Ionizable cationic lipids for RNA delivery

Abstract

This disclosure describes compounds of formula (I) and pharmaceutically acceptable salts thereof. JPEG2026517868000423.jpg69116

Description

[Technical Field]

[0001] The embodiments described herein generally relate to lipids. Specifically, the embodiments described herein relate to novel lipids and lipid compositions that facilitate the intracellular delivery of bioactive and therapeutic molecules. [Background technology]

[0002] Various nucleic acid-based therapeutics for targeted delivery present challenges to lipid-based delivery vehicles. For example, nucleic acids are structurally diverse in size and type. Examples include DNA, plasmids, small interfering nucleic acids (siNAs) used in gene therapy, and microRNAs (miRNAs), antisense molecules, ribozymes, antagonists, and aptamers used in RNA interference (RNAi).

[0003] The design and use of cationic lipids and ionizable cationic lipids for inclusion in such lipid-based delivery vehicles has shown significant advantages. However, the use of these lipids can contribute to significant side effects when administered in vivo. One observed problem is that lipids accumulate in vivo due to their low biodegradability and clearance from target tissues. Another problem is that large amounts of lipids can cause adverse immunogenic effects, leading to discomfort in the target and a reduction in the therapeutic effect of the active ingredient. A third problem associated with many cationic lipids is that the therapeutic effect or potency is relatively low due to the low rate of effective delivery to the target. Finally, it is important that cationic lipids in delivery vehicles have specially adjusted pKas so that they can be formulated with nucleoside therapeutics to protect the nucleoside therapeutic from degradation during administration, as well as to release the therapeutic when the vehicle reaches its target. For this reason, the development of novel lipids that can meet the specific needs of lipid-nucleoside delivery systems is needed in this field.

[0004] Each of the following references is incorporated herein by reference 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 disclosing ionized cationic lipids for RNA delivery. International patent application PCT / US2016 / 069493, published as WO2017117530A1, disclosing ionizable cationic lipids; international patent application PCT / US2019 / 025246, published as WO2019191780A1, disclosing lipid particles for nucleic acid delivery; and U.S. patent application 16 / 823212, published as U.S.2020 / 0297634, disclosing a method for producing lipid-encapsulated RNA nanoparticles. [Overview of the Initiative]

[0005] This 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 obvious to those skilled in the art. Additional features and advantages of the subject art are set forth in the following description and may be partially apparent from the description or learned through the implementation of the subject art. The advantages of the subject art will be realized and achieved by the structures specifically pointed out in the written description and embodiments.

[0006] Both the general description above and the detailed description below should be understood as illustrative and explanatory, and intended to provide further explanation of the subject technology.

[0007] In some embodiments, the present disclosure relates to a compound of formula I, or a pharmaceutically acceptable salt thereof, [ka] (I) During the ceremony, R 1 and R2 each independently being H or C 1-6 being alkyl or R 1 and R 2 being joined to form a saturated heterocyclic ring, R 1 being linear C 1-4 alkylene, R 2 being -(CH2) m (X) n -, X being O, S, or NR 9 and R 9 being H or C 1-6 alkyl, m being 1, 2, 3, or 4, n being 0 or 1, L1 being linear C 1-6 alkylene optionally substituted with 1 to 3 methyl groups, Y being selected from the group consisting of

Chemical formula

[0008] In some embodiments, the present disclosure provides lipid nanoparticles comprising a plurality of ligands, wherein each ligand is independently a compound described herein, and the plurality of ligands self-assemble to form lipid nanoparticles including an internal and external structure.

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

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

[0011] In some embodiments, the present invention provides a method for delivering nucleic acids to a target requiring such delivery, comprising encapsulating a therapeutically effective amount of nucleic acid in lipid nanoparticles described herein, and administering the lipid nanoparticles to the target. [Modes for carrying out the invention]

[0012] I. Overview It will be understood that various configurations of the subject art will be readily apparent to those skilled in the art from this disclosure, in which various configurations of the subject art are shown and described as examples. Naturally, the subject art is capable of other and different configurations, and some of its details can be modified in various other ways, all without departing from the scope of the subject art. Therefore, the outline and the modes for carrying out the invention should be considered essentially illustrative and not as limitations.

[0013] The modes for carrying out the invention described below are intended to describe various configurations of the subject art and are not intended to represent only one configuration in which the subject art can be carried out. The modes for carrying out the invention include specific details for the purpose of providing a complete understanding of the subject art. However, it will be apparent to those skilled in the art that the subject art may be carried out without these specific details.

[0014] II. Definition In various parts of this specification, substituents of the compounds of the Disclosure are disclosed in groups or ranges. This disclosure is specifically intended to include any and all individual partial combinations of members of such groups and ranges. For example, "C 1-6 The term "alkyl" is specifically intended to disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl individually.

[0015] The terms “approximately” or “about” applied to one or more values ​​of interest refer to values ​​similar to the described reference values. In certain embodiments, unless otherwise stated or evident from the context, the terms “approximately” or “about” refer to a range of values ​​that fall 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 (greater than or less than) the described reference value (unless 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 shown to be contrary or otherwise obvious from the context. A claim or description containing “or” between one or more members of a group is deemed satisfied if one, two or more, or all of the members of the group are present in, used in, or otherwise related to a given product or process, unless shown to be contrary or otherwise obvious from the context. This disclosure includes embodiments in which exactly one member of the group is present in, used in, or otherwise related to a given product or process. This disclosure includes embodiments in which two or more, or all, of the members of the group are present in, used in, or otherwise related 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 defined below. The alkoxy group may have the general formula: alkyl-O-. With respect to alkyl groups, the alkoxy group is C 1-6 The alkoxy group may have any suitable number of carbon atoms. Examples of alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, butoxy, 2-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, and hexoxy. The alkoxy group may be further optionally substituted as defined herein.

[0018] As used herein, “alkyl” refers to a fully saturated (i.e., non-double or triple bonded) linear or branched hydrocarbon chain. An alkyl group may have 1 to 20 carbon atoms (wherein it appears herein, numerical ranges such as “1 to 20” refer to each integer within a 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., and may contain up to 20 carbon atoms; however, this definition also includes occurrences of the term “alkyl” where no numerical range is specified). An alkyl group may have 1 carbon, 2 carbon, 3 carbon, 4 carbon, 5 carbon, 6 carbon, 7 carbon, 8 carbon, 9 carbon, 10 carbon, 11 carbon, 12 carbon, 13 carbon, 14 carbon, 15 carbon, 16 carbon, 17 carbon, 18 carbon, 19 carbon, or 20 carbon atoms. Alkyl groups may be linear or branched. For example, an alkyl group may be 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 Alkyl groups can contain any number of carbon atoms, such as C. 1-4 It may be specified as "alkyl" or a similar specification. This is merely an example, but "C 1-4 The term "alkyl" indicates that the alkyl chain contains 1 to 4 carbon atoms. That is, 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 to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, and hexyl.

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

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

[0021] The term "amino" as used herein refers to -N(R N1 ) represents 2, and in the formula, each R N1 These are independently H, OH, NO2, N(R) N2 )2, SO2OR N2 SO2R N2 SOR N2 , N protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl, alkalil, cycloalkyl, alkylcycloalkyl, carboxyalkyl (e.g., optionally substituted with an O-protecting group, e.g., optionally substituted arylalkoxycarbonyl group or any of those specified herein), sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl or any of those specified herein), alkoxycarbonylalkyl (e.g., optionally substituted with an O-protecting group, e.g., optionally substituted arylalkoxycarbonyl group or any of those specified herein), heterocyclyl (e.g., heteroaryl), or alkylheterocyclyl (e.g., alkylheteroaryl), and these listed R N1 Each of the groups may be optionally substituted as defined herein for each group, or two R N1 By combining these, a heterocycline or an N protecting group can be formed, and each R N2 The amino group is independently H, alkyl, or aryl. The amino group of this disclosure may be an unsubstituted amino (i.e., -NH2) or a substituted amino (i.e., -N(R′)2). In preferred embodiments, the amino is -NH2 or -NHR N1 And in the formula, R N1 These 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 as described herein), alkoxycarbonylalkyl (e.g., t-butoxycarbonylalkyl), or aryl, each R N2 H, C1- 20 Alkyl (for example, C1-6 Alkyl), or C 1-10 It could be Ariel.

[0022] The term "anionic lipid" refers to lipids that are 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, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

[0023] As used herein, the term “aryl” means, alone or in combination, a carbocyclic aromatic system comprising one, two, or three rings, which may be joined together in a pendant-like manner or fused together. The term “aryl” encompasses aromatic radicals such as, for example, benzyl, phenyl, naphthyl, anthracenyl, phenanthryl, indanyl, indenyl, annurenyl, azrenyl, tetrahydronaphthyl, and biphenyl. 6-10 Aryls are C6 aryls, C7 aryls, C8 aryls, C9 aryls, or C 10 Contains aryl. In the embodiment, C 6-10 The aryl group is a monocyclic group, such as a phenyl group. In the embodiment, C 6-10 The aryl group is a bicyclic group such as a biphenyl, naphthyl, or indanyl group. The substituents on the aryl ring system described above are selected from the group of acceptable substituents listed below. "Arylene" refers to a divalent radical derived from an aryl group, either alone or as part of another substituent.

[0024] The phrase "at least one," placed before a series of items and accompanied by the terms "and" or "or" to separate any of the items, qualifies the list as a whole, rather than each member of the list (i.e., each item). The phrase "at least one" does not require the selection of at least one of each listed item; rather, it allows for the meaning of including at least one of any item and / or at least one of any combination of items and / or at least one of each of the items. For example, the phrases "at least one of A, B, and C" or "at least one of A, B, or C" refer 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] The terms “include,” “have,” or similar terms are used in the description or claims, and such terms are intended to be comprehensive in the same manner as the term “comprise” is used as a transitional term in the claims.

[0026] The term “cationic lipid” means amphiphilic lipids and salts thereof having a positive hydrophilic head group, one, two, or three or more hydrophobic fatty acid or fatty alkyl chains, and connectors between these two domains. Ionic cationic lipids or protonable cationic lipids are typically protonated (i.e., positively charged) at pH below their pKa and substantially neutral at pH above their pKa. Preferred ionic cationic lipids are lipids having a pKa lower than physiological pH, typically about 7.4. Cationic lipids of this disclosure may also be referred to as titrable cationic lipids. Cationic lipids can be “aminolipids” having a protonable tertiary amine (e.g., pH titrable) head group. Some exemplary aminolipids may include a C18 alkyl chain, as well as an ether linkage, ester linkage, or ketal linkage between the head group and the alkyl chain. Examples of 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 and may, but not require, include additional elements or processes. When the term “comprising” is used herein, the terms “consisting of” and “essentially consisting of” are also consequently included and initiated.

[0028] The term "commercially available chemicals" and the chemicals used in the examples described herein may be obtained from standard commercial sources, such as, for example, 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.), and Riedel de Examples include Haen (Hannover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland, OR), and Wako Chemicals USA, Inc. (Richmond, VAT).

[0029] The phrase "compounds described in chemical literature" can be identified through reference books and databases on chemical compounds and chemical reactions, as is known to those skilled in the art. Suitable reference books and articles that detail the synthesis of reactants useful for the preparation of the compounds disclosed herein, or provide references to articles describing the preparation of the compounds disclosed herein, include, for example, “Synthetic Organic Chemistry”, John Wiley and Sons, Inc. New York; SRSandler et al, “Organic Functional Group Preparations”, 2nd Ed., Academic Press, New York, 1983; HOHouse, “Modern Synthetic Reactions”, 2nd Ed., WABenjamin, Inc. Menlo Park, Calif., 1972; TLGlichrist, “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, 2001 is cited as an example, and specific and similar reactants can also be identified through the index of known chemicals created by the American Chemical Society's Chemical Abstract Service, which is available in most public and university libraries as well as online databases (for more information, contact the American Chemical Society (Washington, DC)). Known chemicals that are not commercially available in catalogs may be prepared by custom chemical synthesis companies, where many standard chemical suppliers (such as those listed above) offer custom synthesis services.

[0030] As used herein, the term “effective dose” of a drug means an amount sufficient to produce a beneficial or desired outcome, such as a clinical outcome, and therefore the “effective dose” depends on the context in which it is applied. For example, in the context of administering a drug to treat cancer, the effective dose of the drug is an amount sufficient to achieve, for example, a treatment of cancer as defined herein, compared to a response obtained without administration of the drug.

[0031] The term "fully encapsulated" means that the nucleic acid (e.g., mRNA) in the nucleic acid-lipid particles is not significantly degraded after exposure to serum or nuclease assays that would significantly degrade free RNA. Fully encapsulated means that, in a process 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%. "Fully encapsulated" also means that the nucleic acid-lipid particles do not rapidly degrade into their component parts upon in vivo administration.

[0032] The term "compound" means that it includes all stereoisomers, geometric isomers, tautomers, and isotopes of the structure shown.

[0033] As used herein, the terms “cycloalkyl” or “carbocyclic” mean, alone or in combination, saturated or partially saturated monocyclic or bicyclic alkyl radicals, where each cyclic portion comprises 3 to 12 carbon-carbon members, which may optionally be benzo-condensed ring systems, which may optionally be substituted as defined herein. In some embodiments, the cycloalkyl may comprise 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, and adamantyl. As used herein, “bicyclic” and “tricyclic” are intended to include, for example, decahydronaphthalene, octahydronaphthalene, and both saturated and partially unsaturated polycyclic (multicentric) condensed ring systems. The latter isomers are generally exemplified by bicyclo[1.1.1]pentane, campha, adamantane, and bicyclo[3.2.1]octane. In the embodiments, the cycloalkyl ring is a monocyclic ring having 3 to 8 carbon atoms. In the embodiments, the monocyclic ring has 3, 4, 5, 6, 7, or 8 carbon atoms. In the embodiments, the cycloalkyl ring is a bicyclic ring having 7 to 12 carbon atoms. In the embodiments, the bicyclic ring has 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, part, shipment, or payload.

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

[0036] The terms “heteroatom” or “ring heteroatom” as used herein mean that they include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), selenium (Se), and silicon (Si). In embodiments, the terms “heteroatom” or “ring heteroatom” mean that they include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0037] The terms “heteroaryl,” “heteroaromatic ring,” or “heteroaromatic group” refer to an aromatic group containing at least one heteroatom, such as N, O, or S, in which a nitrogen atom and a sulfur atom are optionally oxidized and the nitrogen atom is optionally quaternized. For this reason, the term “heteroaryl” includes fused ring heteroaryl groups (i.e., multiple rings fused together, where at least one of the fused rings is a heteroaromatic ring, and the multiple rings are bonded to the parent molecule via any atoms contained within the heteroaromatic rings of the multiple rings). A 5,6-fused ring heteroarylene refers to two rings fused together, one having 5 members and the other having 6 members, with at least one of the rings being a heteroaryl ring. Similarly, a 6,6-fused ring heteroarylene refers to two rings fused together, one having 6 members and the other having 6 members, with at least one of the rings being a heteroaryl ring. Furthermore, a 6,5-fused heteroarylene refers to two rings fused together, where one ring has 6 members and the other has 5 members, and at least one of the rings is a heteroaryl ring. The heteroaryl group can be bonded to the rest of the molecule via carbon or heteroatoms.Non-exclusive examples of heteroaryl groups include pyrrolyl, pyrazolyl, pyridadinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazoyl, benzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrzolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2- Examples include oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Each substituent of the heteroaryl ring system described above is selected from the group of acceptable substituents listed below. "Heteroarylene" means a divalent radical derived from a heteroaryl ring, either alone or as part of another substituent. The heteroaryl group substituent may be -O-bonded to the ring heteroatom nitrogen.

[0038] The term "hydrophobic lipid" refers to compounds having nonpolar groups, including but not limited to long-chain saturated aliphatic hydrocarbon groups and unsaturated aliphatic hydrocarbon groups, as well as such groups optionally substituted with one or more aromatic, alicyclic, or heterocyclic groups. Preferred examples include, but are not limited to, diacylglycerol, dialkylglycerol, NN-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0039] The term "lipid" refers to organic compounds that include fatty acid esters, are insoluble in water, but soluble in many organic solvents. Lipids are generally divided into at least three classes: (1) "simple lipids," which include fats, oils, and waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0040] The term "lipid delivery vehicle" refers to a lipid formulation that can be used to deliver therapeutic nucleic acids (e.g., mRNA) to a target site of interest (e.g., cells, tissues, organs, etc.). Lipid delivery vehicles can be nucleic acid lipid particles that can be formed from cationic lipids, non-cationic lipids (e.g., phospholipids), conjugated lipids to prevent particle aggregation (e.g., PEG lipids), and optionally from cholesterol. Typically, therapeutic nucleic acids (e.g., mRNA) may be encapsulated in the lipid portion of the particle, thereby protecting them from enzymatic degradation.

[0041] The term "lipid encapsulation" refers to lipid particles that provide therapeutic nucleic acids, such as mRNA, having complete encapsulation, partial encapsulation, or both. In preferred embodiments, the nucleic acid (e.g., mRNA) is completely encapsulated within the lipid particle.

[0042] The term "amphipathic lipid" refers to a lipid material in which the hydrophobic portion is oriented towards the hydrophobic phase, while the hydrophilic portion is oriented towards the aqueous phase. Hydrophilic properties derive from polarity or the presence of charged groups such as carbohydrates, phosphates, carboxylic acids, sulfats, aminos, sulfhydryls, nitros, hydroxyls, and other similar groups. Hydrophobicity can be given 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 amphiphilic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0043] As used herein, the term "heteroalkyl" refers, alone or in combination, to a stable linear or branched, or cyclic hydrocarbon radical, or a combination thereof, which may be fully saturated or 1 to 3 degrees unsaturated, and which consists of a specified number of carbon atoms and 1 to 3 heteroatoms selected from the group consisting of O, N, and S, in which case the nitrogen and sulfur atoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized (i.e., bonded to 4 groups). The O, N, and S of the heteroatoms may be located in any internal position of the heteroalkyl group. Up to two heteroatoms may be consecutive, for example, --CH2NHOCH3.

[0044] The term “linker” or “linking portion” refers to a group of atoms, for example, 10 to 100 atoms, which may consist of atoms or groups such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. The linker may be long enough not to interfere with its 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, amide, amino, ether, thioether, ester, alkyl, heteroalkyl, aryl, or heterocyclyl, each of which may be optionally substituted as described herein. Examples of linkers, but not limited to, unsaturated alkanes, polyethylene glycol (e.g., monomer 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, though not limited to them, include cleavable parts within the linker such as disulfide bonds (-SS-) or azo bonds (-N=N-), which can be cleaved using reducing agents or photolysis. Non-limited examples of selectively cleavable bonds include amide bonds that can be cleaved by the use of tris(2-carboxyethyl)phosphine (TCEP) or other reducing agents and / or photolysis, and ester bonds that can be cleaved by, for example, acidic hydrolysis or basic hydrolysis.

[0045] The term "mammal" means human or other mammal, or means human.

[0046] The term "messenger RNA" (mRNA) refers to any polynucleotide that codes for a protein or polypeptide of interest and can be translated in vitro, in vivo, in situ, or ex vivo to produce the coded protein or polypeptide of interest.

[0047] The term "modification" refers to a change in the state or structure of the molecules in this disclosure. Molecules may be modified in many ways, including chemically, structurally, and functionally. In one embodiment, the nucleic acid active ingredient is modified, for example, by the introduction of non-natural nucleosides and / or nucleotides, as it relates to natural ribonucleotides A, U, G, and C. Non-standard nucleotides, such as cap structures, may differ from the chemical structures of A, C, G, and U ribonucleotides, but are not considered "modifications."

[0048] The term "naturally occurring" means existing in nature without artificial assistance.

[0049] The term "patient" refers to a person who seeks or requires treatment, a patient who requires treatment, a person receiving treatment, a person scheduled to receive treatment, or a person receiving care from a professional trained in a particular disease or condition.

[0050] The phrase "arbitrarily substituted X" (e.g., arbitrarily substituted alkyl) is intended to be equivalent to "X, which is arbitrarily substituted" (e.g., "alkyl, which is arbitrarily substituted"). It is not intended to mean that the properties of "X" (e.g., alkyl) itself are arbitrarily substituted.

[0051] The term "pharmaceutically acceptable" is used herein to mean compounds, materials, compositions, and / or dosage forms suitable for use in contact with human and animal tissues, within the bounds of sound medical judgment, without excessive toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0052] When used herein, the term "pharmaceutically acceptable excipient" refers to any component other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving the active compound) that is substantially non-toxic and non-inflammatory in the patient. Examples of excipients include anti-adhesives, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), softeners, emulsifiers, fillers (diluents), film-forming agents or coatings, flavoring agents, tasters, lubricants (flow enhancers), preservatives, printing inks, absorbents, suspending agents or dispersants, sweeteners, and hydration water. Examples of excipients include, but are not limited to, butylhydroxytoluene (BHT), calcium carbonate, dibasic calcium phosphate, calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pre-gelatinized 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.

[0053] The term "pharmaceutically acceptable salt" refers to a derivative of the disclosed compound, where the parent compound is modified by converting an existing acidic or base moiety to its salt form (for example, by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral salts or organic salts of basic residues such as amines, and alkali salts or organic salts of acidic residues such as carboxylic acids. Representative acid addition salts include acetate, adipine, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, sulfonate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptone, glycerophosphate, hemisulfate, heptone, hexanoate, hydrobromide, hydrochloride, hydroiodide, and 2-hydroxyethanesulfone. Examples of alkali or alkaline earth metal salts include acid salts, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malons, methanesulfons, 2-naphthalenesulfons, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectins, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyans, toluenesulfons, undecanoates, and valersates. Typical alkali or alkaline earth metal salts include not only sodium, lithium, potassium, calcium, and magnesium, but also non-toxic ammonium, quaternary ammonium, and amine cations, but are not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine. Examples of pharmaceutically acceptable salts of this disclosure include conventional non-toxic salts of parent compounds formed from non-toxic inorganic or non-toxic organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from a parent compound containing a basic or acidic moiety by conventional chemical methods.Generally, such salts can be prepared by reacting the free acid or free base form of these compounds with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture of the two, generally preferred in non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile. A list 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, PHStahl and CGWermuth (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 whole.

[0054] The term "pharmacokinetics" refers to any one or more properties of a molecule or compound in relation to determining the fate of a substance administered to a living organism. Pharmacokinetics is divided into several areas, including the degree and rate of absorption, distribution, metabolism, and excretion. This is commonly referred to as ADME and includes: (A) Absorption is the process of a substance entering the bloodstream; (D) Distribution is the dispersion or flow of a substance throughout the body's fluids and tissues; (M) Metabolism (or biotransformation) is the irreversible conversion of a parent compound into its daughter metabolites; and (E) Excretion (or elimination) refers to the removal of a substance from the body. Rarely, some drugs accumulate irreversibly in body tissues.

[0055] The term "pharmaceutically acceptable solvate," as used herein, means a compound of the disclosure in which molecules of a preferred solvent are incorporated into the crystal lattice. The preferred solvent is physiologically tolerable at the administered dose. For example, the solvate may be prepared by crystallization, recrystallization, or precipitation from a solution containing an organic solvent, water, or a mixture thereof. Examples of preferred 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, and benzyl benzoate. When water is the solvent, the solvate is called a "hydrate."

[0056] The term "phosphate" is used in its ordinary sense as understood by those skilled in the art, and refers to its protonated form, for example, [ka] Includes.

[0057] As used herein, the terms “monophosphate,” “diphosphate,” and “triphosphate” are used in the ordinary sense as understood by those skilled in the art, and include protonated forms.

[0058] The term “prevention” means 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, functions, or clinical signs of a particular infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, functions, 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 progression of a pathology associated with an infection, disease, disorder, and / or condition.

[0059] The term “RNA” means a molecule containing at least one ribonucleotide residue. “Ribonucleotide” means a nucleotide having a hydroxyl group at the 2' position of the β-D-ribofuranose moiety. This term includes isolated RNA such as double-stranded RNA, single-stranded RNA, and partially purified RNA; essentially pure RNA; synthetic RNA; recombinant-produced RNA; and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations may include the addition of non-nucleotide substances to, for example, one or more nucleotides of RNA, either to the terminal or internal of interfering RNA. Nucleotides in the RNA molecules of this disclosure may also include non-standard nucleotides such as nucleotides not naturally occurring or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs may 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 multivalent RNA.

[0060] The terms “sample” or “biological sample” refer to a subset of its tissues, cells, or component parts (e.g., blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic blood, urine, vaginal fluid, and semen). A sample may further include, for example, plasma, serum, cerebrospinal fluid, lymph, skin, respiratory tract, intestine, and external sections of the reproductive tract, tears, saliva, milk, blood cells, tumors, organs, or a whole organism, or a subset of its tissues, cells, or component parts, or a homogeneous product, lysate, or extract prepared from a fraction or part thereof. A sample may further refer to a culture medium, such as a nutrient broth or gel, which may contain cellular components such as proteins or nucleic acid molecules.

[0061] The terms "significant" or "significantly" are used synonymously with "substantially."

[0062] The term "single dose" refers to a single dose / one administration / single route / single point of contact, i.e., the dose of any therapeutic agent administered in a single administration event.

[0063] The term "siRNA," or small interfering RNA, sometimes known as short interfering RNA or silencing RNA, typically refers to a class of double-stranded RNA non-coding RNA molecules, 18-27 base pairs in length, similar to miRNAs, that operate within the RNA interference (RNAi) pathway. This interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, thereby preventing translation.

[0064] The term solvate refers to the physical association of a compound of this disclosure with one or more solvent molecules. This physical association involves varying degrees of ionic bonding, including hydrogen bonding. In certain cases, solvates have the ability to be isolated, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "Solvate" encompasses both solution phases and isolateable solvates. Non-limiting examples of preferred solvates include ethanolates and methanolates.

[0065] The term "stable" refers to a compound that is robust enough to withstand isolation from a reaction mixture to a useful purity and, preferably, can be formulated into an effective therapeutic agent.

[0066] The terms "stabilize," "stabilized," and "stabilized region" all mean to make something stable or to become stable.

[0067] The term "substitution" means substitution by a specific group other than hydrogen, or substitution by one or more groups, parts, or radicals, each of which may be identical or different, selected independently.

[0068] The term "substantially" refers to a qualitative condition indicating the range or extent of the sum or approximate sum of the characteristic or property of interest. Those skilled in the field of biology will understand that biological and chemical phenomena, if present, rarely complete and / or proceed to completeness, or achieve or avoid absolute results. Therefore, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0069] The phrase "substantially equal" means ±2% in terms of the time difference between doses.

[0070] The phrase "substantially simultaneously" means, in relation to multiple doses, within 2 seconds.

[0071] The phrase “suffering from” refers to an individual who is “suffering from” a disease, disorder, and / or condition, and is diagnosed with or exhibiting one or more symptoms of the disease, disorder, and / or condition.

[0072] The term “susceptibility” refers to individuals who are “highly susceptible” to a disease, and / or condition, even if the disease, disorder, and / or condition is not diagnosed and / or they do not exhibit symptoms of the disease, disorder, and / or condition, but who are prone to developing the disease or its symptoms. In some embodiments, individuals who are highly susceptible to a disease, disorder, and / or condition (e.g., cancer) may be characterized by one or more of the following: (1) gene mutations associated with the development of the disease, disorder, and / or condition; (2) gene polymorphisms associated with the development of the disease, disorder, and / or condition; (3) increased and / or decreased expression and / or activity of proteins and / or nucleic acids associated with the disease, disorder, and / or condition; (4) habits and / or lifestyles associated with the development of the disease, disorder, and / or condition; (5) family history of the disease, disorder, and / or condition; and (6) exposure to and / or infection of microorganisms associated with the development of the disease, disorder, and / or condition. In some embodiments, individuals who are highly susceptible to a disease, disorder, and / or condition develop the disease, disorder, and / or condition. In some embodiments, individuals susceptible to a disease, disorder, and / or condition do not develop the disease, disorder, and / or condition.

[0073] The term "synthesis" means being produced, prepared, and / or manufactured by human hands. The synthesis of the polynucleotides or polypeptides or other molecules in this disclosure may be chemical or enzymatic.

[0074] The term "therapeutic agent" refers to any agent that, when administered to a subject, has a therapeutic effect, a diagnostic effect, and / or a preventive effect, and / or induces a desired biological and / or pharmacological effect.

[0075] The term "therapeutic dose" means the amount of a drug delivered (e.g., nucleic acids, drugs, therapeutic agents, diagnostic agents, prophylactic agents, etc.) that, when administered to a subject suffering from or highly susceptible to an infection, disease, disorder, and / or condition, is sufficient to treat, improve, diagnose, prevent, and / or delay the onset of an infection, disease, disorder, and / or condition.

[0076] The term "therapeutically effective outcome" means an outcome in which, in a person suffering from or highly susceptible to an infection, disease, disorder, and / or condition, the infection, disease, disorder, and / or condition is treated, its symptoms are improved, it is diagnosed, prevented, and / or its onset is delayed.

[0077] The term "total daily dose" refers to the amount given or prescribed over a 24-hour period. This may be administered as a single unit dose.

[0078] The term “treatment” means partial or complete relief, improvement, mitigation, delay of onset, inhibition of progression, reduction of severity, and / or reduction of incidence of one or more symptoms or characteristics of a particular infection, disease, disorder, and / or condition. For example, “treatment” of cancer may mean inhibiting the survival, growth, and / or spread of the tumor. Treatment may be administered to subjects who show no signs of the disease, disorder, and / or condition, and / or subjects who show only early signs of the disease, disorder, and / or condition, for the purpose of reducing the risk of developing pathologies associated with the disease, disorder, and / or condition.

[0079] The term "unmodified" refers to any substance, compound, or molecule before it is altered in any way. Unmodified may, but not necessarily, refer to a wild-type or native biomolecule. A molecule may undergo a series of modifications, in which case each modified molecule may function as an "unmodified" initiation molecule for subsequent modifications.

[0080] The compounds described herein may be asymmetric (e.g., having one or more stereocenters). Unless otherwise indicated, all stereoisomers, such as enantiomers and diastereomers, are intended. Compounds of the disclosure containing asymmetrically substituted carbon atoms can be isolated in optically active forms or racemates. Methods for preparing optically active forms from optically active starting materials are known in the art, such as by racemic mixture resolution or by enantioselective and / or stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, and similar materials can also be present in the compounds described herein, and all such stable isomers are intended in this disclosure. Cis and trans geometric isomers of the compounds of the disclosure are described and may be isolated as mixtures of isomers or as separated isomers.

[0081] The compounds of this disclosure also include tautomers. Tautomers result from the swapping of single bonds with adjacent double bonds and the simultaneous transfer of protons. Tautomers include prototropic tautomers, which are isomeric protonated states having the same empirical formula and total charge. Examples of prototropic tautomers include ketone-enol pairs, amide-imoid acid pairs, lactam-lactim pairs, enamine-imine pairs, and cyclic forms, in which the protons can occupy two or more positions in heterocyclic systems such as 1H- and 3H-imidazoles, 1H-, 2H- and 4H-1,2,4-triazoles, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomers may be in equilibrium or sterically locked into one form by appropriate substitution.

[0082] The compounds of this disclosure also include all isotopes of atoms resulting from intermediate or final compounds. “Isotopes” refer to atoms with the same number of atoms but different mass numbers, resulting from different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium.

[0083] The compounds and salts of this disclosure can be prepared in combination with a solvent or water molecules to form solvates and hydrates by routine methods.

[0084] The term "half-life" refers to the time required for a quantity of nucleic acid or protein, such as its concentration or activity, to decrease to half of its value measured at the beginning of a certain period.

[0085] The term "in vitro" refers to events that occur in an artificial environment, such as a test tube or reaction vessel, a cell culture, or a petri dish, rather than within a living organism (e.g., an animal, plant, or microorganism).

