41% yield) as a yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 2 min, 0.7 min hold): room temperature 1.1 min, m / z (calculated) 1014.68, (observed) 1015.68 (M+H). 1 H NMR (300MHz, CDCl3) δ5.26(m, 2H), 4.31(m, 4H), 4.15(m, 4H), 3.65(t, J=7.2Hz, 4H), 2.93(t, J=7.3Hz , 2H), 2.65(t, J=7.1Hz, 4H), 2.50-2.19(16H), 1.52-1.70(10H), 1.36-1.23(40H), 0.93-0.83(12H).

[0340] Example 7. Synthesis of lipid 7: bis(1,3-bis(octanoyloxy)propan-2-yl) 4-((4-(dimethylamino)butanoyl)thio)heptanedioate [ka] Overall Scheme: [ka]

[0341] 7-1: Synthesis of ((3,3'-(1,3-dithiolane-2,2-diyl)bis(propanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetraoctanoate [ka]

[0342] A solution of 5-2 (16 g, 1.0 equiv.) in CHCl (240 mL) was placed in a 500 mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere. Subsequently, 2-2 (48 g, 2.0 equiv.) and DMAP (23 g, 1.0 equiv.) were added, and the mixture was cooled in an ice-water bath under nitrogen. To this cooled solution, EDCI (36.8 g, 3.0 equiv.) was added in several portions over 45 min at 0 °C. The resulting solution was stirred at room temperature for 16 h. The reaction mixture was adsorbed onto silica gel (110 g, type: ZCX-2, 100-200 mesh) and purified on a silica gel column (800 g, type: ZCX-2, 100-200 mesh) using a 100:0 to 90:10 petroleum ether / ethyl acetate gradient. Fractions containing pure product were pooled and concentrated in vacuo to give 7-1 (60 g, 95% yield) as a yellow oil. ELSD A: water / 0.05% TFA: B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 2 min, 0.7 min hold): room temperature 1.60 min, m / z (calculated) 902.52, (found) 925.50 (M+Na).

[0343] 7-2: Synthesis of bis(1,3-bis(octanoyloxy)propan-2-yl) 4-oxoheptanedioate [ka]

[0344] Acetone (1.5 L) and 7-1 (60 g, 1.0 equiv.) were added to a 3 L three-necked round-bottom flask, the mixture was cooled to -20 °C under nitrogen, and then a solution of NBS (47.3 g, 4.0 equiv.) in acetone (300 mL) was added dropwise to the reaction mixture over 15 minutes. The resulting solution was stirred at -20 °C for 1 hour. The reaction was quenched with water (300 mL), warmed to room temperature, and concentrated in vacuo to remove acetone. The mixture was extracted with EtOAc (600 mL), and the organic phase was dried (NaSO), filtered, and concentrated in vacuo to give crude 7-2. The solvent was removed under reduced pressure. Crude 7-2 was dissolved in CHCl (200 mL), adsorbed onto silica gel (120 g, type: ZCX-2, 100-200 mesh), and purified on a silica gel column (800 g, type: ZCX-2, 100-200 mesh) using a gradient of 100:0 to 90:10 petroleum ether / EtOAc. Fractions containing pure product were pooled and concentrated in vacuo to give 7-2 (44 g, 80% yield) as a colorless oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 2 min, 0.7 min hold): room temperature 1.36 min, m / z (calculated) 826.54, (found) 849.50 (M+Na).

[0345] 7-3: Synthesis of bis(1,3-bis(octanoyloxy)propan-2-yl) 4-hydroxyheptanedioate [ka]

[0346] A 1 L four-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere was charged with a solution of 7-2 (44 g, 1.0 equiv) in THF (400 mL). To this was added HOAc (37 g, 12.0 equiv) at 0 °C. Subsequently, NaBHCN (39 g, 12.0 equiv) was added portionwise at 0 °C. The resulting solution was stirred at 25 °C for 18 h. The reaction was quenched with water (800 mL). The mixture was extracted with EA (800 mL). The organic phase was separated, dried over NaSO, filtered, and concentrated in vacuo to give crude 7-3. Crude 7-3 was dissolved in CHCl (150 mL), adsorbed onto silica gel (80 g, type: ZCX-2, 100-200 mesh), and purified on a silica gel column (800 g, type: ZCX-2, 100-200 mesh) using a gradient of 100:0 to 80:20 petroleum ether / EtOAc. Fractions containing pure product were pooled and concentrated in vacuo to give 7-3 (16 g, 36% yield) as a colorless oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 2 min, 0.7 min hold): room temperature 1.32 min, m / z (calculated) 828.56, (found) 851.50 (M+Na).

[0347] 7-4: Synthesis of bis(1,3-bis(octanoyloxy)propan-2-yl) 4-((methylsulfonyl)oxy)heptanedioate [ka]

[0348] A 500 mL three-necked round-bottom flask was charged with a solution of 7-3 (16 g, 1.0 equiv.) and EtN (2.4 g, 1.2 equiv.) in DCM (160 mL), and the solution was cooled in an ice-water bath under nitrogen. To this cooled solution, MsCl (2.42 g, 1.1 equiv.) was added dropwise with stirring at 0 °C for 20 min. The resulting solution was stirred at room temperature for 3 h. The reaction was then quenched by the addition of water / ice (100 mL). The resulting solution was extracted with dichloromethane (2 × 0.0 mL). The combined organic layers were washed with brine (100 mL). The organic phase was separated, dried over Na SO , filtered, and the solvent was removed under reduced pressure to give crude 7-4. Crude 7-4 was dissolved in CHCl (75 mL), adsorbed onto silica gel (32 g, type: ZCX-2, 100-200 mesh), and purified on a silica gel column (500 g, type: ZCX-2, 100-200 mesh) using a gradient of 100:0 to 80:20 petroleum ether / EtOAc. Fractions containing pure product were pooled and concentrated in vacuo to give 7-4 (10 g, 60% yield) as a colorless oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 2 min, 0.7 min hold): room temperature 1.26 min, m / z (calculated) 906.54, (found) 929.50 (M+Na).

[0349] 7-5: Synthesis of bis(1,3-bis(octanoyloxy)propan-2-yl) 4-((methylsulfonyl)oxy)heptanedioate [ka]

[0350] A 500 mL three-necked round-bottom flask was charged with a solution of 7-4 (200 mg, 1.0 equiv.) in DMF (4 mL), and the solution was cooled in an ice-water bath under nitrogen. Subsequently, NaSH (37.5 mg, 3.0 equiv.) was added at 0°C. The resulting solution was stirred at 0°C for 3 h. The reaction was then quenched by adding water / ice (20 mL). The resulting solution was extracted with ethyl acetate (40 mL). The organic phase was separated and washed with brine (2 × 30 mL). The same scale reaction process was repeated 50 times. The combined mixture (from the reaction replicates) was dried over anhydrous sodium sulfate and concentrated under vacuum to give crude 7-5. Crude 7-5 was dissolved in THF (75 mL), adsorbed onto silica gel (20 g, type: ZCX-2, 100-200 mesh), and purified on a silica gel column (200 g, type: ZCX-2, 100-200 mesh) using a gradient of 100:0 to 80:20 petroleum ether / EtOAc. Fractions containing pure product were pooled and concentrated in vacuo to give 7-5 (3 g, 32% yield) as a light yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 2 min, 0.7 min hold): room temperature 1.26 min, m / z (calculated) 844.54, (found) 845.65 (M+H).

[0351] Lipid 7: Synthesis of bis(1,3-bis(octanoyloxy)propan-2-yl) 4-((4-(dimethylamino)butanoyl)thio)heptanedioate [ka]

[0352] A solution of 7-5 (3 g, 1.0 equiv.) in CHCl (30 mL) was placed in a 100 mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere. 4-(Dimethylamino)butanoic acid HCl salt (0.71 g, 1.2 equiv.) and DMAP (0.43 g, 1.0 equiv.) were added, and the mixture was cooled in an ice-water bath. Following this, EDCI (1.02 g, 1.5 equiv.) was added in several portions at 0 °C. The resulting solution was stirred at room temperature for 16 h. The reaction mixture was adsorbed onto silica gel (250 g, type: ZCX-2, 100-200 mesh) and purified on a silica gel column (250 g, type: ZCX-2, 100-200 mesh) using a gradient of 100:0 to 50:50 n-heptane / acetone. Fractions containing pure product were pooled and concentrated in vacuo to give lipid 7 (1.1 g, 32% yield) as a yellow oil. ELSD A:water / 0.05% TFA; B:CHCN / 0.05% TFA 95:5 to 5:95 A / B, 2 min, 0.7 min hold): room temperature, 0.75 min, m / z (calculated) 957.52, (observed) 958.50 (M+H). 1 H-NMR (300MHz, CDCl3): δ5.25(m, 2H), 4.29(m, 4H), 4.15(m, 4H), 3.54(brm, 1H), 2.62(t, J=7.4Hz, 2H), 2.52-2.21(20H), 2.03(m, 2H), 1.83(m, 4H), 1.61(m, 8H), 1.39-1.17(28H), 0.97-0.80(16H).

[0353] Example 8. Synthesis of lipid 8: ((2,2'-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl)bis(acetyl))bis(oxy))bis(propane-2,1,3-triyl)tetranonanoate [ka] Overall Scheme: [ka]

[0354] 8-1: Synthesis of ((2,2'-((tert-butoxycarbonyl)azanediyl)bis(acetyl))bis(oxy))bis(propane-2,1,3-triyl)tetranonanoate [ka]

[0355] A three-necked round-bottom flask was charged with a solution of 2,2'-((tert-butoxycarbonyl)azanediyl)diacetic acid (1 g, 1.0 equiv.), 1-2 (3.50 g, 2.2 equiv.), and DMAP (0.52 g, 1 equiv.) in DCM (20 mL), and the solution was cooled in an ice-water bath under nitrogen. To this cooled solution, EDCI (1.80 g, 2.2 equiv.) was added in several portions at 0 °C. The resulting solution was stirred at 20 °C for 16 h. The reaction was quenched with 10% aqueous citric acid (10 mL). The organic phase was separated, washed with 10% aqueous citric acid (10 mL, 10 V), brine (10 mL, 10 V), and dried over anhydrous MgSO4. Filtration and concentration under vacuum gave crude 8-1, which was dissolved in CHCl (15 mL), adsorbed onto 5 g of silica gel (type: ZCX-2, 100–200 mesh, 6.43 w / w), and purified on a silica gel column (20 g of silica gel, type: ZCX-2, 100–200 mesh, 32.14 w / w) using a gradient of petroleum ether / EtOAc (volume ratio: 100:0 to 50:1). Fractions containing pure product were analyzed, pooled, combined, and concentrated under reduced pressure to give 8-1 (3.27 g, 81% yield) as a yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature 2.2 min, m / z (calculated) 941.64, (found) 964.60 (M+Na).

[0356] 8-2: Synthesis of ((2,2'-azanediylbis(acetyl))bis(oxy))bis(propane-2,1,3-triyl)tetranonanoate [ka]

[0357] A solution of 8-1 (3 g, 1 equiv.) in CHCl (60 mL) was added to a three-neck flask. The resulting solution was cooled in an ice-water bath under nitrogen, and then TFA (4.5 mL) was added slowly at 0-5°C. The resulting solution was stirred at 20°C for 2 h. The reaction was then quenched by the careful addition of 10% sodium carbonate solution (30 mL). The organic phase was separated, washed with brine (2 × 30 mL), dried over anhydrous MgSO, and filtered. The solvent was removed in vacuo to give 8-2 (2.5 g, 93% yield) as a yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5-5:95 A / B, 3 min, 0.7 min hold): room temperature 1.15 min, m / z (calculated) 841.59, (found) 842.51 (M+H).

[0358] Lipid 8: Synthesis of ((2,2'-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl)bis(acetyl))bis(oxy))bis(propane-2,1,3-triyl)tetranonanoate [ka]

[0359] A solution of 8-2 (2.50 g, 1 equiv.) in CHCl (50 mL) was added in one portion to a three-neck flask at room temperature. The resulting solution was cooled in an ice-water bath under nitrogen. Then, triphosgene (0.88 g, 1 equiv.) was added to the reaction mixture over 5 min at 0–5 °C. Pyridine (1.17 g, 5 equiv.) was slowly added to the reaction mixture over 2 ± 0.5 h. After the addition, the reaction mixture was stirred at room temperature for 2 h. The solvent was evaporated under reduced pressure, and the residue was dissolved in anhydrous pyridine (50 mL) and cooled in an ice-water bath. 3-(dimethylamino)-1-propanethiol hydrochloride (0.42 g, 1.2 equiv.) was added, and the mixture was stirred at room temperature for 18 h. The solvent was removed in vacuo. The residue was dissolved in CHCl (50 mL) and washed with 10% aqueous citric acid (3 × 25 mL). The organic phase was separated, dried over anhydrous MgSO4, and then filtered. Concentration under vacuum gave crude lipid 8. This crude product was dissolved in CHCl2 (15 mL), adsorbed onto silica gel (5 g, type: ZCX-2, 100-200 mesh, 6.43 w / w), and purified on a silica gel column (20 g silica gel, type: ZCX-2, 100-200 mesh, 32.14 w / w) using a CHCl2 / MeOH gradient (volume ratio: 100 / 0 to 98:2). The fractions containing lipid 8 were analyzed, pooled, combined, and concentrated under reduced pressure to give lipid 8 (1.2 g, 41% yield) as a yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 2 min, 0.7 min hold): room temperature 0.88 min, m / z (calculated) 986.65, (observed) 987.95 (M+H). 1 H NMR (300MHz, CDCl3): δ5.28(m, 2H), 4.35(m, 4H), 4.18(m, 8H), 2.95(t, J=7.3Hz, 2H), 2.340-2.212(16H), 1.78(m, 2H), 1.71-1.57(8H), 1.35-1.20(40H), 0.94-0.80(12H).

[0360] Example 9. Synthesis of Lipid 9: Bis(1,3-bis(nonanoyloxy)propan-2-yl) 4-((4-(dimethylamino)butanoyl)thio)heptanedioate [ka] Overall Scheme: [ka]

[0361] 9-1: Synthesis of bis(1,3-bis(nonanoyloxy)propan-2-yl) 4-((methylsulfonyl)oxy)heptanedioate [ka]

[0362] A solution of 5-5 (6 g, 1.0 equiv.) in CHCl (90 mL) was added to a mechanically stirred 250 mL four-necked round bottle flask under N. Subsequently, EtN (2.06 g, 3.0 equiv.) was added, and the resulting solution was cooled in an ice-water bath under nitrogen. To the cooled solution, MsCl (1.16 g, 1.5 equiv.) was added dropwise with stirring at 0 °C. The resulting solution was stirred at room temperature for 12 h. The reaction was then quenched by the addition of H0 (100 mL). The phases were separated, and the aqueous phase was extracted with CHCl (100 mL). The combined organic phase was then dried over anhydrous sodium sulfate and concentrated in vacuo. Crude 9-2 was purified by high-speed preparative HPLC using the following conditions: column, XB-C18 silica gel; mobile phase, i-PrOH in 1 mmol NH4HCO3 in water, 65% to 95% gradient in 30 min; detector, UV ELSD. Concentration to dryness in vacuo afforded 9-1 (5 g, 44% overall yield over two steps) as a colorless oil. ELSD A: water / 0.05% TFA; B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B, 2 min, 0.7 min hold): room temperature 1.66 min, m / z (calculated) 962.60, (found) 985.50 (M+Na).

[0363] 9-2: Synthesis of bis(1,3-bis(nonanoyloxy)propan-2-yl) 4-mercaptoheptanedioate [ka]

[0364] A solution of 9-1 (100 mg, 1.00 equiv.) in DMF (0.5 mL) was placed in a 100 mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere, and the resulting solution was cooled in an ice-water bath under nitrogen. Subsequently, NaSH (29.1 mg, 5.00 equiv.) was added at 0 °C. The resulting solution was stirred at 0 °C for 24 h. This reaction was repeated 49 times to complete the entire mixture. The reaction was then quenched by adding ice / water (200 mL). The resulting solution was extracted with EtOAc (3 × 100 mL), and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give 9-2 (4.8 g, crude) as a colorless oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature 2:08 min, m / z (calculated) 900.60, (observed) 923.50 (M+Na).

[0365] Lipid 9: Synthesis of bis(1,3-bis(nonanoyloxy)propan-2-yl) 4-((4-(dimethylamino)butanoyl)thio)heptanedioate [ka]

[0366] A solution of 9-2 (4.8 g, 1.0 equiv.), 4-(dimethylamino)butanoic acid (1.16 g, 1.3 equiv.), and DMAP (0.46 g, 0.7 equiv.) in CHCl (72 mL) was added to a three-necked round-bottom flask, and the resulting solution was cooled in an ice-water bath under nitrogen. EDCI (1.84 g, 1.4 equiv.) was added portionwise to the reaction mixture at 0 °C. The resulting solution was stirred at room temperature for 12 h. The reaction was quenched with 10% aqueous citric acid (48 mL). The organic phase was separated, washed with 10% aqueous citric acid (48 mL), brine (48 mL, 10 V), and dried over anhydrous MgSO. Filtration and concentration under vacuum gave crude 9, which was dissolved in CHCl (25 mL) and adsorbed onto a silica gel column (10 g, type: ZCX-2, 100-200 mesh). Purification was performed on a silica gel column (50 g, type: ZCX-2, 100-200 mesh) using a gradient of n-heptane / acetone from 100:0 to 75:50. Fractions containing pure 9 were pooled and concentrated under vacuum to give 9 (0.9 g, 18% yield) as a yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature 1.24 min, m / z (calculated) 1013.68, (found) 1015.40 (M+H). 1 H NMR (300MHz, CDCl3): δ5.27(q, J=5.1Hz, 2H), 4.30(m, 4H), 4.16(m, 4H), 3.54(brm, 1H), 2.64(t, J=7.3Hz) , 2H), 2.46-2.23(18H), 2.04(m, 2H), 1.84(m, 4H), 1.35-1.76(13H), 1.00-1.32(37H), 0.96-0.83(12H).

