Ionizable cationic lipids for RNA delivery

Abstract

The present disclosure describes compounds of formula (I) or a pharma- ceutically acceptable salt thereof, wherein R 1 and R 2 are each independently (CH3(CH2) m )2CH-, (CH3(CH2) m )(CH3(CH2) m-1 )CH, (CH3(CH2) m )(CH3(CH2) m-2 )CH, (CH3(CH2) m )2CHCH2-, or (CH3(CH2) m )(CH3(CH2) m-1 ) CHCH2-, m is 4 to 11, and L 1 and L 2 are each independently absent or a linear C 1-5 Alkylene, or (CH2) p -O-(CH2) q p and q are each independently 1 to 3; R 3 is a linear C optionally substituted with one or two methyl groups 2-5 alkylene, R 4 and R 5 are each independently H or C 1-6 alkyl, X is O or S, and n is 0-2. TIFF2024518375000090.tif41100

Description

[Technical field]

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

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

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

[0004] The present disclosure provides lipids of formula (I) described herein that are useful for lipid-based delivery of nucleic acids and other therapeutic agents to treat disease. These and other uses will be apparent to those skilled in the art. Additional features and advantages of the subject technology will be set forth in the description that follows, and in part will be obvious from the description, or may be learned by practice of the subject technology. The advantages of the subject technology will be realized and attained by the structures particularly pointed out in the written description and embodiments herein, as well as the accompanying drawings.

[0005] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the subject technology.

[0006] In some embodiments, the disclosure provides a compound of formula I, or a pharma- ceutically acceptable salt thereof: [ka] In the formula, R 1 and R 2 are each independently (CH 3 (CH 2 ) m ) 2 CH-, (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-1 )CH, (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-2 )CH, (CH 3 (CH 2 ) m ) 2 CHCH 2 - or (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-1 )CHCH 2 -, m is 4 to 11, and L 1 and L2 are each independently absent or a linear C 1-5 Alkylene, or (CH 2 ) p -O-(CH 2 ) q p and q are each independently 1 to 3; R 3 is a linear C optionally substituted with one or two methyl groups 2-5 alkylene, R 4 and R 5 are each independently H or C 1-6 alkyl, X is O or S, and n is 0-2.

[0007] In some embodiments, the present disclosure provides lipid nanoparticles comprising a plurality of ligands, each ligand being independently a compound described herein, and the plurality of ligands self-assemble to form a lipid nanoparticle comprising an interior and an exterior.

[0008] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a compound described herein or a lipid nanoparticle described herein and a pharma- ceutically acceptable excipient.

[0009] In some embodiments, the disclosure provides a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound described herein, a lipid nanoparticle described herein, or a pharmaceutical composition described herein.

[0010] In some embodiments, the present disclosure provides a method of delivering a nucleic acid to a subject in need thereof, the method comprising encapsulating a therapeutically effective amount of a nucleic acid in a lipid nanoparticle described herein and administering the lipid nanoparticle to a subject. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] I. General Various configurations of the subject technology will be readily apparent to those skilled in the art from this disclosure, and it will be understood that various configurations of the subject technology have been shown and described by way of example. It will be understood that the subject technology is capable of other configurations and different configurations, and its several details can all be modified in various other respects without departing from the scope of the subject technology. Accordingly, the Summary and Detailed Description are to be regarded as illustrative in nature, and not restrictive.

[0012] The detailed description of the invention described below is intended as an illustration of various configurations of the subject technology, and is not intended to represent the only configurations in which the subject technology may be practiced. The accompanying drawings are incorporated in this specification and constitute a part of the detailed description of the invention. The detailed description of the invention includes specific details for the purpose of providing a thorough understanding of the subject technology. However, it will be apparent to those skilled in the art that the subject technology may be practiced without these specific details. In some instances, well-known structures and components are shown in block diagram form to avoid obscuring the concepts of the subject technology. Similar components are labeled with the same element numbers for ease of understanding.

[0013] II. Definition At various places in the present specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include any and all individual subcombinations of the members of such groups and ranges. For example, "C 1-6 The term "alkyl" includes methyl, ethyl, C 3 Alkyl, C 4 Alkyl, C 5 Alkyl, and C 6 It is specifically intended to disclose alkyl individually.

[0014] The phrases "administered in combination with" or "administration in combination with" mean that two or more agents are administered to a subject simultaneously or within an interval such that there may be an overlap in the effect of each agent on the patient. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minutes of each other. In some embodiments, administration of the agents is spaced sufficiently close to each other that a combined effect (e.g., a synergistic effect) is achieved.

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

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

[0017] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds). The alkyl group may have 1 to 20 carbon atoms (whenever it appears in this specification, a numerical range such as "1 to 20" refers to each integer within the given range, e.g., "1 to 20 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, but this definition also encompasses occurrences of the term "alkyl" where no numerical range is specified). The alkyl group may also be a medium-sized alkyl having 1 to 9 carbon atoms. The alkyl group may also be a lower alkyl having 1 to 6 carbon atoms. The alkyl group may also be a "C 1-4 By way of example only, "C 1-4 "Alkyl" indicates that there are 1 to 4 carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are not limited in any manner to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like.

[0018] "Alkylene" refers to a straight or branched chain saturated aliphatic radical, i.e., a divalent hydrocarbon radical, having the number of carbon atoms indicated and linking at least two other groups. The two moieties linked to the alkylene can be attached to the same atom or different atoms of the alkylene group. For example, a straight chain alkylene is -(CH 2 ) n - divalent radical, where n is 1, 2, 3, 4, 5, or 6. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, butylene, isobutylene, sec-butylene, pentylene, and hexylene. The alkylene group can be substituted or unsubstituted.

[0019] The term "lower alkyl" means a group having 1 to 6 carbons in the chain which may be straight or branched. Non-limiting examples of suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, and hexyl.

[0020] As used herein, the term “amino” refers to —N(R N1 ) 2 Each R N1 are independently H, OH, and NO 2 , N(R N2 ) 2 , S.O. 2 OR N2 , S.O. 2 R N2 , SOR N2 , an N-protecting group, an alkyl, an alkenyl, an alkynyl, an alkoxy, an aryl, an alkaryl, a cycloalkyl, an alkylcycloalkyl, a carboxyalkyl (e.g., optionally substituted with an O-protecting group, e.g., an optionally substituted arylalkoxycarbonyl group or any as described herein), a sulfoalkyl, an acyl (e.g., acetyl, trifluoroacetyl, or others as described herein), an alkoxycarbonylalkyl (e.g., optionally substituted with an O-protecting group, e.g., an optionally substituted arylalkoxycarbonyl group or any as described herein), a heterocyclyl (e.g., a heteroaryl), or an alkylheterocyclyl (e.g., an alkylheteroaryl), and these enumerated R N1 Each of the groups may be optionally substituted as defined herein for each group, or may have two R N1 can be combined to form a heterocyclyl or N-protecting group, N2 is independently H, alkyl, or aryl. The amino group of the present disclosure includes unsubstituted amino (i.e., -NH 2 ) or substituted amino (i.e., -N(R') 2 In a preferred embodiment, the amino can be -NH 2 or -NHR N1 and RN1 are independently OH, NO 2 , N.H. 2 , N.R. N2 2 , S.O. 2 OR N2 , S.O. 2 R N2 , SOR N2 , alkyl, carboxyalkyl, sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein), alkoxycarbonylalkyl (e.g., t-butoxycarbonylalkyl), or aryl, and each R N2 , H, C 1-20 Alkyl (e.g., C 1-6 alkyl), or C 1-10 It may be aryl.

[0021] 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, palmitoyloleylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

[0022] The phrase "at least one" precedes a list of items with the term "and" or "or" separating any of the items, and modifies the list as a whole, rather than each member (i.e., each item) of the list. The phrase "at least one" does not require the selection of at least one of each of the listed items, rather, the phrase allows for a meaning including any one of the items, and / or at least one of any combination of the items, and / or at least one of each of the items. As an example, the phrase "at least one of A, B, and C" or "at least one of A, B, or C" refers to A only, B only, or C only, any combination of A, B, and C, and / or at least one of each of A, B, and C, respectively.

[0023] When the terms "include," "have," or similar terms are used in the description or claims, such terms are intended to be inclusive in the same manner as the term "comprise" when it is used as a transitional term in a claim.

[0024] Reference to an element in the singular is not intended to mean "one and only one" unless otherwise specified, but rather "one or more". Masculine pronouns (e.g., his) include feminine and neuter genders (e.g., her and its), and vice versa. The term "several" refers to one or more. Underlined and / or italicized headings and subheadings are used for convenience only and do not limit the subject art, and are not referred to in connection with interpreting the description of the subject art. All structural and functional equivalents to the elements of the various configurations described throughout this disclosure that are known or later become known to those skilled in the art are expressly incorporated herein by reference and are intended to be encompassed by the subject art. Furthermore, any disclosures disclosed herein are not intended to be directed to the general public, regardless of whether such disclosures are expressly recited in the description above.

[0025] The term "cationic lipid" refers to amphipathic lipids and their salts having a positive hydrophilic head group, one, two, three or more hydrophobic fatty acid or fatty alkyl chains, and a connector between these two domains. An ionizable cationic lipid or a protonizable cationic lipid is typically protonated (i.e., positively charged) at a pH below its pKa and substantially neutral at a pH above its pKa. Preferred ionizable cationic lipids are lipids with a pKa below physiological pH, typically about 7.4. The cationic lipids of the present disclosure may also be referred to as titratable cationic lipids. The cationic lipids may be "amino lipids" having a protonizable tertiary amine (e.g., pH titratable) head group. Some amino exemplary amino lipids may include C18 alkyl chains, each alkyl chain independently having 0-3 (e.g., 0, 1, 2, or 3) double bonds, and an ether bond, an ester bond, or a ketal bond between the head group and the alkyl chain. Such cationic lipids include, but are not limited to, DSDMA, DODMA, DLinDMA, DLenDMA, γ-DLenDMA, DLin-K-DMA, DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2, and C2K), DLin-K-C3-DMA, 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).

[0026] The term "comprising" is intended to be open, allowing, but not requiring, the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of" is therefore also included and disclosed.

[0027] The term "in combination with" refers to administration of the lipid-formulated mRNA of the present disclosure together with another agent in the therapeutic methods of the present disclosure, and means that the lipid-formulated mRNA of the present disclosure and the other agent are administered sequentially or simultaneously in separate dosage forms, or simultaneously in the same dosage form.

[0028] The term "commercially available chemicals" and 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.), Riedel de Haen (Hannover, Germany), Spectrum Quality Products, Inc. (New Brunswick, NJ), TCI America (Portland, Or.), and Wako Chemicals USA, Inc. (Richmond, Va.).

[0029] The phrase "a compound described in the chemical literature" may be identified through reference books and databases covering chemical compounds and chemical reactions, as known to those of skill in the art. Suitable references and articles detailing the synthesis of reactants useful in the preparation of the compounds disclosed herein or providing references to articles describing the preparation of the compounds disclosed herein include, for example, “Synthetic Organic Chemistry”, John Wiley and Sons, Inc. New York; S.R. Sandler et al., “Organic Functional Group Preparations,” 2nd Ed., Academic Press, New York, 1983; H.O. House, “Modern Synthetic Reactions,” 2nd Ed., W.A. Benjamin, Inc. Menlo Park, Calif., 1972; T.L. Lichrist, “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; specific and similar reactants are also available in most public and university libraries; Chemical Abstract Service of the American Chemicals may be identified through an index of known chemicals prepared by the American Chemical Society, as well as through online databases (further details may be contacted at the American Chemical Society, Washington, DC). Chemicals that are known but not commercially available in catalogs may be prepared by custom chemical synthesis companies, where many of the standard chemical supply companies (such as those listed above) offer custom synthesis services.

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

[0031] The term "fully encapsulated" means that the nucleic acid (e.g., mRNA) in the nucleic acid-lipid particle is not significantly degraded after exposure to serum or nuclease assays that would significantly degrade free RNA.When fully encapsulated, preferably less than 25% of the nucleic acid in the particle is degraded, more preferably less than 10%, and most preferably less than 5% is degraded in a treatment that would normally degrade 100% of free nucleic acid."Fully encapsulated" also means that the nucleic acid-lipid particle does not rapidly degrade into its component parts when administered in vivo.

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

[0033] The term "delivery" refers to the act or method of delivering a compound, substance, entity, moiety, cargo, or payload.

[0034] The term "characteristic" refers to a characteristic, property, or distinctive element.

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

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

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

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

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

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

[0041] The term "linker" or "linking moiety" refers to a group of atoms, e.g., 10-100 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 of sufficient length so as not to interfere with incorporation into the amino acid sequence. Examples of chemical groups that may be incorporated into the linker include, but are not limited to, alkyl, alkenyl, alkynyl, amido, amino, ether, thioether, ester, alkyl, heteroalkyl, aryl, or heterocyclyl, each of which may be optionally substituted as described herein. Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene or propylene glycol monomer units, e.g., diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran polymers. Other examples include, but are not limited to, cleavable moieties in the linker, such as disulfide bonds (-SS-) or azo bonds (-N=N-), which can be cleaved using reducing agents or photolysis. Non-limiting examples of selectively cleavable bonds include amide bonds, which can be cleaved, for example, by the use of tris(2-carboxyethyl)phosphine (TCEP) or other reducing agents, and / or photolysis, as well as ester bonds, which can be cleaved, for example, by acidic or basic hydrolysis.

[0042] The term mammal means a human or other mammalian animal, or means a human.

[0043] The term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a protein or polypeptide of interest and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded protein or polypeptide of interest.

[0044] The term "modification" refers to a change in the state or structure of a molecule of the present disclosure. Molecules may be modified in many ways, including chemically, structurally, and functionally. In one embodiment, a nucleic acid active component is modified by the introduction of non-natural nucleosides and / or nucleotides, for example, as they relate to the natural ribonucleotides A, U, G, and C. Non-standard nucleotides, such as cap structures, may differ from the chemical structure of A, C, G, U ribonucleotides, but are not considered "modified."

[0045] The term "naturally occurring" means existing in nature without artificial assistance.

[0046] The term "non-human vertebrate" includes all vertebrates except Homo sapiens, including wild and domestic species. Examples of non-human vertebrates include, but are not limited to, mammals such as alpacas, bantengs, bison, camels, cats, cows, deer, dogs, donkeys, gayal, goats, guinea pigs, horses, llamas, mules, pigs, guinea pigs, rabbits, reindeer, sheep, buffalo, and yaks.

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

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

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

[0050] As used herein, the phrase "pharmaceutical acceptable excipient" refers to any component other than the compounds described herein (e.g., a vehicle that can suspend or dissolve active compounds) and has the properties of being substantially non-toxic and non-inflammatory in patients. Excipients may include, for example, anti-adhesives, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), softeners, emulsifiers, fillers (diluents), film formers or coatings, flavors, flavorings, glidants (glidants), lubricants, preservatives, printing inks, absorbents, suspending or dispersing agents, sweeteners, and hydration water. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), 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, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

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

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

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

[0054] The term "physicochemical" means or relates to physical and / or chemical properties.

[0055] The term "phosphate" is used in its ordinary sense as understood by those of skill in the art and includes its protonated form, e.g., [ka] Includes. As used herein, the terms "monophosphate," "diphosphate," and "triphosphate" are used in their ordinary sense as understood by those of skill in the art and include the protonated forms.

[0056] The term "prophylaxis" refers to partially or completely delaying the onset of an infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, 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 developing pathology associated with an infection, disease, disorder, and / or condition.

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

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

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

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

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

[0062] The term solvate refers to the physical association of the compound of the present disclosure with one or more solvent molecules. This physical association involves varying degrees of ionic bonding, including hydrogen bonding. In certain cases, the solvate has 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-phase and isolable solvates. Non-limiting examples of suitable solvates include ethanolate, methanolate, etc.

[0063] The term "split dose" refers to the division of a single unit dose or total daily dose into two or more doses.

[0064] The term "stable" refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture and, preferably, is capable of being formulated into an efficacious therapeutic agent.

[0065] The terms "stabilize," "stabilized," and "stabilized region" mean to make stable or to become stable.

[0066] The term "substituted" means substitution with a specified group other than hydrogen, or, for example, with one or more groups, moieties, or radicals, each of which is independently selected and may be the same or different.

[0067] The term "substantially" refers to a qualitative condition that indicates the extent or degree of the total or near total of a characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or completeness, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness that is inherent in many biological and chemical phenomena.

[0068] The phrase "substantially equal" refers to the time difference between doses, the term meaning ±2%.

[0069] The phrase "substantially simultaneously" refers to multiple doses, the term meaning within 2 seconds.

[0070] The phrase "suffering from" relates to an individual "suffering from" a disease, disorder, and / or condition having been diagnosed with or exhibiting one or more symptoms of the disease, disorder, and / or condition.

[0071] The phrase "susceptibility" refers to an individual who is "susceptible" to a disease and / or condition who has not been diagnosed with the disease, disorder, and / or condition and / or does not show symptoms of the disease, disorder, and / or condition, but has a tendency to develop the disease or its symptoms. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., cancer) may be characterized by one or more of the following: (1) a genetic mutation associated with the onset of the disease, disorder, and / or condition, (2) a genetic polymorphism associated with the onset of the disease, disorder, and / or condition, (3) an increase and / or decrease in expression and / or activity of a protein and / or nucleic acid associated with the disease, disorder, and / or condition, (4) habits and / or lifestyle associated with the onset of the disease, disorder, and / or condition, (5) a family history of the disease, disorder, and / or condition, and (6) exposure to and / or infection with a microorganism associated with the onset of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition develops the disease, disorder, and / or condition. In some embodiments, an individual who has a susceptibility to a disease, disorder, and / or condition does not develop the disease, disorder, and / or condition.

[0072] The term "synthetic" means produced, prepared, and / or manufactured by the hand of man. Synthesis of a polynucleotide or polypeptide or other molecule of the disclosure may be chemical synthesis or enzymatic synthesis.

[0073] The term "therapeutic agent" refers to any agent that has a therapeutic, diagnostic, and / or prophylactic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject.

[0074] The term "therapeutically effective amount" means an amount of an agent (e.g., a nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) delivered that, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, is sufficient to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0075] The term "therapeutically effective outcome" means an outcome that is sufficient to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of an infection, disease, disorder, and / or condition in a subject suffering from or susceptible to the infection, disease, disorder, and / or condition.

[0076] The term "total daily dose" is the amount given or prescribed for a 24 hour period. It may be administered as a single unit dose.

[0077] The term "treatment" refers to the partial or complete alleviation, palliation, amelioration, mitigation, delay in onset, inhibition of progression, reduction in severity, and / or reduction in incidence of one or more symptoms or characteristics of a particular infection, disease, disorder, and / or condition. For example, "treatment" of cancer may refer to inhibiting tumor survival, growth, and / or spread. Treatment may be administered to subjects who do not show signs of the disease, disorder, and / or condition and / or to subjects who show only early signs of the disease, disorder, and / or condition, for the purpose of reducing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0078] The term "unmodified" refers to any substance, compound, or molecule before it has been altered in any way. Unmodified may, but does not necessarily, refer to a wild-type or naturally occurring biomolecule. A molecule may undergo a series of modifications, whereby each modified molecule may serve as an "unmodified" starting molecule for subsequent modifications.

[0079] The compounds described herein may be asymmetric (e.g., have one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds of the present disclosure that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods for preparing optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by enantioselective and / or stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, and the like, can also be present in the compounds described herein, and all such stable isomers are contemplated in the present disclosure. Cis and trans geometric isomers of the compounds of the present disclosure are described and may be isolated as a mixture of isomers or as separated isomeric forms.