[0086] The term "in vivo" refers to events that occur within living organisms (for example, animals, plants, or microorganisms, or their cells or tissues).

[0087] The term "monomer" refers to a single unit, such as 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 an unimmobilized nucleic acid, i.e., a UNA monomer.

[0088] The term "neutral lipids" refers to lipid species that exist in either an uncharged or neutral zwitterionic form at a selected pH. Examples of such lipids at physiological pH include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebroside, and diacylglycerol.

[0089] The term "noncationic lipid" means amphiphilic lipid, neutral lipid, or anionic lipid, as used herein.

[0090] The terms “subject” or “patient” refer to any living organism to which the compositions according to this disclosure may be administered, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants.

[0091] The term “translatable” may be used interchangeably with the term “expressable” and refers to the ability of a polynucleotide or 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 polypeptides. In translation, messenger RNA (mRNA) is decoded by tRNA in a ribosome complex to produce a specific amino acid chain or polypeptide. Furthermore, as used herein in relation to oligomers, the term “translatable” means that at least a portion of an oligomer, for example, the coding region of the oligomer sequence (also known as the coding sequence or CDS), has the ability to be converted into a protein or a fragment thereof.

[0092] The abbreviations used herein are defined as follows: "1×" once, "2×" twice, "3×" three times, "℃" degrees Celsius, "eq" equivalent, "g" grams, "mg" milligrams, "L" liters, "mL" milliliters, "μL" microliters, "N" normal, "M" mole, "mmol" millimoles, "min" minutes, "h" hours, "rt" room temperature, "RT" retention time, "RBF" round-bottom flask, "atm" ambient temperature, "psi" pounds per square inch, "conc." concentration, "RCM" ring-closing metathesis, "sat" or "sat'd" saturation, "SFC" supercritical fluid chromatography, "MW" molecular weight, "mp" melting point, "ee" enantiomeric excess, "MS" or "Mass Spec" Mass Spectrometry, ESI "Electrospray Ionization Mass Spectrometry", HR "High Resolution", HRMS "High Resolution Mass Spectrometry", LCMS "Liquid Chromatography Mass Spectrometry", HPLC "High Pressure Liquid Chromatography", RP "Reverse Phase HPLC", TLC or TLC "Thin Layer Chromatography", NMR "Nuclear Magnetic Resonance Spectroscopy", nOe "Nuclear Overhauser Effect Spectroscopy", 1 "H" is a proton, "δ" is a delta, "s" is a single line, "d" is a double line, "t" is a triple line, "q" is a quadruple line, "m" is a multiline, "br" is a broad line, "Hz" is a Hertz, and "α", "β", "R", "S", "E", and "Z" are stereochemical names well known to those skilled in the art. [Table 1-1] [Table 1-2] [Table 1-3]

[0093] This disclosure is described in connection with certain embodiments, and many details are set forth for purposes of illustration. However, the disclosure includes additional embodiments, and it will be apparent to those skilled in the art that some of the details described herein may vary significantly without departing from the disclosure. The disclosure includes such additional embodiments, modifications, and equivalents. In particular, the disclosure includes any combination of features, terms, or elements of various exemplary components and examples.

[0094] III. Compounds In some embodiments, the disclosure is a compound of Formula I, or a pharmaceutically acceptable salt thereof, wherein

Chemical formula

Chemical formula

[0095] In some embodiments, R 1 is H or C 1-6 It is alkyl, R 2 C 1-6 Alkyl or R 1 and R 2 However, they join together to form the saturated heterocyclic ring. In some embodiments, R 1 is H or C 1-6 It is alkyl, R 2 C 1-6 It is alkyl. In some embodiments, R 1 and R 2 However, they join together to form a saturated heterocyclic ring.

[0096] In some embodiments, Y is selected from the group consisting of the following: [ka]

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

[0098] In some embodiments, Y is as follows: [ka]

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

[0100] In some embodiments, at least one of R1 and R2 is H, and L1 is -CH2- or -CH2CH2-.

[0101] In some embodiments, R 1 and R 2 Each of them is independent of C 1-6 It is alkyl.

[0102] In some embodiments, R 1 and R 2 Each of them is independent of C 1-3 It is alkyl.

[0103] In some embodiments, R 1 and R 2 Each of these is methyl.

[0104] In some embodiments, R 1 and R 2 These are joined together to form the complex ring. In some embodiments, the complex ring is selected from the group consisting of the following: [ka] In the formula, each asterisk (*) indicates an atom bonded to L1.

[0105] In some embodiments, the complex ring is selected from the following group: [ka]

[0106] In some embodiments, the complex ring is selected from the following group: [ka]

[0107] In some embodiments, the complex algebra is as follows: [ka]

[0108] In some embodiments, the complex algebra is as follows: [ka]

[0109] In some embodiments, the complex algebra is as follows: [ka]

[0110] In some embodiments, the complex algebra is as follows: [ka]

[0111] In some embodiments, the complex algebra is as follows: [ka]

[0112] In some embodiments, the complex algebra is as follows: [ka]

[0113] In some embodiments, the complex algebra is as follows: [ka]

[0114] In some embodiments, the complex algebra is as follows: [ka]

[0115] In some embodiments, the heterocyclic ring is as follows.

Chemical formula

[0116] In some embodiments, the heterocyclic ring is as follows.

Chemical formula

[0117] In some embodiments, the heterocyclic ring is as follows.

Chemical formula

[0118] In some embodiments, the heterocyclic ring is as follows.

Chemical formula

[0119] In some embodiments, the heterocyclic ring is as follows.

Chemical formula

[0120] In some embodiments, R 5 , R 6 , R 7 , and R 8 are each independently a linear C 1-8 alkyl, and each said linear C 1-8 alkyl is hereinafter C 6-10 aryl, and each said C 6-10 aryl is one or more C 1-6 alkyl optionally substituted with one or more C 6-10A monocyclic or bicyclic aromatic hydrocarbon that is optionally substituted with an aryl group, C 6-10 Aryl, and A 6-10 member heteroaryl, wherein each of the 6-10 member heteroaryls contains one or more C 1-6 It is optionally substituted with one or more substituents selected from 6-10 membered heteroaryls, which are monocyclic or bicyclic aromatic systems optionally substituted with alkyl groups. In some embodiments, R 5 , R 6 , R 7 , and R 8 Each of them is independently a linear C 1-8 It is alkyl. In some embodiments, the nucleus is the linear C 1-8 Alkyl is, as follows, C 6-10 Aaryl, and each of the C 6-10 A aryl is one or more C 1-6 One or more C atoms optionally substituted with alkyl groups. 6-10 C is a monocyclic or bicyclic aromatic hydrocarbon that is optionally substituted with an aryl group. 6-10 Aryls and 6-10 membered heteroaryls, wherein each of the 6-10 membered heteroaryls contains one or more C 1-6 It is substituted with one or more substituents selected from 6- to 10-membered heteroaryls, which are monocyclic or bicyclic aromatic systems optionally substituted with alkyl groups.

[0121] In some embodiments, R 5 , R 6 , R 7 , and R 8 Each is independently C 6-10 It is an aryl, and each C 6-10 Aryls are monocyclic or bicyclic aromatic hydrocarbons. In some embodiments, each C 6-10 A aryl is one or more C 1-6 One or more C atoms optionally substituted with alkyl groups. 6-10 It is a monocyclic or bicyclic aromatic hydrocarbon optionally substituted with an aryl group. In some embodiments, each C 6-10 A aryl is one or more C 1-6One or more C atoms optionally substituted with alkyl groups. 6-10 It is a monocyclic aromatic hydrocarbon optionally substituted with an aryl group. In some embodiments, each C 6-10 A aryl is one or more C 1-6 One or more C atoms optionally substituted with alkyl groups. 6-10 It is a bicyclic aromatic hydrocarbon that is optionally substituted with an aryl group.

[0122] In some embodiments, R 5 , R 6 , R 7 , and R 8 Each of these is independently a 6-10 member heteroaryl, and each of these 6-10 member heteroaryls contains one or more C 1-6 These are monocyclic or bicyclic aromatic systems that are optionally substituted with alkyl groups.

[0123] In some embodiments, R 5 and R 6 They are the same.

[0124] In some embodiments, R 7 and R 8 They are the same.

[0125] In some embodiments, L1 is a linear unsubstituted alkylene.

[0126] In some embodiments, L1 is propylene.

[0127] In some embodiments, L2 and L3 are each independently linear C 1-5 It is alkylene.

[0128] In some embodiments, L2 and L3 are the same.

[0129] In some embodiments, L4 and L5 are the same.

[0130] In some embodiments, L6 and L7 are the same.

[0131] In some embodiments, L8 and L9 are the same.

[0132] In some embodiments, L4, L5, L6, L7, L8, and L9 are each -CH2-.

[0133] In some embodiments, L6, L7, L8, and L9 are each -CH2-, and L4 and L5 are absent.

[0134] In some embodiments, L4, L5, L8, and L9 are each -CH2-, and L6 and L7 are absent.

[0135] In some embodiments, L4, L5, L6, and L7 are each -CH2-, and L8 and L9 are absent.

[0136] 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.

[0137] In some embodiments, the compound is

Chemical formula

[0138] In some embodiments, the present disclosure is

Chemical formula

[0139] In some embodiments, the Disclosure provides a lipid composition comprising a nucleic acid and a compound of the 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 siRNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is self-replicating RNA. In some embodiments, the nucleic acid is DNA plasmid. In some embodiments, the nucleic acid is antisense oligonucleotide.

[0140] In some embodiments, the nucleic acid is mRNA or self-replicating RNA containing a coding region encoding the therapeutic protein of interest. In some embodiments, the nucleic acid is mRNA containing a coding region encoding the therapeutic protein of interest. In some embodiments, the nucleic acid is self-replicating RNA containing a coding region encoding the 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 an enzyme. In some embodiments, the therapeutic protein of interest is an antibody. In some embodiments, the therapeutic protein of interest is an antigen. In some embodiments, the therapeutic protein of interest is a receptor. In some embodiments, the therapeutic protein of interest is a 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 gene-editing enzyme is TALEN. In some embodiments, the gene-editing enzyme is CRISPR. In some embodiments, the gene-editing enzyme is meganuclease. In some embodiments, the gene editing enzyme is a zinc finger nuclease.

[0141] In some embodiments, the lipid composition comprises liposomes, lipoplexes, or lipid nanoparticles. In some embodiments, the lipid composition comprises liposomes. In some embodiments, the lipid composition comprises lipoplexes. In some embodiments, the lipid composition comprises lipid nanoparticles.

[0142] In some embodiments, the Disclosure provides lipid nanoparticles comprising multiple ligands, each ligand independently being a compound of the Disclosure. In some embodiments, the multiple ligands self-assemble to form lipid nanoparticles comprising internal and external components.

[0143] In some embodiments, the average particle size of the lipid nanoparticles is less than approximately 100 nm. In some embodiments, the average particle size of the lipid nanoparticles is approximately 55 nm to approximately 85 nm.

[0144] In some embodiments, the lipid nanoparticles further comprise encapsulated nucleic acids. In some embodiments, the nucleic acid is selected from siRNA, mRNA, self-replicating RNA, DNA plasmids, and antisense oligonucleotides. In some embodiments, the nucleic acid is mRNA or self-replicating RNA containing a coding region encoding the therapeutic protein of interest. In some embodiments, the nucleic acid is siRNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is self-replicating RNA. In some embodiments, the nucleic acid is a DNA plasmid. In some embodiments, the nucleic acid is an antisense oligonucleotide. In some embodiments, the nucleic acid is mRNA containing a coding region encoding the therapeutic protein of interest. In some embodiments, the nucleic acid is self-replicating RNA containing a coding region encoding the 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 an enzyme. In some embodiments, the therapeutic protein of interest is an antibody. In some embodiments, the therapeutic protein of interest is an antigen. In some embodiments, the therapeutic protein of interest is a receptor. In some embodiments, the therapeutic protein of interest is a 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 gene-editing enzyme is TALEN. In some embodiments, the gene-editing enzyme is CRISPR. In some embodiments, the gene-editing enzyme is meganuclease. In some embodiments, the gene-editing enzyme is zinc finger nuclease.

[0145] In some embodiments, the lipid nanoparticles further comprise a helper lipid selected from dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC), and phosphatidylcholine (PC). In some embodiments, the helper lipid is DOPE. In some embodiments, the helper lipid is DMPC. In some embodiments, the helper lipid is DSPC. In some embodiments, the helper lipid is DMPG. In some embodiments, the helper lipid is DPPC. In some embodiments, the helper lipid is PC.

[0146] In some embodiments, the lipid nanoparticles further contain cholesterol.

[0147] In some embodiments, the lipid nanoparticles further comprise polyethylene glycol (PEG)-lipid conjugates. In some embodiments, the PEG-lipid conjugate is PEG-DMG. In some embodiments, the PEG-DMG is PEG2000-DMG.

[0148] In some embodiments, the lipid nanoparticles comprise about 45 mol% to 65 mol% of the compound of the 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.

[0149] In some embodiments, the lipid nanoparticles comprise about 50 mol% to 61 mol% of the compound of the 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.

[0150] In some embodiments, the lipid nanoparticles comprise about 56 mol% to about 58 mol% of the compound of the 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 PEG-lipid conjugate.

[0151] In some embodiments, the lipid nanoparticles have a total lipid:nucleotide weight ratio of about 50:1 to about 10:1. In some embodiments, the lipid nanoparticles have a total lipid:nucleotide weight ratio of about 40:1 to about 20:1. In some embodiments, the lipid nanoparticles have a total lipid:nucleotide weight ratio of about 35:1 to about 25:1. In some embodiments, the lipid nanoparticles have a total lipid:nucleotide weight ratio of about 32:1 to about 28:1. In some embodiments, the lipid nanoparticles have a total lipid:nucleotide weight ratio of about 31:1 to about 29:1.

[0152] In some embodiments, the Disclosure provides a pharmaceutical composition comprising a compound of the Disclosure or lipid nanoparticles of the Disclosure and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is a lyophilized composition. In some embodiments, the pharmaceutical composition comprises a HEPES buffer at a pH of about 7.4. In some embodiments, the HEPES buffer is concentrated at a concentration of about 7 mg / mL to about 15 mg / mL. In some embodiments, the pharmaceutical composition further comprises about 2.0 mg / mL to about 4.0 mg / mL of NaCl. In some embodiments, the pharmaceutical composition further comprises one or more cryoprotective agents. In some embodiments, one or more cryoprotective agents are selected from sucrose, glycerol, or a combination of sucrose and glycerol. In some embodiments, one or more cryoprotective agents are sucrose. In some embodiments, one or more cryoprotective agents are glycerol. In some embodiments, one or more cryoprotective agents are a combination of sucrose and glycerol. In some embodiments, the pharmaceutical composition comprises 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.

[0153] IV. Lipid preparations and nanoparticles Lipid-based preparations Therapies based on intracellular delivery of nucleic acids to target cells face both extracellular and intracellular barriers. Indeed, naked nucleic acid substances cannot be readily administered systemically due to their toxicity, low stability in serum, rapid renal clearance, reduced uptake by target cells, uptake by phagocytic cells, and their ability to activate immune responses—all characteristics that hinder their clinical development. When exogenous nucleic acid substances (e.g., mRNA) enter the human biological system, they are recognized as foreign pathogens by the reticuloendothelial system (RES) and removed from the bloodstream before they have a chance to encounter target cells in or outside the vascular system. The half-life of naked nucleic acids in the bloodstream has been reported to be approximately a few minutes (Kawabata K, Takakura Y, Hashida M Pharm Res. 1995 Jun;12(6):825-30). Chemical modification and appropriate delivery methods can reduce uptake by the RES and protect nucleic acids from degradation by ubiquitous nucleases, thereby increasing the stability and efficacy of nucleic acid-based therapies. In addition, RNA or DNA are anionic hydrophilic polymers that are anionic even on their surface, which is undesirable for cellular uptake. Therefore, the success of nucleic acid-based therapies largely depends on the development of vehicles or vectors that can efficiently and effectively deliver genetic material to target cells and achieve sufficient levels of expression with minimal toxicity and in vivo.

[0154] Furthermore, during internal translocation into target cells, nucleic acid delivery vectors face challenges posed by intracellular barriers, including endosomal uptake, lysosomal degradation, unpacking of nucleic acids from the vector, translocation across the nuclear membrane (for DNA), and release into the cytoplasm (for RNA). Therefore, successful nucleic acid-based therapies depend on the vector's ability to deliver nucleic acids to target sites inside the cell in order to achieve sufficient levels of desired activity, such as gene expression.

[0155] While some gene therapies can successfully utilize 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 mRNA-containing nucleic acid therapeutics via lipid formulations remains 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 that deliver mRNA encoding the COVID-19 spike protein have shown potent protective capabilities. These lipid-based mRNA vaccines include BNT162b2 from Pfizer and BioNtech, and mRNA-1273 from Moderna, which have received emergency use authorization worldwide.

[0156] Some lipid formulation delivery vehicles recognized in the art for nucleic acid therapeutics include, in various embodiments, polymeric carriers (such as polyethyleneimine (PEI), lipid nanoparticles, and liposomes), nanoliposomes, ceramide-containing nanoliposomes, multivesicle liposomes, proteoliposomes, exosomes of both natural and synthetic origin, natural, synthetic, and semi-synthetic layered structures, nanoparticles, micelles, and emulsions. Because these lipid formulations can differ in their structure and composition and are expected to be part of a rapidly developing field, several different terms are used in the art to describe a single type of delivery vehicle. At the same time, the terminology of lipid formulations has changed throughout the scientific literature in terms of their intended meaning, and this inconsistent use has caused confusion regarding the precise meaning of some terms of lipid formulations. Among several potential lipid formulations, liposomes, cationic liposomes, and lipid nanoparticles are described in detail and specifically for the purposes of this disclosure and are defined herein.

[0157] Liposomes Conventional liposomes are vesicles consisting of at least one bilayer and an internal aqueous compartment. The liposome bilayer membrane is typically formed by amphiphilic molecules, such as synthetic or naturally occurring lipids, containing spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16:307-321, 1998). The liposome bilayer membrane 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 liposomal drugs. Methods for preparing liposome compositions are known in the art and are within the skill of those skilled in the art.

[0158] Liposomes with only one bilayer are called monolayers, while liposomes with two or more bilayers are called multilayers. The most common types of liposomes are small monolayer vesicles (SUVs), large monolayer vesicles (LUVs), and multilayer vesicles (MLVs). In contrast to liposomes, lysosomes, micelles, and reverse micelles consist of a single lipid monolayer. Generally, liposomes are thought to have a single internal compartment, but some formulations can be multivesicular liposomes (MVLs) consisting of numerous discontinuous internal aqueous compartments separated by several non-concentric lipid bilayers.

[0159] Liposomes have long been recognized as drug delivery vehicles due to their excellent biocompatibility, considering that they 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 carry hydrophobic and / or hydrophilic molecules. When liposomes are used to support nucleic acids such as RNA, the nucleic acids are contained within the liposome compartment in the aqueous phase.

[0160] 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 properties of liposomes described above, the positively charged moieties of cationic lipids used in cationic liposomes offer several advantages and some unique structural features. For example, the lipophilic moieties of cationic lipids are hydrophobic and therefore orient themselves away from the aqueous interior of the liposome and associate with other nonpolar and hydrophobic species. Conversely, the cationic moieties associate with aqueous media and, more importantly, with polar molecules and species that can complexe within the aqueous interior of cationic liposomes. For these reasons, cationic liposomes are being increasingly studied 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 applications. Cationic lipids suitable for use in cationic liposomes are listed below herein.

[0161] 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 a solid phase. Therefore, unlike liposomes, lipid nanoparticles do not have an aqueous or other liquid phase inside; rather, the 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 dispersion of shape and size. While there is some overlap in the size requirements for classifying lipid particles as nanoparticles, there is some overlap in the fact that lipid nanoparticles can have diameters ranging from 10 nm to 1000 nm. However, more generally, they are considered to be smaller than 120 nm or smaller than 100 nm.

[0162] For lipid nanoparticle nucleic acid delivery systems, the lipid shell can be formulated to contain ionic cationic lipids that can complex with and associate with the negatively charged backbone of the nucleic acid core. Ionic cationic lipids with apparent pKa values ​​less than approximately 7 have the advantage of complexing with the negatively charged backbone of the nucleic acid and providing cationic lipids for loading onto lipid nanoparticles at a pH value less than the pKa of positively charged ionized lipids. Subsequently, at physiological pH values, lipid nanoparticles can adapt to a relatively neutral external environment, enabling a significant increase in the circulating 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 therapeutic drug delivery, and low levels of cytotoxicity and immunogenicity.

[0163] Prior to the development of lipid nanoparticle delivery systems for nucleic acids, cationic lipids have been widely studied as synthetic materials for the delivery of nucleic acid drugs. These early efforts involved mixing nucleic acids together at physiological pH and then condensing them with cationic lipids to form lipid-nucleic acid complexes known as lipoplexes. However, lipoplexes have proven unstable and have been characterized by a wide size distribution ranging from submicron to several microns. Lipoplexes such as LIPOFECTAMINE® reagents have found considerable utility for in vitro transfection. However, these first-generation lipoplexes have not been proven useful in vivo. Their large particle size and positive charge (conferred by cationic lipids) result in rapid plasma clearance, hemolysis, and other toxicities, as well as immune system activation.

[0164] In some embodiments, the lipid nanoparticles are lipids of formula I, [ka] (I) During the ceremony, R 1 and R 2 However, each independently, H or C 1-6 It is alkyl, or R 1 and R 2 However, they join together to form a saturated heterocyclic ring, R 1 However, linear C 1-4 It is alkylene, R 2 However, -(CH2) m (X) n -and, X is O, S, or NR 9 And R 9 However, H or C 1-6 It is alkyl, m is 1, 2, 3, or 4. n is 0 or 1, L1 is a linear C chain that is optionally substituted with 1 to 3 methyl groups. 1-6 It is alkylene, Y is selected from the following group: [ka] During the ceremony, Each asterisk (*) indicates an atom bonded to L2 and L3. R 10 However, H or C 1-6 It is alkyl, L2 and L3 are each independently linear C 1-8 It is alkylene, L4, L5, L6, L7, L8, and L9 are either non-existent or -CH2-, however, At least two of L4, L6, and L8 are -CH2-, At least two of L5, L7, and L9 are -CH2-, R 3 and R 4 However, each is independently H, methyl, or ethyl. R 5 , R 6 , R 7 , and R 8However, each is independent, as follows: Linear C 1-20 Alkyl, and each of the linear C 1-20 Alkyl, below, C 6-10 Aaryl, and each of the C 6-10 A aryl is one or more C 1-6 One or more C atoms optionally substituted with alkyl groups. 6-10 A monocyclic or bicyclic aromatic hydrocarbon that is optionally substituted with an aryl group, C 6-10 Ariel, A 6-10 member heteroaryl, wherein each of the 6-10 member heteroaryls contains one or more C 1-6 Linear carbon atoms are optionally substituted with one or more substituents selected from the group consisting of monocyclic or bicyclic aromatic systems that are optionally substituted with alkyl groups, and 6-10 membered heteroaryl groups. 1-20 Alkyl, C 6-10 Aaryl, and each of the C 6-10 A aryl is one or more C 1-6 One or more C atoms optionally substituted with alkyl groups. 6-10 A monocyclic or bicyclic aromatic hydrocarbon that is optionally substituted with an aryl group, C 6-10 Aryl, and A 6-10 member heteroaryl, wherein each of the 6-10 member heteroaryls contains one or more C 1-6 The present invention provides lipids selected from the group consisting of 6-10 membered heteroaryls, which are monocyclic or bicyclic aromatic systems optionally substituted with alkyl groups.

[0165] In some embodiments, one or more lipids listed herein may be explicitly excluded.

[0166] In some embodiments, the present disclosure provides lipid nanoparticles comprising a plurality of ligands, wherein each ligand is independently a compound described herein, and the plurality of ligands self-assemble to form lipid nanoparticles including an internal and external structure.

[0167] In some embodiments, the average particle size of the lipid nanoparticles is about 100 nm. In some embodiments, the average diameter of the lipid nanoparticles is less than about 100 nm. In some embodiments, the average particle size of the lipid nanoparticles is about 40 nm to about 100 nm. In some embodiments, the average particle size of the lipid nanoparticles is about 50 nm to about 90 nm. In some embodiments, the average particle size of the lipid nanoparticles is about 55 nm to about 85 nm.

[0168] In some embodiments, the lipid nanoparticles further contain nucleic acids. In some embodiments, the nucleic acid is selected from siRNA, mRNA, self-replicating RNA, DNA plasmids, and antisense oligonucleotides. In some embodiments, the nucleic acid is mRNA or self-replicating RNA containing a coding region encoding the therapeutic protein of interest. In some embodiments, the therapeutic protein of interest is an enzyme, and an 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.

[0169] In some embodiments, the lipid nanoparticles further contain siRNA and mRNA.

[0170] In some embodiments, the lipid nanoparticles further comprise the helper lipids described below. In some embodiments, the lipid nanoparticles further comprise the PEG-lipid conjugates described herein.

[0171] In some embodiments, the lipid nanoparticles comprise about 45 mol% to 65 mol% of the compound disclosed, about 2 mol% to about 15 mol% of helper lipids, about 20 mol% to about 42 mol% of cholesterol, and about 0.5 mol% to about 3 mol% of PEG-lipid conjugates. In some embodiments, the lipid nanoparticles comprise about 50 mol% to 61 mol% of the compound disclosed, about 5 mol% to about 9 mol% of helper lipids, about 29 mol% to about 38 mol% of cholesterol, and about 1 mol% to about 2 mol% of PEG-lipid conjugates. In some embodiments, the lipid nanoparticles comprise about 56 mol% to about 58 mol% of the compound disclosed, 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 PEG-lipid conjugates.

[0172] In some embodiments, the lipid nanoparticles comprise about 50 mol% to 61 mol% of the compound of the 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 the compound of the 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 the compound of the 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.

[0173] In some embodiments, the lipid nanoparticles have a total lipid:nucleotide weight ratio of about 50:1 to about 10:1. In some embodiments, the lipid nanoparticles have a total lipid:nucleotide weight ratio of about 40:1 to about 20:1. In some embodiments, the lipid nanoparticles have a total lipid:nucleotide weight ratio of about 35:1 to about 25:1. In some embodiments, the lipid nanoparticles have a total lipid:nucleotide weight ratio of about 32:1 to about 28:1. In some embodiments, the lipid nanoparticles have a total lipid:nucleotide weight ratio of about 31:1 to about 29:1.

[0174] 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.

[0175] In some embodiments, the lipid nanoparticles contain 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 contain about 2.0 mg / mL to about 4.0 mg / mL of NaCl.

[0176] In some embodiments, the lipid nanoparticles further comprise one or more cryoprotective agents. In some embodiments, the one or more cryoprotective agents 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.

[0177] Lipid-nucleic acid preparations Nucleic acids or pharmaceutically acceptable salts thereof can be incorporated into lipid formulations (i.e., lipid-based delivery vehicles).

[0178] In the context of this disclosure, a lipid-based delivery vehicle typically functions to deliver a desired nucleic acid (such as siRNA, plasmid DNA, mRNA, or self-replicating RNA) to a target cell or target 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 lipid nanoparticles containing nucleic acids. In some embodiments, the lipid-based delivery vehicle comprises lipid molecules and nanoparticles or bilayers of nucleic acids. In some embodiments, the lipid bilayer preferably further comprises neutral lipids or polymers. In some embodiments, the lipid formulation preferably comprises a liquid medium. In some embodiments, the formulation preferably further encapsulates nucleic acids. In some embodiments, the lipid formulation preferably further comprises nucleic acids and neutral lipids or polymers. In some embodiments, the lipid formulation preferably encapsulates nucleic acids.

[0179] This specification provides lipid formulations comprising one or more therapeutic nucleic acid molecules encapsulated within a lipid formulation. In some embodiments, the lipid formulation comprises liposomes. In some embodiments, the lipid formulation comprises cationic liposomes. In some embodiments, the lipid formulation comprises lipid nanoparticles.

[0180] In some embodiments, nucleic acids are completely encapsulated within the lipid portion of the lipid formulation, thereby making the nucleic acids in the lipid formulation resistant to nuclease degradation in aqueous solution. In other embodiments, the lipid formulations described herein are substantially nontoxic to mammals such as humans.

[0181] The lipid formulations of this disclosure also typically have total lipid:nucleic acid ratios (mass / mass ratios) 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 ratio) is about 10:1 to about 25:1. The ratio may be any value or sub-value within the enumerated range, including the endpoints.

[0182] The lipid formulations of this disclosure typically have wavelengths of approximately 30nm to 150nm, 40nm to 150nm, 50nm to 150nm, 60nm to 130nm, 70nm to 110nm, 70nm to 100nm, 80nm to 100nm, 90nm to 100nm, 70nm to 90nm, 80nm to 90nm, 70nm to 80nm, or approximately 30nm, 35nm, and 4nm. The nanoparticles have an average diameter of 0 nm, approximately 45 nm, approximately 50 nm, approximately 55 nm, approximately 60 nm, approximately 65 nm, approximately 70 nm, approximately 75 nm, approximately 80 nm, approximately 85 nm, approximately 90 nm, approximately 95 nm, approximately 100 nm, approximately 105 nm, approximately 110 nm, approximately 115 nm, approximately 120 nm, approximately 125 nm, approximately 130 nm, approximately 135 nm, approximately 140 nm, approximately 145 nm, or approximately 150 nm, and are substantially nontoxic. The diameter, including the endpoints, may be any value or sub-value within the enumerated range. In addition, when present in the lipid nanoparticles of this disclosure, the nucleic acids are resistant to degradation by nucleases in aqueous solution.

[0183] In preferred embodiments, the lipid formulation comprises nucleic acids, cationic lipids (e.g., one or more cationic lipids or salts thereof as described herein), phospholipids, and conjugated lipids that inhibit particle aggregation (e.g., one or more PEG-lipid conjugates and / or other lipid conjugates as described herein). The lipid formulation may also contain cholesterol.

[0184] In some embodiments, the lipid nanoparticles further comprise a PEG-lipid conjugate. In some embodiments, the PEG-lipid conjugate is PEG-DMG. In some embodiments, PEG-DMG is PEG2000-DMG. In some embodiments, PEG2000-DMG is the addition of polyethylene glycol to myristoyl diglyceride.

[0185] In nucleic acid-lipid formulations, the nucleic acid may be completely encapsulated within the lipid portion of the formulation, thereby protecting it from nuclease degradation. In a preferred embodiment, a lipid formulation containing nucleic acid is completely encapsulated within the lipid portion of the lipid formulation, thereby protecting the nucleic acid from nuclease degradation. In certain cases, the nucleic acid in the lipid formulation remains substantially undegraded after the particles are exposed to nuclease at 37°C for at least 20, 30, 45, or 60 minutes. In certain other cases, the nucleic acid in the lipid formulation remains substantially undegraded after incubation of the formulation in serum for at least 30, 45, or 60 minutes at 37°C, or for 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, the nucleic acid is complexed with the lipid portion of the formulation.

[0186] In the context of nucleic acids, complete inclusion can be determined by performing a membrane-impermeable fluorescent dye exclusion assay using a dye whose fluorescence is enhanced when associated with the nucleic acid. Inclusion is determined by adding the dye to a lipid preparation, measuring the resulting fluorescence, and comparing it to the fluorescence observed when a small amount of nonionic detergent is added. Detergent-mediated disruption of the lipid layer releases the encapsulated nucleic acid, allowing it to interact with the membrane-impermeable dye. Nucleic acid inclusion may also be calculated as E=(I0-I) / I0, where I and I0 refer to the fluorescence intensity before and after the addition of the detergent.

[0187] In other embodiments, the 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.