[0367] Example 10. Synthesis of lipid 10: ((4,4'-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexylpropanoate) [ka] Overall Scheme: [ka]

[0368] 10-1: Synthesis of 3-cyclohexylpropanoyl chloride [ka]

[0369] A 2000 mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere was charged with a solution of 3-cyclohexylpropanoic acid (100 g, 1.0 equiv.) in CHCl (1 L) and DMF (0.2 mL) was added. Oxalyl chloride (161.00 g, 2.00 equiv.) was added dropwise at room temperature. The mixture was stirred overnight at room temperature under nitrogen. The mixture was concentrated in vacuo to give crude 10-1, which was used directly in the next reaction.

[0370] 10-2: Synthesis of 2-oxopropane-1,3-diyl bis(3-cyclohexylpropanoate) [ka]

[0371] A 2000 mL three-neck round-bottom flask purged and maintained with an inert nitrogen atmosphere was charged with a solution of 10-1 (83.00 g, 2.50 equiv.) and dihydroxy-acetone (17.20 g, 1.00 equiv.) in CHCl (1600 mL), and the solution was cooled in an ice-water bath under nitrogen. To this cooled solution, pyridine (61.00 g, 4.00 equiv.) was added over 40 minutes while maintaining the temperature at 0 °C. The mixture was stirred overnight at room temperature under nitrogen. The formed pyridine hydrochloride salt was removed by filtration and washed with CHCl (200 mL). The combined filtrate was then washed with 5% aqueous NaHCO (2000 mL), 5% aqueous HCl (2000 mL), brine (2000 mL), and dried over NaSO. Concentration under vacuum afforded 65 g (92.8% yield) of 10-2 as a yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature 2.56 min, m / z (calculated) 366.24, (found) 367.40 (M+H).

[0372] 10-3: Synthesis of 2-hydroxypropane-1,3-diyl bis(3-cyclohexylpropanoate) [ka]

[0373] A 2000 mL three-neck round-bottom flask purged and maintained with an inert nitrogen atmosphere was charged with a solution of 10-2 (85.00 g, 1.00 equiv.) in THF (1 L). HOAc (18.00 g, 1.30 equiv.) was added to the solution, and the solution was cooled in an ice-water bath. To this cooled solution, NaBHCN (18.00 g, 1.20 equiv.) was added at 0 °C. The mixture was stirred at room temperature overnight. The reaction mixture was quenched with brine (1 L). The mixture was extracted with EtOAc (3 × 400 mL). The combined organic layer was then washed with 5% aqueous NaHCO (200 mL), 5% aqueous HCl (200 mL), brine (200 mL), and dried over NaSO. Filtration and concentration in vacuo gave crude 10-3, which was dissolved in CHCl (500 mL) and adsorbed onto silica gel (240 g, type: ZCX-2, 100-200 mesh). The crude material was purified on a silica gel column (800 g, type: ZCX-2, 100-200 mesh) using a gradient of 100:0 to 90:10 petroleum ether / EtOAc. Fractions containing pure product were pooled and concentrated in vacuo to give 10-3 (61 g, 70.1% yield) as a yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): RT 2.50 min, m / z (calculated) 368.26, (found) 351.2 (M-HO).

[0374] 10-4: Synthesis of ((4,4'-((tert-butoxycarbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexylpropanoate) [ka]

[0375] To a 250 mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere, 1-5 (4.00 g, 1 equiv.) and 10-3 (10.20 g, 2 equiv.) were added, and the mixture was dissolved in CHCl (80 mL). The solution was cooled in an ice-water bath, and DMAP (1.69 g, 1.00 equiv.) and EDCI (10.60 g, 4.00 equiv.) were added sequentially at 0 °C. The reaction mixture was stirred overnight at room temperature. The reaction was then quenched with 10% aqueous citric acid (200 mL), and the organic phase was separated. The organic phase was washed with 10% aqueous NaHCO (200 mL), brine (200 mL), and dried over anhydrous sodium sulfate. Filtration and concentration under vacuum gave crude 10-4, which was dissolved in CHCl (100 mL), adsorbed onto silica gel (50 g, type: ZCX-2, 100-200 mesh), and purified on a silica gel column (400 g, type: ZCX-2, 100-200 mesh) using a gradient of 100:0 to 90:10 petroleum ether / EtOAc. Fractions containing pure product were pooled and concentrated under vacuum to give 11 g of 10-4 (77.7% yield) as a light yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature 1.79 min, m / z (calculated) 989.64, (found) 1012.50 (M+Na).

[0376] 10-5: Synthesis of bis(4-((1,3-bis((3-cyclohexylpropanoyl)oxy)propan-2-yl)oxy)-4-oxobutyl)ammonium chloride [ka]

[0377] A solution of 10-4 (6.30 g, 1.00 equiv.) in EtOAc (20 mL) was placed in a 250 mL round-bottom flask purged and maintained with an inert nitrogen atmosphere, and the solution was cooled in an ice-water bath. To the cooled solution, a solution of HCl in EtOAc (60 mL, 10 equiv., 2 M) was added dropwise at 0-10 °C. The resulting solution was stirred at room temperature overnight. The resulting mixture was concentrated in vacuo. This afforded 6 g (99% yield) of 10-5 as a light yellow oil. ELSD A: water / 0.05% TFA; B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): RT 1.48 min, m / z (calculated) 889.59, (found) 890.50 (M+H).

[0378] Lipid 10: Synthesis of ((4,4'-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexylpropanoate) [ka]

[0379] A 250 mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere was charged with a solution of 10-5 (6.00 g, 1.00 equiv.) in CHCl (100 mL), and the solution was cooled in an ice-water bath under nitrogen. To the mixture was added triphosgene (1.91 g, 1.62 equiv.) at 0 °C. Pyridine (2.56 g, 5.00 equiv.) was then added dropwise with stirring at 0 °C. The mixture was stirred at room temperature for 4 hours and then concentrated in vacuo. The residue was dissolved in pyridine (100 mL) and cooled in an ice-water bath under nitrogen. To this solution was added 3-(dimethylamino)propane-1-thiol (0.92 g, 1.93 equiv.) dropwise with stirring over 10 minutes at 0 °C. The resulting solution was stirred at room temperature overnight. The resulting mixture was concentrated in vacuo. The residue was diluted with CHCl (500 mL), and the solution was washed with 10% aqueous citric acid (2 × 200 mL), saturated aqueous NaHCO (2 × 200 mL), brine (100 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum to give crude lipid 10. The residue was dissolved in CHCl (100 mL), adsorbed onto silica gel (50 g, type: ZCX-2, 100-200 mesh), and purified on a silica gel column (250 g, type: ZCX-2, 100-200 mesh) with a gradient of 100:0 to 97:3 CHCl / MeOH. Fractions containing the pure product were pooled and concentrated under vacuum. The product was dissolved in n-heptane (40 mL), and 0.22 g of activated carbon powder was added. The mixture was stirred at room temperature for 4 h and then filtered. The filtrate was concentrated in vacuo to give 2 g (48% yield) of lipid 10 as a light yellow oil. ELSD A:water / 0.05% TFA:B:CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature 0.68 min, m / z (calculated) 1034.65, (found) 1035.65 (M+H). 1 H NMR (300MHz, CDCl3): δ5.25 (m, 2H), 4.31 (m, 4H), 4.14 (dd, J=11.9, 5.7Hz, 4H), 3.38 (brm, 4H), 2.94(t, J=7.3Hz, 2H), 2.44-2.22(20H), 1.99-1.47(26H), 1.39-1.17(20H), 0.95-0.79(12H).

[0380] Example 11. Lipid 11: ((4,4'-((((3-(dimethylamino)propyl)thio)carbonyl) Synthesis of (azanediyl)bis(butanoyl)bis(oxy)bis(propane-2,1,3-triyl)tetrakis(4-cyclohexylbutanoate) [ka] Overall Scheme: [ka] [ka]

[0381] 11-1:4-Cyclohexylbutanoyl chloride [ka]

[0382] A 1 L three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere was charged with a solution of 4-cyclohexylbutanoic acid (50 g, 1.0 equiv.) in CHCl3 (500 mL), followed by DMF (0.25 mL). The solution was cooled in an ice-water bath, and then oxalyl chloride (74.1 g, 2.0 equiv.) was added dropwise over 30 minutes. The mixture was stirred overnight at room temperature under nitrogen. The mixture was concentrated under vacuum. This afforded 55.7 g (99.9% yield) of 11-1 as a yellow oil, which was used directly in the next step.

[0383] 11-2: Synthesis of 2-oxopropane-1,3-diyl bis(4-cyclohexylbutanoate) [ka]

[0384] A solution of 11-1 (55.7 g, 2.5 equiv.) was placed in a 2 L three-neck round-bottom flask purged and maintained with an inert nitrogen atmosphere. The solution was cooled in an ice-water bath, and a solution of 1,3-dihydroxyacetone (10.6 g, 1.0 equiv.) in CHCl3 (1114 mL) was added over 1 h. Pyridine (37.3 g, 4.0 equiv.) was added to the mixture over 40 min while maintaining the temperature at 0 °C. The mixture was stirred overnight at room temperature under nitrogen. The reaction mixture was quenched with water (1 L). The organic phase was separated, and the aqueous layer was extracted with CHCl2 (3 x 200 mL). The combined organic phases were washed with 5% aqueous NaHCO3 (300 mL), 5% aqueous HCl (300 mL), and brine (300 mL). The solution was then dried over anhydrous Na2SO4, and the product was obtained by evaporation. This gave 56.4 g of crude 11-2 as a yellow oil, which was used directly in the next reaction.

[0385] 11-3: Synthesis of 2-hydroxypropane-1,3-diyl bis(4-cyclohexylbutanoate) [ka]

[0386] A solution of 11-2 (56.4 g, 1.0 equiv.) in THF (550 mL) was placed in a 1 L three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere, and the mixture was cooled in an ice-water bath. HOAc (11.13 g, 1.3 equiv.) was added to the cooled solution at 0 °C. NaBHCN (10.79 g, 1.2 equiv.) was then added to the mixture at 0 °C. The mixture was stirred overnight at room temperature. The reaction mixture was quenched with water (500 mL). The mixture was extracted with CHCl (3 × 200 mL). The organic layers were combined and washed with 5% aqueous NaHCO (200 mL), 5% aqueous HCl (200 mL), and brine (200 mL). The solution was then dried over anhydrous NaSO and concentrated in vacuo. The residue was dissolved in CHCl (150 mL), silica gel (60 g, type: ZCX-2, 100-200 mesh) was added, and the crude product was adsorbed onto silica gel and purified on a silica gel column (240 g, type: ZCX-2, 100-200 mesh) with a gradient of 100:0 to 80:20 petroleum ether / EtOAc. Fractions containing 11-3 were pooled, concentrated, and dried under vacuum to give 30.3 g (65.0% overall yield for two steps) of 11-3 as a yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature, 0.76 min, m / z (calculated) 396.29, (found) 419.29 (M+Na).

[0387] 11-4: Synthesis of ((4,4'-((tert-butoxycarbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(4-cyclohexylbutanoate) [ka]

[0388] In a 250 mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere, 11-3 (5.46 g, 2.0 equiv.) was added to a solution of 1-5 (2.05 g, 1.0 equiv.) in CHCl (100 mL), and the mixture was cooled in an ice-water bath. To the solution, DMAP (2.3 g, 1.0 equiv.) was added, followed by EDCI (14.51 g, 4.0 equiv.) at 0 °C. The reaction mixture was stirred overnight at room temperature. The reaction was then quenched with 10% aqueous citric acid (200 mL), and the organic phase was separated and washed with 10% aqueous NaHCO (200 mL) and brine (200 mL). The mixture was dried over anhydrous sodium sulfate and concentrated in vacuo. This material was combined with another 7.05 g reaction (based on 11-3), and the combined material was adsorbed onto silica gel (30 g, type: ZCX-2, 100-200 mesh) and purified on a silica gel column (90 g, type: ZCX-2, 100-200 mesh) with a gradient of 100:0 to 65:35 petroleum ether / EtOAc. Fractions containing 11-4 were pooled, concentrated, and dried under vacuum to give 14.2 g (43.0%) of 11-4 as a yellow oil. ELSD A: water / 0.05% TFA; B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature 2.77 min, m / z (calculated) 1045.71, (found) 1068.55 (M+Na).

[0389] 11-5: Synthesis of bis(4-((1,3-bis((4-cyclohexylbutanoyl)oxy)propan-2-yl)oxy)-4-oxobutyl)ammonium chloride [ka]

[0390] A solution of 11-4 (14.17 g, 1.0 equiv.) in EtOAc (71 mL) was placed in a 250 mL round-bottom flask purged and maintained with an inert nitrogen atmosphere. The solution was cooled in an ice-water bath. To this solution, a solution of HCl in EtOAc (142 mL, 2 mol / L) was added dropwise at 0-10 °C. The resulting solution was stirred overnight at room temperature. The resulting mixture was concentrated in vacuo. This afforded 13.6 g (99.9% yield) of 11-5 as a light yellow oil. ELSD A: water / 0.05% TFA; B: CH3CN / 0.05% TFA 95:5-5:95 A / B, 3 min, 0.7 min hold): RT 0.94 min, m / z (calculated) 945.65, (found) 946.60 (M+H).

[0391] Lipid 11: Synthesis of ((4,4'-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(4-cyclohexylbutanoate) [ka]

[0392] A solution of 11-5 (12.0 g, 1.0 equiv.) in CHCl (420 mL) was placed in a 1 L three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere, and the solution was cooled in an ice-water bath. To the solution, triphosgene (5.44 g, 1.5 equiv.) was added at 0 °C. Subsequently, pyridine (4.82 g, 5.0 equiv.) was added dropwise with stirring at 0 °C. The mixture was stirred at room temperature for 4 hours and then concentrated in vacuo. The residue was dissolved in pyridine (240 mL), and the resulting solution was cooled in an ice-water bath. To this solution, 3-(dimethylamino)propane-1-thiol (2.91 g, 2.0 equiv.) was added dropwise over 10 minutes with stirring at 0 °C. The resulting solution was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo. The residue was diluted with DCM (200 mL). The mixture was washed with 10% aqueous NaHCO3 (2 × 200 mL), brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The crude product was adsorbed onto silica gel (20 g, type: ZCX-2, 100-200 mesh) and purified on a silica gel column (80 g, type: ZCX-2, 100-200 mesh) using a gradient of n-heptane / acetone from 100:0 to 65:35. Fractions containing the pure product were pooled, concentrated, and dried under vacuum to give 2 g of 11, which was dissolved in n-heptane (40 mL, 20 V) and activated carbon powder (0.22 g) was added. The mixture was stirred at room temperature for 4 h and then filtered. The filtrate was concentrated under vacuum to give 2 g (14.4% yield) of 11 as a light yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature 1.00 min, m / z (calculated) 1090.71, (found) 1091.60 (M+H). 1 H NMR (300MHz, CDCl3): δ5.24(m, 2H), 4.32(dd, J=11.9, 4.4Hz, 4H), 4.15(dd, J=11.9, 5.7Hz, 4H), 3.39(brm, 4H), 2.92( t, J=7.3Hz, 2H), 2.27-2.38(14H), 2.05(s, 6H), 1.85-2.00(6H), 1.57-1.83(29H), 1.11-1.28(23H), 1.06-1.08(8H).

[0393] Example 12. Synthesis of lipid 12: ((6,6'-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl)bis(hexanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexylpropanoate) [ka] Overall Scheme: [ka]

[0394] 12-1: Synthesis of dimethyl 6,6'-(benzylazanediyl)dihexanoate [ka]

[0395] A solution of KCO (9.5 g, 2.3 equiv.), benzylamine (3.2 g, 1.0 equiv.), and methyl 4-bromobutyrate (15.2 g, 2.3 equiv.) in CHCN (64 mL) was placed in a 250 mL four-neck round-bottom flask at 25 °C under N with mechanical stirring. The mixture was then heated (80 °C) and stirred for 15 h. The mixture was then cooled to 25 °C, poured into water (65 mL), and extracted with EtOAc (2 × 65 mL). The combined organic phases were dried (NaSO), filtered, and concentrated in vacuo to give crude 12-1 (10 g, crude) as a yellow oil, which was used directly in the next reaction.

[0396] 12-2: Synthesis of dimethyl 6,6'-((tert-butoxycarbonyl)azanediyl)dihexanoate [ka]

[0397] A solution of 12-1 (10 g, 1.0 equiv.), (Boc)O (6.6 g, 1.1 equiv.), and Pd / C (1 g, 10% w / w) in EtOH (100 mL) was placed in a 250 mL hydrogenation autoclave at ambient temperature. The mixture was stirred under a hydrogen atmosphere at 5 atm and room temperature for 16 h. The reaction mixture was filtered and concentrated in vacuo at 40 °C. This gave 12-2 (11 g, crude) as a light brown oil, which was used directly in the next reaction.