[0080] The compounds of the present disclosure also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond and the simultaneous migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonation states with the same empirical formula and total charge. Examples of prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and cyclic forms in which protons can occupy two or more positions in heterocyclic ring systems such as 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms may be in equilibrium or sterically locked into one form by appropriate substitution.

[0081] The compounds of the present disclosure also include all isotopes of atoms occurring in intermediate or final compounds. "Isotopes" refers to atoms having the same atomic number but different mass numbers resulting from different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium.

[0082] The compounds and salts of the disclosure can be prepared in combination with a solvent or water molecules to form solvates and hydrates by routine methods.

[0083] The term "half-life" is the time required for a quantity, such as the concentration or activity of a nucleic acid or protein, to decrease to half of its value as measured at the beginning of a period of time.

[0084] The term "in vitro" refers to events that take place not in a living organism (e.g., an animal, plant, or microorganism) but in an artificial environment, e.g., in a test tube or reaction vessel, in a cell culture, in a petri dish, etc.

[0085] The term "in vivo" refers to an event that takes place within a living organism (e.g., an animal, plant, or microorganism, or cells or tissues thereof).

[0086] The term "monomer" refers to a single unit, e.g., a single nucleic acid that can combine with another molecule of the same or different type to form an oligomer. In some embodiments, the monomer may be a non-locked nucleic acid, i.e., a UNA monomer.

[0087] The term "neutral lipid" refers to lipid species that exist in either an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0088] The term "non-cationic lipid" means an amphipathic lipid or a neutral lipid or an anionic lipid, as described herein.

[0089] The term "subject" or "patient" refers to any living organism to which a composition according to the present disclosure may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants.

[0090] The term "translatable" may be used interchangeably with the term "expressible" and refers to the ability of a polynucleotide or a portion thereof to be converted into a polypeptide by a host cell. As understood in the art, translation is the process by which ribosomes in the cytoplasm of a cell generate a polypeptide. In translation, messenger RNA (mRNA) is decoded by tRNA in the ribosomal complex to produce a specific amino acid chain or polypeptide. Furthermore, as used herein with respect to an oligomer, the term "translatable" means that at least a portion of the oligomer, e.g., the coding region (also known as the coding sequence or CDS) of the oligomer sequence, has the ability to be converted into a protein or a fragment thereof.

[0091] Therapeutically Effective Outcome: As used herein, the term "therapeutically effective outcome" means an outcome that is sufficient to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of an infection, disease, disorder, and / or condition in a subject suffering from or susceptible to an infection, disease, disorder, and / or condition.

[0092] The term "unit dose" refers to a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of the active ingredient may generally be equal to the dose of the active ingredient that would be administered to a subject, and / or a convenient fraction of such a dose, including, but not limited to, one-half or one-third of such a dose.

[0093] While the present disclosure has been described in connection with certain specific embodiments and numerous details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the present disclosure includes additional embodiments and that some of the details described herein may vary substantially without departing from the present disclosure. The present disclosure includes such additional embodiments, modifications, and equivalents. In particular, the present disclosure includes any combination of the features, terms, or elements of the various exemplary components and examples.

[0094] III.Compound In some embodiments, the disclosure provides a compound of formula I, or a pharma- ceutically acceptable salt thereof: [ka] In the formula, R 1 and R 2 are each independently (CH 3 (CH 2 ) m ) 2 CH-, (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-1 )CH, (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-2 )CH, (CH 3 (CH 2 ) m ) 2 CHCH 2 - or (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-1 )CHCH 2 -, m is 4 to 11, and L 1 and L 2 are each independently absent or a linear C 1-5 Alkylene, or (CH 2 ) p -O-(CH2 ) q p and q are each independently 1 to 3; R 3 is a linear C optionally substituted with one or two methyl groups 2-5 alkylene, R 4 and R 5 are each independently H or C 1-6 alkyl, X is O or S, and n is 0-2.

[0095] In some embodiments, R 1 and R 2 are each independently (CH 3 (CH 2 ) m ) 2 CH-, (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-1 )CH, (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-2 )CH, (CH 3 (CH 2 ) m ) 2 CHCH 2 - or (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-1 )CHCH 2 In some embodiments, R 1 and R 2 are each independently (CH 3 (CH 2 ) m ) 2 CH-, (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-1 )CH, (CH 3 (CH 2 ) m )2 CHCH 2 - or (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-1 )CHCH 2 In some embodiments, R 1 and R 2 are each independently (CH 3 (CH 2 ) m ) 2 CH- and (CH 3 (CH 2 ) m ) 2 CHCH 2 In some embodiments, R 1 and R 2 are each independently (CH 3 (CH 2 ) m ) 2 In some embodiments, R 1 and R 2 are each independently (CH 3 (CH 2 ) m ) 2 CHCH 2 In some embodiments, R 1 and R 2 are each independently (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-1 )CH, (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-2 )CH, and (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-1 )CHCH 2 In some embodiments, R 1 is (CH3 (CH 2 ) m ) 2 CH- or (CH 3 (CH 2 ) m ) 2 CHCH 2 - and R 2 is (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-1 )CH, (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-2 )CH, and (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-1 )CHCH 2 - is selected.

[0096] In some embodiments, m is 4 to 11. In some embodiments, m is 4 to 9. In some embodiments, m is 4 to 8. In some embodiments, m is 5 to 7. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7.

[0097] In some embodiments, L 1 and L 2 are each independently absent or a linear C 1-5 Alkylene, or (CH 2 ) p -O-(CH 2 ) q In some embodiments, L 1 and L 2 are each independently 1-5 Alkylene or (CH 2 ) p -O-(CH 2 ) qIn some embodiments, L 1 and L 2 are each independently 2-5 Alkylene or (CH 2 ) p -O-(CH 2 ) q In some embodiments, L 1 and L 2 are each independently 2-5 In some embodiments, L is an alkylene. 1 and L 2 Each is propylene. In some embodiments, L 1 and L 2 are each independently 2-5 In some embodiments, L is an alkylene. 1 and L 2 are each independently (CH 2 ) p -O-(CH 2 ) q In some embodiments, L 1 and L 2 do not exist independently of each other.

[0098] In some embodiments, p and q are each independently 1 to 3. In some embodiments, p and q are each independently 1 to 2. In some embodiments, p and q are each independently 1. In some embodiments, p and q are each independently 2. In some embodiments, p and q are each independently 3.

[0099] In some embodiments, R 3 is a linear C optionally substituted with one or two methyl groups 2-5 In some embodiments, R is an alkylene. 3 is a linear C 2-5 In some embodiments, R is an alkylene. 3 is C 3-5 In some embodiments, R is an alkylene. 3 is C 1-3 In some embodiments, R is an alkylene.3 is propylene.

[0100] In some embodiments, R 4 and R 5 are each independently H or C 1-6 In some embodiments, R 4 and R 5 are each independently 1-6 In some embodiments, R 4 and R 5 are each independently 1-3 In some embodiments, R 4 and R 5 are each independently methyl. In some embodiments, R 4 and R 5 are each independently H.

[0101] In some embodiments, X is O or S. In some embodiments, X is O. In some embodiments, X is S.

[0102] In some embodiments, n is 0 to 2. In some embodiments, n is 0 to 1. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2.

[0103] In some embodiments, the compound is [ka] [ka] or a pharma- ceutically acceptable salt thereof.

[0104] In some embodiments, the compound is ATX-193. In some embodiments, the compound is ATX-200. In some embodiments, the compound is ATX-201. In some embodiments, the compound is ATX-202. In some embodiments, the compound is ATX-209. In some embodiments, the compound is ATX-210. In some embodiments, the compound is ATX-230. In some embodiments, the compound is ATX-231. In some embodiments, the compound is ATX-232.

[0105] In some embodiments, the present invention provides a lipid composition comprising a nucleic acid and a compound of the present invention. 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 an mRNA or a self-replicating RNA comprising a coding region encoding a therapeutic protein of interest. In some embodiments, the therapeutic protein of interest is an enzyme, and an antibody, an antigen, a receptor, or 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 lipid composition comprises a liposome, a lipoplex, or a lipid nanoparticle.

[0106] IV. Lipid Formulations and Nanoparticles Lipid-based formulations Therapies based on intracellular delivery of nucleic acids to target cells face both extracellular and intracellular barriers. In fact, naked nucleic acid materials cannot be easily administered systemically due to their toxicity, low stability in serum, rapid renal clearance, reduced uptake by target cells, uptake by phagocytes, and their ability to activate immune responses, all features that hinder their clinical development. When exogenous nucleic acid materials (e.g., mRNA) enter the human biological system, they are recognized as foreign pathogens by the reticuloendothelial system (RES) and are removed from the blood circulation before they have a chance to encounter target cells in or outside the vascular system. It has been reported that the half-life of naked nucleic acids in the bloodstream is approximately several minutes (Kawabata K, Takakura Y, Hashida MPharm Res. 1995 Jun; 12(6): 825-30). Chemical modifications and proper delivery methods can reduce uptake by the RES and protect the nucleic acids from degradation by ubiquitous nucleases, which increases the stability and efficacy of nucleic acid-based therapies. In addition, RNA or DNA are anionic hydrophilic polymers that are also anionic on the surface, which is unfavorable for cellular uptake. Therefore, the success of nucleic acid-based therapy depends primarily on the development of vehicles or vectors that can efficiently and effectively deliver genetic material to target cells and obtain sufficient levels of expression in vivo with minimal toxicity.

[0107] Furthermore, upon internalization into target cells, nucleic acid delivery vectors are challenged by intracellular barriers, including endosomal uptake, lysosomal degradation, unpackaging of the nucleic acid from the vector, translocation across the nuclear membrane (for DNA), and release in the cytoplasm (for RNA). Successful nucleic acid-based therapy therefore relies on the ability of the vector to deliver the nucleic acid to a target site inside the cell to obtain sufficient levels of the desired activity, such as gene expression.

[0108] Although 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 the so-called stable nucleic acid lipid particle (SNALP) transfection technology. Despite the success of patisiran, the delivery of nucleic acid therapeutics, including mRNA, via lipid formulations is still under development. The use of mRNA in lipid delivery vehicles has rapidly come to prominence as a result of the COVID-19 pandemic, and several vaccines delivering mRNA encoding the COVID-19 spike protein have shown strong protective capabilities. Such lipid-based mRNA vaccines include Pfizer and BioNtech's BNT162b2 and Moderna's mRNA-1273, which have received emergency use authorization worldwide.

[0109] Some art-recognized lipid formulation delivery vehicles for nucleic acid therapeutics include polymer-based carriers (such as polyethyleneimine (PEI), lipid nanoparticles and liposomes), nanoliposomes, ceramide-containing nanoliposomes, multivesicular liposomes, proteoliposomes, exosomes of both natural and synthetic origin, natural, synthetic and semi-synthetic lamellar bodies, nanoparticles, micelles, and emulsions, according to various embodiments. Because these lipid formulations can vary in their structure and composition and can be expected in a rapidly developing field, the art uses several different terms to describe a single type of delivery vehicle. At the same time, the terminology of lipid formulations has changed with respect to their intended meaning throughout the scientific literature, and this inconsistent use has caused confusion regarding the exact meaning of some terms of lipid formulations. Among several potential lipid formulations, liposomes, cationic liposomes, and lipid nanoparticles are specifically described in detail for the purposes of this disclosure and are defined herein.

[0110] Liposomes Conventional liposomes are vesicles consisting of at least one bilayer and an internal aqueous compartment. The bilayer membrane of liposomes is typically formed by amphiphilic molecules such as lipids of synthetic or natural origin that contain spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16:307-321, 1998). The bilayer membrane of liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). They generally exist as spherical vesicles and can range in size from 20 nm to several microns. Liposome formulations can be prepared as colloidal dispersions or lyophilized to reduce stability risks and improve the shelf life of liposomal drugs. Methods for preparing liposome compositions are known in the art and are within the skill of the art.

[0111] Liposomes with only one bilayer are called unilamellar, and liposomes with two or more bilayers are called multilamellar. The most common types of liposomes are small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), and multilamellar vesicles (MLVs). In contrast to liposomes, lysosomes, micelles, and reverse micelles are composed of a single layer of lipid. Generally, liposomes are considered to have a single internal compartment, but some formulations can be multivesicular liposomes (MVLs), which consist of multiple discontinuous internal aqueous compartments separated by several non-concentric lipid bilayers.

[0112] Liposomes have long been recognized as drug delivery vehicles due to their excellent biocompatibility, considering that liposomes are essentially analogues 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 that hydrophilic solutes dissolved in the core cannot easily pass through the bilayer, and hydrophobic compounds associate with the bilayer. Thus, liposomes can be loaded with hydrophobic and / or hydrophilic molecules. When liposomes are used to carry nucleic acids such as RNA, the nucleic acid is contained within the liposomal compartment in the aqueous phase.

[0113] 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 that are made entirely or partially from positively charged lipids, more specifically, lipids that contain both cationic groups and lipophilic moieties. In addition to the general characteristics described above for liposomes, the positively charged portion of the cationic lipids used in cationic liposomes offers some advantages and some unique structural features. For example, the lipophilic portion of cationic lipids is hydrophobic and therefore orients itself away from the aqueous interior of the liposome and associates with other non-polar and hydrophobic species. Conversely, the cationic portion associates with polar molecules and species that can be complexed with aqueous medium and, more importantly, with the aqueous interior of cationic liposomes. For these reasons, cationic liposomes are increasingly being investigated for use in gene therapy due to their preference for negatively charged nucleic acids via electrostatic interactions, resulting in complexes that offer biocompatibility, low toxicity, and the potential for large-scale production required for in vivo clinical use. Cationic lipids suitable for use in cationic liposomes are listed below.

[0114] Lipid Nanoparticles In contrast to liposomes and cationic liposomes, lipid nanoparticles (LNPs) have a structure that includes a single monolayer or bilayer of lipids that encapsulates a compound in the solid phase. Thus, unlike liposomes, lipid nanoparticles do not have an aqueous or other liquid phase in their interior, but rather, lipids from the bilayer or monolayer shell are directly complexed to the internal compound, thereby encapsulating it within the solid core. Lipid nanoparticles are typically spherical vesicles with a relatively uniform distribution of shapes and sizes. Sources differ as to the size that qualifies a lipid particle as a nanoparticle, but there is some overlapping agreement that lipid nanoparticles can have diameters ranging from 10 nm to 1000 nm. However, more commonly, they are considered to be smaller than 120 nm, or even smaller than 100 nm.

[0115] For lipid nanoparticle nucleic acid delivery systems, the lipid shell can be formulated to include ionic cationic lipids that can complex and associate with the negatively charged backbone of the nucleic acid core. Ionic cationic lipids with an apparent pKa value of less than about 7 have the advantage that they can complex with the negatively charged backbone of the nucleic acid, providing the cationic lipid for loading into the lipid nanoparticle at pH values ​​below the pKa of the positively charged ionized lipid. Then, at physiological pH values, the lipid nanoparticles can conform a relatively neutral exterior, allowing for a significant increase in the circulatory half-life of the particles after intravenous administration. In the context of nucleic acid delivery, lipid nanoparticles offer many advantages over other lipid-based nucleic acid delivery systems, including high nucleic acid encapsulation efficiency, strong transfection, improved tissue penetration for delivering therapeutic agents, and low levels of cytotoxicity and immunogenicity.

[0116] Prior to the development of lipid nanoparticle delivery systems for nucleic acids, cationic lipids have been widely investigated as synthetic materials for the delivery of nucleic acid drugs. In these early efforts, after mixing together at physiological pH, nucleic acids were condensed by cationic lipids to form lipid-nucleic acid complexes known as lipoplexes. However, lipoplexes proved to be unstable and were characterized by a wide size distribution ranging from the submicron scale to several microns. Lipoplexes such as Lipofectamine® reagent have found considerable utility for in vitro transfection. However, these first generation lipoplexes have not proven useful in vivo. The large particle size and positive charge (imparted by cationic lipids) result in rapid plasma clearance, hemolysis, and other toxicities, as well as immune system activation.

[0117] In some embodiments, the lipid nanoparticle comprises a lipid of formula I: [ka] or a pharma- ceutically acceptable salt or solvate thereof, wherein R 1 and R2 are each independently (CH 3 (CH 2 ) m ) 2 CH-, (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-1 )CH, (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-2 )CH, (CH 3 (CH 2 ) m ) 2 CHCH 2 - or (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-1 )CHCH 2 -, m is 4 to 11, and L 1 and L 2 are each independently absent or a linear C 1-5 Alkylene, or (CH 2 ) p -O-(CH 2 ) q p and q are each independently 1 to 3; R 3 is a linear C optionally substituted with one or two methyl groups 2-5 alkylene, R 4 and R 5 are each independently H or C 1-6 alkyl, X is O or S, and n is 0-2.

[0118] In some embodiments, any one or more of the lipids listed herein may be explicitly excluded.

[0119] In some embodiments, the present disclosure provides lipid nanoparticles comprising a plurality of ligands, each ligand being independently a compound described herein, and the plurality of ligands self-assemble to form a lipid nanoparticle comprising an interior and an exterior.

[0120] In some embodiments, the average size of the lipid nanoparticles is about 100 nm. In some embodiments, the average size of the lipid nanoparticles is less than about 100 nm. In some embodiments, the average particle size of the lipid nanoparticles is 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.

[0121] In some embodiments, the lipid nanoparticle further comprises a nucleic acid therein. In some embodiments, the nucleic acid is selected from siRNA, mRNA, self-replicating RNA, DNA plasmid, and antisense oligonucleotide. In some embodiments, the nucleic acid is an mRNA or a self-replicating RNA that includes a coding region that encodes a therapeutic protein of interest. In some embodiments, the therapeutic protein of interest is an enzyme, and an antibody, an antigen, a receptor, or 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.

[0122] In some embodiments, the lipid nanoparticle further comprises an siRNA or an mRNA therein.In some embodiments, the lipid nanoparticle further comprises an mRNA therein.

[0123] In some embodiments, the lipid nanoparticles further comprise a helper lipid, as described below. In some embodiments, the lipid nanoparticles further comprise a PEG-lipid conjugate, as described herein.

[0124] In some embodiments, the lipid nanoparticles comprise about 45 mol% to about 65 mol% of the compound of the present invention, about 2 mol% to about 15 mol% of the helper lipid, about 20 mol% to about 42 mol% of cholesterol, and about 0.5 mol% to about 3 mol% of the PEG-lipid conjugate. In some embodiments, the lipid nanoparticles comprise about 50 mol% to about 61 mol% of the compound of the present invention, 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. In some embodiments, the lipid nanoparticles comprise about 56 mol% to about 58 mol% of the compound of the present invention, 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.

[0125] In some embodiments, the lipid nanoparticles comprise about 50 mol% to about 61 mol% of the compound of the present invention, 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 present invention, 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 present invention, 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.

[0126] In some embodiments, the lipid nanoparticles have a total lipid:nucleic acid weight ratio of about 50:1 to about 10:1. In some embodiments, the lipid nanoparticles have a total lipid:nucleic acid weight ratio of about 40:1 to about 20:1. In some embodiments, the lipid nanoparticles have a total lipid:nucleic acid weight ratio of about 35:1 to about 25:1. In some embodiments, the lipid nanoparticles have a total lipid:nucleic acid weight ratio of about 32:1 to about 28:1. In some embodiments, the lipid nanoparticles have a total lipid:nucleic acid weight ratio of about 31:1 to about 29:1.

[0127] 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.