[0188] In some embodiments, the lipid formulation contains nucleic acids completely encapsulated within the lipid portion of the formulation, thereby making up approximately 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 30% to 95%, 40% to 95%, 50% to 95%, 60% to 95%, 70% to 95%, 80% to 95%, 85% to 95%, 90% to 95%, and 30% to 95% of the particles. Approximately 90%, approximately 40% to approximately 90%, approximately 50% to approximately 90%, approximately 60% to approximately 90%, approximately 70% to approximately 90%, approximately 80% to approximately 90%, or at least approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, or approximately 99% (or any fraction or range within thereof) contains encapsulated nucleic acids. The amount may be any value or sub-value within the listed range, including the endpoints.

[0189] In some embodiments, the polyvariance index (PDI) is in the range of 0.010 to 1.10. In embodiments, the PDI is 0.010 to 1.05, 0.010 to 1.00, 0.010 to 0.95, 0.010 to 0.90, 0.010 to 0.85, 0.010 to 0.80, 0.010 to 0.75, 0.010 to 0.70, 0.010 to 0.65, 0.010 to 0.60, 0.010 to 0.55, 0.010 to 0.50, 0.010 to 0.45, 0.010 to 0.40, 0.010 to 0.35, 0.010 to 0.30, 0.010 to 0.25, 0.010 to 0.20, 0.010 to 0.15, 0. The range is 0.10~0.10, 0.010~0.09, 0.010~0.08, 0.010~0.07, 0.010~0.06, 0.010~0.05, 0.010~0.04, 0.010~0.03, 0.010~0.02, 0.010~0.019, 0.010~0.018, 0.010~0.017, 0.010~0.016, 0.010~0.015, 0.010~0.014, 0.010~0.013, 0.010~0.012, 0.010~0.011 (or any range within that range). The quantity may be any value or sub-value within the listed range, including the endpoints.

[0190] Depending on the intended use of the lipid formulation, the ratio of its components can be varied, and the delivery efficiency of a particular formulation can be measured using assays known in the art.

[0191] According to several embodiments, expressible polynucleotides, nucleic acid activators, and mRNA constructs can be formulated into lipid formulations. The lipid formulations are preferably selected from, but are not limited to, liposomes, cationic liposomes, and lipid nanoparticles. In one preferred embodiment, the lipid formulation is a cationic liposome or lipid nanoparticles (LNPs). (a) Nucleic acids (mRNA, siRNA, etc.) (b) Lipids of the present disclosure that may be cationic, (c) Selectively, noncationic lipids (neutral lipids, etc.) (d) optionally including sterols.

[0192] Cationic lipids Lipid formulations preferably contain cationic lipids suitable for forming cationic liposomes or lipid nanoparticles. Cationic lipids have been extensively studied for nucleic acid delivery because they can bind to negatively charged membranes and induce uptake. Generally, cationic lipids are amphiphilic substances comprising a positively hydrophilic head group, two (or more) lipophilic tails, or a steroid moiety, and connectors between these two domains. Preferably, cationic lipids carry a net positive charge at approximately physiological pH. Cationic liposomes have traditionally been the most commonly used nonviral delivery system for oligonucleotides, including plasmid DNA, antisense oligonucleotides, 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.

[0193] In the lipid formulations of this disclosure, cationic lipids include, for example, N,N-dioleyl-N,N-di-9-cis-octadecenylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoyltrimethylammoniumpropane 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-γ-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-dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl ), 1,2-dilinoleyl-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 analogue thereof, (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)piperazine-1-yl)ethylazandiyl)didodecane-2-ol (C12-200 Examples include 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-dimethylpropane-1-amine (MC3 ether), 4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutane-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-(sperminecarboxamide)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamideglycylcarboxyspermine (DOGS), 1,2-dioleyl-sn-3-phosphoethanolamine (DOPE), 1,2-dioleyl-3-dimethylammoniumpropane (DODAP), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), and 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3) Dioxolane (XTC) is an example, but it is not limited to these. 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.

[0194] Other suitable cationic lipids are described in International Publications 09 / 086558, 09 / 127060, 10 / 048536, 10 / 054406, 10 / 088537, 10 / 129709, and 2011 / 153493, U.S. Patent Publications 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.

[0195] 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 aminolipids. In some embodiments of the lipid formulations described herein, the cationic lipids are aminolipids. Generally, aminolipids with low-saturation 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 Aminolipids containing unsaturated fatty acids having carbon chain lengths in the range of [specified range] may be used. Other scaffoldings can also be used to separate the amino group from the fatty acid or fatty alkyl portion of the aminolipid.

[0196] In some embodiments, the cationic lipids of this disclosure are ionizable and have at least one protonable or deprotonable 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. Naturally, the addition or removal of protons as a function of pH is an equilibrium process, and references to charged or neutral lipids refer to the properties of the dominant species, and not all lipids are required to exist in charged or neutral form. Lipids having two or more protonable or deprotonable groups, or being zwitterionic, are not excluded from use in this disclosure. In certain embodiments, protonable lipids have a pKa of protonable groups in the range of about 4 to about 11. In some embodiments, ionic cationic lipids have a pKa of about 5 to about 7. In some embodiments, the pKa of ionic cationic lipids is about 6 to about 7.

[0197] In some embodiments, the lipid formulation is a lipid of formula I, [ka] (I) During the ceremony, R 1 and R 2 However, each independently, H or C 1-6 It is alkyl, or R 1 and R 2 However, they join together to form a saturated heterocyclic ring, R 1 However, linear C 1-4 It is alkylene, R 2 However, -(CH2) m (X) n -and, X is O, S, or NR 9 And R 9 However, H or C 1-6 It is alkyl, m is 1, 2, 3, or 4. n is 0 or 1, L1 is a linear C chain that is optionally substituted with 1 to 3 methyl groups. 1-6 It is alkylene, Y is selected from the following group: [ka] During the ceremony, Each asterisk (*) indicates an atom bonded to L2 and L3. R 10 However, H or C 1-6 It is alkyl, L2 and L3 are each independently linear C 1-8 It is alkylene, L4, L5, L6, L7, L8, and L9 are either non-existent or -CH2-, however, At least two of L4, L6, and L8 are -CH2-, At least two of L5, L7, and L9 are -CH2-, R 3 and R 4 However, each is independently H, methyl, or ethyl. R 5 , R 6 , R 7 , and R 8 However, each is independent, as follows: Linear C 1-20 Alkyl, and each of the linear C 1-20 Alkyl, below, C 6-10 Aaryl, and each of the C 6-10 A aryl is one or more C 1-6 One or more C atoms optionally substituted with alkyl groups. 6-10 A monocyclic or bicyclic aromatic hydrocarbon that is optionally substituted with an aryl group, C 6-10 Ariel, A 6-10 member heteroaryl, wherein each of the 6-10 member heteroaryls contains one or more C 1-6Linear carbon atoms are optionally substituted with one or more substituents selected from the group consisting of monocyclic or bicyclic aromatic systems that are optionally substituted with alkyl groups, and 6-10 membered heteroaryl groups. 1-20 Alkyl, C 6-10 Aaryl, and each of the C 6-10 A aryl is one or more C 1-6 One or more C atoms optionally substituted with alkyl groups. 6-10 A monocyclic or bicyclic aromatic hydrocarbon that is optionally substituted with an aryl group, C 6-10 Aryl, and A 6-10 member heteroaryl, wherein each of the 6-10 member heteroaryls contains one or more C 1-6 The present invention comprises lipids selected from the group consisting of 6-10 membered heteroaryls, which are monocyclic or bicyclic aromatic systems optionally substituted with alkyl groups.

[0198] In some embodiments, one or more lipids listed herein may be explicitly excluded.

[0199] Helper lipids and sterols The mRNA lipid formulations of this disclosure may contain helper lipids, which may be referred to as neutral lipids, neutral helper lipids, noncationic lipids, noncationic helper lipids, anionic lipids, anionic helper lipids, or zwitterionic lipids. Lipid formulations, particularly cationic liposomes and lipid nanoparticles, have been found to undergo 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 facilitating than cationic lipids (i.e., facilitate fusion), which may influence the polymorphic characteristics of lipid-nucleic acid complexes, promoting transition from the lamellar phase to the hexagonal phase, and thus inducing cell membrane fusion and disruption. (Nanomedicine (Lond). 2014 Jan;9(1):105-20). In addition, the use of helper lipids may help mitigate any potential adverse effects associated with the use of many common cationic lipids, such as toxicity and immunogenicity.

[0200] Non-limiting examples of non-cationic lipids suitable for the lipid formulations of this disclosure include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl Examples include oil-phosphatidylethanolamine (POPE), palmitoyloleyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dierydoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having a C10-C24 carbon chain, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0201] 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).

[0202] Additional examples of noncationic 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 harmonizes with nucleic acids, thereby more closely matching the charge distribution with 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, 5α-cholestanone, and cholesteryl decanoate; and mixtures thereof. In preferred embodiments, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether.

[0203] In some embodiments, the helper lipids present in the lipid formulation include or consist of a mixture of one or more phospholipids and cholesterol or its derivatives. In other embodiments, the helper lipids present in the lipid formulation include or consist of a lipid formulation that does not contain one or more phospholipids, for example, cholesterol. In yet another embodiment, the helper lipids present in the lipid formulation include or consist of a lipid formulation that does not contain cholesterol or its derivatives, for example, phospholipids. In some embodiments, the lipid nanoparticles further contain cholesterol.

[0204] Other examples of helper lipids include non-phosphorus lipids such as 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.

[0205] In some embodiments, the helper lipids constitute 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 thereof) of the total lipids present in the lipid formulation. In some embodiments, the helper lipids constitute 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%.

[0206] In some embodiments, the total helper lipids in the formulation include two or more helper lipids, and the total amount of helper lipids constitutes 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 thereof) of the total lipids present in the lipid formulation. In some embodiments, the helper lipids are a combination of DSPC and DOTAP. In some embodiments, the helper lipids are a combination of DSPC and DOTMA.

[0207] The cholesterol or cholesterol derivative in the lipid preparation may comprise up to about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, or about 60 mol% of the total lipids present in the lipid preparation. 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 lipids present in the lipid preparation.

[0208] The proportion of helper lipids present in a lipid preparation is the target amount, and the actual amount of helper lipids present in the preparation may vary, for example, by ±5 mol%.

[0209] Mechanism of action for cellular uptake of lipid preparations 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 endocytosis mechanism of the target cell, which is the delivery of the contents of the lipid delivery vehicle into the cytosol of the target cell (Nucleic Acid Therapeutics, 28(3):146-157, 2018). Specifically, in the case of nucleic acid lipid formulations described herein, the lipid formulation enters the cell via receptor-mediated endocytosis. Prior to endocytosis, functionalized ligands, such as the lipid conjugates of this disclosure, can be detached from the surface of the lipid delivery vehicle, thereby inducing internal migration into the target cell. During endocytosis, a portion of the cell's plasma membrane surrounds the vector, engulfs it into a vesicle, then detaches the vesicle from the cell membrane, enters the cytosol, and finally passes through the endolysosome pathway. For delivery vehicles containing ionic or cationic lipids, as endosomes age and acidity increases, a strongly positively charged vehicle is produced on its surface. The interaction between the delivery vehicle and the endosomal membrane then results in a membrane fusion event, leading to cytoplasmic delivery of the payload. For mRNA or self-replicating RNA payloads, the cell's own internal translation process then translates the RNA into an encoded protein. The encoded protein can then undergo further post-translational processing, including transport to a target organelle or intracellular location.

[0210] By controlling the composition and concentration of the lipid conjugate, the rate at which the lipid conjugate exchanges with the lipid formulation and the rate at which the lipid formulation becomes fused can be controlled. In addition, other variable factors, including, for example, pH, temperature, or ionic strength, can be used to change and / or control the rate at which the lipid formulation becomes fused. Other methods that can be used to control the rate at which the lipid formulation becomes fused will become apparent to those skilled in the art upon reading this disclosure. Liposome or lipid particle size can also be controlled by controlling the composition and concentration of the lipid conjugate.

[0211] Lipid product 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). Techniques such as 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 pre-formed liposomes are briefly described herein.

[0212] Thin film hydration In the thin-film hydration (TFH) or Bangham process, lipids dissolve in an organic solvent and are then evaporated through the use of 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 carried, multilayer vesicles (MLVs) are formed, which can be reduced in size to produce small monolayer vesicles (LUVs) or large monolayer vesicles (SUVs) by extrusion through the membrane or by sonication of the initial MLVs.

[0213] Double emulsion Lipid formulations can also be prepared through a biemulsion technique involving lipid dissolution in a water / organic solvent mixture. An organic solution containing water droplets is mixed with an excess of water to form a water-in-oil-in-water (W / O / W) biemulsion. After vigorous mechanical shaking, some of the water droplets disintegrate, yielding large monolayer vesicles (LUVs).

[0214] Reverse-phase evaporation Reverse-phase evaporation (REV) can also achieve nucleic acid-loaded lump-encased phospholipids (LUV). 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 single-phase dispersion. Lipid formulations are achieved after evaporation of the organic solvent under reduced pressure. This technique is used to encapsulate different large and small hydrophilic molecules, including nucleic acids.

[0215] Microfluidic preparation Unlike other bulk techniques, microfluidics offers the possibility of controlling the lipid hydration process. Methods can be classified into continuous flow microfluidics and droplet-based microfluidics, depending on how the flow is manipulated. In microhydrodynamic focusing (MHF) methods operating in continuous flow mode, lipids dissolve in isopropyl alcohol, which is hydrodynamically focused in a microchannel cross-junction between two aqueous buffer flows. Vesicle size can be controlled by adjusting the flow rate, thus controlling the lipid solution / buffer dilution process. Methods can be used to produce oligonucleotide (ON) lipid formulations by using a microfluidic apparatus consisting of three inlet ports and one outlet port.

[0216] Double asymmetric centrifugal separation Double asymmetric centrifugation (DAC) differs from more common centrifugation because it uses an additional rotation around its own vertical axis. Efficient homogenization is achieved by the two overlay movements created: the sample is pushed outward like in a normal centrifuge, and then pushed towards the center of the vial by the additional rotation. By mixing the lipids with the NaCl solution, a viscous vesicular phospholipid gel (VPC) is achieved, which is then diluted to obtain a lipid formulation dispersion. The lipid formulation size can be adjusted by optimizing the DAC speed, lipid concentration, and homogenization time.

[0217] Ethanol injection Ethanol injection (EI) can be used for nucleic acid encapsulation. This method provides rapid injection of an ethanol solution in which lipids are dissolved into an aqueous medium containing nucleic acids to be encapsulated, through the use of a needle. Once the phospholipids are dispersed throughout the medium, vesicles spontaneously form.

[0218] Detergent dialysis Nucleic acids can be encapsulated using detergent dialysis. Briefly, lipids and plasmids are solubilized in a detergent solution of appropriate ionic strength, and after removing the detergent by dialysis, a stabilized lipid formulation is formed. Unencapsulated nucleic acids are then removed by ion-exchange chromatography, and the vesicles are emptied by sucrose density gradient centrifugation. This technique is highly sensitive to the cationic lipid content and the salt concentration of the dialysis buffer, and scaling this method is also difficult.

[0219] Spontaneous vesicle formation due to ethanol dilution Stable lipid formulations can also be produced via a method of spontaneous vesicle formation by ethanol dilution, in which lipids dissolved in ethanol are added in a controlled manner to a rapidly mixing aqueous buffer containing nucleic acids by stepwise or dropwise ethanol dilution, thereby spontaneously forming nucleic acid-loaded vesicles.

[0220] V. Pharmaceutical Compositions and Delivery Methods To promote in vivo nucleic acid activity (e.g., mRNA expression, or knockdown by ASO or siRNA), the delivery vehicles of the lipid formulations described herein may be combined with one or more additional nucleic acids, carriers, targeted ligands, or stabilizing reagents, or may be in a pharmacological composition mixed with suitable excipients. Techniques for drug formulation and administration can be found in "Remington's Pharmaceutical Sciences" (Mack Publishing Co., Easton, Pa, latest edition).

[0221] The lipid formulations and pharmaceutical compositions of this disclosure may be administered and administered in accordance with current medical practice, taking into consideration the clinical condition of the subject, the site and method of administration, the administration schedule, the age, sex, and weight of the subject, and other factors relevant to those skilled in the art. The “effective dose” for the purposes of this specification may be determined by relevant considerations known to those skilled in the art of experimental clinical research, pharmacology, clinical practice, and medical technology. In some embodiments, the dose administered is effective in achieving at least some stabilization, improvement, or elimination of symptoms and other indicators selected by those skilled in the art as appropriate measures of disease progression, regression, or improvement. For example, an appropriate dose and administration regimen would induce the production of at least a transient protein (e.g., an enzyme).

[0222] The pharmaceutical compositions disclosed herein can be formulated using one or more excipients to (1) increase stability, (2) increase cell transfection, (3) allow sustained or delayed release (e.g., from a depot formulation of nucleic acids), (4) alter in vivo distribution (e.g., target nucleic acids to specific tissues or cell types), (5) increase the activity of nucleic acids or proteins expressed therein in vivo, and / or (6) alter the release profile of nucleic acids or encoded proteins in vivo.

[0223] Preferably, the lipid formulation may be administered topically rather than systemically. Topical delivery can affect the target tissue in various ways, depending on the tissue being targeted. For example, an aerosol containing the composition of this disclosure can be inhaled (for nasal, tracheal, or bronchial delivery).

[0224] The pharmaceutical composition may be administered to any desired tissue. In some embodiments, the nucleic acid delivered by the lipid formulation or composition of this disclosure is active in the tissue to which the lipid formulation and / or composition is administered. In some embodiments, the nucleic acid is active in a tissue different from the tissue to which the lipid formulation and / or composition is administered. Examples of tissues to which the nucleic acid may be delivered include, but are not limited to, the lungs, trachea and / or nasal cavity, muscles, liver, eyes, or the central nervous system.

[0225] The pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such preparation methods involve associating an active ingredient (i.e., nucleic acid) with excipients and / or one or more other minor components. The pharmaceutical compositions according to this disclosure may be prepared, packaged and / or sold in bulk as single doses and / or multiple single doses.

[0226] The pharmaceutical composition may further contain pharmaceutically acceptable excipients, which, when used herein, include, but are not limited to, any solvent, dispersion medium, diluent, or other liquid vehicle, dispersant or suspension aid, surfactant, isotonic agent, thickener or emulsifier, preservative, etc., suitable for the desired specific dosage form.

[0227] In addition to conventional excipients such as all kinds of solvents, dispersions, diluents, or other liquid vehicles, dispersants or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, and preservatives, the excipients of the present disclosure 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), hyaluronidases, nanoparticle mimetic bodies, and combinations thereof.

[0228] Accordingly, the formulations described herein may contain one or more excipients in amounts that collectively increase the stability of nucleic acids in the lipid formulation, increase cell transfection by nucleic acids (e.g., mRNA or siRNA), increase the expression of encoded proteins, and / or alter the release profile of encoded proteins, or increase the knockdown of target native nucleic acids. Furthermore, the nucleic acids may be formulated using self-assembling nucleic acid nanoparticles.

[0229] Various excipients for formulating pharmaceutical compositions and techniques for preparing such compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, ARGennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006, which is incorporated herein by reference in its entirety). The use of conventional excipient media may be contemplated within the scope of embodiments of this disclosure, except where any conventional excipient media may be incompatible with the substance or its derivatives by causing any undesirable biological effect or otherwise interacting in an adverse manner with any other component of the pharmaceutical composition.

[0230] The dosage forms of the compositions of this disclosure may be solids that can be reconstituted in a liquid before administration. The solids may be administered as powders. In some embodiments, the pharmaceutical compositions include lyophilized nucleic acid lipid formulations.

[0231] In preferred embodiments, the dosage form of the pharmaceutical composition described herein may be a liquid suspension of 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 sugar and glycerol, or a combination of 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 contains HEPES, sucrose, and glycerol at a pH of 7.4. In some embodiments, the suspension is frozen during storage and thawed before 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 before inhalation administration. In some embodiments, an inhalable administration involves diluting the suspension with about 1 to about 4 volumes of sterile water. In some embodiments, the lyophilized nucleic acid-lipid nanoparticle formulation can be resuspended in the buffer described herein.

[0232] The dosage forms of the compositions of this disclosure may be solids that can be reconstituted in a liquid before administration. The solids may be administered as powders. The solids may be in the form of capsules, tablets, or gels.

[0233] To formulate compositions for pulmonary delivery within this disclosure, nucleic acid lipid formulations can be combined with a variety of pharmaceutically acceptable additives, as well as bases or carriers for dispersion of the nucleic acid lipid formulations. 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), isotonic 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). If the composition for mucosal delivery is a liquid, the tonicity of the formulation, measured by reference to the tonicity of a 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 a solution is adjusted to a value of 1 / 3 to 3, more typically 1 / 2 to 2, and in most cases 3 / 4 to 1.7.

[0234] Nucleic acid lipid formulations may be dispersed in a base or vehicle that contains 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 salts thereof, carboxylic acid anhydrides with other monomers (e.g., methyl (meth)acrylate, acrylic acid, etc.) (e.g., maleic anhydride), 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, for example, polylactic acid, poly(lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid) copolymer, and mixtures thereof. Alternatively, synthetic fatty acid esters such as polyglycerin fatty acid esters and sucrose fatty acid esters may be used as carriers. Hydrophilic polymers and other carriers can be used alone or in combination, and improved structural integrity can be imparted to the carriers by partial crystallization, ionic bonding, crosslinking, etc. Carriers can be provided in various forms, including fluids or viscous solutions, gels, pastes, powders, microspheres, and films for direct application to the nasal mucosa. The use of selected carriers in this context may result in enhanced absorption of nucleic acid lipid formulations.

[0235] Alternatively, the compositions of the present disclosure may include pH adjusters and buffers, tonicity adjusters, and wetting agents, such as pharmaceutically acceptable carrier substances required to approximate physiological conditions, including 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, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and magnesium carbonate.

[0236] According to this disclosure, therapeutically effective doses of the provided compositions, when administered regularly, result in an increase in nucleic acid activity levels in a subject compared to baseline activity levels before treatment. Typically, activity levels are measured in biological samples obtained from a subject, such as blood, plasma or serum, urine, or solid tissue extracts. Baseline levels can be measured immediately before treatment. In some embodiments, administration of the pharmaceutical compositions described herein results in an increase of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in nucleic acid activity levels in a biological sample (e.g., plasma / serum or lung epithelial swab) compared to baseline levels before treatment. In some embodiments, administration of the provided composition results in an increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% in nucleic acid activity levels of a biological sample (e.g., plasma / serum or lung epithelial swab) compared to baseline levels before treatment, 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.

[0237] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a compound or lipid nanoparticle described herein and a pharmaceutically acceptable excipient.

[0238] In some embodiments, the Disclosure provides a method for delivering nucleic acids to a target requiring such delivery, the method comprising encapsulating a therapeutically effective amount of nucleic acid in lipid nanoparticles described herein and administering the lipid nanoparticles to the target.

[0239] In some embodiments, the present disclosure provides a method for delivering mRNA to a subject requiring such delivery, the method comprising encapsulating a therapeutically effective amount of mRNA in lipid nanoparticles described herein and administering the lipid nanoparticles to the subject.

[0240] VI. Treatment method In some embodiments, the Disclosure provides a method for treating a disease in a subject requiring treatment, comprising administering a therapeutically effective amount of a compound, a lipid nanoparticle, or a pharmaceutical composition described herein to the subject. In some embodiments, the compound, composition, or lipid nanoparticle is administered intravenously or intramuscularly. In some embodiments, the compound, composition, or lipid nanoparticle is administered intravenously. In some embodiments, the compound, composition, or lipid nanoparticle is administered intramuscularly.

[0241] In some embodiments, a method is provided for treating a disease in a subject requiring treatment, comprising administering the lipid composition described herein to the subject. 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.

[0242] In some embodiments, methods for treating diseases or disorders in mammalian subjects are provided. A therapeutically effective amount of a composition comprising lipids, particularly cationic lipids, nucleic acids, amphiphilic substances, phospholipids, cholesterol, and PEG-bound cholesterol, disclosed herein may be administered to subjects having diseases or disorders related to the expression or overexpression of genes that can be reduced, decreased, downregulated, or silenced by the composition. The compositions described herein can be used in methods for treating cancer or inflammatory diseases. The diseases may be selected from the group consisting of central nervous system disorders, peripheral nervous system disorders, muscle atrophy, muscular dystrophy, immune disorders, cancer, kidney disease, fibrous disease, genetic abnormalities, inflammation, and cardiovascular disorders.

[0243] In some embodiments, the present disclosure provides a method for delivering nucleic acids to a target requiring such delivery, comprising encapsulating a therapeutically effective amount of nucleic acid in lipid nanoparticles as described herein, and administering the lipid nanoparticles to the target. [Examples]

[0244] VII. Examples Example 1. Synthesis of Lipid 1: ((4,4'-((((3-(dimethylaminopropyl)thio)carbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetranonanoate [ka] General scheme: [ka] [ka] [ka]

[0245] Synthesis of 1-1:2-oxopropane-1,3-diyl diyl oxopropane [ka]

[0246] Under an N2 atmosphere, a CH2Cl2 solution of 1,3-dihydroxyacetone (6.8 g, 1 equivalent) was added to a 500 mL three-necked round-bottom flask reactor. Stirring was started and the temperature was adjusted to room temperature. While maintaining the temperature at 25±5°C, pyridine (17.9 g, 3 equivalents) was added to the reactor. While maintaining the temperature at 25±5°C, 4-dimethylaminopyridine (DMAP) (0.276 g, 0.03 equivalents) was added to the reactor. Nonanoyl chloride (20 g, 1.5 equivalents) was added dropwise to the reactor at 0-5°C. After addition, the temperature was maintained at room temperature and the mixture was stirred for 6 hours. A further 6.66 g of nonanoyl chloride (0.5 equivalents) was added dropwise to the reactor at 0-5°C. The reaction temperature was raised to room temperature and the mixture was stirred overnight under nitrogen. The formed pyridine hydrochloride was removed by filtration and washed with CH2Cl2. Next, the combined filtrate and washing solution were washed with 200 mL each of 5% NaHCO3 aqueous solution, 0.1N HCl, and brine. Then, the solutions were dried over Na2SO4 and concentrated under vacuum. The residue was then crystallized with methanol (50 mL) to obtain a white solid. This yielded 16 g (59.7%) of the white product. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5~5:95 A / B at 3 min, held for 0.7 min): RT 2.26 min, m / z (calculated value) 370.27, (measured value) 371.00 (M+H + ).

[0247] Synthesis of 1-2:2-hydroxypropane-1,3-dinonanoate diyl [ka]

[0248] A 100 mL solution of lipid-1-1 (9.6 g, 1.0 equivalent) in THF was placed in a 250 mL three-necked round-bottom flask. AcOH (2.02 g, 1.3 equivalents) was added to the solution at 0°C. Then NaBH3CN (1.96 g, 1.2 equivalents) was added to the mixture at 0°C. The mixture was stirred at room temperature under a nitrogen atmosphere for 16 hours. The reaction mixture was quenched with 100 mL of water. The mixture was extracted three times with 100 mL of ethyl acetate. The organic layers were combined, washed with brine (300 mL), and concentrated under vacuum. This yielded 9.5 g (crude) of 1-2, which was used in the next step without purification.

[0249] 1-3: Synthesis of dimethyl 4,4'-(benzylazanediyl)dibutyrate [ka]

[0250] In a 1 L three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, acetonitrile (208 mL), K2CO3 (31 g, 2.3 equivalents), benzylamine (10.3 g, 1.0 equivalent), and methyl 4-bromobutyrate (40 g, 2.3 equivalents) were added at 25°C. The mixture was heated to 80°C and stirred for 15 hours. The reaction product was cooled to 25°C. Water (200 mL, 20V) was added to the flask and extracted with RINKAN (2 × 200 mL). The organic phase was dried (Na2SO4) and concentrated under reduced pressure at 35°C. This yielded 1-3 (23 g, crude) as the crude product, which was used thereafter without further purification. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA (95:5~5:95 A / B in 3 minutes, held for 0.7 minutes): RT 2.26 mins, m / z (calculated value) 370.27, (measured value) 371.00 (M+H).

[0251] Synthesis of 1-4:4,4'-((tert-butoxycarbonyl)azandiyl)dimethyl dibutyrate [ka]

[0252] EtOH (230 mL, 10V), 1-3 (23.0 g, 1.0 equivalent), (Boc)2O (18.0 g, 1.1 equivalent), 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 for 16 hours under a hydrogen atmosphere of 5 atm. TLC results showed that 1-3 was completely consumed. The reaction mixture was filtered and concentrated under vacuum at 40°C to obtain 22 g of crude 1-4.

[0253] 1-5: Synthesis of 4,4'-((tert-butoxycarbonyl)azandiyl)dibutyric acid [ka]

[0254] A 500 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with 110 mL of ethanol solution containing 1-4 (22 g, 1.0 equivalent). 110 mL of 6 M NaOH aqueous solution (5V) was added at room temperature. The reaction mixture was stirred at 60°C for 2 hours. The reaction mixture was then diluted with brine (220 mL, 10V) 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 HCl aqueous solution, and then extracted with t-BuOH:n-heptane (2:1) (220 mL). The organic layer was concentrated under reduced pressure. The residue was slurryed with diethyl ether (44 mL, 2V) and filtered. The filtered cake was collected to obtain 1-5 (14 g, 43% yield in 3 steps) as a white solid.

[0255] 1-6: Synthesis of ((4,4'-((tert-butoxycarbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetranonanoate [ka]

[0256] In a 250 mL three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, 100 mL of CH2Cl2 solution containing 1-5 (3.7 g, 1.00 equivalent) was added. To this solution, 1-2 (9.5 g, 2.00 equivalent), DMAP (4.69 g, 3 equivalents), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (7.35 g, 3 equivalents) were added at 0°C. The reaction mixture was stirred overnight at 25°C. The reaction mixture was then quenched with 200 mL of 10% aqueous citric acid solution. The organic layer was washed with 200 mL of brine. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. This yielded 12 g of 1-6, which was used in the next step without further purification. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA. 3 minutes: 95:5~5:95 A / B, held for 0.7 minutes): RT 2.46 mins, m / z (calculated value) 997.71, (measured value) 1020.6 (M+Na).

[0257] 1-7: Synthesis of bis(4-((1,3-bis(nonanoyloxy)propane-2-yl)oxy)-4-oxobutyl)ammonium chloride [ka]

[0258] A 500 mL round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was placed in a solution of 1-6 (12 g, 1.00 equivalent) in siRNA (68 mL). To this solution, siRNA / HCl (15 mL, 5.00 equivalent, 4 M) was added dropwise at 0-10°C. The resulting solution was stirred at room temperature for 5 hours. The resulting mixture was concentrated under vacuum. The residue was diluted with CH2Cl2 (100 mL) and 25 g of silica gel (type: ZCX-2, 100-200 mesh) was added (Note: ZCX-2 is sold by Sincem Silica Gel Co. Ltd). The mixture was concentrated under vacuum and then applied to an atmospheric silica gel column with a CH2Cl2 / MeOH gradient of 1:0-15:1. The product eluate was collected at a ratio of 20:1-15:1 and concentrated under vacuum. This yielded 4.2 g of 1-7 as a light yellow oily substance. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA. 3 minutes: 95:5~5:95 A / B, held for 0.7 minutes): RT 1.50 min, m / z (calculated value) 897.65, (measured value) 898.6 (M+H).