[0398] 12-3: Synthesis of 6,6'-((tert-butoxycarbonyl)azanediyl)dihexanoic acid [ka]

[0399] A round-bottom flask was charged with a solution of 12-2 (11 g, 1.0 equiv.) in ethanol (55 mL) under nitrogen at room temperature. 6 M aqueous NaOH (55 mL) was added at room temperature. After the addition was complete, the mixture was heated to 60 °C for 2 h. The mixture was cooled to room temperature and poured into brine (110 mL). The solution was extracted twice with n-BuOH / n-heptane (2:1, 110 mL) to remove organic impurities. The aqueous phase was acidified to approximately pH 3 by adding 3 mol / L aqueous HCl and then extracted with t-BuOH:n-heptane (2:1) (110 mL x 2). The combined organic phase was concentrated under reduced pressure to give a sticky solid. The residue was slurried with diethyl ether (22 mL) and filtered. The filter cake was collected to give 12-3 (5.6 g, 66% overall yield for three steps) as a white solid. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature 1.64 min, m / z (calculated) 345.22, (found) 368.10 (M+Na).

[0400] 12-4: Synthesis of ((6,6'-((tert-butoxycarbonyl)azanediyl)bis(hexanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexylpropanoate) [ka]

[0401] A 250 mL three-necked round-bottom flask was charged with a solution of 12-3 (5.6 g, 1.0 equiv.), 10-3 (10 g, 2.0 equiv.), and DMAP (1.65 g, 1.0 equiv.) in CHCl (85 mL). The solution was cooled in an ice-water bath under nitrogen. EDCI (7.5 g, 2.2 equiv.) was added to the reaction mixture in several portions over 15 min at 0 °C. After the addition was complete, the reaction was warmed to room temperature and stirred at 20 °C for 16 h. The reaction mixture was poured into 10% aqueous citric acid (112 mL). The organic phase was separated, washed with 10% aqueous citric acid (112 mL), brine (112 mL), dried over anhydrous MgSO4, and then filtered. The solvent was removed under vacuum to give crude 12-4, which was dissolved in CHCl (65 mL). The crude product was adsorbed onto silica gel (30 g, type: ZCX-2, 100-200 mesh) and purified on a silica gel column (150 g, type: ZCX-2, 100-200 mesh) with a gradient of petroleum ether / EtOAc from 100:0 to 88:12. Fractions containing the pure product were pooled, concentrated, and dried under vacuum to give 10.1 g (60%) of 12-4 as a colorless oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 in 3 min. A / B, 0.7 min hold): Room temperature 2.2 min, m / z (calculated) 1045.71, (observed) 1068.65 (M+Na).

[0402] 12-5: Synthesis of ((6,6'-azanediylbis(hexanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexylpropanoate) [ka]

[0403] A 100 mL three-necked round-bottom flask was charged with a solution of 12-4 (5 g, 1.0 equiv.) in CHCl (50 mL), and the solution was cooled in an ice-water bath under nitrogen. TFA (7.5 mL) was then added to the reaction mixture at 0–15 °C. After the addition was complete, the solution was warmed to room temperature and stirred for 2 h. The mixture was concentrated under vacuum at 30 °C, and then n-heptane (100 mL) was added to the reaction mixture. The resulting cloudy mixture was washed with 17% aqueous sodium carbonate (500 mL), brine (250 mL, 3×), and dried over anhydrous MgSO. Filtration and concentration under vacuum gave 12-5 (4.5 g, 90% yield) as a yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature 0.94 min, m / z (calculated) 945.65, (observed) 946.60 (M+H).

[0404] Lipid 12: Synthesis of ((6,6'-((((3(dimethylamino)propyl)thio)carbonyl)azanediyl)bis(hexanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexylpropanoate) [ka]

[0405] To a 250 mL three-necked round-bottom flask was added 12-5 (4.5 g, 1.0 equiv.) and CHCl (68 mL), and the solution was cooled in an ice-water bath under nitrogen. Triphosgene (1.4 g, 1.0 equiv.) was then added to the cooled solution, followed by pyridine (1.88 g, 5.0 equiv.) over 30 minutes. After the addition was complete, the reaction mixture was warmed to room temperature and then stirred for 4 hours. The solvent was removed in vacuo, the residue was dissolved in pyridine (90 mL), and the mixture was cooled in an ice-water bath under nitrogen. To this cooled solution was added 3-(dimethylamino)-1-propanethiol hydrochloride (0.57 g, 1.1 equiv.). After the addition was complete, the reaction mixture was warmed to room temperature and stirred for 18 hours. The mixture was concentrated under vacuum, keeping the temperature below 20 °C, to give crude lipid 12, which was dissolved in CHCl (90 mL). The resulting solution was washed with 10% aqueous citric acid (45 mL), brine (45 mL, 3×), 10% aqueous sodium bicarbonate (45 mL), and brine (45 mL, 2×). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give crude lipid 12. Crude lipid 12 was dissolved in CHCl (30 mL), adsorbed onto silica gel (15 g, type: ZCX-2, 100-200 mesh), and purified on a silica gel column (60 g, type: ZCX-2, 100-200 mesh) using a gradient of 100:0 to 80:20 n-heptane / acetone. Fractions containing pure product were pooled, concentrated, and dried under vacuum to give 1.5 g (29%) of lipid 12 as a light yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 0.7 min hold): room temperature, 0.87 min, m / z (calculated) 1090.71, (found) 1091.60 (M+H). 1 H NMR (300MHz, CDCl3): δ5.27(m, 2H), 4.30(dd, J=11.9, 4.4Hz, 4H), 4.1(dd, J=11.9, 5.8Hz, 4H), 3.27(brm, 4H), 2.90(t , J=7.2Hz, 2H), 2.45-2.20(20H), 1.81(m, 2H), 1.71-1.63(22H), 1.55-1.47(12H), 1.34-1.07(22H), 0.94-0.77(8H).

[0406] Example 13. Lipid 13: Synthesis of nonanoic acid 2-(3-{(3-dimethylamino-propylsulfanylcarbonyl)-[2-(2-nonanoyloxy-1-nonanoyloxymethyl-ethoxycarbonyl)-ethyl]-amino}-propionyloxy)-3-octanoyloxy-propyl ester [ka] Overall Scheme: [ka]

[0407] 13-1: Synthesis of ((3,3'-((tert-butoxycarbonyl)azanediyl)bis(propanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetranonanoate [ka] 1-5 (7.3 g, 28.0 mmol) and 1-2 (18.8 g, 56.0 mmol) were dissolved in CHCl (110 mL) in a 500 mL three-necked round-bottom flask under nitrogen. The solution was cooled in an ice-water bath, and DMAP (3.4 g, 28.0 mmol) and EDCl (21.5 g, 0.112 mol) were added sequentially. After the addition was complete, the mixture was warmed to room temperature and stirred for 16 h. The mixture was poured into 10% aqueous citric acid (200 mL), and the organic phase was separated, washed with brine (200 mL), and dried over anhydrous NaSO. The drying agent was removed by filtration through a sintered glass funnel, and 50 g of silica gel (type: ZCX-2, 100-200 mesh) was added to the filtrate. The solvent was removed in vacuo at a rotary evaporator bath temperature of 35 °C. The silica gel containing adsorbed 13-1 was placed on top of a silica gel column (50 mm OD, 200 g silica gel, type: ZCX-2, 100-200 mesh). The column was eluted with a gradient of petroleum ether: EtOAc from 100:0 to 97:3 in 200 mL fractions. TLC analysis showed that fractions containing 13-1 were combined and concentrated in vacuo to give 13-1 (12.8 g, 13.2 mmol, 47%) as a pale yellow viscous oil. 1 H-NMR (300MHz, DMSO-d6): δ5.18(br m, 2H), 4.26(m, 4H), 4.13(m, 4), 2.29(t, J=7.2Hz, 8H), 1.40-1.60(14H), 1.38(s, 9H), 1.18-1.32(42H), 0.86(m, 12H).

[0408] 13-2: Synthesis of ((3,3'-azanediylbis(propanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetranonanoic acid hydrochloride [ka]

[0409] To a solution of 13-1 (12.8 g, 13.2 mmol) in EtOAc (75 mL) cooled in an ice-water bath under nitrogen was added HCl in EtOAc (2 M, 80 mL, 0.160 mol) at a rate such that the internal temperature remained between 0 and 10 °C. After the addition was complete, the mixture was allowed to warm to room temperature and stirred for 14 h. Concentration in vacuo gave the HCl salt 13-2 (8.1 g, 9.37 mmol, 71%) as a light yellow, viscous oil. 1 H-NMR (300MHz, DMSO-d6): δ5.19(br m, 2H), 4.25(m, 4H), 4.14(m, 4H), 2.73(br t, J=6.9Hz, 4H), 2.40(br t, J=6.9Hz, 4H), 2.29(br t, J=7.2Hz, 8H), 1.38-1.58(8H), 1.16-1.32(40H), 0.87(m, 12H).

[0410] Lipid 13: Synthesis of 2-((3-((((3-(dimethylamino)propyl)thio)carbonyl)(3-((1-(nonanoyloxy)-3-(octanoyloxy)propan-2-yl)oxy)-3-oxopropyl)amino)propanoyl)oxy)propane-1,3-diyl dinonanoate [ka]

[0411] To a solution of 13-2 (8.1 g, 9.37 mmol) in CHCl (280 mL) cooled in an ice-water bath under nitrogen, triphosgene (2.77 g, 9.33 mmol) was added in one portion, followed by the dropwise addition of pyridine (3.68 g, 46.52 mmol). After the addition was complete, the reaction mixture was allowed to warm to room temperature and stirred for 4 h. The solvent was removed in vacuo (bath temperature 25 °C), and the residue was dissolved in pyridine (160 mL). The solution was cooled in an ice-water bath under nitrogen, and 3-dimethylamino-propane-1-thiol (1.32 g, 11.1 mmol) was added dropwise over 10 min. After the addition was complete, the mixture was allowed to warm to room temperature and stirred for 14 h. The solvent was removed in vacuo, and the residue was dissolved in CHCl (200 mL). The solution was washed with 10% aqueous citric acid (100 mL), 5% aqueous NaHCO (100 mL), brine (100 mL), and dried over anhydrous NaSO. The drying agent was removed by filtration through a sintered glass funnel, and silica gel (16 g, type ZCX02, 100-200 mesh) was added to the filtrate. The solvent was removed in vacuo (bath temperature 25 °C), and the silica gel containing adsorbed 13 was placed on top of a silica gel column (81 g silica gel, type ZCX02, 100-200 mesh) using a CombiFlash. The column was eluted with a gradient of 100:0 to 90:10 n-heptane / acetone in 100 mL fractions. The appropriate fractions were identified using TLC, combined, and concentrated in vacuo to give 13 (1.9 g), which was judged to be 90% pure by HPLC. 13 was purified by reverse-phase preparative HPLC (C). 18 Further purification was performed with A: water with 0.1% formic acid, B: acetonitrile, gradient 41% B to 58% B over 8 min. The appropriate fractions were combined and concentrated in vacuo to give 13 (1.01 g, 1.00 mmol, 10.6%) as a clear, pale yellow, viscous oil. HPLC purity: 99.65%; ES-MS (+ mode): calculated 1014.68, found 1015.95 (M+H). + ); 1H-NMR (300MHz, CDCl3): δ5.26(m, 2H), 4.33(m, 4H), 4.16(m, 4H), 3.68(br t, J=7.2Hz, 4H), 2.94(t, J=7.2Hz, 2H), 2.68(br t, J=7.2Hz, 4H), 2.20-2.40(16H), 1,81(m, 2H), 1.52-1.66(8H), 1.16-1.38(40H), 0.90(m, 12H).

[0412] Example 14. Synthesis of lipid 14 [ka] Overall Scheme: [ka]

[0413] 14-1: Synthesis of 2-((benzyloxy)methyl)propane-1,3-diyl dinonanoate [ka]

[0414] 2-[(phenylmethoxy)methyl]-1,3-propanediol (Bioorg.Med.Chem.2017, 25, 4008-4030; 25.0 g, 0.127 mol) was dissolved in CHCl3 (500 mL) and cooled in an ice-water bath under nitrogen. To this solution, nonyl chloride (56.5 g, 0.318 mol) was added in one portion, followed by the dropwise addition of pyridine (40.0 g, 0.508 mol) over 40 minutes. The reaction mixture was allowed to warm to room temperature and then stirred for 14 hours. The cloudy mixture was filtered through a bed of Celite, and the filtrate was washed with 5% aqueous NaHCO3 (250 mL), brine (250 mL), and dried over anhydrous Na2SO4. The drying agent was removed by filtration through a sintered glass funnel, and silica gel (150 g, type: ZCX-2, 100-200 mesh) was added to the filtrate. The solvent was removed in vacuo (bath temperature <35 °C), and the silica gel containing adsorbed 14-1 was added to the top of a Combiflash column (600 g, type: ZCX-2, 100-200 mesh, packed with 99:1 petroleum ether: EtOAc and eluted with 99:1 to 98:2 petroleum ether: EtOAc in 1000 mL fractions). The appropriate fractions were determined by TLC, combined, and concentrated in vacuo to give 14-1 (60.0 g, 0.124 mol, 98%) as a colorless oil. 1 H-NMR (300MHz, CDCl3): δ7.28-7.40(5H), 4.52(s, 2H), 4.19(m, 4H), 3.52(d, J=5.7Hz , 2H), 2.36(m, 1H), 2.29(t, J=7.5Hz, 4H), 1.59(m, 4H), 1.23-1.40(18H), 0.92(m, 6H).

[0415] 14-2: Synthesis of 2-(hydroxymethyl)propane-1,3-diyl dinonanoate [ka]

[0416] In an 11.0 L pressure vessel, a solution of 14-1 (60.0 g, 0.124 mol) in MeOH (600 mL) was flushed with nitrogen 3X, then 10% Pd / C (18.0 g) was added to the vessel and the mixture was placed under hydrogen pressure (3 atm). The mixture was stirred under hydrogen for 14 h, then the vessel was vented and the solution was sparged with nitrogen. The Pd / C was removed by filtration through a bed of Celite, the filter cake was rinsed with MeOH (200 mL), and the combined filtrates were concentrated in vacuo to give 14-2 (32.0 g, 82.8 mmol, 67%) as a clear, colorless oil. 1 H-NMR (300MHz, CDCl3): δ4.16(m, 4H), 3.63(d, J=5.7Hz, 2H), 2.33(t, J=7.5Hz, 4H), 2.20(m, 1H), 1.63(m, 4H), 1.25-1.39(20H), 0.90(m, 6H).

[0417] 14-3: Synthesis of (((3,3'-((tert-butoxycarbonyl)azanediyl)bis(propanoyl))-bis(oxy))bis(methylene))bis(propane-2,1,3-triyl)tetranonanoate [ka]

[0418] A solution of 1-5 (5.00 g, 19.1 mmol) in CHCl (75 mL) was cooled in an ice-water bath under nitrogen, and 14-2 (14.8 g, 38.3 mmol), DMAP (2.34 g, 19.1 mmol), and EDCl (14.7 g, 76.7 mmol) were added in this order. The mixture was allowed to warm to room temperature and then stirred for 14 h. The reaction mixture was poured into 10% aqueous citric acid (125 mL). The organic phase was separated, washed with brine (125 mL), and dried over anhydrous NaSO. Filtration through a sintered glass funnel and concentration in vacuo afforded crude 14-3 (14.0 g, 14.0 mmol, 73%), which was used without further purification.

[0419] 14-4: Synthesis of (((3,3'-azanediylbis(propanoyl))bis(oxy))bis(methylene))-bis(propane-2,1,3-triyl)tetranonanoate [ka]

[0420] To a solution of 14-3 (14.0 g, crude, estimated 14.0 mmol) in CHCl (80 mL) cooled in an ice-water bath under nitrogen, 4.0 M HCl in dioxane (35 mL, 0.140 mol) was added at a rate such that the internal temperature was maintained between 0 and 10 °C. After the addition was complete, the mixture was allowed to stir for 30 min, then warmed to room temperature and stirred for 16 h. The reaction mixture was poured into saturated aqueous NaHCO (100 mL), and the organic phase was separated, washed with saturated aqueous NaHCO (100 mL), brine (100 mL), and dried over anhydrous NaSO. Filtration and concentration under vacuum afforded crude 14-4 as a viscous yellow oil, which was dissolved in CHCl (200 mL) and silica gel (20 g, type ZCX-2, 100-200 mesh) was added. Concentration in vacuo afforded the silica gel containing adsorbed 14-4, which was placed on top of a column of silica gel (100 g, type ZCX-2, 100-200 mesh) eluted with a gradient of 67:33 to 50:50 using a Combiflash. The appropriate fractions were identified by TLC, combined, and concentrated in vacuo to give 14-4 (5.20 g, 5.79 mmol, 30% over two steps) as a clear, pale yellow, viscous oil. 1 H-NMR (300MHz, CDCl3): δ4.12-4.18(12H), 2.93(t, J=6.6Hz, 4H).2.57(t, J=6.6Hz, 4 H), 2.42(m, 2H), 2.33(t, J=7.5Hz, 8H), 1.61(m, 8H), 1.22-1.40(40H), 0.90(m, 12H).