[0128] In some embodiments, the lipid nanoparticles comprise a HEPES buffer at a pH of about 7.4. In some embodiments, the HEPES buffer is at a concentration of about 7 mg / mL to about 15 mg / mL. In some embodiments, the lipid nanoparticles further comprise NaCl at about 2.0 mg / mL to about 4.0 mg / mL.

[0129] In some embodiments, the lipid nanoparticles further comprise one or more cryoprotectants. In some embodiments, the one or more cryoprotectants are selected from sucrose, glycerol, or a combination of sucrose and glycerol. In some embodiments, the lipid nanoparticles comprise a combination of sucrose at a concentration of about 70 mg / mL to about 110 mg / mL and glycerol at a concentration of about 50 mg / mL to about 70 mg / mL.

[0130] Lipid-nucleic acid formulations The nucleic acid or a pharma- ceutically acceptable salt thereof can be incorporated into a lipid formulation (ie, a lipid-based delivery vehicle).

[0131] In the context of the present disclosure, the lipid-based delivery vehicle typically functions to transport a desired nucleic acid (such as siRNA, plasmid DNA, mRNA, self-replicating RNA, etc.) to a target cell or tissue. The lipid-based delivery vehicle can be any suitable lipid-based delivery vehicle known in the art. In some embodiments, the lipid-based delivery vehicle is a liposome, a cationic liposome, or a lipid nanoparticle containing a nucleic acid. In some embodiments, the lipid-based delivery vehicle comprises a nanoparticle or bilayer of lipid molecules and nucleic acid. In some embodiments, the lipid bilayer preferably further comprises a neutral lipid or polymer. In some embodiments, the lipid formulation preferably comprises a liquid medium. In some embodiments, the formulation preferably further encapsulates a nucleic acid. In some embodiments, the lipid formulation preferably further comprises a nucleic acid and a neutral lipid or polymer. In some embodiments, the lipid formulation preferably encapsulates a nucleic acid.

[0132] The present disclosure provides lipid formulations comprising one or more therapeutic nucleic acid molecules encapsulated within the 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.

[0133] In some embodiments, the nucleic acid is fully encapsulated within the lipid portion of the lipid formulation, such that the nucleic acid in the lipid formulation is resistant in aqueous solution to nuclease degradation. In other embodiments, the lipid formulations described herein are substantially non-toxic to mammals, such as humans.

[0134] The lipid formulations of the present disclosure also typically have a total lipid:nucleic acid ratio (mass / mass ratio) 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 subvalue within the recited range, including the endpoints.

[0135] The lipid formulations of the present disclosure typically have a diameter of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, about 35 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 70 nm, ...80 nm, about 80 nm, about 80 nm, about 80 nm, about 80 nm, about 80 nm, about 80 nm, about 80 nm, about 80 nm, about 80 nm, The lipid nanoparticles have an average diameter of about 0 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, or about 150 nm, and are substantially non-toxic. The diameter may be any value or subvalue within the recited range, including the end points. In addition, the nucleic acid is resistant in aqueous solution to degradation by nucleases when present in the lipid nanoparticles of the present disclosure.

[0136] In a preferred embodiment, the lipid formulation comprises a nucleic acid, a cationic lipid (e.g., one or more cationic lipids or salts thereof described herein), a phospholipid, and a conjugated lipid that inhibits particle aggregation (e.g., one or more PEG-lipid conjugates and / or other lipid conjugates of the present disclosure).The lipid formulation can also comprise cholesterol.

[0137] In some embodiments, the lipid nanoparticle further comprises a PEG-lipid conjugate. In some embodiments, the PEG-lipid conjugate is PEG-DMG. In some embodiments, the PEG-DMG is PEG2000-DMG.

[0138] In nucleic acid lipid formulations, nucleic acid may be completely encapsulated in the lipid portion of the formulation, thereby protecting nucleic acid from nuclease degradation.In a preferred embodiment, the lipid formulation containing nucleic acid is completely encapsulated in the lipid portion of the lipid formulation, thereby protecting nucleic acid from nuclease degradation.In certain cases, the nucleic acid in the lipid formulation is not substantially degraded after the particle is 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 is not substantially degraded after the formulation is incubated in serum at 37°C for at least 30, 45, or 60 minutes, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours.In other embodiments, nucleic acid is complexed with the lipid portion of the formulation.

[0139] In the context of nucleic acids, complete encapsulation can be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye whose fluorescence is enhanced when associated with nucleic acid. Encapsulation is determined by adding the dye to the lipid formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of non-ionic detergent. Detergent-mediated disruption of the lipid layer releases the encapsulated nucleic acid, allowing it to interact with the membrane-impermeable dye. Nucleic acid encapsulation may be calculated as E=(I0-I) / I0, where I and I0 refer to the fluorescence intensity before and after the addition of detergent.

[0140] In other embodiments, the present disclosure provides a nucleic acid-lipid composition 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.

[0141] In some embodiments, the lipid formulation comprises nucleic acid fully encapsulated within the lipid portion of the formulation, such that the nucleic acid is encapsulated in about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 30% to about 95%, about 40% to about 95%, about 50% to about 95%, about 60% to about 95%, about 70% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 30% to about 10 ... About 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, or at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% (or any fraction or range therein) have nucleic acid encapsulated therein. The amount may be any value or subvalue within the recited range, including the endpoints.

[0142] Depending on the intended use of the lipid formulation, the ratio of the components can be varied and the delivery efficiency of a particular formulation can be measured using assays known in the art.

[0143] According to some embodiments, the expressible polynucleotides, nucleic acid active agents, and mRNA constructs can be lipid-formulated. The lipid formulation is preferably selected from, but not limited to, liposomes, cationic liposomes, and lipid nanoparticles. In a preferred embodiment, the lipid formulation is a cationic liposome or a lipid nanoparticle (LNP); (a) a nucleic acid (e.g., mRNA, siRNA, etc.); (b) a lipid of the present disclosure, which may be cationic; and (c) optionally a non-cationic lipid (such as a neutral lipid); and (d) optionally, a sterol.

[0144] Cationic lipids The lipid formulation preferably comprises cationic lipid suitable for forming cationic liposome or lipid nanoparticle. Cationic lipids have been widely studied for nucleic acid delivery because they can bind to negatively charged membranes and induce uptake. In general, cationic lipids are amphiphilic substances that contain a positive hydrophilic head group, two (or more) lipophilic tails or steroid moieties, and a connector between these two domains. Preferably, the cationic lipid carries a net positive charge at approximately physiological pH. Cationic liposomes have traditionally been the most commonly used non-viral delivery system for oligonucleotides, including plasmid DNA, antisense oligos, and siRNA / small hairpin RNA-shRNA. Cationic lipids such as DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and DOTMA (N-[1-(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.

[0145] In the lipid formulations of the present disclosure, cationic lipids include, for example, N,N-dioleyl-N,N-di-9-cis-octadecenyl ammonium chloride (DODAC), N,N-distearyl-N,N-dimethyl ammonium bromide (DDAB), 1,2-dioleoyltrimethylammonium propane chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and 1,2-dioleyloxy-3-trimethylaminopropane chloride salt), N-(1-(2,3- Dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-y-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP ), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl) ), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or its analogs, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecane-2-ol (C12-200 ), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1-amine (MC3 ether), 4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutan-1-amine (MC4 ether), or any combination thereof. Other cationic lipids include N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 3P-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamidoglycylcarboxyspermine (DOGS), 1,2-dioleyl-sn-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), and 2,2-dilinoleyl-4-dimethylaminoethyl-[1,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.

[0146] Other suitable cationic lipids are described in WO 09 / 086558, WO 09 / 127060, WO 10 / 048536, WO 10 / 054406, WO 10 / 088537, WO 10 / 129709, and WO 2011 / 153493, U.S. Patent Publication Nos. 2011 / 0256175, 2012 / 0128760, and 2012 / 0027803, U.S. Patent No. 8,158,601, and Love et al., PNAS, 107(5), 1864-69, 2010, the contents of which are incorporated herein by reference.

[0147] Other suitable cationic lipids include those with alternative fatty acid groups and other dialkylamino groups, including those with different alkyl substituents (e.g., N-ethyl-N-methylamino- and N-propyl-N-ethylamino-). These lipids are part of a subcategory of cationic lipids called amino lipids. In some embodiments of the lipid formulations described herein, the cationic lipid is an amino lipid. In general, amino lipids with less saturated alkyl chains are easier to size for filter sterilization purposes, especially when the complex must be sized to less than about 0.3 microns. 14 ~C 22 Amino lipids containing unsaturated fatty acids having carbon chain lengths in the range of 0 to 100 may be used. Other scaffolds may be used to separate the amino group and the fatty acid or fatty alkyl portion of the amino lipid.

[0148] In some embodiments, the cationic lipids of the present disclosure are ionizable and have at least one protonatable or deprotonatable group such that the lipid is positively charged at a pH below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably above physiological pH. Of course, the addition or removal of protons as a function of pH is an equilibrium process, and reference to a charged or neutral lipid refers to the nature of the predominant species, and not all of the lipids need be present in a charged or neutral form. Lipids that have more than one protonatable or deprotonatable group or are zwitterionic are not excluded from use in the present disclosure. In certain embodiments, the protonatable lipid has a pKa of the protonatable group ranging from about 4 to about 11. In some embodiments, the ionic cationic lipid has a pKa of about 5 to about 7. In some embodiments, the pKa of the ionic cationic lipid is about 6 to about 7.

[0149] In some embodiments, the lipid formulation comprises a lipid of formula I: [ka] or a pharma- ceutically acceptable salt or solvate thereof, wherein R 1 and R 2 are each independently (CH 3 (CH 2 ) m ) 2 CH-, (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-1 )CH, (CH 3 (CH 2 ) m )(CH 3 (CH 2 ) m-2 )CH, (CH 3 (CH 2 ) m ) 2 CHCH 2 - or (CH 3 (CH 2 ) m)(CH 3 (CH 2 ) m-1 )CHCH 2 -, m is 4 to 11, and L 1 and L 2 are each independently absent or a linear C 1-5 Alkylene, or (CH 2 ) p -O-(CH 2 ) q p and q are each independently 1 to 3; R 3 is a linear C optionally substituted with one or two methyl groups 2-5 alkylene, R 4 and R 5 are each independently H or C 1-6 alkyl, X is O or S, and n is 0-2.

[0150] In some embodiments, any one or more of the lipids listed herein may be explicitly excluded.

[0151] Helper lipids and sterols The mRNA lipid formulations of the present disclosure can include helper lipids, which can be referred to as neutral lipids, neutral helper lipids, non-cationic lipids, non-cationic helper lipids, anionic lipids, anionic helper lipids, or zwitterionic lipids. Lipid formulations, particularly cationic liposomes and lipid nanoparticles, have been found to have increased cellular uptake when helper lipids are present in the formulation. (Curr. Drug Metab. 2014; 15(9): 882-92). For example, some studies have shown that neutral and zwitterionic lipids, such as 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), di-oleoyl-phosphatidyl-ethanoalamine (DOPE) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), are more fusogenic (i.e., facilitate fusion) than cationic lipids, and may affect the polymorphic characteristics of lipid-nucleic acid complexes, promoting the transition from lamellar to hexagonal phases, thus inducing fusion and disruption of cell membranes. (Nanomedicine (Lond). 2014 Jan; 9 (1): 105-20). In addition, the use of helper lipids may help reduce any potential adverse effects of using many common cationic lipids, such as toxicity and immunogenicity.

[0152] Non-limiting examples of non-cationic lipids suitable for the lipid formulations of the present disclosure include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylcholine ...DPPC), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phosphatidylethanolamine (DPPG), palmitoyloleoyl-phosphatidylcholine (DPPG), palmitoyloleoyl-phosphatidylethanolamine (DPPG), palmitoyloleoyl-phosphatidylcholine ( 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, dielaidoyl-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 C10-C24 carbon chains, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0153] 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).

[0154] Additional examples of non-cationic lipids include sterols such as cholesterol and its derivatives. One study concluded that as a helper lipid, cholesterol increases the charge spacing of the lipid layer that matches the nucleic acid, making the charge distribution more closely match that of the nucleic acid. (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-cholestane, cholestenone, 5α-cholestanone, 5α-cholestanone, and cholesteryl decanoate, and mixtures thereof. In a preferred embodiment, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether.

[0155] In some embodiments, the helper lipid present in the lipid formulation comprises or consists of a mixture of one or more phospholipids and cholesterol or its derivatives.In other embodiments, the helper lipid present in the lipid formulation comprises or consists of one or more phospholipids, for example, cholesterol-free lipid formulations.In yet other embodiments, the helper lipid present in the lipid formulation comprises or consists of cholesterol or its derivatives, for example, phospholipid-free lipid formulations.In some embodiments, the lipid nanoparticle further comprises cholesterol.

[0156] Other examples of helper lipids include non-phosphorus containing lipids such as, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, and sphingomyelin.

[0157] In some embodiments, the helper lipid comprises about 1 mol% to about 50 mol%, about 5 mol% to about 48 mol%, about 5 mol% to about 46 mol%, about 25 mol% to about 44 mol%, about 26 mol% to about 42 mol%, about 27 mol% to about 41 mol%, about 28 mol% to about 40 mol%, or about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, or about 39 mol% (or any fraction or range therein) of the total lipid present in the lipid formulation. In some embodiments, the helper lipid comprises about 1 mol% to about 20 mol%, about 2 mol% to about 12 mol%, about 5 mol% to about 9 mol%, or about 6 mol% to about 8 mol%.

[0158] In some embodiments, the total amount of helper lipids in the formulation includes two or more helper lipids, and the total amount of helper lipids comprises about 20 mol% to about 50 mol%, about 22 mol% to about 48 mol%, about 24 mol% to about 46 mol%, about 25 mol% to about 44 mol%, about 26 mol% to about 42 mol%, about 27 mol% to about 41 mol%, about 28 mol% to about 40 mol%, or about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, or about 39 mol% (or any fraction or range therein) of the total lipids present in the lipid formulation. In some embodiments, the helper lipid is a combination of DSPC and DOTAP. In some embodiments, the helper lipid is a combination of DSPC and DOTMA.

[0159] The cholesterol or cholesterol derivative in the lipid formulation may comprise up to about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, or about 60 mol% of the total lipid present in the lipid formulation. In some embodiments, the cholesterol or cholesterol derivative comprises about 15 mol% to about 45 mol%, about 20 mol% to about 40 mol%, about 30 mol% to about 40 mol%, or about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, or about 40 mol% of the total lipid present in the lipid formulation.

[0160] The percentage of helper lipid present in the lipid formulation is a target amount, and the actual amount of helper lipid present in the formulation may vary, for example, by ±5 mole %.

[0161] Mechanisms of action for cellular uptake of lipid formulations Lipid formulations for intracellular delivery of nucleic acids, particularly liposomes, cationic liposomes, and lipid nanoparticles, are designed for cellular uptake by penetrating the target cell by utilizing the endocytosis mechanism of the target cell, where the contents of the lipid delivery vehicle are delivered to the cytosol of the target cell. (Nucleic Acid Therapeutics, 28(3):146-157, 2018). Specifically, in the case of the 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 the present disclosure, on the surface of the lipid delivery vehicle can be shed from the surface, which triggers internalization into the target cell. During endocytosis, a portion of the cell's plasma membrane surrounds the vector and engulfs it into a vesicle, which then pinches the vesicle out of the cell membrane, enters the cytosol, and finally goes through the endolysosomal pathway. For delivery vehicles containing ionic cationic lipids, the increased acidity as endosomes age results in vehicles with strong positive charges on the surface.The interaction between the delivery vehicle and the endosomal membrane then results in a membrane fusion event that leads 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 the encoded protein.The encoded protein can further undergo post-translational processing, including transport to target organelles or intracellular location.

[0162] By controlling the composition and concentration of lipid conjugate, the rate at which lipid conjugate exchanges from lipid formulation and then the rate at which lipid formulation becomes fusogenic can be controlled.In addition, other variables, including, for example, pH, temperature, or ionic strength, can be used to change and / or control the rate at which lipid formulation becomes fusogenic.Other methods that can be used to control the rate at which lipid formulation becomes fusogenic will be clear to those skilled in the art upon reading this disclosure.Also, by controlling the composition and concentration of lipid conjugate, the size of liposome or lipid particle can be controlled.

[0163] Lipid formulation manufacturing There are many different methods for the preparation of lipid formulations containing nucleic acids. (Curr. Drug Metabol. 2014, 15, 882-892; Chem. Phys. Lipids 2014, 177, 8-18; Int. J. Pharm. Stud. Res. 2012, 3, 14-20). The techniques of thin film hydration, double emulsion, reverse phase evaporation, microfluidic preparation, double asymmetric centrifugation, ethanol injection, detergent dialysis, spontaneous vesicle formation by ethanol dilution, and encapsulation in preformed liposomes are briefly described herein.

[0164] Thin Film Hydration In the thin film hydration method (TFH) or Bangham method, lipids are dissolved in an organic solvent and 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 loaded, multilamellar vesicles (MLVs) are formed, which can be reduced in size by extrusion through a membrane or by sonication of the starting MLVs to produce small unilamellar vesicles (LUVs) or large unilamellar vesicles (SUVs).

[0165] Double Emulsion Lipid formulations can also be prepared through a double emulsion technique, which involves dissolving lipids in a water / organic solvent mixture. An organic solution containing water droplets is mixed with an excess of aqueous medium, resulting in the formation of a water-in-oil-in-water (W / O / W) double emulsion. After vigorous mechanical shaking, some of the water droplets collapse, resulting in large unilamellar vesicles (LUVs).

[0166] Reverse Phase Evaporation Reverse phase evaporation (REV) method can also achieve nucleic acid loaded LUVs. In this technique, a two-phase system is formed by dissolving phospholipids in an organic solvent and an aqueous buffer. The resulting suspension is then sonicated for a short period until the mixture becomes a clear one-phase dispersion. Lipid formulation is 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.

[0167] Microfluidic preparation Microfluidic methods, unlike other bulk techniques, give the possibility to control the lipid hydration process. Methods can be classified into continuous-flow microfluidics and droplet-based microfluidics according to the way the flow is manipulated. In the microhydrodynamic focusing (MHF) method, operating in continuous-flow mode, lipids are dissolved in isopropyl alcohol, which is hydrodynamically focused in a microchannel cross-junction between two aqueous buffer streams. The vesicle size can be controlled by adjusting the flow rate, and thus the lipid solution / buffer dilution process. The method can be used to generate oligonucleotide (ON) lipid formulations by using a microfluidic device consisting of three inlet ports and one outlet port.

[0168] Double asymmetric centrifugation 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; that is, the sample is pushed outward as in a normal centrifuge, and then pushed toward the center of the vial by an additional rotation. By mixing the lipid and NaCl solutions, 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.

[0169] Ethanol injection The ethanol injection (EI) method can be used for nucleic acid encapsulation. This method provides for the rapid injection of an ethanol solution in which lipids are dissolved into an aqueous medium containing the nucleic acid to be encapsulated through the use of a needle. When the phospholipids are dispersed throughout the medium, vesicles spontaneously form.

[0170] Detergent dialysis Nucleic acid can be encapsulated using detergent dialysis. Briefly, lipid and plasmid are solubilized in detergent solution of appropriate ionic strength, and after removing detergent by dialysis, stabilized lipid formulation is formed. Unencapsulated nucleic acid is then removed by ion exchange chromatography, and vesicles are emptied by sucrose density gradient centrifugation. This technique is very sensitive to cationic lipid content and salt concentration of dialysis buffer, and this method is also difficult to scale up.