[0259] Lipid 1: Synthesis of ((4,4'-((((3-(dimethylaminopropyl)thio)carbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetranonanoate [ka]

[0260] A 250 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with 1-7 (4.2 g, 1 equivalent) of CH2Cl2 (150 mL) solution. To this mixture, triphosgene (1.33 g, 1 equivalent) was added at room temperature. Subsequently, pyridine (1.78 g, 5.00 equivalent) was added dropwise 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 (600 mL). To this solution, 3-(dimethylamino)propane-1-thiol (0.92 g, 1.20 equivalent) was added dropwise over 10 minutes with stirring at 0°C. The resulting solution was stirred overnight at room temperature. The resulting mixture was concentrated under vacuum. The residue was diluted with 100 mL of siRNA. The mixture was washed with 2 × 100 mL of 10% citric acid and 2 × 100 mL of NaHCO3. 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 CH2Cl2, and 12 g of silica gel (type: ZCX-2, 100-200 mesh) was added. The mixture was concentrated under vacuum and then applied to a silica gel column at atmospheric pressure using a CH2Cl2 / MeOH gradient of 1:0-15:1. The product eluate was collected at a ratio of 20:1-15:1 and concentrated under vacuum. The product was dissolved in 36 mL of n-heptane (20V), 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 a further 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 to separate the phases, 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 to separate the phases, and the upper phase was collected and concentrated to obtain 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 8 mins (95:5~5:95 A / B, 0.7 min retention): RT 7.07 min, m / z (calculated) 1042.71, (measured) 1043.8 (M+H); 1H NMR(300MHz,CDCl3):δ 5.27(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).

[0261] Example 2. Synthesis of Lipid 2: ((4,4'-((((3-(dimethylaminopropyl)thio)carbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetraoctanoate [ka] General scheme: [ka]

[0262] Synthesis of 2-1:2-oxopropane-1,3-dioctanoate diyl [ka]

[0263] A 2 L three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with a solution of 1,3-dihydroxyacetone (50 g, 1.0 equivalent) and octanoyl chloride (225.8 g, 2.5 equivalents) in CHCl3 (900 mL). Pyridine (175.8 g, 4.0 equivalents) was added to the reactor while maintaining the temperature at 0°C for 40 minutes. The mixture was stirred overnight at room temperature under nitrogen. The formed pyridine hydrochloride was removed by filtration and washed with CH2Cl2. The combined filtrate and washings were then washed with 200 mL of 5% NaHCO3 aqueous solution and brine (200 mL). The solution was then dried over Na2SO4 and concentrated under vacuum. The crude product was slurryed in n-heptane (125 mL) for 30 minutes and filtered. This yielded 98 g (52% yield) of 2-1 as a colorless oil. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA 3 minutes: 95:5~5:95 A / B, held for 0.7 minutes): RT 2.09 min, m / z (calculated value) 342.24, (measured value) 343.45 (M+H + ).

[0264] Synthesis of 2-2:2-hydroxypropane-1,3-dioctanoate diyl [ka]

[0265] A 500 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with 200 mL of THF solution containing 2-1 (20 g, 1.0 equivalent). To this solution, AcOH (4.56 g, 1.3 equivalents) was added at 0°C, followed by NaBH3CN (4.3 g, 1.2 equivalents) at 0°C. The mixture was stirred at room temperature for 4 hours. The reaction mixture was quenched with 200 mL of water. The mixture was extracted with CH2Cl2 (3 × 200 mL), and the combined organic phase was washed with an aqueous solution of 5% NaHCO3 (500 mL) and brine (500 mL), and then dried with Na2SO4. The mixture was filtered and concentrated under vacuum to obtain crude 2-2 (19.5 g), which was used in the next step without purification.

[0266] 2-3: Synthesis of ((4,4'-((tert-butoxycarbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetraoctanoate [ka]

[0267] A 500 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with 1-5 (8.4 g, 1.0 equivalent) of CH2Cl2 (175 mL) solution, followed by 2-2 of CH2Cl2 solution. The solution was cooled in an ice bath. To this solution, DMAP (3.55 g, 1.0 equivalent) and EDCI (22.3 g, 4.0 equivalent) were added at 0°C. The reaction mixture was stirred overnight at 25°C. The reaction mixture was then quenched with 200 mL of 10% citric acid aqueous solution. The organic layer was separated, washed with brine (200 mL), and dried over anhydrous sodium sulfate. Crude 2-3 was obtained by filtration and concentration under vacuum, dissolved in CH2Cl2, and the solution was adsorbed onto 54 g of silica gel (type: ZCX-2, 100-200 mesh, 6.43 w / w). The solution was purified using a silica gel column (270 g of silica gel, type: ZCX-2, 100-200 mesh, 32.14 w / w) with a petroleum ether / ethyl acetate gradient of 100:0 to 90:10. The products containing the fractions were pooled, combined, and concentrated under reduced pressure to obtain 15 g (55% in two steps) of 2-3 as a light yellow oily substance.

[0268] 2-4: Synthesis of bis(4-((1,3-bis(octanoyloxy)propane-2-yl)oxy)-4-oxobutyl)ammonium chloride [ka]

[0269] A 500 mL round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was placed in a solution of 2-3 (15 g, 1.0 equivalent) of  (85.5 mL) and the mixture was cooled in an ice bath. HCl in  (80 mL, 10.0 equivalent, 2 mol / L) was added dropwise to the solution at 0-10°C. The resulting solution was stirred overnight at room temperature. The mixture was concentrated under vacuum. This yielded 13 g (93% yield) of 2-4 as a bright 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 at 3 mins, held for 0.7 mins): RT 0.746 mins, m / z (calculated) 842.60, (measured) 842.71 (M+H + ).

[0270] Lipid 2: Synthesis of ((4,4'-((((3-(dimethylaminopropyl)thio)carbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetraoctanoate [ka]

[0271] In a 500 mL three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, 2-4 (8 g, 1 equivalent) dissolved in CH2Cl2 (280 mL) was added, and the solution was cooled in an ice bath. To this mixture, triphosgene (2.82 g, 1 equivalent) was added, followed by pyridine (3.76 g, 5.00 equivalent) being added dropwise while 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 under nitrogen in an ice bath. To this solution, 3-(dimethylamino)propane-1-thiol (1.356 g, 1.20 equivalent) was added dropwise while 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 CH2Cl2 (200 mL). The mixture was washed with 10% citric acid aqueous solution (2 × 100 mL), 5% NaHCO3 (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 CH2Cl2 (25 mL), adsorbed onto 12 g of silica gel (type: ZCX-2, 100-200 mesh, 6.43 w / w), and purified using a silica gel column (80 g of silica gel, type: ZCX-2, 100-200 mesh, 32.14 w / w) with a CH2Cl2 / MeOH gradient of 100:0-97:3. The fractions containing the pure product were analyzed, pooled, combined, and concentrated under reduced pressure. The resulting product 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 hours and then filtered. The filtrate was concentrated under vacuum. This yielded 3.8 g (42%) of the compound as a viscous, pale yellow oily substance. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA; 95:5~5:95 A / B at 2 mins (held for 0.7 mins): RT 1.48 mins, m / z (calculated value) 986.65, (measured value) 987.4 (M+H); 1H NMR(300MHz,CDCl3)δ 5.26(m,2H), 4.32(dd,J=11.9,4.4Hz,4H), 4.16(dd,J=11.9,5.7Hz,4H), 3.40(brm,4H), 2.9 3(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).

[0272] Example 3. Synthesis of Lipid 3: Bis(1,3-bis(nonanoyloxy)propan-2-yl)5-((4-(dimethylamino)butanoyl)oxy)nonanedioate HCl salt [ka] General scheme: [ka]

[0273] 3-2: Synthesis of bis(1,3-bis(nonanoyloxy)propan-2-yl)5-oxononanedioate [ka]

[0274] A 250 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with a solution of 3-1 (7.04 g, 1.0 equivalent, Chemistry-A European Journal 2017, 23, 12744-12748) in CH2Cl2 (100 mL) and cooled under nitrogen in an ice bath. Subsequently, 1-2 (25.95 g, 2.0 equivalent), DMAP (4.26 g, 1.0 equivalent), and EDCI (20.09 g, 3.0 equivalent) were added at 0°C. The resulting solution was stirred at room temperature for 16 hours. The reaction mixture was adsorbed onto 90 g of silica gel (type: ZCX-2, 100-200 mesh, 6.43 w / w) and purified using a silica gel column (900 g, type: ZCX-2, 100-200 mesh) with a PE / EA gradient of 100:0 to 90:10. The fraction containing the pure product was pooled, concentrated under vacuum, and dried over P2O5 to obtain 19.7 g (62.2%) of 3-2 as a yellow oily substance. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5~5:95 A / B at 3 min, held for 0.7 min): RT 0.87 min, m / z (calculated) 910.63, (measured) 933.35 (M+Na).

[0275] 3-3: Synthesis of bis(1,3-bis(nonanoyloxy)propan-2-yl)5-hydroxynonanediate [ka]

[0276] A 250 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with 3-2 (10.7 g, 1.0 equivalent) of THF (100 mL, 10V) solution and cooled in an ice bath. Subsequently, HOAc (7.96 g, 11.3 equivalents) and NaBH3CN (8.88 g, 12.0 equivalents) were added at 0°C. The resulting solution was stirred at room temperature for 16 hours. The reaction was then quenched by adding water (100 mL, 10V). The resulting solution was extracted with ethyl acetate (3 × 100 mL) and combined with the organic layer. The resulting mixture was washed with brine (2 × 100 mL). The mixture was dried over anhydrous sodium sulfate, and the organic layer was concentrated under vacuum. The reaction mixture was adsorbed onto 40 g of silica gel (Type: ZCX-2, 100-200 mesh, 6.43 w / w) and purified using a silica gel column (400 g, Type: ZCX-2, 100-200 mesh) with a PE / EA gradient of 100:0 to 80:20. The fraction containing the pure product was pooled, concentrated under vacuum, and dried over P2O5 to obtain 7.42 g (69.2%) of 3-3 as a yellow oily substance. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5-5:95 A / B at 3 min, retention 0.7 min): RT 0.87 min, m / z (calculated) 913.27, (measured) 935.35 (M+Na).

[0277] Lipid 3: Synthesis of bis(1,3-bis(nonanoyloxy)propan-2-yl)5-((4-(dimethylamino)butanoyl)oxy)nonanedioate HCl salt [ka]

[0278] A 250 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with a solution of 3-3 (7.42 g, 1.0 equivalent) in CH2Cl2 (110 mL) and cooled in an ice bath. 4-(dimethylamino)butanoic acid-HCl (1.63 g, 1.2 equivalents) and DMAP (0.4 g, 0.4 equivalents) were added, followed by EDCI (3.74 g, 2.4 equivalents) being added in several portions at 0°C. The resulting solution was stirred at room temperature for 16 hours. Silica gel (40 g, type: ZCX-2, 100-200 mesh, 6.43 w / w) was added to the reactants and adsorbed onto the silica gel. The solution was purified using a silica gel column (300 g, type: ZCX-2, 100-200 mesh) with an siRNA / THF gradient of 100:0 to 75:25. The fraction containing the pure product was pooled and concentrated under vacuum to obtain 1.9 g (26.3%) as a yellow oily substance. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA (95:5~5:95 A / B at 3 mins, held for 0.7 mins): RT 1.89 mins, m / z (calculated) 1025.74, (measured) 1026.55 (M+H); 1 H NMR(300MHz,CDCl3):δ 5.21(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).

[0279] Example 4. Synthesis of Lipid 4: Bis(1,3-bis(octanoyloxy)propan-2-yl)5-((4-(dimethylamino)butanoyl)thio)nonanediate [ka] General scheme: [ka] [ka]

[0280] 4-1: Synthesis of bis(1,3-bis(octanoyloxy)propan-2-yl)5-oxonanedioate [ka]

[0281] In a 1 L three-necked round-bottom flask, a solution of 3-1 (20.0 g, 98.909 mmol, 1.00 equivalent), 2-2 (68.2 g, 197.818 mmol, 2 equivalents), and DMAP (36.3 g, 296.727 mmol, 3 equivalents) in CH2Cl2 (600 mL) was added and cooled under nitrogen in an ice bath. Subsequently, EDCI (56.9 g, 296.727 mmol, 3 equivalents) was added in several batches at 0°C. The resulting solution was stirred at room temperature for 16 hours. The reaction was then quenched by adding aqueous HCl (1 mol / L, 75 mL). The resulting solution was extracted with CH2Cl2 (200 mL). The combined organic phases were washed with brine (2 × 200 mL). The mixture was dried over anhydrous sodium sulfate and concentrated. The crude substance was dissolved in CH2Cl2 (200 mL), adsorbed onto silica gel (108 g, type: ZCX-2, 100-200 mesh), and purified using a silica gel column (720 g, type: ZCX-2, 100-200 mesh) with a PE / EA gradient of 100:0 to 90:10. The fraction containing the pure product was pooled and concentrated under vacuum to obtain 50 g (53.2%) of 4-1 as a yellow oily substance. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5-5:95 A / B at 3 min, retention 0.7 min): RT 1.74 min, m / z 854.58 (calculated), (measured) 877.75 (M+Na).

[0282] 4-2: Synthesis of bis(1,3-bis(octanoyloxy)propan-2-yl)5-hydroxynonanediate [ka]

[0283] A 1 L three-necked round-bottom flask was filled with a solution of 4-1 (50.0 g, 58.469 mmol, 1.00 equivalent) in 500 mL of THF. This solution was cooled under nitrogen in an ice bath, and HOAc (35.1 g, 584.686 mmol, 10 equivalents) was added at 0°C. NaBH3CN (36.7 g, 584.686 mmol, 10 equivalents) was added in several portions at 0°C. The resulting solution was stirred at room temperature for 3 hours. The reaction was then quenched by adding water (200 mL). The resulting solution was extracted with dichloromethane (1000 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated. The crude substance was adsorbed onto silica gel (160g, type: ZCX-2, 100-200 mesh) and purified using a silica gel column (400g, type: ZCX-2, 100-200 mesh) with a PE / EA gradient of 100:0 to 90:10. The fraction containing the pure product was pooled and concentrated under vacuum to obtain 20g (40%) of 4-2 as a yellow oily substance. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5-5:95 A / B at 3 min, retention 0.7 min): RT 1.00 min, m / z 856.59 (calculated), (measured) 879.70 (M+Na).

[0284] 4-3: Synthesis of bis(1,3-bis(octanoyloxy)propan-2-yl)5-((methylsulfonyl)oxy)nonanediate [ka]

[0285] A 500 mL three-necked round-bottom flask was filled with a solution of 4-2 (25.0 g, 29.166 mmol, 1.00 equivalent) and Et3N (5.9 g, 58.331 mmol, 2 equivalents) in CH2Cl2 (250 mL) and cooled under nitrogen in an ice bath. Subsequently, MsCl (5.0 g, 43.748 mmol, 1.5 equivalents) was added dropwise while stirring at 0°C for 20 minutes. The resulting solution was stirred at room temperature for 3 hours. The reaction was then quenched by adding water / ice (100 mL). The resulting solution was extracted with CH2Cl2 (2 × 100 mL). The combined organic phase was washed with brine (100 mL) and dried over Na2SO4. After concentration, 24 g (crude) of 4-3 was obtained as a bright yellow oily substance, which was used directly in the next reaction. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA (95:5~5:95 A / B after 2 minutes, held for 0.7 minutes): RT 1.71 min, m / z 934.57 (calculated value), (measured value) 957.65 (M+Na).

[0286] 4-4: Synthesis of bis(1,3-bis(octanoyloxy)propan-2-yl)5-mercaptonananediate [ka]

[0287] A solution of 4-3 (30.0 g, 32.077 mmol, 1.00 equivalent) in DMF (300, 10V) was placed in a 500 mL three-necked round-bottom flask, and the mixture was cooled under nitrogen in an ice bath. Subsequently, NaSH (9.0 g, 160.383 mmol, 5.00 equivalent) was added in three portions over 1.5 hours at 0°C. The resulting solution was stirred at room temperature for 5 hours. The reaction was then quenched by adding water / ice (100 mL). The mixture was extracted with siRNA (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 yielded 10 g (crude) of 4-4 as a bright yellow oily substance. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA (95:5~5:95 A / B after 2 minutes, held for 0.7 minutes): RT 1.17 min, m / z 872.57 (calculated value), (measured value) 895.70 (M+Na).

[0288] Lipid 4: Synthesis of bis(1,3-bis(octanoyloxy)propan-2-yl)5-((4-(dimethylamino)butanoyl)thio)nonanane diate [ka]

[0289] A 120 mL solution of 4-4 (12.0 g, 13.742 mmol, 1.00 equivalent), 4-(dimethylamino)butanoic acid (2.2 g, 16.490 mmol, 1.2 equivalents), and DMAP (2.0 g, 16.490 mmol, 1.2 equivalents) in DCM was placed in a 250 mL three-necked round-bottom flask, and the solution was cooled under nitrogen in an ice bath. Subsequently, EDCI (3.16 g, 16.490 mmol, 1.2 equivalents) was added in several batches at 0°C. The resulting solution was stirred at room temperature for 16 hours. The reaction was then quenched by adding aqueous HCl (1 mol / L, 50 mL). The resulting solution was extracted with dichloromethane (2 × 100 mL). The combined organic phases were washed with brine (2 × 100 mL), dried over anhydrous sodium sulfate, and then concentrated. The crude substance was adsorbed onto silica gel (25g, type: ZCX-2, 100-200 mesh) and purified using a silica gel column (200g, type: ZCX-2, 100-200 mesh) with a CH2Cl2 / MeOH gradient of 100:0-25:1. The fraction containing the pure product was pooled and concentrated under vacuum to obtain 1.5g (6.5% yield in 3 steps) of lipid 4 as a bright yellow oil. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5-5:95 A / B at 2 min, held for 0.7 min): RT 1.15 min, m / z 985.66 (calculated), (measured) 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).

[0290] Example 5. Synthesis of Lipid 5: Bis(1,3-bis(nonanoyloxy)propan-2-yl)4-((4-(dimethylamino)butanoyl)oxy)heptanedioate [ka] General scheme: [ka]

[0291] 5-1:4-Oxoheptanediic acid [ka]

[0292] In a three-necked round-bottom flask, EtOH (25 mL, 5V) and diethyl-4-oxo-heptanthioate (5 g, 1 equivalent) were added under nitrogen at room temperature. The mixture was cooled in an ice bath, and then 25 mL of 6N aqueous sodium hydroxide solution was slowly added to the mixture at 0°C. The resulting solution was then heated and stirred at 60°C for 2 hours. After cooling to room temperature, brine (50 mL) and CH2Cl2 (50 mL) were added to the mixture and stirred for 10 minutes, after which the aqueous phase was separated. The pH of the aqueous phase was adjusted to 3-4 using 3N HCl. The mixture was extracted with CH2Cl2 (100 mL). The organic phase was dried over anhydrous MgSO4 and then filtered. Concentrated under vacuum, 5-1 (3.2 g, yield 84.6%) was obtained as a bright yellow solid. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA (95:5~5:95 A / B at 2 mins, held for 0.7 mins): RT 0.81 min, m / z 174.05 (calculated value), (measured value) 197.06 (M+Na).

[0293] 5-2: Synthesis of 3,3'-(1,3-dithiolan-2,2-diyl)dipropionic acid [ka]

[0294] In a three-necked round-bottom flask, CH2Cl2 (32 mL), 5-1 (3.2 g, 1 equivalent), and ethane-1,2-dithiol (2.1 g, 1.2 equivalents) were added all at once at room temperature. The mixture was cooled under nitrogen in an ice bath, and BF3.Et2O (6.48 g, 2.5 equivalents) was slowly added to the mixture at 0°C. The resulting solution was stirred at 20°C for 16 hours. The solid was collected by filtration. The solid was dried under vacuum to obtain 5-2 (4 g, yield 88%) as a bright yellow solid. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5~5:95 A / B at 2 min, hold for 0.7 min): RT 0.20 min, m / z 250.03 (calculated), (measured) 268.2 (M+Na).

[0295] 5-3: Synthesis of ((3,3'-(1,3-dithiolane-2,2-diyl)bis(propanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetranonanoate [ka]

[0296] CH2Cl2 (100 mL), 5-2 (5 g, 1.0 equivalent), 1-2 (16.37 g, 2.2 equivalents), and DMAP (2.44 g, 1 equivalent) were added sequentially to a three-necked round-bottom flask. The mixture was cooled under nitrogen in an ice bath, and then EDCI (8.42 g, 2.2 equivalents) was added to the reaction mixture in several portions at 0°C. The resulting solution was stirred at 20°C for 16 hours. The reaction system was quenched with 10% citric acid aqueous solution (50 mL). The organic phase was separated, washed with 10% citric acid aqueous solution (50 mL) and brine (50 mL), dried over anhydrous MgSO4, and then filtered. The solution was concentrated under vacuum to obtain crude 5-3, which was dissolved in CH2Cl2 (50 mL). The solution was adsorbed onto silica gel (50 g, type: ZCX-2, 100-200 mesh) and purified using a silica gel column (200 g, type: ZCX-2, 100-200 mesh) with a PE / EA gradient of 100:0 to 98:2. The fraction containing the pure product was pooled and concentrated under vacuum to obtain 5-3 (16.1 g, yield 84%) as a colorless oil. (Due to poor ionization, the mass could not be observed. Therefore, no further characterization was performed, and the molecule was used in the next step).

[0297] 5-4: Synthesis of bis(1,3-bis(nonanoyloxy)propan-2-yl)4-oxoheptanediate [ka]

[0298] In a three-necked round-bottom flask, acetone (400 mL) and 5-3 (16 g, 1.0 equivalent) were added, and the solution was cooled to -20°C under nitrogen. A solution of NBS (11.87 g, 4 equivalents) in acetone (80 mL) was added dropwise to the reaction mixture over 15 minutes at -20°C. The resulting solution was stirred at -20°C for 1 hour. The reaction was quenched with H2O (320 mL) and warmed to room temperature. The acetone was removed by concentration under vacuum, and the mixture was extracted with ELISA (160 mL). The organic phase was dried over Na2SO4, filtered, and the solvent was removed under reduced pressure. The crude substance was dissolved in CH2Cl2 (75 mL), adsorbed onto silica gel (30 g, type: ZCX-2, 100-200 mesh), and purified using a silica gel column (200 g, type: ZCX-2, 100-200 mesh) with a PE / EA gradient of 100:0 to 97:3. The fraction containing the pure product was pooled and concentrated under vacuum to obtain 5-4 (10.3 g, yield 70%) as a colorless oil. 1 H NMR(300MHz,CDCl3)δ 5.23(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).

[0299] 5-5: Synthesis of bis(1,3-bis(nonanoyloxy)propan-2-yl)4-hydroxyheptanediate [ka]

[0300] 5-4 (10.3 g, 1 equivalent) and THF (100 mL) were added to a 500 mL four-necked round-bottom flask that had been purged and maintained under an inert nitrogen atmosphere. AcOH (7.0 g, 10 equivalents) was added, and the solution was cooled in an ice bath. Subsequently, NaBH3CN (7.32 g, 10 equivalents) was added in several batches at 0°C. The resulting solution was stirred at 25°C for 18 hours. The reaction system was quenched with H2O (400 mL). The mixture was extracted with HCl (100 mL) to separate the organic phase, dried with Na2SO4, filtered, and concentrated under vacuum. Crude product 5-5 was dissolved in CH2Cl2 and adsorbed onto silica gel (30 g, type: ZCX-2, 100-200 mesh). The mixture was purified using a silica gel column (150 g, type: ZCX-2, 100-200 mesh) with a PE / EA gradient of 100:0 to 95:5. The fraction containing the pure product was pooled and concentrated under vacuum to obtain 5-5 (6 g, 70% yield) as a colorless oil. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5-5:95 A / B at 5 min, retention 0.7 min): RT 3.48 min, m / z 884.62 (calculated), (measured) 907.35 (M+Na).

[0301] Lipid 5: Synthesis of bis(1,3-bis(nonanoyloxy)propan-2-yl)4-((4-(dimethylamino)butanoyl)oxy)heptanedioate [ka]

[0302] In a three-necked round-bottom flask, 5-5 (4g, 1 equivalent), 4-(dimethylamino)butanoic acid (0.99g, 1.3 equivalents), DMAP (0.39g, 0.7 equivalents), and CH2Cl2 (60mL) were added sequentially. The mixture was cooled under nitrogen in an ice bath, and then EDCI (1.21g, 1.4 equivalents) was added to the reaction mixture in several portions at 0°C. The resulting solution was stirred at 20°C for 16 hours. The reaction system was quenched with 10% citric acid aqueous solution (40mL). The organic phase was separated, washed with 10% citric acid aqueous solution (40mL) and brine (40mL), dried over anhydrous MgSO4, and then filtered. The solvent was removed under vacuum, the residue was dissolved in CH2Cl2 (25 mL), and the crude substance was adsorbed onto silica gel (10 g, type: ZCX-2, 100-200 mesh). Purification was performed using a silica gel column (50 g, type: ZCX-2, 100-200 mesh) with a CH2Cl2 / MeOH gradient of 100:0-80:1. The fraction containing the pure product was pooled and concentrated under vacuum to obtain 5 (1.2 g, yield 27%) as a light yellow oily substance. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5-5:95 A / B at 2 min, 0.7 min retention): RT 1.82 min, m / z 997.71 (calculated), (measured) 998.56 (M+H); 1 H-NMR(300MHz, CDCl3):δ 5.24(m,2H), 4.96(dd,J=8.1,4.2Hz,1H), 4.30(m,4H), 4.15(m,4H), 2.41-2.2 5(20H), 1.80-1.92(6H), 1.67-1.54(10H), 1.02-1.49(40H), 0.94-0.84(12H).

[0303] Example 6. Synthesis of lipid 6a [ka] General scheme: [ka]

[0304] 6-1: Synthesis of (S)-3-(benzyloxy)propane-1,2-diyldinonanate [ka]

[0305] In a three-necked flask, 60 mL of CH2Cl2, 3 g (1 equivalent) of (R)-3-(benzyloxy)propane-1,2-diol, and 6.4 g (2.2 equivalents) of nonanoyl chloride were added all at once at room temperature, and the mixture was cooled under nitrogen in an ice bath. 3.90 g (3 equivalents) of pyridine 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 mixture 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 75 mL of CH2Cl2. The combined organic layers were dried over anhydrous sodium sulfate and then filtered. The solution was concentrated under vacuum to obtain crude 6-1, which was dissolved in CH2Cl2 (50 mL) and adsorbed onto 20 g of silica gel (Type: ZCX-2, 100-200 mesh, 6.43 w / w). Purification was performed using a silica gel column (100 g of silica gel, Type: ZCX-2, 100-200 mesh, 32.14 w / w) with a petroleum ether / siRNA gradient of 100:0-50:1. The fractions containing the pure product were analyzed, pooled, combined, and concentrated under reduced pressure to obtain 6-1 (6.0 g, 80% yield) as a colorless oil. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5-5:95 A / B at 2 min, retention 0.7 min): RT 1.1 min, m / z (calculated) 462.33, (measured) 485.2 (M+Na).

[0306] 6-2: Synthesis of (S)-3-hydroxypropane-1,2-dinonanoate diyl [ka]

[0307] In a three-necked flask, under a nitrogen atmosphere at room temperature, 60 mL of MeOH, 6 g (1 equivalent), and 0.6 g (10 wt%) of Pd / C were added. The flask was evacuated and flushed three times with hydrogen. The mixture was stirred under a hydrogen (balloon) atmosphere at room temperature for 16 hours. After filtration, the filtrate was concentrated to dryness under vacuum to obtain 3.1 g (64% yield) of 6-2 as a colorless oil. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5~5:95 A / B at 2 min, held for 0.7 min): RT 0.89 min, m / z (calculated) 3722.29, (measured) 395.3 (M+Na).

[0308] 6-3: Synthesis of (2R,2'R)-((3,3'-((tert-butoxycarbonyl)azandiyl)bis(propanoyl))bis(oxy))bis(propane-3,1,2-triyl)tetranonanoate [ka]

[0309] In a three-necked round-bottom flask, CH2Cl2 (20 mL), 3,3'-((tert-butoxycarbonyl)azandiyl)dipropionic acid (commercial product, 1 g, 1.0 equivalent), 6-2 (3.10 g, 2.2 equivalents), and DMAP (0.47 g, 1 equivalent) were added sequentially, and the mixture was cooled under nitrogen in an ice bath. EDCI (1.60 g, 2.2 equivalents) was added to the reaction mixture in several portions over 10 minutes at 0°C. The resulting solution was stirred at 20°C for 16 hours. The reaction was quenched with 10% citric acid aqueous solution (10 mL). The organic phase was separated, washed with 10% citric acid aqueous solution (10 mL) and brine (10 mL), dried over anhydrous MgSO4, and then filtered. The solution was concentrated under vacuum to obtain crude 6-3, which was dissolved in CH2Cl2 (10 mL) and adsorbed onto 5 g of silica gel (Type: ZCX-2, 100-200 mesh, 6.43 w / w). The solution was purified using a silica gel column (25 g of silica gel, Type: ZCX-2, 100-200 mesh, 32.14 w / w) with a petroleum ether / siRNA gradient of 100:0-50:1. The fractions containing the pure product were analyzed, pooled, combined, and concentrated under reduced pressure to obtain 6-3 (3 g, yield 81%) as a yellow oily substance. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5-5:95 A / B at 3 min, retention 0.7 min): RT 2.35 min, m / z (calculated) 969.68, (measured) 992.5 (M+Na).

[0310] 6-4: Synthesis of (2R,2'R)-((3,3'-Azandiylbis(propanoyl))bis(oxy))bis(propane-3,1,2-triyl)tetranonanoate [ka]

[0311] In a three-necked flask, CH2Cl2 (60 mL) and 6-3 (3 g, 1 equivalent) were added all at once at room temperature. The mixture was cooled under nitrogen in an ice bath, 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 hours. The reaction was then quenched by adding 5% sodium carbonate aqueous solution (10 wt%, 30 mL). The organic phase was separated. The organic phase was washed with brine (2 × 30 mL), dried over anhydrous MgSO4, filtered, and concentrated to dryness to obtain 6-4 (2.5 g, yield 94%) as a yellow oily substance. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA. 3 minutes: 95:5~5:95 A / B, held for 0.7 minutes): RT 0.87 min, m / z (calculated value) 869.62, (measured value) 892.40 (M+Na).

[0312] Synthesis of lipid 6a [ka]

[0313] In a three-necked flask, CH2Cl2 (50 mL, 20V) and 6-4 (2.50 g, 1 equivalent) were added all at once at room temperature. The mixture was then cooled under nitrogen in an ice bath, and triphosgene (0.85 g, 1 equivalent) was added to the reaction mixture in several portions at 0-5°C. Pyridine (1.13 g, 5 equivalents) was slowly added to the reaction mixture over 2 ± 0.5 hours. After addition, the reaction mixture was stirred at room temperature for 2 hours. The solvent was evaporated under reduced pressure, and the residue was dissolved in anhydrous pyridine (50 ml, 20V) and cooled under nitrogen in an ice bath. 3-(dimethylamino)-1-propanthell hydrochloride (0.41 g, 1.2 equivalents) was added at 0°C. After addition, the mixture was stirred at room temperature for 18 hours. The solvent was removed by rotary evaporation under vacuum. The mixture was diluted with CH2Cl2 (50 mL). The organic phase was washed with a 10% citric acid aqueous solution (3 × 25 mL). The organic phase was dried over anhydrous MgSO4 and then filtered. The solution was concentrated under vacuum to obtain crude lipid 6a, which was adsorbed onto 10 g of silica gel (type: ZCX-2, 100-200 mesh, 6.43 w / w) and purified using a silica gel column with a CH2Cl2 / MeOH gradient of 100:0-98:2 (50 g of silica gel, type: ZCX-2, 100-200 mesh, 32.14 w / w). The fractions containing the pure product were analyzed, pooled, combined, and concentrated under reduced pressure to obtain 6 (1.2 g, yield 41%) as a yellow oily substance. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA (95:5~5:95 A / B, held for 0.7 minutes): RT 1.1 min, m / z (calculated value) 1014.68, (measured value) 1015.68 (M+H); 1 H NMR(300MHz,CDCl3)δ 5.26(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).