[0421] Synthesis of lipid 14 [ka]

[0422] To a solution of 14-4 (5.20 g, 5.79 mmol) in CHCl (175 mL) cooled in an ice-water bath under nitrogen, triphosgene (1.72 g, 5.75 mmol) was added in one portion, followed by pyridine (2.29 g, 28.9 mmol, 2.34 mL) at a rate such that the temperature remained between 0 and 5 °C. After the addition was complete, the mixture was stirred for 30 min, then warmed to room temperature and stirred for 4 h. The solvent was removed in vacuo, and the residue was dissolved in pyridine (100 mL), and the solution was cooled in an ice-water bath under nitrogen. To this stirred solution, 3-dimethylamino-propane-1-thiol (0.82 g, 6.88 mmol) was added dropwise over 10 min. After the addition was complete, the mixture was stirred for 30 min, then warmed to room temperature and stirred for 14 h. The solvent was removed in vacuo, and the residue was dissolved in CHCl (200 mL) and washed with 10% aqueous citric acid (2 × 100 mL), saturated aqueous NaHCO (2 × 100 mL), brine (2 × 100 mL), and dried over NaSO. Filtration and concentration in vacuo gave crude 14 as a viscous yellow oil, which was purified by reverse-phase Combiflash chromatography (A: water + 0.1% CFCOH, B: acetonitrile; gradient 60% B to 80% B over 20 min, then 100% B for 20 min). The appropriate fractions were combined and concentrated in vacuo to give 14 (1.12 g, 1.07 mmol, 18.5%) as a clear, pale yellow oil. 1 H-NMR (300MHz, CDCl3): δ4.12-4.20(12H), 3.65(brt, J=7.2Hz, 4H), 3.11(m, 2H), 2.93(t, J=6.9Hz, 2H), 2. 82(s, 6H), 2.65(brm, 4H), 2.45(m, 4H), 2.31(t, J=7.5Hz, 8H), 1.61(m, 8H), 1.18-1.35(40H, 0.88(m, 12H).

[0423] Example 15. Synthesis of lipid 15: ((4,4'-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(2-(4-methylcyclohexyl)acetate) [ka] Overall Scheme: [ka]

[0424] 15-1: Synthesis of ethyl 2-(4-methylcyclohexylidene)acetate [ka]

[0425] To a suspension of NaH (12.5 g, 60% in oil) cooled in an ice-water bath under nitrogen and washed with a solution of hexane (2 × 25 mL, 0.312 mol) in anhydrous THF (600 mL), triethyl phosphonate (70.0 g, 0.312 mol) was added dropwise over 30 min. The mixture was stirred in the ice-water bath for 2 h, and then 4-methyl-cyclohexanone (35.0 g, 0.312 mol) was added over 30 min. The mixture was stirred for 30 min, then allowed to warm to room temperature and stirred for 14 h. The mixture was poured into water (1.2 L) and EtOAc (600 mL). The organic phase was separated, silica gel (200 g, type: ZCX-2, m100-200 mesh) was added, and the solvent was removed under vacuum (bath temperature <35 °C) to give the silica gel containing the adsorbed crude 1. The silica gel was placed on top of a column of silica gel (1000 g, type: ZCX-2, m100-200 mesh) eluted with a gradient of petroleum ether: EtOAc 100:0 to 95:5, and 1000 mL fractions were collected using a CombiFlash. Appropriate fractions were identified by TLC, combined, and concentrated under vacuum to give 15-1 (45.0 g, 0.247 mol, 79%) as a clear, colorless oil. LC-MS (+ mode): RT 1.804, 183.2 (M+H) + ); 1 H-NMR (300MHz, CDCl3): δ5.61(s, 1H), 4.14(q, J=7.2Hz, 2H), 3.75(m, 1H), 2.14-2.30(2H), 1.90 (m, 1H), 1.80 (m, 2H), 1.62 (m, 1H), 1.28 (t, J=7.2Hz, 3H), 1.00-1.15 (2H), 0.82 (d, J=9.0Hz, 3H).

[0426] 15-2: Synthesis of ethyl 2-(4-methylcyclohexyl)acetate [ka]

[0427] To ester 15-1 (45.0 g, 0.247 mol) dissolved in EtOH (450 mL) at room temperature under nitrogen, 10% Pd / C (13.5 g) was added. Hydrogen was then bubbled through the reaction mixture for 16 h. The solvent was then sparged with nitrogen for 1 h, the catalyst was removed by filtration through a bed of Celite, and the filter cake was rinsed with EtOH (450 mL). The combined filtrate was concentrated in vacuo to give 15-2 (35.0 g, 0.190 mol, 77%) as a pale yellow oil. 1 H-NMR (300MHz, CDCl3): δ4.14(q, J=7.2Hz, 2H), 2.25-2.40(2H), 2.18(m, 1H), 1.52-1.78(3H), 1.28(t, J=7.2Hz, 3H), 0.78-1.03(9H).

[0428] 15-3: Synthesis of 2-(4-methylcyclohexyl)acetic acid [ka]

[0429] To a solution of 15-2 (35.0 g, 0.190 mol) in THF:HO (350 mL, 50:50) at room temperature under nitrogen, solid NaOH (84.0 g, 2.10 mol) was added over 30 min. The mixture was stirred for 16 h and then concentrated in vacuo to remove THF. The aqueous solution was then adjusted to pH 3.0 by the addition of 3 N aqueous HCl. The reaction mixture was extracted with EtOAc (350 mL), and the organic phase was dried over NaSO. Filtration and concentration in vacuo gave 15-3 (25.0 g, 0.160 mol, 84%) as a white solid.

[0430] 15-4: Synthesis of 2-(4-methylcyclohexyl)acetyl chloride [ka]

[0431] To a solution of 15-3 (25.0 g, 0.160 mol) in CHCl (250 mL) containing DMF (1.0 mL) cooled in an ice-water bath under nitrogen, oxalyl chloride (40.7 g, 0.321 mol) was added dropwise over 20 min. After the addition was complete, the mixture was stirred for 30 min, then warmed to room temperature and stirred for 14 h. Concentration in vacuo with a bath temperature <30 °C gave 15-4 (25.2 g, 0.144 mol, 90%) as a clear, colorless oil. 1 H-NMR (300MHz, CDCl3): δ2.75 (m, 2H), 1.30-1.64 (5H), 1.18-1.28 (2H), 0.80-1.05 (6H).

[0432] 15-5: Synthesis of 2-oxopropane-1,3-diyl bis(2-(4-methylcyclohexyl)acetate) [ka]

[0433] To a solution of 1,3-dihydroxyacetone (5.90 g, 65.5 mmol) in CHCl (500 mL) at room temperature under nitrogen, DMAP (2.40 g, 19.6 mmol) and pyridine (11.4 g, 0.144 mol) were added, followed by dropwise addition of 15-4 (25.2 g, 0.144 mol) over 30 min. The mixture was stirred at room temperature for 16 h and then poured into water (400 mL). The organic phase was separated, washed with water (400 mL), brine (400 mL), and dried over NaSO. The desiccant was removed by filtration, and silica gel (60 g, type: ZCX-2, 100-200 mesh) was added to the filtrate. The solvent was removed under vacuum to give the silica gel containing the crude 15-5 adsorbed. The silica gel was placed on top of a column of silica gel (300 g, type: ZCX-2, 100-200 mesh) and eluted using a Combiflash with a gradient of 100:0 to 90:10 petroleum ether: EtOAc in 500 mL fractions. Appropriate fractions were identified using TLC, combined, and concentrated in vacuo to give 15-5 (18.0 g, 49.1 mmol, 75%) as a clear, colorless oil. LC-MS (+-mode): RT 0.36 min, 367.3 (M+H) + ); 1 H-NMR (300MHz, CDCl3): δ4.76 (s, 4H), 2.40 (d, J=6.0Hz, 1H), 2.00-2.30 (4H), 0.75-1.10 (25H).

[0434] 15-6: Synthesis of 2-hydroxypropane-1,3-diyl bis(2-(4-methylcyclohexyl)acetate) [ka]

[0435] To a solution of 15-5 (18.0 g, 49.1 mmol) in THF under nitrogen, cooled in an ice-water bath, was added CHCOH (25.0 g, 0.42 mol). To this stirred solution, NaBHCN (12.9 g, 0.205 mol) was added portionwise over 20 min. After the addition was complete, the mixture was stirred for 30 min, then warmed to room temperature and stirred for 2 h. The mixture was poured into water (100 mL) and extracted with EtOAc (3 × 100 mL). The combined organic phase was dried over NaSO, the drying agent was removed by filtration, and silica gel (50 g, type: ZCX-2, 100-200 mesh) was added to the filtrate. Concentration under vacuum gave the silica gel containing the crude adsorbed 15-6, which was placed on a silica gel column (250 g, Type: ZCX-2, 100-200 mesh) eluted with a gradient of 100:0 to 92:8 petroleum ether: EtOAc, and 500 mL fractions were collected using CombiFlash. Appropriate fractions were identified using TLC, then combined and concentrated under vacuum to give 15-6 (17.0 g, 46.1 mmol, 94%) as a clear, colorless oil. LC-MS (+-mode): 1.47 min RT, 391.2 (M+Na+); 1 H-NMR (300MHz, CDCl3): δ5.30(s, 1H), 4.00-4.20(4H), 2.30(m, 1H), 2.18(m, 2H), 1.90-2.05(2H), 1.18-1.75(14H), 0.80-1.00(12H).

[0436] 15-7: Synthesis of ((4,4'-((tert-butoxycarbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(2-(4-methylcyclohexyl)acetate) [ka]

[0437] To a solution of 1-5 (6.10 g, 21.1 mmol) in CHCl (120 mL) cooled in an ice-water bath under nitrogen, DMAP (1.00 g, 8.18 mmol) and 15-6 (17.0 g, 46.1 mmol) were added in one portion, followed by EDCl (9.70 g, 50.6 mmol) in several portions over 30 min. After the addition was complete, the mixture was stirred for 30 min, then warmed to room temperature and stirred for 16 h. The mixture was poured into brine (120 mL), and the organic phase was separated, washed with brine (120 mL), and dried over NaSO. The drying agent was removed by filtration, and silica gel (60 g, type: ZCX-2, 100-200 mesh) was added to the filtrate. Concentration in vacuo gave the silica gel containing the crude adsorbed 15-7, which was placed on top of a silica gel column (300 g, Type: ZCX-2, 100-200 mesh) eluted with a gradient of 100:0 to 70:30 petroleum ether: EtOAc, and 500 mL fractions were collected using CombiFlash. Appropriate fractions were identified using TLC, then combined and concentrated in vacuo to give 15-7β (10.0 g, 10.1 mmol, 48%) as a clear, colorless oil. LC-MS (+-mode): 1.654 min, 1012.9 (M+Na+) at room temperature. 1 H-NMR (300MHz, CDCl3): δ5.25(m, 2H), 4.31(m, 4H), 4.18(m, 4H), 3.20(brm, 4H), 2.31-2.40(6H), 2.15-2.23(5H ), 2.00(m, 1H), 1.78(m, 4H), 1.50-1.75(14H), 1.30-1.50(3H), 1.35(s, 9H), 1.18-1.30(9H), 0.77-1.00(26H).

[0438] 15-8: Synthesis of bis(4-((1,3-bis(2-(4-methylcyclohexyl)acetoxy)propan-2-yl)oxy)-4-oxobutyl)ammonium trifluoroacetate [ka]

[0439] To a solution of 15-7 (10.0 g, 10.1 mmol) in CHCl (40 mL) cooled in an ice-water bath under nitrogen, CFCOH (5.00 g, 43.9 mmol, 3.36 mL) was added in one portion. After the addition, the mixture was stirred for 30 min, then warmed to room temperature and stirred for 4 h. The mixture was concentrated in vacuo to give crude 15-8 (5.60 g, 5.58 mmol, 55%) as a colorless viscous oil. LC-MS (+-mode): RT 0.608 min, 890.6 (M+H) + ); 1 H-NMR (300MHz, CDCl3): δ11.10(brs, 2H), 5.26(m, 2H), 4.45(m, 4H), 4.18(m, 4H), 3.18(m, 4H), 2.40(t, J= 6.5Hz, 4H), 2.31(t, J=7.2Hz, 2H), 2.16-2.25(6H), 1.82-2.08(10H), 1.14-1.67(22H), 0.75-1.00(24H).

[0440] 15-9: Synthesis of ((4,4'-((1H-imidazole-1-carbonyl)azanediyl)bis(butanoyl))-bis(oxy))bis(propane-2,1,3-triyl)tetrakis(2-(4-methylcyclohexyl)acetate) [ka]

[0441] To a solution of 15-8 (5.60 g, 5.58 mmol) in CHCl (100 mL) under nitrogen, EtN (1.30 g, 12.8 mmol, 1.21 mL) was added, followed by carbonyldiimidazole (CDI, 2.00 g, 12.33 mmol). The mixture was stirred at room temperature for 14 h and then diluted with n-heptane (100 mL). The solution was washed with water (3 × 100 mL), and the organic phase was dried over NaSO. Filtration and concentration under vacuum gave crude 15-9 (4.00 g, 4.06 mmol, 73%) as a viscous yellow oil. LC-MS (+ mode): RT 0.645 min, 984.9 (M+H) + ); 1H-NMR (300MHz, CDCl3): δ7.96(m, 1H), 7.26(m, 1H), 7.13(m, 1H), 4.32(m, 4H), 4.16(m, 4H), 3.41(m, 4H), 2.21-2.32(6H), 2.08-2.16(6H), 1.81-2.00(6H), 1.50-1.72(14H), 1.14-1.50(12H), 0.75-1.00(24H).

[0442] Lipid 15: Synthesis of ((4,4'-((((3-(dimethylamino)propyl)thio)carbonyl)-azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(2-(4-methylcyclohexyl)acetate) [ka]

[0443] To a solution of 15-9 (4.00 g, 4.06 mmol) in CHCl (80 mL) cooled in an ice-water bath under nitrogen, CFSOCH (0.70 g, 4.27 mmol) was added over 5 min. After the addition was complete, the mixture was stirred for 1 h, and then EtN (0.80 g, 7.90 mmol, 1.10 mL) and 3-dimethylamino-propane-1-thiol HCl salt (0.76 g, 4.87 mmol) were added sequentially in one portion. After the addition was complete, the mixture was stirred for 30 min. The mixture was then warmed to room temperature and stirred for 16 h. Silica gel (15 g, Type: ZCX-2, 100-200 mesh) was added to the solution. Concentration under vacuum gave the silica gel containing the adsorbed crude 15, which was placed on top of a silica gel column (75 g, type: ZCX-2, 100-200 mesh) eluted with a gradient of 100:0 to 96:4 CHCl:MeOH, and 300 mL fractions were collected using a CombiFlash. Appropriate fractions were identified using TLC, then combined and concentrated under vacuum to give lipid 15 (1.70 g), which was further purified by SFC (column: Torus 2-PIC, 4.6 x 100 mm, 5 μm, mobile phase B: i-PrOH, flow rate: 4 mL / min, gradient: isocratic 10% B, wavelength: 220 nM) to give lipid 15 (1.00 g, 0.965 mmol, 23.8%) as a clear, light yellow oil after concentration under vacuum. ES-MS: 1035.7 (M+H). + ); HPLC purity: 98.47%, 1 H-NMR (300MHz, CDCl3): δ5.24(m, 2H), 4.31(m, 4H), 4.14(m, 4H), 3.38(brm, 4H), 2.92(t , J=7.3Hz, 2H), 2.11-2.52(18H), 1.56-2.11(23H), 1.12-1.56(14H), 0.75-1.11(23H).

[0444] Example 16. Synthesis of lipid 16: ((4,4'-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(4-ethylcyclohexane-1-carboxylate) [ka] Overall Scheme: [ka]

[0445] 16-1: Synthesis of 4-ethylcyclohexane-1-carbonyl chloride [ka]

[0446] To a solution of 4-ethyl-cyclohexanecarboxylic acid (22.5 g, 0.144 mol) in CHCl (225 mL) cooled in an ice-water bath under nitrogen, DMF (0.5 mL) was added followed by oxalyl chloride (36.6 g, 0.288 mol) over 25 min. After the addition was complete, the mixture was stirred for 30 min, then warmed to room temperature and stirred for 16 h. Concentration in vacuo afforded crude 16-1 (22.6 g, 0.129 mol, 90%) as a clear, colorless oil. 1 H-NMR (300MHz, CDCl3): δ2.62 (m, 1H), 2.62 (m, 2H), 1.37-1.53 ​​(2H), 1.10-1.27 (4H), 0.80-1.00 (6H).

[0447] 16-2: Synthesis of 2-oxopropane-1,3-diyl bis(4-ethylcyclohexane-1-carboxylate) [ka]

[0448] To a solution of dihydroxyacetone (5.30 g, 58.8 mmol) in CHCl (500 mL) cooled in an ice-water bath under nitrogen, DMAP (0.36 g, 2.94 mmol) and pyridine (10.24 g, 0.129 mol) were added in one portion, followed by 16-1 (22.6 g, 0.129 mol) over 10 min. After the addition was complete, the mixture was stirred for 30 min, then warmed to room temperature and stirred for 14 h. The solvent was removed in vacuo, and the residue was dissolved in CHCl (100 mL). Silica gel (25 g, type ZCX-2, 100-200 mesh) was added to the solution, and the solvent was then removed in vacuo to yield silica gel impregnated with adsorbed 16-2. The silica gel was placed on top of a silica gel column (250 g, type ZCX-2, 100-200 mesh) and purified using a Combiflash by eluting with a petroleum ether: EtOAc gradient from 100:0 to 90:10, collecting 300 mL fractions. The appropriate fractions were identified by TLC, combined, and concentrated in vacuo to give 16-2 (20.4 g, 55.6 mmol, 94%) as a clear, colorless oil. LC-MS (+ mode): RT 0.450 min, 367.3 (M+H) + ); 1 H-NMR (300MHz, DMSO-d6): δ4.83(s, 4H), 2.27(m, 2H), 2.15(m, 1H), 1.77-1.90(5H), 1.48(m, 4H), 1.00-1.33(10H), 0.80-0.95(8H).