[0171] Spontaneous vesicle formation upon ethanol dilution Stable lipid formulations can also be generated via the spontaneous vesicle formation by ethanol dilution method, in which stepwise or dropwise ethanol dilution provides for the spontaneous formation of nucleic acid-loaded vesicles by controlled addition of lipids dissolved in ethanol to a rapidly mixing aqueous buffer containing the nucleic acid.

[0172] V. Pharmaceutical Compositions and Delivery Methods To promote in vivo nucleic acid activity (e.g., mRNA expression, or knockdown by ASO or siRNA), the lipid formulation delivery vehicle described herein can be combined with one or more additional nucleic acids, carriers, targeting ligands, or stabilizing reagents, or can be in a pharmacological composition in which it is mixed with a suitable excipient. Techniques for drug formulation and administration can be found in "Remington's Pharmaceutical Sciences" (Mack Publishing Co., Easton, Pa, latest edition).

[0173] The lipid formulations and pharmaceutical compositions of the present disclosure may be administered and dosed according to current medical practice, taking into account the subject's clinical condition, the administration site and method, the administration schedule, the subject's age, sex, weight, and other factors relevant to those skilled in the art. For purposes herein, the "effective amount" may be determined by experimental clinical studies, pharmacological, clinical, and relevant considerations as known to those skilled in the art of medical technology. In some embodiments, the amount administered is effective to achieve at least some stabilization, improvement, or elimination of symptoms and other indicators selected by those skilled in the art as an appropriate measure of disease progression, regression, or improvement. For example, an appropriate amount and administration regimen is one that causes at least transient production of a protein (e.g., an enzyme).

[0174] The pharmaceutical compositions described herein can be inhalable compositions. Suitable routes of administration include, for example, intratracheal, inhalation, or intranasal. In some embodiments, administration results in delivery of the nucleic acid to lung epithelial cells. In some embodiments, administration shows selectivity for lung epithelial cells over other types of lung cells and airway cells.

[0175] The pharmaceutical compositions disclosed herein can be formulated using one or more excipients to (1) increase stability, (2) increase cell transfection, (3) allow for sustained or delayed release (e.g., from a depot formulation of the nucleic acid), (4) alter biodistribution (e.g., to target the nucleic acid to a particular tissue or cell type), (5) increase the activity of the nucleic acid or protein expressed therefrom in vivo, and / or (6) alter the release profile of the nucleic acid or encoded protein in vivo.

[0176] Preferably, lipid formulations can be administered in a localized manner rather than systemically.Local delivery can affect in various ways depending on the tissue targeted.For example, aerosols containing the compositions of the present disclosure can be inhaled (for nasal, tracheal or bronchial delivery).

[0177] Pharmaceutical compositions can be administered to any desired tissue. In some embodiments, the nucleic acid delivered by lipid formulations or compositions of the present disclosure is active in the tissue to which lipid formulations and / or compositions are administered. In some embodiments, the nucleic acid is active in a tissue different from the tissue to which lipid formulations and / or compositions are administered. Examples of tissues to which nucleic acid can be delivered include, but are not limited to, lung, trachea, and / or nasal cavity, muscle, liver, eye, or central nervous system.

[0178] The pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparation methods include the step of bringing into association the active ingredient (i.e., nucleic acid) with an excipient and / or one or more other accessory ingredients. Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses.

[0179] Pharmaceutical compositions may additionally contain pharma- ceutically acceptable excipients, which as used herein include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surface active agents, isotonicity agents, thickening or emulsifying agents, preservatives, and the like, appropriate for the particular dosage form desired.

[0180] In addition to conventional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surface active agents, isotonicity agents, thickening or emulsifying agents, preservatives, excipients of the present disclosure can include, but are not limited to, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with primary DNA constructs, or mRNA (e.g., for implantation into a subject), hyaluronidase, nanoparticle mimics, and combinations thereof.

[0181] Thus, the formulations described herein can include one or more excipients in an amount that together increase the stability of the nucleic acid in the lipid formulation, increase cell transfection with the nucleic acid (e.g., mRNA or siRNA), increase expression of the encoded protein, and / or alter the release profile of the encoded protein, or increase knockdown of the target native nucleic acid. Additionally, the nucleic acid may be formulated using self-assembling nucleic acid nanoparticles.

[0182] Various excipients for formulating pharmaceutical compositions and techniques for preparing the compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, AR Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006, which is incorporated herein by reference in its entirety). The use of conventional excipient vehicles may be contemplated within the scope of the embodiments of the present disclosure, except insofar as any conventional excipient vehicle may be incompatible with the substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition.

[0183] The dosage form of the composition of the present disclosure can be a solid that can be reconstituted in liquid before administration.The solid can be administered as a powder.In some embodiments, the pharmaceutical composition comprises a lyophilized nucleic acid-lipid formulation.

[0184] In preferred embodiments, the dosage form of the pharmaceutical compositions described herein can be a liquid suspension of the nucleic acid-lipid nanoparticles described herein. In some embodiments, the liquid suspension is in a buffer solution. In some embodiments, the buffer solution comprises a buffer selected from the group consisting of HEPES, MOPS, TES, and TRIS. In some embodiments, the buffer has a pH of about 7.4. In some preferred embodiments, the buffer is HEPES. In some further embodiments, the buffer solution further comprises a cryoprotectant. In some embodiments, the cryoprotectant is selected from a combination of a sugar and glycerol, or a sugar and glycerol. In some embodiments, the sugar is a dimeric sugar. In some embodiments, the sugar is sucrose. In some preferred embodiments, the buffer comprises HEPES, sucrose, and glycerol at a pH of 7.4. In some embodiments, the suspension is frozen during storage and thawed prior to administration. In some embodiments, the suspension is frozen at a temperature less than about -70°C. In some embodiments, the suspension is diluted with sterile water prior to inhalable administration. In some embodiments, inhalable administration comprises diluting the suspension with about 1 volume to about 4 volumes of sterile water. In some embodiments, the lyophilized nucleic acid-lipid nanoparticle formulation can be resuspended in a buffer solution described herein.

[0185] The compositions and methods of the present disclosure may be administered to a subject by various mucosal administration modes, including intranasal and / or intrapulmonary. In some aspects of the present disclosure, the mucosal tissue layer comprises an epithelial cell layer. The epithelial cells can be pulmonary, tracheal, bronchial, alveolar, nasal, and / or oral. The compositions of the present disclosure can be administered using conventional actuators, such as mechanical spray devices, as well as pressurized, electrically actuated, or other types of actuators.

[0186] The compositions of the present disclosure may be administered in aqueous solution as a nasal or pulmonary spray, or may be dispensed in spray form by various methods known to those skilled in the art. Pulmonary delivery of the compositions of the present disclosure is achieved by administering the compositions in the form of droplets, particles, or sprays, which can be, for example, aerosolized, atomized, or nebulized. The particles of the composition, spray, or aerosol can be either liquid or solid form, for example, lyophilized lipid formulations. A preferred system for dispensing liquids as nasal sprays is disclosed in U.S. Pat. No. 4,511,069. Such formulations can be conveniently prepared by dissolving the compositions according to the present disclosure in water to produce an aqueous solution, and sterilizing the solution. The formulations can be presented in a multi-drug container, for example, in a closed dispensing system disclosed in U.S. Pat. No. 4,511,069. Other suitable nasal spray delivery systems are described in TRANSDERMAL SYSTEMIC MEDICATION, YW Chien ed., Elsevier Publishers, New York, 1985, and U.S. Pat. No. 4,778,810. Additional aerosol delivery forms may include, for example, compressed air, jet, ultrasonic, and piezoelectric nebulizers, which deliver the nucleic acid-lipid formulation or are suspended in a pharmaceutical solvent, such as water, ethanol, or mixtures thereof.

[0187] The nasal and pulmonary spray solutions of the present disclosure typically contain a surface active agent, such as a non-ionic surfactant (e.g., polysorbate-80), and the nucleic acid optionally formulated with one or more buffers, provided that the inclusion of the surfactant does not disrupt the structure of the lipid formulation. In some embodiments of the present disclosure, the nasal spray solution further comprises a propellant. The pH of the nasal spray solution may be pH 6.8 to 7.2. The pharmaceutical solvent used may also be a slightly acidic aqueous buffer with a pH of 4 to 6. Other ingredients, including preservatives, surfactants, dispersants, or gases, may be added to enhance or maintain chemical stability.

[0188] In some embodiments, the present disclosure provides a pharmaceutical product comprising a solution containing a composition of the present disclosure and an actuator for a pulmonary, mucosal, or intranasal spray or aerosol.

[0189] The dosage form of the composition of the present disclosure can be in the form of a liquid, droplets or emulsion, or in the form of an aerosol.

[0190] The dosage form of the composition of the present disclosure can be a solid that can be reconstituted in a liquid prior to administration. The solid can be administered as a powder. The solid can be in the form of a capsule, tablet, or gel.

[0191] To formulate a composition for pulmonary delivery within the present disclosure, the nucleic acid-lipid formulation can be combined with various pharma- ceutically acceptable additives, as well as bases or carriers for dispersing the nucleic acid-lipid formulation. Examples of additives include pH adjusters, such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and mixtures thereof. Other additives include local anesthetics (e.g., benzyl alcohol), isotonicity agents (e.g., sodium chloride, mannitol, sorbitol), adsorption inhibitors (e.g., Tween 80), solubility enhancers (e.g., cyclodextrin and its derivatives), stabilizers (e.g., serum albumin), and reducing agents (e.g., glutathione). When the composition for mucosal delivery is liquid, the tonicity of the formulation, measured with reference to the tonicity of 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 site of administration. Generally, the tonicity of the solution is adjusted to a value of 1 / 3 to 3, more typically 1 / 2 to 2, and most often 3 / 4 to 1.7.

[0192] The nucleic acid-lipid formulation may be dispersed in a base or vehicle, which may include a hydrophilic compound capable of dispersing the nucleic acid-lipid formulation and any desired additives. The base may be selected from a wide range of suitable carriers, including, but not limited to, copolymers of polycarboxylic acids or their salts, carboxylic acid anhydrides (e.g., maleic anhydride) with other monomers (e.g., methyl (meth)acrylate, acrylic acid, etc.), hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives such as hydroxymethylcellulose, hydroxypropylcellulose, and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and their non-toxic metal salts. Biodegradable polymers are often selected as bases or carriers, such as, for example, polylactic acid, poly(lactic acid-glycolic acid) copolymers, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid) copolymers, and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters, etc. can be used as carriers. Hydrophilic polymers and other carriers can be used alone or in combination, and improved structural integrity can be imparted to the carrier by partial crystallization, ionic bonding, crosslinking, etc. The carrier can be provided in various forms, including fluid or viscous solutions, gels, pastes, powders, microspheres, and films for direct application to the nasal mucosa. The use of selected carriers in this context can result in enhanced absorption of nucleic acid-lipid formulations.

[0193] Alternatively, the compositions of the present disclosure may contain pharma- ceutically acceptable carrier materials required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, and wetting agents, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and mixtures thereof. For solid compositions, conventional non-toxic pharma-ceutically acceptable carriers can be used, including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like.

[0194] In certain embodiments of the present disclosure, nucleic acid-lipid formulations may be administered in time-release formulations, for example, compositions that contain slow-release polymers.Nucleic acid-lipid formulations can be prepared with carriers that protect against rapid release, such as controlled release vehicles, for example, polymers, microencapsulated delivery systems, or bioadhesive gels.Long-term delivery of nucleic acid-lipid formulations in various compositions of the present disclosure can be achieved by including them in compositions that delay absorption, for example, aluminum monostearate hydrogels and gelatin.

[0195] Nucleic acids can be delivered to the lungs by intratracheal administration of a liquid suspension of the nucleic acid composition and inhalation of an aerosol mist produced by a liquid nebulizer, or by use of dry powder equipment such as that described in U.S. Pat. No. 5,780,014, incorporated herein by reference.

[0196] In certain embodiments, the compositions of the present disclosure may be formulated such that they may be aerosolized or otherwise delivered as particulate liquids or solids prior to or upon administration to a subject. Such compositions may be administered with the aid of one or more suitable devices for administering such solid or liquid particulate compositions (e.g., aerosolized aqueous solutions or suspensions, etc.) to generate particles that are easily respirable or inhalable by a subject. In some embodiments, such devices (e.g., metered dose inhalers, jet nebulizers, ultrasonic nebulizers, dry powder inhalers, propellant-based inhalers, or pneumoperitoneum devices) facilitate administration of a predetermined mass, volume, or dose (e.g., about 0.010 to about 0.5 mg / kg of nucleic acid per dose) of the composition to a subject. For example, in certain embodiments, the compositions of the present disclosure are administered to a subject using a metered dose inhaler that contains a suspension or solution comprising the composition and a suitable propellant. In certain embodiments, the compositions of the present disclosure may be formulated as particulate powders (e.g., respirable dry particles) intended for inhalation. In certain embodiments, compositions of the present disclosure formulated as respirable particles are appropriately sized (e.g., average D50 or D90 particle size of about 500 μm, 400 μm, 300 μm, 250 μm, 200 μm, 150 μm, 100 μm, 75 μm, 50 μm, 25 μm, 20 μm, 15 μm, 12.5 μm, 10 μm, 5 μm, 2.5 μm or less) so that they may be respirable by a subject or delivered using a suitable device. In yet other embodiments, compositions of the present disclosure are formulated to include one or more pulmonary surfactants (e.g., lamellar bodies).In some embodiments, the compositions of the present disclosure provide a bolus of at least 0.010 mg / kg, at least 0.015 mg / kg, at least 0.020 mg / kg, at least 0.025 mg / kg, at least 0.030 mg / kg, at least 0.035 mg / kg, at least 0.040 mg / kg, at least 0.045 mg / kg, at least 0.05 mg / kg, at least 0.1 mg / kg, at least 0.5 mg / kg, at least 1.0 mg / kg, at least 2.0 mg / kg, at least 3.0 mg / kg, at least 4.0 mg / kg, at least 5.0 mg / kg, at least 6.0 mg / kg, at least 7.0 mg / kg, at least 8.0 mg / kg, at least 9.0 mg / kg, at least 10.0 mg / kg, at least 11.0 mg / kg, at least 12.0 mg / kg, at least 13.0 mg / kg, at least 14.0 mg / kg, at least 15.0 mg / kg, at least 16.0 mg / kg, at least 17.0 mg / kg, at least 18.0 mg / kg, at least 19.0 mg / kg, at least 20.0 mg / kg, at least 21.0 mg / kg, at least 22.0 mg / kg, at least 23.0 mg / kg, at least 24.0 mg / kg, at least 25.0 mg / kg, at least 26.0 mg / kg, at least 27.0 mg / kg, at least 28.0 mg / kg, at least 29.0 mg / kg, at least 30.0 mg / kg, at least 31.0 mg / kg, at least 32.0 mg / kg, at least 33.0 mg / kg, at least 34.0 mg / kg, at least The subject is administered a concentration of at least 8.0 mg / kg, at least 9.0 mg / kg, at least 10 mg / kg, at least 15 mg / kg, at least 20 mg / kg, at least 25 mg / kg, at least 30 mg / kg, at least 35 mg / kg, at least 40 mg / kg, at least 45 mg / kg, at least 50 mg / kg, at least 55 mg / kg, at least 60 mg / kg, at least 65 mg / kg, at least 70 mg / kg, at least 75 mg / kg, at least 80 mg / kg, at least 85 mg / kg, at least 90 mg / kg, at least 95 mg / kg, or at least 100 mg administered in a single dose. In some embodiments, compositions of the present disclosure are administered to a subject such that a total amount of at least 0.1 mg, at least 0.5 mg, at least 1.0 mg, at least 2.0 mg, at least 3.0 mg, at least 4.0 mg, at least 5.0 mg, at least 6.0 mg, at least 7.0 mg, at least 8.0 mg, at least 9.0 mg, at least 10 mg, at least 15 mg, at least 20 mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 40 mg, at least 45 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 90 mg, at least 95 mg, or at least 100 mg of nucleic acid is administered in one or more doses.

[0197] In some embodiments, the pharmaceutical composition of the present disclosure is administered to a subject once a month. In some embodiments, the pharmaceutical composition of the present disclosure is administered to a subject twice a month. In some embodiments, the pharmaceutical composition of the present disclosure is administered to a subject three times a month. In some embodiments, the pharmaceutical composition of the present disclosure is administered to a subject four times a month.

[0198] According to the present disclosure, a therapeutically effective dose of the provided compositions, when administered periodically, results in an increase in nucleic acid activity levels in a subject, compared to baseline activity levels before treatment. Typically, activity levels are measured in 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 in nucleic acid activity levels in a biological sample (e.g., plasma / serum or lung epithelial swab) of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to baseline levels before treatment. In some embodiments, administration of provided compositions results in an increase in nucleic acid activity levels in a biological sample (e.g., plasma / serum or lung epithelial swab) of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to pre-treatment baseline levels for at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, or at least about 15 days.

[0199] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a compound described herein or a lipid nanoparticle described herein and a pharma- ceutically acceptable excipient.

[0200] In some embodiments, the present disclosure provides a method of delivering a nucleic acid to a subject in need thereof, the method comprising encapsulating a therapeutically effective amount of a nucleic acid in a lipid nanoparticle described herein and administering the lipid nanoparticle to a subject.

[0201] In some embodiments, the present disclosure provides a method of delivering mRNA to a subject in need thereof, the method comprising encapsulating a therapeutically effective amount of mRNA in a lipid nanoparticle described herein and administering the lipid nanoparticle to the subject.

[0202] VI. Treatment method In some embodiments, the disclosure provides a method of treating a disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound described herein, a lipid nanoparticle described herein, or a pharmaceutical composition described herein. In some embodiments, the compound or lipid nanoparticle is administered intravenously or intramuscularly. In some embodiments, the compound or lipid nanoparticle is administered intravenously. In some embodiments, the compound or lipid nanoparticle is administered intramuscularly.

[0203] In some embodiments, a method of treating a disease in a subject in need thereof is provided, the method comprising administering to the subject a lipid composition described herein. In some embodiments, the lipid composition is administered intravenously or intramuscularly. In some embodiments, the lipid composition is administered intravenously. In some embodiments, the lipid composition is administered intramuscularly.

[0204] In some embodiments, a method for treating a disease or disorder in a mammalian subject is provided. A therapeutically effective amount of the composition comprising the lipids disclosed herein, particularly cationic lipids, nucleic acids, amphiphiles, phospholipids, cholesterol, and PEG-linked cholesterol, can be administered to a subject having a disease or disorder associated with the expression or overexpression of a gene that can be reduced, decreased, downregulated, or silenced by the composition. The compositions described herein can be used in a method for treating cancer or inflammatory disease. The disease can be selected from the group consisting of central nervous system disorder, peripheral nervous system disorder, muscle atrophy, muscular dystrophy, immune disorder, cancer, kidney disease, fibrotic disease, genetic abnormality, inflammation, and cardiovascular disorder.