[0314] Example 7. Synthesis of Lipid 7: Bis(1,3-bis(octanoyloxy)propan-2-yl)4-((4-(dimethylamino)butanoyl)thio)heptanedioate [ka] General scheme: [ka]

[0315] 7-1: Synthesis of ((3,3'-(1,3-dithiolane-2,2-diyl)bis(propanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetraoctanoate [ka]

[0316] A 500 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with a solution of 5-2 (16 g, 1.0 equivalent) in CH2Cl2 (240 mL). Subsequently, 2-2 (48 g, 2.0 equivalent) and DMAP (23 g, 1.0 equivalent) were added, and the mixture was cooled under nitrogen in an ice bath. To this cooled solution, EDCI (36.8 g, 3.0 equivalent) was added in several portions over 45 minutes at 0°C. The resulting solution was stirred at room temperature for 16 hours. This reaction mixture was adsorbed onto silica gel (110 g, type: ZCX-2, 100-200 mesh) and purified using a silica gel column (800 g, type: ZCX-2, 100-200 mesh) with a petroleum ether / ethyl acetate gradient of 100:0 to 90:10. The fraction containing the pure product was pooled and concentrated under vacuum to obtain 7-1 (60 g, 95% yield) as a yellow oily substance. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5~5:95 A / B at 2 min, held for 0.7 min): RT 1.60 min, m / z 902.52 (calculated), (measured) 925.50 (M+Na).

[0317] 7-2: Synthesis of bis(1,3-bis(octanoyloxy)propan-2-yl)4-oxoheptanediate [ka]

[0318] In a 3 L three-necked round-bottom flask, acetone (1.5 L) and 7-1 (60 g, 1.0 equivalent) were added, and the mixture was cooled to -20°C under nitrogen. Then, a solution of NBS (47.3 g, 4.0 equivalent) 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 mixture was quenched with water (300 mL), heated to room temperature, and concentrated under vacuum to remove the acetone. The mixture was extracted with HCl (600 mL), the organic phase was dried to (Na2SO4), filtered, and concentrated under vacuum to obtain crude 7-2. The solvent was removed under reduced pressure. Crude 7-2 was dissolved in CH2Cl2 (200 mL), adsorbed onto silica gel (120 g, type: ZCX-2, 100-200 mesh), and purified using a silica gel column (800 g, type: ZCX-2, 100-200 mesh) with a petroleum ether / siRNA gradient of 100:0 to 90:10. The fraction containing the pure product was pooled and concentrated under vacuum to obtain 7-2 (44 g, yield 80%) as a colorless oil. ELSD A: water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5-5:95 A / B at 2 min, retention 0.7 min): RT 1.36 min, m / z 826.54 (calculated), (measured) 849.50 (M+Na).

[0319] 7-3: Synthesis of bis(1,3-bis(octanoyloxy)propan-2-yl)4-hydroxyheptanediate [ka]

[0320] A 1 L four-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with a solution of 7-2 (44 g, 1.0 equivalent) in THF (400 mL). HOAc (37 g, 12.0 equivalents) was added at 0°C. Then, NaBH3CN (39 g, 12.0 equivalents) was added in several batches at 0°C. The resulting solution was stirred at 25°C for 18 hours. The reaction was quenched with water (800 mL). The mixture was extracted with EA (800 mL). The organic phase was separated, dried over Na2SO4, filtered, and concentrated under vacuum to obtain crude 7-3. Crude 7-3 was dissolved in CH2Cl2 (150 mL), adsorbed onto silica gel (80 g, type: ZCX-2, 100-200 mesh), and purified using a silica gel column (800 g, type: ZCX-2, 100-200 mesh) with a petroleum ether / siRNA gradient of 100:0 to 80:20. The fraction containing the pure product was pooled and concentrated under vacuum to obtain 7-3 (16 g, yield 36%) as a colorless oil. ELSD A: water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5-5:95 A / B at 2 min, retention 0.7 min): RT 1.32 min, m / z 828.56 (calculated), (measured) 851.50 (M+Na).

[0321] 7-4: Synthesis of bis(1,3-bis(octanoyloxy)propan-2-yl)4-((methylsulfonyl)oxy)heptanediate [ka]

[0322] A solution of 7-3 (16 g, 1.0 equivalent) and Et3N (2.4 g, 1.2 equivalents) in DCM (160 mL) was placed in a 500 mL three-necked round-bottom flask, and the solution was cooled under nitrogen in an ice bath. MsCl (2.42 g, 1.1 equivalents) was added dropwise to this cooled solution while stirring at 0°C for 20 minutes. The resulting solution was stirred at room temperature for 3 hours. The reaction was then quenched by adding water / ice (100 mL). The resulting solution was extracted with dichloromethane (2 × 00 mL). The combined organic phase was washed with brine (100 mL). The organic phase was separated, dried over Na2SO4, filtered, and the solvent was removed under reduced pressure to obtain crude 7-4. Crude 7-4 was dissolved in CH2Cl2 (75 mL), adsorbed onto silica gel (32 g, type: ZCX-2, 100-200 mesh), and purified using a silica gel column (500 g, type: ZCX-2, 100-200 mesh) with a petroleum ether / siRNA gradient of 100:0 to 80:20. The fraction containing the pure product was pooled and concentrated under vacuum to obtain 7-4 (10 g, yield 60%) as a colorless oil. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5-5:95 A / B at 2 min, retention 0.7 min): RT 1.26 min, m / z 906.54 (calculated), (measured) 929.50 (M+Na).

[0323] 7-5: Synthesis of bis(1,3-bis(octanoyloxy)propan-2-yl)4-((methylsulfonyl)oxy)heptanedioate [ka]

[0324] A solution of 7-4 (200 mg, 1.0 equivalent) in DMF (4 ml) was placed in a 500 mL three-necked round-bottom flask, and the solution was cooled under nitrogen in an ice bath. Subsequently, NaSH (37.5 mg, 3.0 equivalent) was added at 0°C. The resulting solution was stirred at 0°C for 3 hours. 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 repeated reactions) was dried over anhydrous sodium sulfate and concentrated under vacuum to obtain 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 using a silica gel column (200 g, type: ZCX-2, 100-200 mesh) with a petroleum ether / siRNA gradient of 100:0 to 80:20. The fraction containing the pure product was pooled and concentrated under vacuum to obtain 7-5 (3 g, yield 32%) as a light yellow oil. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5-5:95 A / B at 2 min, retention 0.7 min): RT 1.26 min, m / z 844.54 (calculated), (measured) 845.65 (M+H).

[0325] Lipid 7: Synthesis of bis(1,3-bis(octanoyloxy)propan-2-yl)4-((4-(dimethylamino)butanoyl)thio)heptanedioate [ka]

[0326] A 100 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with a solution of 7-5 (3 g, 1.0 equivalent) in CH2Cl2 (30 mL). 4-(dimethylamino)butanoate HCl (0.71 g, 1.2 equivalents) and DMAP (0.43 g, 1.0 equivalent) were added, and the mixture was cooled in an ice bath. Subsequently, EDCI (1.02 g, 1.5 equivalents) was added in several portions at 0°C. The resulting solution was stirred at room temperature for 16 hours. The reaction mixture was adsorbed onto silica gel (250 g, type: ZCX-2, 100-200 mesh) and purified using a silica gel column (250 g, type: ZCX-2, 100-200 mesh) with an n-heptane / acetone gradient of 100:0 to 50:50. The fraction containing the pure product was pooled and concentrated under vacuum to obtain lipid 7 (1.1 g, yield 32%) as a yellow oil. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5~5:95 A / B at 2 min, held for 0.7 min): RT 0.75 min, m / z 957.52 (calculated), (measured) 958.50 (M+H). 1 H-NMR(300MHz, CDCl3):δ 5.25(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).

[0327] Example 8. Synthesis of Lipid 8: ((2,2'-((((3-(dimethylaminopropyl)thio)carbonyl)azandiyl)bis(acetyl))bis(oxy))bis(propane-2,1,3-triyl)tetranonanoate [ka] General scheme: [ka]

[0328] 8-1: Synthesis of ((2,2'-((tert-butoxycarbonyl)azandiyl)bis(acetyl))bis(oxy))bis(propane-2,1,3-triyl)tetranonanoate [ka]

[0329] In a three-necked round-bottom flask, a solution of 2,2'-((tert-butoxycarbonyl)azandiyl)diacetic acid (1 g, 1.0 equivalent), 1-2 (3.50 g, 2.2 equivalents), and DMAP (0.52 g, 1 equivalent) in DCM (20 mL) was added, and the solution was cooled under nitrogen in an ice bath. To this cooled solution, EDCI (1.80 g, 2.2 equivalents) was added in several portions at 0°C. The resulting solution was stirred at 20°C for 16 hours. The reaction was quenched with 10% citric acid aqueous solution (10 mL). The organic phase was separated, washed with 10% citric acid aqueous solution (10 mL, 10V) and brine (10 mL, 10V), and dried over anhydrous MgSO4. Crude 8-1 was obtained by filtration and concentration under vacuum, dissolved in CH2Cl2 (15 mL), adsorbed onto 5 g of silica gel (type: ZCX-2, 100-200 mesh, 6.43 w / w), and purified using a silica gel column (20 g silica gel, type: ZCX-2, 100-200 mesh, 32.14 w / w) with a petroleum ether / siRNA (volume ratio) (gradient from 100:0 to 50:1). The fractions containing the pure product were analyzed, pooled, combined, and concentrated under reduced pressure to obtain 8-1 (3.27 g, yield 81%) as a yellow oil. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA. 3 minutes: 95:5~5:95 A / B, held for 0.7 minutes): RT 2.2 minutes, m / z (calculated value) 941.64, (measured value) 964.60 (M+Na).

[0330] 8-2: Synthesis of ((2,2'-Azandiylbis(acetyl))bis(oxy))bis(propane-2,1,3-triyl)tetranonanoate [ka]

[0331] In a three-necked flask, 3 g (1 equivalent) of 8-1 was added to a solution of 60 mL of CH2Cl2. The resulting solution was cooled in an ice bath under nitrogen, and then 4.5 mL of TFA was slowly added at 0-5°C. The resulting solution was stirred at 20°C for 2 hours. The reaction was then quenched by carefully adding 30 mL of 10% sodium carbonate solution. The organic phase was separated, washed with brine (2 × 30 mL), dried over anhydrous MgSO4, and filtered. The solvent was removed under vacuum to obtain 8-2 (2.5 g, 93% yield) as a yellow oil. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA (3 minutes, 95:5~5:95 A / B, held for 0.7 minutes): RT 1.15 min, m / z (calculated value) 841.59, (measured value) 842.51 (M+H).

[0332] Lipid 8: Synthesis of ((2,2'-((((3-(dimethylaminopropyl)thio)carbonyl)azandiyl)bis(acetyl))bis(oxy))bis(propane-2,1,3-triyl)tetranonanoate [ka]

[0333] In a three-necked flask, a solution of 8-2 (2.50 g, 1 equivalent) in 50 mL of CH2Cl2 was added all at once at room temperature. The resulting solution was cooled under nitrogen in an ice bath, and then triphosgene (0.88 g, 1 equivalent) was added to the reaction mixture over 5 minutes at 0-5°C. Pyridine (1.17 g, 5 equivalents) was slowly added to the reaction mixture over 2 ± 0.5 hours. After addition, the reaction mixture was stirred at room temperature for 2 hours. The solvent was evaporated under reduced pressure, and the residue was dissolved in anhydrous pyridine (50 mL) and cooled in an ice bath. 3-(dimethylamino)-1-propanthell hydrochloride (0.42 g, 1.2 equivalents) was added, and the mixture was stirred at room temperature for 18 hours. The solvent was removed under vacuum. The residue was dissolved in CH2Cl2 (50 mL) and washed with 10% citric acid aqueous solution (3 × 25 mL). The organic phase was separated, dried over anhydrous MgSO4, and then filtered. The solution was concentrated under vacuum to obtain crude lipid 8. This crude product was dissolved in CH2Cl2 (15 mL), adsorbed onto silica gel (5 g, type: ZCX-2, 100-200 mesh, 6.43 w / w), and purified using a silica gel column (20 g silica gel, type: ZCX-2, 100-200 mesh, 32.14 w / w) with a CH2Cl2 / MeOH gradient (volume ratio, gradient of 100 / 0 to 98:2). The fraction containing lipid 8 was analyzed, pooled, and combined to obtain lipid 8 (1.2 g, yield 41%) as a yellow oily substance. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA (95:5~5:95 A / B, held for 0.7 minutes): RT 0.88 min, m / z (calculated value) 986.65, (measured value) 987.95 (M+H); 1 H NMR(300MHz,CDCl3):δ 5.28(m,2H), 4.35(m,4H), 4.18(m,8H), 2.95(t,J=7.3Hz,2H), 2.340-2.21 2(16H), 1.78(m,2H), 1.71-1.57(8H), 1.35-1.20(40H), 0.94-0.80(12H).

[0334] Example 9. Synthesis of Lipid 9: Bis(1,3-bis(nonanoyloxy)propan-2-yl)4-((4-(dimethylamino)butanoyl)thio)heptanedioate [ka] General scheme: [ka]

[0335] 9-1: Synthesis of bis(1,3-bis(nonanoyloxy)propan-2-yl)4-((methylsulfonyl)oxy)heptanedioate [ka]

[0336] In a 250 mL four-necked round-bottle flask, mechanically stirred under N2, a solution of 5-5 (6 g, 1.0 equivalent) CH2Cl2 (90 mL) was added. Subsequently, Et3N (2.06 g, 3.0 equivalents) was added, and the resulting solution was cooled under nitrogen in an ice bath. To the cooled solution, MsCl (1.16 g, 1.5 equivalents) was added dropwise while stirring at 0°C. The resulting solution was stirred at room temperature for 12 hours. The reaction was then quenched by adding H2O (100 mL). The phases were separated, and the aqueous phase was extracted with CH2Cl2 (100 mL). The combined organic phase was then dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. Crude 9-2 was purified by high-speed preparative HPLC using the following conditions: column, XB-C18 silica gel; mobile phase, i-PrOH in an aqueous solution of 1 mmol NH4HCO3, gradient from 65% to 95% over 30 minutes; detector, UV ELSD. The mixture was concentrated to dryness under vacuum to obtain 9-1 (5 g, 44% total yield in 2 steps) as a colorless oil. ELSD A: water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5~5:95 A / B over 2 minutes, retention 0.7 minutes): RT 1.66 min, m / z 962.60 (calculated), (measured) 985.50 (M+Na).

[0337] 9-2: Synthesis of bis(1,3-bis(nonanoyloxy)propan-2-yl)4-mercaptoheptane diate [ka]

[0338] A 100 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with a solution of 9-1 (100 mg, 1.00 equivalent) in DMF (0.5 mL). The resulting solution was cooled under nitrogen in an ice bath. Subsequently, NaSH (29.1 mg, 5.00 equivalent) was added at 0°C. The resulting solution was stirred at 0°C for 24 hours. This reaction was repeated 49 times, and the entire mixture was combined to complete the reaction. The reaction was then quenched by adding ice / water (200 mL). The resulting solution was extracted with ELISA (3 × 100 mL), the organic phase was combined, dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum to obtain 9-2 (4.8 g, crude) as a colorless oil. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA. 3 minutes: 95:5~5:95 A / B, held for 0.7 minutes): RT 2.08 min, m / z 900.60 (calculated value), (measured value) 923.50 (M+Na).

[0339] Lipid 9: Synthesis of bis(1,3-bis(nonanoyloxy)propan-2-yl)4-((4-(dimethylamino)butanoyl)thio)heptane diate [ka]

[0340] In a three-necked round-bottom flask, a solution of 9-2 (4.8 g, 1.0 equivalent), 4-(dimethylamino)butanoic acid (1.16 g, 1.3 equivalents), and DMAP (0.46 g, 0.7 equivalents) in CH2Cl2 (72 mL) was added, and the resulting solution was cooled under nitrogen in an ice bath. EDCI (1.84 g, 1.4 equivalents) was added to the reaction mixture in several portions at 0°C. The resulting solution was stirred at room temperature for 12 hours. The reaction system was quenched with 10% citric acid aqueous solution (48 mL). The organic phase was separated, washed with 10% citric acid aqueous solution (48 mL) and brine (48 mL, 10V), and dried over anhydrous MgSO4. Crude 9 was obtained by filtration and concentration under vacuum, dissolved in CH2Cl2 (25 mL), adsorbed onto a silica gel column (10 g, type: ZCX-2, 100-200 mesh), and purified using a silica gel column (50 g, type: ZCX-2, 100-200 mesh) with an n-heptane / acetone gradient of 100:0 to 75:50. The fraction containing pure 9 was pooled and concentrated under vacuum to obtain 9 (0.9 g, yield 18%) as a yellow oil. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5-5:95 A / B at 3 min, held for 0.7 min): RT 1.24 min, m / z 10¹3.68 (calculated), (measured) 10¹5.40 (M+H); 1 H NMR(300MHz,CDCl3):δ 5.27(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).

[0341] Example 10. Synthesis of Lipid 10: ((4,4'-((((3-(dimethylaminopropyl)thio)carbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexylpropanoate) [ka]

[0342] General scheme: [ka]

[0343] Synthesis of 10-1:3-cyclohexylpropanoyl chloride [ka]

[0344] A 2000 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was placed in a solution of 3-cyclohexylpropanoic acid (100 g, 1.0 equivalent) in CH2Cl2 (1 L), and DMF (0.2 mL) was added. Oxalyl chloride (161.00 g, 2.00 equivalent) was added dropwise at room temperature. The mixture was stirred overnight under nitrogen at room temperature. The mixture was concentrated under vacuum to obtain crude 10⁻¹. This was used directly in the next reaction.

[0345] Synthesis of 10-2:2-oxopropane-1,3-diylbis(3-cyclohexylpropanoate) [ka]

[0346] A 2000 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with a solution of 10-1 (83.00 g, 2.50 equivalents) and dihydroxyacetone (17.20 g, 1.00 equivalent) in CHCl3 (1600 mL). The solution was cooled under nitrogen in an ice bath. To this cooled solution, pyridine (61.00 g, 4.00 equivalents) was added over 40 minutes while maintaining the temperature at 0°C. The mixture was stirred overnight under nitrogen at room temperature. The formed pyridine hydrochloride was removed by filtration and washed with CH2Cl2 (200 mL). The combined filtrate was then washed with 5% NaHCO3 aqueous solution (2000 mL), 5% HCl aqueous solution (2000 mL), and brine (2000 mL), and dried over Na2SO4. The solution was concentrated under vacuum to obtain 65 g (yield 92.8%) of 10-2 as a yellow oil. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA (95:5~5:95 A / B in 3 minutes, held for 0.7 minutes): RT 2.56 min, m / z 366.24 (calculated), (measured) 367.40 (M+H).

[0347] Synthesis of 10-3:2-hydroxypropane-1,3-diyrbis(3-cyclohexylpropanoate) [ka]

[0348] A 2000 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with a solution of 10⁻² (85.00 g, 1.00 equivalent) of THF (1 L). HOAc (18.00 g, 1.30 equivalent) was added to the solution, and the solution was cooled in an ice bath. To this cooled solution, NaBH₃CN (18.00 g, 1.20 equivalent) was added at 0°C. The mixture was stirred overnight at room temperature. The reaction mixture was quenched with brine (1 L). The mixture was extracted with RINKAN (3 × 400 mL). The combined organic phase was then washed with 5% NaHCO₃ aqueous solution (200 mL), 5% HCl aqueous solution (200 mL), and brine (200 mL), and dried over Na₂SO₄. Crude 10-3 was obtained by filtration and concentration under vacuum, which was dissolved in CH2Cl2 (500 mL) and adsorbed onto silica gel (240 g, type: ZCX-2, 100-200 mesh). The crude substance was purified using a silica gel column (800 g, type: ZCX-2, 100-200 mesh) with a petroleum ether / siRNA gradient of 100:0 to 90:10. The fraction containing the pure product was pooled and concentrated under vacuum to obtain 10-3 (61 g, yield 70.1%) as a yellow oily substance. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5-5:95 A / B at 3 min, held for 0.7 min): RT 2.50 min, m / z 368.26 (calculated), (measured) 351.2 (M-H2O).

[0349] 10-4: Synthesis of ((4,4'-((tert-butoxycarbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexylpropanoate) [ka]

[0350] In a 250 mL three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, 1-5 (4.00 g, 1 equivalent) and 10-3 (10.20 g, 2 equivalents) were added, and the mixture was dissolved in CH2Cl2 (80 mL). The solution was cooled in an ice bath, and DMAP (1.69 g, 1.00 equivalent) and EDCI (10.60 g, 4.00 equivalent) were added sequentially at 0°C. The reaction mixture was stirred overnight at room temperature. The reaction was then quenched with 10% citric acid aqueous solution (200 mL) to separate the organic phase. The organic phase was washed with 10% NaHCO3 aqueous solution (200 mL) and brine (200 mL), and dried over anhydrous sodium sulfate. Crude 10-4 was obtained by filtration and concentration under vacuum, dissolved in CH2Cl2 (100 mL), adsorbed onto silica gel (50 g, type: ZCX-2, 100-200 mesh), and purified by silica gel column (400 g, type: ZCX-2, 100-200 mesh) with a petroleum ether / siRNA gradient of 100:0 to 90:10. The fraction containing the pure product was pooled and concentrated under vacuum to obtain 11 g of 10-4 as a light yellow oil (yield 77.7%). ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5-5:95 A / B at 3 min, held for 0.7 min): RT 1.79 min, m / z 989.64 (calculated), (measured) 1012.50 (M+Na).

[0351] 10-5: Synthesis of bis(4-((1,3-bis((3-cyclohexylpropanoyl)oxy)propan-2-yl)oxy)-4-oxobutyl)ammonium chloride [ka]

[0352] A 250 mL round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was placed in a 20 mL solution of 10⁻⁴ (6.30 g, 1.00 equivalent) of  (20 mL), and the solution was cooled in an ice bath. To the cooled solution, a HCl- solution (60 mL, 10 equivalents, 2 M) was added dropwise at 0-10°C. The resulting solution was stirred overnight at room temperature. The resulting mixture was concentrated under vacuum. This yielded 6 g (99% yield) of 10⁻⁵ as a bright yellow oil. ELSD A: Water / 0.05% TFA: B: CH₃CN / 0.05% TFA 95:5~5:95 A / B at 3 mins, held for 0.7 mins): RT 1.48 mins, m / z 889.59 (calculated), (measured) 890.50 (M+H).

[0353] Lipid 10: Synthesis of ((4,4'-((((3-(dimethylaminopropyl)thio)carbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexylpropanoate) [ka]

[0354] A 250 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with a solution of 10⁻⁵ (6.00 g, 1.00 equivalent) of CH₂Cl₂ (100 mL) and cooled under nitrogen in an ice bath. Triphosgene (1.91 g, 1.62 equivalents) was added to the mixture at 0°C. Pyridine (2.56 g, 5.00 equivalents) was then added dropwise at 0°C with stirring. The mixture was stirred at room temperature for 4 hours and then concentrated under vacuum. The residue was dissolved in pyridine (100 mL) and cooled under nitrogen in an ice bath. 3-(dimethylamino)propane-1-thiol (0.92 g, 1.93 equivalents) was added dropwise to this solution over 10 minutes at 0°C with stirring. The resulting solution was stirred overnight at room temperature. The resulting mixture was concentrated under vacuum. The residue was diluted with CH2Cl2 (500 mL), the solution was washed with 10% citric acid aqueous solution (2 × 200 mL), saturated NaHCO3 aqueous solution (2 × 200 mL), and brine (100 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum to obtain crude lipid 10. The residue was dissolved in CH2Cl2 (100 mL), adsorbed onto silica gel (50 g, type: ZCX-2, 100-200 mesh), and purified using a silica gel column (250 g, type: ZCX-2, 100-200 mesh) with a CH2Cl2 / MeOH gradient of 100:0 to 97:3. The fraction containing the pure product was 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 hours and then filtered. The filtrate was concentrated under vacuum to obtain 2 g (48% yield) of lipid 10 as a bright yellow oily substance. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA; 95:5~5:95 A / B at 3 mins, held for 0.7 mins); RT 0.68 mins, m / z 1034.65 (calculated value), (measured value) 1035.65 (M+H); 1 H NMR(300MHz,CDCl3):δ 5.25(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).

[0355] Example 11. Lipid 11: ((4,4'-((((3-(dimethylaminopropyl)thio)carbonyl) Synthesis of azandiyl bis(butanoyl)bis(oxy)bis(propane-2,1,3-triyl)tetrakis(4-cyclohexylbutanoate) [ka] General scheme: [ka] [ka]

[0356] Synthesis of 11-1:4-cyclohexylbutanoyl chloride [ka]

[0357] A 1 L three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with a solution of 4-cyclohexylbutanoic acid (50 g, 1.0 equivalent) in CHCl3 (500 ml), followed by the addition of DMF (0.25 ml). The solution was cooled in an ice bath, and then oxalyl chloride (74.1 g, 2.0 equivalents) was added dropwise over 30 minutes. The mixture was stirred overnight under nitrogen at room temperature. The mixture was concentrated under vacuum. This yielded 55.7 g (99.9% yield) of 11-1 as a yellow oil, which was used directly in the next step.

[0358] Synthesis of 11-2:2-oxopropane-1,3-diylbis(4-cyclohexylbutanoate) [ka]

[0359] A 2 L three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with 11-1 (55.7 g, 2.5 equivalents) of solution. The solution was cooled in an ice bath, and a solution of 1,3-dihydroxyacetone (10.6 g, 1.0 equivalent) in CHCl3 (1114 mL) was added over 1 hour. Pyridine (37.3 g, 4.0 equivalents) was added to the mixture over 40 minutes 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 CH2Cl2 (3 × 200 mL). The organic phases were combined and washed with 5% NaHCO3 aqueous solution (300 mL), 5% HCl aqueous solution (300 mL), and brine (300 mL). The solution was then dried over anhydrous Na2SO4, and the product was obtained by evaporation. This yielded 56.4 g of crude 11-2 as a yellow oily substance, which was used directly in the next reaction.

[0360] Synthesis of 11-3:2-hydroxypropane-1,3-diyrbis(4-cyclohexylbutanoate) [ka]

[0361] A 1 L three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with a solution of 11-2 (56.4 g, 1.0 equivalent) in THF (550 ml), and the mixture was cooled in an ice bath. To the cooled solution, HOAc (11.13 g, 1.3 equivalents) was added at 0°C. Then NaBH3CN (10.79 g, 1.2 equivalents) was 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 CH2Cl2 (3 × 200 ml). The organic layers were combined and washed with 5% NaHCO3 aqueous solution (200 ml), 5% HCl aqueous solution (200 ml), and brine (200 ml). The solution was then dried over anhydrous Na2SO4 and concentrated under vacuum. The residue was dissolved in CH2Cl2 (150 mL), silica gel (60 g, type: ZCX-2, 100-200 mesh) was added, and the crude product was adsorbed onto the silica gel. The mixture was then purified using a silica gel column (240 g, type: ZCX-2, 100-200 mesh) with a petroleum ether / siRNA gradient of 100:0 to 80:20. The fraction containing 11-3 was pooled, concentrated, and dried under vacuum to obtain 30.3 g of 11-3 (65.0% total yield in two steps) as a yellow oily substance. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5-5:95 A / B at 3 min, held for 0.7 min): RT 0.76 min, m / z 396.29 (calculated), (measured) 419.29 (M+Na).

[0362] 11-4: Synthesis of ((4,4'-((tert-butoxycarbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(4-cyclohexylbutanoate) [ka]

[0363] In a 250 ml three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, 11-3 (5.46 g, 2.0 equivalent) was added to a solution of 1-5 (2.05 g, 1.0 equivalent) in CH2Cl2 (100 mL), and the mixture was cooled in an ice bath. DMAP (2.3 g, 1.0 equivalent) and then EDCI (14.51 g, 4.0 equivalent) were added to the solution at 0°C. The reaction mixture was stirred overnight at room temperature. The reaction mixture was then quenched with 10% citric acid aqueous solution (200 mL) to separate the organic phase, which was washed with 10% NaHCO3 aqueous solution (200 mL) and brine (200 mL). The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. This substance was combined with another 7.05 g scale reactant (based on 11-3), and the combined substance was adsorbed onto silica gel (30 g, type: ZCX-2, 100-200 mesh) and purified using a silica gel column (90 g, type: ZCX-2, 100-200 mesh) with a petroleum ether / siRNA gradient of 100:0 to 65:35. The fraction containing 11-4 was pooled, concentrated, and dried under vacuum to obtain 14.2 g (43.0%) of 11-4 as a yellow oily substance. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5-5:95 A / B at 3 min, held for 0.7 min): RT 2.77 min, m / z 1045.71 (calculated), (measured) 1068.55 (M+Na).

[0364] 11-5: Synthesis of bis(4-((1,3-bis((4-cyclohexylbutanoyl)oxy)propan-2-yl)oxy)-4-oxobutyl)ammonium chloride [ka]

[0365] A 250 ml round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was placed in a solution of 11-4 (14.17 g, 1.0 equivalent) in HCl (71 mL), and the solution was cooled in an ice bath. To this solution, a solution of HCl with HCl (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 under vacuum. This yielded 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 at 3 mins, held for 0.7 mins): RT 0.94 mins, m / z 945.65 (calculated), (measured) 946.60 (M+H).

[0366] Lipid 11: Synthesis of ((4,4'-((((3-(dimethylaminopropyl)thio)carbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(4-cyclohexylbutanoate) [ka]

[0367] A 1 L three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was placed in a solution of 11-5 (12.0 g, 1.0 equivalent) in CH2Cl2 (420 mL), and the solution was cooled in an ice bath. Triphosgene (5.44 g, 1.5 equivalents) was added to the solution at 0°C. Subsequently, pyridine (4.82 g, 5.0 equivalents) was added dropwise at 0°C with stirring. The mixture was stirred at room temperature for 4 hours, and then concentrated under vacuum. The residue was dissolved in pyridine (240 mL), and the resulting solution was cooled in an ice bath. To this solution, 3-(dimethylamino)propane-1-thiol (2.91 g, 2.0 equivalents) 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 under vacuum. The residue was diluted with DCM (200 ml). The mixture was washed with 10% NaHCO3 aqueous solution (2 × 200 mL) and 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 using a silica gel column (80 g, type: ZCX-2, 100-200 mesh) with an n-heptane / acetone gradient of 100:0 to 65:35. The fraction containing the pure product was pooled, concentrated, and dried under vacuum to obtain 2 g of 11, which was dissolved in n-heptane (40 ml, 20V) and activated carbon powder (0.22 g) was added. The mixture was stirred at room temperature for 4 hours and then filtered. The filtrate was concentrated under vacuum. This yielded 2 g (yield 14.4%) of 11 as a light yellow oil. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA (3 minutes, 95:5~5:95 A / B, held for 0.7 minutes): RT 1.00 min, m / z 1090.71 (calculated value), (measured value) 1091.60 (M+H); 1 H NMR(300MHz,CDCl3):δ 5.24(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).

[0368] Example 12. Synthesis of Lipid 12: ((6,6'-((((3-(dimethylamino)propyl)thio)carbonyl)azandiyl)bis(hexanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexylpropanoate) [ka] General scheme: [ka]

[0369] 12-1: Synthesis of dimethyl 6,6'-(benzylazandiyl)dihexanoate [ka]

[0370] A solution of K2CO3 (9.5 g, 2.3 equivalents), benzylamine (3.2 g, 1.0 equivalent), and methyl 4-bromo-butyrate (15.2 g, 2.3 equivalents) in CH3CN (64 mL) was placed in a 250 ml four-necked round-bottom flask at 25°C under N2 with mechanical stirring. The mixture was then heated (80°C) and stirred for 15 hours. The mixture was then cooled to 25°C, added to water (65 mL), and extracted with RINKAN (2 × 65 mL). The combined organic phase was dried (Na2SO4), filtered, and concentrated under vacuum to obtain crude 12-1 (10 g, crude) as a yellow oil, which was used directly in the next reaction.