[0449] 16-3: Synthesis of 2-hydroxypropane-1,3-diyl bis(4-ethylcyclohexane-1-carboxylate) [ka]

[0450] To a solution of 16-2 (20.4 g, 55.66 mmol) in THF (400 mL) cooled in an ice-water bath, HOAc (33.4 g, 0.556 mol) was added in one portion, followed by NaBHCN (17.5 g, 0.278 mol) in several portions over 30 min. After the addition was complete, the mixture was stirred for 30 min, then warmed to room temperature and stirred for 2 h. The mixture was poured into water (2.0 L), and the resulting solution was extracted with EtOAc (3 × 200 mL). The combined organic phase was dried (NaSO), filtered, and concentrated in vacuo, and the residue was dissolved in CHCl (100 mL). Silica gel (50 g, type ZCX-2, 100–200 mesh) was added to the solution of crude 16-3, and the solvent was removed in vacuo to give the silica gel containing adsorbed crude 16-3. Crude 16-3 was purified by placing silica gel on a silica gel column (250 g, type ZCX-2, 100-200 mesh) and using a Combiflash to elute with a petroleum ether: EtOAc gradient from 100:0 to 92:8, collecting 300 mL fractions. Appropriate fractions were identified by TLC, combined, and concentrated in vacuo to give 16-3 (16.0 g, 43.42 mmol, 78%) as a clear, colorless oil. LC-MS (+ mode): 1.463 min, 391.3 (M+Na) + ); 1 H-NMR (300MHz, CDCl6): δ4.00-4.25(5H), 2.32(brs, 1H), 2.25(m, 2H), 1.91(m, 4H), 1.77(m, 4H), 1.37(m, 4H), 1.00-1.25(8H), 0.75-0.95(8H).

[0451] 16-4: Synthesis of ((4,4'-((tert-butoxycarbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(4-ethylcyclohexane-1-carboxylate) [ka]

[0452] Under nitrogen, to a solution of 1-5 (5.42 g, 18.73 mmol) in CHCl (100 mL) were added DMAP (0.91 g, 7.49 mmol) and 16-3 (15.2 g, 41.21 mmol) in that order. The resulting solution was cooled in an ice-water bath, and then EDCl (8.60 g, 44.96 mmol) was added in five portions over 30 min. After the addition was complete, the mixture was stirred for 30 min, and then the solution was warmed to room temperature and stirred for 14 h. The mixture was poured into brine (100 mL), and the organic phase was separated and dried over NaSO. Filtration gave a solution of crude 16-4, to which silica gel (15 g, type ZCX-2, 100-200 mesh) was added, and the solvent was removed under vacuum to give the silica gel containing adsorbed crude 16-4. The silica gel was placed on top of a silica gel column (75 g, type ZCX-2, 100-200 mesh) and purified using a Combiflash by eluting with a petroleum ether: EtOAc gradient from 100:0 to 80:20, collecting 300 mL fractions. The appropriate fractions were identified by TLC, combined, and concentrated in vacuo to give 16-4 (12.98 g, 13.10 mmol, 70%) as a clear, colorless oil. LC-MS (+ mode): RT 1.703 min, 890.6 (M-Boc + 2H + ); 1 H-NMR (300MHz, CDCl3): δ5.50 (m, 2H), 4.32 (m, 4H), 4.21 (m, 4H), 3.20 (brm, 4H), 2.16-2 .35(7H), 1.82(m, 8H), 1.75-1.80(9H), 1.38(s, 9H), 1.00-1.38(24H), 0.75-0.90(20H).

[0453] 16-5: Synthesis of bis(4-((1,3-bis((4-ethylcyclohexane-1-carbonyl)oxy)propan-2-yl)oxy)-4-oxobutyl)ammonium trifluoroacetate [ka]

[0454] To a solution of 16-4 (12.98 g, 13.11 mmol) in CHCl (50 mL) cooled in an ice-water bath under nitrogen, CFCOH (7.47 g, 65.54 mmol) was added over 10 min. After the addition was complete, the mixture was stirred for 15 min, then warmed to room temperature and stirred for 16 h. Concentration in vacuo gave crude 16-5 (14.82 g) as a colorless oil. LC-MS (+ mode): RT 0.677 min, 890.6 (M+H) + ); 1 H-NMR (300MHz, CDCl3): δ5.16 (m, 2H), 4.41 (m, 4H), 4.11 (m, 4H), 3.19 (brm, 4H), 2.42(m, 4H), 2.25(m, 4H), 1.75-2.20(18H), 1.00-1.50(22H), 0.75-0.95(20H).

[0455] 16-6: Synthesis of ((4,4'-((1H-imidazole-1-carbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(4-ethylcyclohexane-1-carboxylate) [ka]

[0456] A solution of 16-5 (14.82 g, crude) was dissolved in CHCl (300 mL) and cooled in an ice-water bath under nitrogen. To this solution, EtN (6.74 g, 66.59 mmol) and carbonyldiimidazole (5.39 g, 33.30 mmol) were added sequentially. After the addition was complete, the mixture was stirred for 30 min, and then the solution was warmed to room temperature and stirred for 3 h. The solvent was removed in vacuo, and the residue was dissolved in n-heptane (300 mL), and water was added to the flask. With vigorous stirring, the pH of the aqueous phase was adjusted to approximately 6.0 by adding 3% aqueous citric acid. After the target pH was achieved, the organic phase was separated and dried over NaSO. Filtration and concentration in vacuo gave crude 16-6 (13.77), which was used in the next step without further purification. LC-MS (+ mode): Room temperature, 0.773 min, 984.5 (M+H + );1 H-NMR (300MHz, CDCl3): δ7.99(m, 1H), 7.27(m, 1H), 7.12(m, 1H), 5.20(m, 2H), 4.31(m, 4H), 4.18(m, 4H), 3 .41(m, 4H), 2.31(m, 4H), 2.23(m, 4H), 1.82-2.00(12H), 1.79(m, 8H), 1.00-1.40(20H), 0.75-0.92(20H).

[0457] Lipid 16: Synthesis of ((4,4'-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(4-ethylcyclohexane-1-carboxylate) [ka]

[0458] A solution of 16-6 (13.77 g, crude) in CHCl (150 mL) was cooled in an ice-water bath under nitrogen. To this cooled solution, methyl trifluoromethanesulfonate (2.52 g, 20.99 mmol) was added over 10 min. The mixture was stirred in the ice-water bath for 1 h, and then EtN (4.24 g, 41.97 mmol) was added over 5 min, followed by 3-dimethylamino-propane-1-thiol (2.49 g, 20.99 mmol) over 5 min. The mixture was stirred for 30 min, then warmed to room temperature, and stirred for 8 h. The mixture was concentrated in vacuo, and the residue was dissolved in CHCl (100 mL). Silica gel (30 g, type ZCX-2, 100-200 mesh) was added, and the solvent was removed in vacuo to give the silica gel containing adsorbed crude 16. Lipid 16 was purified by placing silica gel on a silica gel column (80 g, type ZCX-2, 100-200 mesh) and using a CombiFlash to elute with a gradient of CHCl:MeOH from 100:0 to 96:4, collecting 300 mL fractions. Appropriate fractions were identified by TLC, combined, and concentrated in vacuo to give lipid 16 (10.14 g, 9.797 mmol, 75% yield over three steps) as a clear, light yellow oil. ES-MS: 1036.0 (M+H) + );HPLC purity 94.24%; 1 H-NMR (300MHz, CDCl3): δ5.24(m, 2H), 4.30(dd, J=11.9, 4.6Hz, 4H), 4.14(dd, J=11.9, 5.7Hz, 4H), 3.38(brm, 4H), 2.94(t, J=7.1Hz, 2H), 2.46(s, 6H), 2.34(brm, 4H), 2.20(m, 4H), 1.72-1.91(20H), 1.37(m, 8H), 1.08-1.22(16H), 0.80-0.91(20H).

[0459] Example 17. Synthesis of lipid 17: ((4,4'-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexyl-2-methylpropanoate) [ka] Overall Scheme: [ka] [ka]

[0460] 17-1: Synthesis of ethyl (E)-3-cyclohexyl-2-methylacrylate [ka]

[0461] A 2 L four-neck round-bottom flask purged and maintained with an inert nitrogen atmosphere was charged with NaH (60%, 21.42 g, 0.534 mol, 1.0 equiv.) and THF (822 mL). Ethyl 2-(diethoxyphosphoryl)propanoate (127.2 g, 0.536 mol, 1.0 equiv.) was then added dropwise over 30 min at room temperature, and the mixture was stirred for 1.5 h after the addition was complete. A solution of cyclohexanecarboxaldehyde (60.0 g, 0.536 mol, 1.0 equiv.) in THF (318 mL) was added dropwise over 30 min, and the mixture was stirred at room temperature for 2 h. The reaction was quenched with saturated aqueous NH4Cl (1.5 L) and extracted with MTBE (2 × 0.75 L). The combined organic layers were washed with HO (0.75 L), brine (0.75 L, 12.5 V), dried over anhydrous NaSO, filtered, and concentrated in vacuo to give 105 g of 17-1 (crude) as a yellow oil, which was used in the next step without further purification.

[0462] 17-2: Synthesis of ethyl 3-cyclohexyl-2-methylpropanoate [ka]

[0463] A solution of 17-1 (120.0 g, 1.0 equiv.) in EtOH (1.2 L) was placed in a nitrogen-flushed 2 L round-bottom flask. Then, 10 wt.% Pd / C (36.0 g, 30% w / w) was added in one portion. The mixture was then stirred at room temperature under an H atmosphere for 4 h. It was filtered, and the filter cake was washed with CHCl (1.2 L). The filtrate was concentrated under vacuum to give crude 17-2. Crude 17-2 was dissolved in CHCl (1 L), and 200 g of silica gel (type: ZCX-2, 100-200 mesh, 1.67 w / w) was added. The solvent was removed under vacuum while maintaining the temperature below 35 °C. A column was packed with 1 kg of silica gel (type: ZCX-2, 100-200 mesh, 8.33 w / w), followed by the dried silica gel prepared in the last step, which adsorbed the reaction mixture. The product was purified using Combiflash by eluting with a gradient of 100:0 to 95:5 petroleum ether / EtOAc, collecting 1000 mL fractions. Samples were taken for TLC analysis, and the appropriate products were combined to give 94 g (76% yield) of 17-2 as a yellow oil.

[0464] 17-3: Synthesis of 3-cyclohexyl-2-methylpropanoic acid [ka]

[0465] A solution of 17-2 (57.0 g, 0.288 mol, 1.0 equiv) in EtOH (285 mL) was placed in a 2 L three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere at room temperature. A solution of NaOH (17.3 g, 0.433 mol, 1.5 equiv) in HO (285 mL) was then added in one portion. The resulting solution was then warmed to 70 °C and stirred for 3 h. The reaction was cooled to room temperature and extracted with n-heptane (2 × 200 mL). The HO layer was adjusted to pH = 2 with aqueous HCl (12 mol / L) and then extracted with MTBE (2 × 300 mL). The combined organic layers were washed with HO (2 × 150 mL), dried over anhydrous NaSO, filtered, and concentrated in vacuo. This afforded 47 g (0.276 mol, 96% yield) of 17-3 as a colorless oil. ELSD A: Water / 5mM NH4 + HCO3 - B: CH3CN 90:10 to 10:9 in 2 min, 1 min hold): Room temperature 0.56 min, m / z (calculated) 170.1, (observed) 169.13 (M−H).

[0466] 17-4: Synthesis of 2-oxopropane-1,3-diyl bis(3-cyclohexyl-2-methylpropanoate) [ka]

[0467] A solution of 1,3-dihydroxyacetone (17.5 g, 0.194 mol, 1.0 equiv.) and 17-3 (66.0 g, 0.388 mol, 2.0 equiv.) in CHCl (350 mL) was added to a 1 L three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere. The temperature was lowered to 0 °C in an ice / water bath. To the cooled solution, DMAP (12.0 g, 0.098 mol, 0.5 equiv.) and EDCI (112 g, 0.583 mol, 3.0 equiv.) were added at 0 °C. The ice / water bath was removed, and the temperature was allowed to rise gradually. The reaction mixture was stirred overnight at room temperature. Directly to the reaction mixture was added 200 g of silica gel (type: ZCX-2, 100-200 mesh, 11.4 w / w), and the solvent was removed under vacuum while maintaining the temperature below 35 °C. A column was packed with 1 kg of silica gel (type: ZCX-2, 100-200 mesh, 57.1 w / w), followed by the dried silica gel prepared in the last step, which adsorbed the reaction mixture. The product was purified by CombiFlash elution using a gradient of 100:0 to 90:10 petroleum ether / EtOAc, collecting 1000 fractions. Samples were taken for TLC analysis, and the appropriate products were combined. This gave 73 g (0.184 mol, 95% yield) of 17-4 as a light yellow oil. The product had no MS signal and was used directly in the next step.

[0468] 17-5: Synthesis of 2-hydroxypropane-1,3-diyl bis(3-cyclohexyl-2-methylpropanoate) [ka]

[0469] A solution of 17-4 (56.0 g, 0.142 mol, 1.0 equiv) in THF (560 mL) was placed in a 1 L three-neck round-bottom flask purged and maintained with an inert nitrogen atmosphere. The temperature was lowered to 0 °C in an ice / water bath. HOAc (12.8 g, 0.213 mol, 1.5 equiv) was added to the solution at 0 °C, and then NaBHCN (12.5 g, 0.199 mol, 1.4 equiv) was added to the mixture at 0 °C. The ice / water bath was removed, and the temperature was allowed to gradually increase. The reaction mixture was stirred at room temperature for 8 h. The reaction mixture was quenched with HO (1.1 L) and extracted with CHCl (1.6 L). The organic layer was washed with aqueous NaHCO (560 mL), HO (2 × 280 mL), dried over anhydrous NaSO, and filtered. The filtrate was used directly in the next step.

[0470] 17-6: Synthesis of ((4,4'-((tert-butoxycarbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexyl-2-methylpropanoate) [ka]

[0471] To a 2 L three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere were added 1-5 (20.0 g, 0.069 mol, 1.0 equiv.) and 17-5 (from the above solution, 0.138 mol, 2.0 equiv.). The temperature was lowered to 0 °C in an ice / water bath. To the solution, DMAP (8.4 g, 0.069 mol, 1.0 equiv.) and EDCI (53 g, 0.277 mol, 4.0 equiv.) were added at 0 °C. The ice / water bath was removed, and the temperature was allowed to rise gradually. The reaction mixture was stirred overnight at room temperature. Directly to the reaction mixture was added 200 g of silica gel (type: ZCX-2, 100-200 mesh, 10.0 w / w), and the mixture was concentrated under vacuum while maintaining the temperature below 35 °C. A column was packed with 1.5 kg of silica gel (type: ZCX-2, 100-200 mesh, 75.0 w / w), followed by the dried silica gel prepared in the last step, which adsorbed the reaction mixture. The product was purified by CombiFlash elution using a gradient of 100:0 to 90:10 petroleum ether / EtOAc, collecting 500 mL fractions. Samples were taken for TLC analysis, and the appropriate products were combined. This afforded 30.8 g (59.6 mmol, 42% over two steps) of 17-6 as a light yellow oil. ELSD A: water / 0.05% TFA; B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B, 3 min, 1.0 min hold): room temperature 2.08 min, m / z (calculated) 1045.7, (found) 946.6 (M-Boc+H).

[0472] 17-7: Synthesis of bis(4-((1,3-bis((3-cyclohexyl-2-methylpropanoyl)oxy)propan-2-yl)oxy)-4-oxobutyl)ammonium chloride [ka]

[0473] A solution of 17-6 (48.0 g, 0.046 mol, 1.0 equiv) in 1,4-dioxane (240 mL) was placed in a 1 L round-bottom flask purged and maintained with an inert nitrogen atmosphere, and the solution was cooled in an ice-water bath. 4 M HCl in 1,4-dioxane (240 mL) was added dropwise over 10 min at 0–10 °C. The resulting solution was stirred at room temperature overnight. The mixture was concentrated in vacuo. This afforded 48 g (crude) of 17-7 as a yellow oil. ELSD A: water / 0.05% TFA; B: CH3CN / 0.05% TFA 95:5–5:95 A / B, 3 min, 1.0 min hold): room temperature 0.92 min, m / z (calculated) 945.6, (found) 946.6 (M+H).