[0205] In some embodiments, the disclosure provides a method of expressing a protein or polypeptide in a target cell, the method comprising contacting the target cell with a lipid nanoparticle described herein, or a pharmaceutical composition described herein, in some embodiments, the protein or polypeptide is an antigen, and expression of the antigen provides an in vivo immunogenic response. EXAMPLES

[0206] VII. Examples Example 1. Synthesis of ATX-193 [ka] General scheme: [ka]

[0207] Synthesis of ATX-193-1 [ka]

[0208] A 1 L three-necked round bottom flask purged and maintained with an inert nitrogen atmosphere was charged with 1,3 cyclohexanedione (20 g, 1.00 equiv.) and dimethylformamide (200 mL). Ethyl acrylate (21.45 g, 1.20 equiv.) and Cs 2 CO 3 (35.00 g, 0.60 equiv.) was added and the resulting solution was stirred at 80° C. for 16 h. The reaction was then quenched by adding 600 mL of water / ice. The pH value of the solution was adjusted to 6 with HCl (1 mol / L). The resulting solution was extracted with 2×1 L of ethyl acetate and the organic layers were combined. The combined organic layers were washed with 2×1 L of brine. The organic layers were diluted with anhydrous MgSO 4 The mixture was dried over 1000 ml of ethyl 3-(2,6-dioxocyclohexyl)propanoate, filtered, and concentrated in vacuo. This gave 29 g (78%) of ethyl 3-(2,6-dioxocyclohexyl)propanoate as a yellow solid. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH 3 CN / 0.05% TFA 95:5 to 5:95 A / B, 2.00 min, 0.7 min hold): RT 1.01 min, m / z (calculated) 212.10, (observed) 213.10 (M+H).

[0209] Synthesis of ATX-193-2 [ka]

[0210] A 1 L three-necked round bottom flask purged and maintained with an inert nitrogen atmosphere was charged with ethyl 3-(2,6-dioxocyclohexyl)propanoate (29 g, 1.00 equiv.) and HCl (1 mol / L, 300 mL aqueous solution). The resulting solution was stirred at 95° C. for 16 h. The resulting mixture was concentrated under vacuum. The residue was dissolved in 1 L of EtOAc. Undissolved solids were removed by filtration. The resulting EA phase was concentrated to dryness under vacuum. This gave 23 g (crude) of 5-oxononanedioic acid as a yellow solid.

[0211] Synthesis of ATX-193-3 [ka]

[0212] A 1 L three-necked round bottom flask purged and maintained with an inert nitrogen atmosphere was charged with 5-oxononanedioic acid (23 g, 1.00 equiv.) and DCM (345 mL) at room temperature. This was followed by the addition of pentadecan-8-ol (62 g, 2.2 equiv.) and DMAP (13 g, 1.00 equiv.) at room temperature, followed by the addition of EDCI (52 g, 2.20 equiv.) at 0° C. The resulting solution was stirred at room temperature for 16 hours. The reaction was then quenched by the addition of 75 mL of aqueous HCl (1 mol / L). The resulting solution was extracted with 2×1 L of DCM and the organic layers were combined. The organic layers were washed with 2×1 L of brine and concentrated with MgSO 4 The mixture was dried at 70° C., filtered and concentrated under vacuum to approximately 500 mL. To this, 100 g of silica gel (type: ZCX-2, 100-200 mesh) was added and the mixture was concentrated under vacuum. This silica gel was applied onto a silica gel column (1 Kg, type: ZCX-2, 100-200 mesh) and the product was eluted with a gradient of PE / EA, 1 / 0 to 80 / 1. Fractions were collected and the product fractions were concentrated under vacuum. This gave 26 g (38%) of 1,9-bis(pentadecan-8-yl) 5-oxononanedioate as a yellow oil. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH 3 CN / 0.05% TFA 95:5 to 5:95 A / B, 2.00 min, 0.7 min hold): RT 2.99 min, m / z (calculated) 623.02, (observed) 645.35 (M+Na).

[0213] Synthesis of ATX-193-4 [ka]

[0214] Into a 500 mL three-neck round bottom flask purged and maintained with an inert nitrogen atmosphere was added 1,9-bis(pentadecan-8-yl) 5-oxononanedioate (18 g, 1.00 equiv.) and THF / H 22H2O (10:1, 180 mL) was added. This was followed by NaBH 4 (1.08 g, 1.00 equiv.) was added at 0° C. The resulting solution was stirred at room temperature for 4 h. The reaction was then quenched by adding 200 mL of water / ice. The resulting solution was extracted with 3×300 mL of ethyl acetate and the organic layers were combined. The organic layers were washed with anhydrous MgSO 4 The extract was dried at 40° C., filtered, and concentrated in vacuo to give 17.3 g (95%) of 1,9-bis(pentadecan-8-yl) 5-hydroxynonanedioate as a pale yellow oil.

[0215] Synthesis of ATX-193-6 [ka]

[0216] In a 1L four-neck round flask, add N 2 486 mL of bromo(heptyl)magnesium (1 mol / L) in THF (180 mL) was charged at 25° C. with mechanical stirring under 0° C. Ethyl formate (18.00 g, 1.00 equiv.) was charged dropwise with stirring at 0° C. in 30 min. The resulting solution was stirred at room temperature for 15 h. Then, 500 mL of saturated NH 4 The reaction was quenched by adding aqueous Cl. The phases were separated and the aqueous layer was extracted with 2×500 mL of ethyl acetate. The combined organic layers were then washed with anhydrous MgSO. 4 The solid residue was extracted with 60 mL of CH 3 The solid was collected by filtration and dried under vacuum to give 50 g (78%) of pentadecan-8-ol as a white powder, which was used directly in the next reaction step.

[0217] Synthesis of ATX-193 [ka]

[0218] A solution of 1,9-bis(pentadecan-8-yl)5-hydroxynonanedioate (17.3 g, 1.00 equiv.) in DCM (180 mL) was placed in a 250 mL three-necked round bottom flask purged and maintained with an inert nitrogen atmosphere at room temperature. 4-(Dimethylamino)butanoic acid (5.58 g, 1.20 equiv.) and DMAP (0.69 g, 0.20 equiv.) were added at room temperature, followed by EDCI (6.39 g, 1.20 equiv.) in small portions at 0° C. The resulting solution was stirred at room temperature for 16 hours. The reaction was then quenched by adding 300 mL of HCl (1 mol / L). The resulting solution was extracted with 2×500 mL of DCM and the organic layers were combined. The organic layers were washed with 2×500 mL of brine. The resulting organic layer was concentrated under vacuum and 40 g of the resulting crude product was adsorbed onto 80 g of silica gel. The residue was purified on a silica gel column (800 g, type: ZCX-2, 100-200 mesh) using a gradient of DCM / ME, 100:0 to 90:10. The product containing fractions were concentrated under vacuum. The product was then dissolved in heptane (300 mL, 20 V) and the organic layer was then washed with MeOH / H 2 The mixture was washed with 300 mL (20 V) of 3:1 H2O. The heptane phase was concentrated under vacuum. This gave 10.5 g (49%) of 1,9-bis(pentadecan-8-yl) 5-[[4-(dimethylamino)butanoyl]oxy]nonanedioate as a colorless oil. ELSD A: Water / 0.05% TFA: B: CH 3 CN / 0.05%TFA 95:5-5:95 A / B, 2.00 min, 0.7 min hold):RT 2.76 min, m / z (calculated) 737.6, (observed) 738.5 (M+H);H-NMR: (300 MHz, chloroform-d, ppm): δ 4.881 (h, 3H), 2.332 (dt, 8H), 2.241 (s, 6H), 1.812 (m, 2H), 1.710-1.413 (m, 16H), 1.282 (s, 40H), 0.952-0.844 (m, 12H).

[0219] Example 2. Synthesis of ATX-200 [ka] General scheme: [ka]

[0220] Synthesis of ATX-200-4 [ka]

[0221] A 500 mL four-neck round bottom flask purged and maintained with an inert nitrogen atmosphere was charged with methyltriphenylphosphanium bromide (4540.31 mg, 12.456 mmol, 1.60 equiv, 98%), THF (150.00 mL, 99%). This was followed by the addition of t-BuOK (1323.54 mg, 11.677 mmol, 1.50 equiv, 99%) in several batches at 0° C. in 10 min. To this was added 1,9-bis(pentadecan-8-yl) 5-oxononanedioate (5.00 g, 7.785 mmol, 1.00 equiv, 97%) in THF (25 ml) at 0° C. in 20 min. The resulting solution was stirred at 25° C. for 18 h. The resulting mixture was concentrated. The residue was applied onto a silica gel column with ethyl acetate / petroleum ether (1:50). This gave 3.7 g (75.00%) of 1,9-bis(pentadecan-8-yl) 5-methylidenenonane dioate as a colorless oil.

[0222] Synthesis of ATX-200-5 [ka]

[0223] A 100 mL 4-neck round bottom flask purged and maintained with an inert nitrogen atmosphere was charged with 1,9-bis(pentadecan-8-yl) 5-methylidene nonanedioate (3.7 g, 5.779 mmol, 1.00 equiv, 97%), THF (3.70 mL). This was followed by the dropwise addition of 9-BBN (14.90 mL, NaN mmol, 1.25 equiv) at 18° C. in 20 min with stirring. The mixture was stirred at 18° C. for 18 h, after which water (1 mL) and 3N NaOH (5 mL) were added successively. The mixture was then cooled to 30° C. with 30% H 2 O while maintaining the temperature below 50° C. 2 O 2 (10 mL) was added dropwise. After stirring at room temperature for 18 h, the resulting solution was extracted with 2×50 mL of ethyl acetate. The resulting mixture was washed with 3×50 mL of brine. The mixture was dried over anhydrous sodium sulfate and concentrated. The residue was applied onto a silica gel column with ethyl acetate / petroleum ether (1:20). This gave 2.6 g (68.29%) of 1,9-bis(pentadecan-8-yl) 5-(hydroxymethyl)nonanedioate as a white oil. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH 3 CN / 0.05% TFA 95:5 to 5:95 A / B, 2.00 min, 0.7 min hold): RT 3.19 min, m / z (calculated) 638.5, (observed) 661.5 (M+Na).

[0224] Synthesis of ATX-200 [ka]

[0225] A 100 mL 4-neck round bottom flask purged and maintained with an inert nitrogen atmosphere was charged with 1,9-bis(pentadecan-8-yl)5-(hydroxymethyl)nonanedioate (2.60 g, 3.946 mmol, 1.00 equiv, 97%), THF (15.00 mL). This was followed by the addition of 4-(dimethylamino)butanoic acid (633.88 mg, 4.736 mmol, 1.20 equiv, 98%) in several batches over 10 min at 0° C. To this was added EDCI (926.37 mg, 4.736 mmol, 1.20 equiv, 98%) in several batches over 10 min at 0° C. To the mixture was added DMAP (98.39 mg, 0.789 mmol, 0.20 equiv, 98%) in several batches over 10 min at 0° C. The resulting solution was stirred at 18° C. for 18 hours. The resulting mixture was concentrated. The residue was applied onto a silica gel column with ethyl DCM:MeOH (30:1). The crude product was purified by Flash-Prep-HPLC (IntelFlash-1) with the following conditions: column, C18 silica gel; mobile phase, 2-propanol:H within 30 min. 2 O=60:40, 2-propanol:H 2 O=80:20, detector, evaporation light. The product was obtained. The product was then dissolved in heptane (30 mL, 20 V), and the organic layer was then washed with MeOH / H 2 20 V. The heptane phase was concentrated under vacuum. This gave 1.5 g (50.02%) of 1,9-bis(pentadecan-8-yl) 5-([[4-(dimethylamino)butanoyl]oxy]methyl)nonanedioate as a pale yellow oil. ELSD A: water / 0.05% TFA: B: CH 3 CN / 0.05% TFA 95:5 to 5:95 A / B, 2.00 min, 0.7 min hold):RT 3.19 min, m / z (calculated) 751.67, (observed) 752.50 (M+H);H-NMR: (400 MHz, chloroform-d, ppm): 4.847-4.909 (m, 2H), 4.001-4.015 (m, 2H), 2.241-2.380 (m, 14H), 1.392-1.845 (m, 69H).

[0226] Example 3. Synthesis of ATX-201 [ka] General scheme: [ka]

[0227] Synthesis of ATX-201-1 [ka]

[0228] In a three-necked round-bottom flask, EtOH (25 mL, 5 V) and ATX-201-SM (5 g, 1 equiv.) were added at room temperature and stirred. 6N NaOH (25 mL, 5 V) was slowly added to the mixture at 0 °C. The resulting solution was stirred at 60 °C for 2 h, and TLC showed complete consumption of ATX-201-SM. Brine (10 wt%, 50 mL, 10 V) and DCM (50 mL, 10 V) were added to the mixture and stirred for 10 min, the phases were cut, the aqueous phase was collected, and the pH was adjusted to 3-4 with 3N HCl. The mixture was extracted with DCM (100 mL, 20 V). The organic phase was washed with anhydrous MgSO 4 The mixture was dried at rt, filtered, concentrated and dried under vacuum to give ATX-205-1 (3.2 g, 84.6% yield) as a pale yellow solid.

[0229] Synthesis of ATX-201-2 [ka]

[0230] Into a three-necked round-bottom flask was added DCM (100 mL, 10V), ATX-201-1 (3.2 g, 1 equiv.), and ethane-1,2-dithiol (2.1 g, 1.2 equiv.) at room temperature. 3 . Et 2O (2.5 equiv.) was slowly added to the mixture at 0° C. The resulting solution was stirred at 20° C. for 16 h, and TLC showed complete consumption of ATX-201-1. The solid was collected by filtration. The solid was dried under vacuum to give ATX-201-2 (4 g, 88% yield) as a pale yellow solid, which was used as such in the next reaction.

[0231] Synthesis of ATX-201-3 [ka]

[0232] In a three-necked round-bottom flask, DCM (80 mL, 20 V), ATX-201-2 (4 g, 1.0 equiv), ATX-193-6 (8 g, 2.2 equiv), and DMAP (2 g, 1 equiv) were added successively. EDCI (6.7 g, 2.2 equiv) was added in small portions to the reaction mixture at 0 °C. The resulting solution was stirred at 20 °C for 16 h, and TLC showed complete consumption of ATX-201-2. The reaction was quenched with 10% citric acid solution (40 mL, 10 V). The organic phase was collected and the organic phase was washed with 10% citric acid solution (40 mL, 10 V) and brine (40 mL, 10 V). The organic phase was washed with anhydrous MgSO 4 The crude product was adsorbed onto 20 g of silica gel and purified on a 100 g silica gel column (Type: ZCX-2, 100-200 mesh, 8.00 w / w.) eluted with a PE / EA gradient of 100:0 to 99:1. The relevant fractions were combined, concentrated, and dried under vacuum to give ATX-201-3 (8 g, 75% yield) as a colorless oil. 1 H NMR(300MHz,chloroform-d)δ 4.86(p,J=6.2Hz,2H),3.26(s,4H),2.67-2.56(m,4H),2.30-2.15(m,4H) ,1.50(t,J=6.3Hz,8H),1.28(d,J=11.2Hz,41H),0.88(d,J=6.3Hz,12H).

[0233] Synthesis of ATX-201-4 [ka]

[0234] Acetone (160 mL, 20 V), ATX-201-3 (8 g, 1.0 equiv.) were added successively to a three-necked round-bottom flask. NBS (4.25 g, 2 equiv.) was added to the reaction mixture in small portions at 0 °C. The resulting solution was stirred at room temperature for 2 h, and TLC showed complete consumption of ATX-201-3. The solvent was removed under reduced pressure. The crude product was adsorbed onto 20 g of silica gel and purified on a 100 g silica gel (Type: ZCX-2, 100-200 mesh, 8.00 w / w.) column using a Combi-flash system. The product was eluted with a PE / EA gradient from 100:0 to 97:3. The eligible product was pooled and concentrated under vacuum to give ATX-201-4 (2.5 g, 35% yield) as a colorless oil. 1 H NMR(400MHz,chloroform-d)δ 4.84(p,J=6.3Hz,2H),2.76(t,J=6.7Hz,4H),2.59(t,J=6.7Hz,4H),1.50(q,J=6.4,6.0Hz,8H),1.31-1.21(m,40H),0.91-0.84(m,12H).

[0235] Synthesis of ATX-201-5 [ka]

[0236] A 500 mL 4-neck round bottom flask purged and maintained with an inert nitrogen atmosphere was charged with methyltriphenylphosphanium bromide (1.9 g, 1.6 equiv.), THF (75 mL, 30 V). This was followed by the addition of t-BuOK (0.7 g, 1.5 equiv.) in several batches at 0° C. in 10 min. To this was added ATX-201-4 (2.5 g, 1 equiv.) in THF (25 ml) at 0° C. in 20 min. The resulting solution was stirred at 25° C. for 18 h. The resulting mixture was concentrated. The product was adsorbed onto 5 g of silica gel and purified on a 25 g silica gel (Type: ZCX-2, 100-200 mesh, 8.00 w / w.) column on a Combi-Flash system by eluting with a PE / EA gradient from 100:0 to 99:1. The relevant products were combined, concentrated and dried under vacuum to give ATX-201-5 (1.9 g, 76% yield) as a colorless oil. 1 H NMR(300MHz,chloroform-d)δ 4.88(p,J=6.2Hz,2H),4.78(s,2H),2.47(ddd,J=8.5,6.2,1.8Hz,4H),2.37(dd,J=8.6,5.9Hz,4H),1.53(s,8H),1.27(,40H),0.88(m,12H).

[0237] Synthesis of ATX-201-6 [ka]

[0238] In a three-necked round-bottom flask, ATX-201-5 (1.9 g, 1 equiv.) and THF (3.70 mL) were added successively. This was followed by dropwise addition of 0.5 mol of 9-BBN in THF (8 mL, 1.25 equiv.) with stirring in 20 min at 18° C. After the mixture was stirred at 18° C. for 18 h, water (0.47 mL, 0.25 V) and 3N NaOH (2.8 mL, 1.5 V) were added successively. Then, the mixture was cooled to 30° C. with 30% H 2 O while maintaining the temperature below 50° C. 2 O 2(4.75 ml, 2.5 V) was added dropwise. After stirring at room temperature for 18 h, the resulting solution was extracted with 2×20 mL of ethyl acetate. The combined organic phase was washed with 3×20 mL of brine. The mixture was dried over anhydrous sodium sulfate and filtered. The product was purified on a 25 g silica gel (Type: ZCX-2, 100-200 mesh, 8.00 w / w.) column on a Combi-Flash system by adsorbing onto 5 g of silica gel and eluting with PE. The eligible products were combined, concentrated, and dried under vacuum to give ATX-201-6 (1.4 g, 76% yield) as a colorless oil. 1 H NMR(300MHz,chloroform-d)δ 4.87(p,J=6.3Hz,2H),4.12(q,J=7.1Hz,1H),3.52(d,J=4.8Hz,2H),2.40-2.27(m,4H),1.7 5-1.61(m,3H),1.51(d,J=6.4Hz,9H),1.27(t,J=3.7Hz,40H),1.23(m,40H),0.87(m,12H).

[0239] Synthesis of ATX-201 [ka]

[0240] In a three-necked round-bottom flask, DCM (28 mL, 20 V), ATX-201-6 (1.4 g, 1.0 equiv), 4-(dimethylamino)butanoic acid (380 mg, 1 equiv), and DMAP (168 mg, 0.6 equiv) were added successively. EDCI (526 mg, 1.2 equiv) was added in small portions to the reaction mixture at 0 °C. The resulting solution was stirred at 20 °C for 16 h, and TLC showed complete consumption of 4-(dimethylamino)butanoic acid. The reaction mixture was quenched with 10% citric acid solution (14 mL, 10 V). The organic phase was collected, washed with 10% citric acid solution (14 mL, 10 V), and washed with brine (14 mL, 10 V). The organic phase was washed with anhydrous MgSO 4The product was adsorbed onto 5 g of silica gel and purified on a 25 g silica gel (Type: ZCX-2, 100-200 mesh, 8.00 w / w.) column on a Combi-Flash system by eluting with a DCM / MeOH gradient from 100:0 to 95:5. The eligible product was combined, concentrated and dried under vacuum to give ATX-201 (1.3 g, 78% yield) as a pale yellow oil. ELSD A: Water / 0.05% TFA: B: CH3CN / 0.05% TFA 95:5 to 5:95 A / B, 2.00 min, 0.7 min hold): RT 3.19 min, m / z (calculated) 723.6, (observed) 724.7 1 H NMR(300MHz,chloroform-d)δ 4.831(p,J=6.2Hz,2H),4.013(d,J=4.5Hz,2H),3.000-2.881(m,2H),2.70(s,6H),2.464(t,J=6.7Hz,2H),2.310(t,J=7.5H z,4H),2.112(dq,J=13.7,6.8Hz,2H),1.653(t,J=7.2Hz,5H),1.492(d,J=6.3Hz,8H),1.242(m,40H),0.910-0.762(m,12H).