[0371] 12-2: Synthesis of dimethyl 6,6'-((tert-butoxycarbonyl)azandiyl)dihexanoate [ka]

[0372] A solution of 12-1 (10 g, 1.0 equivalent), (Boc)2O (6.6 g, 1.1 equivalent), 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 hours. The reaction mixture was filtered and concentrated under vacuum at 40°C. This yielded 12-2 (11 g, crude) as a light brown oily substance. This was used directly in the next reaction.

[0373] Synthesis of 12-3:6,6'-((tert-butoxycarbonyl)azandiyl)dihexanoic acid [ka]

[0374] A solution of 12-2 (11 g, 1.0 equivalent) in ethanol (55 mL) was placed in a round-bottom flask under nitrogen at room temperature. 55 mL of 6 M NaOH aqueous solution was added at room temperature. After the addition, the mixture was heated to 60°C for 2 hours. The mixture was cooled to room temperature and placed in 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 HCl aqueous solution, and then extracted with t-BuOH:n-heptane (2:1) (110 mL x 2). The combined organic phase was concentrated under reduced pressure to obtain a viscous solid. The residue was slurryed with diethyl ether (22 mL) and filtered. The filtered cake was collected to obtain 12-3 (5.6 g, 66% total yield in 3 steps) as a white solid. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA (95:5~5:95 A / B after 3 minutes, held for 0.7 minutes): RT 1.64 min, m / z 345.22 (calculated value), (measured value) 368.10 (M+Na).

[0375] 12-4: Synthesis of ((6,6'-((tert-butoxycarbonyl)azandiyl)bis(hexanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexylpropanoate) [ka]

[0376] In a 250 ml three-necked round-bottom flask, a solution of 12-3 (5.6 g, 1.0 equivalent), 10-3 (10 g, 2.0 equivalent), and DMAP (1.65 g, 1.0 equivalent) in CH2Cl2 (85 mL) was added, and the solution was cooled under nitrogen in an ice bath. EDCI (7.5 g, 2.2 equivalents) was added to the reaction mixture in several portions over 15 minutes at 0°C. After the addition was complete, the reaction mixture was warmed to room temperature and stirred at 20°C for 16 hours. The reaction mixture was then added to a 10% citric acid aqueous solution (112 mL). The organic phase was separated and dried with 112 mL of 10% citric acid aqueous solution and brine (112 mL), then filtered. The solvent was removed under vacuum to obtain crude 12-4, which was dissolved in CH2Cl2 (65 mL). The crude product was adsorbed onto silica gel (30 g, type: ZCX-2, 100-200 mesh) and purified by silica gel column (150 g, type: ZCX-2, 100-200 mesh) with a petroleum ether / siRNA gradient of 100:0-88:12. The fraction containing the pure product was pooled, concentrated, and dried under vacuum to obtain 10.1 g (60%) of 12-4 as a colorless oil. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5-5:95 A / B, held for 0.7 min at 3 min):RT 2.2 min, m / z 1045.71 (calculated value), (measured value) 1068.65 (M+Na).

[0377] 12-5: Synthesis of ((6,6'-Azandiylbis(hexanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexylpropanoate) [ka]

[0378] A solution of 12-4 (5g, 1.0 equivalent) in CH2Cl2 (50mL) was placed in a 100mL three-necked round-bottom flask, and the solution was cooled under nitrogen in an ice bath. Then, TFA (7.5mL) was 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 hours. The mixture was concentrated at 30°C under vacuum, and then n-heptane (100mL) was added to the reaction mixture. The resulting turbid mixture was washed with 17% sodium carbonate aqueous solution (500mL) and brine (250mL, 3×), and dried over anhydrous MgSO4. The mixture was filtered and concentrated under vacuum to obtain 12-5 (4.5g, yield 90%) as a yellow oily substance. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA (3 minutes, 95:5~5:95 A / B, held for 0.7 minutes): RT 0.94 min, m / z 945.65 (calculated value), (measured value) 946.60 (M+H).

[0379] Lipid 12: Synthesis of ((6,6'-((((3(dimethylamino)propyl)thio)carbonyl)azandiyl)bis(hexanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexylpropanoate) [ka]

[0380] In a 250 mL three-necked round-bottom flask, 12-5 (4.5 g, 1.0 equivalent) and CH2Cl2 (68 mL) were added, and the solution was cooled under nitrogen in an ice bath. Next, triphosgene (1.4 g, 1.0 equivalent) was added to the cooled solution, followed by pyridine (1.88 g, 5.0 equivalents) 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 under vacuum, and the residue was dissolved in pyridine (90 mL), and the mixture was cooled under nitrogen in an ice bath. To this cooled solution, 3-(dimethylamino)-1-propanthell hydrochloride (0.57 g, 1.1 equivalents) was added. After the addition was complete, the reaction mixture was warmed to room temperature and stirred for 18 hours. The mixture was concentrated under vacuum and kept at a temperature below 20°C to obtain crude lipid 12, which was dissolved in CH2Cl2 (90 mL). The resulting solution was washed with 10% citric acid aqueous solution (45 mL), brine (45 mL, 3 ×), 10% sodium bicarbonate aqueous solution (45 mL), and brine (45 mL, 2 ×). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude lipid 12. Crude lipid 12 was dissolved in CH2Cl2 (30 mL), adsorbed onto silica gel (15 g, type: ZCX-2, 100-200 mesh), and purified using a silica gel column (60 g, type: ZCX-2, 100-200 mesh) with an n-heptane / acetone gradient of 100:0 to 80:20. The fraction containing the pure product was pooled, concentrated, and dried under vacuum to obtain 1.5 g (29%) of lipid 12 as a light yellow oily substance. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA (3 minutes, 95:5~5:95 A / B, held for 0.7 minutes): RT 0.87 min, m / z 1090.71 (calculated value), (measured value) 1091.60 (M+H); 1 H NMR(300MHz,CDCl3):δ 5.27(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).

[0381] Example 13. Synthesis of Lipid 13: Nonanoic Acid 2-(3-{(3-dimethylaminopropylsulfanylcarbonyl)-[2-(2-nonanoyloxy-1-nonanoyloxymethyl-ethoxycarbonyl)-ethyl]-amino}propionyloxy)-3-octanoyloxypropyl ester [ka] General scheme: [ka]

[0382] 13-1: Synthesis of ((3,3'-((tert-butoxycarbonyl)azandiyl)bis(propanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetranonanoate [ka] 1-5 (7.3g, 28.0 mmol) and 1-2 (18.8g, 56.0 mmol) were dissolved in CH2Cl2 (110 mL) in a 500 mL three-necked round-bottom flask under nitrogen. The solution was cooled in an ice bath, and DMAP (3.4g, 28.0 mmol) and EDCl (21.5g, 0.112 mol) were added in sequence. After adding the compounds, the mixture was warmed to room temperature and stirred for 16 hours. The mixture was placed in a 10% citric acid aqueous solution (200 mL) to separate the organic phase, washed with brine (200 mL), and dried over anhydrous Na2SO4. The desiccant 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 under vacuum at a rotating evaporator temperature of 35°C. Silica gel containing adsorbed 13-1 was placed on a silica gel column (50 mm OD, 200 g silica gel, type: ZCX-2, 100-200 mesh). The column was eluted with petroleum ether:siRNA at a gradient of 100:0 to 97:3 for a 200 mL fraction. TLC analysis showed a fraction containing 13-1, which was combined and concentrated under vacuum to obtain 13-1 (12.8 g, 13.2 mmol, 47%) as a pale yellow, viscous oil. 1 H-NMR(300MHz,DMSO-d6):δ 5.18(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).

[0383] 13-2: Synthesis of ((3,3'-Azandiylbis(propanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetranonanoate hydrochloride [ka]

[0384] A solution of 13-1 (12.8 g, 13.2 mmol) of HCl (75 mL), cooled in an ice bath under nitrogen, was to which a solution of HCl-HCl HCl (2 M, 80 mL, 0.160 mol) was added at a rate that kept the internal temperature between 0 and 10°C. After the addition, the mixture was warmed to room temperature and stirred for 14 hours. The mixture was concentrated in vacuum to obtain the HCl salt 13-2 (8.1 g, 9.37 mmol, 71%) as a bright yellow, viscous oil. 1 H-NMR (300MHz, DMSO-d6): δ 5.19(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).

[0385] 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)propan-1,3-diyldinonanoate [ka]

[0386] To a solution of 13-2 (8.1 g, 9.37 mmol) in CH2Cl2 (280 mL), cooled under nitrogen in an ice bath, triphosgene (2.77 g, 9.33 mmol) was added all at once, followed by the dropwise addition of pyridine (3.68 g, 46.52 mmol). After the addition was complete, the reaction mixture was warmed to room temperature and stirred for 4 hours. The solvent was removed under vacuum (bath temperature 25°C), and the residue was dissolved in pyridine (160 mL). The solution was cooled under nitrogen in an ice bath, and 3-dimethylaminopropane-1-thiol (1.32 g, 11.1 mmol) was added dropwise over 10 minutes. After the addition was complete, the mixture was warmed to room temperature and stirred for 14 hours. The solvent was removed under vacuum, and the residue was dissolved in CH2Cl2 (200 mL). The solution was washed with 10% citric acid aqueous solution (100 mL), 5% NaHCO3 aqueous solution (100 mL), and brine (100 mL), and dried over anhydrous Na2SO4. The desiccant 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 under vacuum (bath temperature 25°C), and the silica gel containing the adsorbed 13 was placed on a silica gel column using CombiFlash (81 g silica gel, type ZCX02, 100-200 mesh). The column was eluted with an n-heptane / acetone gradient of 100:0 to 90:10, with a fraction of 100 mL. A suitable fraction was identified using TLC, and these were combined and concentrated under vacuum to obtain 13 (1.9 g), which was determined to be 90% pure by HPLC. 13 was subjected to reverse-phase preparative HPLC (C 18 The solution was further purified by A: water containing 0.1% formic acid, B: acetonitrile, with a gradient of 41%B to 58%B over 8 minutes. The suitable fractions were combined and concentrated under vacuum to obtain 13 (1.01 g, 1.00 mmol, 10.6%) as a clear, pale yellow, viscous oil. HPLC purity: 99.65%; ES-MS (+ mode): calculated value 10¹4.68, measured value 10¹5.95 (M+H + ); 1H-NMR (300MHz, CDCl3): δ 5.26(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).

[0387] Example 14. Synthesis of Lipid 14 [ka] General scheme: [ka]

[0388] Synthesis of 14-1:2-((benzyloxy)methyl)propane-1,3-dinonanoate diyl [ka]

[0389] 2-[(phenylmethoxy)methyl]-1,3-propanediol (Bioorg.Med.Chem.2017,25,4008-4030;25.0g, 0.127mol) was dissolved in CHCl3 (500mL) and cooled under nitrogen in an ice bath. Nonyl chloride (56.5g, 0.318mol) was added all at once to this solution, followed by pyridine (40.0g, 0.508mol) being added dropwise over 40 minutes. The reaction mixture was heated to room temperature and then stirred for 14 hours. The turbid mixture was filtered through a Celite pad, and the filtrate was washed with 5% NaHCO3 aqueous solution (250mL) and brine (250mL), and dried over anhydrous Na2SO4. The desiccant 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 under vacuum (bath temperature <35°C), and the silica gel containing the adsorbed 14-1 was added to the top of a combiflash column (600 g, type: ZCX-2, 100-200 mesh, packed with petroleum ether:siRNA 99:1, with petroleum ether:siRNA eluted at 99:1-98:2 in 1000 mL fractions). Qualified fractions were determined by TLC and concentrated under vacuum to obtain 14-1 (60.0 g, 0.124 mol, 98%) as a colorless oil. 1 H-NMR(300MHz, CDCl3):δ 7.28-7.40(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).

[0390] Synthesis of 14-2:2-(hydroxymethyl)propane-1,3-diyldinonanoate [ka]

[0391] 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 3x nitrogen, 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 hours, then the vessel was aerated and the solution was sparged with nitrogen. Pd / C was removed by filtration through a Celite pad, the filter cake was rinsed with MeOH (200 mL), and the combined filtrate was concentrated under vacuum to obtain 14-2 (32.0 g, 82.8 mmol, 67%) as a clear, colorless oil. 1 H-NMR (300MHz, CDCl3): δ 4.16(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).

[0392] 14-3: Synthesis of (((3,3'-((tert-butoxycarbonyl)azandiyl)bis(propanoyl))-bis(oxy))bis(methylene))bis(propane-2,1,3-triyl)tetranonanoate [ka]

[0393] A solution of 1-5 (5.00 g, 19.1 mmol) in CH2Cl2 (75 mL) was cooled under nitrogen in an ice bath, 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 that order. The mixture was heated to room temperature and then stirred for 14 hours. The reaction mixture was placed in 10% citric acid aqueous solution (125 mL). The organic phase was separated, washed with brine (125 mL), and dried over anhydrous Na2SO4. Filtering through a sintered glass funnel and concentration under vacuum was performed to obtain crude 14-3 (14.0 g, 14.0 mmol, 73%), which was used without further purification.

[0394] 14-4: Synthesis of (((3,3'-Azandiylbis(propanoyl))bis(oxy))bis(methylene))-bis(propane-2,1,3-triyl)tetranonanoate [ka]

[0395] To a solution of 14-3 (14.0 g, crude, estimated 14.0 mmol) in CH2Cl2 (80 mL), cooled in an ice bath under nitrogen, 4.0 M HCl in dioxane (35 mL, 0.140 mol) was added at a rate that maintained the internal temperature at 0-10°C. After the addition, the mixture was stirred for 30 minutes, then heated to room temperature and stirred for 16 hours. The reaction mixture was placed in saturated NaHCO3 aqueous solution (100 mL) to separate the organic phase, washed with saturated NaHCO3 aqueous solution (100 mL) and brine (100 mL), and dried over anhydrous Na2SO4. Crude 14-4 was obtained as a viscous yellow oil by filtration and concentration under vacuum, which was dissolved in CH2Cl2 (200 mL), and silica gel (20 g, type ZCX-2, 100-200 mesh) was added. Concentration under vacuum yielded silica gel containing adsorbed 14-4, which was placed on a silica gel column (100 g, type ZCX-2, 100-200 mesh) eluting at a gradient of 67:33 to 50:50 using CombiFlash. Qualified fractions were identified by TLC and concentrated under vacuum to obtain 14-4 (5.20 g, 5.79 mmol, 30% in two steps) as a clear, pale yellow, viscous oil. 1 H-NMR (300MHz, CDCl3): δ 4.12-4.18 (12H), 2.93 (t, J = 6.6Hz, 4H). 2.57(t,J=6.6Hz,4H), 2.42(m,2H), 2.33(t,J=7.5Hz,8H), 1.61(m,8H), 1.22-1.40(40H), 0.90(m,12H).

[0396] Lipid 14 synthesis [ka]

[0397] To a solution of 14-4 (5.20 g, 5.79 mmol) in CH2Cl2 (175 mL), cooled under nitrogen in an ice bath, triphosgene (1.72 g, 5.75 mmol) was added all at once, followed by pyridine (2.29 g, 28.9 mmol, 2.34 mL) at a rate that maintained the temperature at 0-5°C. After the addition, the mixture was stirred for 30 minutes, then warmed to room temperature and stirred for 4 hours. The solvent was removed under vacuum, the residue was dissolved in pyridine (100 mL), and the solution was cooled under nitrogen in an ice bath. To this stirred solution, 3-dimethylaminopropane-1-thiol (0.82 g, 6.88 mmol) was added dropwise over 10 minutes. After the addition, the mixture was stirred for 30 minutes, then warmed to room temperature and stirred for 14 hours. The solvent was removed under vacuum, and the residue was dissolved in CH2Cl2 (200 mL). The mixture was washed with 10% citric acid aqueous solution (2 × 100 mL), saturated NaHCO3 aqueous solution (2 × 100 mL), and brine (2 × 100 mL), and dried over Na2SO4. Filtering and concentration were performed under vacuum to obtain crude 14 as a viscous yellow oil, which was purified by reverse-phase combiflash chromatography (A: water + 0.1% CF3CO2H, B: acetonitrile; gradient 60%B to 80%B over 20 minutes, then 100% over 20 minutes). The suitable fractions were combined and concentrated under vacuum to obtain 14 (1.12 g, 1.07 mmol, 18.5%) as a clear pale yellow oil. 1 H-NMR(300MHz, CDCl3):δ 4.12-4.20(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).

[0398] Example 15. Synthesis of Lipid 15: ((4,4'-((((3-(dimethylaminopropyl)thio)carbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(2-(4-methylcyclohexyl)acetate) [ka] General scheme: [ka]

[0399] Synthesis of 15-1:2-(4-methylcyclohexylidene)ethyl acetate [ka]

[0400] To a suspension of NaH (12.5 g, 60% in oil) washed with a solution of hexane (2 × 25 mL, 0.312 mol) in anhydrous THF (600 mL) and cooled under nitrogen in an ice bath, triethyl phosphoneacetate (70.0 g, 0.312 mol) was added dropwise over 30 minutes. The mixture was stirred in an ice bath for 2 hours, and then 4-methylcyclohexanone (35.0 g, 0.312 mol) was added over 30 minutes. The mixture was stirred for 30 minutes, then heated to room temperature and stirred for 14 hours. The mixture was placed in water (1.2 L) and siRNA (600 mL). The organic phase was separated, silica gel (200 g, type: ZCX-2, m100-200 mesh) was added, and then the solvent was removed under vacuum (bath temperature < 35°C) to obtain silica gel containing adsorbed crude 1. Silica gel was placed on a silica gel column (1000 g, type: ZCX-2, m100-200 mesh) and eluted with a petroleum ether:siRNA gradient of 100:0 to 95:5. A 1000 mL fraction was collected using CombiFlush. Qualified fractions were identified by TLC, combined, and concentrated under vacuum to obtain 15-1 (45.0 g, 0.247 mol, 79%) as a clear, colorless oil. LC-MS (+ mode): RT 1.804, 183.2 (M+H + ); 1H-NMR(300MHz, CDCl3):δ 5.61(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).

[0401] Synthesis of 15-2:2-(4-methylcyclohexyl)ethyl acetate [ka]

[0402] Ester 15-1 (45.0 g, 0.247 mol) was dissolved in EtOH (450 mL) at room temperature under nitrogen, and 10% Pd / C (13.5 g) was added. Hydrogen was then passed through the reaction mixture for 16 hours. The solvent was then sparged with nitrogen for 1 hour, the catalyst was removed by filtration through a Celite pad, and the filtrate cake was rinsed with EtOH (450 mL). The combined filtrate was concentrated under vacuum to obtain 15-2 (35.0 g, 0.190 mol, 77%) as a pale yellow oil. 1 H-NMR (300MHz, CDCl3): δ 4.14 (q, J = 7.2 Hz, 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).

[0403] Synthesis of 15-3:2-(4-methylcyclohexyl)acetic acid [ka]

[0404] At room temperature under nitrogen, solid NaOH (84.0 g, 2.10 mol) was added over 30 minutes to a solution of 15-2 (35.0 g, 0.190 mol) in THF:H2O (350 mL, 50:50). The mixture was stirred for 16 hours, then concentrated under vacuum to remove THF. The aqueous solution was then adjusted to pH 3.0 by adding 3N HCl aqueous solution. The reaction mixture was extracted with siRNA (350 mL), and the organic phase was dried with Na2SO4. By filtration and concentration under vacuum, 15-3 (25.0 g, 0.160 mol, 84%) was obtained as a white solid.

[0405] Synthesis of 15-4:2-(4-methylcyclohexyl)acetyl chloride [ka]

[0406] To a solution of 15-3 (25.0 g, 0.160 mol) in CH2Cl2 (250 mL) containing DMF (1.0 mL), cooled in an ice bath under nitrogen, oxalyl chloride (40.7 g, 0.321 mol) was added dropwise over 20 minutes. After the addition was complete, the mixture was stirred for 30 minutes, then heated to room temperature and stirred for 14 hours. The mixture was concentrated in a vacuum at a bath temperature <30°C to obtain 15-4 (25.2 g, 0.144 mol, 90%) as a clear, colorless oil. 1 H-NMR (300MHz, CDCl3): δ 2.75 (m, 2H), 1.30-1.64 (5H), 1.18-1.28 (2H), 0.80-1.05 (6H).

[0407] Synthesis of 15-5:2-oxopropane-1,3-diylbis(2-(4-methylcyclohexyl)acetate) [ka]

[0408] To a solution of 1,3-dihydroxyacetone (5.90 g, 65.5 mmol) in CH2Cl2 (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 the dropwise addition of 15-4 (25.2 g, 0.144 mol) over 30 minutes. The mixture was stirred at room temperature for 16 hours and then poured into water (400 mL). The organic phase was separated, washed with water (400 mL) and brine (400 mL), and dried over Na2SO4. The drying agent 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 obtain silica gel containing adsorbed crude 15-5. Silica gel was placed on a silica gel column (300 g, type: ZCX-2, 100-200 mesh) and eluted using a CombiFlush with a petroleum ether:siRNA gradient of 100:0 to 90:10 for a 500 mL fraction. Qualifying fractions were identified by TLC and combined and concentrated under vacuum to obtain 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): δ 4.76 (s, 4H), 2.40 (d, J=6.0Hz, 1H), 2.00-2.30 (4H), 0.75-1.10 (25H).

[0409] 15-6: Synthesis of 2-hydroxypropane-1,3-diyrbis(2-(4-methylcyclohexyl)acetate) [ka]

[0410] To a solution of 15-5 (18.0 g, 49.1 mmol) THF cooled in an ice bath under nitrogen, CH3CO2H (25.0 g, 0.42 mol) was added. To this stirred solution, NaBH3CN (12.9 g, 0.205 mol) was added in several portions over 20 minutes. After the addition was complete, the mixture was stirred for 30 minutes, then warmed to room temperature and stirred for 2 hours. The mixture was poured into water (100 mL) and extracted with siRNA (3 × 100 mL). The combined organic phase was dried with Na2SO4, the desiccant was removed by filtration, and silica gel (50 g, type: ZCX-2, 100-200 mesh) was added to the filtrate. The silica gel containing adsorbed crude 15-6 was concentrated in a vacuum and placed on a silica gel column (250 g, type: ZCX-2, 100-200 mesh) eluted with a petroleum ether:siRNA gradient of 100:0 to 92:8. A 500 mL fraction was collected using CombiFlush. Qualified fractions were identified by TLC, then combined and concentrated in a vacuum to obtain 15-6 (17.0 g, 46.1 mmol, 94%) as a clear, colorless oil. LC-MS (+ / - mode): RT 1.47 min, 391.2 (M+Na+); 1 H-NMR (300MHz, CDCl3): δ 5.30(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).

[0411] 15-7: Synthesis of ((4,4'-((tert-butoxycarbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(2-(4-methylcyclohexyl)acetate) [ka]

[0412] To a solution of 1-5 (6.10 g, 21.1 mmol) in CH2Cl2 (120 mL), cooled under nitrogen in an ice bath, DMAP (1.00 g, 8.18 mmol) and 15-6 (17.0 g, 46.1 mmol) were added all at once, followed by the addition of EDCl (9.70 g, 50.6 mmol) in several portions over 30 minutes. After the addition was complete, the mixture was stirred for 30 minutes, then heated to room temperature and stirred for 16 hours. The mixture was poured into brine (120 mL) to separate the organic phase, washed with brine (120 mL), and dried over Na2SO4. The desiccant was removed by filtration, and silica gel (60 g, type: ZCX-2, 100-200 mesh) was added to the filtrate. Concentration in vacuum was performed to obtain silica gel containing adsorbed crude 15-7, which was placed on a silica gel column (300 g, type: ZCX-2, 100-200 mesh) eluted with a petroleum ether:siRNA gradient of 100:0 to 70:30. A 500 mL fraction was collected using CombiFlush. Qualified fractions were identified by TLC, then combined and concentrated in vacuum to obtain 15-7β (10.0 g, 10.1 mmol, 48%) as a clear, colorless oil. LC-MS (+ / - mode): RT 1.654 min, 1012.9 (M+Na+); 1 H-NMR(300MHz, CDCl3):δ 5.25(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,1) H), 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).

[0413] 15-8: Synthesis of bis(4-((1,3-bis(2-(4-methylcyclohexyl)acetoxy)propan-2-yl)oxy)-4-oxobutyl)ammonium trifluoroacetate [ka]

[0414] To a solution of 15-7 (10.0 g, 10.1 mmol) in CH2Cl2 (40 mL), cooled under nitrogen in an ice bath, CF3CO2H (5.00 g, 43.9 mmol, 3.36 mL) was added all at once. After addition, the mixture was stirred for 30 minutes, then heated to room temperature and stirred for 4 hours. The mixture was concentrated under vacuum to obtain 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):δ 11.10(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).

[0415] 15-9: Synthesis of ((4,4'-((1H-imidazole-1-carbonyl)azandiyl)bis(butanoyl))-bis(oxy))bis(propane-2,1,3-triyl)tetrakis(2-(4-methylcyclohexyl)acetate) [ka]

[0416] Under nitrogen, a solution of 15-8 (5.60 g, 5.58 mmol) in CH2Cl2 (100 mL) was mixed with Et3N (1.30 g, 12.8 mmol, 1.21 mL), followed by carbonyl diimidazole (CDI, 2.00 g, 12.33 mmol). The mixture was stirred at room temperature for 14 hours and then diluted with n-heptane (100 mL). The solution was washed with water (3 × 100 mL), and the organic phase was dried over Na2SO4. By filtration and concentration under vacuum, crude 15-9 (4.00 g, 4.06 mmol, 73%) was obtained as a viscous yellow oil. LC-MS (+ mode): RT 0.645 min, 984.9 (M+H + ); 1H-NMR(300MHz, CDCl3):δ 7.96(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).

[0417] Lipid 15: Synthesis of ((4,4'-((((3-(dimethylaminopropyl)thio)carbonyl)-azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(2-(4-methylcyclohexyl)acetate) [ka]

[0418] A solution of 15-9 (4.00 g, 4.06 mmol) CH2Cl2 (80 mL), cooled in an ice bath under nitrogen, is mixed with CF3SO2OCH2. 3(0.70 g, 4.27 mmol) was added over 5 minutes. After addition, the mixture was stirred for 1 hour, and then Et3N (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 in succession, one at a time. After addition, the mixture was stirred for 30 minutes, then the mixture was heated to room temperature and stirred for 16 hours. Silica gel (15 g, type: ZCX-2, 100-200 mesh) was added to the solution. The solution was concentrated in vacuum to obtain silica gel containing adsorbed crude 15, which was placed on a silica gel column (75 g, type: ZCX-2, 100-200 mesh) eluted with a CH2Cl2:MeOH gradient of 100:0 to 96:4, and a 300 mL fraction was collected using CombiFlash. A suitable fraction was identified using TLC, then combined and concentrated under vacuum to obtain lipid 15 (1.70 g). This was further purified by SFC (column: Torus 2-PIC, 4.6 × 100 mm 5 μm, mobile phase B: i-PrOH, flow rate 4 mL / min, gradient: isocratic 10% B, wavelength 220 nM), and after concentration under vacuum, lipid 15 (1.00 g, 0.965 mmol, 23.8%) was obtained as a clear, light yellow oil. ES-MS: 1035.7 (M+H + );HPLC purity: 98.47%; 1 H-NMR(300MHz, CDCl3):δ 5.24(m,2H), 4.31(m,4H), 4.14(m,4H), 3.38(brm,4H), 2.92(t,J=7.3Hz,2 H), 2.11-2.52(18H), 1.56-2.11(23H), 1.12-1.56(14H), 0.75-1.11(23H).

[0419] Example 16. Synthesis of Lipid 16: ((4,4'-((((3-(dimethylaminopropyl)thio)carbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(4-ethylcyclohexane-1-carboxylate) [ka] General scheme: [ka]

[0420] Synthesis of 16-1:4-ethylcyclohexane-1-carbonyl chloride [ka]

[0421] To a solution of 4-ethyl-cyclohexanecarboxylic acid (22.5 g, 0.144 mol) in CH2Cl2 (225 mL), cooled under nitrogen in an ice bath, DMF (0.5 mL) was added, followed by oxalyl chloride (36.6 g, 0.288 mol) over 25 minutes. After the addition was complete, the mixture was stirred for 30 minutes, then heated to room temperature and stirred for 16 hours. By concentration in vacuum, crude 16-1 (22.6 g, 0.129 mol, 90%) was obtained as a clear, colorless oil. 1 H-NMR (300MHz, CDCl3): δ 2.62 (m, 1H), 2.62 (m, 2H), 1.37-1.53 ​​(2H), 1.10-1.27 (4H), 0.80-1.00 (6H).

[0422] Synthesis of 16-2:2-oxopropane-1,3-diylbis(4-ethylcyclohexane-1-carboxylate) [ka]

[0423] To a solution of dihydroxyacetone (5.30 g, 58.8 mmol) in CH2Cl2 (500 mL), cooled under nitrogen in an ice bath, DMAP (0.36 g, 2.94 mmol) and pyridine (10.24 g, 0.129 mol) were added all at once, followed by the addition of 16-1 (22.6 g, 0.129 mol) over 10 minutes. After the addition was complete, the mixture was stirred for 30 minutes, then heated to room temperature and stirred for 14 hours. The solvent was removed under vacuum, and the residue was dissolved in CH2Cl2 (100 mL). Silica gel (25 g, type ZCX-2, 100-200 mesh) was added to the solution, and the solvent was removed under vacuum to obtain silica gel impregnated with adsorbed 16-2. Crude 16-2 was purified by placing silica gel on a silica gel column (250 g, type ZCX-2, 100-200 mesh) and eluting with a petroleum ether:siRNA gradient of 100:0-90:10 using a combiflush, and collecting a 300 mL fraction. The qualified fractions were identified by TLC, combined, and concentrated under vacuum to obtain 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): δ 4.83(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).

[0424] Synthesis of 16-3:2-hydroxypropane-1,3-diyrbis(4-ethylcyclohexane-1-carboxylate) [ka]

[0425] To a solution of 16-2 (20.4 g, 55.66 mmol) in THF (400 mL), cooled under nitrogen in an ice bath, HOAc (33.4 g, 0.556 mol) was added all at once, followed by the addition of NaBH3CN (17.5 g, 0.278 mol) in several portions over 30 minutes. After the addition was complete, the mixture was stirred for 30 minutes, then heated to room temperature and stirred for 2 hours. The mixture was poured into water (2.0 L), and the resulting solution was extracted with siRNA (3 × 200 mL). The combined organic phase was dried (Na2SO4), filtered, concentrated under vacuum, and the residue was dissolved in CH2Cl2 (100 mL). Silica gel (50 g, type ZCX-2, 100-200 mesh) was added to the crude 16-3 solution, and the solvent was removed under vacuum to obtain 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 eluting with petroleum ether:siRNA gradient 100:0-92:8 using a combiflush, and collecting 300 mL of fraction. Qualified fractions were identified by TLC, combined, and concentrated under vacuum to obtain 16-3 (16.0 g, 43.42 mmol, 78%) as a clear, colorless oil. LC-MS (+ mode): RT 1.463 min, 391.3 (M+Na) + ); 1 H-NMR (300MHz, CDCl6): δ 4.00-4.25(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).