[0474] 17-8: Synthesis of ((4,4'-((1H-imidazole-1-carbonyl)azanediyl)bis(butanoyl))-bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexyl-2-methylpropanoate) [ka]

[0475] Under a nitrogen atmosphere, a solution of 17-7 (48.0 g, 0.046 mol, 1.0 equiv) in CHCl (1.06 L) was placed in a 2 L three-neck round-bottom bottle. Carbonyldiimidazole (15.9 g, 0.098 mol, 2.1 equiv) was then added, followed by pyridine (15.4 g, 0.196 mol, 4.26 equiv), and the mixture was stirred at room temperature overnight. The resulting solution was washed with 3% aqueous citric acid (2 × 500 mL), HO (3 × 500 mL), and brine (500 mL), dried over anhydrous NaSO, and concentrated under vacuum at 35 °C. The crude mixture was dissolved in CHCl (800 mL), 100 g of silica gel (type: ZCX-2, 100-200 mesh, 2.08 w / w) was added, and the solvent was removed under vacuum while maintaining the temperature below 35 °C. A column was packed with 300 g of silica gel (type: ZCX-2, 100-200 mesh, 6.25 w / w), followed by the dried silica gel prepared in the last step, which adsorbed the reaction mixture. The product was purified by CombiFlash elution using a gradient of petroleum ether / EtOAc from 100:0 to 70:30, collecting 400 mL fractions. Samples were taken for TLC analysis, and the appropriate products were combined. This afforded 39 g (37.5 mmol, 81% yield) of 17-8 as an oil. ELSD A: water / 0.05% TFA; B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B, 2 min, 0.6 min hold): room temperature 1.0 min, m / z (calculated) 1039.6, (found) 1040.6 (M+H).

[0476] Lipid 17: Synthesis of ((4,4'-((((3-(dimethylamino)propyl)thio)carbonyl)-azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexyl-2-methylpropanoate) [ka]

[0477] A solution of 17-8 (20.0 g, 0.019 mol, 1.0 equiv.) in CHCl (200 mL) was placed in a 500 mL three-neck round-bottom flask purged and maintained with an inert nitrogen atmosphere. The reaction temperature was lowered to 0 °C in an ice / water batch. Methyl trifluoromethanesulfonate (3.3 g, 0.020 mol, 1.05 equiv.) was added to the mixture at 0 °C. After the addition, the reaction was continued to stir at 0 °C for 3 h. A solution of methylamine 2.0 M in THF (28.9 mL, 0.058 mol, 3.0 equiv.) was added to the reactor at 0 °C. After the addition, the reaction was continued to stir at 0 °C for 0.5–1 h. 3-(dimethylamino)propane-1-thiol (3.0 g, 0.025 mol, 1.3 equiv.) was added to the reactor at 0 °C. After the addition, the reaction mixture was allowed to reach room temperature and continued to stir for 5.0 hours. Aqueous sodium chloride solution (10.0 wt%, 200 mL) and 10% aqueous citric acid solution (10.0 wt%, 200 mL) were added to the reactor. Stirring was continued for 15 minutes, after which the reactor was allowed to stand for 15 minutes to allow phase separation at room temperature. The organic phase was collected. This procedure was repeated once more. Aqueous sodium chloride solution (10.0 wt%, 200 mL) was added, and aqueous sodium bicarbonate solution (5.0 wt%, 200 mL) was added to the reactor. Stirring was continued for 15 minutes, after which the reactor was allowed to stand for 15 minutes to allow phase separation at room temperature. The organic phase was collected. This procedure was repeated once more. Aqueous sodium chloride solution (10.0 wt%, 400 mL) was added to the reactor. Stirring was continued for at least 15 minutes, after which the reactor was allowed to stand for at least 15 minutes to allow phase separation at room temperature. The organic phase was collected. n-Heptane (250 mL) was added to the reactor. The solution was concentrated under vacuum to approximately 300 mL while maintaining the temperature at 20-40°C. A 10.0 wt% citric acid solution in methanol / water (10:1, 200 mL) was added to the reactor. After addition, the mixture was stirred for 15 minutes, and then the reactor was allowed to stand for 15 minutes to allow phase separation at 36±5°C. The MeOH / HO phase was collected. n-Heptane (250 mL) was added to the reactor to wash the MeOH / HO phase. This n-heptane washing procedure was repeated eight times. The reactor was charged with n-heptane (500 mL), 15.0 wt% sodium carbonate solution (250 mL), and 10.0 wt% sodium chloride solution (250 mL).The mixture was stirred for 15 minutes, then the reactor was allowed to stand for 15 minutes to allow for phase separation at room temperature. The organic phase was collected. 400 mL of 5.0 wt. % sodium bicarbonate solution was added to the reactor. The mixture was stirred for 15 minutes, then the reactor was allowed to stand for 15 minutes to allow for phase separation at room temperature. The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated under vacuum. 30 g of silica gel (type: ZCX-2, 100-200 mesh, 1.5 wt. / wt.) was added to the residue in 300 mL of CHCl, and the mixture was concentrated under vacuum while maintaining the temperature below 35°C. 200 g of silica gel (type: ZCX-2, 100-200 mesh, 10.0 wt. / wt.) was packed into a column, followed by the dried silica gel prepared in the last step, which adsorbed the reaction mixture. Purify the product by CombiFlash elution using a gradient of 100:0 to 90:10 CHCl / MeOH, collecting 400 mL fractions. Samples were taken for TLC analysis, and the appropriate products were combined. This afforded 11.5 g (55% yield) of lipid 17 as a yellow oil. ELSD A: Water / 0.05% TFA; B: CHCN / 0.05% TFA 80:20 to 20:80 A / B in 3 min, 1 min hold): room temperature 0.97 min, m / z (calculated) 1090.7, (found) 1091.7 (M+H). 1 H-NMR (300MHz, CDCl3): δ5.25(m, 2H), 4.34(dt, J=11.9, 4.0Hz, 4H), 4.15(m, 4H), 3.38(brm, 4H), 2.92(t, J=7.3 Hz, 2H), 2.67-2.49(4H), 2.48-2.22(12H), 1.89-1.84(6H), 1.78-1.51(24H), 1.35-1.07(32H), 0.99-0.76(8H).

[0478] Example 18. Synthesis of lipid 18: ((4,4'-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(2-methyloctanoate) [ka] Overall Scheme: [ka]

[0479] 18-1: Synthesis of 2-oxopropane-1,3-diyl bis(2-methyloctanoate) [ka]

[0480] To a 50 mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere was added a solution of 1,3-dihydroxyacetone (1.42 g, 1.0 equiv.) and 2-methyloctanoic acid (5.0 g, 2.0 equiv.; Org. Biomol. Chem. 2014, 12, 3649-3663) in CHCl (30 mL). The solution was cooled in an ice-water bath, and then DMAP (0.96 g, 0.5 equiv.) and EDCI (12.1 g, 4.0 equiv.) were added at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred overnight. To the mixture was added 20 g of silica gel (type: ZCX-2, 100-200 mesh, 15.5 w / w), and the solvent was removed under vacuum while maintaining the temperature below 35 °C. A column was packed with 100 g of silica gel (type: ZCX-2, 100-200 mesh, 77.5 w / w), followed by the dried silica gel prepared in the last step, which adsorbed the reaction mixture. The crude product was purified using a Combi-Flash purification system eluting with a gradient of 100:0 to 90:10 petroleum ether / EtOAc, collecting 200 ± 50 mL aliquots. A sample was taken for TLC analysis, and the appropriate fractions were combined and concentrated under vacuum. This afforded 4.9 g (85% yield) of 18-1 as a light yellow oil. ELSD A: water / 0.05% TFA; B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B in 2 min, 1.3 min hold): room temperature, 1.6 min, m / z (calculated) 370.3, (found) 371.3 (M+H).

[0481] 18-2: Synthesis of 2-hydroxypropane-1,3-diyl bis(2-methyloctanoate) [ka]

[0482] A 100 mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere was charged with a solution of 18-1 (4.9 g, 1.0 equiv.) in THF (50 mL), and the solution was cooled in an ice-water bath. HOAc (1.03 g, 1.3 equiv.) was added to the solution at 0 °C, followed by NaBHCN (1.0 g, 1.2 equiv.) at 0 °C. The ice-water bath was removed after all reagents were added. The reaction mixture was stirred overnight at room temperature. The reaction mixture was quenched with HO (100 mL) and extracted with CHCl (150 mL). The organic layer was washed with 5% aqueous NaHCO (50 mL), HO (2 × 50 mL), dried over anhydrous NaSO, and filtered. The filtrate containing 18-2 was used directly in the next step.

[0483] 18-3: Synthesis of ((4,4'-((tert-butoxycarbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(2-methyloctanoate) [ka]

[0484] A solution of 18-2 (8.8 g, 2.3 equiv.) and 1-5 (3.0 g, 1.0 equiv.) in CHCl (60 mL) was added to a 100 mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere, and the mixture was cooled in an ice-water bath. To the solution, DMAP (1.26 g, 1.0 equiv.) and EDCI (7.96 g, 4.0 equiv.) were added at 0 °C. The ice-water bath was removed after all reagents were added. The reaction mixture was stirred overnight at room temperature. To the mixture, 20 g of silica gel (type: ZCX-2, 100-200 mesh, 6.7 w / w) was added, and the solvent was removed under vacuum while maintaining the temperature below 35 °C. A column was packed with 120 g of silica gel (type: ZCX-2, 100-200 mesh, 40.0 w / w), followed by the dried silica gel prepared in the last step, which adsorbed the reaction mixture. The product was purified using CombiFlash by eluting with a gradient of 100:0 to 90:10 petroleum ether / EtOAc, collecting 200 mL fractions. Samples were taken for TLC analysis, and the appropriate products were combined. This afforded 4.3 g (42% yield) of 18-3 as a light yellow oil. ELSD A: water / 0.05% TFA; B: CH3CN / 0.05% TFA 80:20 to 20:80 A / B in 3 min, 1 min hold): room temperature 1.97 min, m / z (calculated) 997.7, (found) 1020.6 (M+Na).

[0485] 18-4: Synthesis of bis(4-((1,3-bis((2-methyloctanoyl)oxy)propan-2-yl)oxy)-4-oxobutyl)ammonium chloride [ka]

[0486] A solution of 18-3 (4.3 g, 1.0 equiv.) in 1,4-dioxane (21 mL) was placed in a 100 mL round-bottom flask purged and maintained with an inert nitrogen atmosphere, and the solution was cooled in an ice-water bath. To the cooled solution, 4 M HCl in 1,4-dioxane (21 mL) was added dropwise over 10 min at 0–10 °C. The resulting solution was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo. This afforded 4 g (crude) of 18-4 as a yellow oil. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B in 2 min, 1.3 min hold): RT 1.6 min, m / z (calculated) 897.6, (found) 898.6 (M+H).

[0487] Lipid 18: Synthesis of ((4,4'-((((3-(dimethylamino)propyl)thio)carbonyl)-azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(2-methyloctanoate) [ka]

[0488] A solution of 18-4 (3.8 g, 1.0 equiv.) in CHCl (80 mL) was placed in a 250 mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere, and the solution was cooled in an ice-water bath. Triphosgene (1.26 g, 1.0 equiv.) was added to the mixture at 0 °C, followed by the dropwise addition of pyridine (1.67 g, 5.0 equiv.) with stirring at 0 °C. The ice-water bath was removed after all reagents were added. The mixture was stirred at room temperature for 4 h and then concentrated under vacuum (temperature below 30 °C). The residue was dissolved in pyridine (80 mL) and cooled in an ice-water bath under nitrogen, followed by the dropwise addition of 3-(dimethylamino)propane-1-thiol (1.0 g, 2.0 equiv.) with stirring at 0 °C for 10 min. The resulting solution was stirred at room temperature overnight. The reaction mixture was concentrated under vacuum, and the residue was diluted with CHCl (80 mL). The solution was washed with 10% aqueous citric acid (40 mL), HO (40 mL), saturated NaHCO (2 × 40 mL), and brine (40 mL, 10 V). The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. To the residue dissolved in CHCl (60 mL), 8 g of silica gel (type: ZCX-2, 100-200 mesh, 2.11 w / w) was added, and the solvent was removed under vacuum while maintaining the temperature below 35 °C. 100 g of silica gel (type: ZCX-2, 100-200 mesh, 26.3 w / w) was packed into a column, followed by the dried silica gel prepared in the last step, which adsorbed the reaction mixture. The product was purified by CombiFlash, eluting with a gradient of 100:0 to 90:10 CHCl / MeOH, collecting 100 mL fractions. A sample was taken for TLC analysis, and the appropriate products were combined. This gave 1.3 g (29% yield over two steps) of lipid 18 as a yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 80:20 to 20:80 A / B in 3 min, 2.1 min hold): room temperature, 1.1 min, m / z (calculated) 1042.7, (found) 1043.6 (M+H). 1H-NMR (300MHz, CDCl3): δ5.25(m, 2H), 4.34(m, 4H), 4.16(m, 4H), 3.38(brm, 4H), 2.92(m, 4H), 2.66(s, 6H), 2.46(m, 4H), 2.35(brs, 4H), 2.09(m, 2H), 1.90(brs, 4H), 1.64(m, 4H), 1.47-1.20(38H), 1.15-1.13(12H), 0.95-0.81(12H).

[0489] Example 19. Synthesis of lipid 19: ((4,4'-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(2,2-dimethylheptanoate) [ka] Overall Scheme: [ka]

[0490] 19-1: Synthesis of ethyl 2,2-dimethylheptanoate [ka]

[0491] A solution of ethyl isobutyrate (40.0 g, 1.0 equiv.) in THF (400 mL) was placed in a 500 mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere. The resulting solution was cooled to -78 °C, and then LDA (205.6 mL, in hexane, 1.05 equiv.) was added dropwise, and the resulting solution was stirred at -78 °C for 1 hour. 1-Iodopentane (92.8 g, 1.2 equiv.) was then added dropwise, and the resulting solution was stirred at -78 °C for 5 hours. The cooling bath was removed, and the solution was stirred at room temperature overnight. The pH of the solution was adjusted to 6 using aqueous HCl (1 mol / L). The resulting solution was extracted with ethyl acetate (2 × 300 mL), and the organic layers were combined. The resulting mixture was washed with brine (500 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum. This gave 51 g (75.6%) of 19-1 as a yellow oil, which was used in the next step without further purification.

[0492] 19-2: 2,2-dimethylheptanoic acid [ka]

[0493] A solution of 19-1 (70.0 g, 1.0 equiv.) in MeOH (700 mL) was placed in a 2 L three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere. A solution of NaOH (49.0 g, 3.0 equiv.) in HO (350 mL) was added dropwise to the solution at room temperature. The resulting solution was warmed and stirred at 60 °C for 4 h. After cooling to room temperature, the resulting mixture was concentrated under vacuum. The residue was dissolved in HO (200 mL) and extracted with MTBE (200 mL), and the aqueous layer was separated. The pH of the aqueous phase was adjusted to 5 with aqueous HCl (1 mol / L). The resulting solution was extracted with ethyl acetate (2 × 100 mL), and the organic layers were combined. The resulting mixture was washed with brine (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. This gave 32 g (68%) of 19-2 as a yellow oil, which was used without purification.

[0494] 19-3: Synthesis of 2-oxopropane-1,3-diyl bis(2,2-dimethylheptanoate) [ka]

[0495] A solution of 1,3-dihydroxyacetone (12.0 g, 1.0 equiv.) in CHCl (240 mL) was added to a 500 mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere. The solution was cooled to 0 °C in an ice / water bath. 19-2 (44.0 g, 2.1 equiv.), DMAP (16.3 g, 1.0 equiv.), followed by EDCI (76.7 g, 3.0 equiv.) were added to the solution at 0 °C. The ice / water bath was removed, and the reaction mixture was stirred at room temperature overnight. 25 g of silica gel (type: ZCX-2, 100-200 mesh, 2.08 w / w) was added to the reaction solution, and the mixture was concentrated under vacuum while maintaining the temperature below 35 °C. A column was packed with 500 g of silica gel (type: ZCX-2, 100-200 mesh, 41.7 w / w), followed by the dried silica gel prepared in the last step, which adsorbed the reaction mixture. The product was purified by CombiFlash elution using a gradient of 95:5 to 90:10 petroleum ether / EtOAc, collecting 1000 fractions. Samples were taken for TLC analysis, and the appropriate product was combined. Concentration in vacuo afforded 40.6 g (75.8%) of 19-3 as a colorless oil. ELSD A: water / 0.05% TFA; B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B in 2 min, 1.3 min hold): room temperature 1.8 min, m / z (calculated) 370.2, (found) 393.2 (M+Na).

[0496] 19-4: Synthesis of 2-hydroxypropane-1,3-diyl bis(2,2-dimethylheptanoate) [ka]

[0497] A 500 mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere was charged with a solution of 19-3 (15.5 g, 1.0 equiv.) in THF (155 mL). The solution was cooled to 0 °C in an ice / water bath. HOAc (3.26 g, 1.3 equiv.) was added to the solution at 0 °C, and then NaBHCN (3.16 g, 1.2 equiv.) was added to the mixture in one portion at 0 °C. The ice / water bath was removed, and the mixture was stirred at room temperature for 16 h. The reaction was quenched with water (200 mL). The mixture was extracted with CHCl (3 × 200 mL). The combined organic phase was washed with brine (500 mL) and then dried over NaSO. Filtration and concentration under vacuum gave crude 19-4, which was dissolved in CHCl (75 mL). 30 g of silica gel (type: ZCX-2, 100-200 mesh, 1.94 w / w) was added to the solution, and the mixture was concentrated under vacuum while maintaining the temperature below 35 °C. 200 g of silica gel (type: ZCX-2, 100-200 mesh, 12.9 w / w) was packed into a column, followed by the dried silica gel prepared in the last step, which adsorbed the reaction mixture. The product was purified by CombiFlash elution using a gradient of 90:10 to 85:15 petroleum ether / EtOAc, collecting 400 mL fractions. A sample was taken for TLC analysis, and the appropriate product was combined. Concentration under vacuum gave 12.3 g (79.3% yield) of 19-4 as a yellow oil. ELSD A: water / 0.05% TFA: B: CHCN / 0.05% TFA 95:5 to 5:95 A / B, 1.2 min hold, 2 min at room temperature, 1.5 min, m / z (calculated) 372.2, (found) 395.2 (M+Na).