[0241] Example 4. Synthesis of ATX-202 [ka] General scheme: [ka]

[0242] Synthesis of ATX-202-5 [ka]

[0243] In a 250 mL four-neck round flask, add N 25 g of 1,9-bis(pentadecan-8-yl) 5-hydroxynonanedioate in DCM (50 mL) was charged at 0° C. with mechanical stirring under 100° C. This was followed by dropwise addition of TEA (1.6 g, 2 eq.) and MsCl (1.35 g, 1.5 eq.) with stirring at 0° C. The resulting solution was stirred at room temperature for 5 h. Then, 100 mL of H 2 The reaction was quenched by the addition of O. The phases were separated and the aqueous layer was extracted with 1×100 mL of DCM. The organic layers were then combined. The solvent was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. This gave 4.8 g (85%) of di(pentadecan-8-yl) 5-((methylsulfonyl)oxy)nonanedioate. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH 3 CN / 0.05% TFA 95:5 to 5:95 A / B, 2.00 min, 0.7 min hold): RT 4.76 min, m / z (calculated) 703.12, (observed) 725.3 (M+Na).

[0244] Synthesis of ATX-202-6 [ka]

[0245] A 100 mL three-necked round bottom flask purged and maintained with an inert nitrogen atmosphere was charged with 1,9-bis(pentadecan-8-yl) 5-oxononanedioate (4.8 g, 1.00 equiv.) in DMF (48 mL). This was followed by the addition of NaHS (2 g, 5.00 equiv.) at 0 °C. The resulting solution was stirred at room temperature for 5 h. The reaction was then quenched by adding 200 mL of water / ice. The resulting solution was extracted with 3 x 100 mL of ethyl acetate and the organic layers were combined. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. This gave 2.8 g (64%) of di(pentadecan-8-yl) 5-mercaptononanedioate as a pale yellow oil. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH 3CN / 0.05% TFA 95:5 to 5:95 A / B, 2.00 min, 0.7 min hold): RT 4.84 min, m / z (calculated) 640.5, (observed) 663.4 (M+Na+H).

[0246] Synthesis of ATX-202 [ka]

[0247] A 100 mL three-necked round bottom flask purged and maintained with an inert nitrogen atmosphere was charged with a solution of di(pentadecan-8-yl) 5-mercaptononanedioate (2.8 g, 1.00 equiv.) in DCM (28 mL). 4-(Dimethylamino)butanoic acid (0.87 g, 1.20 equiv.), DMAP (0.1 g, 0.20 equiv.) were added followed by EDCI (0.95 g, 1.20 equiv.) in small portions at 0° C. The resulting solution was stirred at room temperature for 16 hours. The reaction was then quenched by adding 100 mL of HCl (1 mol / L). The resulting solution was extracted with 2×100 mL of DCM and the organic layers were combined. The resulting mixture was washed with 2×100 mL of brine. The resulting mixture was concentrated under vacuum to give 6 g of crude product. The product was dissolved in 30 mL of DCM and 10 g of silica gel (type: ZCX-2, 100-200 mesh) was added. The mixture was concentrated under vacuum. The residue was applied onto an atmospheric silica gel column (800 g, type: ZCX-2, 100-200 mesh) with a gradient of DCM / ME, 1 / 0 to 30 / 1, and the product eluate (50 / 1 to 30 / 1) was collected. The collected product phase was concentrated under vacuum. The product was then dissolved in heptane (30 mL, 20 V) and the organic layer was then washed with MeOH / H 2 The mixture was washed with 30 mL (20 V) of HO (3:1). The heptane phase was concentrated under vacuum. This gave 1.3 g (45%) of 1,9-bis(pentadecan-8-yl) 5-[[4-(dimethylamino)butanoyl]oxy]nonanedioate as a colorless oil. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH 3CN / 0.05% TFA 95:5 to 5:95 A / B, 2.00 min, 0.7 min hold): RT 2.83 min, m / z (calculated) 754.25, (observed) 754.45 (M); 1 H-NMR (300MHz, chloroform-d, ppm):δ 4.83-4.87(m,2H),3.51-3.54(s,1H),2.55-2.60(m,2H),2.21-2.30(m,12H) ,1.40-1.91(m,19H),1.11-1.30(m,41H),1.28(s,40H),0.82-0.91(m,12H).

[0248] Example 5. Synthesis of ATX-209 [ka] General scheme: [ka]

[0249] Synthesis of ATX-209-1 [ka]

[0250] In a three-necked round-bottom flask, DMSO (38 mL, 15 V), ATX-209-SM1 (2.5 g, 1 eq.), and 1((isocyanomethyl)sulfonyl)-4-methylbenzene (1 g, 0.5 eq.) were added at room temperature. To the mixture, NaH (0.30 g, 1.2 eq.) and tetrabutylammonium iodide (0.37 g, 0.1 eq.) were added successively and slowly at 0 °C. The resulting solution was stirred at 60 °C for 2 h, and TLC showed complete consumption of ATX-209-SM1. The reaction was then quenched by adding 25 mL of water. The solution was extracted with DCM (3 × 25 mL). The organic phase was washed with 2 × 25 mL of saturated brine. The organic phase was dried over anhydrous magnesium sulfate. The organic phase was diluted with anhydrous MgSO 4The mixture was dried over 1000 ml of ethyl acetate, then filtered, concentrated, and dried under vacuum to give ATX-209-1 (3.2 g, 60% yield) as a colorless oil. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH 3 CN / 0.05% TFA 95:5 to 5:95 A / B, 2.00 min, 0.7 min hold): RT 0.84 min, m / z (calculated) 535.30, (observed) 558.20 (M+Na).

[0251] Synthesis of ATX-209-2 [ka]

[0252] To a three-necked round-bottom flask, DCM (30 mL, 10 V), ATX-209-1 (3 g, 1 equiv.) were added in one portion at room temperature. HCl (6 ml, 2 V) was added slowly to the mixture at 0° C., and the resulting solution was stirred at 0° C. for 2 h, when TLC showed complete consumption of ATX-209-1. The reaction was then quenched by adding 30 mL of sodium bicarbonate. The organic phase was washed with 2×30 mL of saturated brine. The organic phase was diluted with anhydrous MgSO 4 The mixture was dried at 37° C. and then filtered. The filtrate was added with 5 g of silica gel (type: ZCX-2, 100-200 mesh, 2.00 w / w) and concentrated under vacuum to no fractions while maintaining the temperature below 35° C. 25 g of silica gel (type: ZCX-2, 100-200 mesh, 8.00 w / w) was packed into a column, followed by the dried silica gel prepared in the last step, which absorbed the reaction mixture. The product was purified using Combi-flash. It was eluted with DCM / MeOH (volume ratio). (100:0 to 20:1 gradient, collecting every 100±50 mL). A sample was taken for TLC analysis. The eligible products were combined. It was concentrated to dryness under vacuum to give ATX-209-2 (1.15 g, 80% yield) as a white solid. 1 H NMR(300MHz,DMSO-d6) δ 2.36(t,J=7.2Hz,4H),2.16(t,J=7.3Hz,4H),1.43(,8H),1.21-1.12(m,4H).

[0253] Synthesis of ATX-209-3 [ka]

[0254] In a three-necked round-bottom flask, DCM (20 mL, 20 V), ATX-209-2 (1 g, 1.0 equiv), ATX-209-5 (1.71 g, 2.2 equiv), and DMAP (0.47 g, 1 equiv) were added successively. EDCI (1.63 g, 2.2 equiv) was added in small portions to the reaction mixture at 0 °C. The resulting solution was stirred at 20 °C for 16 h, and TLC showed complete consumption of ATX-209-2. The reaction was quenched with 10% citric acid solution (10 mL, 10 V). The organic phase was collected and the organic phase was washed with 10% citric acid solution (10 mL, 10 V) and with brine (10 mL, 10 V). The organic phase was washed with anhydrous MgSO 4 The mixture was dried at 37° C. and then filtered. The filtrate was added with 5 g of silica gel (type: ZCX-2, 100-200 mesh, 2.00 w / w) and concentrated under vacuum to no fractions while maintaining the temperature below 35° C. 25 g of silica gel (type: ZCX-2, 100-200 mesh, 8.00 w / w) was packed into a column, followed by the dried silica gel prepared in the last step, which absorbed the reaction mixture. The product was purified using Combi-flash. It was eluted with PE / EA (volume ratio). (100:0 to 50:1 gradient, collected every 20±10 ml). A sample was taken for TLC analysis. The eligible products were combined. Concentrated to dryness under vacuum to give ATX-209-3 (1.68 g, 70% yield) as a colorless oil. LCMS(Schimadzu 2020;ELSD A:Water / 0.05%TFA:B:CH 3 CN / 0.05% TFA 95:5 to 5:95 A / B, 5.00 min, 0.7 min hold): RT 4.83 min, m / z (calculated) 622.55, (observed) 645.3 (M+Na).

[0255] Synthesis of ATX-209-4 [ka]

[0256] In a 100 ml three-neck flask, MeOH (20 ml, 10 V), ATX-209-3 (2 g, 1 equiv.) were added at room temperature. NaBH 4 (0.18 g, 1.5 equiv.) was added in small portions to the reaction mixture at 0° C. The resulting solution was stirred at 0° C. for 2 h, and TLC showed complete consumption of ATX-209-3. The reaction was then quenched by adding 20 mL of water. The system was extracted again with METB (2×10 ml, 10 V). The organic phase was washed with anhydrous MgSO 4 The mixture was dried over 1000 ml of ethyl acetate and then filtered. Concentration to dryness in vacuo afforded ATX-209-3 (1.5 g, 75% yield) as a colorless oil. LCMS (Schimadzu 2020; ELSD A: water / 0.05% TFA: B: CH 3 CN / 0.05% TFA 95:5 to 5:95 A / B, 5.50 min, 0.7 min hold): RT 4.83 min, m / z (calculated) 624.57, (observed) 647.35 (M+Na).

[0257] Synthesis of ATX-209-5 [ka]

[0258] In a 100 ml four-neck round flask, add N 2 ATX-209-SM2 (1 mol / L, 31 ml) in THF (10 mL) was added at 25° C. with mechanical stirring under 50° C. Ethyl formate (1 g, 1.00 equiv.) was added dropwise with stirring at 0° C. The resulting solution was stirred at room temperature for 15 h. Then, NH 4 The reaction was quenched by adding Cl solution (20 mL, 20V). The phases were separated and the aqueous layer was extracted with ethyl acetate (2×20 mL). The organic layers were then combined. The solvent was dried over anhydrous sodium sulfate. It was filtered and concentrated under vacuum. The residue was slurried with 6 mL of ACN. The solid was collected by filtration. This gave ATX-209-5 (2 g, 74% yield) as a white powder.

[0259] Synthesis of ATX-209 [ka]

[0260] In a three-necked round-bottom flask, DCM (30 mL, 20 V), 4-(dimethylamino)butanoic acid (0.45 g, 1.1 equiv), ATX-209-4 (1.5 g, 1 equiv), and DMAP (0.29 g, 1 equiv) were added successively. EDCI (0.60 g, 1.3 equiv) was added in small portions to the reaction mixture at 0 °C. The resulting solution was stirred at 20 °C for 16 h, and TLC showed complete consumption of ATX-209-4. The reaction was quenched with 10% citric acid solution (15 mL, 10 V). The organic phase was collected and the organic phase was washed with 10% citric acid solution (15 mL, 10 V) and with brine (15 mL, 10 V). The organic phase was washed with anhydrous MgSO 4 The mixture was dried at 37°C for 1 hour and then filtered. The filtrate was added with 5 g of silica gel (type: ZCX-2, 100-200 mesh, 2.00 w / w) and concentrated under vacuum to no fractions while maintaining the temperature below 35°C. 25 g of silica gel (type: ZCX-2, 100-200 mesh, 8.00 w / w) was packed into a column, followed by the dried silica gel prepared in the last step, which absorbed the reaction mixture. The product was purified using Combi-flash. Elute with PE / EA (volume ratio). (100:0 to 50:1 gradient, collecting every 20±10 ml). Take a sample for TLC analysis. The eligible product was combined. Concentrated to dryness under vacuum to give ATX-209 (1.2 g, 75% yield) as a colorless oil. ELSD A: Water / 0.05% TFA: B: CH 3 CN / 0.05% TFA 95:5 to 5:95 A / B, 5.50 min, 0.7 min hold): RT 4.83 min, m / z (calculated) 737.5, (observed) 738.3 (M+H); 1H NMR (300 MHz, chloroform-d) δ 4.860 (t, J = 6.2 Hz, 3H), 2.370-2.201 (m, 14H), 1.801 (q, J = 7.4 Hz, 2H), 1.611-1.470 (m, 16H), 1.272 (m, 40H), 0.920-0.821 (m, 12H).

[0261] Example 6. Synthesis of ATX-210 [ka] General scheme: [ka]

[0262] Synthesis of ATX-210-4 [ka]

[0263] A 1 L three-necked round bottom flask purged and maintained with an inert nitrogen atmosphere was charged with 5-oxononanedioic acid (6 g, 1.00 equiv), DCM (90 mL). This was followed by the addition of pentadecan-8-ol (6.77 g, .0 equiv), DMAP (0.72 g, 0.2 equiv) and to this was added EDCI (6.84 g, 1.2 equiv) at 0° C. The resulting solution was stirred at room temperature for 16 hours. The reaction was then quenched by adding 75 mL of HCl (1 mol / L). The resulting solution was extracted with 2×100 ml of DCM and the organic layers were combined. The resulting mixture was washed with 2×100 ml of NaCl. The organic layer was concentrated under vacuum. The product was dissolved in 60 mL of DCM and 40 g of silica gel (type: ZCX-2, 100-200 mesh) was added. The mixture was concentrated under vacuum. The residue was applied onto an atmospheric silica gel column (400 g, type: ZCX-2, 100-200 mesh) with a gradient of MeOH / DCM, 0 / 1 to 1 / 10, and the product eluate (1 / 20 to 1 / 10) was collected. The collected product phase was concentrated under vacuum. This gave 7.2 g (58.8%) of ATX-210-4 as a yellow oil. ELSD A: Water / 0.05% TFA: B: CH 3 CN / 0.05% TFA 95:5 to 5:95 A / B, 2 min, 0.7 min hold): RT 1.60 min, m / z (calculated) 412.32, (observed) 435.15 (M+Na).

[0264] Synthesis of ATX-210-5 [ka]

[0265] In a 1L four-neck round flask, add N 2 540 mL of pentylmagnesium bromide (1 mol / L) in THF (200 mL) was charged at 25° C. with mechanical stirring under 5° C. Ethyl formate (20.0 g, 1.0 equiv.) was charged dropwise with stirring at 0° C. The resulting solution was stirred at room temperature for 15 h. Then, 500 mL of NH 4The reaction was quenched by adding Cl. The phases were separated and the aqueous layer was extracted with 2×500 mL of ethyl acetate. The organic layers were then combined. The solvent was dried over anhydrous sodium sulfate. It was filtered and concentrated under vacuum. This gave 38.9 g (83.6%) of undecan-6-ol as a yellow oil.

[0266] Synthesis of ATX-210-6 [ka]

[0267] A 1 L three-necked round bottom flask purged and maintained with an inert nitrogen atmosphere was charged with ATX-210-4 (7.2 g, 1.00 equiv), DCM (108 mL). This was followed by the addition of undecan-6-ol (3.0 g, 1.0 equiv), DMAP (0.43 g, 0.2 equiv), to which EDCI (4.1 g, 1.2 equiv) was added at 0° C. The resulting solution was stirred at room temperature for 16 hours. The reaction was then quenched by adding 75 mL of HCl (1 mol / L). The resulting solution was extracted with 2×100 ml of DCM and the organic layers were combined. The resulting mixture was washed with 2×100 ml of NaCl. The mixture was dried over anhydrous sodium sulfate and concentrated under vacuum. This gave 10 g (99.9%) of ATX-210-6 as a yellow oil, which was used directly in the next step without further purification. ELSD A: Water / 0.05% TFA: B: CH 3 CN / 0.05% TFA 95:5 to 5:95 A / B, 5 min, 0.7 min hold): RT 3.62 min, m / z (calculated) 566.49, (observed) 589.40 (M+Na).

[0268] Synthesis of ATX-210-7 [ka]

[0269] Into a 250 mL three-neck round bottom flask purged and maintained with an inert nitrogen atmosphere was added ATX-210-6 (10 g, 1.0 equiv.), THF / H 22H2O (10:1, 100 mL) was added. This was followed by NaBH 4 (1.34 g, 2.0 equiv.) was added at 0° C. The resulting solution was stirred at room temperature for 16 h. The reaction was then quenched by adding 100 mL of water / ice. The resulting solution was extracted with 3×100 mL of ethyl acetate and the organic layers were combined. The resulting mixture was washed with 2×100 ml of NaCl. The mixture was then dried over anhydrous sodium sulfate and the organic layers were concentrated under vacuum. The product was dissolved in 10 mL of DCM and 40 g of silica gel (type: ZCX-2, 100-200 mesh) was added. The mixture was concentrated under vacuum. The residue was applied onto an atmospheric silica gel column (400 g, type: ZCX-2, 100-200 mesh) with a gradient of PE / EA from 1 / 0 to 10 / 1 and the product eluate (20 / 1 to 10 / 1) was collected. The collected product phase was concentrated under vacuum. This gave 7.1 g (70.7%) of ATX-210-7 as a yellow oil, which was used directly in the next step without further purification. ELSD A: Water / 0.05% TFA: B: CH 3 CN / 0.05% TFA 95:5 to 5:95 A / B, 5 min, 0.7 min hold): RT 3.64 min, m / z (calculated) 568.50, (observed) 591.35 (M+Na).

[0270] Synthesis of ATX-210 [ka]

[0271] A solution of ATX-210-7 (3.3 g, 1.00 equiv.) in DCM (50 mL) was placed in a 100 mL three-necked round bottom flask purged and maintained with an inert nitrogen atmosphere. 4-(Dimethylamino)butanoic acid (1.16 g, 1.20 equiv.), DMAP (0.14 g, 0.20 equiv.) were added, followed by EDCI (1.34 g, 1.20 equiv.) in small portions at 0° C. The resulting solution was stirred at room temperature for 16 h. Then, 50 mL of NaHCO 3The reaction was quenched by adding (1 mol / L). The resulting solution was extracted with 3×50 mL of DCM and the organic layers were combined. The resulting mixture was washed with 2×50 mL of brine. The organic layers were concentrated under vacuum. The product was dissolved in 5 mL of DCM and 15 g of silica gel (type: ZCX-2, 100-200 mesh) was added. The mixture was concentrated under vacuum. The residue was applied onto an atmospheric silica gel column (150 g, type: ZCX-2, 100-200 mesh) with a gradient of PE / EA from 1 / 0 to 10 / 1 and the product eluate (20 / 1 to 10 / 1) was collected. The collected product phase was concentrated under vacuum. The product was dissolved in heptane (60 mL, 20 V) and the heptane phase was concentrated under vacuum. This gave 2.3 g (60.0%) of ATX-210 as a yellow oil. ELSD A: Water / 0.05% TFA: B: CH 3 CN / 0.05%TFA 95:5 to 5:95 A / B, 2 min, 0.7 min hold):RT 1.84 min, m / z (calculated) 681.59, (observed) 682.40 (M+H);H-NMR-PH-ARC-Lipid-210-0: (300 MHz, chloroform-d): δ 4.821-4.904 (3H, m), 2.235-2.357 (8H, m), 2.187-2.204 (6H, s), 1.571-1.831 (16H, m), 1.261 (32H, s), 0.855-0.899 (12H, m).