[0426] 16-4: Synthesis of ((4,4'-((tert-butoxycarbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(4-ethylcyclohexane-1-carboxylate) [ka]

[0427] Under nitrogen, DMAP (0.91 g, 7.49 mmol) and 16-3 (15.2 g, 41.21 mmol) were sequentially added to a solution of 1-5 (5.42 g, 18.73 mmol) in CH2Cl2 (100 mL). The resulting solution was cooled in an ice bath, and then EDCl (8.60 g, 44.96 mmol) was added in five portions over 30 minutes. After the addition was complete, the mixture was stirred for 30 minutes, then the solution was heated to room temperature and stirred for 14 hours. The mixture was poured into brine (100 mL) to separate the organic phase, and dried with Na2SO4. A crude 16-4 solution was obtained by filtration, to which silica gel (15 g, type ZCX-2, 100-200 mesh) was added, and the solvent was removed under vacuum to obtain silica gel containing adsorbed crude 16-4. Crude 16-4 was purified by placing silica gel on a silica gel column (75 g, type ZCX-2, 100-200 mesh) and eluting with a petroleum ether:siRNA gradient of 100:0-80:20 using a combiflush, and collecting 300 mL of fraction. The qualified fractions were identified by TLC, combined, and concentrated under vacuum to obtain 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):δ 5.50(m,2H), 4.32(m,4H), 4.21(m,4H), 3.20(brm,4H), 2.16-2.35(7H), 1.8 2(m,8H), 1.75-1.80(9H), 1.38(s,9H), 1.00-1.38(24H), 0.75-0.90(20H).

[0428] 16-5: Synthesis of bis(4-((1,3-bis((4-ethylcyclohexane-1-carbonyl)oxy)propan-2-yl)oxy)-4-oxobutyl)ammonium trifluoroacetate [ka]

[0429] To a solution of 16-4 (12.98 g, 13.11 mmol) in CH2Cl2 (50 mL), cooled in an ice bath under nitrogen, CF3CO2H (7.47 g, 65.54 mmol) was added over 10 minutes. After the addition, the mixture was stirred for 15 minutes, then heated to room temperature and stirred for 16 hours. The mixture was concentrated under vacuum to obtain 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):δ 5.16(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).

[0430] 16-6: Synthesis of ((4,4'-((1H-imidazole-1-carbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(4-ethylcyclohexane-1-carboxylate) [ka]

[0431] A solution of 16-5 (14.82 g, crude) was dissolved in CH2Cl2 (300 mL) and cooled in an ice bath under nitrogen. To this solution, Et3N (6.74 g, 66.59 mmol) and carbonyldiimidazole (5.39 g, 33.30 mmol) were added in sequence. After the additions were completed, the mixture was stirred for 30 minutes, then the solution was warmed to room temperature and stirred for 3 hours. The solvent was removed under vacuum, and the residue was dissolved in n-heptane (300 mL), and water was added to the flask. The pH of the aqueous phase was adjusted to approximately 6.0 by adding 3% citric acid aqueous solution while stirring vigorously. After the target pH was achieved, the organic phase was separated and dried over Na2SO4. Crude 16-6 (13.77) was obtained by filtration and concentration under vacuum, and this was used in the next step without further purification. LC-MS (+ mode): RT 0.773 min.984.5 (M+H +); 1 H-NMR(300MHz, CDCl3):δ 7.99(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).

[0432] Lipid 16: Synthesis of ((4,4'-((((3-(dimethylaminopropyl)thio)carbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(4-ethylcyclohexane-1-carboxylate) [ka]

[0433] A solution of 16-6 (13.77 g, crude) in CH2Cl2 (150 mL) was cooled under nitrogen in an ice bath. Methyl trifluoromethanesulfonate (2.52 g, 20.99 mmol) was added to the cooled solution over 10 minutes. The mixture was stirred in an ice bath for 1 hour, then Et3N (4.24 g, 41.97 mmol) was added over 5 minutes, followed by 3-dimethylaminopropane-1-thiol (2.49 g, 20.99 mmol) over 5 minutes. The mixture was stirred for 30 minutes, then heated to room temperature and stirred for 8 hours. The mixture was concentrated in vacuum, the residue was dissolved in CH2Cl2 (100 mL), silica gel (30 g, type ZCX-2, 100-200 mesh) was added, and the solvent was removed in vacuum to obtain 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 eluting it with a CH2Cl2:MeOH gradient of 100:0-96:4 using a combiflush, and collecting 300 mL of the fraction. The suitable fractions were identified by TLC, combined, and concentrated under vacuum to obtain lipid 16 (10.14 g, 9.797 mmol, 75% yield in 3 steps) as a clear, light yellow oil. ES-MS: 1036.0(M+H + );HPLC purity 94.24%; 1 H-NMR(300MHz, CDCl3):δ 5.24(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).

[0434] Example 17. Synthesis of Lipid 17: ((4,4'-((((3-(dimethylaminopropyl)thio)carbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexyl-2-methylpropanoate) [ka] General scheme: [ka] [ka]

[0435] 17-1: Synthesis of (E)-3-cyclohexyl-2-methylacrylate ethyl [ka]

[0436] In a 2 L four-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, NaH (60%, 21.42 g, 0.534 mol, 1.0 equivalent) and THF (822 mL) were added. Then, ethyl 2-(diethoxyphosphoryl)propanoate (127.2 g, 0.536 mol, 1.0 equivalent) was added dropwise over 30 minutes at room temperature, and the mixture was stirred for 1.5 hours. A solution of cyclohexanecarboxaldehyde (60.0 g, 0.536 mol, 1.0 equivalent) in THF (318 mL) was added dropwise over 30 minutes, and the mixture was stirred at room temperature for 2 hours. The reaction product was quenched with saturated NH4Cl aqueous solution (1.5 L) and extracted with MTBE (2 × 0.75 L). The combined organic layers were washed with H2O (0.75 L) and brine (0.75 L, 12.5 V), dried with anhydrous Na2SO4, filtered, and concentrated under vacuum. This yielded 105 g of 17-1 (crude) as a yellow oily substance, which was used in the next step without further purification.

[0437] Synthesis of 17-2:3-Ethyl Cyclohexyl-2-Methylpropanoate [ka]

[0438] A solution of 17-1 (120.0 g, 1.0 equivalent) in EtOH (1.2 L) was placed in a 2 L round-bottom flask flushed with nitrogen. Then, 10% wt of Pd / C (36.0 g, 30% w / w) was added all at once. The mixture was then stirred at room temperature under an H2 atmosphere for 4 hours. The solution was filtered, and the filter cake was washed with CH2Cl2 (1.2 L). The filtrate was concentrated under vacuum to obtain crude 17-2. Crude 17-2 was dissolved in CH2Cl2 (1 L), 200 g of silica gel (type: ZCX-2, 100-200 mesh, 1.67 w / w) was added, and the solvent was removed under vacuum while maintaining the temperature below 35°C. 1 kg of silica gel (type: ZCX-2, 100-200 mesh, 8.33 w / w) was packed into a column, followed by the dried silica gel prepared in the final step, which adsorbed the reaction mixture. Using a combiflash, the product was purified by elution with a petroleum ether / siRNA gradient of 100:0 to 95:5, and a 1000 mL fraction was collected. Samples were taken for TLC analysis, and qualified products were combined. This yielded 94 g (76% yield) of 17-2 as a yellow oily substance.

[0439] Synthesis of 17-3:3-cyclohexyl-2-methylpropanoic acid [ka]

[0440] A 2 L three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was placed in a solution of 17-2 (57.0 g, 0.288 mol, 1.0 equivalent) in EtOH (285 mL) at room temperature. Then, a solution of NaOH (17.3 g, 0.433 mol, 1.5 equivalents) in H2O (285 mL) was added all at once. The resulting solution was then heated to 70°C and stirred for 3 hours. The reaction mixture was cooled to room temperature and extracted with n-heptane (2 × 200 mL). The H2O layer was adjusted to pH=2 using HCl aqueous solution (12 mol / L) and then extracted with MTBE (2 × 300 mL). The combined organic layers were washed with H2O (2 × 150 mL), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum. This yielded 47 g (0.276 mol, yield 96%) of 17-3 as a colorless oil. ELSD A: Water / 5mM NH4 + HCO3 - :B:CH3CN 2 minutes 90:10~10:9 A / B, 1 minute hold):RT 0.56 min, m / z 170.1 (calculated value), (measured value) 169.13 (MHz).

[0441] Synthesis of 17-4:2-oxopropane-1,3-diylbis(3-cyclohexyl-2-methylpropanoate) [ka]

[0442] A 1 L three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with a solution of 1,3-dihydroxyacetone (17.5 g, 0.194 mol, 1.0 equivalent) and 17-3 (66.0 g, 0.388 mol, 2.0 equivalents) in CH2Cl2 (350 mL). 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 equivalents) and EDCI (112 g, 0.583 mol, 3.0 equivalents) were added at 0°C. The ice / water bath was removed, and the temperature was gradually increased. The reaction mixture was stirred overnight at room temperature. 200 g of silica gel (type: ZCX-2, 100-200 mesh, 11.4 w / w) was added directly to the reaction mixture, and the solvent was removed under vacuum while maintaining the temperature below 35°C. 1 kg of silica gel (type: ZCX-2, 100-200 mesh, 57.1 w / w) was packed into the column, followed by the dried silica gel prepared in the final step, which adsorbed the reaction mixture. Elution was performed using a 100:0 to 90:10 petroleum ether / siRNA gradient, and the product was purified by combiflash, collecting 1000 fractions. Samples were taken for TLC analysis, and qualified products were combined. This yielded 73 g (0.184 mol, 95% yield) of 17-4 as a light yellow oily substance. The product showed no MS signal and was used directly in the next step.

[0443] Synthesis of 17-5:2-hydroxypropane-1,3-diyrbis(3-cyclohexyl-2-methylpropaneate) [ka]

[0444] A 1 L three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with a solution of 17-4 (56.0 g, 0.142 mol, 1.0 equivalent) in 560 mL of THF. The temperature was lowered to 0°C in an ice / water bath. HOAc (12.8 g, 0.213 mol, 1.5 equivalents) was added to the solution at 0°C, followed by NaBH3CN (12.5 g, 0.199 mol, 1.4 equivalents) at 0°C. The ice / water bath was removed, and the temperature was gradually increased. The reaction mixture was stirred at room temperature for 8 hours. The reaction mixture was quenched with H2O (1.1 L) and extracted with CH2Cl2 (1.6 L). The organic layer was washed with NaHCO3 aqueous solution (560 mL) and H2O (2 × 280 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was used directly in the next step.

[0445] 17-6: Synthesis of ((4,4'-((tert-butoxycarbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexyl-2-methylpropaneate) [ka]

[0446] To a 2 L three-necked round-bottom flask purged and maintained under an inert nitrogen atmosphere, 1-5 (20.0 g, 0.069 mol, 1.0 equivalent) and 17-5 (the above solution, 0.138 mol, 2.0 equivalents) were added. The temperature was lowered to 0°C in an ice / water bath. DMAP (8.4 g, 0.069 mol, 1.0 equivalent) and EDCI (53 g, 0.277 mol, 4.0 equivalents) were added to the solution at 0°C. The ice / water bath was removed and the temperature was gradually increased. The reaction mixture was stirred overnight at room temperature. 200 g of silica gel (type: ZCX-2, 100-200 mesh, 10.0 w / w) was added directly to the reaction mixture, and the mixture was concentrated under vacuum while maintaining the temperature below 35°C. 1.5 kg of silica gel (type: ZCX-2, 100-200 mesh, 75.0 w / w) was packed into the column, followed by the dried silica gel prepared in the final step, which adsorbed the reaction mixture. Elution was performed using a 100:0-90:10 petroleum ether / siRNA gradient, and the product was purified by combiflash, collecting a 500 mL fraction. A sample for TLC analysis was taken, and the qualified product was combined. This yielded 30.8 g (59.6 mmol, 42% in two steps) of 17-6 as a light yellow oily substance. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5-5:95 A / B at 3 min, hold for 1.0 min): RT 2.08 min, m / z (calculated) 1045.7, (measured) 946.6 (M-Boc+H).

[0447] 17-7: Synthesis of bis(4-((1,3-bis((3-cyclohexyl-2-methylpropanoyl)oxy)propan-2-yl)oxy)-4-oxobutyl)ammonium chloride [ka]

[0448] A 1 L round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was placed in a solution of 17-6 (48.0 g, 0.046 mol, 1.0 equivalent) in 1,4-dioxane (240 mL), and the solution was cooled in an ice bath. 4 M HCl from the 1,4-dioxane (240 mL) was added dropwise to the solution over 10 minutes at 0-10°C. The resulting solution was stirred overnight at room temperature. The mixture was concentrated under vacuum. This yielded 48 g (crude) of 17-7 as a yellow oily substance. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5-5:95 A / B at 3 mins, held for 1.0 min): RT 0.92 min, m / z (calculated) 945.6, (measured) 946.6 (M+H).

[0449] 17-8: Synthesis of ((4,4'-((1H-imidazole-1-carbonyl)azandiyl)bis(butanoyl))-bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexyl-2-methylpropanoate) [ka]

[0450] Under a nitrogen atmosphere, a solution of 17-7 (48.0 g, 0.046 mol, 1.0 equivalent) in CH2Cl2 (1.06 L) was placed in a 2 L three-necked round-bottom bottle. Next, carbonyl diimidazole (15.9 g, 0.098 mol, 2.1 equivalents) was added, followed by pyridine (15.4 g, 0.196 mol, 4.26 equivalents), and the mixture was stirred overnight at room temperature. The resulting solution was washed with 3% citric acid aqueous solution (2 × 500 mL), H2O (3 × 500 mL), and brine (500 mL), dried over anhydrous Na2SO4, and concentrated under vacuum at 35°C. The crude mixture was dissolved in CH2Cl2 (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. 300 g of silica gel (type: ZCX-2, 100-200 mesh, 6.25 w / w) was packed into the column, followed by the dried silica gel prepared in the final step, which adsorbed the reaction mixture. Elution was performed using a 100:0 to 70:30 petroleum ether / siRNA gradient, and the product was purified by combiflash, collecting a 400 mL fraction. Samples were taken for TLC analysis, and qualified products were combined. This yielded 39 g (37.5 mmol, 81% yield) of 17-8 as an oily substance. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5 to 5:95 A / B at 2 min, retention 0.6 min): RT 1.0 min, m / z (calculated) 1039.6, (measured) 1040.6 (M+H).

[0451] Lipid 17: Synthesis of ((4,4'-((((3-(dimethylaminopropyl)thio)carbonyl)-azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(3-cyclohexyl-2-methylpropaneate) [ka]

[0452] A 500 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with a solution of 17-8 (20.0 g, 0.019 mol, 1.0 equivalent) in CH2Cl2 (200 mL). The reaction temperature was reduced to 0°C in an ice / water batch. Methyl trifluoromethanesulfonate (3.3 g, 0.020 mol, 1.05 equivalent) was added to the mixture at 0°C. After addition, the reaction mixture was stirred for 3 hours at 0°C. A solution of 2.0 M methylamine in THF (28.9 mL, 0.058 mol, 3.0 equivalent) was added to the reactor at 0°C. After addition, the reaction mixture was stirred for 0.5 to 1 hour at 0°C. 3-(dimethylamino)propane-1-thiol (3.0 g, 0.025 mol, 1.3 equivalent) was added to the reactor at 0°C. After adding the reagents, the reaction mixture was allowed to come to room temperature and stirred for 5.0 hours. A 10.0% by weight sodium chloride aqueous solution (200 mL) and a 10% by weight citric acid aqueous solution (200 mL) were added to the reactor. The mixture was stirred for 15 minutes, then the reactor was allowed to stand for 15 minutes to allow phase separation at room temperature. The organic layer was collected. This procedure was repeated one more time. A 10.0% by weight sodium chloride aqueous solution (200 mL) was added, followed by a 5.0% by weight sodium bicarbonate aqueous solution (200 mL) in the reactor. The mixture was stirred for 15 minutes, then the reactor was allowed to stand for 15 minutes to allow phase separation at room temperature. The organic layer was collected. This procedure was repeated one more time. A 10.0% by weight sodium chloride aqueous solution (400 mL) was added to the reactor. The mixture was stirred for at least 15 minutes, then the reactor was allowed to stand for at least 15 minutes to allow phase separation at room temperature. The organic layer was collected. 250 mL of n-heptane was added to the reactor. The solution was concentrated to approximately 300 mL under vacuum while maintaining the temperature at 20-40°C. 200 mL of 10.0 wt% methanol citrate / water (10:1) solution was added to the reactor. After addition, the mixture was stirred for 15 minutes, then the reactor was allowed to stand for 15 minutes, and phase separation was performed at 36±5°C. The MeOH / H2O phase was collected. The MeOH / H2O phase was washed by adding 250 mL of n-heptane to the reactor. This n-heptane washing operation was repeated 8 times. 500 mL of n-heptane, 250 mL of 15.0 wt% sodium carbonate solution, and 250 mL of 10.0 wt% sodium chloride solution were added to the reactor.The mixture was stirred for 15 minutes, then allowed to stand for 15 minutes to allow phase separation at room temperature. The organic layer was collected. 400 mL of 5.0 wt% sodium bicarbonate solution was added to the reactor. The mixture was stirred for 15 minutes, then allowed to stand for 15 minutes to allow 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 w / w) was added to the residue in 300 ml of CH2Cl2, 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 w / w) was packed into a column, followed by the dried silica gel prepared in the final step, which adsorbed the reaction mixture. Elution was performed using a CH2Cl2 / MeOH gradient from 100:0 to 90:10, and the product was purified by combiflash. A 400 mL fraction was collected. A sample for TLC analysis was taken, and suitable products were combined. This yielded 11.5 g (55% yield) of lipid 17 as a yellow oily substance. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (80:20~20:80 A / B at 3 mins, 1 min hold): RT 0.97 min, m / z (calculated) 1090.7, (measured) 1091.7 (M+H). 1 H-NMR(300MHz, CDCl3):δ 5.25(m,2H), 4.34(dt,J=11.9,4.0Hz,4H), 4.15(m,4H), 3.38(brm,4H), 2.92(t,J=7.3Hz,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).

[0453] Example 18. Synthesis of Lipid 18: ((4,4'-((((3-(dimethylaminopropyl)thio)carbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(2-methyloctanoate) [ka] General scheme: [ka]

[0454] Synthesis of 18-1:2-oxopropane-1,3-diirbis(2-methyloctanoate) [ka]

[0455] A 50 ml three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with a solution of 1,3-dihydroxyacetone (1.42 g, 1.0 equivalent) and 2-methyloctanoic acid (5.0 g, 2.0 equivalent, Org. Biomol. Chem. 2014, 12, 3649-3663) in CH2Cl2 (30 mL). The solution was cooled in an ice bath, and then DMAP (0.96 g, 0.5 equivalent) and EDCI (12.1 g, 4.0 equivalent) were added at 0°C. The reaction mixture was brought to room temperature and stirred overnight. 20 g of silica gel (type: ZCX-2, 100-200 mesh, 15.5 w / w) was added to the mixture, 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, 77.5 w / w) was packed into the column, followed by the dried silica gel prepared in the final step, which adsorbed the reaction mixture. The crude product was purified using a combiflush purification system eluting with a petroleum ether / Âde gradient of 100:0 to 90:10, and collected in 200 ± 50 mL portions. Samples were taken for TLC analysis, qualified fractions were combined, and concentrated under vacuum. This yielded 4.9 g (85% yield) of 18-1 as a light yellow oily substance. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5-5:95 A / B at 2 min, retention 1.3 min): RT 1.6 min, m / z (calculated) 370.3, (measured) 371.3 (M+H).

[0456] Synthesis of 18-2:2-hydroxypropane-1,3-diyrbis(2-methyloctanoate) [ka]

[0457] A 100 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was placed in a solution of 18-1 (4.9 g, 1.0 equivalent) in THF (50 mL), and the solution was cooled in an ice bath. HOAc (1.03 g, 1.3 equivalents) was added to the solution at 0°C, followed by NaBH3CN (1.0 g, 1.2 equivalents) at 0°C. The ice bath was removed after all reagents had been added. The reaction mixture was stirred overnight at room temperature. The reaction mixture was quenched with H2O (100 mL) and extracted with CH2Cl2 (150 mL). The organic layer was washed with 5% NaHCO3 aqueous solution (50 mL) and H2O (2 × 50 mL), dried over anhydrous Na2SO4, and filtered. The filtrate containing 18-2 was used directly in the next step.

[0458] 18-3: Synthesis of ((4,4'-((tert-butoxycarbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(2-methyloctanoate) [ka]

[0459] A 100 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was mixed with 18-2 (8.8 g, 2.3 equivalents) and 1-5 (3.0 g, 1.0 equivalent) solutions in 60 mL of CH2Cl2, and the mixture was cooled in an ice bath. DMAP (1.26 g, 1.0 equivalent) and EDCI (7.96 g, 4.0 equivalents) were added to the solution at 0°C. The ice bath was removed after all reagents had been added. The reaction mixture was stirred overnight at room temperature. 20 g of silica gel (type: ZCX-2, 100-200 mesh, 6.7 w / w) was added to the mixture, and the solvent was removed under vacuum while maintaining the temperature below 35°C. 120 g of silica gel (type: ZCX-2, 100-200 mesh, 40.0 w / w) was packed into the column, followed by the dried silica gel prepared in the final step, which adsorbed the reaction mixture. Using a combiflash, the product was purified by elution with a petroleum ether / siRNA gradient of 100:0 to 90:10, and a 200 mL fraction was collected. A sample for TLC analysis was taken, and the qualified product was combined. This yielded 4.3 g (42% yield) of 18-3 as a bright yellow oily substance. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (80:20-20:80 A / B at 3 min, 1 min retention): RT 1.97 min, m / z (calculated) 997.7, (measured) 1020.6 (M+Na).

[0460] 18-4: Synthesis of bis(4-((1,3-bis((2-methyloctanoyl)oxy)propan-2-yl)oxy)-4-oxobutyl)ammonium chloride [ka]

[0461] A 100 mL round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was placed in a solution of 18-3 (4.3 g, 1.0 equivalent) in 1,4-dioxane (21 mL), and the solution was cooled in an ice bath. To the cooled solution, 4 M HCl in the 1,4-dioxane (21 mL) was added dropwise over 10 minutes at 0-10°C. The resulting solution was stirred overnight at room temperature. The reaction mixture was concentrated under vacuum. This yielded 4 g (crude) of 18-4 as a yellow oily substance. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5~5:95 A / B at 2 mins, held for 1.3 mins): RT 1.6 mins, m / z (calculated) 897.6, (measured) 898.6 (M+H).

[0462] Lipid 18: Synthesis of ((4,4'-((((3-(dimethylaminopropyl)thio)carbonyl)-azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(2-methyloctanoate) [ka]

[0463] A 250 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, contains 18-4 (3.8 g, 1.0 equivalent) CH2Cl. 2(80 mL of the solution was added and the solution was cooled in an ice bath. Triphosgene (1.26 g, 1.0 equivalent) was added to the mixture at 0°C, followed by pyridine (1.67 g, 5.0 equivalents) being added dropwise while stirring at 0°C. The ice bath was removed after all reagents had been added. The mixture was stirred at room temperature for 4 hours, then concentrated under vacuum (below 30°C). The residue was dissolved in pyridine (80 mL), cooled under nitrogen in an ice bath, and then 3-(dimethylamino)propane-1-thiol (1.0 g, 2.0 equivalents) was added dropwise while stirring at 0°C for 10 minutes. The resulting solution was stirred at room temperature overnight. The reaction mixture was concentrated under vacuum, and the residue was diluted with CH2Cl2 (80 mL). The solution was washed with 10% citric acid aqueous solution (40 mL), H2O (40 mL), saturated NaHCO3 (2 × 40 mL), and brine (40 mL, 10V). The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. To the residue dissolved in CH2Cl2 (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 the column, followed by the dried silica gel prepared in the final step, which adsorbed the reaction mixture. The product was purified by combiflash after elution using a CH2Cl2 / MeOH gradient of 100:0 to 90:10, and a 100 mL fraction was collected. Samples for TLC analysis were taken, and qualified products were combined. This yielded 1.3 g of lipid 18 (29% yield in 2 steps) as a yellow oily substance. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA 80:20~20:80 A / B at 3 mins, held for 2.1 mins): RT 1.1 min, m / z (calculated value) 1042.7, (measured value) 1043.6 (M+H). 1H-NMR(300MHz, CDCl3):δ 5.25(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(br s,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).

[0464] Example 19. Synthesis of Lipid 19: ((4,4'-((((3-(dimethylaminopropyl)thio)carbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(2,2-dimethylheptanoate) [ka] General scheme: [ka]

[0465] Synthesis of 19-1:2,2-dimethylheptanoate ethyl [ka]

[0466] A 500 ml three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with a solution of ethyl isobutyrate (40.0 g, 1.0 equivalent) in THF (400 mL). The resulting solution was cooled to -78 °C, and then LDA (205.6 ml, 1.05 equivalent in hexane) was added dropwise. The resulting solution was stirred at -78 °C for 1 hour. Next, 1-iodopentane (92.8 g, 1.2 equivalents) was added dropwise, and the resulting solution was stirred at -78 °C for 5 hours. The condenser was removed, and the solution was stirred at room temperature overnight. The pH of the solution was adjusted to 6 using an aqueous HCl solution (1 mol / L). The resulting solution was extracted with ethyl acetate (2 × 300 mL), and the organic layer was combined. The resulting mixture was washed with brine (500 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum. This yielded 51 g (75.6%) of 19-1 as a yellow oily substance, which was used in the next step without further purification.

[0467] 19-2:2,2-dimethylheptanoic acid [ka]

[0468] A 2 L three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was placed in a solution of 19-1 (70.0 g, 1.0 equivalent) in MeOH (700 mL). An aqueous solution of NaOH (49.0 g, 3.0 equivalent) in H2O (350 mL) was added dropwise to the solution at room temperature. The resulting solution was heated and stirred at 60°C for 4 hours. After cooling to room temperature, the resulting mixture was concentrated under vacuum. The residue was dissolved in H2O (200 mL), extracted with MTBE (200 mL), and the aqueous layer was separated. The pH of the aqueous phase was adjusted to 5 using an aqueous HCl solution (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 yielded 32 g (68%) of 19-2 as a yellow oily substance, which was used without purification.

[0469] Synthesis of 19-3:2-oxopropane-1,3-diirbis(2,2-dimethylheptanoate) [ka]

[0470] A 500 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with a solution of 1,3-dihydroxyacetone (12.0 g, 1.0 equivalent) in CH2Cl2 (240 mL). The solution was cooled to 0°C in an ice / water bath. To the solution, 19-2 (44.0 g, 2.1 equivalents), DMAP (16.3 g, 1.0 equivalent), followed by EDCI (76.7 g, 3.0 equivalents) were added at 0°C. The ice / water bath was removed, and the reaction mixture was stirred overnight at room temperature. 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. 500 g of silica gel (type: ZCX-2, 100-200 mesh, 41.7 w / w) was packed into the column, followed by the dried silica gel prepared in the final step, which adsorbed the reaction mixture. Elution was performed using a 95:5-90:10 petroleum ether / siRNA gradient, and the product was purified by combiflash, collecting 1000 fractions. Samples were taken for TLC analysis, and qualified products were combined. Concentration under vacuum yielded 40.6 g (75.8%) of 19-3 as a colorless oil. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5-5:95 A / B at 2 min, retention 1.3 min): RT 1.8 min, m / z (calculated) 370.2, (measured) 393.2 (M+Na).

[0471] Synthesis of 19-4:2-hydroxypropane-1,3-diirbis(2,2-dimethylheptanoate) [ka]

[0472] A 500 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was placed in a solution of 19-3 (15.5 g, 1.0 equivalent) in THF (155 mL). The solution was cooled to 0°C in an ice / water bath. HOAc (3.26 g, 1.3 equivalents) was added to the solution at 0°C, followed by the addition of NaBH3CN (3.16 g, 1.2 equivalents) to the mixture at 0°C in a single addition. The ice / water bath was removed, and the mixture was stirred at room temperature for 16 hours. The reaction was quenched with water (200 mL). The mixture was extracted with CH2Cl2 (3 × 200 mL). The combined organic phases were washed with brine (500 mL) and then dried over Na2SO4. The mixture was filtered and concentrated under vacuum to obtain crude 19-4, which was dissolved in CH2Cl2 (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 final step, which adsorbed the reaction mixture. The product was purified by elution using a 90:10-85:15 petroleum ether / siRNA gradient and combiflash, and a 400 mL fraction was collected. Samples were taken for TLC analysis, and qualified products were combined. Concentration under vacuum yielded 12.3 g (79.3% yield) of 19-4 as a yellow oily substance. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA. 95:5~5:95 A / B after 2 minutes (hold for 1.2 minutes): RT 1.5 minutes, m / z (calculated value) 372.2, (measured value) 395.2 (M+Na).

[0473] 19-5: Synthesis of ((4,4'-((tert-butoxycarbonyl)azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(2,2-dimethylheptanoate) [ka]

[0474] A 250 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with a solution of 1-5 (4.77 g, 1.0 equivalent) in CH2Cl2 (80 mL). The solution was cooled to 0°C in an ice / water bath, and then 19-4 (12.3 g, 2.0 equivalent), DMAP (2.0 g, 1.0 equivalent) were added in sequence, followed by EDCI (9.5 g, 3.0 equivalent) at 0°C. The ice / water bath was removed, and the resulting solution was stirred at room temperature for 16 hours. 15 g of silica gel (type: ZCX-2, 100-200 mesh, 3.14 w / w) was added to the reaction 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, 41.9 w / w) was packed into the column, followed by the dried silica gel prepared in the final step, which adsorbed the reaction mixture. Elution was performed using a 90:10-86:14 petroleum ether / siRNA gradient, and the product was purified by combiflash, collecting a 400 mL fraction. A sample for TLC analysis was taken, and the qualified product was then combined. This yielded 15.8 g (96.3%) of 19-5 as a yellow oily substance.

[0475] 19-6: Synthesis of bis(4-((1,3-bis((2,2-dimethylheptanoyl)oxy)propan-2-yl)oxy)-4-oxobutyl)ammonium chloride [ka]

[0476] A 250 mL round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was placed in a solution of 19-5 (6.0 g, 1.0 equivalent) in CH2Cl2 (30 mL). The solution was cooled to 0°C in an ice / water bath. To this solution, a HCl dioxane solution (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 under vacuum to obtain 6.2 g (crude) of 19-6 as a light yellow oil. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA (95:5~5:95 A / B at 2 mins, held for 1.2 mins): RT 1.5 min, m / z (calculated) 897.6, (measured) 898.5 (M+H).

[0477] Lipid 19: Synthesis of ((4,4'-((((3-(dimethylaminopropyl)thio)carbonyl)-azandiyl)bis(butanoyl))bis(oxy))bis(propane-2,1,3-triyl)tetrakis(2,2-dimethylheptanoate) [ka]

[0478] A 500 mL three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was placed in a solution of 19-6 (6.0 g, 1.0 equivalent) in CH2Cl2 (210 mL). The solution was cooled to 0°C in an ice / water bath. Triphosgene (2.69 g, 1.5 equivalents) was added to the mixture at 0°C. Pyridine (2.53 g, 5.0 equivalents) was then added dropwise with stirring at 0°C. The ice / water batch was removed, and the mixture was stirred at room temperature for 4 hours, then concentrated under vacuum (at a temperature below 30°C). The residue was dissolved in pyridine (120 mL, 20V), and the solution was cooled to 0°C in an ice / water bath. 3-(dimethylamino)propane-1-thiol (1.53 g, 2.0 equivalents) was added dropwise to this solution with stirring at 0°C for 10 minutes. The ice / water batch was removed, and the resulting solution was stirred overnight at room temperature. The reaction mixture was concentrated under vacuum, crude 19 was dissolved in CH2Cl2 (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 final step, which adsorbed the reaction mixture. The product was purified using CombiFlash and eluted with a CH2Cl2 / acetone gradient of 75 / 25 to 70 / 30, and a 200 mL fraction was collected. Samples were taken for TLC analysis, and qualified products were combined. This yielded 1.0 g (yield 13.8%) of 19 as a yellow oil. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA. 95:5~5:95 A / B in 2 minutes (hold for 1.2 minutes): RT 1.5 minutes, m / z (calculated value) 1042.7, (measured value) 1043.9 (M+H). 1 H-NMR(300MHz, CDCl3):δ 5.28(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).