[0498] 19-5: Synthesis of ((4,4'-((tert-butoxycarbonyl)azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(2,2-dimethylheptanoate) [ka]

[0499] A solution of 1-5 (4.77 g, 1.0 equiv.) in CHCl (80 mL) was placed in a 250 mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere. The solution was cooled to 0 °C in an ice / water bath, and then 19-4 (12.3 g, 2.0 equiv.), DMAP (2.0 g, 1.0 equiv.), and EDCI (9.5 g, 3.0 equiv.) were added sequentially at 0 °C. The ice / water bath was removed, and the resulting solution was stirred at room temperature for 16 h. To the reaction solution was added 15 g of silica gel (type: ZCX-2, 100-200 mesh, 3.14 w / w), and the mixture was concentrated under vacuum while maintaining the temperature below 35 °C. A column was packed with 200 g of silica gel (type: ZCX-2, 100-200 mesh, 41.9 w / w), followed by the dried silica gel prepared in the last step, which adsorbed the reaction mixture. The product was purified by CombiFlash elution using a gradient of 90:10 to 86:14 petroleum ether / EtOAc, collecting 400 mL fractions. Samples were taken for TLC analysis, and the appropriate products were combined. This yielded 15.8 g (96.3%) of 19-5 as a yellow oil.

[0500] 19-6: Synthesis of bis(4-((1,3-bis((2,2-dimethylheptanoyl)oxy)propan-2-yl)oxy)-4-oxobutyl)ammonium chloride [ka]

[0501] A solution of 19-5 (6.0 g, 1.0 equiv.) in CHCl (30 mL) was placed in a 250 mL round-bottom flask purged and maintained under an inert nitrogen atmosphere. The solution was cooled to 0 °C in an ice / water bath. To the solution, a solution of HCl in dioxane (60 mL, 4 mol / L) was added dropwise at 0–10 °C. The ice / water bath was removed, and the resulting solution was stirred overnight at room temperature. The reaction mixture was concentrated in vacuo to give 6.2 g (crude) of 19-6 as a light yellow oil. ELSD A: water / 0.05% TFA; B: CHCN / 0.05% TFA 95:5 to 5:95 A / B in 2 min, 1.2 min hold): room temperature 1.5 min, m / z (calculated) 897.6, (found) 898.5 (M+H).

[0502] Lipid 19: Synthesis of ((4,4'-((((3-(dimethylamino)propyl)thio)carbonyl)-azanediyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(2,2-dimethylheptanoate) [ka]

[0503] A 500 mL three-necked round-bottom flask purged and maintained with an inert nitrogen atmosphere was charged with a solution of 19-6 (6.0 g, 1.0 equiv.) in CHCl (210 mL). The solution was cooled to 0 °C in an ice / water bath. To the mixture, triphosgene (2.69 g, 1.5 equiv.) was added at 0 °C. Subsequently, pyridine (2.53 g, 5.0 equiv.) was added dropwise with stirring at 0 °C. The ice / water batch was removed, and the mixture was stirred at room temperature for 4 h and then concentrated under vacuum (temperature below 30 °C). The residue was dissolved with pyridine (120 mL, 20 V), and the solution was cooled to 0 °C in an ice / water bath. To this solution, 3-(dimethylamino)propane-1-thiol (1.53 g, 2.0 equiv.) was added dropwise with stirring at 0 °C for 10 min. The ice / water batch was removed, and the resulting solution was stirred at room temperature overnight. The reaction mixture was concentrated under vacuum, and crude 19 was dissolved in CHCl (100 mL). 12 g of silica gel (type: ZCX-2, 100-200 mesh) was added, and the mixture was concentrated under vacuum while maintaining the temperature below 35 °C. 100 g of silica gel (type: ZCX-2, 100-200 mesh) was packed into a column, followed by the dried silica gel prepared in the last step, which adsorbed the reaction mixture. The product was purified using CombiFlash, eluting with a gradient of 75 / 25 to 70 / 30 CHCl / acetone, collecting 200 mL fractions. A sample was taken for TLC analysis, and the appropriate product was combined. This gave 1.0 g (13.8% yield) of 19 as a yellow oil. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B, 1.2 min hold, 2 min at room temperature, 1.5 min, m / z (calculated) 1042.7, (found) 1043.9 (M+H). 1 H-NMR (300MHz, CDCl3): δ5.28(m, 2H), 4.32(dd, J=11.9, 4.4Hz, 4H), 4.11(dd, J=11.9, 5.7Hz, 4H), 3.38(brm, 4H), 2 .91(m, 2H), 2.18-2.33(6H), 2.23(s, 6H), 1.78(brm, 4H), 1.55-1.42(8H), 1.35-1.10(50H), 0.87(t, J=6.9Hz, 12H).

[0504] Example 20. Synthesis of lipid 20: ((3,3'-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl)bis(propanoyl))bis(oxy))bis(methylene))bis(2-methylpropane-2,1,3-triyl)tetrakis(3-(4-methylcyclohexyl)propanoate) [ka] Fat 20

[0505] Overall Scheme: [ka]

[0506] 20-2: Synthesis of 3-(4-methylcyclohexyl)propanoyl chloride [ka]

[0507] To a 25 mL three-necked round-bottom flask was added SOCl (100 mL, 1378.615 mmol, 4.69 equiv.) and commercially available 3-(4-methylcyclohexyl)propanoic acid (50 g, 293.682 mmol, 1 equiv.) at room temperature. The resulting mixture was stirred at room temperature for 10 h and concentrated under reduced pressure to give 3-(4-methylcyclohexyl)propanoyl chloride (51 g, 92.03%) as a yellow oil, which was used directly without further purification or characterization.

[0508] 20-4: Synthesis of 2-((benzyloxy)methyl)-2-methylpropane-1,3-diyl bis(3-(4-methylcyclohexyl)-propanoate) [ka]

[0509] To a 1 L three-necked round-bottom flask, commercially available 2-[(benzyloxy)methyl]-2-methylpropane-1,3-diol (20-3, 27.1 g, 128.7 mmol, 1.00 equiv), pyridine (25.45 g, 321.7 mmol, 2.5 equiv), and CHCl (500 mL, 20V) were added at room temperature. The mixture was cooled to 0 °C. To the above mixture, 20-2 (51.00 g, 270.2 mmol, 2.1 equiv) was added dropwise at 0 °C. The resulting mixture was stirred at room temperature for an additional 4 h. The reaction was quenched by adding water (1 L) at 0 °C. The resulting mixture was extracted with CHCl (3 × 500 mL). The combined organic layer was dried over anhydrous NaSO, filtered, and the filtrate was concentrated under reduced pressure. The residue was adsorbed onto 540 g of silica gel (type: ZCX-2, 100-200 mesh, 2.00 w / w) and purified on a 2700 g silica gel column using a Combi-Flash purification system. The product was eluted with PE / EA (100:0 to 70:30 gradient, collected every 500 ± 10 mL). After TLC analysis (EA:PE = 1:10), the appropriate fractions were combined, concentrated, and dried under vacuum to give 20-4 (45 g, 67.9%) as a colorless oil. ELSD A: water / 0.05% TFA; B: CH3CN 95:5 to 5:95 A / B in 2 min, 1.2 min hold): room temperature 1.48 min, m / z (calculated) 514.4, (found) 537.5 (M+Na).

[0510] 20-5: Synthesis of 2-(hydroxymethyl)-2-methylpropane-1,3-diyl bis(3-(4-methylcyclohexyl)-propanoate) [ka]

[0511] To a solution of 20-4 (45 g, 87.4 mmol, 1 equiv) in MeOH (500 mL, 11 V) was added Pd / C (10%, 4.5 g) in a 1 L three-necked round-bottom flask under nitrogen. The mixture was hydrogenated at room temperature for 10 h under a hydrogen atmosphere using a hydrogen balloon. The reaction was filtered through a bed of Celite and concentrated under reduced pressure to give 20-5 (35 g, 94.3%) as a colorless oil. ELSD A: water / 0.05% TFA; B: CH3CN 95:5 to 5:95 A / B in 2 min, 1.2 min hold): RT 1.29 min, m / z (calculated) 424.3, (found) 447.4 (M+Na).

[0512] 20-7: Synthesis of (((3,3'-((tert-butoxycarbonyl)azanediyl)bis(propanoyl))bis(oxy))bis(methylene))bis(2-methylpropane-2,1,3-triyl)tetrakis(3-(4-methylcyclohexyl)propanoate) [ka]

[0513] To a 1 L three-necked round-bottom flask was added 20-5 (35.04 g, 82.5 mmol, 2.2 equiv.), 3-[(tert-butoxycarbonyl)(2-carboxyethyl)amino]propanoic acid (20-6, 9.8 g, 37.5 mmol, 1.00 equiv.), EDCI (14.38 g, 75.0 mmol, 2 equiv.), DCM (700 mL, 20V), and DMAP (4.58 g, 37.48 mmol, 1.00 equiv.) at room temperature. The resulting mixture was stirred at room temperature for 10 h and diluted with water (500 mL). The resulting mixture was extracted with CHCl (3 × 500 mL), and the combined organic layer was dried over anhydrous NaSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was adsorbed onto 540 g of silica gel (type: ZCX-2, 100-200 mesh, 2.00 w / w) and purified on a 2.7 kg silica gel column using a Combi-Flash purification system. The product was eluted with PE / EA (100:0 to 50:50 gradient, collected every 500 ± 10 mL). After TLC analysis (EA:PE = 1:10), the appropriate fractions were combined, concentrated, and dried under vacuum to give 20-7 as a colorless oil (37 g, 91.8%), which was purified by HPLC. 1 Based on purity and structure by 1 H NMR it was used in the next step.

[0514] 20-8: Synthesis of (((3,3'-azanediylbis(propanoyl))bis(oxy))bis(methylene))bis(2-methylpropane-2,1,3-triyl)tetrakis(3-(4-methylcyclohexyl)propanoate) trifluoroacetate [ka]

[0515] To a 500 mL three-necked round-bottom flask, 20-7 (37 g, 34.4 mmol, 1 equiv.), DCM (370 mL), and trifluoroacetic acid (150 mL) were added at room temperature. The resulting mixture was stirred at room temperature for 10 h. The resulting mixture was concentrated under reduced pressure to give 20-8 (40 g, crude) as a colorless oil. 1Both H NMR and HPLC showed approximately 94% pure product, which was used directly in the next reaction after drying under vacuum.

[0516] Lipid 20: Synthesis of ((3,3'-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl)bis(propanoyl))bis(oxy))bis(methylene))bis(2-methylpropane-2,1,3-triyl)tetrakis(3-(4-methylcyclohexyl)propanoate) [ka]

[0517] To a 1 L three-necked round-bottom flask, 20-8 (37 g, 34 mmol, 1 equiv), TEA (10.32 g, 102 mmol, 3 equiv), CDI (6.06 g, 37.4 mmol, 1.1 equiv), and DCM (1.48 L, 40V) were added at room temperature. The resulting mixture was stirred at room temperature for 10 hours. The mixture was cooled to 0 °C. To the above mixture, methyl trifluoromethanesulfonate (6.14 g, 37.4 mmol, 1.1 equiv) was added dropwise at 0 °C. The resulting mixture was stirred at 0 °C for 1 hour, and 3-(dimethylamino)propane-1-thiol (4.46 g, 37.4 mmol, 1.1 equiv) was added dropwise at 0 °C. The resulting mixture was stirred at room temperature for an additional 10 hours, diluted with water (300 mL), and extracted with CHCl (3 × 300 mL). The combined organic phase was dried over anhydrous Na2SO4, filtered, and evaporated. The residue was adsorbed onto 74 g of silica gel (type: ZCX-2, 100-200 mesh, 2.00 w / w) and purified on a 370 g silica gel column using a Combi-Flash purification system. The product was eluted with PE / EA (100:0 to 90:10 gradient, collected every 500 ± 10 mL). After TLC analysis (EA:PE = 1:10), the appropriate fractions were combined, concentrated, and dried under vacuum to give lipid 20 (5.1 g, 13.3%) as a yellow oil. ELSD A: water / 0.05% TFA: B: CHCN / 0.05% TFA 95:5 to 5:95 in 25 min. A / B): room temperature 10.3 min, m / z (calculated) 1118.8, (observed) 1119.9 (M+H). 1 H NMR (300MHz, chloroform-d) δ4.005(d, J=6.6Hz, 12H), 3.654(t, J=7.2Hz, 4H), 2.956(t, J=7.0Hz, 2H), 2.755-2.598(m, 6H), 2. 519(s, 6H), 2.373-2.248(m, 8H), 2.023-1.899(m, 2H), 1.769-1.092(m, 46H), 1.044-0.995(m, 6H), 0.941-0.830(m, 16H).

[0518] Example 21: Synthesis of lipid 21: ((3,3'-((((3-(dimethylamino)propyl)thio)carbonyl)azanediyl)bis(propanoyl))bis(oxy))bis(methylene))bis(2-methylpropane-2,1,3-triyl)tetrakis(2-(4-methylcyclohexyl)acetate) [ka]

[0519] Fat 21

[0520] Overall Scheme [ka]

[0521] 21-2: Synthesis of 2-(4-methylcyclohexyl)acetyl chloride [ka]

[0522] Under nitrogen, a 2 L three-necked round-bottle flask was charged with a solution of 21-1 (80 g, 512.0 mmol, 1.00 equiv) in DCM (800 mL, 10V). The solution was cooled to 0 °C in an ice / water bath, and oxalyl chloride (130 g, 1024.2 mmol, 2.00 equiv) was added dropwise at 0 °C. The ice / water bath was removed, and the reaction was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo to give 21-2 (82 g, 91.7%) as a colorless oil, which was used directly in the next reaction.

[0523] 21-3: Synthesis of 2-((benzyloxy)methyl)-2-methylpropane-1,3-diyl bis(2-(4-methylcyclohexyl)acetate) [ka]

[0524] A solution of 20-3 (40 g, 190.2 mmol, 1.00 equiv) in DCM (800 mL) was treated with pyridine (60.19 g, 760.9 mmol, 4.00 equiv) and DMAP (6.97 g, 57.0 mmol, 0.30 equiv) under nitrogen at 0 °C, followed by the dropwise addition of 21-2 (83.1 g, 475.6 mmol, 2.50 equiv) at 0 °C. The mixture was stirred at room temperature for 16 h. The resulting mixture was diluted with water (500 mL) and acidified to pH 6 with HCl (aq). The aqueous layer was extracted with DCM (2 × 200 mL). The combined organic layers were washed with brine (1 × 300 mL), dried over anhydrous NaSO, filtered, and the filtrate was concentrated under reduced pressure to give 21-3 (67 g, 72.4%) as a colorless oil. ELSD A:water / 0.05% TFA:B:CH3CN 95:5 to 5:95 in 3 min. A / B): room temperature 2.49 min, m / z (calculated) 486.3, (found) 509.4 (M+Na).

[0525] 21-4: Synthesis of 2-(hydroxymethyl)-2-methylpropane-1,3-diyl bis(2-(4-methylcyclohexyl)acetate) [ka]

[0526] A solution of 21-3 (67 g, 137.66 mmol, 1.00 equiv) in MeOH (670 mL 10V) was placed in a 2 L four-neck round-bottom flask, and Pd / C (20.1 g, 18.9 mmol, 0.14 equiv, 10% wt) was added in one portion. The resulting mixture was stirred under H2 at room temperature for 16 h. The reaction mixture was filtered, and the filter cake was washed with MeOH (1 × 300 mL). The filtrate was concentrated in vacuo to give 21-4 (53 g, 97.1%) as a colorless oil. ELSD A: water / 0.05% TFA; B: CH3CN 95:5 to 5:95 A / B: RT 2.11 min, m / z (calculated) 396.3, (found) 397.2 (M+H).

[0527] 21-5: Synthesis of (((3,3'-(...