[0272] Example 7. Synthesis of ATX-230 [ka] General scheme: [ka]

[0273] Synthesis of ATX-230-1 [ka]

[0274] A 100 mL three-neck round bottom flask was charged with ATX-230-SM (2.5 g, 1.0 equiv.) in THF (50 mL, 20 V). NaH (560 mg, 60% in mineral oil, 1.2 equiv.) was added to the reaction mixture in portions at 0° C. and stirred for 30 min. Benzyl bromide (2.4 g, 1.0 equiv.) and tetra-n-butylammonium iodide (TBAI) (1.5 g, 0.1 equiv.) were added to the reaction mixture at 0° C. The resulting solution was stirred at room temperature for 2 h and HPLC showed complete consumption of ATX-230-SM. The reaction was quenched by carefully adding ice water to the system and stirred for 10 min. The organic solvent was evaporated in vacuum and the aqueous phase was extracted with DCM (2×25 mL, 20 V). The organic solvent was concentrated under vacuum. The residue was dissolved in THF (25 mL, 10 V) and 6 mol / L aqueous HCl (25 mL, 10 V) was added at room temperature. The resulting solution was stirred at room temperature for 30 min. The pH value of the solution was adjusted using NaHCO 3 The acid concentration was adjusted to 7-8 with aqueous solution. The resulting solution was extracted with ethyl ether (2×25 mL, 20 V). The organic layers were combined and washed with anhydrous MgSO 4 The mixture was dried at 37° C. and then filtered. The filtrate was loaded with 8 g of silica gel (type: ZCX-2, 100-200 mesh, 3.20 w / w) and concentrated under vacuum to no fractions while maintaining the temperature below 20° C. 40 g of silica gel (type: ZCX-2, 100-200 mesh, 16.00 w / w.) was loaded into a column, followed by the dried silica gel prepared in the last step, which absorbed the reaction mixture. The product was purified using Combi-flash. Eluted with PE / EA. (volume ratio, gradient 100:0 to 95:5). The product fraction was concentrated under vacuum to give ATX-230-1 (1.5 g, 60% yield) as a white solid. ELSD A: Water / 0.05% TFA: B: CH 3 CN / 0.05%TFA 95:5 to 5:95 A / B, 2 min, 0.7 min hold):RT 0.79 min, m / z (calculated) 182.09, (observed) 205.10 (M+Na);H-NMR-PH-ARC-Lipid-230-1: (300 MHz, chloroform-d): δ 7.40-7.29 (5H,m), 4.66 (2H,s), 3.83-3.71 (4H,m), 3.64-3.58 (1H,m).

[0275] Synthesis of ATX-230-2 [ka]

[0276] Step 1: Then, N 2 To a solution of pentadecan-8-ol (150.0 g, 1.0 equiv.) in DCM (3 L, 20 V) in a dry three-neck flask containing 100% CO, TEA (266.0 g, 4.0 equiv.) was added in one portion, followed by bromoacetyl bromide (526.0 g, 4.0 equiv.) at 0° C. The reaction was stirred at room temperature for 3 days and diluted with saturated NH 4 It was quenched by adding aqueous Cl (10L, 66.7V) at 0°C. The crude compound was extracted with DCM (10L*3, 200V). The combined organic fractions were washed with brine (10L, 66.7V) and anhydrous MgSO 4 Dry at 400 C and filter. Add 500 g of silica gel (type: ZCX-2, 100-200 mesh, 3.33 w / w.) to the filtrate and concentrate under vacuum to no fractions while maintaining the temperature below 35°C. Pack 2.5 kg of silica gel (type: ZCX-2, 100-200 mesh, 16.67 w / w.) into a column followed by the dried silica gel prepared in the last step, which absorbed the reaction mixture. Purify the product using Combi-flash. Elute with PE / EA (volume ratio). (100:0 gradient, collecting every 3±0.5 L). Take a sample for TLC (PE:EA=8:1, Rf=0.2) analysis. Combine the relevant fractions and concentrate to dryness. ELSD A: Water / 0.05% TFA: B: CH 3 CN / 0.05% TFA 80:20-20:80 A / B, 3 min, 0.98 min hold): RT 0.98 min, m / z (calculated) 348.17, (observed) 390.30 (M+Na+H 2 O), H-NMR-PH-ARC-lipid-230-4:(300MHz,chloroform-d):δ 5.01-4.87(1H,m),3.81(2H,s),1.57(4H,m),1.34(22H,m),0.88(6H,t).

[0277] Step 2: A 100 mL three-neck round bottom flask was charged with ATX-230-1 (1.5 g, 1.0 equiv.) in THF (30 mL, 20 V). t-BuOK (1.38 g, 1.5 equiv.) was added in portions to the reaction mixture at 0° C. and stirred for 30 min. ATX-230-4 (4.3 g, 1.5 equiv.) was added to the reaction mixture in portions at 0° C. The resulting solution was stirred at room temperature for 16 h. Additional t-BuOK (1.38 g, 1.5 equiv.) and ATX-230-4 (4.3 g, 1.5 equiv.) were added to the reaction mixture at room temperature. The resulting solution was stirred at room temperature for 16 h. LCMS showed complete consumption of ATX-230-1. The reaction was then quenched by adding ammonium chloride solution (15 mL, 10 V). The resulting solution was dissolved in Et 2 O (2*30 mL, 40 V). The organic layers were combined and washed with anhydrous MgSO 4 The mixture was dried at 40° C. and then filtered. The filtrate was loaded with 3 g of silica gel (type: ZCX-2, 100-200 mesh, 2.00 w / w) and concentrated under vacuum while maintaining the temperature below 20° C. to adsorb the compound. The material was purified on a 20 g Combi-flash silica gel column using PE / EA (volume ratio, gradient 100 / 0 to 95:5) to elute the product. The fractions were pooled and concentrated under vacuum to give ATX-230-2 (2.1 g, 35.5% yield) as a yellow solid. ELSD A: Water / 0.05% TFA: B: CH 3 CN / 0.05%TFA 80:20~20:80 A / B, 3 min, 2.6 min hold): RT 2.62 min, m / z (calculated value) 718.57, (actual value) 741.50 (M+Na);H-NMR-PH-ARC-lipid-230-2:(300MHz,chloroform-d):δ 7.39-7.27(5H,m),4.99-4.93(2H,m),4.52(2H,s),4.10(4H,s),3.99-3.68(m,5H),1.53(9H,m),1.49(42H,m),1.26-1.24(12H,t).

[0278] Synthesis of ATX-230-3 [ka]

[0279] ATX-230-2 (2.1 g, 1.0 equiv.) and 20% Pd(OH) in EA (21 mL, 10 V) 2 A solution of ATX-230-3 (0.63 g, 30 wt%) was charged into an autoclave at room temperature. Stirred under hydrogen atmosphere (50 atm) at 35° C. for 16 h. TLC showed complete conversion of ATX-230-2. The reaction mixture was filtered and concentrated under vacuum at 40° C. to give ATX-230-3 (1.7 g, 95% yield) as a white solid. ELSD A: Water / 0.05% TFA: B: CH 3 CN / 0.05%TFA 80:20~20:80 A / B, 3 min, 2.6 min hold): RT 1.90 min, m / z (calculated value) 628.53, (actual value) 651.50 (M+Na);H-NMR-PH-ARC-lipid-230-3:(300MHz,chloroform-d):δ 4.99-4.91(2H,dd),4.03(4H,s),3.67-3.37(4H,m),1.56-1.49(9H,m),1.29-1.25(40H,m),0.97-0.85(12H,t).

[0280] Synthesis of ATX-230 [ka]

[0281] A solution of ATX-230-3 (1.7 g, 1.0 equiv.), 4(dimethylamino)butanoic acid hydrochloride (450 mg, 1.0 equiv.), and DMAP (198 mg, 0.6 equiv.) in DCM (34 mL, 20 V) was added to a 100 mL three-neck round-bottom flask. EDCI (620 mg, 1.2 equiv.) was added to the reaction mixture in portions at 0° C. The resulting solution was stirred at 20° C. for 16 h. The reaction was quenched with 10% aqueous citric acid (17 mL, 10 V) and the organic phase was collected. The organic solution was washed with 10% aqueous citric acid (17 mL, 10 V) followed by brine (17 mL, 10 V). The organic phase was washed with anhydrous MgSO 4The mixture was purified on a Combi-flash silica gel column (40 g) by adsorbing onto 5 g of silica gel (type: ZCX-2, 100-200 mesh, 2.94 w / w) and eluting with a 100:0 to 98:2 DCM / MeOH gradient. The product containing fractions were pooled and concentrated under vacuum to give 1.2 g (65% yield) of ATX-230 as a pale yellow oil. ELSD A: Water / 0.05% TFA: B: CH 3 CN / 0.05% TFA 80:20-20:80 A / B, 3 min, 2.6 min hold): RT 0.96 min, m / z (calculated) 741.62, (observed) 742.6 [M+1] + ;H-NMR-PH-ARC-ATX-230-0:(400MHz,CDCl 3 ,ppm)δ 5.181(quint,J=5.0Hz,1H),4.931(quint,J=6.3Hz,2H),4.081(s,4H),3.830-3.700(m,4H),2.341(dt,J=41.4,7 .4Hz,4H),2.212(s,6H),1.800(quint,J=7.4Hz,2H),1.530(d,J=3.9Hz,8H),1.25(m,40H),0.900-0.830(m,12H).

[0282] Example 8. Synthesis of ATX-231 [ka] General scheme: [ka]

[0283] Synthesis of ATX-231-1 [ka]

[0284] To a 1 L three-necked round bottom flask was added ethyl ATX-231-SM1 (50.0 g, 1.0 equiv.) and sodium iodide (180 g, 4.4 equiv.) in acetone (500 mL, 10 V). The reaction was stirred at room temperature overnight. The reaction mixture was diluted with water (400 mL, 8 V) and extracted with diethyl ether (400 mL, 8 V). The organic fraction was washed with water, dried over anhydrous magnesium sulfate, filtered, and the solvent was removed. Sodium ethoxide (10.8 g, 2.1 equiv.) was dissolved in absolute ethanol (90 mL, 2 V). Diethyl acetone dicarboxylate (36.0 g, 1.12 equiv.) was added and the solution was heated to reflux. Ethyl 6-iodocaproate (24.0 g, 1.0 equiv.) was then added slowly and the solution was refluxed for 1 h. A solution of sodium ethoxide (10.8 g, 2.1 equiv.) in ethanol (90 mL, 2 V) was added followed by ethyl 6-iodovalerate (24.0 g, 1.0 equiv.). The solution was refluxed overnight. The reaction mixture was cooled, diluted with water (400 mL, 8 V) and extracted with diethyl ether (400 mL, 8 V). Concentration in vacuo afforded 47.5 g (crude) of ATX-231-1 as a yellow oil.

[0285] Synthesis of ATX-231-2 [ka]

[0286] A 100 mL three-necked round bottom flask was charged with ATX-231-1 (40.0 g, 1.0 equiv.) in citric acid (40 mL, 1V) and HCl (80 mL, 2V, 12 mol / L). The reaction solution was refluxed overnight. The solution was cooled, diluted with water, and extracted with dichloromethane. The solvent was removed and the residue was recrystallized from acetone and dried under vacuum to give 4 g (14%) of ATX-231-2 as a white solid. ELSD A: Water / 5 mM NH 4 HCO 3 :B:CH 3 CN 80:20-90:10 A / B, 2 min): RT 0.16 min, m / z (calculated) 258.15, (observed) 257.30 [M+1] +;H-NMR-PH-ARC-ATX-231-1:(400MHz,CDCl 3 ,ppm)δ 2.5-2.49(m,2H),2.42-2.32(m,2H),2.19-2.15(m,4H),2.00-1.98(m,8H),1.51-1.47(m,4H).

[0287] Synthesis of ATX-231-3 [ka]

[0288] Step 1: [ka]

[0289] DCM (300 ml, 20V), ATX-209-5 (15 g, 1 eq.), and pyridinium chlorochromate (PCC, 40 g, 2.5 eq.) were added to a 500 ml three-neck flask. The resulting solution was stirred at room temperature for 5 h. TLC observation showed complete conversion of ATX-209-5. The solvent was removed by distillation under vacuum. The crude product was applied onto a silica gel column and the product was eluted with ethyl acetate / petroleum ether (1:10) gradient to give ATX-231-8 (13 g, 88% yield) as a colorless clear oil. 1 H NMR(300MHz,DMSO-d6) δ 2.38(t,J=7.3Hz,4H),1.53-1.36(m,4H),1.34-1.15(m,12H),0.89-0.80(m,6H).

[0290] Step 2: [ka]

[0291] THF (260 ml, 20 V) and (methoxymethy)triphenylphosphonium chloride (32 g, 1.6 equiv.) were added to a 500 ml three-neck flask, followed by t-BuOK (11.8, 1.6 equiv.) in a batch to the mixture at 0° C. Stirred at 0° C. for 1 h. ATX-231-8 (13 g, 1 equiv.) was added to the reaction mixture. Stirred at room temperature for 15 h. The system was quenched by adding aqueous ammonium chloride (10 wt.%, 260 ml, 20 V). MTBE (260 ml, 20 V) was added to extract the reaction mixture and the organic phase was collected. After concentration of the organic phase, the mixture was applied onto a silica gel column with ethyl acetate / petroleum ether (2:98). ATX-231-7 (10 g, 70% yield) was obtained as an oil. 1 H NMR (300 MHz, chloroform-d) δ 5.74 (s, 1H), 3.51 (s, 3H), 2.03 (t, J = 7.3 Hz, 2H), 1.88-1.81 (m, 2H), 1.42-1.20 (m, 16H), 0.93-0.83 (m, 6H).

[0292] Step 3: [ka]

[0293] THF (50 ml, 5V), ATX-231-7 (10 g, 1 eq.), and 6N HCl (20 ml, 2V) were added to a 250 ml three-neck flask at room temperature. Stirred at 50° C. for 5 h. 3N NaOH (40 ml, 4V) and MTBE (100 ml, 10V) were added to the reaction mixture and the product was extracted into the ether phase. The ether phase was collected and concentrated under vacuum to give ATX-231-6 (6.57 g, 71% yield) as an oil. 1 H NMR (300 MHz, chloroform-d) δ 9.49 (d, J = 3.1 Hz, 1H), 3.62 (m, 1H), 1.22 (m, 20H), 0.88-0.78 (m, 6H).

[0294] Step 4: [ka]

[0295] MeOH (65 ml, 10V) and ATX-231-6 (6.57 g, 1 eq.) were added to a 100 ml three-neck flask at room temperature. NaBH 4 (1.76, 1.5 equiv.) was added in batches to the reaction mixture at 0° C. and stirred for 2 h at 0° C. Citric acid solution (10 wt %, 65.7 ml, 10 V) was added to the reaction mixture at 0° C. The product was extracted into methyl tert-butyl ether (MTBE, 65 ml, 10 V) and the organic phase was collected and concentrated under vacuum to give ATX-231-5 (5.2 g, 79% yield) as an oil. 1 H NMR (300 MHz, chloroform-d) δ 3.53 (d, J = 5.4 Hz, 2H), 3.48 (s, 1H), 1.28 (m, 20H), 0.93-0.83 (m, 6H).

[0296] Step 5: A 250 mL three-neck round bottom flask was charged with ATX-231-2 (3.0 g, 1.0 equiv), ATX-231-5 (4.97 g, 2.0 equiv), and DMAP (1.42 g, 1.0 equiv) in DCM (60 mL, 20 V). EDCI (4.9 g, 2.2 equiv) was then added to the reaction mixture in portions at 0 °C. The resulting solution was stirred at 20 °C for 16 h, and TLC showed complete consumption of ATX-231-2. The reaction was quenched with 10% aqueous citric acid (30 mL, 10 V). The isolated organic phase was washed once more with 10% aqueous citric acid (30 mL, 10 V), followed by brine (30 mL, 10 V). The organic phase was washed with anhydrous MgSO 4 The crude product was adsorbed onto 6 g of silica gel (type: ZCX-2, 100-200 mesh, 2.00 w / w) and purified on a 30 g silica gel column using a 100:0 to 98:2 petroleum ether / ethyl acetate gradient. The eligible fractions after TLC analysis (10:1 PE:EA) were pooled and concentrated to dryness under vacuum to give 4 g (53% yield) of ATX-231-3 as a colorless oil. ELSD A: water / 0.05% TFA: B: CH 3CN / 0.05%TFA 80:20~20:80A / B 3.5 min): RT 2.89 min, m / z (calculated value) 650.58, (actual value) 673.50 (M+Na);H-NMR-PH-ARC-lipid-230-2:(300MHz,chloroform-d):δ 3.97-3.96(d,J=2.4Hz,4H),2.45-2.43(m,4H),2.38-2.28(m,4H),1.66-1.60(9H,m),1.49(48H,m),0.86-0.88(12H,t).

[0297] Synthesis of ATX-231-4 [ka]

[0298] A 100 mL three-neck flask was charged with ATX-231-3 (4.0 g, 1 equiv.) in MeOH (40 mL, 10 V) at room temperature. Then, NaBH 4 (0.34 g, 1.5 equiv.) was added to the reaction mixture in portions at 0° C. The resulting solution was stirred at 0° C. for 2 h. TLC analysis showed complete consumption of ATX-231-3. The reaction was quenched by adding water (40 mL, 10 V). The product was extracted twice with MTBE (2×20 ml, 10 V). The organic phase was washed with anhydrous MgSO 4 The mixture was dried over 1000 ml of ethyl acetate, filtered, and concentrated to dryness in vacuo to give 3.4 g (85% yield) of ATX-231-4 as a colorless oil. ELSD A: Water / 0.05% TFA: B: CH 3 CN / 0.05% TFA 80:20-20:80 A / B 3.5 min): RT 3.03 min, m / z (calculated) 652.60, (observed) 675.50 (M+Na); H-NMR-PH-ARC-Lipid-230-2: (300 MHz, chloroform-d): δ 4.00-3.98 (d, J=7.6 Hz, 4H), 3.59-3.51 (m, 1H), 2.35-2.30 (m, 4H), 1.68-1.63 (m, 6H), 1.55-1.29 (m, 55H), 0.92-0.88 (12H, t).