[0479] Example 20. Synthesis of Lipid 20: ((3,3'-((((3-(dimethylamino)propyl)thio)carbonyl)azandiyl)bis(propanoyl))bis(oxy))bis(methylene))bis(2-methylpropane-2,1,3-triyl)tetrakis(3-(4-methylcyclohexyl)propanoate) [ka] Lipids 20

[0480] General scheme: [ka]

[0481] Synthesis of 20-2:3-(4-methylcyclohexyl)propanoyl chloride [ka]

[0482] In a 25 mL three-necked round-bottom flask, SOCl2 (100 mL, 1378.615 mmol, 4.69 equivalents) and commercially available 3-(4-methylcyclohexyl)propanoic acid (50 g, 293.682 mmol, 1 equivalent) were added at room temperature. The resulting mixture was stirred at room temperature for 10 hours and concentrated under reduced pressure to obtain 3-(4-methylcyclohexyl)propanoyl chloride (51 g, 92.03%) as a yellow oily substance, which was used as is without further purification or characterization.

[0483] Synthesis of 20-4:2-((benzyloxy)methyl)-2-methylpropane-1,3-diylbis(3-(4-methylcyclohexyl)-propanoate) [ka]

[0484] In 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 equivalent), pyridine (25.45 g, 321.7 mmol, 2.5 equivalents), and CH2Cl2 (500 mL, 20V) were added at room temperature. The mixture was cooled to 0°C. 20-2 (51.00 g, 270.2 mmol, 2.1 equivalents) was added dropwise to the mixture at 0°C. The resulting mixture was stirred at room temperature for a further 4 hours. The reaction was quenched by adding water (1 L) at 0°C. The resulting mixture was extracted with CH2Cl2 (3 × 500 mL). The combined organic layers were dried over anhydrous Na2SO4, 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 using a CombiFlash purification system with a 2700 g silica gel column. The product was eluted with PE / EA (gradient from 100:0 to 70:30, collected every 500 ± 10 mL). After TLC analysis (EA:PE = 1:10), suitable fractions were combined, concentrated, and dried under vacuum to obtain 20-4 (45 g, 67.9%) as a colorless oil. ELSD A: Water / 0.05% TFA: B: CH3CN 95:5~5:95 A / B at 2 min, retention 1.2 min): RT 1.48 min, m / z (calculated) 514.4, (measured) 537.5 (M+Na).

[0485] 20-5: Synthesis of 2-(hydroxymethyl)-2-methylpropane-1,3-diyrbis(3-(4-methylcyclohexyl)-propaneate) [ka]

[0486] To a solution of 20-4 (45 g, 87.4 mmol, 1 equivalent) in MeOH (500 mL, 11V), Pd / C (10%, 4.5 g) was added under a nitrogen atmosphere in a 1 L three-necked round-bottom flask. The mixture was hydrogenated at room temperature for 10 hours under a hydrogen atmosphere using a hydrogen balloon. The reaction product was filtered through a Celite pad and concentrated under reduced pressure to obtain 20-5 (35 g, 94.3%) as a colorless oil. ELSD A: Water / 0.05% TFA: B: CH3CN 95:5~5:95 A / B at 2 mins, hold for 1.2 mins): RT 1.29 mins, m / z (calculated) 424.3, (measured) 447.4 (M+Na).

[0487] 20-7: Synthesis of (((3,3'-((tert-butoxycarbonyl)azandiyl)bis(propanoyl))bis(oxy))bis(methylene))bis(2-methylpropane-2,1,3-triyl)tetrakis(3-(4-methylcyclohexyl)propanoate) [ka]

[0488] In a 1 L three-necked round-bottom flask, 20-5 (35.04 g, 82.5 mmol, 2.2 equivalents), 3-[(tert-butoxycarbonyl)(2-carboxyethyl)amino]propanoic acid (20-6, 9.8 g, 37.5 mmol, 1.00 equivalent), EDCI (14.38 g, 75.0 mmol, 2 equivalents), DCM (700 mL, 20V), and DMAP (4.58 g, 37.48 mmol, 1.00 equivalent) were added at room temperature. The resulting mixture was stirred at room temperature for 10 hours and diluted with water (500 mL). The resulting mixture was extracted with CH2Cl2 (3 × 500 mL), and the combined organic layers were dried over anhydrous Na2SO4. 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 using a CombiFlash purification system with a 2.7 kg silica gel column. The product was eluted with PE / EA (gradient from 100:0 to 50:50, collected every 500 ± 10 mL). After TLC analysis (EA:PE = 1:10), the suitable fractions were combined, concentrated, and dried under vacuum to obtain (37 g, 91.8%) 20-7 as a colorless oil. 1 Based on the purity and structure determined by 1H NMR, it was used in the following steps.

[0489] 20-8: Synthesis of (((3,3'-Azandiylbis(propanoyl))bis(oxy))bis(methylene))bis(2-methylpropane-2,1,3-triyl)tetrakis(3-(4-methylcyclohexyl)propanoate)trifluoroacetate [ka]

[0490] 20-7 (37 g, 34.4 mmol, 1 equivalent), DCM (370 mL), and trifluoroacetic acid (150 mL) were added to a 500 mL three-necked round-bottom flask at room temperature. The resulting mixture was stirred at room temperature for 10 hours. The resulting mixture was concentrated under reduced pressure to obtain 20-8 (40 g, crude) as a colorless oil. 1Both 1H NMR and HPLC showed a product with approximately 94% purity. This product was dried under vacuum and then used directly in the next reaction.

[0491] Lipid 20: Synthesis of ((3,3'-((((3-(dimethylaminopropyl)thio)carbonyl)azandiyl)bis(propanoyl))bis(oxy))bis(methylene))bis(2-methylpropane-2,1,3-triyl)tetrakis(3-(4-methylcyclohexyl)propanoate) [ka]

[0492] In a 1 L three-necked round-bottom flask, 20-8 (37 g, 34 mmol, 1 equivalent), TEA (10.32 g, 102 mmol, 3 equivalents), CDI (6.06 g, 37.4 mmol, 1.1 equivalents), 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. Methyl trifluoromethanesulfonate (6.14 g, 37.4 mmol, 1.1 equivalents) was added dropwise to the mixture 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 equivalents) was added dropwise at 0°C. The resulting mixture was stirred at room temperature for a further 10 hours, diluted with water (300 mL), and extracted with CH2Cl2 (3 × 300 mL). The combined organic layers were 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 using a CombiFlash purification system with a 370 g silica gel column. The product was eluted with PE / EA (gradient of 100:0 to 90:10, collected every 500 ± 10 mL). After TLC analysis (EA:PE = 1:10), the qualified fractions were combined, concentrated, and dried under vacuum to obtain lipid 20 (5.1 g, 13.3%) as a yellow oil. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05% TFA. 95:5~5:95 in 25 minutes. A / B): RT 10.3 mins, m / z (calculated value) 1118.8, (measured value) 1119.9 (M+H). 1 H NMR(300MHz,chloroform-d)δ 4.005(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).

[0493] Example 21. Synthesis of Lipid 21: ((3,3'-((((3-(dimethylamino)propyl)thio)carbonyl)azandiyl)bis(propanoyl))bis(oxy))bis(methylene))bis(2-methylpropane-2,1,3-triyl)tetrakis(2-(4-methylcyclohexyl)acetate) [ka]

[0494] Lipid 21

[0495] General Scheme [ka]

[0496] 21-2: Synthesis of 2-(4-methylcyclohexyl)acetyl chloride [ka]

[0497] Under nitrogen, a solution of 21-1 (80 g, 512.0 mmol, 1.00 equivalent) in DCM (800 mL, 10V) was added to a 2 L three-necked round-bottle flask. The solution was cooled to 0°C in an ice / water bath, and oxalyl chloride (130 g, 1024.2 mmol, 2.00 equivalent) was added dropwise at 0°C. The ice / water bath was removed, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was concentrated under vacuum to obtain 21-2 (82 g, 91.7%) as a colorless oil, which was used directly in the next reaction.

[0498] 21-3: Synthesis of 2-((benzyloxy)methyl)-2-methylpropane-1,3-diylbis(2-(4-methylcyclohexyl)acetate) [ka]

[0499] A solution of 20-3 (40 g, 190.2 mmol, 1.00 equivalent) in DCM (800 mL) was treated with pyridine (60.19 g, 760.9 mmol, 4.00 equivalent) and DMAP (6.97 g, 57.0 mmol, 0.30 equivalent) under nitrogen at 0°C, followed by the dropwise addition of 21-2 (83.1 g, 475.6 mmol, 2.50 equivalent) at 0°C. The mixture was stirred at room temperature for 16 hours. The resulting mixture was diluted with water (500 mL) and acidified to pH 6 with HCl (aqueous solution). The aqueous layer was extracted with DCM (2 × 200 mL). The combined organic layers were washed with brine (1 × 300 mL), dried over anhydrous Na₂SO₄, filtered, and the filtrate was concentrated under reduced pressure to obtain 21-3 (67 g, 72.4%) as a colorless oil. ELSD A: Water / 0.05% TFA: B: CH3CN 3 minutes 95:5~5:95 A / B):RT 2.49 min, m / z (calculated value) 486.3, (measured value) 509.4 (M+Na).

[0500] 21-4: Synthesis of 2-(hydroxymethyl)-2-methylpropane-1,3-diylbis(2-(4-methylcyclohexyl)acetate) [ka]

[0501] In a 2 L four-necked round-bottom flask, a solution of 21-3 (67 g, 137.66 mmol, 1.00 equivalent) in MeOH (670 mL 10V) was added, and Pd / C (20.1 g, 18.9 mmol, 0.14 equivalent, 10% by weight) was added all at once. The resulting mixture was stirred under H2 at room temperature for 16 hours. The reaction mixture was filtered, and the filter cake was washed with MeOH (1 × 300 mL). The filtrate was concentrated under vacuum to obtain 21-4 (53 g, 97.1%) as a colorless oil. ELSD A: Water / 0.05% TFA: B: CH3CN 95:5~5:95 A / B):RT 2.11 min, m / z (calculated) 396.3, (measured) 397.2 (M+H).

[0502] 21-5: Synthesis of (((3,3'-((tert-butoxycarbonyl)azandiyl)bis(propanoyl))bis(oxy))bis(methylene))bis(2-methylpropane-2,1,3-triyl)tetrakis(2-(4-methylcyclohexyl)acetate) [ka]

[0503] Under nitrogen, 21-4 (50 g, 126.1 mmol, 1.00 equivalent), followed by DMAP (15.40 g, 126.1 mmol, 1.00 equivalent) and 20-6 (72.47 g, 277.4 mmol, 2.20 equivalents) were added to a 2 L four-necked round-bottom flask in a 1000 mL DCM (20V) solution. The solution was cooled to 0°C in an ice / water bath, and EDCI (96.68 g, 504.3 mmol, 4.00 equivalent) was added. The ice / water batch was removed, and the mixture was stirred at room temperature for 16 hours. The mixture was washed with brine (1 × 1 L, 20V), the organic layer was dried over Na₂SO₄, filtered, and evaporated. The crude product was adsorbed onto 300 g of silica gel (type: ZCX-2, 100-200 mesh, 2.00 w / w.) and purified using a CombiFlash purification system with a 900 g silica gel column. The product was eluted with PE / EA (gradient from 100:0 to 70:30, collected every 500 ± 10 mL). After TLC analysis (EA:PE 1:8), the qualified fractions were combined, concentrated, and dried under vacuum to obtain 21-5 (33 g, 25.7%) as a colorless oil. Purity and identity ( 1 After verifying the 1H NMR spectrum, the substance was used in the following reaction.

[0504] 21-6: Synthesis of (((3,3'-Azandiylbis(propanoyl))bis(oxy))bis(methylene))bis(2-methylpropane-2,1,3-triyl)tetrakis(2-(4-methylcyclohexyl)acetate)trifluoroacetate [ka]

[0505] Under nitrogen, a solution of 21-5 (33 g, 32.4 mmol, 1.00 equivalent) in DCM (150 mL, 5V) was added to a 250 mL three-necked round-bottom flask. The solution was cooled to 0°C in an ice / water bath. Trifluoroacetic acid (15.88 g, 162.0 mmol, 5.00 equivalent) was added. The ice / water bath was removed, and the mixture was stirred at room temperature for 4 hours. The reaction product was concentrated under vacuum to obtain 21-6 (24.5 g, 74.4%) as a colorless oil, which was used directly in the next reaction.

[0506] Lipid 21: Synthesis of ((3,3'-((((3-(dimethylaminopropyl)thio)carbonyl)azandiyl)bis(propanoyl))bis(oxy))bis(methylene))bis(2-methylpropane-2,1,3-triyl)tetrakis(2-(4-methylcyclohexyl)acetate) [ka]

[0507] A 1 L three-necked round-bottom flask, purged and maintained under an inert nitrogen atmosphere, was filled with 21-6 (24.5 g, 24.1 mmol, 1.00 equivalent) in a 500 mL, 20V solution of DCM. TEA (9.76 g, 96.4 mmol, 4.00 equivalent) was added, followed by CDI (7.82 g, 48.21 mmol, 2.00 equivalent). The mixture was stirred overnight at room temperature. The solution was cooled to 0°C in an ice / water bath. Methyl trifluoromethanesulfonate (4.35 g, 26.5 mmol, 1.10 equivalent) was then added, and the mixture was stirred at 0°C for 1 hour. 3-(dimethylamino)propane-1-thiol (3.45 g, 28.9 mmol, 1.20 equivalent) was then added to the solution, the ice / water bath was removed, and the mixture was stirred overnight at room temperature. The crude compound was adsorbed onto 50 g of silica gel (type: ZCX-2, 100-200 mesh, 3.75 w / w) and purified using a combiflush system with 200 g of silica gel (type: ZCX-2, 300-400 mesh, 18.8 w / w). The product was eluted under a DCM / MeOH gradient of 100:0 to 96:4 and collected in 300 ± 50 mL increments. The fractions were analyzed (TLC, DCM:MeOH = 10:1, Rf = 0.5), and the qualified fractions were combined and concentrated to obtain lipid 21 (6 g, 23.4%) as a bright yellow oily substance. ELSD A: Water / 0.05% TFA; B: CH3CN / 0.05 TFA. 75:25~25:75 A / B): RT 10.9 min, m / z (calculated value) 1062.7, (measured value) 1063.8 (M+H). 1 H-NMR-Lipid 21:(400MHz,CDCl3,ppm):δ 4.02(d,J=8.2Hz,12H), 3.67(t,J=7.3Hz,4H), 2.95(t,J=7.2Hz,2H), 2.67(t,J=7.2Hz,4H), 2.53(s,2H), 2.45-2.28(m,8H), 2.24-2.16(m, 6H), 2.01(d,J=3.4Hz,1H), 1.90(t,J=7.3Hz,2H), 1.71(dd,J=5.8,3.1Hz,14H), 1.56-1.46(m,5H), 1.44-1.20(m,9H), 1.05-0.84(m,30H).

[0508] Example 22. Synthesis of Lipid 22: ((3,3'-((((3-(dimethylamino)propyl)thio)carbonyl)azandiyl)bis(propanoyl))bis(oxy))bis(methylene))bis(2-methylpropane-2,1,3-triyl)tetrakis(2-(4-ethylcyclohexyl)acetate) [ka]

[0509] Lipids 22

[0510] General Scheme [ka]

[0511] 22-2: Synthesis of 2-((benzyloxy)methyl)-2-methylpropane-1,3-diylbis(4-ethylcyclohexane-1-carboxylate) [ka]

[0512] In a 3 L three-necked round-bottom flask, 20-3 (70.0 g, 0.33 mol, 1.00 equivalent), CHCl3 (1400 mL, 20V), and pyridine (105.0 g, 4.0 equivalents) were added at room temperature under an N2 atmosphere. Subsequently, 22-1 (127.0 g, 0.73 mol, 2.20 equivalents) was added dropwise at 0°C. The resulting mixture was stirred overnight at room temperature. The reaction was quenched with water (700 mL, 10V) at room temperature, and the organic layer was washed with saturated NaHCO3 aqueous solution (1000 mL, 15V), HCl (1000 mL, 15V, 1 mol / L), and brine (1000 mL, 15V). The organic phase was dried over anhydrous Na2SO4 and then filtered. After filtration, the filtrate was concentrated under vacuum. This yielded 22-2 (120g, 0.25mol, yield 74.0%) as a yellow oily substance. ELSD A:Water / 0.05%TFA:B:CH3CN 95:5~5:95 A / B):RT 1.8min, m / z (calculated) 486.3, (measured) 509.5 (M+Na).

[0513] 22-3: Synthesis of 2-(hydroxymethyl)-2-methylpropane-1,3-diylbis(4-ethylcyclohexane-1-carboxylate) [ka]

[0514] A solution of Pd / C (36.0 g, 0.3 w / w) in MeOH (1.2 L, 10V) was added to a 3 L three-necked round-bottom flask at room temperature. Then, 22-2 (120.0 g, 0.25 mol, 1.00 equivalent) was added to the reaction mixture at room temperature. The reaction system was changed with H2 three times. The resulting solution was stirred under H2 at room temperature overnight. LC-MS showed complete consumption of 22-2. The resulting mixture was filtered, and the filter cake was washed with MeOH (2 × 1000 mL, 8V). The filtrate was concentrated and dried under vacuum. This yielded 22-3 (90 g, 0.23 mol, 92.0% yield) as a yellow oil, which was used without further purification. ELSD A: Water / 0.05% TFA: B: CH3CN 2 minutes 95:5~5:95 A / B):RT 1.5 minutes, m / z (calculated value) 396.3, (measured value) 397.3 (M+H).

[0515] 22-4: Synthesis of (((3,3'-((tert-butoxycarbonyl)azandiyl)bis(propanoyl))bis(oxy))bis(methylene))bis(2-methylpropane-2,1,3-triyl)tetrakis(4-ethylcyclohexane-1-carboxylate) [ka]

[0516] In a 5 L four-necked round-bottom flask, 22-3 (90 g, 0.22 mol, 2.20 equivalents), DCM (1.8 L, 20V), and 20-6 (26.96 g, 0.10 mol, 1.00 equivalent) were added at room temperature under an ...

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt thereof, 【Chemistry 1】 (I) During the ceremony, R １ and R ２ However, each independently, H or C １－６ It is alkyl, or R １ and R ２ However, they join together to form a saturated heterocyclic ring, R １ However, linear C １－４ It is alkylene, R ２ is -(CH ２ ) ｍ (X) ｎ -, and X is O, S, or NR ９ And R ９ However, H or C １－６ It is alkyl, m is 1, 2, 3, or 4. n is 0 or 1, L1 is a linear C molecule in which L1 is optionally substituted with 1 to 3 methyl groups. １－６ It is alkylene, Y is selected from the following group: 【Chemistry 2】 During the ceremony, Each asterisk (*) indicates an atom bonded to L2 and L3. R １０ However, H or C １－６ It is alkyl, L2 and L3 are each independently linear C １－８ It is alkylene, L4, L5, L6, L7, L8, and L9 are either not present or -CH, independently of each other. ２ -However, 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 However, each is independently H, methyl, or ethyl. R ５ , R ６ , R ７ , and R ８ However, each is independent, as follows: Linear C １－２０ Alkyl, and each of the linear C １－２０ Alkyl, below, C ６－１０ A aryl, and each of the C ６－１０ Aaryl is one or more C １－６ One or more C atoms optionally substituted with alkyl groups. ６－１０ C is a monocyclic or bicyclic aromatic hydrocarbon that is optionally substituted with an aryl group. ６－１０ Ariel, A 6- to 10-membered heteroaryl, wherein each of the 6- to 10-membered heteroaryls contains one or more C １－６ Linear carbon atoms are optionally substituted with one or more substituents selected from the group consisting of monocyclic or bicyclic aromatic systems that are optionally substituted with alkyl groups, and 6- to 10-membered heteroaryl groups. １－２０ Alkyl, C ６－１０ A aryl, and each of the C ６－１０ Aaryl is one or more C １－６ One or more C atoms optionally substituted with alkyl groups. ６－１０ C is a monocyclic or bicyclic aromatic hydrocarbon that is optionally substituted with an aryl group. ６－１０ Aryl, and A 6- to 10-membered heteroaryl, wherein each of the 6- to 10-membered heteroaryls contains one or more C １－６ A compound of formula I, selected from the group consisting of 6-10 membered heteroaryls, which are monocyclic or bicyclic aromatic systems optionally substituted with alkyl groups, or a pharmaceutically acceptable salt thereof.

2. R １ However, H or C １－６ It is alkyl, R ２ However, C １－６ It is alkyl, or R １ and R ２ The compound according to claim 1, wherein the compounds are joined to form the saturated heterocyclic ring.

3. The compound according to claim 1 or 2, wherein Y is selected from the group consisting of the following. 【Transformation 3】

4. The compound according to claim 1 or 2, wherein Y is as follows: 【Chemistry 4】

5. The compound according to claim 1 or 2, wherein Y is as follows: 【Transformation 5】

6. The compound according to claim 1 or 2, wherein Y is as follows: 【Transformation 6】

7. R １ and R ２ At least one of them is H, L1 is -CH ２ - or -CH ２ CH ２ - The compound according to claim 5 or 6.

8. R １ and R ２ However, each is independent, C １－６ A compound according to any one of claims 1 to 6, wherein it is alkyl.

9. R １ and R ２ However, each is independent, C １－３ The compound according to claim 8, wherein it is alkyl.

10. R １ and R ２ The compound according to claim 9, wherein each of the compounds is methyl.

11. R １ and R ２ The compound according to any one of claims 1 to 6, wherein the compound is joined to form the heterocyclic ring.

12. The aforementioned complex ring is selected from the group consisting of the following: 【Transformation 7】 The compound according to claim 11, wherein each asterisk (*) in the formula represents an atom bonded to L1.

13. The compound according to claim 12, wherein the heterocyclic ring is selected from the group consisting of the following. 【Transformation 8】

14. The compound according to claim 13, wherein the heterocyclic ring is selected from the group consisting of the following. 【Chemistry 9】

15. R ５ , R ６ , R ７ , and R ８ However, each independently, linear C １－８ It is alkyl, and each of the linear C １－８ Alkyl is as follows: C ６－１０ A aryl, and each of the C ６－１０ Aaryl is one or more C １－６ One or more C atoms optionally substituted with alkyl groups. ６－１０ C is a monocyclic or bicyclic aromatic hydrocarbon that is optionally substituted with an aryl group. ６－１０ Aryl, and A 6- to 10-membered heteroaryl, wherein each of the 6- to 10-membered heteroaryls contains one or more C １－６ The compound according to any one of the prior claims, which is optionally substituted with one or more substituents selected from 6-10 membered heteroaryls, which are monocyclic or bicyclic aromatic systems optionally substituted with alkyl groups.

16. R ５ , R ６ , R ７ , and R ８ However, each is independent, C ６－１０ It is an aryl, and each of the C ６－１０ Aaryl is one or more C １－６ One or more C atoms optionally substituted with alkyl groups. ６－１０ The compound according to any one of claims 1 to 14, which is a monocyclic or bicyclic aromatic hydrocarbon that is optionally substituted with an aryl group.

17. R ５ , R ６ , R ７ , and R ８ However, each is independently a 6-10 member heteroaryl, and each of the 6-10 member heteroaryls contains one or more C １－６ The compound according to any one of claims 1 to 14, which is a monocyclic or bicyclic aromatic system optionally substituted with an alkyl group.

18. R ５ and R ６ is the same, a compound according to any one of the preceding claims.

19. R ７ and R ８ The compound according to any one of the prior claims, which is the same as the compound.

20. The compound according to any one of the prior claims, wherein L1 is a linear unsubstituted alkylene.

21. The compound according to any one of the prior claims, wherein L1 is propylene.

22. L2 and L3 are each independently linear C １－５ A compound according to any one of the prior claims, which is an alkylene.

23. A compound according to any one of the prior claims, wherein L2 and L3 are the same.

24. A compound according to any one of the prior claims, wherein L4 and L5 are the same.

25. The compound according to any one of the prior claims, wherein L6 and L7 are the same.

26. A compound according to any one of the prior claims, wherein L8 and L9 are the same.

27. L4, L5, L6, L7, L8, and L9 are each -CH ２ - The compound according to any one of the prior claims.

28. L6, L7, L8, and L9 are each -CH ２ - and L4 and L5 are absent, the compound according to any one of claims 1 to 26.

29. L4, L5, L8, and L9 are each -CH ２ - and L6 and L7 are absent, the compound according to any one of claims 1 to 26.

30. L4, L5, L6, and L7 are each -CH ２ -, and L8 and L9 are absent, a compound according to any one of claims 1 to 26.

31. R ３ and R ４ The compound according to any one of the prior claims, wherein each is independently H or methyl.

32. R ３ and R ４ The compound according to any one of the prior claims, wherein each is H.

33. R ３ and R ４ The compound according to any one of the prior claims, wherein each is methyl.

34. below, 【Chemistry 10】 A compound according to claim 1, selected from the group consisting of the following: or a pharmaceutically acceptable salt thereof.

35. below, 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 A compound selected from the group consisting of, or a pharmaceutically acceptable salt thereof.

36. A lipid composition comprising a nucleic acid and a compound according to any one of the prior claims.

37. The lipid composition according to claim 36, wherein the nucleic acid is selected from siRNA, mRNA, self-replicating RNA, DNA plasmid, and antisense oligonucleotide.

38. The lipid composition according to claim 36 or 37, wherein the nucleic acid is mRNA or self-replicating RNA containing a coding region that encodes a target therapeutic protein.

39. The lipid composition according to claim 38, wherein the therapeutic protein for the purpose is an enzyme, and an antibody, antigen, receptor, or transporter.

40. The lipid composition according to claim 38 or 39, wherein the therapeutic protein for the purpose is a gene editing enzyme.

41. The lipid composition according to claim 40, wherein the gene editing enzyme is selected from TALEN, CRISPR, meganuclease, or zinc finger nuclease.

42. The lipid composition according to any one of claims 36 to 41, wherein the lipid composition comprises liposomes, lipoplexes, or lipid nanoparticles.

43. Lipid nanoparticles comprising a plurality of ligands, wherein each ligand is independently a compound according to any one of claims 1 to 35, and the plurality of ligands self-assemble to form the lipid nanoparticles, which include an interior and an exterior.

44. The lipid nanoparticles according to claim 43, wherein the average particle size of the lipid nanoparticles is less than approximately 100 nm.

45. The lipid nanoparticles according to claim 43 or 44, wherein the average particle size of the lipid nanoparticles is about 55 nm to about 85 nm.

46. The lipid nanoparticles according to any one of claims 43 to 45, wherein the lipid nanoparticles further comprise nucleic acids encapsulated therein.

47. The lipid nanoparticle according to claim 46, wherein the nucleic acid is selected from siRNA, mRNA, self-replicating RNA, DNA plasmid, and antisense oligonucleotide.

48. The lipid nanoparticle according to claim 46 or 47, wherein the nucleic acid is mRNA or self-replicating RNA containing a coding region encoding a target therapeutic protein.

49. The lipid nanoparticles according to claim 48, wherein the therapeutic protein for the purpose is an enzyme, and an antibody, antigen, receptor, or transporter.

50. The lipid nanoparticle according to claim 48 or 49, wherein the therapeutic protein for the purpose is a gene editing enzyme.

51. The lipid nanoparticle according to claim 50, wherein the gene editing enzyme is selected from TALEN, CRISPR, meganuclease, or zinc finger nuclease.

52. The lipid nanoparticles according to any one of claims 43 to 51, further comprising a helper lipid selected from dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC), and phosphatidylcholine (PC).

53. The lipid nanoparticle according to claim 52, wherein the helper lipid is distearoylphosphatidylcholine (DSPC).

54. Lipid nanoparticles according to any one of claims 43 to 53, further comprising cholesterol.

55. Lipid nanoparticles according to any one of claims 43 to 54, further comprising polyethylene glycol (PEG)-lipid conjugate.

56. The lipid nanoparticle according to claim 55, wherein the PEG-lipid conjugate is PEG-DMG.

57. The lipid nanoparticle according to claim 56, wherein the PEG-DMG is PEG2000-DMG.

58. Lipid nanoparticles according to any one of claims 43 to 57, wherein the lipid nanoparticles comprise about 45 mol% to 65 mol% of the compound according to any one of claims 1 to 35, 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.

59. The lipid nanoparticles according to claim 58, wherein the lipid nanoparticles comprise about 50 mol% to about 61 mol% of the compound according to any one of claims 1 to 35, about 5 mol% to about 9 mol% of the helper lipid, about 29 mol% to about 38 mol% of cholesterol, and about 1 mol% to about 2 mol% of the PEG-lipid conjugate.

60. The lipid nanoparticles according to claim 59, wherein the lipid nanoparticles comprise about 56 mol% to about 58 mol% of the compound according to any one of claims 1 to 35, 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 the PEG-lipid conjugate.

61. The lipid nanoparticles according to any one of claims 43 to 60, wherein the lipid nanoparticles have a total lipid:nucleic acid weight ratio of about 50:1 to about 10:

1.

62. The lipid nanoparticles according to claim 61, wherein the lipid nanoparticles have a total lipid:nucleic acid weight ratio of about 40:1 to about 20:

1.

63. The lipid nanoparticles according to claim 61, wherein the lipid nanoparticles have a total lipid:nucleotide weight ratio of about 35:1 to about 25:

1.

64. The lipid nanoparticles according to claim 61, wherein the lipid nanoparticles have a total lipid:nucleic acid weight ratio of about 32:1 to about 28:

1.

65. The lipid nanoparticles according to claim 61, wherein the lipid nanoparticles have a total lipid:nucleic acid weight ratio of about 31:1 to about 29:

1.

66. A pharmaceutical composition comprising a compound according to any one of claims 1 to 35 or lipid nanoparticles according to any one of claims 43 to 65, and a pharmaceutically acceptable excipient.

67. The pharmaceutical composition according to claim 66, wherein the pharmaceutical composition is a freeze-dried composition.

68. The pharmaceutical composition according to claim 66 or 67, wherein the pharmaceutical composition comprises a HEPES buffer solution with a pH of about 7.

4.

69. The pharmaceutical composition according to claim 68, wherein the HEPES buffer solution has a concentration of about 7 mg / mL to about 15 mg / mL.

70. The pharmaceutical composition according to any one of claims 66 to 69, wherein the pharmaceutical composition further comprises about 2.0 mg / mL to about 4.0 mg / mL of NaCl.

71. The pharmaceutical composition according to any one of claims 66 to 70, wherein the pharmaceutical composition further comprises one or more cryoprotective agents.

72. The pharmaceutical composition according to claim 71, wherein the one or more cryoprotective agents are selected from sucrose, glycerol, or a combination of sucrose and glycerol.

73. The pharmaceutical composition according to claim 72, wherein the pharmaceutical composition comprises 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.

74. A method for treating a disease in a subject requiring treatment, comprising administering to the subject a therapeutically effective amount of lipid nanoparticles according to any one of claims 43 to 65 or a pharmaceutical composition according to any one of claims 66 to 73.

75. The method according to claim 74, wherein the pharmaceutical composition or lipid nanoparticles are administered intravenously or intramuscularly.

76. A method for expressing a protein or polypeptide in target cells, comprising contacting the target cells with lipid nanoparticles according to any one of claims 43 to 65 or a pharmaceutical composition according to any one of claims 66 to 73.

77. The method according to claim 76, wherein the protein or polypeptide is an antigen, and the expression of the antigen elicits an in vivo immunogenic response.

78. A method for delivering nucleic acids to a subject requiring such delivery, comprising: encapsulating a therapeutically effective amount of the nucleic acid in lipid nanoparticles described in any one of claims 43 to 65; and administering the lipid nanoparticles to the subject.

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