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt thereof, 【Chemistry 1】 (I) During the ceremony, R １ and R ２ are each independently H or C １－６ is alkyl, or R １ and R ２ are joined to form a saturated heterocyclic ring, R １ is a linear C １－４ is alkylene, and R ２ is -(CH ２ ) ｍ (X) ｎ -, wherein X is O, S or NR ９ wherein R ９ is H or C １－６ is alkyl, m is 1, 2, 3, or 4, and n is 0 or 1; L1 is a linear C optionally substituted with 1 to 3 methyl groups. １－６ is alkylene, Y is selected from the group consisting of: 【Chemistry 2】 During the ceremony, Each asterisk (*) indicates an atom bonded to L2 and L3, and R １０ is H or C １－６ is alkyl, L2 and L3 each independently represent a linear C １－８ is alkylene, L4, L5, L6, L7, L8 and L9 each independently represent absent or -CH ２ - but, At least two of L4, L6 and L8 are —CH ２ - and At least two of L5, L7 and L9 are —CH ２ - and R ３ and R ４ are each independently H, methyl, or ethyl; and R ５ , R ６ , R ７ , and R ８ are each independently selected from the group consisting of: Linear C １－２０ alkyl, wherein each of said linear C １－２０ The alkyl is optionally Substituted with one or more substituents selected from the group consisting of: C １－６ Alkyl, C １－６ Alkoxy, and -F, wherein １－６ Each of the alkyl substituents is C １－３ optionally substituted with one or more groups selected from the group consisting of alkoxy and -F; C ３－８ monocycloalkyl, wherein said C ３－８ Each monocycloalkyl is C １－６ Alkyl, C １－６ optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C ７－１２ bicycloalkyl, wherein said C ７－１２ Each of the bicycloalkyls is C １－６ Alkyl, C １－６ optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F, and C ６－１０ aryl, wherein said C ６－１０ Each of the aryls is C １－６ Alkyl, C １－６ a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C ３－８ monocycloalkyl, wherein said C ３－８ Each monocycloalkyl is C １－６ Alkyl, C １－６ optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; C ７－１２ bicycloalkyl, wherein said C ７－１２ Each of the bicycloalkyls is C １－６ Alkyl, C １－６ optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F, and C ６－１０ aryl, wherein said C ６－１０ Each of the aryls is C １－６ Alkyl, C １－６ A compound which is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F.

2. R １ is H or C １－６ alkyl, and R ２ But C １－６ is alkyl, or R １ and R ２ or a pharmaceutically acceptable salt thereof, wherein:

3. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein Y is selected from the group consisting of: 【Transformation 3】

4. 4. The compound of claim 3, wherein Y is: or a pharmaceutically acceptable salt thereof. 【Chemistry 4】

5. R １ and R ２ However, each independently, C １－６ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: R is alkyl;

6. R １ and R ２ 6. The compound of claim 5, or a pharmaceutically acceptable salt thereof, wherein each is methyl.

7. R １ and R ２ or a pharmaceutically acceptable salt thereof.

8. The heterocyclic ring is 【Transformation 5】 8. The compound of claim 7, or a pharmaceutically acceptable salt thereof, wherein each asterisk (*) represents an atom bonded to L1.

9. 9. The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein the heterocyclic ring is selected from the group consisting of: 【Transformation 6】

10. R ５ , R ６ , R ７ , and R ８ are each independently selected from the group consisting of: Linear C １－８ alkyl, wherein the linear C １－８ The alkyl is optionally Substituted with one or more substituents selected from the group consisting of: C １－３ Alkyl, C １－３ Alkoxy, and -F, wherein １－３ Each of the alkyl substituents is C １－３ optionally substituted with one or more groups selected from the group consisting of alkoxy and -F; saturated C ３－６ monocycloalkyl, wherein the saturated C ３－６ Each monocycloalkyl is C １－６ Alkyl, C １－３ optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; saturated C ７－１２ bicycloalkyl, wherein the saturated C ７－１２ Each of the bicycloalkyls is C １－６ Alkyl, C １－３ optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F, and C ６－１０ aryl, wherein said C ６－１０ Each of the aryls is C １－６ Alkyl, C １－３ a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; saturated C ３－６ monocycloalkyl, wherein said C ３－６ Each monocycloalkyl is C １－６ Alkyl, C １－３ optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F; saturated C ７－１２ bicycloalkyl, wherein the saturated C ７－１２ Each of the bicycloalkyls is C １－６ Alkyl, C １－３ optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F, and C ６－１０ aryl, wherein said C ６－１０ Each of the aryls is C １－６ Alkyl, C １－３ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, which is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F.

11. R ５ , R ６ , R ７ , and R ８ are each independently a linear C １－８ alkyl, and the linear C １－８ Each alkyl is optionally selected from C １－３ Alkyl, C １－３ substituted with one or more substituents selected from the group consisting of alkoxy and -F; Said C １－３ The alkyl substituents each optionally include C １－３ 11. The compound of claim 10, or a pharmaceutically acceptable salt thereof, substituted with one or more groups selected from the group consisting of alkoxy and -F.

12. R ５ , R ６ , R ７ , and R ８ each independently represent one or more C １－３ Linear C optionally substituted with alkyl １－８ 12. The compound of claim 11, or a pharmaceutically acceptable salt thereof, wherein:

13. R ５ , R ６ , R ７ , and R ８ are each independently a linear C optionally substituted with 1 to 3 methyl groups ６－８ The compound v of claim 12, wherein v is alkyl.

14. R ５ , R ６ , R ７ , and R ８ is independently n-heptyl or n-octyl, or a pharmaceutically acceptable salt thereof.

15. R ５ , R ６ , R ７ , and R ８ are each independently, saturated C ３－６ Linear C optionally substituted with monocycloalkyl １－８ alkyl, and the saturated C ３－６ Each monocycloalkyl is optionally C １－６ Alkyl, C １－３ 11. The compound of claim 10, or a pharmaceutically acceptable salt thereof, substituted with one or more substituents selected from the group consisting of alkoxy, and -F.

16. The saturation C ３－６ 16. The compound of claim 15, or a pharmaceutically acceptable salt thereof, wherein each monocycloalkyl is optionally substituted with 1 to 3 methyl groups.

17. R ５ , R ６ , R ７ , and R ８ are each independently, saturated C ７－１２ A linear C optionally substituted with bicycloalkyl １－８ alkyl, wherein the saturated C ７－１２ Each bicycloalkyl is optionally selected from C １－６ Alkyl, C １－３ 11. The compound of claim 10, or a pharmaceutically acceptable salt thereof, substituted with one or more substituents selected from the group consisting of alkoxy, and -F.

18. The saturation C ７－１２ 18. The compound of claim 17, or a pharmaceutically acceptable salt thereof, wherein each bicycloalkyl is optionally substituted with 1 to 3 methyl groups.

19. R ５ , R ６ , R ７ , and R ８ are each independently, C ６－１０ Linear C optionally substituted with aryl １－８ alkyl, wherein said C ６－１０ Each aryl is C １－６ Alkyl, C １－３ 11. The compound of claim 10, or a pharmaceutically acceptable salt thereof, which is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F.

20. Said C ６－１０ 20. The compound of claim 19, or a pharmaceutically acceptable salt thereof, wherein each aryl is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with 1 to 3 methyl groups.

21. R ５ , R ６ , R ７ , and R ８ are each independently, saturated C ３－６ monocycloalkyl, wherein the C ３－６ Each monocycloalkyl is C １－６ Alkyl, C １－３ 11. The compound of claim 10, or a pharmaceutically acceptable salt thereof, optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F.

22. Said C ３－６ Each monocycloalkyl optionally has one or more C １－３ 22. The compound of claim 21, or a pharmaceutically acceptable salt thereof, which is substituted with alkyl.

23. R ５ , R ６ , R ７ , and R ８ are each independently, saturated C ７－１２ bicycloalkyl, wherein the saturated C ７－１２ Each bicycloalkyl is C １－６ Alkyl, C １－３ 11. The compound of claim 10, or a pharmaceutically acceptable salt thereof, optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F.

24. Said C ７－１２ 24. The compound of claim 23, or a pharmaceutically acceptable salt thereof, wherein each bicycloalkyl is optionally substituted with 1 to 3 methyl groups.

25. R ５ , R ６ , R ７ , and R ８ are each independently C ６－１０ aryl, wherein said C ６－１０ Each aryl is C １－６ Alkyl, C １－３ 11. The compound of claim 10, or a pharmaceutically acceptable salt thereof, which is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with one or more substituents selected from the group consisting of alkoxy, and -F.

26. i.R. ５ , R ６ , R ７ , and R ８ are each independently, 【Transformation 7】 is a monocycloalkyl selected from the group consisting of: Each asterisk (*) indicates an atom bonded to the carbonyl carbon, Each R １１ is independent, C １－６ is alkyl, Each R １２ is independent, C １－３ is an alkoxy, Each R １３ is -F, each p is independently 0 to 11; each q is independently 0 to 11; and each r is independently 0 to 11; wherein the sum of p, q and r is 11 or less; ii. R 5 , R 6 , R 7 , and R 8 are each independently 【Transformation 8】 is a bicycloalkyl selected from the group consisting of: Each asterisk (*) indicates an atom bonded to a carbonyl carbon, and Each bicycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: (R 11 ) p , wherein each R 11 is independently C 1-6 alkyl and each p is independently 0 to 13; (R 12 ) q , where each R 12 is independently C 1-3 alkoxy and each q is independently 0 to 13; and (R 13 ) r , wherein each R 13 is —F and each r is independently 0 to 13; In the formula, the sum of p, q, and r is 13 or less, or iii. R 5 , R 6 , R 7 , and R 8 are each independently 【Chemistry 9】 is a bicycloalkyl selected from the group consisting of: Each asterisk (*) indicates an atom bonded to a carbonyl carbon, and Each bicycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: (R 11 ) p , wherein each R 11 is independently C 1-6 alkyl and each p is independently 0 to 15; (R 12 ) q , where each R 12 is independently C 1-3 alkoxy and each q is independently 0 to 15; and (R 13 ) r , wherein each R 13 is —F and each r is independently 0 to 15; wherein the sum of p, q and r is 15 or less; iv. R 5 , R 6 , R 7 , and R 8 are each independently 【Chemistry 10】 is a bicycloalkyl selected from the group consisting of: Each asterisk (*) indicates an atom bonded to a carbonyl carbon, and Each bicycloalkyl is optionally substituted with one or more substituents selected from the group consisting of: (R 11 ) p , wherein each R 11 is independently C 1-6 alkyl and each p is independently 0 to 17; (R 12 ) q , where each R 12 is independently C 1-3 alkoxy and each q is independently 0 to 17; and (R 13 ) r , wherein each R 13 is —F and each r is independently 0 to 17; wherein the sum of p, q and r is 17 or less; v. R 5 , R 6 , R 7 , and R 8 are each independently 【Chemistry 11】 wherein: Each asterisk (*) indicates an atom bonded to the carbonyl carbon, each R 14 is independently H or C 1-6 alkyl; and each R 15 is independently H or C 1-6 alkyl; vi. R 5 , R 6 , R 7 , and R 8 are each independently 【Chemistry 12】 wherein: Each asterisk (*) indicates an atom bonded to the carbonyl carbon, each R 11 is independently C 1-6 alkyl; each R 12 is independently C 1-3 alkoxy; each R 13 is —F; each p is independently 0 to 5; each q is independently 0 to 5, and each r is independently 0 to 5; wherein the sum of p, q and r is 5 or less; or vii. R 5 , R 6 , R 7 , and R 8 are each independently 【Chemistry 13】 wherein: Each asterisk (*) indicates an atom bonded to a carbonyl carbon, and Each bicyclic aromatic hydrocarbon is optionally substituted with one or more substituents selected from the group consisting of: (R 11 ) p , wherein each R 11 is independently C 1-6 alkyl and each p is independently 0 to 7; (R 12 ) q , wherein each R 12 is independently C 1-3 alkoxy and each q is independently 0 to 7; and (R 13 ) r , wherein each R 13 is —F and each r is independently 0 to 7; wherein the sum of p, q and r is 7 or less.

16. The compound of claim 15 or a pharmaceutically acceptable salt thereof.

27. ​​The sum of p, q and r is 0 or the sum of p, q and r is 1, or 27. The compound of claim 26, or a pharmaceutically acceptable salt thereof, wherein each R<14> is independently H or methyl and R<15> is H.

28. R ５ and R ６ are the same, or R 7 and R 8 are the same, or a pharmaceutically acceptable salt thereof.

29. R ５ , R ６ , R ７ and R ８ The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein:

30. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein L1 is a straight-chain unsubstituted alkylene.

31. 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein L1 is propylene.

32. L2 and L3 are each independently a linear C １－５ 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, which is alkylene.

33. The compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized by one or more of the following: L2 and L3 are the same; L4 and L5 are the same; L6 and L7 are the same, and L8 and L9 are the same.

34. L4, L5, L6, L7, L8 and L9 are each —CH ２ - or L6, L7, L8 and L9 are each —CH 2 —, and L4 and L5 are absent; L4, L5, L8 and L9 are each —CH 2 —, and L6 and L7 are absent; 2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein L4, L5, L6 and L7 are each -CH 2 -, and L8 and L9 are absent.

35. R ３ and R ４ 10. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein each is independently H or methyl.

36. The compound of claim 1 selected from the group consisting of: 【Chemistry 14-1】 【Chemistry 14-2】 【Chemistry 14-3】 【Chemistry 14-4】 【Chemistry 14-5】 【Chemistry 14-6】 【Chemistry 14-7】 【Chemistry 14-8】 or a pharmaceutically acceptable salt thereof.

37. The compound is 【Chemistry 15】 or a pharmaceutically acceptable salt thereof.

38. The compound is 【Chemistry 16】 or a pharmaceutically acceptable salt thereof.

39. The compound is 【Chemistry 17】 or a pharmaceutically acceptable salt thereof.

40. The compound is [Chemistry 18] or a pharmaceutically acceptable salt thereof.

41. The compound is 【Chemistry 19】 37. The compound of claim 36, wherein:

42. The compound is 【Chemistry 20】 or a pharmaceutically acceptable salt thereof.

43. The compound is 【Chemistry 21】 or a pharmaceutically acceptable salt thereof.

44. The compound is 【Chemistry 22】 or a pharmaceutically acceptable salt thereof.

45. The compound is 【Chemistry 23】 or a pharmaceutically acceptable salt thereof.

46. The compound is 【Chemistry 24】 or a pharmaceutically acceptable salt thereof.

47. The compound is 【Chemistry 25】 or a pharmaceutically acceptable salt thereof.

48. A lipid composition comprising a nucleic acid and the compound of claim 1 or a pharmaceutically acceptable salt thereof.

49. The lipid composition of claim 48, wherein the nucleic acid is selected from siRNA, mRNA, self-replicating RNA, DNA plasmid, and antisense oligonucleotide, and the mRNA or the self-replicating RNA comprises a coding region encoding a therapeutic protein of interest.

50. 50. The lipid composition of claim 49, wherein the therapeutic protein of interest is an enzyme, an antibody, an antigen, a receptor, or a transporter.

51. 49. The lipid composition of claim 48, wherein the lipid composition comprises a liposome, a lipoplex, or a lipid nanoparticle.

52. The lipid composition of claim 48, wherein the lipid composition comprises lipid nanoparticles, the lipid nanoparticles comprising a plurality of ligands, each ligand independently being a compound described in claim 1 or a pharmaceutically acceptable salt thereof, and the plurality of ligands self-assemble to form the lipid nanoparticles comprising an interior and an exterior.

53. 53. The lipid composition of claim 52, wherein the lipid nanoparticles have an average particle size of less than about 100 nm.

54. 53. The lipid composition of claim 52, wherein the nucleic acid is in the interior.

55. 53. The lipid composition of claim 52, wherein the lipid composition further comprises one or more of the following: a. a helper lipid selected from dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC), and phosphatidylcholine (PC); b. Cholesterol, c. polyethylene glycol (PEG)-lipid conjugates, and d. One or more cryoprotectants.

56. The lipid composition of claim 48, comprising lipid nanoparticles comprising about 45 mol% to 65 mol% of a compound described in any one of claims 1 to 47, about 2 mol% to about 15 mol% of a helper lipid, about 20 mol% to about 42 mol% of cholesterol, and about 0.5 mol% to about 3 mol% of a PEG-lipid conjugate.

57. 52. The lipid composition of claim 51, wherein the lipid composition comprises lipid nanoparticles having a total lipid:nucleic acid weight ratio of about 50:1 to about 10:

1.

58. 49. The lipid composition of claim 48, wherein the lipid composition comprises lipid nanoparticles having a total lipid:nucleic acid weight ratio of about 31:1 to about 29:

1.

59. A pharmaceutical composition comprising the lipid composition of claim 48 and a pharmaceutically acceptable excipient.

60. 60. The pharmaceutical composition of claim 59, wherein the lipid composition comprises one or more of HEPES buffer at a pH of about 7.4 and about 2.0 mg / mL to about 4.0 mg / mL NaCl.

61. 57. The lipid composition of claim 56, wherein the lipid nanoparticles further comprise one or more cryoprotectants.

62. 62. The lipid composition of claim 61, wherein the one or more cryoprotectants are selected from sucrose, glycerol, or a combination of sucrose and glycerol.

63. 63. The lipid composition of claim 62, wherein the lipid nanoparticles comprise a combination of sucrose at a concentration of about 70 mg / mL to about 110 mg / mL and glycerol at a concentration of about 50 mg / mL to about 70 mg / mL.

64. 60. The lipid composition of claim 48, or the pharmaceutical composition of claim 59, for use in treating a disease in a subject in need thereof.

65. 65. The lipid or pharmaceutical composition of claim 64, wherein the lipid or pharmaceutical composition is administered intravenously or intramuscularly.

66. 60. The lipid composition of claim 48, or the pharmaceutical composition of claim 59, for use in expressing a protein or polypeptide in a target cell.

67. 67. The lipid or pharmaceutical composition of claim 66, wherein the protein or polypeptide is an antigen and expression of the antigen provides an in vivo immunogenic response.

68. 49. The lipid composition of claim 48 for use in delivering a nucleic acid to a subject in need thereof, wherein the nucleic acid is encapsulated in the lipid particle.

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