[0299] Synthesis of ATX-231 [ka]

[0300] A solution of ATX-231-4 (2.0 g, 1.0 equiv.), 4-(dimethyl-amino)butanoic acid hydrochloride (0.81 g, 1.6 equiv.), and DMAP (0.4 g, 1.1 equiv.) in DCM (60 mL, 30 V) was added to a 100 mL three-neck round-bottom flask. EDCI (1.0 g, 1.7 equiv.) was then added to the reaction mixture in portions at 0 °C. The resulting solution was stirred at room temperature for 16 h, and TLC showed complete consumption of ATX-231-4. The reaction was quenched with 10% aqueous citric acid (20 mL, 10 V) and the organic phase was isolated. The organic phase was washed with additional 10% aqueous citric acid (20 mL, 10 V), followed by brine (20 mL, 10 V) and anhydrous MgSO 4 The crude product was adsorbed onto 6 g of silica gel (type: ZCX-2, 100-200 mesh, 3.00 w / w.) and purified on a Combi-flash system using a 30 g silica gel column. The product was eluted with a gradient of 100:0 to 98:2 petroleum ether ethyl acetate. Fractions were analyzed (TLC, EA:PE=1:10), pooled and concentrated to dryness under vacuum to give 1.5 g (75% yield) of ATX-231 as a pale yellow oil. ELSD A: water / 0.05% TFA: B: CH 3 CN / 0.05% TFA 80:20 to 20:80 A / B 3.5 min): RT 1.90 min, m / z (calculated) 766.25, (observed) 767.23 (M+H). 1 H-NMR-PH-ARC-ATX-231-0:(300MHz,CDCl 3 ,ppm)δ 4.892-4.851(m,1H),3.988-3.969(d,J=5.8Hz,4H),2.957-2.905(t,J=8.2Hz,2H),2.713(s,6H),2.445(t,J=6.8Hz,2 H),2.308(t,J=7.4Hz,4H),2.117(quint,J=6.9Hz,2H),1.650-1.521(m,10H),1.288(bs,48H),0.921-0.900(m,12H).

[0301] Example 9. Synthesis of ATX-232 [ka] General scheme: [ka]

[0302] Synthesis of ATX-232-4:

[0303] Step 1: [ka]

[0304] Into a 250 mL three-neck round bottom flask purged and maintained with an inert nitrogen atmosphere was added Et 2 ATX-232-SM3 (10.0 g, 1.0 equiv.) in 20O (100 mL, 10 V) was added at room temperature. This was followed by LiAlH 4 (1.48 g, 1.0 equiv) was charged at 0° C. The resulting solution was stirred at room temperature for 16 h. The reaction was then quenched by adding ice water (50 mL, 5 V). The resulting solution was extracted with EA (3*200 mL, 60 V) and the organic layers were combined. The organic layers were washed with brine (2*100 mL, 20 V) and diluted with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated in vacuo to give 7.0 g (75% yield) of ATX-232-10 as a yellow oil. 1 H NMR (300 MHz, chloroform-d) δ 4.18-4.11 (m, 1H), 3.57-3.55 (d, J=8 Hz, 2H), 1.44-1.28 (m, 25H), 0.93-0.85 (m, 6H).

[0305] Step 2: [ka]

[0306] A 250 mL three-necked round bottom flask, purged and maintained with an inert nitrogen atmosphere, was charged with ATX-210-4 (5.0 g, 1.0 equiv.) and DCM (75 mL, 15 V) at room temperature. This was followed by the addition of ATX-232-10 (2.91 g, 1.0 equiv.) and DMAP (0.3 g, 0.2 equiv.) at room temperature, followed by the addition of EDCI (2.74 g, 1.2 equiv.) at 0° C. The resulting solution was stirred at room temperature for 16 hours. The reaction was then quenched by the addition of 1 mol / L aqueous HCl (25 mL, 5 V). The resulting solution was extracted with DCM (3*165 mL, 100 V) and the organic layers were combined. The organic layers were washed with brine (2*150 mL, 60 V) and anhydrous Na 2 SO 4 The crude mixture was purified on a 100 g silica gel column by adsorbing onto 10 g silica gel (type: ZCX-2, 100-200 mesh, 2.00 w / w.) and eluting with a 100:0 to 90:10 DCM / MeOH gradient. Fractions were pooled after TLC analysis. (DCM:MeOH=10:1) and concentrated under reduced pressure to give 5.5 g (72% yield) of ATX-232-4 as a yellow oil. ELSD A: water / 0.05% TFA: B: CH 3 CN / 0.05%TFA 80:20~20:80A / B 3.5 min): RT 2.81 min, m / z (calculated value) 636.57, (actual value) 659.55 (M+Na);H-NMR-PH-ARC-lipid-230-2:(300MHz,chloroform-d):δ 4.89-4.85(m,1H),3.99-3.97(d,J=8Hz,2H),2.50-2.46(m,4H),2.36-2.29(m ,4H),1.95-1.85(m,4H),1.52-1.51(6H,m),1.28(48H,m),0.91-0.84(m,12H).

[0307] Synthesis of ATX-232-5 [ka]

[0308] In a 100 mL three-neck round bottom flask purged and maintained with an inert nitrogen atmosphere, add THF / H 2 ATX-232-4 (5.5 g, 1.0 equiv.) in 20O (10 / 1, 55 mL, 10 V) was added at room temperature. This was followed by NaBH 4 (0.88 g, 2.0 equiv.) was added in several batches at 0° C. The resulting solution was stirred at room temperature for 16 h. The reaction was then quenched by adding ice water (27.5 mL, 5 V). The resulting solution was extracted with ethyl acetate (3*90 mL, 50 V) and the organic layers were combined. The organic layers were washed with brine (2*110 mL, 40 V) and diluted with anhydrous Na 2 SO 4 The crude mixture was purified on a 60 g silica gel column by adsorbing onto 11 g silica gel (type: ZCX-2, 100-200 mesh, 2.00 w / w.) and eluting with a 100:0-90:10 DCM / MeOH gradient. Fractions were pooled after TLC analysis (DCM:MeOH=10:1) and concentrated under reduced pressure to give 5.1 g (93% yield) of ATX-232-5 as a yellow oil. ELSD A: water / 0.05% TFA: B: CH 3 CN / 0.05% TFA 80:20-20:80 A / B 3.5 min): RT 2.81 min, m / z (calculated) 638.58, (observed) 661.55 (M+Na); H-NMR-PH-ARC-Lipid-230-2: (300 MHz, chloroform-d): δ 4.93-4.83(m,1H),4.00-3.98(d,J=8Hz,2H),3.66-3.62(m,12H),2.50-2.46(m,4H),2.64-2.69(m,4 H),1.88-1.85(m,6H),1.95-1.85(m,4H),1.58-1.52(m,7H),1.47-1.44(m,44H),0.96-0.88(m,12H).

[0309] Synthesis of ATX-232 [ka]

[0310] A 100 mL three-necked round bottom flask purged and maintained with an inert nitrogen atmosphere was charged with ATX-232-5 (5.1 g, 1.0 equiv.) in DCM (80 mL, 15 V) at room temperature. This was followed by the addition of ATX-232-7 (1.6 g, 1.2 equiv.) and DMAP (0.21 g, 0.2 equiv.) at room temperature, followed by the addition of EDCI (1.92 g, 1.2 equiv.) at 0° C. The resulting solution was stirred at room temperature for 16 h. The reaction was then quenched by the addition of ice water (25 mL, 5 V). The resulting solution was extracted with DCM (3*80 mL, 50 V) and the organic layers were combined. The organic layers were washed with brine (2*100 mL, 40 V) and anhydrous Na 2 SO 4 The crude mixture was purified on a 60 g silica gel column by adsorbing onto 11 g silica gel (type: ZCX-2, 100-200 mesh, 2.00 w / w.) and eluting with a 100:0 to 90:10 DCM / MeOH gradient. The fractions were pooled after TLC analysis (DCM:MeOH=10:1) and concentrated under reduced pressure to give 1.1 g (18.3% yield) of ATX-232 as a yellow oil. LC-MS-PH-ARC-ATX-232-0: (ES, m / z): 752 [M+1] + ;H-NMR-PH-ARC-ATX-232-0:(300MHz,CDCl 3 ,ppm):δ 4.997-4.858(m,2H),3.983(d,J=5.7Hz,2H),2.386-2.261(m,6H),2.261(s,6H),1.8 23(quint,J=7.2Hz,2H),1.799-1.512(m,13H),1.289(s,46H),0.922-0.882(m,12H).

[0311] Example 10. Biological data of the compounds of the present invention To evaluate the effectiveness of the lipids of the present disclosure, various assays were performed. A description of these assays follows.

[0312] Protocol for evaluating factor VII knockdown Lipid formulations containing FVII siRNA, as described further below, were evaluated for knockdown activity using the protocol of this example. For FVII evaluation, 7-8 week old female Balb / C mice were purchased from Charles River Laboratories (Hollister, CA). Mice were kept in a pathogen-free environment and all procedures involving mice were performed in accordance with guidelines established by the Institutional Animal Care and Use Committee (IACUC). Lipid nanoparticles containing Factor VII siRNA were administered intravenously at a dose volume of 10 mL / kg and two dose levels (0.03 mg / kg and 0.01 mg / kg). After 48 hours, mice were anesthetized with isoflurane and bled retro-orbitally into Microtainer® tubes coated with 0.109 M sodium citrate buffer (BD Biosciences, San Diego, CA) and processed to plasma. Plasma specimens were either tested immediately for Factor VII levels or stored at -80°C for later analysis. Measurement of FVII protein in plasma was determined using a colorimetric Biophen VII assay kit (Aniara Diagnostica, USA). Absorbance was measured at 405 nm and a calibration curve was generated using serially diluted control plasma to determine levels of factor VII in plasma from treated animals compared to saline-treated control animals.

[0313] Protocol for evaluating hEPO mRNA expression The following lipid formulations containing hEPO mRNA were evaluated for their ability to express hEPO in vivo according to the protocol of this example. All animal experiments were performed using an institutionally approved protocol (IACUC). For this protocol, female Balb / c mice at least 6-8 weeks old were purchased from Charles River Laboratory. Mice were intravenously injected via the tail vein with hEPO-LNPs with one of two dose levels of hEPO (0.1 and 0.03 mg / kg). After 6 hours, blood was collected in serum separator tubes and serum was isolated by centrifugation. Serum hEPO levels were then measured using an ELISA assay (Human Erythropoietin Quantikine IVD ELISA Kit, R&D Systems, Minneapolis, MD).

[0314] Mouse plasma stability A lipid stock solution was prepared by dissolving lipids in isopropanol at a concentration of 5 mg / mL. The required volume of lipid-isopropanol solution was then diluted in 50:50 (v / v) ethanol / water to a concentration of 100 μM in a total volume of 1.0 mL. Ten microliters of this 100 μM solution was added to 1.0 mL of mouse plasma (BioIVT, Catalog Number: MSE00PLNHUNN, CD-1 mouse, Anticoagulant: Sodium Heparin, Unfiltered) pre-warmed to 37° C. and stirred at 50 rpm with a magnetic stir bar. Thus, the starting concentration of lipid in plasma was 1 μM. At 0, 15, 30, 45, 60, and 120 min, 0.1 mL of plasma was removed from the reaction mixture and proteins were precipitated by adding 0.9 mL of ice-cold 4:1 (v / v) acetonitrile / methanol spiked with 1 μg / mL of selected internal standard lipid. After filtration through a 0.45 micron 96-well filtration plate, the filtrate was analyzed by LC-MS (Vanquish UHPLC-LTQ XL linear ion trap Mass Spectrometer from Thermo Fisher) on a Waters XBridge BEH Shield RP18 2.5 μm (2.1 × 100 mm) column with its matching guard column. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in 1:1 (v / v) acetonitrile / methanol. The flow rate was 0.5 min / min. The elution gradient was: time 0–1 min: 10% B, 1–6 min: 10%–95% B, 6–8.5 min: 95% B, 8.5–9 min: 95%–10% B, 9–10 min: 10% B. Mass spectrometry was in positive scan mode from 600 to 1100 m / z. Lipid molecular ion peaks were integrated into extracted ion chromatography (XIC) using Xcalibur software (Thermo Fisher). Relative peak areas compared to T=0 were used as the percentage of lipid remaining at each time point after normalization with the peak area of ​​the internal standard. 1 / 2 Values ​​were calculated using a first-order decay model.

[0315] In vivo biodegradation assay An in vivo biodegradation assay was performed to evaluate the biodegradability of lipids in LNPs. Briefly, mice were injected with either 0.1 or 0.03 mg / Kg doses, and mouse livers were harvested 24 or 48 hours later. To measure the concentration of lipids in mouse livers, liver samples were homogenized in 1-10 dilutions of appropriate buffer and mixed with an equal volume of stabilized plasma. Samples were then mixed with organic solvent spiked with internal standards to precipitate proteins. After centrifugation, the supernatants were further diluted with organic solvent before the samples were analyzed by LC-MS. For LC-MS analysis, positive electrospray ionization was used, and multiple reaction monitoring (MRM) parameters were set to specifically target lipid analytes and internal standards. Calibration standards were prepared in stabilized plasma and mixed with an equal volume of homogenization buffer prior to protein precipitation. Quality control samples with known amounts of lipids were prepared in blank liver homogenates to monitor the precision and accuracy of the assay. [Table 1] [Table 2]

[0316] Compound-10111 is shown below and listed on page 243 of WO2021 / 030701. [ka]

[0317] Table 3 below shows the calculated LogD (cLogD) and calculated pKa (cpKa) of ATX compounds, with the measured pKa in parentheses. The cLogD and cpKa values ​​are generated by ACD Labs Structure Designer v12.0. [Table 3]

[0318] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, those skilled in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. Furthermore, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. In the event of a conflict between this application and a reference provided herein, this application shall control.

Claims

1. Formula I: 【Chemistry 1】 [During the ceremony, R １ and R ２ Each independently represents (CH ３ (CH ２ ) ｍ ) ２ CH-, (CH ３ (CH ２ ) ｍ ) (CH ３ (CH ２ ) ｍ－１ )CH, (CH ３ (CH ２ ) ｍ ) (CH ３ (CH ２ ) ｍ－２ )CH, (CH ３ (CH ２ ) ｍ ) ２ CHCH ２ - or (CH ３ (CH ２ ) ｍ ) (CH ３ (CH ２ ) ｍ－１ ) CHCH ２ -, m is 4 to 11, L １ and L ２ are each independently absent or a linear C １－５ Alkylene, or (CH ２ ) ｐ -O-(CH ２ ) ｑ p and q are each independently 1 to 3; R ３ is a linear C optionally substituted with one or two methyl groups ２－５ is alkylene, R ４ and R ５ are each independently H or C １－６ is alkyl, X is O or S; n is 0 to 2. or a pharma- ceutically acceptable salt thereof.

2. R １ and R ２ Each independently represents (CH ３ (CH ２ ) ｍ ) ２ CH-, and (CH ３ (CH ２ ) ｍ ) ２ CHCH ２ 2. The compound of claim 1, selected from:

3. R １ and R ２ Each independently represents (CH ３ (CH ２ ) ｍ ) (CH ３ (CH ２ ) ｍ－１ )CH, (CH ３ (CH ２ ) ｍ ) (CH ３ (CH ２ ) ｍ－２ ) CH, and (CH ３ (CH ２ ) ｍ ) (CH ３ (CH ２ ) ｍ－１ ) CHCH ２ 2. The compound of claim 1, selected from:

4. 2. The compound according to claim 1, or a pharma- ceutically acceptable salt thereof, wherein m is 4 to 8.

5. L １ and L ２ Each independently, C ２－５ Alkylene or (CH ２ ) ｐ -O-(CH ２ ) ｑ 2. The compound of claim 1, wherein:

6. L １ and L ２ or a pharma- ceutically acceptable salt thereof. The compound of claim 1, or a pharma- ceutically acceptable salt thereof, wherein each of

7. R ３ But, C ３－５ 2. The compound of claim 1, or a pharma- ceutically acceptable salt thereof, which is alkylene.

8. R ４ and R ５ Each independently, C １－６ 2. The compound of claim 1, or a pharma- ceutically acceptable salt thereof, wherein R is an alkyl group.

9. The compound according to claim 1, or a pharma- ceutically acceptable salt thereof, wherein n is 0 to 1.

10. 【Chemical 2】 【Chemistry 3】 2. The compound of claim 1, or a pharma- ceutically acceptable salt thereof, selected from the group consisting of: and pharma- ceutically acceptable salts thereof.

11. The compound is ATX-193: 【Chemistry 4】 11. The compound of claim 10, wherein:

12. The compound is ATX-200: 【Chemistry 5】 11. The compound of claim 10, wherein:

13. The compound is ATX-201: 【Chemistry 6】 11. The compound of claim 10, wherein:

14. The compound is ATX-202: 【Chemistry 7】 11. The compound of claim 10, wherein:

15. The compound is ATX-209: 【Chemistry 8】 11. The compound of claim 10, wherein:

16. The compound is ATX-210: 【Chemistry 9】 11. The compound of claim 10, wherein:

17. The compound is ATX-230: 【Chemistry 10】 11. The compound of claim 10, wherein:

18. The compound is ATX-231: 【Chemistry 11】 11. The compound of claim 10, wherein:

19. The compound is ATX-232: 【Chemistry 12】 11. The compound of claim 10, wherein:

20. A lipid composition comprising a nucleic acid and the compound according to any one of claims 1 to 19 or a pharma- ceutically acceptable salt thereof.

21. 21. The lipid composition of claim 20, wherein the nucleic acid is selected from siRNA, mRNA, self-replicating RNA, DNA plasmids, and antisense oligonucleotides.

22. 21. The lipid composition of claim 20, wherein the nucleic acid is an mRNA or a self-replicating RNA comprising a coding region encoding a therapeutic protein of interest.

23. 23. The lipid composition of claim 22, wherein the therapeutic protein of interest is an enzyme, an antibody, an antigen, a receptor, or a transporter.

24. The lipid composition of claim 22, wherein the therapeutic protein of interest is a gene editing enzyme.

25. The lipid composition of claim 24, wherein the gene editing enzyme is selected from a TALEN, a CRISPR, a meganuclease, and a zinc finger nuclease.

26. 21. The lipid composition of claim 20, wherein the lipid composition comprises a liposome, a lipoplex, or a lipid nanoparticle.

27. The lipid composition of claim 26, wherein the lipid composition comprises lipid nanoparticles encapsulating the nucleic acid.

28. 28. The lipid composition of claim 27, wherein the lipid nanoparticles have an average particle size of less than about 100 nm.

29. 28. The lipid composition of claim 27, wherein the average particle size of the lipid nanoparticles is from about 55 nm to about 85 nm.

30. 28. The lipid composition of claim 27, wherein the lipid nanoparticles further comprise a helper lipid selected from dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylcholine (DMPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), dipalmitoylphosphatidylcholine (DPPC), and phosphatidylcholine (PC).

31. The lipid composition of claim 30, wherein the helper lipid is distearoylphosphatidylcholine (DSPC).

32. 28. The lipid composition of claim 27, further comprising cholesterol.

33. 28. The lipid composition of claim 27, further comprising a polyethylene glycol (PEG)-lipid conjugate.

34. The lipid composition of claim 27, wherein the lipid nanoparticle comprises about 45 mol% to 65 mol% of the compound of any one of claims 1 to 19 or a pharma- ceutically acceptable salt thereof, 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.

35. 28. The lipid composition of claim 27, wherein the lipid nanoparticles have a total lipid:nucleic acid weight ratio of about 50:1 to about 10:

1.

36. A medicament comprising the lipid composition of claim 27 for treating a disease in a subject in need of such treatment.

37. The pharmaceutical composition described in claim 36, which is administered intravenously or intramuscularly.

38. A pharmaceutical composition for expressing a protein or polypeptide in a target cell, comprising the lipid composition according to claim 27, contacting said target cells; The said medicine.

39. 39. The method of claim 38, wherein the protein or polypeptide is an antigen, and expression of the antigen provides an in vivo immunogenic response.

40. A pharmaceutical for delivering a nucleic acid to a subject in need thereof, comprising the lipid composition of claim 27.

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